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Drosophila Neural Development: From Stem Cells to Circuits
Research Summary
Chris Doe investigates central nervous system (CNS) development. His lab is currently interested in (1) asymmetric cell division and self-renewal/differentiation of Drosophila neural stem cells, (2) temporal identity programs used to generate an ordered series of neural progeny from a single progenitor, (3) the generation of interneuron diversity and establishment of neural circuits that drive larval locomotion, and (4) the use of TU tagging—a method for covalently labeling nascent RNA in specific cell types within intact tissues—to identify temporally regulated or activity-regulated RNAs in the mouse CNS.
Cell Polarity, Asymmetric Cell Division, and Stem Cell Self-Renewal Drosophila neural stem cells (called neuroblasts) divide asymmetrically to generate neurons while maintaining their undifferentiated state, and thus they can be used as a model system to study stem cell self-renewal versus differentiation.
Work from our lab and others has identified a suite of asymmetrically localized proteins and RNAs that promote either self-renewal or neuronal differentiation. We have completed transcriptomic and genetic screens for genes that are required for neuroblast self-renewal and neuronal differentiation. We have identified dozens of mutants with too few or too many larval neuroblasts; some of these are regulators of cortical polarity proteins, and many have yet to be characterized. Possible functions for these genes include regulation of neuroblast cell polarity, spindle orientation (misoriented spindles lead to increased neuroblast numbers), neuronal differentiation, and/or neuroblast quiescence.
Three areas of interest in the Doe lab: cell polarity (left), temporal identity (middle), and neural diversity (right). Left, confocal image of a single Drosophila larval neuroblast during metaphase stained for Miranda (red crescent), aPKC (green crescent), tubulin (blue spindle), and phosphohistone H3 (green DNA).
Right, DiI labeling of individual neuroblasts reveals clones containing a diverse population of neurons with complex but stereotyped axonal projections.
Left, Karsten Siller. Middle, see also Pearson, B.J., and Doe, C.Q. 2003. Nature 425:624–628. Right, see also Schmid, A., Chiba, A., and Doe, C.Q. 1999. Development 126:4653–4689.
Figure 2: Confocal image of a Drosophila larval brain lobe showing multiple neuroblasts at different places in the cell cycle; the left neuroblast is in metaphase, the right neuroblast is in anaphase, and the center neuroblast is in telophase. The markers shown are Miranda (red crescent), aPKC (blue crescent), tubulin (green spindle), and phosphohistone H3 (blue DNA).
Image: Sarah Siegrist
Figure 3: Transcription factor switching.
Transcription factor switching controls temporal identity in neuroblast (NB) lineages. Top, embryonic neuroblasts sequentially express transcription factors (Hunchback, Krüppel, Pdm, Castor) that specify temporal identity of their neuronal progeny. Bottom, 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.
Figure 4: Gal4 lines expressed in small subsets of interneurons.
Gal4 lines (Janelia Farm Research Campus) are expressed in small subsets of interneurons; these lines can be used to construct a three-dimensional atlas of interneurons, and allow optogenetic manipulation of single interneurons to determine their function in circuits and behavior.
Image: Ellie Heckscher
Figure 5: TU tagging is a genetic/chemical intersectional method for transcriptional profiling. It can be used in Drosophila or mouse to identify newly transcribed RNAs within specific cell types at specific time points.
Temporal Identity in Neural Progenitor Lineages
The generation of neuronal diversity requires spatial patterning (anteroposterior and dorsoventral differences in progenitor pools), but also requires 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. Previously we showed that Drosophila embryonic neuroblasts sequentially express four transcription factors: Hunchback (Ikaros class) —> Krüppel (zinc finger class) —> Pdm (Pou/homeodomain class) —> Castor (zinc finger class). Each transcription factor specifies the "temporal identity" of the neurons born during its expression window. We are currently identifying transcription factors expressed after Castor in neuroblast lineages.
We have shown that neuroblasts can be used as a model for progressive restriction of competence, which is a feature of cortical and retinal mammalian progenitors. Neuroblasts are only able to make early-born neurons in response to Hunchback during their first five divisions (~5 hours). The end of the competence window is triggered by the synchronous movement of a key Hunchback target gene (hunchback itself) to the nuclear lamina, resulting in this target gene becoming uninducible and thus unable to specify early-born neuronal identity. In future experiments, we will investigate neuroblast competence to respond to later temporal identity factors (Krüppel, Pdm, Castor), and investigate the existence, regulation, and function of the "second competence window."
Most recently, our lab and others have discovered a novel population of type II neuroblasts. These differ from canonical type I neuroblasts by dividing asymmetrically to produce transit-amplifying cells called intermediate neural progenitors (INPs). INPs may be a genetically tractable model for understanding human outer subventricular zone (OSVZ) progenitors, which also generate INPs. Drosophila INPs divide asymmetrically to generate ~12 neural progeny, raising the question of whether INPs merely expand the population of a neural subtype or undergo temporal patterning to generate an ordered series of distinct neural subtypes. We have recently shown that Drosophila INPs sequentially generate distinct neural subtypes; that INPs sequentially express Dichaete —> Grainyhead —> Eyeless transcription factors; and that these transcription factors are required for the production of distinct neural subtypes. Moreover, parental type II neuroblasts also sequentially express 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. Currently there are many experiments to identify and functionally characterize both neuroblast and INP transcriptional cascades and to determine if these conserved factors play a similar role during primate neurogenesis.
Neural Circuits Driving Larval Locomotion: Identification of Component Interneurons and the Developmental Logic of Circuit Assembly
Interneurons are the most common cell type in the Drosophila and mammalian CNS, yet little is known about how interneuronal diversity is generated, or how interneurons choose synaptic partners during neural circuit assembly. We have identified several hundred Gal4 lines expressed in 1–5 interneurons (from Gerald Rubin's collection, Janelia Farm Research Campus) and mapped them into a three-dimensional atlas that allows us to uniquely identify more than 50 percent of all interneurons in the ventral CNS (in collaboration with the JFRC labs of Eugene Myers [now Max Planck Institute of Molecular Cell Biology and Genetics] and Hanchuan Peng [now Allen Institute for Brain Science]). We are using these tools to (1) characterize interneuronal diversity by transcriptomics and morphology; (2) use optogenetics to determine the function of these interneurons in larval locomotion (in collaboration with the lab of Shawn Lockery, University of Oregon); and (3) locate these cells within a TEM (transmission electron microscopy) serial reconstruction of the entire larval CNS (in collaboration with Albert Cardona's lab, JFRC) to identify pre- and postsynaptic partners. We are testing three 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.
Neuronal Diversity in the Mouse
We previously developed a method of covalent labeling of newly transcribed RNA from specific cell types within intact tissues, called TU tagging. TU tagging expresses uracil phosphoribosyltransferase (UPRT) from Toxoplasma gondii in a cell type-specific pattern, and then RNAs are covalently labeled by addition of 4-thiouracil (4TU). Only cells expressing UPRT and exposed to 4TU produce thio-labeled RNA, which can be purified from whole tissue for RNA sequencing (RNAseq). We have used TU tagging to identify Drosophila glial transcripts, neuroblast transcripts, and temporally distinct type II neuroblast transcripts. TU tagging only labels newly transcribed RNA (not bulk RNA), making this method ideal for detecting acute changes in gene expression due to developmental events, small-molecule exposure, or behavioral changes.
We have recently established TU tagging in the mouse. We made a CA-lox-GFP-stop-lox-HA:UPRT transgene that only produces UPRT in cells exposed to Cre recombinase. We have shown that TU tagging in endothelial cells (Tie2:Cre driver) results in excellent purification of endothelial-specific transcripts. Our next step is to use TU tagging to explore interneuronal diversity, temporal patterning in neural lineages, and activity-dependent changes in neuronal gene expression.
Grants from the National Institutes of Health provided support for portions of this work.
