TRANSCRIPTIONAL REGULATION OF EARLY PROGENITOR COMPETENCE IN
THE DROSOPHIIA CENTRAL NERVOUS SYSTEM
by
KHOA DANG TRAN
A DISSERTATION
Presented to the Department of Biology
and the Graduate School of the University of Oregon
in partial fulfillment of the requirements
for the degree of
Doctor of Philosophy
September 2010
11
University of Oregon Graduate School
Confirmation of Approval and Acceptance of Dissertation prepared by:
Khoa Tran
Title:
"Transcriptional Regulation of Early Progenitor Competence in the Drosophila Central Nervous
System"
This dissertation has been accepted and approved in partial fulfillment ofthe requirements for
the Doctor of Philosophy degree in the Department of Biology by:
Victoria Herman, Chairperson, Biology
Christopher Doe, Advisor, Biology
Judith Eisen, Advisor, Biology
Charles Kimmel, Member, Biology
Hui Zong, Member, Biology
Kenneth Prehoda, Outside Member, Chemistry
and Richard Linton, Vice President for Research and Graduate Studies/Dean of the Graduate
School for the University of Oregon.
September 4,2010
Original approval signatures are on file with the Graduate School and the University of Oregon
Libraries.
© 2010 Khoa Dang Tran
III
iv
An Abstract of the Dissertation of
Khoa Dang Tran for the degree of Doctor of Philosophy
in the Department of Biology to be taken September 2010
Title: TRANSCRIPTIONAL REGULATION OF EARLY PROGENITOR
COMPETENCE IN THE DROSOPHILA CENTRAL NERVOUS SYSTEM
Approved:
Dr. Christopher Doe, Advisor
Neurogenesis in Drosophila and mammals requires the precise integration of
spatial and temporal cues. In Drosophila, embryonic neural progenitors, called
neuroblasts, sequentially express the transcription factors Hunchback, Kruppel,
Pdml/Pdm2 (Pdm) and Castor as they divide to generate a stereotyped sequence of
neuronal and glial progeny. Hunchback is necessary and sufficient to specify the first-
born cell identity in many neuroblast lineages. Additionally, Hunchback is able to
maintain an early-competence state in which early-born cells are generated. Furthermore,
the Hunchback mammalian ortholog, Ikaros, possesses a similar ability to specify early-
born cells in the vertebrate nervous system. However, the mechanisms underlying the
function of HunchbacklIkaros are unknown.
vPdm and Castor are expressed later in many neuroblasts and can specify late-born
neuronal cell identities in a model neuroblast lineage, NB7-1. Previous work studying
their function in the .t\TB7-1 lineage showed that Pdm and Castor act as repressors of
Kruppel gene expression and inhibit the generation of the Kruppel-dependent cell
identity. It is not known if the functions of Pdm and Castor are conserved across multiple
neuroblast lineages during neurogenesis or whether these factors impart any restrictions
on the ability of a factor like Hunchback to maintain early competence.
To investigate the transcriptional mechanisms regulating early neuroblast
competence in Drosophila, I have focused my dissertation research on two aims. The
first is to examine the function of Pdm and Castor across multiple neuroblast lineages to
characterize their potential roles as competence restricting factors; the second is to
determine how Hunchback maintains early neuroblast competence and specifies early-
born cell identities (e.g. as a transcriptional activator, repressor, or both). My work
demonstrates that Pdm and Castor control the timing of Kruppel gene expression, and
possibly the timing of other genes, in neuroblasts. Furthermore, I have shown that
Hunchback acts as a transcriptional repressor of multiple target genes, including pdm and
castor, to maintain early neuroblast competence. Because Hunchback must repress at
least one additional unknown factor that can restrict neuroblast competence, I have
piloted a screen to identify and characterize novel Hunchback target genes in the nervous
system.
This dissertation includes previously published and unpublished co-authored
materials.
VI
CURRICULUM VITAE
NAME OF AUTHOR: Khoa Dang Tran
GRADUATE AND UNDERGRADUATE SCHOOLS ATTENDED:
University of Oregon, Eugene, Oregon
University of California, Berkeley, California
DEGREES AWARDED:
Doctor of Philosophy in Biology, 2010, University of Oregon
Bachelor of Science in Bio-Engineering, 2005, University of California, Berkeley
AREAS OF SPECIAL INTEREST:
Stem Cell Biology
Developmental Biology
Cell Biology
PROFESSIONAL EXPERIENCE:
Graduate Research Fellow, Department of Biology, University of Oregon,
Eugene, Oregon, 2005-2010
Graduate Teaching Fellow, Department of Biology, University of Oregon,
Eugene, Oregon, 2005-2006
Undergraduate Research Associate, Dr. Nipam Patel Lab, University of
California, Berkeley California, 2003-2005
Undergraduate Research Associate, Dr. Michael Levine Lab, University of
California, Berkeley California, 2002-2003
Vll
GRANTS, AWARDS AND HONORS:
American Heart Association Predoctoral Fellowship, 2009
Genetics Training Grant, National Institutes of Health (NIH), University of
Oregon, 2008
Integrative Graduate Education and Research Traineeship (IGERT) Associate
Award, National Science Foundation (NSF), University of Oregon, 2007
Howard Hughes Medical Institute (HHMI) Predoctoral Funding, University of
Oregon, 2006
Howard Hughes Medical Institute (HHMI) Summer Research Traineeship,
University of California, Berkeley, 2004 & 2005
PUBLICATIONS:
Tran, K D., Miller, M.R., and Doe, C.Q. (2010). Recombineering Hunchback
identifies two conserved domains required to maintain neuroblast competence
and specify early-born neuronal identity. Development 137,1421-30.
Liubicich, D.M., Serano, J.M., Pavlopoulos, A., Kontarakis, Z., Protas, M.E.,
Kwan, E., Chatterjee, S. Tran, KD., Averof, M., and Patel, N.H. (2009).
Knockdown of Parhyale Ultrabithorax recapitulates evolutionary changes in
crustacean appendage morphology. Proc Natl Acad Sci USA 106, 13892-6.
Tran, KD. and Doe, C.Q. (2008). Pdm and Castor close successive temporal
identity windows in the NB3-1Iineage. Development 135, 3491-9.
Lee, C.Y., Andersen, R.O., Cabernard, C., Manning, L., Tran, KD., Lanskey,
MJ., Bashirullah, A., and Doe, C.Q. (2006). Drosophila Aurora-A kinase
inhibits neuroblast self-renewal by regulating aPKClNumb cortical polarity
and spindle orientation. Genes Dev 20,3464-74.
Vlll
ACKNOWLEDGMENTS
I would like to sincerely thank my advisor, Dr. Chris Q. Doe, for all of his support
during the course of my graduate training at the University of Oregon. His patience and
generosity gave me the freedom to explore my research interests, and his guidance kept
me focused on my development as a young scientist. I would also like to thank all Doe
lab members, past and present, for providing an exciting work environment that nurtures
both academic and personal growth. In particular, I would like to thank Drs. Jason
Boone, Yves Chabu, Minoree Kohwi, and Sen-Lin Lai for the intellectually stimulating
conversations over the years. I will miss having you all to discuss "hot off the confocal"
results. Furthermore, I would also like to thank my co-advisor Dr. Judith Eisen for the
many opportunities to present my work to her and the members of her lab, whose
comments, suggestions, and perspectives in the field of neurobiology will always be with
me. In addition, I would like to thank Janet Hanawalt, Peg Morrow, Ellen McCumsey
and Donna Overall for all of their help in keeping me sane during my graduate career by
taking care of all administrative issues including, but not limited to, making sure I meet
deadlines that I get paid each month. I would like to thank the National Institutes of
Health, the Howard Hughes Medical Institute, and the American Heart Association for
funding this research. Lastly, I would like to thank my family and friends for their
support over the years. Without you all, this would not be possible.
ix
I dedicate my dissertation and all of my scientific publications to my parents, who taught
me the values of hard work, and whose many sacrifices made all of my achievements
possible.
Toi danh lu~n an nay va t~t Cll cac ~n phAm khoa hQc cua toi cho cha m(f toi, nguai da dSlY
cho Wi nhtIng gia tri cua vi~c lam kh6 khan, va c6 nhieu S\f hy sinh de cho Wi c6 t~t Cll
cac thanh tich t6t hom nay.
xTABLE OF CONTENTS
Chapter Page
I. INTRODUCTION TO THE DROSOPHILA CENTRAL NERVOUS SYSTEM AS
A MODEL FOR TEMPORAL REGULATION OF NEURAL
PROGENITORS 1
II. PDM AND CASTOR ARE TIIVIING ELEMENTS THAT CLOSE SUCESSIVE
TEMPORAL IDENTITY WINDOWS IN THE NEUROBLAST 3-1
LINEAGE 10
Introduction 10
Materials and Methods. 13
Results 15
Discussion............................................................................................................... 27
III. HUNCHBACK REPRESSES MlJLTIPLE DOWN-STREAM FACTORS TO
MAINTAIN NEUROBLAST COMPETENCE AND SPECIFY EARLY-
BORN NEURONAL IDENTITy..... 39
Introduction 39
Materials and Methods........................................................................................... 41
Results..................................................................................................................... 45
Discussion............................................................................................................... 62
IV. IDENTIF1CATION OF NOVEL HUNCHBACK TARGET GENES THAT ARE
CANDIDATES FOR RESTRICTING EARLY NEUROBLAST
COMPETENCE AND SPECIFY LATE-BORN NEURONAL IDENTITIES... 71
Introduction.................................................................................... 71
Materials and Methods......................... 73
Results..................................................................................................................... 75
Discussion............................................................................................................... 76
V. CONCLUSIONS 80
Chapter
xi
Page
APPENDICES
A. SUPPLEMENTAL MATERIALS FOR CHAPTER II 83
B. SUPPLEMENTAL MATERIALS FOR CHAPTER III 88
C. SUPPLEMENTAL MATERIALS FOR CHAPTER N 91
REFERENCES 97
xu
LIST OF FIGURES
Figure Page
Chapter II
1. Temporal identity gene expression in NB3-1............................................ 18
2. Molecular markers and cell position can be used to identify each RP motor
neuron in the NB3-1 lineage 19
3. Hb is necessary and sufficient to specify the first temporal identity................ 22
4. Kr is necessary and sufficient to specify the second temporal identity............ 23
5. Pdm closes the second temporal identity window in the NB3-1Iineage.......... 31
6. Cas closes the third temporal identity window in the NB3-1 lineage 33
7. Hb and Kr specify early temporal identity, whereas Svp, Pdm, and Cas act
as timer elements within the NB3-1Iineage............................................... 38
Chapter III
1. VPI6::Hb acts as a constitutive transcriptional activator 47
2. VPI6::Hb activates Hb direct and indirect targets in the CNS......................... 53
3. Hb maintains early neuroblast competence through the repression of
multiple target genes 56
4. The Hunchback D and DMZ domains are required for transcriptional
repression and maintenance of neuroblast competence.............................. 59
5. The Hb D and DMZ domains are required for the first-temporal identity....... 64
6. Models for Hb mediated transcriptional regulation of neuroblast
competence................................................................................................. 68
Chapter IV
1. Cluster analysis identifies potential Hb CNS targets........................................ 78
APPENDIX A
S1. Misexpression ofHb generates ectopic RPIIRP4 motor neurons and a
thicker motor nerve root............................................................................. 84
S2. Ectopic IsI+ HB9+ cells that are superficial to RP5 in cas mutant embryos
do not express the motor neuron marker, Late Bloomer 86
S3. NB7-3 progeny are unaffected inpdm mutants and after Pdm
misexpression 87
APPENDIXB
S1. The D and DMZ domains are required for Hb repression of CNS targets ....... 89
S2. Overexpression of HbLill and Hb LillMZ does not alter U neuron identities......... 90
APPENDIXC
S1. Distribution of candidate genes based on established expression database ..... 91
xiii
LIST OF TABLES
Table Page
Chapter II
1. Summary of phenotypes in the NB3-1lineage.................................................. 21
Chapter III
1. Summary of U neuron identity specified by Hb proteins 63
APPENDIXC
S1. Candidate Hb-target gene cluster......... 92
1CHAPTER I
INTRODUCTION TO THE DROSOPHILA CENTRAL NERVOUS SYSTEM AS A
MODEL FOR TEMPORAL REGULATION OF NEURAL PROGENITORS
"The brain is a wonderfUl organ. It starts working the moment
you get up in the morning and does not stop
until you get into the office. "
-- Robert Frost
INTRODUCTION
Background on temporal regulation of vertebrate neural stem cells during nervous
system development
Normal development of the central nervous system (CNS) depends on both the
spatial patterning of progenitor domains, as well as the tempo at which individual
progenitors generate distinct subtypes of neurons and glia (Berry and Rogers, 1965;
Cepko, 1999; Doe and Skeath, 1996; Harris, 1997; Livesey and Cepko, 2001; Rapaport et
aI., 2001; Reid et aI., 1997; Walsh and Reid, 1995). In the mammalian cerebral cortex,
2individual neural stem cells generate progeny capable of populating all laminar layers
(Reid et al., 1997; Walsh and Reid, 1995). Birth-dating studies revealed that each layer is
occupied by neurons of similar birth-order, so that early-born neurons occupy the deepest
layers and late-born neurons occupy the most superficial layers (McConnell, 1995). It has
been shown that the transcription factor Foxg1 is required at a precise moment during
neural stem cell proliferation to repress the first-born cell fates and allow progenitors to
specify later-born cells in the lineage (Hanashima et aI., 2007; Hanashima et aI., 2004).
These findings highlight the importance of temporal cues during the development and
diversification of the mammalian nervous system.
Background on nervous system development in Drosophila
Neural stem cells in the Drosophila embryonic nerve cord, called neuroblasts,
delaminate from the epithelium to the interior of the embryo marking the start of neural
differentiation. Individual neuroblasts can be identified based on the time at which they
are formed, their position within each hemisegment (for example, NB7-1 is positioned in
the seventh row, first column of the neuroblast array), and their pattern of gene
expression (Broadus et aI., 1995; Doe, 1992). In addition, each neuroblast generates a
unique and invariant cell lineage (Bossing et aI., 1996; Karcavich and Doe, 2005; Lundell
and Hirsh, 1998; Pearson and Doe, 2003; Schmid et aI., 1999; Schmidt et aI., 1997)
resulting from a series of asymmetric cell divisions where neuroblasts "bud-off' ganglion
mother cells (OMCs) that typically undergo an additional division to produce two post-
mitotic neurons. These neurons can differentiate as motoneurons, interneurons, or glia;
3and all three cell types can arise from a single parent neuroblast. The unique cell lineage
that is specified by each individual neuroblast depends on the identity of that neuroblast;
and that identity is conferred by positional information and spatial gene expression
(Broadus et aI., 1995; Doe, 1992). While the spatial patterning cues that give rise to
individual progenitor identity has been well characterized, less is known about the
regulation of these progenitors over time.
In this dissertation, I discuss the mechanisms that promote the production of
diverse cell identities by single progenitors over time. I will show that unique temporal
identities are a product of well orchestrated interactions between temporal identity factors
and timing factors; and that these timely interactions are critical to the normal
development of the Drosophila central nervous system.
Background on temporal regulation ofDrosophila neural progenitors
Each of the 30 neuroblasts in a hemisegment divides to produce an invariant order
of neurons and/or glia whose identity is determined by the sequential expression of the
transcription factors: Hunchback (Hb) ~ Krupple (Kr) ~ Pdm1 (Nubbin, Flybase)/Pdm2
(henceforth Pdm) ~ Castor (Cas) ~ Grainyhead (Grh) (Baumgardt et al., 2009; Isshiki
et aI., 2001; Maurange et aI., 2008). In this manner, neurons resulting from first-born
GMC division are pushed to deep positions in the CNS and express molecular markers
for early-born cells, while later-born neurons occupy more ventral positions and express
late-born fate markers (Isshiki et aI., 2001). The early- and late-born markers are
excellent candidates for genes that specify cell identity based on birth-order, also called
4temporal identity genes (Grosskortenhaus et al., 2006; Isshiki et aI., 2001; Novotny et aI.,
2002; Pearson and Doe, 2003).
Two transcription factors are known to have critical roles in specifying temporal
identity: Hunchback (Hb) and Kriippel (Kr). Hb is anlkaros-type zinc finger protein
expressed in newly formed neuroblasts and in their early-born GMCs and neuronal
progeny; it is necessary and sufficient to specify the first temporal identity in multiple
neuroblast lineages (Cleary and Doe, 2006; Isshiki et aI., 2001; Kambadur et aI., 1998;
Novotny et al., 2002; Pearson and Doe, 2003). Note that we define the "first" temporal
identity as the neuronal fates specified dming the window of Hb expression; this can be
just one GMC and its sibling neurons as in the NB7-3 lineage, or two GMCs and their
neuronal progeny as in the NB7-1Iineage. In the latter case, high Hb specifies the fIrst
GMC/Ul neuron fate, and low Hb specifIes the second GMC/U2 fate (reviewed in
Pearson and Doe, 2004). Kr is a zinc fmger protein that is detected at low levels together
with Hb, and at high levels in neuroblasts and their progeny immediately following Hb
down-regulation; it is necessary and suffIcient to specify the second temporal identity in
both the NB7-1 and NB7-3 lineages (lsshiki et al., 2001). We defIne the second temporal
identity to be that following the Hb-dependent fIrst temporal identity; this can be the
second-born or the third-born GMC in a lineage.
It is unknown what factors specify the temporal identity that comes after Kruppei.
In NB7-1, Pdm and Castor specify the U4 neuronal identity, and Castor alone specifies
the U5 neuronal identity. It is not known whether Pdm and Castor are specifying
successive temporal identities following the Kruppel-dependent second temporal identity,
5or whether they have a lineage specific role of specifying the U4 and U5 neurons in NB7-
1. Furthermore, to classify Pdm and Castor as bonafide temporal identity factors, they
must specify the same temporal identity in multiple neuroblast lineages. In Chapter II, I
present and discuss results from my investigation of Pdm and Castor function in a
neuroblast lineage where the temporal identity of progeny neurons have never been
characterized, NB3-l. My work shows that Pdm and Castor act as timing factors that
regulate the gene expression window of factors that precede them in many neuroblast
lineages.
Background on the regulation of early neuroblast competence in Drosophila
Small pools of multipotent neural progenitors give rise to a large number of
neurons and glia to allow proper assembly of a functional nervous system (Cepko, 1999;
Doe and Skeath, 1996; Rapaport et al., 2001; Walsh and Reid, 1995). However, as
progenitors change over time to accommodate the production of different tissues, they
also undergo a progressive restriction and lose their competence to produce the full
assortment of cell types (Desai and McConnell, 2000; Rapaport et aI., 2001). The ability
to maintain progenitors in their early competent states yields the capacity to generate any
desired tissues for use in future cell therapy applications. In recent years, substantial
progress has been made in the identification of new factors involved in regulating
progenitor competence, bringing with it the need to understand the molecular basis
underlying their function.
6The identification of genes regulating neural progenitor competence in both
vertebrates and insects has provided an entry point for investigating the molecular
mechanism of progenitor competence (Elliott et aI., 2008; Hanashima et aI., 2004; Isshiki
et aI., 2001; Novotny et aI., 2002). The zinc-finger transcription factor lkaros (lk) is both
necessary and sufficient to specify early-progenitor competence, leading to the
production of early-born cell types in the mouse retina (Elliott et aI., 2008). This function
of lkaros mimics that of its Drosophila ortholog, the zinc-finger transcription factor
Hunchback (Hb), which has also been shown to promote early progenitor competence
during Drosophila neurogenesis (Isshiki et al., 2001; Novotny et aI., 2002; Pearson and
Doe, 2003).
Hb is expressed early in many neuroblasts and is required for the specification of
the first-born cell identity, or first-temporal identity, in those lineages. In addition, Hb
can also confer the early-competence state where early-born progeny are specified in
many neuroblast lineages. Interestingly, the ability of Hb to specify and extend the early-
competence window declines over time (Cleary and Doe, 2006; Pearson and Doe, 2003).
When Hb is reintroduced to NB7-1 at progressively later time points, its ability to specify
ectopic U1/U2 neurons is greatly reduced. Eventually, NB7-1 is unable to respond to Hb
to specify early-born cells after the fifth neuroblast division. It is possible that some
unknown factor or factors expressed later in the neuroblast lineage may act to advance
neuroblast programming beyond the early-competence state where the neuroblast
normally specifies early-born cell identities. Two likely candidates that may function to
restrict early neuroblast competence are Pdm and Castor, as they are expressed later in
7many neuroblast lineages; and in NB7-1, they are expressed around the time when the
neuroblast loses the ability to respond to Hb. In NB7-1, the lost of Castor leads to the
specification of ectopic US neurons and extends the window of motoneurons production.
This provides some evidence that Castor is required to properly advance NB7-1 from a
motoneurons producing state to an interneuron producing state. In Chapter II, I
characterize the roles of Pdm and Castor in the NB3-1lineage and provide more evidence
suggesting that Pdm and Castor can impose restrictions on the ability of a neuroblast to
specify early-born cell identities.
While it is known that Hb can maintain progenitor competence in the nervous
system, the molecular mechanisms underlying its function remains unknown. Hunchback
is a transcription factor previously characterized to have both activator and repressor
activities in the early fly embryo (Kraut and Levine 1991; Schulz and Tautz 1994;
Papatsenko and Levine 2008). During the first few hours of development, very high
levels of Hb repress Kr, while low levels activate Kr. Hb can also repress the posterior
gap genes Knirps (Kni) and Giant (Gt), at low concentrations. While the mechanisms of
Hb-mediated gene regulation in the early embryo have been well characterized, the
mechanisms underlying Hb function in the CNS have yet to be described. In Chapter III,
I investigate the mechanisms of Hb-mediated gene regulation in the nervous system, and
address the following questions: First, how does Hb regulate gene expression to maintain
early-neuroblast competence? And second, why does this ability decline over time? I
present and discuss the results of my investigation, and show that Hb must repress
multiple late-expressing genes in order to maintain early neuroblast competence. One
8such target is the previously discussed gene, Pdm. My work suggests that factors such as
Pdm can advance neuroblast timing beyond the early-competence window, and that Hb
must normally act as a repressor of these factors to create a unique window of
opportunity for neuroblasts to specify early-born cells.
For results presented in the proceeding chapters, Khoa D. Tran and Chris Q. Doe
designed research; Khoa D. Tran performed research; Khoa D. Tran and Chris Q. Doe
analyzed data. Michael R. Miller provided new reagents for the work presented in
Chapter III.
Bridge to Chapter n
In the following chapter, I present the findings from my investigation of the roles
of Pdm and Castor in specifying neuronal identity during early neurogenesis in the
Drosophila embryo. Because Pdm and Castor have only been assayed in one lineage, it
is unknown whether their function is restricted to only the NB7-1 lineage, or whether
they function more broadly as late temporal identity genes in all neuroblast lineages. To
investigate the roles of Pdm and Castor outside of the NB7-1 lineage, I identify the
neuronal birth-order and molecular markers within the NB3-1 cell lineage, and then use
this lineage to assay Pdm and Castor function. I show that Hunchback and Kruppe1
specify first and second temporal identities in the NB3-1 lineage, similar to their roles in
other neuroblast lineages; surprisingly, Pdm does not specify the third temporal identity,
but rather acts as a timing factor to close the second temporal identity window. Similarly,
Castor closes the third temporal identity window. This work lead us to conclude that
Hunchback/Kruppel specify the first/second temporal identity, an unknown factor
specifies the third temporal identity, and PdmlCastor are timing factors that close the
second/third temporal identity windows in the NB3-1 lineage. These results provide a
new neuroblast lineage for investigating temporal identity; and reveal the importance of
Pdm and Castor as timing factors that regulate precise temporal identity windows during
which unique cell identities are specified. Furthermore, these results suggest that late-
onset genes such as Pdm and Castor may also have a role in regulating the early-
competence window in many neuroblasts.
9
10
CHAPTER II
PDM AND CASTOR ARE TIMING ELEMENTS THAT CLOSE SUCESSIVE
TEMPORAL IDENTITY WINDOWS IN THE NEUROBLAST 3-1 LINEAGE
"The only reasonfor time is so that everything doesn't happen at once."
-- Albert Einstein
Khoa D. Tran and Chris Q. Doe designed research; Khoa D. Tran performed research;
Khoa D. Tran and Chris Q. Doe analyzed the data and wrote the manuscript published
below.
Reproduced with the permission from Tran, K.D. and Doe, C.Q. Development, 2008,
135,3491-3499. Copyright 2008, The Company of Biologists.
INTRODUCTION
While previous studies have identified temporal identity factors that specify the
first and the second temporal identities, it remains unknown what factor or factors specify
the cell identities following Kruppel. The best candidate for a multi-lineage third
11
temporal identity factor is Pdm (a pair of co-expressed, redundantly-functioning POU
domain proteins, Pdm1/Nubbin and Pdm2). Pdm is expressed immediately after Kr in
many neuroblasts, and is known to specify the third temporal identity (U4 neuron) within
the NB7-1lineage (Grosskortenhaus et al., 2006). However, the analysis of just one
neuroblast lineage does not resolve whether Pdm has a specific function in specifying U4
motoneuron identity, or a more general function as a multi-lineage third temporal identity
factor. This is a crucial distinction, because many transcription factors are likely to
regulate different neuronal subtype specification without having anything to do with
temporal patterning. In fact, Pdm is also required for specification of the first-born
progeny in the NB4-2lineage (Yang et aI., 1993; Yeo et al., 1995), raising some doubt
about its role as a multi-lineage temporal identity gene.
The best candidate for a multi-lineage fourth temporal identity factor is the zinc
finger protein Castor (Cas), which is detected in neuroblasts just as Pdm fades away, and
together with Pdm specifies the fourth temporal identity (U5 neuron) in the NB7-1
lineage (Grosskortenhaus et aI., 2006; Isshiki et aI., 2001). As with Pdm, it is impossible
to know whether Cas has a specific role in specifying U5 identity or a general role as a
fourth temporal identity gene without analyzing its function in additional neuroblast
lineages. This has been difficult, because most neuroblast lineages have not been
characterized past the first or second cell division, and few molecular markers are known
for late-born neurons. For example, NB7-3 generates neurons with well-characterized
molecular markers (Isshiki et aI., 2001; Lundell et aI., 1996; Lundell and Hirsh, 1998;
Novotny et aI., 2002), but it only divides three times and never expresses Cas; in contrast,
12
NB2-4 divides many times and expresses Pdm and Cas (Isshiki et al., 2001) but there are
no molecular markers available to identify late-born neurons in this lineage. Thus, to test
the role ofPdm and Cas as multi-lineage late temporal identity factors, and to test any
new candidate late-born temporal identity factors, it is necessary to characterize a new
neuroblast lineage for both birth-order lineage data as well as neuronal molecular
markers.
Here we trace the birth-order of the first four divisions in the NB3-1 lineage and
develop molecular markers to distinguish early-born and late-born neuronal identity,
allowing us to use this lineage to assay late temporal identity gene expression and
function. We find that Hb and Kr specify early temporal identity in this lineage,
extending their role as multi-lineage temporal identity factors to a different spatial
domain of the CNS. Surprisingly, we fmd that Pdm is not required to specify the third
temporal identity, but rather that Pdm is required to repress Kr and thus close the second
temporal identity window. Similarly, we find that Cas is required to close the third
temporal identity window in this lineage. We conclude that Hb and Kr are multi-lineage
temporal identity factors, while Pdm and Cas are timing factors that close successive
temporal identity windows in the NB3-1 lineage.
13
MATERIAL AND METHODS
Fly stocks
We used the following fly stocks to analyze wild-type and mutant phenotypes at
23°C: hbPl, hbFB /TM3 hb-IacZ to remove Hb CNS expression (Hulskamp et aI., 1994;
Isshiki et aI., 2001); Kr1, KrCD / CyO hb-IacZ to remove Kr CNS expression (Isshiki et
aI., 2001; Romani et aI., 1996); Df(2L)ED773 which removes bothpdml andpdm2
(Grosskortenhaus et aI., 2006), cai4/TM3 ftz-IacZ (formerly called minl4 (Cui and
Doe, 1992)), red e spdOZZ27 / TM3 and numb2pr cn Bc / Cyo ftz-IacZ (Skeath and Doe,
1998); cas-IacZ (Cui and Doe, 1992); svpe22/svpz4 (Miller et aI., 2008; Mlodzik et al.,
1990). Unless otherwise noted, for misexpression experiments we crossed insc-gal4
(l407-gaI4, Bloomington Stock Center) on chromosome II to UAS-hb on chromosomes II
and III (Wimmer et al., 2000), UAS-Kr on chromosomes II and III (Hoch and Jackle,
1998), UAS-HA:pdm2 on chromosomes II and III (Grosskortenhaus et aI., 2006), and
UAS-cas on chromosomes II and III (W. Odenwald, NIH, Washington, DC) at 29°C.
Recombinant clones were generated using flies with the following genotype: y w hs-FLP
/ + ; X-15-33 / X-15-29 (courtesy of Allan C. Spradling, Carnegie Inst., Washington DC).
Molecular markers and immunostaining
Antibodv staining: was oerformed according: to standard methods. Primarv
01 '-" ~ '-' 01
antibodies, dilutions, and sources: rabbit HB9, 1:1000 (Odden et aI., 2002); guinea pig
HB9, 1:500 and rat Islet, 1:500 (Broihier and Skeath, 2002); mouse Islet, 1:200, mouse
14
FasII, 1: 100, mouse FasIII, 1:5 (Developmental Studies Hybridoma Bank, Iowa); rabbit
Hb, 1:200 (this work); guinea pig Kr, 1:500 (East Asian Distribution Center for
Segmentation Antibodies); rat Pdm2, 1:10 (Grosskortenhaus et ai., 2006); rabbit Cas,
1:1000 (Kambadur et ai., 1998); rat Ztb2, 1:400 (M. Lundell, UT San Antonio); mouse
En 4D9, 1:5 (Patel et ai., 1989); mouse Late Bloomer, 1:4 (C. Goodman, UC Berkeley);
mouse Beta-galactosidase, 1:500 (Promega, Madison, WI). Secondary antibodies were
purchased conjugated to Alexa 488, Rhodamine RedX, or Cy5 (Jackson, West Grove,
PA), biotin (Vector, Burlingame, CA), or alkaline phosphatase (Southern Biotechnology,
Birmingham, AL) and used at 1:400. Confocal image stacks were collected using a Leica
SP2 confocal microscope, processed using ImageJ (NIH), and shown as two-dimensional
projections. Histochemical preparations were acquired using a Zeiss Axioplan.
Identification of NB3-1 and the RP motor neurons
We staged embryos using standard methods (Campos-Ortega and Hartenstein,
1985), and identified NB3-1 using spatial and morphological features along with the
expression of Engrailed (marking neuroblast in rows six and seven). RPl, RP4, RP3, and
RP5 neurons were identified as Isl+ HB9+ neurons in the dorsal-medial region of each
hemisegment. The only other nearby Isl+ HB9+ neurons are the more posteriorllateral EW
neurons from the NB7-3lineage, which can be distinguished from the RP neurons by
their expression of Engrailed (Isshiki et ai., 2001; Lundell et al., 1996). RPI and RP4 are
often shown as insets as they appear directly ventral to RP3 and are often obstructed from
VIew.
15
RESULTS
Temporal identity gene expression and neuronal birth-order in the NB3-1lineage
To identify a new lineage ideal for temporal identity analysis, we focused on one
that was outside neuroblast row 7 (where the previously characterized NB7-1 and NB7-3
reside), and contained several well-characterized neurons. We chose NB3-1 by virtue of
its position within the anterior region of the segment, far from the posterior row 7, and
because it was known to generate the well-characterized RP1, RP4, RP3, and RP5 motor
neurons (Bossing et al., 1996; Landgraf et aI., 1997; Schmid et al., 1999). We first
characterized the expression of the known and candidate temporal identity genes Hb, Kr,
Pdm, and Cas in NB3-1 as it begins its cell lineage. We detect Hb and Kr expression in
the newly formed NB3-1 at stage 10; Kr alone during early stage 11; Pdm alone at mid
stage 11; and Cas expression from late stage 11 into stage 12 (Fig. lA, B). We conclude
that NB3-1 sequentially expressed Hb/Kr~ Kr~ Pdm~ Cas during the initial phase of
its cell lineage, and that this lineage is appropriate for investigating the role of all four
genes in specifying temporal identity.
Next, we characterized the birth-order of the RP neurons and determined which
known or candidate temporal identity gene was expressed at the time of their birth. We
use both molecular markers and cell body position to identify and distinguish the RP
neurons (Fig. 2A,B; see methods for details). Motor neuron backfills show that RPl/4 are
the most dorsal, RP3 is intermediate, and RP5 is most ventral in position within the CNS
(Landgraf et aI., 1997; Schmid et al., 1999). Early-born neurons occupy deeper layers
16
(Isshiki et a1., 2001), consistent with a birth-order of RP11RP4 ~ RP3 ~ RP5. Here we
used molecular markers to identify the NB3-l derived RP neurons, and assayed their Hb,
Kr, Pdm, and Cas expression profile. We find that RPl/4 are Hb+ Ki'+, RP3 is Hb-Kr+, and
RP5 is Hb- Kr- (Fig. 2A). This precisely matches the sequence of gene expression within
NB3-l as it goes through the early portion of its lineage (Fig. 1). We conclude that RPl/4
are born during the early Hb+Kr+neuroblast expression window, RP3 is born during the
Hb- Kr+ neuroblast expression window, and RP5 is generated after Hb and Kr expression
is lost from the neuroblast.
The RPI and RP4 neurons express the same molecular markers (Fig. 2A) and
have identical axon projections (Landgraf et al., 1997), raising the possibility that they
are sibling neurons. To determine whether RPI and RP4 are sibling neurons, we used
sanpodo and numb mutants to equalize sibling cell fate (Skeath and Doe, 1998). We
found that sanpodo mutants typically generate a pair of RPI neurons and a pair of RP4
neurons, and numb mutants show the opposite phenotype (Fig. 2B; Table 1). These
results show that the RPI and RP4 neurons are not siblings; but rather that they each have
a non-RP sibling that assumes the RP fate in sanpodo mutants. Furthermore, we have
generated clones by mitotic recombination that label the entire NB3-l lineage except RPI
(n=4; Fig. 2C); as all four neurons are definitively produced by NB3-l based on DiI
labeling (Bossing et al., 1996; Schmid et a1., 1999), this proves that RPI is derived from
the first-born GMC in the lineage. We conclude that NB3-l sequentially generates the
RPI~ RP4~RP3~RP5 neurons (and their non-RP siblings), followed by a pool of
local interneurons (Fig. 2D).
17
Hunchback specifies the first temporal identity in the NB3-1lineage
Hb is known to specify the first temporal identity in two closely positioned
lineages, NB7-1 and NB7-3, located within the Engrailed+ posterior region of the
neuromere (Broadus et aI., 1995). Here we test whether Hb has a similar function in the
NB3-1 lineage, which is located in the anterior region of the neuromere. We used hb
mutants that were rescued for Hb segmentation expression (Isshiki et aI., 2001), and
found a loss of the early-born RP1 and RP4 neurons; the later-born RP3 and RP5 neurons
were unaffected (Fig. 3A, Table 1). Thus, Hb is required for the specification and/or
survival of the early-born RP1 and RP4 neurons. To determine if Hb is sufficient to
induce the first-born RP11RP4 temporal identity, we used insc-gal4 UAS-hb to prolong
expression ofHb in NB3-1 beyond its normal expression window. We observed as many
as nine RP neurons per lineage, and all appeared to take the early-born RP11RP4 identity
based on molecular markers (Fig. 3B-C, Table 1). Consistent with the increase in
RP11RP4 motor neurons, we observe a thickening of FasII+ and FasIII+ motor axon
fascicles exiting the CNS and entering the ventral longitudinal muscle fields (Fig. Sl).
Late-born RP3 and RP5 neurons expressing Zfh2 or Cut were never detected. We
conclude that Hb is necessary and sufficient to specify early-born RP11RP4 temporal
identity within the NB3-1 lineage, paralleling to its role in specifying the first temporal
identity in the NB7-1 and NB7-3 lineages (Isshiki et al., 2001; Novotny et aI., 2002).
18
E10 Ll0 Ell Mll Lll
A
Hb
Kr
Pdm
Cas
B
""'I
M
I:C
Z
~ Pdm ~ Cas ~
Time
Figure 1. Temporal identity gene expression in NB3-1.
(A) NB3-1 (black circle) sequentially expresses Hb, Kr, Pdm, and Cas. A portion of one
hemisegment is shown, with Engrailed marking the most posterior NB rows 617
(blue); midline, left; anterior, up. Embryonic staging from Campos-Ortega and
Hariellsiein (1985): eariy stage 10 (E10) when NB3-1 forms; late stage i 0 (LlO);
early stage 11 (Ell); mid stage 11 (MIl); late stage 1] (Lll). Scale bar is 10 ~m.
(B) Summary of gene expression in NB3-1.
19
Figure 2. Molecular markers and cell position can be used to identify each RP motor
neuron in the NB3-1lineage.
(A) RPI and RP4 are Hb+, Kr+, cur, Zfh2- and are often shown in insets as they are
usually obstructed in the projection (n > 100). RP3 is Hb-, Kr+, cur, Zfh2+ (n > 100).
RP5 is Hb-, Kr-, Cut, Zfh2+ (n > 100). A single representative hemisegment of a
stage-16 CNS is shown as a maximum intensity projection; midline, left; anterior, up;
phenotype summary, top panel; scale bar, 3 J.lm.
(B) Top row: RP motor neurons are Islet HB9+ (white outline). RPI and RP4 occupy the
deepest layer; RPI is more dorsal and expresses HB9 at higher levels than RP4 after
stage 15 (n> 100). RP3 is directly ventral to RPI and RP4 (n > 100). RP5 is ventral
and anterior to RP3 (n> 100). NB7-3 derived EW interneurons (white arrowheads);
midline, dashed vertical line; ventral views of two segments are shown from deep
(left) to superficial (right) focal planes. Bottom row: Each RP neuron has a non-RP
neuron sibling, based the duplication of each RP neuron in sanpodo mutants. Eight
Islet HB9+ Late Bloomer+ RP motor neurons are observed in each hemisegment;
quantified in Table 1. Midline, between each pair of panels.
(C) Recombination-induced activation of the lacZ gene in NB3-1labeled RP4, RP3, RP5
and the late-born interneurons but not RPl, showing that RPI is the first-born neuron
in the lineage. HB9 is green, beta-gal is magenta, and RP neurons and NB3-1 are
outlined and labeled. One hemisegment of a stage-16 CNS is shown as a maximum
intensity projection; midline, left; anterior, up. A summary of the clone is to the right.
Scale bar is 3 J.lm.
(D) Schematic of NB3-1 gene expression and cell lineage. The vertical dashed line
represents the transition between RP neuron and subsequent interneuron specification.
Nb, sibling cell fate specified by Numb; N, sibling cell fate requires Sanpodo and
active Notch signaling.
20
Hb- Kr+
Cut" Zfh2+e
O Hb+Kr+o Cut"Zfh2-
RP neurons
»»»»»»»»»»»»»»»»»»
A
B
0 Sib Sib Sib Sib Sib
RP Neurons 0 0 0 00 00
N;;-v'N N;;-v'N N~~ N;;-v'N "'-/
GMC 0 0
t t
NB3-1 ~ Kr ~ Pdm ... Cas ~
Time
Figure 2
c
21
Table 1 Summary of phenotypes in the NB3-1lineage
Genotype'" Protein gain RP neuron identity Ii Conclusion
orloss (+1-) Total#ofRP (n) RPI RP4 RP3 RP5
wild-type 4 (100) 1 1 1 1 wild type
spdo mutant - Sanpodo 8.0 (70) 2 2 2 2 Spdo specifies RP sibling fates
lIumb mutant - Nwnb 1.0 (88) 1! 0 0 Numb specifies RP fates
hb mutant - Hb (CNS) 2 (163) 0 0 1 1 Hb is required for RPI/RP4 identity
2x VAS-hb + Hb 6.9 (198) 6.9' 0 0 Hb sufficient for RPl/4 identity
Krmutant - Kr (CNS) 2.7 (152) 0 0.7 Kr is required for RP3 identity
2xVAS-Kr +Kr 4.3 (147) 2.3 0 Kr is sufficient for RP3 identity
pdm mutant - Pdm1l2 5.1 (172) 1.8 1.3 Pdm closes the RP3 temporal window
2x VAS-pdm2 + Pdm2 2.9 (142) 0.6 0.3 Pdm represses RP3/RP5 identity
cas mutant - Cas 5.6 (183) 1 2.6 Castor closes the RP5 temporal window
2x VAS-cas + Cas 2.8 (156) 0.8 0 Cas represses RP5 identity
p «0.01 for all experiments.
*
II
:j:
All genotypes are described in Materials and Methods.
Average number of each cell type present per hemi-segment based on markers
described in Figure 1.
We currently lack the markers to distinguish between RP1 and RP4 in functional
analysis.
22
A
c
'1l0~
••••
• 1/4
••
Figure 3. Hb is necessary and sufficient to specify the first temporal identity.
(A) hb CNS mutants lack the RP]/RP4 neurons, but have normal RP3 and RPS neurons;
quantified in Table 1. Wild type has RP], RP4, RP3, and RPS neurons (Fig. 2).
(B) hb misexpression (insc-gaI4 UAS-hb) generates ectopic RP ]/4 neurons based on
molecular markers; quantified in Table 1.
(e) Top row: Wild type RP motor neurons express the pan-motor neuron markers pMAD
and Late Bloomer. Bottom row: hb misexpression (insc-gaI4 UAS-hb) generates
ectopic RPI/4 neurons that are pMAD and Late Bloomer double positive (100%, n =
48). For all panels, a single representative hemisegment of a stage-I6 CNS is shown
as a maximum intensity projection; midline, left; anterior, UIJ; IJhtIlOlypt sUlnmary
shown in right panel, with Late Bloomer expression indicated by dashed circles.
Scale bar is 3 ~m.
23
A
I
~
151+ or HB9+ RP neurons
Cut Zfh2 '1lTl~
47%
00
30%
00
ee
e
00
Figure 4. Kr is necessary and sufficient to specify the second temporal identity.
(A) In Kr CNS mutant embryos, RP3 is missing in the majority of hemisegments
examined (both rows), and RP5 is occasionally missing (second row); RPI/RP4 are
normal; quantified in Table 1. Wild type has RPI, RP4, RP3, and RP5 neurons
(Fig. 2).
(B) Kr misexpression (insc-gaf4 UAS-Kr) generates ectopic ofRP3 neurons, RP5 is
usually absent, and RPI/RP4 are normal; quantified in Table 1. For all panels, a
single representative hemisegment of a stage-I 6 CNS is shown as a maximum
intensity projection; midline, left; anterior, up; phenotype surnrnary, right.
Scale bar is 3 /.lID.
24
Kruppel specifies the second temporal identity in the NB3-1lineage
Kr is known to specify the second temporal identity in the NB7-1 and NB7-3
lineages (i.e. the fate of the GMC born immediately after Hb downregu1ation) (Cleary
and Doe, 2006; Isshiki et aI., 2001). Here we test whether Kr has a similar function in the
NB3-11ineage. We used Kr mutants that were rescued for early segmentation expression
(Isshiki et aI., 2001), and found a loss of the RP3 neuron; the early-born RP1/RP4
neurons and the late-born RP5 neuron were mostly unaffected (Fig. 4A, Table 1). Thus,
Kr is required for the specification and/or survival of the RP3 neuron; this is the RP
neuron born during the Kr neuroblast expression window. To determine if Kr is sufficient
to induce the RP3 identity, we used insc-gal4 UAS-Kr to prolong expression of Kr in
NB3-1 for the entire length of its cell lineage. We observed a maximum of three RP3
neurons per lineage (Fig. 4B, Table 1). We saw no deleterious effect on the specification
ofRP1 and RP4, but tlle Cut+ RP5 neuron was typically missing (Fig. 4B). We conclude
that Kr is necessary to specify the second temporal identity in the NB3-1 lineage (RP3) --
within a competence window -- similar to its role in specifying the second temporal
identity in the NB7-1 and NB7-3 lineages (Isshiki et aI., 2001).
Pdm is required to close the second temporal identity window, but not for specifying
the third temporal identity in the NB3-1lineage
Pdm expression follows Hb and Kr in most neuroblasts, and thus is an excellent
candidate for specifying the third temporal identity. Indeed, Pdm is necessary and
sufficient to specify the third temporal identity (U4 neuron) within the NB7-1lineage
25
(Grosskortenhaus et al., 2006). To determine ifPdm is a multi-lineage temporal identity
gene, we assay its loss of function and misexpression phenotype in the NB3-1 lineage.
We assayed embryos homozygous for the deficiency Df(2L)ED773 (which eliminates
both pdml and pdm2; henceforth called pdm mutant embryos). In pdm mutant embryos,
we observed normal timing ofHb expression in NB3-1 and other neuroblasts (data not
shown), a modest extension of Kr expression, and a similar delay in Cas expression (Fig.
5A). Consistent with this change in neuroblast gene expression, pdm mutant embryos
show normal specification of the early-born Hb+ RPl and RP4 neurons, possess extra Kr+
RP3 neurons, followed by an apparently normal Cut+ late-born RP5 (Fig. 5C, Table 1).
We conclude that Pdm is not required to specify the third temporal identity (the Cut+ RP5
neuron), but it is required to limit Kr expression in the neuroblast and thus close the
second temporal identity window after the birth of just one Kr+ RP3 neuron.
We next determined if continuous expression ofPdm in NB3-1 was sufficient to
induce ectopic RP5 neurons (i.e. extend the third temporal identity window). We used
insc-ga14 UAS-pdm2 to generate continuous Pdm expression in neuroblasts, and observed
normal timing of Hb expression in NB3-1 and other neuroblasts (data not shown), but
premature loss of Kr expression and precocious Cas expression (Fig. 5B). Consistent with
this change in neuroblast gene expression, we observe normal specification of the early-
born Hb+RPl and RP4 neurons, but lack Kr+RP3 neurons; there is also a loss of the Cut
late-born RP5 neuron (Fig. 5D, Table 1). We conclude that Pdm is not sufficient to
specify the third temporal identity (RP5), but rather it acts as a timer element to define
the window of Kr expression, and thus the length of the second temporal identity
26
window. The precocious expression of Cas in these Pdm misexpression embryos may
result in the precocious formation of Cas+ interneurons at the expense of the RP5 neuron
(see below).
Castor is required to close the third temporal identity window in the NB3-1lineage
Cas is expressed in NB3-1 following Hb, Kr, and Pdm, but it is not detected in
any of the post-mitotic RP1-RP5 motor neurons (Figs. lA, 2A). In addition, we
examined flies carrying the cas-IacZ reporter transgene (Cui and Doe, 1992) and found
no residual ~-galactosidase expression in any NB3-1 derived RP neurons (data not
shown). This suggests that Cas expression is initiated after NB3-1 has made its fourth
GMC, at the time it shifts to producing local interneurons (Fig. 2D). Thus, we can test
whether Cas is important for closing the third (RP5) temporal identity window, but due to
the lack of interneuronal markers we are unable to assay for a Cas function in specifying
the fourth (interneuron) temporal identity.
To test whether Cas is required to close the third temporal identity window, we
assayed cas null mutant embryos (Cui and Doe, 1992). We find that cas mutants have
normal Hb and Kr expression in neuroblasts (data not shown) but prolonged Pdm
expression (Fig. 6A), consistent with previous work showing that Cas is required to
repress pdm (Grosskortenhaus et aI., 2006; Kambadur et aI., 1998). At the neuronal level,
we find that cas mutants have normal early-born RPl, RP4, and RP3 neurons but possess
ectopic RP5 neurons (Fig. 6B, Table 1), consistent with a prolonged third temporal
identity window. The ectopic RP5 neurons are not specified by the persistent Pdm
27
protein, because pdm mutants still form apparently normal RP5 neurons (Fig. 5), and pdm
cas double mutants still form Cut+ RP5 neurons (data not shown). Interestingly, cas
mutants have a few RP-like (lslet+ HB9+) neurons that lack expression of the motor
neuron marker Late Bloomer and thus may have a mixed interneuronIRP motor neuron
identity (Fig. S2). We next examined insc-gal4 VAS-cas embryos which have continuous
expression of Cas in NB3-1. We find that RP5 is often missing, but the early-born RP1,
RP4 and RP3 are normal (Fig. 6C, Table 1). We conclude that the precocious expression
of Cas is sufficient to close the third temporal identity window, in which RP5 is
specified. Taken together, our results suggest that Cas is necessary and sufficient to close
the third temporal identity window in the NB3-1 lineage.
DISCUSSION
We have characterized the neuronal birth-order of the first four motor neurons within
the NB3-1 lineage, described the temporal identity gene expression pattern within NB3-1
and its motor neuronal progeny, and performed functional analysis of all four known and
candidate temporal identity genes. Our results confirm and extend previous conclusions
that Hb and Kr are multi-lineage temporal identity genes, and reveal novel aspects
regarding the role of Pdm during the specification of temporal identity. We find that both
Pdm and Cas play essential roles as part of the neuroblast gene expression timer: Pdm
closing the second temporal identity window, and Cas closing the third temporal identity
window.
28
Hunchback and Kruppel are multi-lineage temporal identity factors
We have shown that Hb and Kr are necessary and sufficient to specify the first
and second temporal identities in the NB3-1Iineage. We can now conclude that Hb and
Kr function as temporal identity factors in many spatial domains of the CNS - anterior-
medial (NB3-l), posterior-medial (NB7-1), posterior-lateral regions (NB7-3) - showing
that temporal identity and spatial identity are independent with regards to Hb and Kr.
Furthermore, Hb and Kr maintain similar functions in neuroblasts that form at distinct
times during embryogenesis - early (NB7-1), middle (NB3-1), and late (NB7-3) - thus
confirming that temporal identity is a lineage-autonomous event that is not coordinated
by embryo-wide timing events (Brody and Odenwald, 2000; Grosskortenhaus et aI.,
2005). Overall, our data strongly support the conclusion that Hb and Kr are multi-lineage
temporal identity genes.
Our data also provide insight into neuroblast competence. When we misexpressed
Hb in the NB3-1 lineage, we can generate up to nine RP motor neurons; if each has a
non-RP sibling, it would be near the expected number of cells for the entire lineage
(Schmid et aI., 1999). Thus, Hb seems capable of maintaining at least three very different
neuroblast lineages (NB3-1, NB7-1, and NB7-3) in a "young" state for their entire
lineage. In contrast, misexpression of Kr produces only a few RP3 motor neurons before
NB3-1 proceeds to make the later-born neurons. The inability of Kr to maintain a second
temporal identity state may be due to the initiation of progressive restriction in neuroblast
competence in NB3-1, as occurs in NB7-1 (Cleary and Doe, 2006; Pearson and Doe,
2003).
29
Pdm closes the second temporal identity window in the NB3-1lineage
Our findings show that Pdm is not required to specify the third temporal identity
in the NB3-1Iineage, but rather that Pdm is a timer element that represses Kr expression
and closes the second temporal identity window. Loss of Pdm allows for a transient
extension of the Kr expression window leading to a few ectopic Kr-specified RP3
followed by a Cut+ RP5. We hypothesize that the production of the RP5 cell is possible
because Kr is not permanently maintained in the neuroblast. In contrast, permanent
expression of Kr in NB3-1 (insc-gaI4 UAS-Kr) also leads to extra RP3 neurons but does
not allow production of a Cut+ RP5, perhaps due to the continuous expression of Kr. Pdm
is not the first transcription factor known to act as a timing element. The orphan nuclear
hormone receptor Seven-up (Svp) is required for repressing Hb to close the first temporal
identity window in the NB7-1 and NB7-3lineages (Kanai et aI., 2005; Mettler et aI.,
2006), and in the NB3-1 lineage (data not shown). It should be noted that Svp represses
Hb expression in all neuroblasts tested to date, whereas Pdm represses Kr expression in
some but not all neuroblasts.
Pdm does not act as a timer element in all neuroblast lineages. For example, pdm
mutants do not show extended Kr expression in the NB7-1 or NB7-3 lineages, based on
the lack of ectopic Kr+ neurons in these lineages (Grosskortenhaus et aI., 2006)(Fig. S3).
These results suggest that the spatial identity of a neuroblast can alter its response to
timing factors such as Pdm. While this is counter to the simple model that spatial and
temporal factors are independent and act combinatorially to specify birth-order identity
within each lineage (Pearson and Doe, 2004), it is consistent with the finding that spatial
30
identity occurs at the time of neuroblast formation (Chu-LaGraff and Doe, 1993; Prokop
and Technau, 1994; Skeath et aI., 1995), prior to the expression of temporal factors.
Taken together, these data suggest that spatial cues allow individual neuroblasts to
respond differently to a temporal identify factor expressed at a similar time in all
lineages.
The prior expression of early temporal identity factors is also likely to alter the
response of a neuroblast to later temporal identity factors. Previous work has shown that
misexpression of later temporal factors such as Kr, Pdm, or Cas has no detectable effect
on the fate of first-born Hb+ neurons in the NB7-1lineage (Cleary and Doe, 2006;
Grosskortenhaus et al., 2006; Isshiki et aI., 2001; Pearson and Doe, 2003). Consistent
with these results, we find that in the NB3-1Iineage, Pdm misexpression cannot repress
Kr or activate Cas during the early Hb+ expression window (Fig. 5B). Just as prior spatial
patterning cues may alter the response to a later temporal identity factor, so to may prior
temporal identity factor expression alter the response of a neuroblast to later temporal
factors. The mechanism by which spatial and temporal factors confer heritable changes to
neuroblasts remains a mystery. One entry into this mechanism could be the investigation
of how Hb blocks Pdm from repressing Kr gene expression.
If Pdm does not specify temporal identity in the J\TB3-1 lineage, what is the third
temporal identity factor in this lineage? It has recently been reported that the SoxB family
member Dichaete is expressed immediately prior to Cas in many embryonic neuroblast
lineages (Maurange et aI., 2008); however Dichaete is only transiently expressed in
31
Figure 5. Pdm closes the second temporal identity window in the NB3-1lineage.
(A-B) Neuroblast expression of Kr and Cas in pdm mutant and Pdm misexpression
embryos. One hemisegment shown; Kr or Cas, brown; the positional marker
Engrailed, blue. NB3-1 is outlined in black. Scale bar is 10 /..lm. (A) In pdm mutants,
Kr expression persists through late stage 11 (normally off by mid stage 11; see Fig.
lA) and Cas expression is delayed until mid stage 12. (B) In pdm misexpression
embryos (insc-gal4 UAS-pdm2), Kr expression is lost prematurely and Cas is
expressed precociously (compare to Fig. lA).
(C-D) RP neuron specification in pdm mutant and Pdm misexpression embryos. One
hemisegment of a stage 16 CNS is shown as a maximum intensity projection;
midline, left; anterior, up; phenotype summary, right; scale bar, 3 /..lm. (C) In pdm
mutant embryos there are 2-3 ectopic RP3 neurons; RPl, RP4, and RP5 neurons are
usually normal; quantified in Table 1. (D) pdm misexpression (insc-gal4 UAS-pdm2)
results in the frequent loss of the RP5 neuron (both rows) and occasional loss of the
RP3 neuron (second row); RPl/RP4 are normal; quantified in Table 1.
32
Kr+ Neuroblasts II
Lll E12
Cas+ Neuroblasts
Lll M12
27%
54%
00
800
00
800
1111~
=-_.........
Cas+ Neuroblasts
Ell
Isl+ or HB9+ RP neurons
Cut Zfh2Hb
Kr+ Neuroblasts
Ll0 11
A
C
o
Figure 5
33
Figure 6. Cas closes the third temporal identity window in the NB3-1lineage.
(A) cas mutants have persistent Pdm expression in NB3-1 until at least mid stage 12
(MI2); in wild type Pdm is gone from NB3-1 by late stage 11 (Fig lA). At mid stage
16, neuroblasts in the medial column no longer expressed Pdm (white arrowheads);
one of these neuroblasts is likely to be NB3-1. Scale bar, 10 Jlm.
(B) In cas mutants, there are up to four ectopic Cut+ RP5 neurons; RPl, RP4 and RP3 are
normal; quantified in Table 1. Wild type has RPl, RP4, RP3, and RP5 neurons
(Fig. 2).
(C) cas misexpression (insc-ga14 VAS-cas) results in frequent loss of RP5 and occasional
loss ofRP3; RPl, RP4 are normal; quantified in Table 1. For all panels, a single
representative hemisegment of a stage-16 CNS is shown as a maximum intensity
projection; midline, left; anterior, up; phenotype summary, right.
Scale bar is 3 Jlm in Band C.
34
A
B
Pdm+ Neuroblasts
Lll E12 M12
Isl+ or HB9+ RP neurons
C t IIlTl~
e
eO
e00
Figure 6
35
medial column neuroblasts such as NB3-1 at their time of formation (Zhao and Skeath,
2002) and thus does not have the proper timing for a third temporal identity factor in this
lineage. Alternatively, the absence of Hb, Kr, and Cas may specify the third temporal
identity, with Pdm acting solely as a timing factor to establish a gap between Kr and Cas
expression. Another possibility is that a currently unknown factor specifies the third
temporal identity in the NB3-1lineage. Finally, Pdm may specify aspects ofRP5 identity
that we are not able to detect with our limited number of markers; unfortunately due to
severe morphological defects in late-stage pdm mutant embryos, we have been unable to
assay the RP5 axon projection to its target muscle, which is a sensitive read-out of proper
neuronal identity.
Castor closes the third temporal identity window
Cas is expressed right after Pdm in most neuroblasts, and at the time NB3-1 is
generating its fourth temporal identity (interneurons). We find that cas mutants have an
extended window of Pdm neuroblast expression and production of ectopic RP5 neurons.
Thus, Cas is required to close the third (RP5) temporal identity window. In addition, we
find that precocious expression of Cas can prematurely close the third temporal identity
window and repress the specification ofRP5. We observed comparable phenotypes in the
NB7-1lineage where loss of Cas leads to ectopic U4 formation and gain of Cas results in
the repression of that identity (Grosskortenhaus et aI., 2006). Based on these
observations, we predict that Cas functions in multiple neuroblast lineages to close the
third temporal identity window. Does Cas specify the fourth temporal identity? We
36
cannot answer this question in the NB3-1 lineage due to lack of interneuron markers, but
Cas does specify the fourth temporal identity (together with Pdm) in the NB7-1 lineage
(Grosskortenhaus et al., 2006). In the future, the role of Cas in the NB3-1 lineage could
be examined by making CD8::GFP-marked cas mutant clones and assaying neuronal
identity by axon projections, or by developing molecular markers for interneurons within
the lineage.
Temporal identity genes, timing factors, and neuronal cell type specification
We propose that there are two classes of genes that regulate neuroblast temporal
identity. One class of genes encodes temporal identity factors that are necessary and
sufficient to directly specify a particular temporal identity in multiple neuroblast lineages
(lsshiki et aI., 2001); Hb and Kr are good examples. A second class of genes encodes
timing factors that establish the timing of temporal identity gene expression, but do not
directly specify temporal identity (Fig. 7). Timing factors, however, may indirectly
influence the specification of temporal identities as seen in NB3-1 where pdm is required
to restrict the specification of RP3 and properly advance the neuroblast to the Cas-
positive state (Fig. 5). Previously only one timing factor had been identified, Seven-up,
which down-regulates Hb protein levels, and along with cytokinesis, closes the first
temporal identity window to facilitate the Hb~Kr transition (Grosskortenhaus et aI.,
2005; Kanai et al., 2005). The Kr~Pdm~Cas transitions are independent of cell cycle
progression (Grosskortenhaus et aI., 2005). Here we show that Pdm closes the second
temporal identity window by repressing Kr expression and activating Cas in NB3-1.
37
Taken together, our observations suggest that Kr and Pdm are involved in a negative-
feedback loop: where Kr activates Pdm which in turns represses Kr and activates Cas to
advance neuroblast timing independent of cell cycle progression. Through its role as a
regulator of Kr and Cas timing, Pdm can restrict the production of neuronal cell types and
advance the NB3-1 lineage.
Bridge to Chapter III
In Chapter III, I present the results from my investigation of the molecular
mechanisms underlying Hunchback function to maintain early neuroblast competence
and specify early neuronal-identity. Hunchbackf[karos proteins can directly activate or
repress target gene transcription during early insect development, but their mode of
action in during neural development is unknown. Previous studies show that Hunchback
can specify early-born neuronal identity and maintain "young" neural progenitor
(neuroblast) competence. In this chapter, I use recombineering to generate a series of
Hunchback domain deletion variants and assay their function during neurogenesis, in the
absence of endogenous Hunchback. I identify two conserved domains required for
Hunchback-mediated transcriptional repression, and show that transcriptional repression
is necessary and sufficient to induce early-bom neuronal identity and maintain neuroblast
competence. I identify one direct target gene that must be repressed to maintain
competence, pdm-2, but show that additional genes must also be repressed. Base on these
findings, we propose that Hunchback maintains early neuroblast competence by silencing
a suite of late-expressed genes.
38
Svp Pdm Cas/' y
--.- Hb ----+- Kr ----+- ? ----.. Cas? NB temporal
identity genes
Neuronal
identity
NB timer genes
IN
INsibsib
___ lThird TI) (Fourth T1)
.sib·
o
First TI
sib
o
NB3-1
forms
"-.. H
~
Spatial
cues
Figure 7. Hb and Kr specify early temporal identity, whereas Svp, Pdm, and Cas act
as timer elements within the NB3-11ineage.
Left: Spatial cues specify the NB3-] identity and formation, which a])ows the neuroblast
to respond in a potentially unique way to subsequently expressed timer genes and
temporal identity genes.
Top row: Timer elements include Seven-up (Svp), Pdm, and Cas; these factors close
successive temporal identity windows. Timer genes indirectly control cell fate
through regulation of temporal identity genes.
Middle row: Temporal identity genes include Hb and Kr, which specify first and second
temporal identities respectively in this and other lineages.
Bottom row: Neuronal identity within the lineage. GMC-l makes RPlIsibling neurons;
GMC-2 makes RP4/sibling neurons; GMC-3 makes RP3/sibling neurons; GMC-4
makes RPS/sibling neurons; GMC-S makes interneurons.
39
CHAPTER III
HUNCHBACK REPRESSES MULTIPLE DOWN-STREAM FACTORS TO
MAINTAIN NEUROBLAST COMPETENCE AND SPECIFY
EARLY-BORN NEURONAL IDENTITY
"Age is an issue ofmind over matter. Ifyou don't mind, it doesn't matter."
-- Mark Twain
Khoa D. Tran and Chris Q. Doe designed the research; Khoa D. Tran perlormed research;
Michael R. Miller contributed new reagents; Khoa D. Tran and Chris Q. Doe analyzed
the data and wrote the manuscript published below.
Reproduced with the permission from Tran, K.D., Miller, M.R., and Doe, C.Q.
Development, 2010,137,1421-1430. Copyright 2010, The Company of Biologists.
INTRODUCTION
Hb is expressed early in many neuroblasts and is required for the specification of
the first-born cell identity, or first-temporal identity, in those lineages (Isshiki et al.,
2001; Novotny et aI., 2002). In addition, Hb can also confer the early competent state in
40
many neuroblast lineages (Cleary and Doe, 2006; Pearson and Doe, 2003). This is
achieved by maintaining Hb expression in neuroblasts throughout neurogenesis, or by re-
introducing Hb in neuroblasts after its normal expression window. The ectopic Hb
expression results in the specification of extra early-born progeny.
Interestingly, the ability of Hb to specify and extend the early-competence
window declines over time (Cleary and Doe, 2006; Pearson and Doe, 2003). When Hb is
reintroduced to NB7-1 at progressively later time points, its ability to specify ectopic
U11U2 neurons is greatly reduced. Eventually, Hb is unable to specify early-born cells
after the fifth neuroblast division. This raises two interesting questions regarding
neuroblast competence. First, how does the Hb regulate gene expression to maintain
early-neuroblast competence? And second, why does this ability decline over time?
Hb regulates gene expression via multiple, well-characterized modes during the
formation of the Drosophila body plan; however, little is known about its modes of
operation in the CNS. In the cellular blastoderm, the Hb protein gradient initiates and
establishes the spatial expression domains of the gap genes Kruppel (Kr), knirps (kni) and
giant (gt). Rigorous genetic and molecular analyses have shown that Hb acts a
concentration dependent transcriptional activator and repressor of gene expression during
embryonic segmentation (Berman et aI., 2002; Hoch et aI., 1991; Pankratz et aI., 1992;
Rivera-Pomar et aI., 1995; Schulz and Tautz, 1994; StmW et aI., 1992). Furthermore, Hb
can induce permanent repression of target genes through its interaction with Mi2 and
recruitment of Polycomb complex proteins (Kehle et al., 1998). As a result, the multi-
functional Hb protein is a potent regulator of gene expression in the early embryo. Here,
41
we extend the analysis of Hb-mediated gene regulation to include its role of maintaining
neuroblast competence during nervous system development.
MATERIALS AND .METHODS
Generation ofVP16::Hb chimera protein
We generated the VP16::Hb chimera by PCR amplifying the VP16 activation
domain (Lai and Lee, 2006) using primers with a 3' -tail that contained hb 5' sequence,
and ligating to PCR amplified full length hb coding sequence. Primer sequences are
available upon request. The chimeric gene was verified by sequencing, cloned into the
pUASTvector (Brand and Perrimon, 1993), and transgenic flies were made
(GenetiVision, Houston, Texas).
Generation of tagged Hb deletion proteins
We generated hb genes deleted for the six previously described conserved
domains (Tautz, 1987), as well as two additional domains (B' and E) that we identified as
conserved in at least eight sequenced Drosophila species using EvoPrinter (Odenwald et
aI., 2005). Each hb deletion construct (except the D domain deletion) was generated using
recombineering by targeted insertion and replacement of the galK expression cassette
(Warming et al., 2005). GalK targeting cassettes were prepared by PCR amplification of
the galK expression cassette using primers containing homology to hb. To insert galK,
SW102 cells containing BAC clone BACROlF13 were electroporated with the
42
appropriate targeting cassette and plated on minimal medium with galactose and
chloramphenicol. To replace galK, SW102 cells were electroporated with the appropriate
replacement cassette and plated on minimal medium with glycerol, 2-deoxy-galactose,
and chloramphenicol. The replacement cassette for epitope tagging (3xFLAG::3xHA)
was prepared by PCR amplification using homology primers. Primer sequences are
available upon request. The D domain deletion was generated by two-step PCR. Each
construct was sequenced to confirm that Hb was modified correctly. Deletions were
cloned into a pUAST(attB) vector (Bischof et aI., 2007) and sent to GenetiVision for
injections into flies carrying the attP40 docking site on Chromosome 2 (Markstein et aI.,
2008). In addition to the deletions, we also generated flies carrying the same epitope
tagged wild type Hb in the attP40 locus as a standard control. The fly stocks generated
are described below.
Fly stocks
The following pre-existing fly stocks were used: yw (wild type); v32a-gal4 for
ubiquitous embryonic expression (Siegrist and Doe, 2005); UAS-hb (Wimmer et al.,
2000); engrailed-gal4 for expression in the posterior compartment of each segment
(Harrison et aI., 1995; Isshiki et aI., 2001; Pearson and Doe, 2003); worniu-gal4 for
expression in neuroblasts (Albertson et aI., 2004); UAS-HA is UAS-HA::UPRT (Miller et
al., 2009), this transgene was used as a VAS control so that each misexpression
experiments had two VAS transgenes; it does not change the number of V neurons when
expressed alone.
43
The following fly stocks were generated in this work:
UAS-VP16::hb on Chromosomes 2 and 3
UAS-VP16::hb; hbFB hbP1ITM3 Jtz-IacZ hbFB hbPiITM3 Jtz-IacZ [hbFB is a null-mutant, hb
Pi is a segmentation rescue construct (Hulskamp et aI., 1994; Isshiki et aI., 2001)]
UAS-VP16::hb; Df(2L)ED773/CyOJtz-IacZ [DJ(2L)ED773 removes bothpdml and
pdm2, (Grosskortenhaus et aI., 2006)]
UAS_hbwild-type/CyO and UAS_hbwild-type/CyO; hbFB hbPiITM3 Ubx-IacZ
UAS-hbMB/CyO and UAS-hbMB/CyO; hbFB hbP1ITM3 Ubx-IacZ
UAS-hb£JBiCyO and UAS-hb£JBiCyO; hbFB hbP1ITM3 Ubx-IacZ
UAS-hbLJDBD/CyO and UAS-hbLJDBD/CyO; hbFB hbPiITM3 Ubx-IacZ
UAS-hb.tJ.c/CyO and UAS-hb.tJ.c/CyO; hbFB hbP1ITM3 Ubx-IacZ
UAS-hbLJD/CyO and UAS-hbLJD/CyO; hbFB hbP1ITM3 Ubx-IacZ
UAS-hbLJE/CyO and UAS-hbLJE/CyO; hbFB hbP1ITM3 Ubx-IacZ
UAS-hbLJDMZ/CyO and UAS-hbLJDMZ/CyO; hbFB hbP1ITM3 Ubx-IacZ
engrailed-GaI4/CyO; hbFB hbP1ITM3 Ubx-IacZ
Molecular markers and immunostaining
Antibody staining was performed according to standard methods. Polyclonal
antisera against Zfu2 was made by expressing a GST-fusion protein containing amino
acids 1641-2149 of the Zf112 protein (pZFH-2c, (Lai et aI., 1991)), and purified proteins
were sent to Alpha Diagnostic International (San Antonia, TX) for injections into rats.
Primary antibodies, dilutions, and sources: rat Zfu2, 1:400 (this work; M. Lundell, UT
44
San Antonio); mouse Eve 2B8, 1:10 (Patel et al., 1994); guinea pig Eve, 1:1000, rat Eve,
1:000, guinea pig Kr, 1:800 (Kosman et al., 1998); rabbit Hb, 1:200 (Tran and Doe,
2008); rabbit Kr, 1:500 (Gaul et al., 1987); rat Pdm2, 1:10 (Grosskortenhaus et al., 2006);
rabbit Cas, 1:2000 (Kambadur et al., 1998); mouse En 4D9, 1:5 (Patel et al., 1989);
mouse Beta-galactosidase, 1:500 (Promega, Madison, WI); rabbit Beta-galactosidase,
1:1000 (MP Biomedicals, Solon, OH); mouse HA, 1:500 (Roche, Palo Alto, CAy; rabbit
HA, 1:1000 (Sigma, St. Louis, MO). Secondary antibodies were purchased conjugated to
Alexa 488, Rhodamine RedX, Cy5 (Jackson, West Grove, PAy, biotin (Vector,
Burlingame, CAy, or alkaline phosphatase (Southern Biotechnology, Birmingham, AL)
and used at 1:400.
Microscopy and statistical analysis
Confocal image stacks were collected using Leica SP2 or Biorad Radiance 2000
confocal microscopes and shown as two-dimensional projections. Histochemical
preparations were acquired using a Zeiss AxioCam HRc camera on an Axioplan
microscope. Two-tailed T tests were used to determine the significance in the differences
in cell number.
45
RESULTS
VP16::Hb activates both positively- and negatively-regulated Hb direct target genes
in the early embryo
To address the molecular mechanism underlying Hb maintenance of neuroblast
competence, we began by examining whether Hb achieves this role through the
transcriptional activation or repression of target genes. Because no transcriptional
activation or repression domains have been identified within Hb, we began by converting
Hb to act solely as a transcriptional activator by fusing the VP16 trans-activation domain
(Triezenberg et aI., 1988) to the N-terminus of full-length Hb (Fig. 1A). We then tested
whether VP16::Hb could induce competence or first-born temporal identity.
Before assaying the effects of VP16::Hb in the eNS, we first examined the ability
of VP16::Hb to recognize and activate known Hb direct target genes in the early embryo.
In the embryonic blastoderm, Hb is a direct activator of the gap gene Kr (Hoch et aI.,
1991). Expression of Hb throughout the embryo using a heat-shock promoter can
activate Kr and expand the Kr central gap domain to the posterior of the embryo (Hoch et
aI., 1991; Hulskamp et aI., 1990; Struhl et aI., 1992). We expressed wild type Hb
throughout the embryo using the Gal4IUAS gene expression system (Brand and
Perrimon, 1993) with a maternal Gal4 driver (v32a-ga14 UAS-hb) and were able to
reproduce this expansion of the Kr central gap domain (Fig. 1B). Next, we examined
v32a-ga14 UAS-VP16::hb embryos and found a dramatic increase in Kr expression
46
throughout the embryo (Fig. IB). The activation of Kr throughout the embryo indicates
that VPI6::Hb is a potent activator of Kr gene expression.
We further tested the capacity ofVPI6::Hb as an activator by examining its
effects on genes that are normally directly repressed by Hb. Hb expression in the
posterior regions of the embryonic blastoderm directly represses the gap genes knirps
(kni) and giant (gt) (Fig. lC, ID) (Berman et al., 2002; Berman et aI., 2004; Pankratz et
al., 1992; Pe1egri and Lehmann, 1994). However, VPI6::Hb overexpression activated
both kni and gt, expanding their domains anteriorly and posteriorly into the domains of
their respective repressors (Fig. lC, ID). Based on these observations, we conclude that
VPI6::Hb is also sufficient to activate genes that are normally repressed by Hb (Fig. IE).
The strong activation and expansion of the Kr, Kni, and Gt gap domains indicates that
VPI6::Hb acts as a potent transcriptional activator that overcomes or eliminates the
normal transcriptional repression function of Hb. Taken together, we conclude that
VPI6::Hb has the capacity to recognize and activate Hb target genes whether they are
normally activated or repressed by Hb in the early embryo.
VP16::Hb activates all known Hb-regulated genes in the eNS
Because VPI6: :Hb acts as a strong activator of Hb direct target genes in the early
embryo, we next examined the expression ofHb CNS-targets in response to VPI6::Hb
expression. Previous studies suggest that Hb regulates its own transcription in the
blastoderm, but not in the CNS (Grosskortenhaus et aI., 2005; Treisman and Desplan,
1989). However, it is possible that the VPI6::Hb chimera will activate endogenous Hb
47
Figure 1. VP16: :Hb acts as a constitutive transcriptional activator.
(A) Schematic of wild type Hb protein and the VP16::Hb protein chimera with previously
characterized conserved domains. DBD, DNA-binding domain; DMZ, dimerization
domain.
(B-D) Expression of gap genes in the Drosophila embryonic blastoderm in wild type,
v32a-gal4 UAS-hb, and v32a-gal4 UAS-VP16::hb embryos.
(E) Summary of gene interactions.
48
25 68
DMZ
705 758466350
DBD
240
I
~~~----
A Hb
VP16::Hb
B
K
Anterior Posterior
.'\ JL\/ ~,
. \ . ,
i \ " i \
~~~~
c
I{.) (l 1(;)
"
fl; \
~ " i
,.r-/ "--'"y--.,.,/
o G
.\:
~ "
:" ~\( ;
.....:
.~
:/ '. "•. II ;
" ; \, ..,..
.'-./ I'" ' .... ~.
- ..
"
. "" "
\ '
.' ~: ~
Kr
Kni, Gt
---------I"r Kr, Kni, Gt
Hb-------
Hb--------
VP16::Hb
E
Figure 1
49
transcription via the VP16 activation domain, leading to cells containing both a
transcription activating Hb protein (VPI6::Hb) and a potential transcription repressing
Hb protein (endogenous Hb). To test whether VPI6::Hb can activate endogenous Hb
transcription, we petformed in-situ hybridizations against the 3' UTR of endogenous hb
mRNA. In engrailed-gal4 UAS-hb embryos, we found that Hb cannot induce its own
transcription in neuroblasts (Fig. 2A, B), consistent with previous findings
(Grosskortenhaus et aI., 2005). In contrast, engrailed-gal4 UAS-VP16::hb embryos show
strong activation of endogenous Hb transcription in neuroblasts (Fig. 2C). Based on
these observations, we conclude that VPI6::Hb can activate endogenous hb transcription
in the CNS.
We next examined whether VPI6::Hb can activate pdm, a gene normally
repressed by Hb through well-characterized Hb-binding sites in its CNS enhancer
element (Kambadur et al., 1998). In stage 15 embryos, only a few neuroblasts express
Pdm (Fig. 2D), and we found minimal change in Pdm following the expression of wild
type Hb protein (Fig. 2E), probably because most neuroblasts can no longer respond to
Hb at this stage (Cleary and Doe, 2006). However, the overexpression of VPI6::Hb in
neuroblasts resulted in the up-regulation ofPdm (Fig. 2F). We conclude that VPI6::Hb
can activate the direct target pdm in the CNS.
Having shown that VPI6::Hb can activate Hb direct target genes, we extended
our analyses of VPI6::Hb activity to all other known Hb CNS target genes (Fig. 2G). Hb
is known to activate Kr, and repress :zfh2, cut, runt, and cas (Fig. 2H) (Grosskortenhaus et
aI., 2006; Isshiki et al., 2001; Kambadur et al., 1998), although it is not known whether
50
Hb acts directly or indirectly to regulate the expression of these genes. We
overexpressed VP16::Hb in a hb mutant background and found that it can activate Kr in
neurons similar to wild type Hb (Fig. 21). Additionally, we found that VP16::Hb
expression also results in the activation of the normally repressed target genes zjh2, cut,
runt, and cas in neurons (Fig. 21). Based on these observations, we conclude that
VP16::Hb can activate all known Hb CNS targets, whether they are normally activated or
repressed by Hb. Because Hb normally acts as a transcriptional repressor of most CNS
targets (Fig. 2J), we suggest that this repression may play an essential role in maintaining
neuroblast competence, and we test this prediction below.
Overexpression of VP16::Hb in the eNS reveals that Hb maintains neuroblast
competence by transcriptional repression of multiple target genes
Because VP16::Hb is a potent transcriptional activator with little ability to repress
gene expression, we can use it to test whether Hb-mediated transcriptional activation of
target genes is sufficient for maintaining neuroblast competence. Overexpression of wild
type Hb can extend neuroblast competence and production of early-born neuronal cell
types (Isshiki et aI., 2001; Novotny et aI., 2002; Tran and Doe, 2008). In NB7-1, Hb
misexpression produces approximately 18-20 Eve+ U motoneurons. If Hb maintains
early-competence by acting solely as a transcriptional activator, then VP16::Hb should
mimic Hb function and specify the same or more U neurons. In wild-type embryos,
NB7-1 generates five Eve+U motoneurons (Fig. 3A, A"') (Pearson and Doe, 2003;
Schmid et aI., 1999). Overexpression of wild type Hb in a hb mutant NB7-1 gives an
51
average of 12 V neurons (range=6-18, n=100; Fig. 3A'). However, the overexpression of
VP16::Hb in a hb mutant NB7-1 only generates an average of six V neurons (range=2-12,
n:::88; Fig. 3A"). We conclude that the constitutive activator VP16::Hb is not as good as
wild type Hb at maintaining the early-competence window necessary for Eve+V neuron
production; and suggest that the repression of Hb downstream targets in neuroblasts may
be essential to maintain early-competence.
Because Hb normally represses downstream targets such as pdm and cas, and
VP16::Hb can activate these targets, we tested whether the repression of these
downstream genes is required to maintain neuroblast competence. First, we examined
engrailed-ga14 UAS-hb, hb mutant embryos at early stage 12 and found that neuroblasts
in the engrailed-ga14 domain expressed Kr but not pdm and cas (Fig. 3B) (lsshiki et aI.,
2001). In contrast, we found that the majority of neuroblasts in the engrailed-ga14
domain of engrailed-ga14 UAS- VP16::hb, hb mutant embryos expressed Kr, pdm, and
cas (Fig. 3C). We conclude that VP16::Hb can ectopically induce pdm and cas
expression in neuroblasts.
Next, we tested whether the co-expression of wild type Hb plus Pdm or Hb plus
Cas can lead to the same reduction in ectopic cells as seen in VP16::Hb overexpression
experiments. In control engrailed-ga14 UAS-hb UAS-HA embryos, NB7-1 generated
about 17 Eve+ V-neurons (range=13-22, n=87; Fig. 3D). We next examined engrailed-
ga14 UAS-hb UAS-pdm2 embryos for the total number of V neurons generated and found
a large decrease in the number of ectopic V neurons compared to our control embryos
(average=9, range=3-14, n=70; Fig. 3D). engrailed-ga14 UAS-hb UAS-cas embryos
52
showed a slight decrease in U neurons generated (average=15, range=8-20, n=118; Fig.
3D). Taken together, we conclude that Hb normally represses down-stream targets such
as pdm and cas to maintain neuroblast competence.
Because Pdm2 is sufficient to block Hb-induced neuroblast competence, we
tested if Pdm2 was also necessary to terminate neuroblast competence. Ifpdm2 is the
only factor activated by VP16::Hb that limits neuroblast competence, then the
overexpression of VP16: :Hb in a pdm mutant background should have extended
neuroblast competence and result in the generation of many Eve+U neurons. On the
other hand, if VP16::Hb activates multiple factors, then VP16::Hb may not be able to
extend competence and make large numbers of U neurons even in the absence of Pdm.
Control prospero-gal4 UAS-hb embryos can generate an average of nine Eve+ U neurons
per hemisegment (range=5-16, n=100; Fig. 3E). In contrast, prospero-gal4 UAS-
VP16::hb embryos generated only 5.6 Eve+ U neurons (range=4-9, n=100, p«.OOl; Fig.
3E), presumably due to the transcriptional activation of factors limiting competence.
Strikingly, performing the same experiments in a pdm mutant embryo (lacking both pdml
and pdm2 genes) did not increase the number of Eve+ U neurons (average=4.3, range=2-
8, n=90, p«.OOl; Fig. 3E). We conclude that Hb must normally repress multiple factors,
in addition to pdml/pdm2, to maintain early neuroblast competence.
Identification of two domains required for Hb transcriptional repression
Because Hb repression of target genes is essential to the maintenance of
neuroblast competence, we next sought to identify the Hb protein domain(s) required for
53
Figure 2. VP16::Hb activates Hb direct and indirect targets in the eNS.
(A-C) VPI6::Hb activates endogenous hb. In-situ hybridization against endogenous hb
mRNA. Each panel shows the neuroblast layer of a stage 12 embryo with the anterior
to the left. Lines in Band C indicate the engrailed-ga14 expression domain.
(D-F) VPI6::Hb activates the direct target,pdm. Histochemical detection ofPdm
(brown) and the segment boundary marker Engrailed (purple) proteins. Each panel
shows the neuroblast layer of a stage 15 embryo with anterior to the left. wornui-ga14
drives gene expression in all neuroblasts.
(G-I) Expression of Kr, zlh2, cut, runt, and cas in wild-type, engrailed-ga14 UAS-hb,
and engrailed-ga14 UAS-VPI6::hb, hb mutant embryos. Each panel shows a 2D-
projection of approximately two segments of the ventral nerve cord of a stage 16
embryo. Anterior is up. Lines in Band C indicate the engrailed-ga14 expression
domain.
(J) Summary of gene interactions in the CNS.
54
A-C
D-F
wild type UAS-hb
UAS-hb
UAS-VP16::hb
UAS-VP16::hb
G
Q)
0-
~
:E
.~
H 1
Q) l.t;j0-
~ \"';=
"tl 1(1,)
~ ~I:§~I
I -gl
:-:::I.Q
.... ~ I ~c: Ol <D$ c: 1 ....
::l Q)I§;E
.Q Ich
"= 1:3
1
1
J Hb
Hb
VP16::Hb
Kr
Pdm, Zlh2, Cut, Runt, Cas
Hb, Kr, Pdm, Zfh2, Cut, Runt, Cas
Figure 2
55
transcriptional repression. We generated a series hb transgenes, each deleted for one or
more evolutionarily-conserved domains (Fig. 4A, B). Hb has eight conserved domains:
the first group of zinc-finger domains bind DNA (DNA-binding domain; DBD), the
second group of zinc-finger domains allow for Hb dimetization (dimerization domain;
DMZ), four previously identified conserved domains A, B, C, and D (Tautz, 1987), and
two additional domains containing short DNA sequences conserved in at least eight
sequenced Drosophila species (B' and E; Fig. 4A). We deleted each of these domains in a
series of UAS-HA::Hb transgenes, which we placed in the same att? site on chromosome
2 so that the results of each transgene could be directly compared.
We tested each protein for its ability to activate the direct target Kr (Hoch et aI.,
1991) or repress the direct target pdm (Kambadur et aI., 1998) within the CNS. Wild type
embryos at stage 11 are Kf Pdm+ in the neuroblast layer (Grosskortenhaus et aI., 2005;
Isshiki et al., 2001; Tran and Doe, 2008). Overexpression of the full length wild type Hb
protein resulted in the activation of Kr and repression ofpdm (Fig. 4B, C), consistent
with previous findings (Isshiki et aI., 2001; Kambadur et aI., 1998). Similarly,
overexpression ofHb proteins lacking either the A+B domains, the B' domain, or the E
domain also activated Kr and repressed pdm (Fig. 4B, C), showing that none of these
domains are required for transcriptional activation or repression. Overexpression of Hb
proteins lacking the DBD or the C domain cannot activate Kr or repress pdm (Fig. 4B,
C), suggesting that they are non-functional, although they are nuclear localized and have
similar stability to wild type Hb protein in neuroblasts (Fig. 4C). In contrast,
overexpression ofHb proteins lacking the D domain (which includes the Mi2-binding
56
Figure 3. Hb maintains early neuroblast competence through the repression of
multiple target genes.
(A-AI/') Quantification ofU neurons (dashed box) specified in various genetic
backgrounds. Wild-type, avg=5; engrailed-ga14 UAS-hb hb mutant, avg=12 ± 2
(standard deviation), range=6-18; engrailed-ga14 UAS-VP16::hb hb mutant, avg=6 ±
2, range=2-12. p«.OOl for all experiments.
(B-C) Neuroblast expression profile at early stage 12. One hemisegment is shown.
Dashed circles outline neuroblasts, solid lines indicate engrailed-ga14 expression
domain. Scale bar equals 10 [lm. Hb activates Kr and represses pdm and cas in
neuroblasts, whereas VP16::Hb activates all three factors.
(D-D') Co-misexpression of Hb + Pdm and Hb + Cas results in less ectopic U neurons.
UAS-hb UAS-HA control, avg=17 ± 2, range=13-22; UAS-hb UAS-pdm2, avg=9 ± 2,
range=3-14; UAS-hb UAS-cas, avg=15 ± 2, range=8-20. p«.OOl for all experiments.
(E) Overexpression ofVP16::Hb in apdm mutant embryo does not result in the recovery
of ectopic U neurons. pros-ga14 UAS-hb, avg=9 ± 2.5, range=5-16; pros-ga14 UAS-
VP16::hb, 5.6 ± 1, range=4-9;pros-ga14 UAS-VP16::hb pdm mutant, avg=4.3 ± 1,
range=2-8. p«.001 for all experiments.
(F) Summary and comparison of the competence window generated by Hb and
VP16::Hb.
57
A
(J)
en<>.
~ 4:
~ ::>0.~ c
A'
-c ..0
.J'l
-<;:
"E (f)
..0 §
-t:
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..0
-
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.J'l
" ~E
..0 ch
.<::
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~
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-
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~ <0
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..0 ch
-t: §
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..0
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-t: c .
ch ~,§
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-' ... ('-
•~.. ~
-"~a'Eve .
F
D'
10 OJ ~'"coe 15~ IT]~10+G>>
w
HA pr/m2 cas
eIJ(Jrailed·gaI4 J( UAS·Hb; VAS·
pros-gElf4 x VAS·
-6
~::~
17·10
I.. c .. .:.:..:....-_--------J~nceUAS·hb
wild lypc
UAS·VPI6::hb
VP16::hbI'b
VPI6::llb;
pdm-
-----------------
'"
10
co
e
~ 15
W
co
::>
+ 10
G> [j] ~>wn~
I'. hb; VP16::hb;Ilb- lIb·
-----------
ef/gra/fed-ga/4 x VAS·
~ 20
~ 15
c:
::>
+ 10
G>
>
W
Figure 3
58
sites) fails to repress pdm, but still weakly activates Kr; an identical result was observed
for Hb protein lacking the DMZ domain (Fig. 4B, C). The sparse and intermittent
activation of Kr may be due to Pdm repression of Kr (Grosskortenhaus et aI., 2006; Tran
and Doe, 2008), which would be expected to counteract Hb-induced Kr activation. We
next tested whether the HbL'.D and HbL'.DMZ proteins fail to repress other known Hb target
genes. Whereas wild type Hb protein can efficiently repress zfh2, cut, runt, and cas (Fig.
3C), the HbL'.D and HbL'.DMZ proteins failed to repress all of these genes (Fig. SI). We
conclude that both the D and DMZ domains are required for Hb-mediated transcriptional
repression.
Hb repression domains are required for maintenance of neuroblast competence
We showed above that overexpression of the constitutive transcriptional activator
VPI6::Hb was not sufficient to extend neuroblast competence, suggesting that this
function may require Hb-mediated transcriptional repression. Here we test whether the D
or DMZ Hb repression domains are required for extending competence. We assay
neuroblast competence by measuring the number of Eve+U neurons that can be induced
by overexpression ofHb within the NB7-1lineage using the engrailed-ga14 driver
(Isshiki et aI., 2001; Pearson and Doe, 2003). Wild type embryos have 5 Eve+ U neurons
per hemisegment (Fig. 3A)(Isshiki et aI., 2001), whereas overexpression of wild type Hb
can extend neuroblast competence to allow the formation of approximately 14 Eve+U
neurons (n=100; Fig. 4D; Table 1). Similarly, overexpression ofHb proteins lacking
either the A+B domains, the B' domain, or the E domain generated approximately 16
59
A
HA
118 125
I \I¢I @ i -!
235 355
I ,
_,.1·1
DNA Binding Domain
450
I
@I
Mi2 Domain
583
I
670 700 758
1m: '-PNA
Dimerization Domain
D
lOW;
c
758
I
235
Hb Proteins
':49 ~3
---------< 1-0
zS2 7ot)
---------< >--<
JS!I 4~O
-----~ o----~
22-4 355
---~I 1>---------<
B
Hb wild lype
~ 1
.,
I
~
<h
~
'"t
LU Hb DAB
cO 118
cO I
~ Hb 0.8'
~ 117
~ ~Hbl.\E
~
'"~
~
a
Hb ,-,OBD
~
0
., Hb (~c..,
""~
Vl
~
~§
'"
Hbc.O
"C
c:i
<'
~ Hb6.0MZ
~
~
i§
Figm'e 4. The Hunchback D and DMZ domains are required for transcriptional
repression and maintenance of neuroblast competence.
(A) Schematic of Hb protein showing conserved domains and deletion breakpoints (bold).
(B) Deletions grouped by functional phenotype.
(C) In each panel set, two segments of a stage 11 embryo are shown with neuroblasts
stained for Kr and Pdm proteins. Dashed lines indicate engrailed-gaf4 expression
domains. Wild type Hb, HbMB, HbL>B', and Hb6.E can activate Kr and repress pdm;
HbL>DllD and Hb6c are non-functional in the eNS; HbL>D and HbL>DMZ can activate Kr,
but cannot repress pdm.
(D) U neurons specified by Hb and each deletion construct.
60
Eve+ U neurons (n>100 each; Fig. 4D; Table 1), showing that none ofthese domains is
required for Hb-mediated extension of neuroblast competence. As expected,
overexpression of the non-functional Hb proteins lacking the DBD or the C domains
cannot specify ectopic U neurons or alter the identity of the existing neurons (Fig. 4D,
S2; Table 1). Interestingly, overexpression of Hb proteins that lack transcriptional
repressor activity, i.e. those lacking the D or DMZ domain, both fail to extend neuroblast
competence, generating only 5-6 Eve+ U neurons (n>200, Fig. 4D; Table 1). The identity
of the Eve+ U neurons will be addressed in the next section, but based simply on the
change in the number of Eve+U neurons, we conclude that Hb-mediated transcriptional
repression using the D and DMZ domains is required to extend neuroblast competence.
Hb repression domains are required for the specification of first-born neuronal
identity
In addition to its role in regulating neuroblast competence, Hb has an essential
role in the specification of early-born neuronal identity in multiple neuroblast lineages
(lsshiki et aI., 2001; Novotny et aI., 2002; Pearson and Doe, 2003; Tran and Doe, 2008).
To determine whether Hb transcriptional repression is required to specify early-born
neuronal identity, we expressed Hb domain deletion proteins in the NB7-1lineage, either
in wild type embryos or hb mutant embryos. Wild type embryos have five U neurons per
hemisegment: the first-born UI neuron is Hb+Kr+, the second-born U2 neuron is Hb+ Kr+
Zfu2+, and the later-born neurons are Zfu2+ Kr+ Cut+ (U3), Zfu2+ Cut+ Runt+ (U4) or
Zfu2+ Cut+ Runt+ Cas+ (U5) (Fig. SA).
61
Overexpression of wild type Hb generated approximately 14 early-born U11U2
neurons when endogenous Hb is present (Table 1), and approximately 12 U11U2 neurons
in a hb mutant background (Fig. 5B; Table 1), consistent with previous reports (Isshiki et
aI., 2001; Novotny et aI., 2002; Pearson and Doe, 2003). Similarly, overexpression ofHb
proteins lacking either the A+B domains, the B' domain, or the E domain also generated
about 12 Eve+ U11U2 neurons in a hb mutant background (Table 1), showing that none of
these domains is required for Hb-mediated specification of early-born neuronal identity.
Overexpression of the Hb deletion proteins that lack transcriptional repression
activity (Hb"'D and Hb"'DMZ ) have 5-6 Eve+U neurons when endogenous Hb is present
(Fig. 5C, E; Table 1). The ectopic neuron is typically the Kr+ U3 neuron, which is
consistent with the Hb"'D and Hb"'DMZ proteins having the ability to transcriptionally
activate Kr (Fig. 5C) and thus specify U3 identity (Cleary and Doe, 2006; Isshiki et aI.,
2001). Consistent with this result, overexpression of Hb"'D and Hb"'DMZ deletion proteins
in a hb mutant background results in loss of the Hb-dependent early-born U11U2 neurons,
while still generating an ectopic U3 neuron (Fig. 5D, F). The only difference we have
observed between the Hb"'D and Hb"'DMZ proteins is that the Hb"'D, but not Hb"'DMz,
frequently generated an ectopic U5 neuron (Fig. 5C, D; Table 1; see Discussion).
Because both the Hb"'D and Hb"'DMZ proteins retain the ability to activate Kr expression,
we conclude that Hb-mediated transcriptional repression through the D and DMZ
domains is required for the specification of first-born neuronal identity.
62
DISCUSSION
We have shown that Hb acts as an activator and repressor of gene expression in
the eNS, but only its transcriptional repressor function is essential for maintaining
neuroblast competence and specifying early-born neuronal identity. We have identified
two repression domains within the Hb protein: the Mi2-binding D domain and the DMZ
dimerization domain.
How do the D and DMZ domains repress gene expression? It is interesting to
note that the D and DMZ domains are not dedicated repression domains such as the one
found in Engrailed (Han and Manley, 1993; Jaynes and O'Farrell, 1991). Instead, both
are known to mediate protein-protein interactions. The DMZ allows Hb dimerization,
leading to the proposal that high Hb levels promote dimerization and thus transcriptional
repression ability (Papatsenko and Levine, 2008). For example, at cellular blastoderm
stages, high levels of Hb in the anterior of the embryo are required to repress Kr, whereas
low Hb levels activate Kr (Hulskamp et aI., 1990; Schulz and Tautz, 1994; Strohl et al.,
1992), and mutations in the DMZ lead to an anterior expansion of the Kr expression
domain (Hulskamp et al., 1994). Yet it remains unknown how Hb dimerization leads to
gene repression. The D domain is also involved in protein-protein interactions. The
region of Hb containing the D domain is known to bind the chromatin regulator Mi2, and
this interaction promotes epigenetic silencing of the Hb target gene Ubx during early
embryonic patterning (Kehle et aI., 1998). Our results suggest that D and DMZ domains
Table I Summary of U neuron identity specified by Hb proteins
Genetic U- neuron identity 11*
Ectopic protein Background Total' n Ul U2 U3 U4 US
wt 5 100
None
hb mutant 3 100 0 0
wt 14 116 8 6 0 0 0
Hb (wild type)
hb mutant 11.5 77 5.5 6 0 0 0
wt 16 114 8 8 0 0 0
HbMB
hb mutant 11.8 65 5 6.8 0 0 0
wt 16.2 102 9 7.2 0 0 0
HbMl'
hb mutant 12.1 62 5 7.1 0 0 0
HbADBD
wt 5 185
hb mutant 3 62 0 0
HbAC
wt 5 180
hb mutant 3 68 0 0
wt 5.5 242 1.25 1.25 0 2
HbAD
hb mutant 4 150 0 0 2 0 2
wt 16 112 8.8 7.2 0 0 0
HbAE
hb mutant 13.2 76 6 7.2 0 0 0
wt 5.3 237 1.15 1.15
HbADMZ
hb mutant 3.6 184 0 0 1.6
P « 0.001 for all experiments.
II Average number of each cell present per hemisegment based on markers
described in Fig. 5.
* Cell fate markers: UI, Hb+ Kr+ Zfh2-; U2, Hb+ Kr+ Zfh2+; U3, Kr+ Cut+;
U4, Runt+ Cas-; U5, Runt+ Cas+.
:j: Total number of U neurons.
n =Total number of hemisegments analyzed.
63
64
Figure 5. The Hb D and DMZ domains are required for the first-temporal identity.
Each panel shows a 2D-projection of U neurons from one hemisegment of a stage 16
embryo; medial is to the left and anterior is on top. Quantification U neuron identity can
be found in Table 1. Scale bar equals 3 [Am.
(A) Wild type embryo. The UI-U5 neurons can be uniquely identified based on the
indicated molecular markers.
(B) engrailed-ga14 UAS-hb embryo. Ectopic early-born UIIU2 neurons are specified.
Arrowheads indicate weak Zfh2+ cells.
(C) engrailed-ga14 UAS-hbLID embryo. An ectopic U2 or U3 neuron can be found
(arrowhead) in 50% ofhemisegments. All hemisegments contain two Cas+ U5
neurons.
(D) engrailed-ga14 UAS-hbLID in a hb mutant embryo. Most hemisegments contain an
ectopic U3 neuron, and no Ul or U2 neurons are specified.
(E) engrailed-ga14 UAS_hbLIDMZ embryo. An ectopic U2 or U3 neuron can be found
(arrowheads) in 25% of hemisegments. All other U neurons differentiate as in wild-
type.
(F) engrailed-ga14 UAS-hbLIDMzin a hb mutant embryo. Most hemisegments contain an
ectopic U3 neuron, and no Ul or U2 neurons are specified.
6S
c
E
F _
C
ttl
:;
E
.Q
.:::
Figure 5
66
could act in different processes that are both required for transcriptional repression
(Fig. 6B, C), or they could act in a common pathway such as dimerization-dependent
recruitment ofMi2 and/or other repressor proteins to the D domain (Fig. 6D).
Hb proteins lacking the D or the DMZ domain have very similar phenotypes in
the CNS (this study). Although both the D and DMZ domains appear to be required for
Hb-mediated transcriptional repression, they do not have identical functions.
Overexpression of HbL1D leads to the specification of two U5 neurons at the expense of
the U4 cell identity, whereas overexpression of HbL1DMZ results in normal U4 and U5
identities (Fig. 5). Perhaps HbL1DMZ retains some ability to repress cas expression,
allowing the production of the Cas- U4 identity. Alternatively, Hb may use the D and
DMZ domains to repress different target genes. Currently we can't distinguish these
models due to the limited number of known Hb direct target genes.
Both Hb and the related mammalian protein Ikaros have major roles as
transcriptional repressors, but are also weak transcriptional activators. How does Hb
activate gene expression within the CNS? We were unable to identify a discrete
activation domain despite the fact that our deletion series covered the entire protein. We
can rule out the possibility that the activation domain maps to the D region, similar to its
location in the closely related Ikaros protein (Sun et aI., 1996), because Hb~D protein has
no effect on Kr transcriptional activation or the specification of U3 neuronal identity
(Figure 4). We can also rule out the presence of a single activation domain within the A,
B, B', E, or DMZ domains for the same reason. Mechanisms for Hb-mediated
transcriptional activation consistent with our data are: Hb activates transcription
67
indirectly by blocking DNA binding of a repressor (Fig. 6A), Hb has multiple activation
domains, or the Hb activation domain is tighly linked to an essential domain like the
DBD. In any case, our Hb:VP16 experiments together with our repression domain
deletion experiments show that Hb-mediated transcriptional repression -- not
transcriptional activation -- is essential for maintaining neuroblast competence and
specifying early-born neuronal identity.
What are the Hb-repressed target genes involved in extending neuroblast
competence? At least one negatively regulated target is pdm, because we find that co-
expression of Pdm with wild type Hb fails to extend neuroblast competence. However,
overexpression of VP16: :Hb in a pdm mutant background (lacking both pdml and pdm2)
was incapable of extending neuroblast competence, showing that Hb must repress
multiple genes to extend competence. In the future, further characterization of Hb CNS
function will require a genomic analyses, such as chromatin immunoprecipitation to
identify Hb binding sites within the genome or TU-tagging (Miller et al., 2009)
experiments to identify all the genes regulated by Hb within the CNS. This type of
comparative analyses may help elucidate the complex gene interactions involved in
regulating neuroblast competence.
Bridge to Chapter IV
In Chapter III, I have shown that Hb act as a transcriptional repressor of multiple
targets to maintain early neuroblast competence and specify the first temporal identity.
To better understand the mechanism by which Hb functions, I have piloted a screen to
68
Figure 6. Models for Hb mediated transcriptional regulation of neuroblast
competence.
(A-D) Proposed molecular interactions underlying Hb eNS function. Magenta ovals,
Hunchback; smaller ovals, Hb dimerization domain; magenta squares, Hb binding
sights; dark-green line, genomic DNA; green arrow, transcription start site; dark-
green boxes, gene; A, activator; green square, activator binding sites; R, repressor;
red squares, repressor binding sites.
(A) Hb binds to its consensus sequence and recruits co-activators (and/or out compete
repressors) to promote gene expression.
(B) Hb monomers bind to genomic DNA and recruit repressor complexes.
(C) Hb monomer bind to the regulatory region of a target gene, which is repressed by Hb
dimerization with a Hb monomer bound in a heterochromatin domain.
(D) Hb dimerization is required for the recruitment of repressor complexes which may
include Mi2 and other repressor proteins.
69
Gene activation
A
c
o
Gene repression (individual)
o
Gene repression (cooperative)
(
Figure 6
70
identify Hb target genes that may: 1) function to restrict the early-competence window, or
2) specify late-born temporal identities. I have performed a microarray analysis to
compare the gene expression profiles of wild-type and Hb misexpression embryos during
early neurogenesis. Gene transcripts that appear less abundant in Hb misexpression
embryos are potentially interesting as they may be Hb targets that must be kept off in
order to maintain early-competence. Furthermore, genes that appear to be repressed
similar to pdm and castor can potentially be factors that specify late-born neuronal
identities. The initial arrays have generated a list of approximately 170 candidate genes.
In chapter IV, I will discuss the rationale, methods, and my preliminary results.
71
CHAPTER IV
IDENTIFICATION OF NOVEL HUNCHBACK TARGET GENES THAT ARE
CANDIDATES FOR RESTRICTING EARLY NEUROBLAST COMPETENCE
AND SPECIFY LATE-BORN NEURONAL IDENTITIES
"IfEdison had a needle to find in a haystack, he would proceed at once with the
diligence of the bee to examine straw after straw until he found the
object ofhis search. I was a sorry witness ofsuch doings,
knowing that a little theory and calculation would
have saved him ninety per cent ofhis labor. "
-- Nikola Tesla
Khoa D. Tran and Chris Q. Doe designed the research; Khoa D. Tran performed research;
Khoa D. Tran and Chris Q. Doe analyzed the data.
INTRODUCTION
The mechanisms governing stem-cell multipotentiality and the ability to specify
the correct cells at the right time and place is an active area of research in both
developmental and stem-cell biology. Yet, little is known about the cell-autonomous
72
mechanisms regulating a progenitor's decision to produce different cells over time. The
ability to manipulate progenitors and to coerce them to produce specific cells when
needed may one day prove useful in therapeutic treatments for tissue repair after a life
altering event such as a stroke, or damage due to neurodegenerative diseases.
The Drosophila C2H2 zinc-finger transcription factor Hunchback (Hb) is an
essential regulator of anterior-posterior patterning and neurogenesis (lsshiki et al., 2001;
Pearson and Doe, 2004; Schulz and Tautz, 1994; Struhl et al., 1992; Tran et al., 2010).
During early Drosophila development, Hb transcriptional regulation of HOX genes
establishes the anterior-posterior axis of the organism. During neurogenesis, Hb is
expressed early in Drosophila neuroblasts and is necessary and sufficient to specify the
"young" neuroblast state in which first-born progeny are produced (Isshiki et aI., 2001).
Loss of Hb in the CNS results in embryos lacking many first-born neurons; and
prolonged Hb expression produces a supernumerary of cells that differentiate with first-
born identity (Isshiki et al., 2001; Novotny et al., 2002).
Previous studies have shown that Hb must act as a transcriptional repressor of
multiple target genes to maintain early neuroblast competence and specify early born
cells (Tran et aI., 2010). Misexpression of a Hb chimera protein (VPI6::Hb) that act as a
strong transcriptional activator revealed that Hb transcriptional repressor activity is
necessary for its function to maintain neuroblast competence. One gene that must be
repressed is the Hb-direct target, pdm. Co-misexpression of Hb and Pdm results in the
repression of the Hb misexpression phenotype, generating less ectopic U neurons than the
misexpression ofHb alone (Isshiki et aI., 2001; Novotny et aI., 2002; Tran et al., 2010).
73
However, Pdm is not the only factor that must normally be repressed by Hunchback, as
the misexpression of VP16: :Hb in a pdm mutant did not result in the recovery of ectopic
U neurons. Therefore, Hb must be repressing at least one other factor to maintain
neuroblast competence.
To identify novel Hb target genes that may act as competence restricting factors,
or factors that may specify late-born neuronal identity, I have piloted a screen to compare
the gene expression profile of wild-type embryos and Hb misexpression using microarray
analysis. Additionally, I have compared the gene expression profile of wild-type
embryos to those that overexpress different Hb-deletions discussed in the previous
chapter. Using a gene cluster analysis, I have identified approximately 175 candidate
Hb-target genes that behave similar to pdm across multiple Hb misexpression genotypes.
In the following months, I will conduct follow-up studies from this dataset. Below, I
describe my progress and future directions.
MATERIALS AND METHODS
Fly stocks
Wild-type (yw) embryos were collected at 23°C and fixed using standard fixation
methods as previously described (Grosskortenhaus et aI., 2005; Tran and Doe, 2008).
For misexpression experiments, flies carrying insc-gal4 (l407-gaI4, Bloomington Stock
Center) on chromosome II were crossed to flies carrying UAS-hb on chromosomes II and
74
III (Wimmer et aI., 2000), UAS_HbI':.DBD, UAS-HbI':.C, UAS-HbI':.D, and UAS_HbfJDMZ on
chromosome II at 25°C (Tran et aI., 2010).
Microarray experiments
Stage 11-12 embryos were collected and total RNA were extracted according to
standard methods. RNA was processed using the Quick-Amp Labeling Kit from Agilent
Techonologies and hybridized to aD. melanogaster oligonucleotide microarray
representing all 14,141 genes from the Flybase release 5.4 genome according to Agilent's
protocol (Agilent Technologies, Santa Clara, CA). Microarray slides were scanned using
an Axon GenePix 4000B scanner and fluorescent ratios for each microarray element were
recovered and normalized using GenePix Pro 6.0. For arrays comparing wild-type
embryos to wild-type Hb rnisexpression embryos, we quantified and averaged the change
in transcript levels of each gene over four biological replicates. Experiments comparing
wild-type embryos to various Hb-deletions rnisexpression embryos were performed in
triplicates.
In-situ probe production and in-situ hybrization
In-situ probes were prepared and in-situs were performed according to the
Berkeley Drosophila Genome Project (BDGP) protocols. In summary, probe templates
were obtained by PCR from cDNA clones or a cDNA library, transcribed and labeled
with Digoxigenin (DIG) (Roche, Pleasonton, CA), and detected using alkaline-
phosphatase conjugated sheep anti-DIG antibodies (Roche, Pleasonton, CA). In-situ
75
images were captured using a Zeiss AxioCam HRc camera on an Axioplan microscope.
Images were processed in Adobe Photoshop and figures were prepared using Adobe
lllustrator (Adobe Systems, San Jose, CA).
RESULTS
Transcriptional profiling and cluster analysis of multiple Hb misexpression
genotypes identify potential Hunchback target genes
To identify potential Hb targets that may act to restrict early neuroblast
competence or specify late-born temporal identities, we have performed microarray
experiments to compare the transcriptional profile of wild-type embryos to those of
various Hb misexpression genotypes. In addition to wild-type Hb, misexpression
experiments were conducted with Hb-deletion proteins that lack the repression domains
(HbL1D and HbL1DMZ ) and Hb-deletion proteins that appear to be non-functional in the
nervous system (HbL1DBD and HbL).c) (Tran et ai., 2010). To filter our microarray data and
minimize background changes in the transcription profile of experimental embryos, we
employed a clustering algorithm to identify genes with similar expression profiles across
all misexpression genotypes.
Because pdm is a Hb target that must be repressed to maintain neuroblast
competence, and because pdm can specify late-born cell identify in at least one neuroblast
lineage, we focus our attention on genes that behave similarly to pdm in microarray
experiments across multiple genotypes. We have identified approximately 175 candidate
76
genes that cluster closely with pdm, in that they are repressed by wild-type Hb but not
repressed by Hb-deletions lacking the repression domains (Fig. 1, Table Sl). One known
Hb CNS target, the gene castor, also clustered closely to pdm, suggesting that our array
experiments can identify genes normally repressed by Hb in neuroblasts (Fig. 1)
(Grosskortenhaus et aI., 2006; Isshiki et aI., 2001; Tran et aI., 2010).
To further filter our array results, we searched the BDGP embryonic gene
expression database to look for expression of our candidate genes in the nervous system.
This search identified 26 genes with expression in the nervous system, 30 genes that are
not expressed in the nervous system, 75 genes with no expression data, and 44 genes that
require further analysis due to insufficient data in the database (Fig. S1, Table S1). Our
in-silico search reduced the list of candidate Hb target to 145 genes, 83 of which are
conserved in vertebrates.
DISCUSSION
We have identified 145 candidate Hb CNS target genes that cluster closely to
known Hb-targets such as pdm and castor. Further characterization is required to
determine whether these genes are expressed in neuroblasts at the right stages. In the
future, whole mount in-situ hybridization for these candidate genes in wild-type and Hb-
misexpression embryos will confirm whether they are repressed by Hb during
neurogenesis.
77
At this time, we are currently examining the 145 candidate genes from a relatively
small cluster containingpdm and castor (Fig. 1). Factors that are identified as Rb targets
will be functional examined for their roles during neurogenesis. This includes the
potential role for specifying unique temporal identities in multiple neuroblast lineages, as
well as the potential role for as factors that restrict neuroblast competence to specify
early-born cell types.
Although our "core" cluster of potential Hb-target genes contains only 170
candidate genes, this analysis can be expanded to include an additional 230 genes in
adjacent clusters. We will examine this second tier of candidate genes after completing
our initial investigation of the core cluster. And, while we have limited our initial efforts
to genes that are repressed by Rb, this data set can also be used to identify factors that are
activated by Rb. But, that will be another story for another time and person.
78
Figure 1. Cluster analysis identifies potential Hb CNS targets.
Microarray experiments and cluster analysis comparing the transcriptional profiles of
wild-type and multiple Hb-misexpression phenotypes. Each column represents an
average over multiple biological replicates (UAS_hbWild-type, four; all others, three). Blue
represents up-regulated genes, and yellow represents down-regulation genes.
(A) Cluster of all genes that are differentially expressed. Only genes that are present in
all biological replicates are included in this analysis.
(B) Cluster containing genes that behave similarly to known Hb CNS targets across all
genotypes.
79
<1J ~~ a N a N
:g en ~ :g en ~() a a a () a a as <J <J <J <l S <J <J <J <J
.Q .Q .Q .Q .Q .Q .Q .Q .Q .Q
:r: :r: :r: :r: :r: :r: :r: :r: :r: :r:
, ,
CI) CI) CI) CI) CI) CI) CI) CI) CI) CI)
§ § § § § § <l: § § §:::,
A B
Figure 1
80
CHAPTER V
CONCLUSIONS
My dissertation work has focused on the temporal regulation of Drosophila
neuroblasts with an emphasis on the molecular mechanisms and genetic interactions
underlying the specification of neuronal cell identities over developmental time. I, and
others, have shown that temporally regulated genes encode for factors that specify
temporal identities as well as factors that regulate the timing of gene expression in
neuroblasts. In this text, I have highlighted the impOltance of precise cross-regulation
between the temporal identity factors Hb and Kr, and timing factors Pdm and Cas; and
how they must function in perfect unison to promote the proper specification of neuronal
identities at the correct time, and in the correct place.
In Chapter II, I discussed the findings from my investigation of the functions of
Pdm and Cas in a new neuroblast lineage. While previous studies conducted in the NB7-
1 lineage shows that Pdm and Cas specifies late-born cell identities (Grosskortenhaus et
aI., 2006), my studies in the NB3-1 lineage revealed that they act to regulate the timing of
the temporal identity windows under which late-born cell identities are specified. Since
my initial characterization of Pdm and Cas as regulator of temporal identity windows in
81
NB3-1, others have indentified important roles for Cas as the initiator of important gene
regulation cascades required for the specification of late-born cell identities in an
additional neuroblast lineage, NB5-6 (Baumgardt et al., 2009; Karlsson et aI., 2010).
These findings underscore the need to investigate the role of temporally regulated factors
in multiple lineages in order to gain a more comprehensive understanding of temporal
regulation during neural development. In the future, as more tools are development to aid
in the analysis of single neuroblast lineages, it will be possible to identify and compare
the multi-faceted roles of factors such as Pdm and Cas in many neuroblasts.
In Chapter III, I discussed in detail the findings from my investigation of how Hb
maintains early neuroblast competence. It has been well documented, by myself and
others, that Hb is necessary and sufficient to maintain early neuroblast competence and
specify early-born cells in many neuroblast lineages (lsshiki et aI., 2001; Novotny et aI.,
2002; Tran and Doe, 2008). However, the mechanisms underlying Hb function are
unknown; and this understanding is crucial for helping to identify novel factors involved
in specifying the early-competence state. Therefore, I conducted a molecular dissection
of Hb functions during early neurogenesis. I found that although Hb is capable of
activating and repressing gene expression in the embryonic nervous system, it is the
transcriptional repression of multiple target genes that is necessary to maintain early-
competence and specify early-born neuronal identities. Further, I found that this
repression is mediated through two well conserved protein-protein interaction domains,
suggesting that Hb may regulate gene expression by recruiting one or more co-factors. In
82
the future, it will be important to identify these binding partners and characterize how
they regulate gene expression together with Hb.
Another question that stems from the work I described in Chapter TIl is "What are
the factors that must be repressed in order to maintain early neuroblast competence?"
Thus, in Chapter IV I describe my initial attempt to identify Hb CNS targets that must be
repressed to maintain early neuroblast competence. I have identified a list of candidate
genes and will proceed to characterize their function during nervous system development
upon confirmation that they are indeed targets of Hb.
CLOSING REMARKS
Recent studies have also shown that the Hb ortholog, Ikaros, is necessary and
sufficient to specify early neural progenitor competence and early-born cells in the
vertebrate retina. The orthologs of Kntppel and pdm are also present in vertebrates and
have important roles in stem-cell biology. The Kruppel-like factor 4 and Oct4 (POD
domain protein family which includes Pdm) are sufficient to create induced pluripotent
stem cells from fully differentiated cells in mouse models (Kim et aI., 2009; Kim et aI.,
2008). Thus, a fundamental understanding of how different states of neural progenitor
competence in Drosophila is specified and maintained may have further reaching
implications than merely that of a conceptual understanding of how progenitors can stay
"young".
APPENDIX A
SUPPLEMENTAL MATERIALS FOR CHAPTER II
83
84
Figure 81. Misexpression of Hb generates ectopic RPl/RP4 motor neurons and a
thicker motor nerve root.
(A) Wild type embryo stained for HB9 and Fasciclin III (FasIII). RP motor neurons
express FasIII (white chevron) and exit the CNS via the ISNa nerve root (white
arrowhead). Dashed vertical lines show the edge of CNS in A and B.
(B) Misexpression ofHb (worniu-gaI4 UAS-hb) results in more RP motor neurons and a
thicker ISNa fascicle (white arrowhead). Scale bar is 3 j..tm in A and B.
(C) Wild type RP motor neurons project to the indicated lateral muscles (muscle names in
white, RP axons labeled in yellow text). Nerve roots, white arrowheads. Image is one
dissected hemi-embryo shown in mirror image, with muscles shown on the right side.
(D) Misexpression ofHb (worniu-gaI4 UAS-hb) shown with the same orientation and
labeling as in C. Nerve roots are thicker (white arrowheads) and RP motor neurons
reach their normal muscle field (yellow labels). Scale bar is 10 j..tm in C and D.
8S
HB9 HB9
c
o
Figure 81
86
cas-
e
o
Figure S2. Ectopic Isl+ HB9+ cells that are superficial to RPS in cas mutant embryos
do not express the motor neuron marker, Late Bloomer.
Islet, green; HB9, magenta; Late Bloomer, red. RP neurons, white arrowheads; ectopic
Isl+ HB9+ cells (yellow arrowhead; 1-2 per hemisegment, 90%, n= 183). For all panels, a
single representative hemisegment of a stage-] 6 eNS is shown as a maximum intensity
projection; midline, left; anterior, up; phenotype summary, right (Late Bloomer, red
olltlines); scale bar, 3/-lm.
87
A
B
c
Isl+ or HB9+ EW
Hb Kr
(
neurons
Zfh2
Figure 83. NB7-3 pl'ogeny are unaffected in pdm mutants and after Pdm
misexpression.
(A-C) Interneurons of the NB7-3 lineage. Molecular markers shown at top; genotypes
shown to left; schematic of neuronal identity shown to right. The first GMC makes
the Hb+Kr+ EWJ interneuron (and the GW motoneuron, not shown), the second GMC
makes the Hb-Kr+ EW2 interneuron, and the third GMC makes the Zfh2+ EW3
interneuron (Isshiki et al., 200 I; Novotny et al., 2002). Pdm is expressed in the EW3
interneuron (Isshiki et al., 200 1) and possibly transiently in other neurons in the
lineage (Novotny et al., 2002). Anterior, up, midline, left. Scale bar is 3 !lm.
(A) Wild type (wt) embryos have EWI-EW3 neurons.
(B) pdm mutant embryos have norma] EW neuron fates.
(C) Pdm misexpression embryos (insc-gaI4 lJAS-pdm2) have normal FW neuron fMP.S.
APPENDIXB
SUPPLEMENTAL MATERIALS FOR CHAPTER III
88
89
A
B
c
-C
nl
-::lE
.0
-I:
'e1<1
• .0
lot::I
It/)
:§
I
,!,I
Cli'
0)1
"61
~:~0)'
c:: 'lil',
'N
':i!;
'e
'<1
'.0
lot::
lei,
10:(
l:::i
I
I
I
1
1
1
Figure SI: The D and DMZ domains are required for Hb repression of eNS
targets.
(A-C) The overexpression of Hb6D and HbADMZ fail to repress Hb CNS targets normally
(A) Expression of Zfh2, Cut, Runt, and Cas in hb mutants.
(B-C) There is no noticeable change in the expression of Hb CNS target genes.
90
~IA:.Hb
\~ 1t
.-~~'~~'4 5. "- '.-'.. _,: .... )..-tt...
3 '2 ,., 4
1 ,-,:,.: ~-, 5
.. ,-~ )'-~.l~'~~1-' .'-'lWl'-' ,.'!\ '-'
... -
'Figure 82: Overexpression of HbLill and HbLillMZ does not alter U neuron identities.
Each panel shows a 2D-projection of U neurons from one hemisegment of a stage 16
embryo; medial is to the left and anterior is on top. Scale bar equals 3 lAm.
(A-B) UJ -U5 neurons differentiate normally.
91
APPENDIX C
SUPPLEMENTAL MATERIALS FOR CHAPTER IV
Distribution of expression data from BDGP expression database
In:,unicien
dal0, .:\4
C 50 e,:presslc ,_6
Figure Sl. Distribution of candidate genes based on established expression database.
The application of an in-silica filter identifies 26 genes with known CNS expression and
removes 30 genes with no CNS expression,
92
Table Sl Candidate Hb-target gene cluster
Fold
Gene Name Change Known Expression Pattern Vertebrate Homolog
1 Klp61F -1.4 CNS Brain Yes
2 l(l)sc -1.6 CNS Brain Gut Strip Yes
3 ena -1.4 CNS Yes
4 asp -1.4 CNS Brain Yes
5 Mcm7 -1.5 CNS Brain Yes
6 dnk -1.5 CI\JS Brain Gut Yes
7 Ect3 -4.5 CNS Yes
8 hb -1.5 CNS Yes
9 cas -2.0 CNS Brain Yes
10 Chrac-16 -2.0 CNS Brain Yes
11 pdm2 -1.4 CNS Brain Gut Yes
12 Nub -2.7 CNS Brain Yes
13 CGl1398 -1.5 maternal Yes
14 CG11403 -1.4 maternal Yes
15 CG12863 -1.6 maternal Yes
16 CG13277 -2.6 maternal Yes
17 CG13398 -1.4 maternal Yes
18 CG15747 -2.0 maternal Yes
19 CGl7219 -1.6 maternal Yes
20 CG17470 -1.8 maternal Yes
21 CG31120 -1.4 maternal Yes
22 CG40191 -1.5 maternal Yes
23 CG5857 -1.5 maternal Yes
24 CG5969 -1.5 maternal Yes
25 CG5987 -1.6 maternal Yes
26 CG6071 -2.1 maternal Yes
27 CG7597 -1.4 maternal Yes
28 CG8441 -1.4 maternal Yes
29 CG9723 -1.7 maternal Yes
30 fh -1.4 maternal Yes
31 RpL17 -2.3 maternal Yes
32 RpL37a -1.5 maternal Yes
..,..,
scpr-C -1.5 maternal Yes;);)
34 Sse -1.5 maternal Yes
35 mit(1)15 -1.4 Muscle Brain Yes
36 CGl1327 -2.0 No expression Yes
37 CG11436 -1.6 No expression Yes
Fold
Gene Name Change Known Expression Pattern Vertebrate Homolog
38 CG4680 -1.4 No expression Yes
39 CG7102 -1.4 No expression Yes
40 Clk -1.7 No expression Yes
41 Rala -4.0 No expression Yes
42 Rh7 -1.7 No expression Yes
43 pxb -2.1 Stripes Yes
44 Aats-gln -1.5 no data Yes
45 ac -1.7 CNS,4 neuroblasts Yes
46 amos -1.4 PNS, sensory neurons Yes
47 ato -1.5 PNS, sensory neurons Yes
48 baf -1.5 no data Yes
49 SobA -2.0 no data Yes
50 CG10469 -1.4 no data Yes
51 CG10581 -1.4 no data Yes
52 CG10859 -2.1 no data Yes
53 CG11839 -1.4 no data Yes
54 CG12269 -2.3 no data Yes
55 CG12325 -1.8 no data Yes
56 CG13083 -2.1 no data Yes
57 CG1311 -1.5 no data Yes
58 CG13361 -27.2 no data Yes
59 CG13427 -1.4 no data Yes
60 CG13454 -1.8 no data Yes
61 CG13465 -2.0 no data Yes
62 CG13794 -12.1 no data Yes
63 CG14057 -1.4 no data Yes
64 CG14966 -1.4 no data Yes
65 CG15456 -1.5 no data Yes
66 CG15816 -1.6 no data Yes
67 CG17118 -1.7 no data Yes
68 CGl7233 -1.6 no data Yes
69 CGl7264 -1.6 no data Yes
70 CG18190 -1.5 no data Yes
71 CG1946 -2.0 no data Yes
72 CG2453 -2.0 no data Yes
73 CG30272 -1.8 no data Yes
74 CG3071 -1.7 no data Yes
75 CG31012 -1.4 no data Yes
93
Fold
Gene Name Change Known Expression Pattern Vertebrate Homolog
76 CG31606 -21.1 no data Yes
77 CG32150 -1.7 no data Yes
78 CG32182 -1.4 no data Yes
79 CG32572 -27.2 maternal Yes
80 CG32581 -2.8 no data Yes
81 CG33791 -1.7 no data Yes
82 CG3781 -1.4 no data Yes
83 CG4645 -2.6 no data Yes
84 Esp -1.4 Ectoderm Yes
85 HLHm5 -1.7 Ectoderm Yes
86 SAK -1.7 Ectoderm Yes
87 Toll-6 -1.5 Ectoderm Yes
88 slp2 -1.4 Ectoderm Stripes Yes
89 CG6910 -2.2 fat body Yes
90 Dhpr -1.6 fat body Yes
91 boi -1.5 Gut Yes
92 bwa -1.5 Gut Yes
93 CG18600 -1.6 Gut Yes
94 CG6512 -1.4 Gut Yes
95 CG9279 -4.2 Gut Yes
96 tst -1.4 Gut Yes
97 CG9281 -1.4 Gut Muscles Yes
98 Docl -1.5 Guts Muscles Yes
99 Doc2 -1.4 Guts Muscles Yes
100 Doc3 -1.4 Guts Muscles Yes
101 CG15628 -1.4 Gut Yes
102 CG30431 -1.5 Gut, Yolk Sac Yes
103 CG8436 -1.4 muscle Yes
104 bin -1.4 muscle Yes
105 CG12022 -1.7 Ubi Yes
106 CG8191 -1.5 Ubi Yes
107 CG11407 -1.7 Yolk nuclei Yes
109 CG3085 -1.7 not interesting Yes
110 CG3597 -2.1 not interesting Yes
111 CG30043 -2.1 not interesting Yes
112 Sry-alpha -1.7 not interesting Yes
113 CG7768 -1.4 CNS Not identified
114 CG14937 -1.5 CNS Brain Not identified
94
95
Fold
Gene Name Change Known Expression Pattern Vertebrate Homolog
115 CG17321 -1.5 CNS Brain Not identified
116 Sr-CII -1.7 CNS Ectoderm Not identified
117 CG12236 -1.4 CNS (faint) Not identified
118 ial -1.5 CNS Brain Not identified
119 mod(mdg4) -1.5 CNS Brain Not identified
120 Nrt -1.5 CNS Brain Not identified
121 BEAF-32 -1.4 CNS Brain Gut Not identified
122 Mes2 -1.4 CNS Brain Gut Not identified
123 CG3036 -1.5 CNS Not identified
124 msbll -1.5 CNS Ubi Not identified
125 mira -1.4 CNS Not identified
126 Gte -1.4 CNS Brain Not identified
127 CG4676 -1.5 no data Not identified
128 CG5167 -1.5 no data Not identified
129 Reck -1.4 no data Not identified
130 CG5602 -1.4 no data Not identified
131 Npc2f -1.4 no data Not identified
132 Ir68a -1.4 no data Not identified
133 CG6475 -1.8 no data Not identified
134 CG6520 -1.4 no data Not identified
135 CG6729 -1.6 no data Not identified
136 CG7763 -3.0 no data Not identified
137 CG7912 -12.9 no data Not identified
138 CG8783 -1.5 no data Not identified
139 CG9270 -1.7 no data Not identified
140 cmet -1.4 no data Not identified
141 CS-2 -1.4 no data Not identified
142 DNApol-epsiion -1.4 no data Not identified
143 Dspl -1.4 no data Not identified
144 Fancd2 -1.4 no data Not identified
145 fz -1.4 no data Not identified
146 Grip84 -1.5 no data Not identified
147 gsb -1.4 Subset of neuroblasts Not identified
1110 UI U"""",'"7
-1.6 .__ -1_ .... - Not identified.L"to IILnlll1 IIUUdLd
149 Hsp70Bc -1.5 no data Not identified
150 ind -1.4 no data Not identified
Fold
Gene Name Change Known Expression Pattern Vertebrate Homolog
151 Kap3 -5.6 no data Not identified
152 kek5 -1.6 no data Not identified
153 Klp54D -10.5 no data Not identified
154 1(2)05714 -1.4 Ectoderm, maybe CNS Not identified
155 HipHop -1.5 no data Not identified
156 malpha -1.4 no data Not identified
157 MED7 -1.4 faint ubi Not identified
158 MRP -1.7 no data Not identified
159 mus205 -1.4 no data Not identified
160 nmdyn-D7 -1.4 no data Not identified
161 Notum -1.4 no data Not identified
162 pad -1.4 no data Not identified
163 Pen -1.4 no data Not identified
164 polo -1.5 no data Not identified
165 Pptl -1.4 no data Not identified
166 RfC3 -1.5 no data Not identified
167 RpL37A -1.4 maternal Not identified
168 sc -1.4 Ectoderm, not CNS Not identified
169 sca -1.5 no data Not identified
170 sens -1.4 Ectoderm, not CNS Not identified
171 Six4 -1.6 no data Not identified
172 spn-E -1.9 no data Not identified
173 sqz -1.4 CNS Brain Not identified
174 wnd -2.0 no data Not identified
175 Cyt-b5-r -2.2 Gut Not identified
Gene expression data were collected from the Berkeley Drosophila Genome Project
database (as of June 1,2010).
96
97
REFERENCES
Albertson, R., Chabu, c., Sheehan, A. and Doe, C. Q. (2004). Scribble protein domain
mapping reveals a multistep localization mechanism and domains necessary for
establishing cortical polarity. J Cell Sci 117,6061-70.
Baumgardt, M., Karlsson, D., Terriente, J., Diaz-Benjumea, :F. J. and Thor, S.
(2009). Neuronal subtype specification within a lineage by opposing temporal feed-
forward loops. Cell 139, 969-82.
Berman, B. P., Nibu, Y., Pfeiffer, B. D., Tomancak, P., Celniker, S. E., Levine, M.,
Rubin, G. M. and Eisen, M. B. (2002). Exploiting transcription factor binding site
clustering to identify cis-regulatory modules involved in pattern formation in the
Drosophila genome. Proc Natl Acad Sci USA 99, 757-62.
Berman, B. P., Pfeiffer, B. D., Laverty, T. R., Salzberg, S. L., Rubin, G. M., Eisen,
M. B. and Celniker, S. E. (2004). Computational identification of developmental
enhancers: conservation and function of transcription factor binding-site clusters in
Drosophila melanogaster and Drosophila pseudoobscura. Genome BioI 5, R61.
Berry, M. and Rogers, A. W. (1965). The migration of neuroblasts in the developing
cerebral cortex. J Anat 99,691-09.
Bischof, J., Maeda, R. K., Hediger, M., Karch, F. and Basler, K. (2007). An
optimized transgenesis system for Drosophila using germ-line-specific phiC31 integrases.
Proc Natl Acad Sci USA 104, 3312-7.
Bossing, T., Udolph, G., Doe, C. Q. and Technau, G. M. (1996). The embryonic
central nervous system lineages of Drosophila melanogaster. I. Neuroblast lineages
derived from the ventral half of the neuroectoderm. Dev BioI 179, 41-64.
Brand, A. H. and Perrimon, N. (1993). Targeted gene expression as a means of altering
cell fates and generating dominant phenotypes. In Development, voL 118 (ed., pp. 401-
15.
Broadus, J., Skeath, J. B., Spana, E. P., Bossing, T., Technau, G. and Doe, C. Q.
(1995). New neuroblast markers and the origin of the aCC/pCC neurons in the
Drosophila central nervous system. Mech Dev 53, 393-402.
Brody, T. and Odenwald, W. F. (2000). Programmed transformations in neuroblast
gene expression during Drosophila CNS lineage development. Dev BioI 226, 34-44.
-------------
98
Broihier, H. T. and Skeath, J. B. (2002). Drosophila homeodomain protein dHb9
directs neuronal fate via crossrepressive and cell-nonautonomous mechanisms. Neuron
35,39-50.
Campos-Ortega, J. A. and Hartenstein, V. (1985). The embryonic development of
Drosophila melanogaster: Springer-Verlag, Berlin.
Cepko, C. L. (1999). The roles of intrinsic and extrinsic cues and bHLH genes in the
determination of retinal cell fates. Curr Opin Neurobiol9, 37-46.
Chu-LaGraff, Q. and Doe, C. Q. (1993). Neuroblast specification and formation
regulated by wingless in the Drosophila CNS. Science 261, 1594-7.
Cleary, M. D. and Doe, C. Q. (2006). Regulation of neuroblast competence: multiple
temporal identity factors specify distinct neuronal fates within a single early competence
window. Genes Dev 20, 429-34.
Cui, X. and Doe, C. Q. (1992). ming is expressed in neuroblast sublineages and
regulates gene expression in the Drosophila central nervous system. Development 116,
943-52.
Desai, A. R. and McConnell, S. K. (2000). Progressive restriction in fate potential by
neural progenitors during cerebral cortical development. Development 127, 2863-72.
Doe, C. Q. (1992). Molecular markers for identified neuroblasts and ganglion mother
cells in the Drosophila central nervous system. Development 116,855-63.
Doe, C. Q. and Skeath, J. B. (1996). Neurogenesis in the insect central nervous system.
Curr Opin Neurobiol6, 18-24.
Elliott, J., Jolicoeur, C., Ramamurthy, V. and Cayouette, M. (2008). Ikaros confers
early temporal competence to mouse retinal progenitor cells. Neuron 60, 26-39.
Gaul, D., Seifert, K, Schuh, R. and JackIe, H. (1987). Analysis of Kruppel protein
distribution during early Drosophila development reveals posttranscriptional regulation.
Cell 50, 639-47.
Grosskortenhaus, R., Pearson, B. J., Marusich, A. and Doe, C. Q. (2005). Regulation
of temporal identity transitions in Drosophila neuroblasts. Dev CellS, 193-202.
Grosskortenhaus, R., Robinson, K. J. and Doe, C. Q. (2006). Pdm and Castor specify
late-born motor neuron identity in the NB7-1Iineage. Genes Dev 20, 2618-27.
Han, K. and Manley, J. L. (1993). Functional domains ofthe Drosophila Engrailed
protein. EMBO J 12, 2723-33.
99
Hanashima, c., Fernandes, M., Hebert, J. M. and Fishell, G. (2007). The role of
Foxg1 and dorsal midline signaling in the generation of Cajal-Retzius subtypes. J
Neurosci 27, 11103-11.
Hanashima, C., Li, S. C., Shen, L., Lai, E. and Fishell, G. (2004). Foxg1 suppresses
early cortical cell fate. Science 303, 56-9.
Harris, W. A. (1997). Cellular diversification in the vertebrate retina. Curr Opin Genet
Dev 7,651-8.
Harrison, D. A., Binari, R., Nahreini, T. S., Gilman, M. and Perrimon, N. (1995).
Activation of a Drosophila Janus kinase (JAK) causes hematopoietic neoplasia and
developmental defects. EMBO J 14,2857-65.
Hoch, M. and JackIe, H. (1998). Kruppel acts as a developmental switch gene that
mediates Notch signalling-dependent tip cell differentiation in the excretory organs of
Drosophila. EMBO J 17, 5766-75.
Hoch, M., Seifert, E. and JackIe, H. (1991). Gene expression mediated by cis-acting
sequences of the Kmppel gene in response to the Drosophila morphogens bicoid and
hunchback. EMBO J 10, 2267-78.
Hulskamp, M., Lukowitz, W., Beermann, A., Glaser, G. and Tautz, D. (1994).
Differential regulation of target genes by different alleles of the segmentation gene
hunchback in Drosophila. Genetics 138, 125-34.
Hulskamp, M., Pfeifle, C. and Tautz, D. (1990). A morphogenetic gradient of
hunchback protein organizes the expression of the gap genes Kruppel and knirps in the
early Drosophila embryo. Nature 346, 577-80.
Isshiki, T., Pearson, B., Holbrook, S. and Doe, C. Q. (2001). Drosophila neuroblasts
sequentially express transcription factors which specify the temporal identity of their
neuronal progeny. Cell 106, 511-21.
Jaynes, J. B. and O'Farrell, P. H. (1991). Active repression of transcription by the
engrailed homeodomain protein. EMBO J 10,1427-33.
Kambadur, R., Koizumi, K., Stivers, C., Nagle, J., Poole, S. J. and Odenwald, W. F.
(1998). Regulation of POD genes by castor and hunchback establishes layered
compartments in the Drosophila CNS. Genes Dev 12, 246-60.
Kanai, M. I., Okabe, M. and Hiromi, Y. (2005). seven-up Controls switching of
transcription factors that specify temporal identities of Drosophila neuroblasts. Dev Cell
8,203-13.
100
Karcavich, R. and Doe, C. Q. (2005). The Drosophila neuroblast 7-3 cell lineage: a
model system for studying programmed cell death, NotchfNumb signaling, and sequential
specification of GMC identity. J Comp Neuro1481, 240-51.
Karlsson, D., Baumgardt, M. and Thor, S. (2010). Segment-specific neuronal subtype
specification by the integration of anteroposterior and temporal cues. PLoS Biol8,
e1000368.
KeWe, J., Beuchle, D., Treuheit, S., Christen, B., Kennison, J. A., Bienz, M. and
Muller, J. (1998). dMi-2, a hunchback-interacting protein that functions in polycomb
repression. Science 282, 1897-900.
Kim, J. B., Sebastiano, V., Wu, G., Arauzo-Bravo, M. J., Sasse, P., Gentile, L., Ko,
K., Ruau, D., Ehrich, M., van den Boom, D., Meyer, J., Hubner, K., Bernemann, C.,
Ortmeier, C., Zenke, M., Fleischmann, B.K., Zaehres, H., Scholer, H.R. (2009).
Oct4-induced pluripotency in adult neural stem cells. Cell 136, 411-9.
Kim, J. B., Zaehres, H., Wu, G., Gentile, L., Ko, K., Sebastiano, V., Arauzo-Bravo,
M. J., Ruau, D., Han, D. W., Zenke, M., Scholer, H.R. (2008). Plm1potent stem cells
induced from adult neural stem cells by reprogramming with two factors. Nature 454,
646-50.
Kosman, D., Small, S. and Reinitz, J. (1998). Rapid preparation of a panel of
polyclonal antibodies to Drosophila segmentation proteins. Dev Genes Evol208, 290-4.
Lai, S. L. and Lee, T. (2006). Genetic mosaic with dual binary transcriptional systems in
Drosophila. Nat Neurosci 9, 703-9.
Lai, Z. c., Fortini, M. E. and Rubin, G. M. (1991). The embryonic expression patterns
of zfh-1 and zfh-2, two Drosophila genes encoding novel zinc-finger homeodomain
proteins. Mech Dev 34, 123-34.
Landgraf, M., Bossing, T., Technau, G. M. and Bate, M. (1997). The origin, location,
and projections of the embryonic abdominal motorneurons of Drosophila. J Neurosci 17,
9642-55.
Livesey, R. and Cepko, C. (2001). Neurobiology. Developing order. Nature 413, 471,
473.
Lundell, M. J., Chu-LaGraff, Q., Doe, C. Q. and Hirsh, J. (1996). The engrailed and
huckebein genes are essential for development of serotonin neurons in the Drosophila
CNS. Mol Cell Neurosci 7, 46-61.
101
Lundell, M. J. and Hirsh, J. (1998). eagle is required for the specification of serotonin
neurons and other neuroblast 7-3 progeny in the Drosophila eNS. Development 125, 463-
72.
Markstein, M., Pitsouli, C., Villalta, C., Celniker, S. E. and Perrimon, N. (2008).
Exploiting position effects and the gypsy retrovirus insulator to engineer precisely
expressed transgenes. Nat Genet 40,476-83.
Maurange, c., Cheng, L. and Gould, A. P. (2008). Temporal transcription factors and
their targets schedule the end of neural proliferation in Drosophila. Cell 133, 891-902.
McConnell, S. K. (1995). Strategies for the generation of neuronal diversity in the
developing central nervous system. J Neurosci 15, 6987-98.
Mettler, U., Vogler, G. and Urban, J. (2006). Timing of identity: spatiotemporal
regulation of hunchback in neuroblast lineages of Drosophila by Seven-up and Prospero.
Development 133, 429-37.
Miller, A. C., Seymour, H., King, C. and Herman, T. G. (2008). Loss of seven-up
from Drosophila RIIR6 photoreceptors reveals a stochastic fate choice that is normally
biased by Notch. Development 135, 707-15.
Miller, M. R., Robinson, K. J., Cleary, M. D. and Doe, C. Q. (2009). TU-tagging: cell
type-specific RNA isolation from intact complex tissues. Nat Methods 6,439-41.
NIlodzik, M., Hiromi, Y., Weber, U., Goodman, C. S. and Rubin, G. M. (1990). The
Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls
photoreceptor cell fates. Cell 60, 211-24.
Novotny, T., Eiselt, R. and Urban, J. (2002). Hunchback is required for the
specification of the early sublineage of neuroblast 7-3 in the Drosophila central nervous
system. Development 129, 1027-36.
Odden, J. P., Holbrook, S. and Doe, C. Q. (2002). Drosophila HB9 is expressed in a
subset of motoneurons and interneurons, where it regulates gene expression and axon
pathfinding. J Neurosci 22,9143-9.
Odenwald, W. F., Rasband, W., Kuzin, A. and Brody, T. (2005). EVOPRINTER, a
multigenomic comparative tool for rapid identification of functionally important DNA.
Proc NatlAcad Sci USA 102, 14700-5.
Pankratz, M. J., Busch, M., Hoch, M., Seifert, E. and JackIe, H. (1992). Spatial
control of the gap gene knirps in the Drosophila embryo by posterior morphogen system.
Science 255,986-9.
102
Papatsenko, D. and Levine, M. S. (2008). Dual regulation by the Hunchback gradient in
the Drosophila embryo. Proc Natl Acad Sci USA 105, 2901-6.
Patel, N. H., Condron, B. G. and Zinn, K. (1994). Pair-rule expression patterns of
even-skipped are found in both short- and long-germ beetles. Nature 367, 429-34.
Patel, N. H., Martin-Blanco, K, Coleman, K. G., Poole, S. J., Ellis, M. C., Kornberg,
T. B. and Goodman, C. S. (1989). Expression of engrailed proteins in arthropods,
annelids, and chordates. Cell 58, 955-68.
Pearson, B. and Doe, C. Q. (2004). Specification of temporal identity in the developing
nervous system. Annu Rev Cell Dev Biol20, 619-47.
Pearson, B. J. and Doe, C. Q. (2003). Regulation of neuroblast competence in
Drosophila. Nature 425,624-28.
Pelegri, F. and Lehmann, R. (1994). A role of polycomb group genes in the regulation
of gap gene expression in Drosophila. Genetics 136, 1341-53.
Prokop, A. and Technau, G. M. (1994). Early tagma-specific commitment of
Drosophila CNS progenitor NB 1-1. Development 120, 2567-78.
Rapaport, D. H., Patheal, S. L. and Harris, W. A. (2001). Cellular competence plays a
role in photoreceptor differentiation in the developing Xenopus retina. J Neurobiol49,
129-41.
Reid, C. B., Tavazoie, S. F. and Walsh, C. A. (1997). Clonal dispersion and evidence
for asymmetric cell division in felTet cortex. Development 124,2441-50.
Rivera-Pomar, R., Lu, X., Perrimon, N., Taubert, H. and Jackie, H. (1995).
Activation of posterior gap gene expression in the Drosophila blastoderm. Nature 376,
253-6.
Romani, S., Jimenez, F., Hoch, M., Patel, N. H., Taubert, H. and Jackie, H. (1996).
Kruppel, a Drosophila segmentation gene, participates in the specification of neurons and
glial cells. Mech Dev 60, 95-107.
Schmid, A., Chiba, A. and Doe, C. Q. (1999). Clonal analysis of Drosophila embryonic
neuroblasts: neural cell types, axon projections and muscle targets. Development 126,
4653-89.
Schmidt, H., Rickert, C., Bossing, T., Vee, 0., Urban, J. and Technau, G. M. (1997).
The embryonic central nervous system lineages of Drosophila melanogaster. II.
Neuroblast lineages derived from the dorsal part of the neuroectoderm. Dev Biol189,
186-204.
------ -----
103
Schulz, C. and Tautz, D. (1994). Autonomous concentration-dependent activation and
repression of Kruppel by hunchback in the Drosophila embryo. Development 120, 3043-
9.
Siegrist, S. E. and Doe, C. Q. (2005). Microtubule-induced Pins/Galphai cortical
polarity in Drosophila neuroblasts. Cell 123, 1323-35.
Skeath, J. B. and Doe, C. Q. (1998). Sanpodo and Notch act in opposition to Numb to
distinguish sibling neuron fates in the Drosophila CNS. Development 125, 1857-65.
Skeath, J. B., Zhang, Y., Holmgren, R., Carroll, S. B. and Doe, C. Q. (1995).
Specification of neuroblast identity in the Drosophila embryonic central nervous system
by gooseberry-distal. Nature 376, 427-30.
Struhl, G., Johnston, P. and Lawrence, P. A. (1992). Control of Drosophila body
pattern by the hunchback morphogen gradient. Cell 69, 237-49.
Sun, L., Liu, A. and Georgopoulos, K. (1996). Zinc finger-mediated protein
interactions modulate Ikaros activity, a molecular control of lymphocyte development.
EMBO J 15, 5358-69.
Tautz, D., Lehman, R., Schnurh, H., Schuh R., Seifert E., Kienlin, A., Jones, K.,
JackIe, H. (1987). Finger protein of novel structure encoded by Huchback, a second
member of the gap class of Drosophila Segmentation genes. Nature 327,383-89.
Tran, K. D. and Doe, C. Q. (2008). Pdm and Castor close successive temporal identity
windows in the NB3-1Iineage. Development 135,3491-9.
Tran, K. D., Miller, M. R. and Doe, C. Q. (2010). Recombineering Hunchback
identifies two conserved domains required to maintain neuroblast competence and
specify early-born neuronal identity. Development 137, 1421-30.
Treisman, J. and Desplan, C. (1989). The products of the Drosophila gap genes
hunchback and Kruppel bind to the hunchback promoters. Nature 341, 335-7.
Triezenberg, S. J., Kingsbury, R. C. and McKnight, S. L. (1988). Functional
dissection of VP16, the trans-activator of herpes simplex virus immediate early gene
expression. Genes Dev 2, 718-29.
Walsh, C. and Reid, C. (1995). Cell lineage and patterns of migration in the developing
cortex. Ciba Found Symp 193,21-40; discussion 59-70.
Warming, S., Costantino, N., Court, D. L., Jenkins, N. A. and Copeland, N. G.
(2005). Simple and highly efficient BAC recombineering using galK selection. Nucleic
Acids Res 33, e36.
104
Wimmer, E. A., Carleton, A., Harjes, P., Turner, T. and Desplan, C. (2000). Bicoid-
independent formation of thoracic segments in Drosophila. Science 287, 2476-9.
Yang, X., Yeo, S., Dick, T. and Chia, W. (1993). The role of a Drosophila POD homeo
domain gene in the specification of neural precursor cell identity in the developing
embryonic central nervous system. Genes Dev 7, 504-16.
Yeo, S. L., Lloyd, A., Kozak, K., Dinh, A., Dick, T., Yang, X., Sakonju, S. and Chia,
W. (1995). On the functional overlap between two Drosophila POD homeo domain genes
and the cell fate specification of a eNS neural precursor. Genes Dev 9, 1223-36.
Zhao, G. and Skeath, J. B. (2002). The Sox-domain containing gene Dichaete/fish-hook
acts in concert with vnd and ind to regulate cell fate in the Drosophila neuroectoderm.
Development 129, 1165-74.