Professor, Department of Biology
Member & Co-Director, ION
Ph.D. Stanford University
B.S. New College
Research Interests: Drosophila Neural Development: From Stem Cells to Circuits
Overview: Our lab investigates central nervous system development in Drosophila. His lab is currently interested in (1) temporal identity programs used to generate an ordered series of neural progeny from a single progenitor, (2) how spatial patterning and temporal identity are integrated to generate heritable neuronal identity, (3) how neuronal progenitors change competence to respond to intrinsic and extrinsic cues over time, and (4) the developmental mechanisms driving neural circuit assembly, with a focus on larval locomotor circuits and adult central complex circuits.
The role of “temporal transcription factors” in generating an ordered series of neural subtypes
Neuronal diversity is generated in a stepwise fashion. In the first step, neuroectodermal spatial patterning cues (homeodomain proteins expressed in columns and rows) assign a unique identity to every neural progenitor (called neuroblasts in Drosophila). This results in 100 unique neuroblasts in each brain lobe, and 30 unique neuroblasts in each hemisegment of the ventral nerve cord (VNC). The second step of generating neuronal diversity is temporal patterning (the ordered production of different neural subtypes from a single progenitor). Although much is known about spatial patterning mechanisms, relatively little is known about temporal patterning mechanisms.
Embryonic neuroblasts sequentially express transcription factors (Hunchback, Krüppel, Pdm, Castor) that specify temporal identity of their neuronal progeny. Isshiki et al., Cell (2001).
There are five “temporal transcription factors” (TTFs) sequentially expressed in embryonic VNC neuroblasts: Hunchback (Ikaros class) > Krüppel (zinc finger class) > Pdm (Pou/homeodomain class) > Castor (Casz1 class) > Grainy head (CP2 class). Each of these factors is sequentially expressed by neuroblasts, and maintained in the neural progeny born during each expression window. Each temporal transcription factor is necessary to specify the neuronal identity produced during its expression window, and early TTFs can suppress the expression of later TTFs to generate ectopic early-born neurons (see Kohwi and Doe, 2014, Nature Reviews Neuroscience).
It is unknown if larval neuroblasts have a similar temporal transcription factor cascade to increase the diversity of neurons in the adult CNS. There are two types of larval neuroblasts: canonical type I neuroblasts bud off progeny called GMCs that differentiate into a pair of neurons, whereas type II neuroblasts produce transit-amplifying cells called intermediate neural progenitors (INPs) that themselves bud off ~6 GMCs to generate ~12 neural progeny. Thus, type II neuroblasts generate much larger, and possibly more complex, cell lineages than type I neuroblasts. Interestingly, type II neuroblasts produce most of the intrinsic neurons of the adult central complex (a brain region with beautiful laminar/columnar organization that is used for multimodal sensorimotor processing).
We are interested in identifying temporal transcription factors in larval type II lineages (both in neuroblasts and in the INPs). We have recently shown that INPs sequentially express Dichaete (Sox family) > Grainyhead > Eyeless (Pax6) transcription factors, and that these temporal transcription factors are required for the production of distinct neural subtypes. Moreover, young type II neuroblasts also transiently express at least three transcription factors and generate different neuronal/glial progeny over time, providing a second temporal identity axis. Thus, neuroblast and INP temporal patterning axes act combinatorially to generate increased neural diversity within the adult brain (Bayraktar and Doe, 2013, Nature). How these two “axes of information” are integrated to generate the specific neurons of the adult central complex remains an open question.
Currently there are many experiments ongoing to identify and functionally characterize both neuroblast and INP transcriptional cascades. For example, we are using TU-tagging (Miller et al., 2009, Nature Methods) to identify novel TTFs during larval type II neuroblast lineages, and to determine their role in assembling the adult central complex. We are also investigating the embryonic origin of type II neuroblasts, to determine (a) if they form as type II neuroblasts “de novo” or by transition from a simpler type I neuroblast; (b) if they use the Hunchback>Kruppel>Pdm>Cas>Grh cascade of TTFs during their embryonic lineages, and if these factors are maintained in INP lineages; and (c) to identify the neurons produced by embryonic type II neuroblasts and determine if they are the pioneers of the adult central complex neuropil.
The integration of spatial and temporal patterning to generate heritable neuronal identity
Spatial patterning to specify neuroblast identity occurs in the neuroectoderm, just prior to neuroblast delamination and initiation of its cell lineage. Yet these transient spatial patterning cues somehow generate a heritable cell fate that is maintained by neuroblasts cultured in isolation or transplanted into a new spatial location within the embryo. It remains unknown how transient spatial cues lead to heritable neuroblast identity. Even more interesting, the spatial cues that specify unique neuroblast identity must be combined with the sequential expression of temporal transcription factors to generate lineage-specific cell fates. For example, NB7-1 is specified by the combination of Engrailed (row 6/7) and Ventral nervous system defective (Vnd; row 1); the first temporal transcription factor Hunchback induces a motor neuron identity (U1) in this lineage. In contrast, NB7-2 has a different spatial code: Engrailed (row 6/7) and Intermediate nervous system defective (Ind; row 2) and the first temporal transcription factor Hunchback induces an interneuron identity in this lineage. How do the spatial patterning genes shift the output of Hunchback from making a motor neuron in one lineage (NB7-1) and an interneuron in the adjacent lineage (NB7-2)? Or a serotonergic neuron in the next adjacent lineage (NB7-3)?
The question of how transient spatial patterning cues impart a heritable neuroblast identity, and how this neuroblast identity “flavors” the output of subsequent temporal transcription factors, are two of the most important open questions for understanding the generation of neuronal identity.
Larval type II neuroblasts undergo changes in gene expression that indicate temporal patterning, while their intermediate neural progenitors (INPs) sequentially express three transcription factors (Dichaete, Grainyhead, Eyeless). The combination of neuroblast and INP temporal identity axes increases the neural diversity generated by a single progenitor.
The development and function of locomotor circuits
Over the past 30 years we have learned a great deal about the specification and connectivity of motor neurons to their target body wall muscles. To determine how these motor neurons are used to generate larval locomotion it is essential to identify the interneurons in the locomotor circuits. Yet almost nothing is known about interneuron specification (just a few exemplar interneurons have been characterized, out of the ~270 interneurons per hemisegment of the VNC), and even less is known about interneuron function in locomotor behavior.
Confocal image of an entire Drosophila larva stained using the Multicolor Flip Out (MCFO) method of Nern et al. (2015) PNAS 112:E2967. Note that staining the intact larva required development of a novel fixation method (Manning and Doe, unpublished) Image: Laurina Manning.
To initiate a comprehensive analysis of interneuron diversity, including their role in locomotion, we have identified several hundred Gal4 lines expressed in 1–5 interneurons (Manning et al., 2012, Cell Reports). We have used these lines in two ways. First, we have mapped them into a three-dimensional atlas that allows us to uniquely identify more than 50 percent of all interneurons in the ventral CNS (Heckscher et al., 2014, Development), and are currently linking each neuron to its developmental origin by adding lineage and TTF markers to the atlas. Second, we are using these lines to screen the interneurons for a role in larval behavior. We have expressed the warmth-activated TrpA1 channel in each “sparse interneuron Gal4 line” and found lines where neuronal activation leads to behavioral defects such as reverse locomotion, turning only, feeding only, left-right uncoordinated locomotion, pausing, and rigid paralysis (Matt Clark, submitted). We have investigated one phenotype (left-right uncoordination), showing that the affected interneurons are Even-skipped (Eve)+ contralateral ascending interneurons (that are conserved in mouse; Evx1/2). We used thermogenetics and optogenetics to determine the function of these interneurons in larval locomotion and used a TEM (transmission electron microscopy) serial reconstruction of the entire larval CNS to identify pre- and postsynaptic partners to define a proprioceptive sensorimotor circuit (Heckscher et al., 2015, Neuron).
The characterization of interneurons in other phenotypic categories remains to be studied. In addition, we are using the circuits we identify as an entry point for testing hypotheses for how interneurons and motor neurons assemble locomotor circuits: common transcriptional programs, common birth order, or common lineage. Future directions include studying plasticity and compensation within these circuits, as well as their remodeling and participation in adult locomotor circuits.
Neural connectivity linking proprioception (ddaD neuron) to motor output (RP5 neuron) in the larval CNS. The Even-skipped+ interneurons (e.g. Eve e1, shown) are responsible for maintaining left/right balanced motor output during forward locomotion. See: Heckscher ES, Zarin AA, Faumont S, Clark MQ, Manning L, Fushiki A, Schneider-Mizell CM, Fetter RD, Truman JW, Zwart MF, Landgraf M, Cardona A, Lockery SR, Doe CQ (2015) Even-skipped+ interneurons are core components of a sensorimotor circuit that maintains left-right symmetric muscle contraction amplitude. Neuron, 88, 314-329.
TU-Tagging: A Method for Identifying Layer-Enriched Neuronal Genes in Developing Mouse Visual Cortex.
eNeuro. 2017 Sep-Oct;4(5):
Authors: Tomorsky J, DeBlander L, Kentros CG, Doe CQ, Niell CM
Thiouracil (TU)-tagging is an intersectional method for covalently labeling newly transcribed RNAs within specific cell types. Cell type specificity is generated through targeted transgenic expression of the enzyme uracil phosphoribosyl transferase (UPRT); temporal specificity is generated through a pulse of the modified uracil analog 4TU. This technique has been applied in mouse using a Cre-dependent UPRT transgene, CA>GFPstop>HA-UPRT, to profile RNAs in endothelial cells, but it remained untested whether 4TU can cross the blood-brain barrier (BBB) or whether this transgene can be used to purify neuronal RNAs. Here, we crossed the CA>GFPstop>HA-UPRT transgenic mouse to a Sepw1-cre line to express UPRT in layer 2/3 of visual cortex or to an Nr5a1-cre line to express UPRT in layer 4 of visual cortex. We purified thiol-tagged mRNA from both genotypes at postnatal day (P)12, as well as from wild-type (WT) mice not expressing UPRT (background control). We found that a comparison of Sepw1-purified RNA to WT or Nr5a1-purified RNA allowed us to identify genes enriched in layer 2/3 of visual cortex. Here, we show that Cre-dependent UPRT expression can be used to purify cell type-specific mRNA from the intact mouse brain and provide the first evidence that 4TU can cross the BBB to label RNA in vivo.
PMID: 29085897 [PubMed - indexed for MEDLINE]
Temporal Patterning in the Drosophila CNS.
Annu Rev Cell Dev Biol. 2017 Oct 06;33:219-240
Authors: Doe CQ
A small pool of neural progenitors generates the vast diversity of cell types in the CNS. Spatial patterning specifies progenitor identity, followed by temporal patterning within progenitor lineages to expand neural diversity. Recent work has shown that in Drosophila, all neural progenitors (neuroblasts) sequentially express temporal transcription factors (TTFs) that generate molecular and cellular diversity. Embryonic neuroblasts use a lineage-intrinsic cascade of five TTFs that switch nearly every neuroblast cell division; larval optic lobe neuroblasts also use a rapid cascade of five TTFs, but the factors are completely different. In contrast, larval central brain neuroblasts undergo a major molecular transition midway through larval life, and this transition is regulated by a lineage-extrinsic cue (ecdysone hormone signaling). Overall, every neuroblast lineage uses a TTF cascade to generate diversity, illustrating the widespread importance of temporal patterning.
PMID: 28992439 [PubMed - indexed for MEDLINE]
Drosophila Nucleostemin 3 is required to maintain larval neuroblast proliferation.
Dev Biol. 2018 Apr 18;:
Authors: Johnson PW, Doe CQ, Lai SL
Stem cells must maintain proliferation during tissue development, repair and homeostasis, yet avoid tumor formation. In Drosophila, neural stem cells (neuroblasts) maintain proliferation during embryonic and larval development and terminate cell cycle during metamorphosis. An important question for understanding how tissues are generated and maintained is: what regulates stem cell proliferation versus differentiation? We performed a genetic screen which identified nucleostemin 3 (ns3) as a gene required to maintain neuroblast proliferation. ns3 is evolutionarily conserved with yeast and human Lsg1, which encode putative GTPases and are essential for organism growth and viability. We found NS3 is cytoplasmic and it is required to retain the cell cycle repressor Prospero in neuroblast cytoplasm via a Ran-independent pathway. NS3 is also required for proper neuroblast cell polarity and asymmetric cell division. Structure-function analysis further shows that the GTP-binding domain and acidic domain are required for NS3 function in neuroblast proliferation. We conclude NS3 has novel roles in regulating neuroblast cell polarity and proliferation.
PMID: 29679561 [PubMed - as supplied by publisher]
Neural circuits driving larval locomotion in Drosophila.
Neural Dev. 2018 Apr 19;13(1):6
Authors: Clark MQ, Zarin AA, Carreira-Rosario A, Doe CQ
More than 30 years of studies into Drosophila melanogaster neurogenesis have revealed fundamental insights into our understanding of axon guidance mechanisms, neural differentiation, and early cell fate decisions. What is less understood is how a group of neurons from disparate anterior-posterior axial positions, lineages and developmental periods of neurogenesis coalesce to form a functional circuit. Using neurogenetic techniques developed in Drosophila it is now possible to study the neural substrates of behavior at single cell resolution. New mapping tools described in this review, allow researchers to chart neural connectivity to better understand how an anatomically simple organism performs complex behaviors.
PMID: 29673388 [PubMed - in process]
The Hunchback temporal transcription factor establishes, but is not required to maintain, early-born neuronal identity.
Neural Dev. 2017 Jan 31;12(1):1
Authors: Hirono K, Kohwi M, Clark MQ, Heckscher ES, Doe CQ
BACKGROUND: Drosophila and mammalian neural progenitors typically generate a diverse family of neurons in a stereotyped order. Neuronal diversity can be generated by the sequential expression of temporal transcription factors. In Drosophila, neural progenitors (neuroblasts) sequentially express the temporal transcription factors Hunchback (Hb), Kruppel, Pdm, and Castor. Hb is necessary and sufficient to specify early-born neuronal identity in multiple lineages, and is maintained in the post-mitotic neurons produced during each neuroblast expression window. Surprisingly, nothing is currently known about whether Hb acts in neuroblasts or post-mitotic neurons (or both) to specify first-born neuronal identity.
METHODS: Here we selectively remove Hb from post-mitotic neurons, and assay the well-characterized NB7-1 and NB1-1 lineages for defects in neuronal identity and function.
RESULTS: We find that loss of Hb from embryonic and larval post-mitotic neurons does not affect neuronal identity. Furthermore, removing Hb from post-mitotic neurons throughout the entire CNS has no effect on larval locomotor velocity, a sensitive assay for motor neuron and pre-motor neuron function.
CONCLUSIONS: We conclude that Hb functions in progenitors (neuroblasts/GMCs) to establish heritable neuronal identity that is maintained by a Hb-independent mechanism. We suggest that Hb acts in neuroblasts to establish an epigenetic state that is permanently maintained in early-born neurons.
PMID: 28137283 [PubMed - indexed for MEDLINE]
Playing Well with Others: Extrinsic Cues Regulate Neural Progenitor Temporal Identity to Generate Neuronal Diversity.
Trends Genet. 2017 Dec;33(12):933-942
Authors: Syed MH, Mark B, Doe CQ
During neurogenesis, vertebrate and Drosophila progenitors change over time as they generate a diverse population of neurons and glia. Vertebrate neural progenitors have long been known to use both progenitor-intrinsic and progenitor-extrinsic cues to regulate temporal patterning. In contrast, virtually all temporal patterning mechanisms discovered in Drosophila neural progenitors (neuroblasts) involve progenitor-intrinsic temporal transcription factor cascades. Recent results, however, have revealed several extrinsic pathways that regulate Drosophila neuroblast temporal patterning: nutritional cues regulate the timing of neuroblast proliferation/quiescence and a steroid hormone cue that is required for temporal transcription factor expression. Here, we discuss newly discovered extrinsic cues regulating neural progenitor temporal identity in Drosophila, highlight conserved mechanisms, and raise open questions for the future.
PMID: 28899597 [PubMed - indexed for MEDLINE]