Temporal Progression of Drosophila Neural Stem Cell Promoting Neuronal Diversity 

 

 

 

by  

 

Noah Robert Dillon 

 

 

A dissertation accepted and approved in partial fulfillment of the   

requirements for the degree of  

Doctor of Philosophy   

in Biology 

 

 

Dissertation Committee:  

Dr. Adam Miller, Chair  

Dr. Chris Doe, Advisor  

Dr. Tory Herman, Core Member  

Dr. Kryn Stankunas, Core Member  

Dr. Matt Smear, Institutional Representative  

 

 

University of Oregon  

 

Fall 2024 



   
 

 2  
 

 

 

 

 

 

 

 

 

 

 

 

 

 

© 2024 Noah Robert Dillon 

This work is openly licensed via CC BY-NC 4.0. 

  



   
 

 3  
 

Dissertation Abstract 

Noah Robert Dillon 

Doctor of Philosophy in Biology 

Title: Temporal Progression of Drosophila Neural Stem Cells Promoting Neuronal Diversity 

 

How are complex nervous systems generated? During development, a small pool of neural stem 

cells generates a diverse array of cell type diversity that forms a functional brain. Remarkably, 

this neuronal diversity is generated in a predictable order. In this dissertation, I report my work in 

understanding how neural stem cells of the developing Drosophila melanogaster, known as 

neuroblasts, are temporally patterned. My work has established a single-cell RNA sequencing 

atlas of the early larval stages of neurogenesis that identified key regulators of how neuroblasts 

progress from a quiescent to a proliferative state. My subsequent studies focused on neuroblast 

lineages that generate the central brain of the adult. I show that the transcription factor Seven-up 

is required for switching the production of early to late neuron identities and progressing Type 2 

neuroblasts to the end of their lineage (i.e. death). Finally, I show the temporal transcription 

factor Castor is required for specifying neuron identities born in early larval Type 2 neuroblast 

lineages. My work shows significant advancements in understanding how the fly brain is 

generated and provides fruitful future directions to pursue.  

 

This dissertation includes previously published and unpublished co-authored material. 

  



   
 

 4  
 

Curriculum vitae 

Name of author: Noah Robert Dillon 

 

Graduate and undergraduate schools attended: 

 University of Oregon, Eugene, OR 

 University of Puget Sound, Tacoma, WA 

 

Degrees awarded: 

 Doctor of Philosophy, Biology, 2024, University of Oregon 

 Bachelor of Science, Biology, 2020, University of Puget Sound  

 

Areas of special interest: 

 Developmental Biology 

 Neurogenetics  

 Stem cells  

 

Professional experience: 

Graduate Researcher, University of Oregon, 2021-2024 

 Laboratory of Dr. Chris Q. Doe  

Graduate Employee – Teaching Assistant, University of Oregon, 2020-2021 

Associate Editor – Elements magazine, University of Puget Sound 2019-2020  

Lab Accessibility and Biology Instructor Assistant, University of Puget Sound 2018-2020 

Undergraduate Research Intern, University of Puget Sound, 2017-2020 

 Laboratory of Dr. Oscar Sosa, 2019-2020 

 Laboratory of Dr. Siddhartha Ramakrishnan, 2018-2019 

 Laboratory of Dr. Alyce DeMarais, 2017-2019  

Museum Docent, Puget Sound Natural History Museum, 2016-2020 

 Director Dr. Peter Wimberger  

 

Grants, awards, and honors: 



   
 

 5  
 

Talk Platform Prize, Seven-up acts in neuroblasts to specify adult Drosophila central 

complex neuron identity and initiate neuroblast decommissioning, Society for 

Developmental Biology, 2024 

Developmental Biology Training Grant, University of Oregon / National Institute of 

Child Health and Human Disease, 2021-2024 

 University Enrichment Committee Funds for Research, University of Puget Sound, 2018 

 Mellam Independent Research Scholarship, University of Puget Sound, 2018 

LIASE: Biodiversity in Borneo, Henry Luce Foundation, 2018 

  

Publications  

Dillon, N. R. and Doe, C. Q. (2024). Castor is a temporal transcription factor that 

specifies early born central complex neuron identity. 2024.08.22.609207. Under review at 

Development.  

 

Dillon, N. R., Manning, L., Hirono, K. and Doe, C. Q. (2024). Seven-up acts in 

neuroblasts to specify adult central complex neuron identity and initiate neuroblast 

decommissioning. Development 151, dev202504. 

 

Epiney, D., Chaya, G. N. M., Dillon, N. R., Lai, S.-L. and Doe, C. Q. (2023). 

Transcriptional complexity in the insect central complex: single nuclei RNA sequencing 

of adult brain neurons derived from type 2 neuroblasts. bioRxiv. 2023.12.10.571022. 

 

Dillon, N., Cocanougher, B., Sood, C., Yuan, X., Kohn, A. B., Moroz, L. L., Siegrist, S. 

E., Zlatic, M. and Doe, C. Q. (2022). Single cell RNA-seq analysis reveals temporally-

regulated and quiescence-regulated gene expression in Drosophila larval neuroblasts. 

Neural Development 17, 7. 

  



   
 

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Acknowledgements 

My adventure into the sciences has been an unexpected but marvelous journey. My original 

inspiration for pursing biology came from reading books by Sean B. Carroll and Richard 

Dawkins while ditching school in rural Wyoming. I have my high school science teachers to 

thank for keeping me from dropping all my classes. I am grateful to have found a home in the 

Pacific Northwest for the past eight years attending the University of Puget Sound and then the 

University of Oregon. My mentors during undergrad were gracious enough to steer me towards a 

path in scientific research. I am indebted to Dr. Alyce DeMarais, Dr. Siddhartha Ramakrishnan, 

Dr. Oscar Sosa, Dr. Peter Wimberger, and Dr. Joel Elliott (just to name a few).  

 

I thank Dr. Chris Q. Doe for his mentorship and guidance during my dissertation as I have 

worked in his lab pursing projects built on a passion for understanding developmental biology. 

The Doe Lab members (Sarah Ackerman, Nathan Anderson, Elena Barth, Ben Brissette, Arnaldo 

Carreira-Rosario, Kasey Drake, Derek Epiney, Katie Fisher, Josmarie Graciani, Janet Hanawalt, 

Emily Heckman, Keiko Hirono, Sen-Lin Lai, Kristen Lee, Laurina Manning, Gonzalo Morales 

Chaya, Jordan Munroe, Peter Newstein, Heather Pollington, Megan Radler, Tyler Ramos, Natalie 

Rico Carvajal, Rishi Sastry, Austin Seroka, Alanna Sowles, and Chundi Xu) have, over the past 

four years, been an incredible support structure that I have cherished in helping me produce work 

that I am proud to publish. I also thank Dr. Adam Miller, and the Miller Lab, for guidance when I 

first joined the program in setting me up for success the moment I started at the University of 

Oregon. I extend gratitude to the rest of my dissertation advisory committee members Dr. Tory 

Herman, Dr. Matt Smear, and Dr. Kryn Stankunas for their guidance.  

 

For the work in Chapter II, I thank Dr. Sen-Lin Lai, Dr. Cheng-Yu, and Dr. Sarah Ackerman for 

comments on the manuscript. Funding for this work was provided by NIH Training Grant 5-T32-

HD07348, NIH HD27056, and Howard Hughes Medical Institute (Noah Dillon and Chris Doe). I 

thank my co-authors: Ben Cocanougher, Chhavi Sood, Xin Yuan, Andrea Kohn, Leonid Moroz, 

Dr. Sarah Siegrist, and Dr. Marta Zlatic for their help in making this work possible. Funding in 

part was also covered by NIH R35 GM141886 (Sarah Siegrist). 

 



   
 

 7  
 

For work in Chapter III, I thank Dr. Kristen Lee, Peter Newstein, Dr. Megan Radler, and Dr. 

Chundi Xu for internal comments on the manuscript. I also thank Dr. Josie Clowny, Dr. Tzumin 

Lee and Dr. Mubarak Syed for feedback on the project. Funding for this work was provided by 

NIH Training Grant 5-T32-HD07348, NIH HD27056, and Howard Hughes Medical Institute 

(Noah Dillon and Chris Doe). I thank my co-authors Laurina Manning and Keiko Hirono as it 

was a privilege to work with these gifted scientists.  

 

For work in Chapter IV, I thank Dr. Tory Herman, Derek Epiney and Gonzalo Morales Chaya for 

comments on the manuscript. I thank Laurina Manning and Jordan Munroe for assistance on the 

EdU experiments and Dr. Sen-Lin Lai for advice on antibodies. Funding for this work was 

provided by NIH Training Grant 5-T32-HD07348, NIH HD27056, and Howard Hughes Medical 

Institute. I thank Chris Doe for motivating me in getting this last paper ready in record time.  

 

I would like to acknowledge the resource centers that make doing high quality science possible: 

FlyBase (http://flybase.org/) for a wonderful repository of Drosophila genetic information; 

Developmental Studies Hybridoma Bank, created by the NICHD of the NIH and maintained at 

the University of Iowa, Department of Biology, Iowa City, IA for antibodies; Bloomington 

Drosophila Stock Center (NIH P40OD018537); and Vienna Drosophila Resource Center for fly 

stocks; Institute of Molecular Biology Media Room at the University of Oregon for prepping the 

abundance of fly food used to maintain stocks and for cleaning the lab glassware.  

 

This work was only made possible by the contributions of many in supporting the pursuit of 

knowledge. I will forever be grateful and indebted to the people who have shown me kindness 

and respect through my scientific career.  

  



   
 

 8  
 

Table of Contents 

Chapter Page 

I. An introduction to generating neuronal diversity and a complex 

   brain in Drosophila ................................................................................................. 17 

 Patterning during Drosophila neurogenesis creates neural diversity ..................... 17 

 An introduction to neuroblasts and the developing nervous system ................ 17 

 The discovery of temporal transcription factors .............................................. 20 

 Temporal progression in the larval neuroblasts ............................................... 23 

 Establishing complexity within the Drosophila central brain ............................... 27 

 The diversity and function of the Central Complex ......................................... 27 

 Tools for accessing the Central Complex ........................................................ 30 

 Bridge ..................................................................................................................... 32 

 References .............................................................................................................. 33 

II. A single-cell RNA-seq analysis reveals temporally-regulated and 

 quiescence-regulated gene expression in larval neuroblasts ............................ 44 

 Author contributions .............................................................................................. 44 

 Introduction ............................................................................................................ 44 

 Results .................................................................................................................... 45 

 Larval atlas shows distinct cell identities and differentiating neural  

 progenitor axis ................................................................................................. 45 

 Quiescent neuroblasts and associated glia are enriched for expression  

 of genes regulating the TOR and insulin pathways ......................................... 49 

 Proliferating neuroblasts shows candidate novel markers and temporal  

 transcription factors ......................................................................................... 52 

 INPs express candidate novel cell type markers .............................................. 56 

GMCs, new‐born neurons and immature neurons express candidate  

novel cell type markers .................................................................................... 58 

Mature neurons show temporally distinct groups of transcription  

factors and cell surface molecules ................................................................... 61 

 Discussion .............................................................................................................. 63 

 Quiescent neuroblasts and glial signaling ........................................................ 64 



   
 

 9  
 

 TTFs in type I and type II neuroblasts ............................................................. 65 

 Intermediate neural progenitors ....................................................................... 65 

 The transition from progenitor to post‐mitotic neurons ................................... 66 

 Mature neurons show novel temporal changes ................................................ 66 

 Conclusions ............................................................................................................ 67 

 Materials and methods  .......................................................................................... 67 

 Single cell isolation and sequencing ................................................................ 67 

 scRNA‐seq analysis ......................................................................................... 68 

 Subclustering for further Seurat analysis ......................................................... 69 

 Data and code availability ................................................................................ 69 

 Protein localization .......................................................................................... 69 

 Supplementary information ............................................................................. 70 

 Bridge ..................................................................................................................... 71 

 References .............................................................................................................. 72 

III. Seven-up acts in neuroblasts to specify adult central complex neuron 

      identity and initiate neuroblast decommissioning ............................................ 81 

 Author contributions .............................................................................................. 81 

 Introduction ............................................................................................................ 81 

 Results .................................................................................................................... 84 

 Columnar neurons P-EN and P-FN are born from larval T2NBs  

 in different temporal windows ......................................................................... 84 

 Svp is expressed transiently and asynchronously in all larval T2NB 

 lineages between 18 h and 24 h ALH .............................................................. 85 

 Cut expression distinguishes molecular identities of adult P-EN  

 neurons from P-FN neurons ............................................................................. 88 

 Loss of Svp decreases the number of late born P-FN adult neurons ............... 89 

 Loss of Svp extends the production of early born P-EN adult neurons ........... 91 

 Loss of Svp extends T2NB lineages into the adult .......................................... 95 

 Discussion .............................................................................................................. 97 

 Columnar neurons are born at different times in the T2NB lineage ................ 97 

 Svp expression in larval T2NB lineages .......................................................... 98 



   
 

 10  
 

 Svp is required for late born fates in the T2NB lineages ................................. 98 

 Svp specification of other CX neuron subtypes ............................................... 99 

 Svp-mediated regulation of Type 2 neuroblast temporal progression ............. 100 

 Conserved role of Svp in vertebrate temporal patterning ................................ 101 

 Materials and methods ........................................................................................... 101 

 Animal preparation .......................................................................................... 101 

 EdU experiments .............................................................................................. 102 

 Larval experiments ........................................................................................... 102 

 Adult experiments ............................................................................................ 102 

 Hybridization chain reaction (HCR) RNA fluorescent in situ  

 hybridization .................................................................................................... 102 

 Larval brain sample preparation ...................................................................... 102 

 Adult brain sample preparation ........................................................................ 103 

 EdU adult brain sample preparation ................................................................ 103 

 Antibodies ........................................................................................................ 103 

 Confocal microscopy ....................................................................................... 103 

 Image processing and analysis ......................................................................... 103 

  Cell counting and neuropil target scoring ............................................ 104 

  Imaris neuropil reconstructions ............................................................ 104 

  Figure preparation ................................................................................ 104 

 Statistical analyses ........................................................................................... 104 

 Data availability ............................................................................................... 105 

 Supplementary information ............................................................................. 105 

 Bridge ..................................................................................................................... 106 

 References .............................................................................................................. 107 

IV. Castor is a temporal transcription factor that specifies early born 

      central complex neuron identity ......................................................................... 113 

 Author contributions .............................................................................................. 113 

 Introduction ............................................................................................................ 113 

 Results .................................................................................................................... 116 

 E-PG neurons are born early in Type 2 neuroblast lineages ...........................  116 



   
 

 11  
 

 E-PG and P-EN neurons have distinct molecular identities ............................    117 

 Seven-up is required to restrict E-PG production in early Type 2 

 neuroblast lineages ..........................................................................................    118 

 Castor expression is transient in the Type 2 larval neuroblast lineages .........    120 

 Generating Type 2 lineage specific Castor knockout and  

            misexpression lines ..........................................................................................    122 

 Castor is required to specify early born P-EN and E-PG adult neuron 

 molecular identities ..........................................................................................    123 

 Castor is sufficient to produce ectopic adult P-EN neurons but not 

 E-PG neurons ...................................................................................................    125 

 Discussion ..............................................................................................................    127 

 Castor expression in larval Type 2 neuroblast lineages ..................................    127 

 Castor is a narrowly expressed early temporal transcription factor in  

 larval Type 2 lineages ......................................................................................    128 

 Columnar neurons born in the same Type 2 neuroblast window have  

 distinct adult molecular identities ....................................................................    129 

 Conserved role of Castor in vertebrate neurogenesis ......................................    129 

 Materials and methods ...........................................................................................    130 

 Animal preparation ..........................................................................................    130 

 EdU experiment ...............................................................................................    130 

 Larval experiments...........................................................................................    130 

 Adult experiments.............................................................................................    130 

 Immunohistochemistry .....................................................................................    131 

 Larval brain sample preparation .....................................................................    131 

 Adult brain sample preparation .......................................................................    131 

 EdU adult brain sample preparation ...............................................................    131 

 Confocal microscopy ........................................................................................    132 

 Image processing and analysis ........................................................................    132 

  Figure preparation ...............................................................................    132 

  Statistical analyses ...............................................................................    132 

 Supplementary information ..............................................................................    132 



   
 

 12  
 

 Data availability ..............................................................................................    133 

 Bridge .....................................................................................................................    134 

 References ..............................................................................................................    135 

V. Discussion ...............................................................................................................    140 

 Future directions in understanding neuroblast quiescence and reactivation ..........    140 

 Insulin signaling and cell cycle in quiescence .................................................    140 

 Connecting quiescence to the temporal cascade .............................................    142 

 Future directions in understanding larval neuroblast temporal progression ..........    143 

 Extrinsic signaling over time ...........................................................................    143 

 Switching factors and the shifting of competence ............................................    146 

 Temporal factors and the Central Complex .....................................................    147 

 Conservation of temporal patterning mechanisms in Drosophila and  

 mammalian central nervous systems .....................................................................    152 

 An introduction to mammalian neural stem cells that generate the cortex .....    153 

 A brief introduction to mammalian retinal progenitor cells ............................    165 

 A comparison between the fly and mammalian neurogenesis .........................    165 

 Concluding remarks ...............................................................................................    158 

 References ..............................................................................................................    159 

Appendices ..................................................................................................................    169 

 A. Chapter III supplementary figures and tables ...................................................    169 

 B. Chapter IV supplementary figures and tables ...................................................    178  



   
 

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List of Figures 

Figure Page 

1.1 Spatial patterning of neuroblasts in a single hemisegment of the developing  

 Drosophila embryonic ventral nerve cord ............................................................. 17 

1.2 Division patterns of Drosophila neuroblast lineages ............................................. 18 

1.3 Spatial positioning of Type 2 neuroblasts and cell lineage markers ...................... 19 

1.4 The canonical temporal transcription factor cascade of embryonic  

 Type 1 neuroblasts in the ventral nerve cord ......................................................... 21 

1.5 The candidate temporal transcription factor cascade of larval Type 2  

 intermediate neural progenitors ............................................................................. 22 

1.6 Neuroblast progression of neurogenesis across fly development .......................... 23 

1.7 Temporal patterning of the Type 2 neuroblast in larval stages .............................. 25 

1.8 The adult Central Complex neuropils and neurons ................................................ 27 

2.1 Larval atlas shows distinct cell identities and differentiating neural  

 progenitor axis ....................................................................................................... 46 

2.2 Quiescent neuroblasts and glial cells show enriched markers for  

 regulating the TOR and insulin pathway ............................................................... 50 

2.3 Type I neuroblasts shows candidate novel markers and temporal  

 transcription factors ............................................................................................... 53 

2.4 Type II progenitor cluster contains type II neuroblasts that show  

 candidate temporal transcription factors ................................................................ 68 

2.5 INPs show candidate novel markers ...................................................................... 57 

2.6 GMCs, new-born neurons and immature neurons show candidate  

 novel markers ......................................................................................................... 59 

2.7 Mature neuron conclusion ...................................................................................... 62 

3.1 Columnar neuron subtypes are born from larval T2NBs at distinct  

 temporal windows .................................................................................................. 82 

3.2 Svp is expressed early in all larval T2NB lineages ................................................ 86 

3.3 Cut expression distinguishes P-EN and P-FN molecular identities ....................... 89 

3.4 Svp is required for the late born P-FN neuron identity .......................................... 90 

3.5 Svp restricts the early born P-EN neuron molecular identity and  



   
 

 14  
 

 birth window .......................................................................................................... 92 

3.6 Svp regulates the early born P-EN adult neuron morphology ............................... 94 

3.7 Svp is required for timely onset of T2NB decommissioning ................................ 96 

4.1 E-PG neurons are born early in Type 2 neuroblast lineages .................................. 115 

4.2 E-PG and P-EN neurons have distinct molecular identities .................................. 118 

4.3 Seven-up is required to restrict E-PG production in early Type 2  

 neuroblast lineages ................................................................................................. 119 

4.4 Castor expression is transient in Type 2 neuroblast lineages ................................. 121 

4.5 Castor is required to specify early born P-EN and E-PG adult neuron  

 molecular identities ................................................................................................ 124 

4.6 Castor is sufficient to produce ectopic adult P-EN neurons but not  

 EPG neurons .......................................................................................................... 126 

5.1 Model of temporal progression in larval neuroblasts by  

 regulation from extrinsic signaling pathways. ....................................................... 145 

5.2 Model for Type 2 neuroblast generation of Central Complex  

 neuron identities ..................................................................................................... 149 

5.3 Current understanding of temporal patterning in the Type 2 lineages  

 generating Central Complex neurons ..................................................................... 151 

S3.1 Svp mRNA is expressed early in all larval T2NB lineages similar 

   to protein expression ............................................................................................ 170 

S3.2 Svp CRISPR/Cas9 knockout Svp in T2NBs and prevents temporal  

   expression of the late factor E93 .......................................................................... 171 

S3.3 Adult P-EN and P-FN neurons do not express Svp ............................................. 172 

S3.4 Svp in T2NBs regulates adult CX neuropil development ................................... 174 

S4.1 Loss of Seven-up in Type 2 neuroblasts leads to altered E-PG  

    ellipsoid body morphology ................................................................................. 178 

S4.2 Generating Type 2 lineage specific Castor knockout and  

    misexpression lines ............................................................................................. 179 

S4.3 Castor and Seven-up do not cross regulate in Type 2 neuroblasts ....................... 180 

  



   
 

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List of Tables 

Table Page 

1.1 Markers distinguishing the Type 2 neuroblast lineage progenitor cell types ......... 20 

2.1 Validated markers for progenitors and young neurons .......................................... 48 

2.2 Validated markers for glial cell types ..................................................................... 48 

2.3 Validated markers for mature neuron cell types .................................................... 49 

S3.1 Transgenes and Drosophila melanogaster stock lines used in  

    Dillon et al., 2024................................................................................................ 175 

S3.2 Genetic crosses for each experiment in Dillon et al., 2024 ................................. 176 

S3.3 Antibodies used in Dillon et al., 2024.................................................................. 177 

S4.1 Transgenes and Drosophila melanogaster stock lines used in  

    Dillon and Doe 2024 ........................................................................................... 181 

S4.2 Genetic crosses for each experiment in Dillon and Doe 2024 ............................. 182 

S4.3 Antibodies used in Dillon and Doe 2024. ............................................................ 184



   
 

 16  
 

Chapter I 

 

An introduction to generating neuronal diversity and a complex brain in Drosophila 

 

Patterning during Drosophila neurogenesis creates neural diversity  

Animal behaviors require complex neural circuits, which requires a diverse set of neuronal cell 

types, to function. An open question in developmental neurobiology remains: How are diverse 

neurons generated from a small pool of neural stem cells? The primary focus of this chapter will 

be on the developing Drosophila central nervous system. The secondary focus will shift briefly 

to the matured adult central brain and how it is generated during development. Primary attention 

is placed on the developing ventral nerve cord and brain lobes with brief mentions of the optic 

lobes when relevant. The following chapters will cover my contributions to the field. The last 

chapter will discuss future directions and connect work done in Drosophila to mammalian neural 

development.  

 

An introduction to neuroblasts and the developing nervous system 

The neural stem cells in Drosophila are known as neuroblasts (NBs). NBs delaminate from the 

neuroectoderm with each NB having a distinct identity (Broadus et al., 1995; Doe, 1992; 

Hartenstein and Campos-Ortega, 1984; Wheeler, 1891; Wheeler, 1893) (reviewed in: Hartenstein 

and Wodarz, 2013; Skeath and Thor, 2003). Early lineage tracing studies show that each NB 

generates distinct and reproducible cell lineages (Bossing et al., 1996; Doe, 1992; Schmid et al., 

1999; Schmidt et al., 1997). The identity of each NB in the ventral nerve cord is controlled by 

spatial factors that impart a unique molecular profile based on a ‘column and row’ logic 

(reviewed in Skeath and Thor, 2003) (Fig 1.1). For example, the row factor Gsb is required for 

row 5 NB identities (Bhat, 1996; Skeath et al., 1995) (Fig 1.1). The column factor Msh is 

required for the lateral most column of NB identities (Isshiki et al., 1997) (Fig 1.1). Recent work 

has shown these combinations of spatial factors to establish NB identity-specific chromatin 

accessibility (Sen et al., 2019). It remains to be seen if this NB identity chromatin profile is 

required for establishing unique cell lineages. Similar spatial patterning mechanisms have been 

seen in the optic lobe NBs (reviewed Rossi et al., 2021). These findings established the first level 

of generating diverse neuronal cell types in that each NB has a unique identity and produces a 



   
 

 17  
 

distinct lineage with a defined set of cell types.  

 

 

Fig. 1.1 Spatial patterning of neuroblasts in a single hemisegment of the developing 

Drosophila embryonic ventral nerve cord. Neuroblasts have unique spatial identities 

determined by cross-regulating spatial factors in a row and column logic. Row spatial factors 

include: Hh, En, Wg, and Gsb (Bhat, 1996; Chu-LaGraff and Doe, 1993; Deshpande et al., 

2001; Matsuzaki and Saigo, 1996; McDonald and Doe, 1997; Skeath et al., 1995). Column 

spatial factors include: Vnd, Ind, and Msh (Isshiki et al., 1997; McDonald et al., 1998; von 

Ohlen and Doe, 2000; Weiss et al., 1998). A, Anterior; P, Posterior; M, Medial; L, Lateral.  

 

All NBs undergo asymmetric cell divisions to generate smaller progeny while self-renewing for 

subsequent divisions. There are three NB division patterns (Fig 1.2). Type 0 NBs produce a 

progeny cell that directly differentiates into its postmitotic neuronal fate (Baumgardt et al., 2014) 

(Fig 1.2, left). These Type 0 divisions have not been extensively studied and therefore will not be 

discussed further. Type 1 NBs (T1NBs) produce a transitional progeny cell type known as a 

ganglion mother cell (GMC) that will divide once to produce a pair of postmitotic cells, neurons 

or glia (Fig 1.2, middle). Type 2 NBs (T2NBs) have a more complex division with each NB 

division generating an intermediate neural progenitor (INP) that will asymmetrically divide to 

produce 4-6 GMCs that each will divide once to produce a pair of postmitotic cells (Bello et al., 

2008; Boone and Doe, 2008; Bowman et al., 2008) (Fig 1.2, right). Thus, T2NBs have more 

neurogenic potential from a single stem cell as each T2NB division will generate a total of 8-12 

postmitotic cells compared to the 2 cells generated each T1NB division.  

 



   
 

 18  
 

 

Fig. 1.2 Division patterns of Drosophila neuroblasts. Type 0 division (left) is a mode of 

direct neurogenesis with the NB producing one progeny cell per division. Type 1 division 

(middle) is indirect neurogenesis with the NB producing one GMC that will divide once to 

produce two progeny cells per NB division. Type 2 division (right) has the most neurogenic 

potential with the production of INPs that produces 4-6 GMCs that in total generate 8-12 

progeny cells per T2NB division. NB, Neuroblast; GMC, Ganglion Mother Cell; INP, 

intermediate neural progenitor.  

 

T1NBs have been extensively studied as they comprise the majority of NB lineages in the central 

nervous system. The ventral nerve cord is divided into 14 repeated segments known are 

hemisegments that are bilaterally symmetrical across the midline and contain ~30 NBs each for a 

total of ~420 NBs per ventral nerve cord (Broadus et al., 1995; Hartenstein and Campos-Ortega, 

1984; Hartenstein et al., 1994). The central brain lobes contain ~100 T1NBs (Pereanu and 

Hartenstein, 2006; Younossi-Hartenstein et al., 1996). The optic lobes contain >800 NBs (Bertet 

et al., 2014; Li et al., 2013; Yasugi et al., 2008). These T1NB lineages generate most of the 

Drosophila central nervous system; thus, it is no surprise that these NB lineages have been the 

most studied.  

 

T2NBs have been recently discovered and offer an exciting division pattern that closely 

resembles some primate cortical neural stem cell lineages (reviewed in El-Danaf et al., 2023; 

Holguera and Desplan, 2018) (discussed more in Chapter V). There are 8 T2NB lineages 

localized to each central brain lobe; each having a unique spatial identity with 6 lineages that are 

dorsal medial and 2 lineages dorsal lateral (Bello et al., 2008; Boone and Doe, 2008; Bowman et 

al., 2008) (Fig 1.3, left). Each T2NB produces a unique cell lineage with morphologically 

distinct progeny cells that populate the adult brain (Andrade et al., 2019; Pereanu and 



   
 

 19  
 

Hartenstein, 2006; Riebli et al., 2013; Yang et al., 2013). Moreover, each progenitor cell type 

within the Type 2 lineages have been identified to express unique markers (Fig 1.3, right; Table 

1.1). Thus, the Type 2 lineage has proven to be an excellent model for precise lineage 

manipulations for understanding the development of neural stem cells and neurons (Bayraktar 

and Doe, 2013; Dillon et al., 2024; Hamid et al., 2024; Li et al., 2016; Li et al., 2017b; Munroe et 

al., 2022; Rives-Quinto et al., 2020; San-Juán and Baonza, 2011; Sullivan et al., 2019; Syed et 

al., 2017; Zhu et al., 2011; Zhu et al., 2012). Type 2 lineages will be discussed in length below 

and are the focus in Chapters III and IV. 

 

 
Fig. 1.3 Spatial positioning of Type 2 neuroblast and cell lineage markers. The eight Type 

2 neuroblast lineages are bilaterally symmetrical with stereotyped spatial positions in the 

brain lobes (left). Type 2 neuroblasts generate identifiable lineage clusters of cell types 

(right). See Table 1.1 for references. A, Anterior; P, Posterior; M, Medial; L, Lateral; D, 

Dorsal; V, Ventral; DM, Dorsal-medial; DL; Dorsal-lateral; T2NB, Type 2 Neuroblast; INP, 

Intermediate Neural Progenitor; GMC, Ganglion Mother Cell. PntP1, Pointed; Dpn, 

Deadpan; Tll, Tailless; Ase, Asense; Ham, Hamlet; Erm, Earmuff; Tap, Target of poxn; Dap, 

Dacapo; Mir, Miranda; Elav, Rmbryonic lethal abnormal vision; Repo; Reverse polarity; Brp, 

Bruchpilot; Fne, Found in neurons; Ncad, Neural cadherin.   

  



   
 

 20  
 

Table 1.1 Markers distinguishing the Type 2 neuroblast lineage progenitor cell types. 

Cell type Marker References 

Type 2 

neuroblast 

(T2NB) 

Deadpan (Dpn) + (Bello et al., 2008; Bier et al., 1992; Boone 

and Doe, 2008; Bowman et al., 2008; 

Rives-Quinto et al., 2020; Zhu et al., 2011) 

Pointed (Pnt) + 

Tailless (Tll) + 

Asense (Ase) - 

Intermediate 

neural 

progenitor 

(INP) 

Deadpan (Dpn) + (Bello et al., 2008; Boone and Doe, 2008; 

Bowman et al., 2008; Li et al., 2016; 

Rives-Quinto et al., 2020; Zhu et al., 2011) 

Asense (Ase) + 

Hamlet (Ham) + 

Earmuff (Erm) + 

Ganglion 

mother cell 

(GMC) 

Target of Poxn (Tap) + (Ikeshima-Kataoka et al., 1997; Lane et al., 

1996; Michki et al., 2021) Dacapo (Dap) + 

Deadpan (Dpn) - 

Miranda (Mir) - 

Glia Reverse polarity (Repo) + (Campbell et al., 1994; Xiong et al., 1994) 

Neuron Embyonic lethal abnormal vision (Elav) + (Robinow and White, 1991; Samson and 

Chalvet, 2003; Wagh et al., 2006; Young 

and Armstrong, 2010)  

Bruchpilot (Brp) + 

Found in neurons (Fne) + 

Neural cadherin (Ncad) + 

 

The discovery of temporal transcription factors  

In addition to the spatial factors determining the unique NB identities, temporal patterning within 

the lineages have been extensively studied as a mechanism for generating neuronal diversity. 

Embryonic NBs were first observed to sequentially express a series of transcription factors that 

were hypothesized to regulate temporal fate decisions (Brody and Odenwald, 2000; Kambadur et 

al., 1998). Isshiki et al. first discovered the canonical embryonic temporal transcription factor 

(TTF) cascade where narrow windows of transiently expressed factors, TTFs, in the NBs are 

required and sufficient to specify individual neuron identities (Isshiki et al., 2001) (Fig 1.4). 

Subsequent work has shown this TTF cascade to be a widely used mechanism across most 

embryonic T1NB lineages, persists in larval NBs, and is cross-regulating (Almeida and Bray, 

2005; Baumgardt et al., 2014; Benito-Sipos et al., 2011; Cenci and Gould, 2005; Cleary and Doe, 

2006; Grosskortenhaus, 2006; Grosskortenhaus et al., 2005; Meng et al., 2019; Meng et al., 

2020; Moris-Sanz et al., 2014; Novotny et al., 2002; Pearson and Doe, 2003; Seroka and Doe, 

2019; Tran and Doe, 2008) (Fig 1.4).  



   
 

 21  
 

 

Fig. 1.4 The canonical temporal transcription factor cascade of embryonic Type 1 

neuroblasts in the ventral nerve cord. Most embryonic T1NBs of the ventral nerve cord 

will transiently express the series of Hb, Kr, Pdm, Cas, and Grh. Variations of this temporal 

cascade is seen across some NB lineages (reviewed in: Doe, 2017; Pearson and Doe, 2004; 

Pollington et al., 2023). Hemilineages are formed during the GMC division when one 

daughter cell receives a Non cue and the other cell a Noff signal. Dashed lines indicate cross-

regulatory relationship between the TTFs. Hb, Hunchback; Kr, Krüppel; Pdm, POU Domain 

protein; Cas, Castor; Grh, Grainyhead; NB, Neuroblast; GMC, Ganglion Mother Cell; N, 

Notch; TTF, Temporal Transcription Factor.  

 

The most extensively studied T1NB lineage for understanding TTFs has been NB 7-1 with the 

earliest TTF Hb studied the most within this lineage (Grosskortenhaus et al., 2005; Hirono et al., 

2017; Isshiki et al., 2001; Kanai et al., 2005; Kohwi et al., 2011; Meng et al., 2019; Meng et al., 

2020; Pearson and Doe, 2003; Seroka and Doe, 2019; Seroka et al., 2020; Seroka et al., 2022) 

(reviewed in Doe, 2017; Pollington et al., 2023). Hb was reported to be required and sufficient 

for specifying the first two neuron cell fates born from NB 7-1, motor neurons U1 and U2 

(Isshiki et al., 2001). Recent studies have demonstrated that Hb is required for proper U motor 

neuron dendritic morphology and targeting to body wall muscles (Meng et al., 2019; Meng et al., 

2020; Seroka and Doe, 2019). Interestingly, after more than 20 years since its discovery as a 

TTF, a mechanism for Hb in specifying the U1 and U2 motor neurons has not been identified 

(i.e., U1/U2 specific identity genes activated by Hb). Previous work has shown that Hb binds 

differentially across NB lineages 5-6 and 7-4 due to a difference in lineage-specific chromatin 

accessibility imparted by spatial factors (Sen et al., 2019). These studies suggest that determining 

the DNA-binding targets of TTFs across NB lineages will provide insight into the mechanism for 

specifying diverse cell fates. It remains an open question how any TTF acts within any NB 

lineage to specify unique identities.  



   
 

 22  
 

 

Larval INPs, derived from T2NBs, display a similar temporal patterning progression as 

embryonic T1NBs. INPs express a series of cross-regulatory TTFs as INPs age (Bayraktar and 

Doe, 2013; Tang et al., 2022) (Fig 1.5). Most INP TTFs have been validated for being required to 

specify neuronal fates based on molecular markers and adult neuron morphology (Bayraktar and 

Doe, 2013; Sullivan et al., 2019). Neurons derived from INPs express markers tied to their INP 

temporal window. Young INP derived neurons generally express Dichaete, Runt, or Bsh 

(Bayraktar and Doe, 2013; Sullivan et al., 2019) (Fig 1.5). Old INP derived glia express Repo 

and the neurons Toy (Bayraktar and Doe, 2013) (Fig 1.5). It remains unknown if some of these 

markers are maintained from the larval to adult stages. Interestingly, the glia (Repo+) are 

restricted to an early T2NB window and Bsh+ neurons to a late T2NB window (Bayraktar and 

Doe, 2013). These data suggest that the temporal patterning in INPs can act in combination with 

temporal patterning of T2NBs to generate significant cell type diversity (discussed below). It 

remains unknown if Type 2 GMCs produce Non and Noff hemilineages, as seen in the embryonic 

T1NB lineages. If hemilineages exist in the Type 2 lineage, this would provide a fourth 

mechanism (1 - Spatial, 2 – T2NB temporal, 3 - INP temporal, 4 - hemilineage) for generating 

the neuronal diversity seen in the adult brain.  

 

 

Fig. 1.5 The temporal transcription factor cascade of larval Type 2 intermediate neural 

progenitors. Larval INPs express a series of TTFs as they age from young (recently derived 

from the T2NB) INPs that produce D+, Runt+, and Bsh+ progeny to old (final divisions for 

the INP) INPs that produce Repo+ and Toy+ progeny (Bayraktar and Doe, 2013; Sullivan et 

al., 2019; Tang et al., 2022). Dashed lines indicate cross-regulatory relationship between the 

TTFs. D, Dichaete; Hbn, Homeobrain; Grh, Grainyhead; Ey, Eyeless; Scro, Scarecrow; INP, 

Intermediate Neural Progenitor; GMC, Ganglion Mother Cell; N, Notch; TTF, Temporal 

Transcription Factor.  



   
 

 23  
 

 

Temporal progression in the larval neuroblasts  

Most late-stage embryonic T1NBs and T2NBs will enter quiescence, cell cycle arrest when the 

NB does not divide, and reactivate ~12-24h after larval hatching (Munroe et al., 2022; Prokop 

and Technau, 1991; Truman and Bate, 1988) (Fig 1.6). The reactivation of larval NBs requires 

insulin signaling with quiescent NBs shown to be primed to respond to Insulin-like peptides to 

reactive proliferation (Chell and Brand, 2010; Dillon et al., 2022; Otsuki and Brand, 2018; 

Sousa-Nunes et al., 2011) (Fig 1.6; see Chapter II). Additionally, glia secrete the signaling 

molecules Ana and Trol to inhibit NB proliferation and promote NB proliferation, respectively, 

to temporally control neurogenesis in the early larvae (Datta, 1995; Datta, 1999; Ebens et al., 

1993) (Fig 1.6). Quiescent NBs have been shown to repress Artichoke (Atk) and reactivate 

proliferation based on a dorsal-ventral positioning due to a heterogeneity in arrested cell stated of 

G0 or G2 (Otsuki and Brand, 2018; Otsuki and Brand, 2019). Additionally, Hh has been 

suggested to play a role in regulating quiescence downstream of the embryonic TTF cascade 

(Chai et al., 2013). Recent work has shown that the RNA-binding protein IGF-II mRNA-binding 

protein (Imp) is required for T2NBs to exit from quiescence (Munroe et al., 2022). It remains to 

be seen if this cue is also required for other NB lineages. Ongoing work aims to understand the 

mechanisms controlling the reactivation of NBs in the larval stages with the transcription factor 

Foxo suspected to be an essential regulator (see Dr. Sarah Siegrist’s lab: 

https://siegristlab.org/research/; personal communication). See Chapter V for further discussion. 

 

 

Fig. 1.6 Neuroblast progression of neurogenesis across fly development. Embryonic NBs 

proliferate until late stages when most lineages enter quiescence. Larval NBs express Trbls 

during quiescence with Ana secreted by glia to inhibit proliferation (Datta, 1995; Datta, 

1999; Ebens et al., 1993). NBs express InR and are reactivated by signaling molecules Trol 

and Ilps (Chell and Brand, 2010; Datta, 1995; Datta, 1999; Dillon et al., 2022; Sousa-Nunes 

et al., 2011). Green indicates proliferating NB, magenta a quiescent NB, and grey a 



   
 

 24  
 

decommissioning NB. Dashed lines indicate regulatory inputs. NB, Neuroblast; Trbl, 

Tribbles; Ana, Anachronism; InR, Insulin Receptor; Trol, Terribly reduce optic lobes; Ilps, 

Insulin like peptides.  

 

Larval NBs, excluding the optic lobes, have been less studied than embryonic NBs with an open 

question remaining about how temporal patterning occurs during larval stages. Mushroom body 

T1NBs have been shown to express opposing temporal gradients of the RNA-binding proteins 

Imp and Syncrip that are required for proper specification of neuronal fates (Liu et al., 2015). 

Imp is expressed at high levels in early larval NBs and decreases in expression level as Syncrip 

increases in expression level during later stages (Liu et al., 2015). The temporal gradient of Imp 

and Syncrip has been seen in the T2NB lineages (Ren et al., 2017; Syed et al., 2017) (Fig 1.7). 

This suggests that larval NB lineages may use the broad protein gradients as a mechanism for 

temporal patterning instead of the narrow windows of TTF expression. This is supported by 

several studies showing that Imp and Syncrip gradients in the T2NBs and INPs are required for 

specifying some neuronal fates (Hamid et al., 2024; Munroe and Doe, 2023; Ren et al., 2017; 

Syed et al., 2017). Alternatively, candidate TTFs have been identified as temporally expressed 

within T2NBs and may be required to specify neuronal identities, similar to the embryonic T1NB 

lineages (Bayraktar and Doe, 2013; Ren et al., 2017; Syed et al., 2017). Recent unpublished 

studies indicate the these candidate TTFs are indeed necessary for specifying neuronal identities 

(Wani et al., 2023 preprint) (Dillon and Doe, 2024, preprint; see Chapter IV). Regardless of the 

mechanism, the temporal progression in T2NBs is required for specifying birth order-dependent 

neuronal identities (Dillon et al., 2024). These data suggest that both protein gradients and TTFs 

regulate temporal patterning in the larval T2NBs.  

 



   
 

 25  
 

 

Fig. 1.7 Temporal patterning of the Type 2 neuroblast in larval stages. T2NBs express 

opposing gradients of the RNA-binding proteins Imp and Syncrip (Ren et al., 2017; Syed et 

al., 2017). Seven-up is known to be required for switching the temporal progression from 

early-to-late (Dillon et al., 2024; Ren et al., 2017; Syed et al., 2017). Candidate TTFs are 

hypothesized to specify neuronal fates (Bayraktar and Doe, 2013; Dillon et al., 2024; Ren et 

al., 2017; Syed et al., 2017). Green indicates a relationship to an early temporal fates and 

magenta a relationship to a late temporal fates (Bayraktar and Doe, 2013; Dillon et al., 2024; 

Hamid et al., 2024; Ren et al., 2017; Syed et al., 2017) (Dillon and Doe, 2024, preprint). 

White indicates an unvalidated relationship. T2NB, Type 2 Neuroblast; Imp, IGF-II mRNA-

binding protein; EcR-B1, Ecdysone Receptor isoform 1B; E93, Ecdysone induced protein 93; 

TTF, Temporal Transcription Factor.  

 

The mechanism for temporal progression in proliferating larval NBs remains unknown. Recent 

work has shown that the early factor Imp is required for reactivation of proliferation (Munroe et 

al., 2022). Additionally, the Imp and Syncrip gradients are cross-regulatory for the early-to-late 

progression (Liu et al., 2015; Ren et al., 2017). The switching factor Seven-up has been 

identified as being required for regulating this transition and the T2NB production switch from 

early-to-late identity neurons (Dillon et al., 2024; Ren et al., 2017; Syed et al., 2017) (Fig 1.7). 

This role for Seven-up as a switching has also been seen in the embryonic and larval T1NBs 

(Benito-Sipos et al., 2011; Kanai et al., 2005; Kohwi et al., 2011; Maurange et al., 2008; Mettler 

et al., 2006). The ecdysone receptor isoform B1 (EcR-B1) is also required for initiating the early-

to-late switch and acts downstream of Seven-up in T2NBs (Syed et al., 2017). These data suggest 

that both intrinsic (Imp, Syncrip, and Seven-up) and extrinsic (EcR-B1 and ecdysone signaling) 

are required for temporal progression in proliferating T2NBs. It remains unknown what other 

factors are involved. See Chapter V for further discussion. 



   
 

 26  
 

 

Larval NBs go through decommissioning, a process where the NB will stop proliferating to 

either differentiate into a postmitotic cell fate or undergo cell death (Fig 1.6). Most larval NBs 

decommission in the late larval or early pupal stages (Homem et al., 2014; Ito and Hotta, 1992; 

Maurange et al., 2008; Narbonne-Reveau et al., 2016; Siegrist et al., 2010; Yang et al., 2017). 

This process in the T1NBs is initiated by the Imp-to-Syncrip transition with atypically high Imp 

expression sustaining mushroom body NBs to continue proliferating longer than other T1NB 

lineages (Yang et al., 2017). Thus, it is no surprise that Seven-up is required for T1NB and T2NB 

lineages to initiate the timely onset of decommissioning as this switching factor acts upstream of 

the Imp-to-Syncrip transition (Dillon et al., 2024; Maurange et al., 2008; Narbonne-Reveau et 

al., 2016). Interestingly, the expression of Seven-up is early in the larval T2NBs at ~24h yet 

decommissioning occurs in the early pupal stages, days after Seven-up is no longer expressed 

(Bayraktar and Doe, 2013; Dillon et al., 2024; Homem et al., 2014; Ren et al., 2017; Syed et al., 

2017). It remains unknown how Seven-up acts to initiate temporal progression for the onset of 

decommissioning in T2NB. See Chapter V for further discussion.  

  



   
 

 27  
 

Establishing complexity within the Drosophila central brain 

As discussed in the previous section, neurogenesis in the developing Drosophila central nervous 

system produces diverse neuronal cell types. The integration of spatial patterning of distinct NB 

lineages and the subsequent temporal patterning provide a robust framework for understanding 

neurogenesis that has been extensively reviewed (reviewed in Doe, 2017; El-Danaf et al., 2023; 

Pearson and Doe, 2004; Pollington et al., 2023; Rossi et al., 2021; Skeath and Thor, 2003). The 

result of these 5 days of neurogenesis leads to the adult fruit fly that contains the complex neural 

circuitry to allow context-dependent behavior. This section will focus on the adult central brain 

with brief mentions of some circuits. The cell type diversity and function of the larval nervous 

system will not be discussed here.  

 

The diversity and function of the Central Complex 

An extensively studied region of the adult Drosophila brain is the Central Complex (CX). The 

CX is comprised of four neuropils, regions of high synaptic connectivity, known as the 

Protocerebral Bridge (PB), Fan-shaped Body (FB), Ellipsoid Body (EB), and Noduli (NO) 

(Hanesch et al., 1989) (Fig 1.8, left). The main two classes of neurons in the CX are the 

tangential neurons that are transversally oriented and the columnar neurons that are 

longitudinally oriented (Hanesch et al., 1989). These broad neuron classes have distinct functions 

in the CX and arise from distinct NB lineages.  

 

 
Fig. 1.8 The adult Central Complex neuropils and neurons. The CX is comprised of four 

main neuropils: PB, FB, EB, and NO. Other neuropils to note are the GA and BU (left). Most 



   
 

 28  
 

studied neurons of the CX include the tangential Ring neurons and the columnar neurons 

(right). CX, Central Complex; PB, Protocerebral Bridge; FB, Fan-shaped Body; EB, 

Ellipsoid Body; NO, Noduli; GA, Gall; BU, Bulb. Figure diagrams inspired and adapted 

from: Wolff and Rubin, 2018; Seeling and Jayaraman 2013. 

 

Ring neurons have been the most studied of the tangential neurons. These neurons bridge the 

anterior optic tubercle, the dominant visual input region, to columnar neurons to form the 

anterior visual pathway circuit (Omoto et al., 2017; Seelig and Jayaraman, 2013). Importantly, 

Ring neurons provide spatially organized visual information to the CX circuitry (Omoto et al., 

2017; Timaeus et al., 2020). These Ring neurons are characterized by their unique morphology in 

targeting the Bulb (BU), their concentric rings within the EB, and laterally located cell bodies 

(Renn et al., 1999; Young and Armstrong, 2010) (Fig 1.8, right). The Ring neurons are derived 

from two T1NB lineages with other tangential neurons derived from both T1NB and T2NB 

lineages (Bridi et al., 2019; Larsen et al., 2009; Omoto et al., 2017; Wong et al., 2013; Yang et 

al., 2013). One lineage tracing study has demonstrated that Ring neuron subtypes are born in a 

birth order-dependent manner with unique molecular markers across embryonic, larval, and 

pupal stages (Bridi et al., 2019). It will be important work investigating the mechanisms behind 

the specification of tangential neurons. Interest should be placed in the Ring neurons as they 

provide a system of two T1NB lineages that generate a diverse class of neurons across all of 

Drosophila neurogenesis.  

 

The majority of the CX is generated from T2NBs starting from late-stage embryo and into the 

pupal stages (Riebli et al., 2013; Walsh and Doe, 2017). The four T2NB lineages DM1-4 (Fig 

1.3, left) generate all the adult columnar neurons (Andrade et al., 2019; Ito and Hotta, 1992; 

Pereanu and Hartenstein, 2006; Riebli et al., 2013; Yang et al., 2013) These columnar neurons 

are characterized by their apical cell body location with the majority innervating the PB, a subset 

of other CX neuropils, and occasionally neuropils outside of the CX (reviewed in Pfeiffer and 

Homberg, 2014) (Fig 1.8, right). Lineage tracing studies have shown that columnar neuron 

subtypes are born in a birth order-dependent manner (Andrade et al., 2019; Riebli et al., 2013; 

Walsh and Doe, 2017). Thus, columnar neurons provide an excellent model for understanding 

temporal patterning in the T2NB that give rise to this unique class of neurons.  

 



   
 

 29  
 

Due to the diversity in CX neuron morphology, these neurons are named after the neuropils they 

innervate for a dendrite-axon naming convention (Wolff and Rubin, 2018; Wolff et al., 2015). 

For example, E-PG neurons are named for their dendrite targeting to the EB and axonal targeting 

to the PB and GA. Similarly, P-EN neurons are named for their dendritic targeting of the PB and 

axonal targeting to the EB and NO. Connectomes of the CX show hundreds of morphologically 

distinct neuron subtypes with complex synaptic connectivity (Franconville et al., 2018; Hulse et 

al., 2021; Scheffer et al., 2020; Zheng et al., 2018). Studies in other insect specicies has shown 

conserved and evolutionarily divergent neuroarchitecture with the Drosophila CX (reviewed in 

Pfeiffer and Homberg, 2014). These studies show that the CX is primed as a model for 

understanding not only developmental biology but also probe questions about evolution and 

systems neuroscience.  

 

Research on the circuitry of the CX has focused on the integration of visual sensory input and the 

locomotor output with some recent work aiming to understand the CX’s role in sleep (reviewed 

in: Fisher, 2022; Helfrich-Förster, 2018; Strauss, 2002; Turner-Evans and Jayaraman, 2016). The 

most studied role of the CX has been in navigational behavior. The EB can be considered the 

fly’s compass as the directional heading of the adult fly is encoded in the EB and can be 

maintained even in the absence of a visual stimulus (Green et al., 2017; Green et al., 2019; Seelig 

and Jayaraman, 2013; Turner-Evans et al., 2017; Turner-Evans et al., 2020). The two columnar 

identities of E-PG and P-EN neurons are integrated to maintain and continuously update 

directional heading from sensory cues (Green et al., 2017; Green et al., 2019; Turner-Evans et al., 

2017; Turner-Evans et al., 2020). Surprisingly, the P-EN neurons were found to contain two 

subtypes, P-ENa and P-ENb neurons, based on behavioral differences (Green et al., 2017). 

Subsequent EM reconstructions have identified morphological differences in synaptic 

connectivity initially missed by light microscopy (Scheffer et al., 2020; Turner-Evans et al., 

2020; Wolff et al., 2015). The visual inputs for the directional heading circuit is encoded by the 

Ring neurons (discussed above), and other similar neuron classes not discussed here, connecting 

the visual system to the CX via the EB (Omoto et al., 2017; Seelig and Jayaraman, 2013; 

Timaeus et al., 2020). It remains an open question how these CX circuits are initially formed and 

if there is an underlining developmental mechanism that connects these neuron subtypes. For 

example: Are the CX neurons that are wired together born in the same temporal windows and are 



   
 

 30  
 

their identities specified by shared temporal factors? See Chapters III and IV for studies on the 

development of E-PG and P-EN neurons and Chapter V for further discussion. 

 

Tools for accessing the Central Complex 

While connectomes of the CX provide unparallel access to the morphology and connectivity of 

neurons, they fail to provide data on the genetic programs underlining the brain’s development. 

Recent single-cell/ single-nuclei RNA sequencing (scRNAseq) datasets of the adult Drosophila 

brain demonstrate that neurons are equally diverse in their transcriptomes as they are in 

morphology (Abruzzi et al., 2017; Croset et al., 2018; Davie et al., 2018; Epiney et al., 2023, 

preprint; Janssens et al., 2021; Li et al., 2017a). The Doe lab (https://www.doelab.org/) has 

ongoing work with a comprehensive dataset on adult neurons and glia derived from the T2NB 

lineages (Epiney et al., 2023, preprint). This work has identified unique molecular markers that 

distinguish CX neuron identities and provide support that combinatorial expression patterns 

define diverse neuronal cell types (Epiney et al., 2023, preprint). Importantly, these data sets 

provide access to the transcriptomes of individual neuronal subtypes. These data support a model 

similar to that found in C. elegans where combinations of homeodomain transcription factors 

delineate all neuronal cell types (Reilly et al., 2020). It remains to be seen if this is consistent 

across NB lineages in Drosophila much less vertebrate nervous systems, both of which contain 

significantly more neuronal diversity. These large transcriptomic datasets are valuable tools for 

understanding neuron diversity and building testable hypotheses prior to running experiments.  

 

One of the greatest genetic tools in Drosophila neurobiology has been the development and wide 

use of the UAS/Gal4 and LexA/LexAop systems (Brand and Perrimon, 1993; Fischer et al., 

1988; Lai and Lee, 2006; Szüts and Bienz, 2000). These tools allow selective expression of 

genetic constructs in cell-specific patterns to provide spatial and temporal control, a geneticist’s 

dream. Combined with the recent advancements in CRISPR/Cas9 tools to use in flies, tissue-

specific genetic knockouts offer a tool to study null mutations with precision of the affected cell 

types (Ewen-Campen et al., 2017; Port and Bullock, 2016; Port et al., 2020). Most relevant for 

the CX has been the extensive Gal4 and LexA lines that label hundreds of CX neuron types 

(Jenett et al., 2012; Wolff and Rubin, 2018; Wolff et al., 2015). These genetic stocks have 

allowed for the precise labeling of CX neuron molecular identity (i.e., pattern of Gal4 and LexA 



   
 

 31  
 

expression) and labeling of neuron morphology. In addition to these genetic markers, several 

studies have identified unique transcription factors for CX cell types that can be identified 

through immunohistochemistry (Bayraktar and Doe, 2013; Dillon et al., 2024; Epiney et al., 

2023; Sullivan et al., 2019) (Dillon and Doe, 2024, preprint). The tools described approve have 

been used extensively throughout the following work to investigate the development of the CX.  

  



   
 

 32  
 

Bridge 

This chapter covered the development of the Drosophila central nervous system starting at the 

delamination of the NB from embryonic neuroectoderm. I describe how NBs are spatially 

patterned to produce unique NBs identities and how each NB identity generates unique cell 

lineages. I focused on how temporal patterning by TTFs are required for generating neuronal 

diversity in embryonic T1NB lineages. It remains an open question whether temporal protein 

gradients or TTFs act as temporal patterning mechanisms in the larval T1NB and T2NB lineages. 

Furthermore, it is unknown how the larval NB lineages temporally progress in exiting from 

quiescence to proliferating and eventually decommissioning. I shifted focus to discuss how the 

adult CX is comprised of diverse cell types that are apart of important behavioral neural circuits. 

I finished the chapter by discussing the available tools used to study the CX.  

 

In the following chapters, I present my previously published and unpublished work investigating 

the development of the Drosophila central nervous system. Chapter II will cover my 2022 

publication that investigated a large scRNAseq dataset across multiple stages of early larval 

development. Chapter III will cover my 2024 publication that shows Seven-up is required in 

T2NB lineages for important temporal progression of a) switching production from early-to-late 

neuron identities and b) initiating T2NB decommissioning. Chapter IV covers my recent work, 

under review at Development, on how Castor acts as a TTF in T2NBs to specify early born CX 

identities. The last chapter, Chapter V, will discuss future directions from my work and others. 

Additionally, I will cover the similarities and differences between neurogenesis in the fly and 

mammalian brains.   



   
 

 33  
 

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Chapter II 

 

A single-cell RNA-seq analysis reveals temporally-regulated and quiescence-regulated gene 

expression in larval neuroblasts 

 

Dillon, N., Cocanougher, B., Sood, C., Yuan, X., Kohn, A. B., Moroz, L. L., Siegrist, S. E., 

Zlatic, M. and Doe, C. Q. (2022). Single cell RNA-seq analysis reveals temporally-regulated and 

quiescence-regulated gene expression in Drosophila larval neuroblasts. Neural Development 17, 

7. 

 

Author contributions 

ND performed all scRNAseq analysis, generated all figures, and wrote the manuscript. BC, 

LLM, ABK, and MZ generated the scRNAseq data; CS, XY, and SES generated Fig. 2.2H and 

provided comments on the manuscript. CQD supervised the project and edited figures and text. 

The authors read and approved the final manuscript.  

 

Introduction 

A major question in neuroscience is how neural diversity is generated, which underlies complex 

neural circuits and behavioral output of the central nervous system (CNS). In the past, neuronal 

diversity was commonly defined by morphological features (axon/dendrite processes), 

biochemical features (neurotransmitter choice), and physiological features (distinct ion channels 

and membrane properties) [1]. In addition, “low throughput” assays for molecular differences 

among neurons, typically for transcription factor (TF) expression, have been crucial for finding 

insights into the generation of neural diversity for decades [2, 3]. Taken together, these 

approaches resulted in the definition various classes or subtypes of motor neurons, interneurons, 

sensory neurons and peptidergic neurons, but they are ill-suited to address the question of how 

many unique types of neurons exist within the CNS, and the subsequent question of how each 

cell type contributes to the function of the CNS. 

 

The advent of single cell RNA sequencing (scRNA-seq) allowed a more complete inventory of 

gene expression profiles within individual neurons, with the expression of “validated cell type 



   
 

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genes” used as a framework to identify transcriptionally related neurons [4–8]. Further analysis 

has revealed novel cell types based on common gene expression, but also that trajectories 

between cell types to be more gradual and less saltatory than previously appreciated, in part due 

to transcriptional priming [9–11]. 

 

In Drosophila, neuronal scRNA-seq has been done on adult brain [12–16], pupae [17–22], larvae 

[23–25], and blastoderm-stage embryos [26]. These experiments have provided valuable insight 

into the number of distinct neuronal types and identified gene candidates for regulating neural 

subtype function or connectivity. However, no studies to date have focused on identifying and 

characterizing the transcriptional diversity of neural progenitors, nor has any study mapped 

progenitor transcriptional profile at multiple larval stages. In this study, we identify multiple 

progenitor subtypes across several larval stages with differential gene expression to provide 

candidate genes as cell type specific markers and functional roles during development. 

 

Results 

Larval atlas shows distinct cell identities and differentiating neural progenitor axis 

To identify single cell gene expression profiles throughout larval development, we used scRNA-

seq data collected by [27] from dissociated brain and ventral nerve cord (VNC) – together 

termed the CNS – from larvae at 1h, 24h and 48h (all times in hours after larval hatching; ALH). 

We used the 10X Genomics pipeline for scRNA-seq analysis and used Cell Ranger Aggregation 

to aggregate multiple samples from the same timepoint. We used the standard Seurat integration 

pipeline to filter out low quality cells and clustered 97,845 cells from all larval stages (see 

methods; Fig. 2.1a). Within our atlas we identified clusters enriched for cell types in the CNS: 

neural progenitors, immature and mature neurons, glia, trachea, hemocytes and insulin-producing 

cells (IPCs; Fig. 2.1a; Supp. Table 2. 1). Representative examples of a progenitor marker 

(Deadpan; dpn), a new-born neuron marker (Hey), a maturing neuron marker (nSyb), and a glia