The nervous system contains densely packed cell bodies, yet the role of neuronal cell body position in circuit function is poorly understood. Here we show that four Drosophila Moonwalker Descending Neurons (MDNs), command neurons for backward locomotion, must maintain cell body contact to allow gap junction-dependent synchronous activity necessary to initiate backward walking. MDNs express the transcription factor Hunchback, which drives expression of the Lar cell adhesion molecule; Hunchback, Lar, and its ligand Dlp promote MDN cell body clustering and backward walking. When clustered, the gap junction protein Inx8 allows synchronous firing of MDNs, which is required to initiate backward walking. These findings reveal a previously unappreciated role for cell body clustering and synchronous firing in neural circuit function.

Neural diversity is required for the brain to generate complex behaviors. During development, neural progenitors are exposed to different combination of transient spatial cues for their identity specification. This identity is then interpreted by their progeny to activate terminal selector genes to become lineage-specific neurons. After spatial cues fade, it remains unclear how progenitors maintain their unique identity so that their progeny express the accurate, lineage-specific terminal selector genes. Using single cell RNA sequencing in Drosophila, we identified a Forkhead domain transcription factor, Fd4, that is exclusively expressed in a single neural progenitor (neuroblast) and its new-born progeny. This neuroblast (NB), named NB7-1, forms at the intersection of the transient spatial cues Vnd (columnar expression) and En (row expression). We show that Fd4 expression overlaps spatial factor expression and terminal selector gene expression, thereby making Fd4 an excellent candidate for bridging transient spatial factors to lineage-specific terminal selector genes. We show that Fd4 is required for expression of terminal selector genes that maintain neuronal identity. Conversely, Fd4 misexpression generates ectopic NB7-1 progeny at the expense of Fd4-negative progenitor lineages. We conclude that Fd4 is continuously expressed in the NB7-1 and its new-born neuronal progeny where it activates terminal selector genes to produce lineage-specific neurons. We propose that Fd4 is a pioneering member of a class of “lineage identity genes” that translate transient spatial cues into a long-term lineage identity.

Understanding how neuronal diversity is generated is a major goal of neuroscience. Here we characterize the first step in generating neuronal diversity in the Drosophila embryo: spatial transcription factors (STFs) expressed in orthogonal rows and columns of neural progenitors. These factors give spatial identity to neural progenitors (neuroblasts, NBs), and are highly conserved in mammals. Here we investigate the roles of Engrailed (En+; posterior row) and Vnd+ (medial column) in specifying the well-characterized progenitor: neuroblast 7-1 (NB7-1). We show that NB7-1 is located at the intersection of Vnd and En, and we identify NB7-1 using a newly characterized gene, fd4, that we show is specifically expressed in NB7-1 and its progeny, giving us a specific assay for NB7-1 identity. We show that En and Vnd are both required for Fd4 expression, and that Vnd and En co-expression is sufficient to induce ectopic Fd4 expression in other NBs and their lineages. Finally, we show that NBs gradually lose competence to respond to En or Vnd. We conclude that En and Vnd are STFs that act combinatorially to specify the identity of an individual progenitor, NB7-1.

During neurodevelopment, a single progenitor cell can generate many different neuron types. As these neurons mature, they form unique morphologies, integrate into neural circuits, and contribute to behavior. However, the integration of these developmental events is understudied. Here, we show that the same transcription factor is important for both the generation of neuronal diversity and maintaining mature neuronal identity, providing novel insights into how the generation of neuronal identity and morphology are coordinated. We utilized a previously characterized larval locomotor circuit in Drosophila, where activation of the moonwalker descending neuron (MDN) triggers backward locomotion via its presynaptic connection with the premotor neuron A18b. The MDN expresses the temporal transcription factor Hunchback (Hb), which has a well-characterized role in neural progenitors. Loss of Hb in the postmitotic MDN increases axon/dendrite branching, leading to additional functional synapses on A18b and increasing backward locomotion. We conclude that the endogenous function of Hb is to restrain axon/dendrite outgrowth, including limiting MDN-A18b synapses, thereby dampening backward locomotion. Our work provides insights into how a transcription factor can have different functions throughout life; that is, Hb generates neuronal diversity in the progenitor and regulates neuronal connectivity in the mature neuron to generate an appropriately tuned behavior.

Sensory-motor integration requires proper connectivity of interneurons within the central nervous system (CNS). Unique interneuron identity, defined by a collection of features that includes expression of transcription factors (TFs; molecular identity) and connectivity, is essential for proper circuit assembly. In Drosophila and mammals, interneuron identity is generated by sequential expression of a temporal transcription factor (TTF) cascade in progenitors which is then transiently maintained in post-mitotic progeny. In Drosophila, neural progenitors (neuroblasts; NBs) sequentially express the TTFs Hunchback (Hb) > Krüppel (Kr) > Pdm > Castor > Grainy head (Grh). While many studies have investigated the role of Hb in specifying early-born neuron fate, our understanding of whether late TTFs specify aspects of late-born interneuron identity is severely lacking. Here we investigate the role of Castor in specifying late-born interneuron molecular identity and connectivity using the NB5-2 lineage. NB5-2 predominantly produces interneuron progeny and late-born interneurons, Saaghi1-3 (SA1-3) and Jaam1-3 (JA1-3), contribute to a well characterized proprioceptive circuit. We find that NB5-2 Castor is required to close the Pdm expression window and promote Grh expression, consistent with other VNC NB lineages. Lineage specific misexpression of Castor in NB5-2 results in an increase in progeny expressing the late-born TFs, Runt and Nab. We identify SA1, SA3, and JA3 as Castor expressing NB5-2 progeny and that SA1 presynapses localize to discrete neuropil subregions. Presynapse number significantly increases in two SA1 subregions following NB5-2 Castor misexpression and disrupts proprioceptive circuit behavior. We conclude that NB5-2 Castor functions to specify late-born interneuron molecular identity and connectivity.

Both mammalian and invertebrate nervous systems contain densely packed neuronal cell bodies in stereotyped locations, which express cell surface molecules and gap junction proteins. An open question that has rarely been addressed is: What role does cell body position play in neuronal circuit function? Here we show that the four adult Drosophila Moonwalker Descending Neurons (MDNs), command neurons for backward locomotion, must maintain cell body contact to initiate backward walking. MDNs express the transcription factor Hunchback, which drives expression of the cell adhesion molecule Lar. Hunchback, Lar, and its ligand Dlp, promote MDN cell body clustering, which blocks initiation of backward walking. When the cell bodies are clustered, electrical synapses form between MDN cell bodies. The gap junction protein Innexin 8 (also known as Shaking B) maintains a more depolarized resting membrane potential and allows synchronous firing of MDNs. Together, these electrophysiological properties are necessary to initiate backward walking. These findings reveal a previously unappreciated role for cell body location, and the properties it mediates, in neural circuit function.

Neural progenitors generate distinct neuronal populations over time. Drosophila larval neural progenitors, neuroblasts (NBs), generate neuronal diversity by expressing temporal gradients of transcription factors and RNA-binding proteins, including early factors Imp and Chinmo and late factors Syp, Mamo, and Broad. These factors have been well characterized in the larval central nervous system (CNS), yet nothing is known about their expression or function in the embryonic CNS. We show that embryonic Imp is expressed in a low-to-high temporal gradient, the opposite of the larval Imp gradient. Embryonic Chinmo is expressed in all post-mitotic neurons, but not in a gradient, while the late larval factors Mamo, E93, Syp, and Broad show little embryonic expression. We show that Imp is required for Chinmo expression in postmitotic neurons, and loss of Chinmo - but not Imp - derepresses Syp. Finally, we tested whether Imp and Chinmo are required for motor neuron molecular identity or morphology. Although neither is required to specify temporal or molecular neuronal identity, both are required for axon targeting to the correct body wall muscle, and downregulating dendrite outgrowth. We conclude that temporal factors are regulated differently in embryos and larvae, and that Imp and Chinmo are required for proper neuronal axon and dendrite projections.

In the Drosophila nervous system, neuroblasts (NBs) divide to produce a clone of neurons that establish distinct fates via precise timing and patterning of transcription factors (TFs). The final step in neurogenesis is a Notch/Numb asymmetric division that produces one daughter neuron that has active Notch signaling (NotchON) and one that does not (NotchOFF). NotchON neurons are well characterized, but the NotchOFF progeny are understudied due to lack of molecular markers. Here, we have identified Fer3 (forty-eight related 3) as a NotchOFF-specific transcription factor expressed in the NB7-1, NB6-1 and NB5-2 lineages. Fer3 is inhibited by Notch signaling in post-mitotic neurons, thereby restricting its expression to NotchOFF neurons. In some contexts, Fer3 is a transcriptional repressor, but we find that Fer3 misexpression generates ectopic Dbx+ neurons, and this is more penetrant when we misexpress a Fer3:activation domain fusion protein. Moreover, Fer3 is sufficient to induce Dbx expression in a neuroblast lineage that does not endogenously express Dbx or Fer3. This work presents the first known NotchOFF exclusive TF in the developing embryonic Drosophila ventral nerve cord.

Interneuron diversity within the central nervous system (CNS) is essential for proper circuit assembly. Functional interneurons must integrate multiple features, including combinatorial transcription factor (TF) expression, axon/dendrite morphology, and connectivity to properly specify interneuronal identity. Yet, how these different interneuron properties are coordinately regulated remains unclear. Here we used the Drosophila neural progenitor, NB5-2, known to generate late-born interneurons in a proprioceptive circuit, to determine if the early-born temporal transcription factor (TTF), Hunchback (Hb), specifies early-born interneuron identity, including molecular profile, axon/dendrite morphology, presynapse targeting, and behavior. We found that prolonged Hb expression in NB5-2 increases the number of neurons expressing early-born TFs (Nervy, Nkx6, and Dbx) at the expense of late-born TFs (Runt and Zfh2); thus, Hb is sufficient to promote interneuron molecular identity. Hb is also sufficient to transform late-born neuronal morphology to early-born neuronal morphology. Furthermore, prolonged Hb promotes the relocation of late-born neuronal presynapses to early-born neuronal presynapse neuropil locations, consistent with a change in interneuron connectivity. Finally, we found that prolonged Hb expression led to defects in proprioceptive behavior, consistent with a failure to properly specify late-born interneurons in the proprioceptive circuit. We conclude that the Hb TTF is sufficient to specify multiple aspects of early-born interneuron identity, as well as disrupt late-born proprioceptive neuron function.

In both Drosophila and mammals, the brain contains the most diverse population of cell types of any tissue. It is generally accepted that transcriptional diversity is an early step in generating neuronal and glial diversity, followed by the establishment of a unique gene expression profile that determines morphology, connectivity, and function. In Drosophila, there are two types of neural stem cells, called Type 1 (T1) and Type 2 (T2) neuroblasts. The diversity of T2-derived neurons contributes a large portion of the central complex (CX), a conserved brain region that plays a role in sensorimotor integration. Recent work has revealed much of the connectome of the CX, but how this connectome is assembled remains unclear. Mapping the transcriptional diversity of T2-derived neurons is a necessary step in linking transcriptional profile to the assembly of the adult brain. Here we perform single nuclei RNA sequencing of T2 neuroblast-derived adult neurons and glia. We identify clusters containing all known classes of glia, clusters that are male/female enriched, and 161 neuron-specific clusters. We map neurotransmitter and neuropeptide expression and identify unique transcription factor combinatorial codes for each cluster. This is a necessary step that directs functional studies to determine whether each transcription factor combinatorial code specifies a distinct neuron type within the CX. We map several columnar neuron subtypes to distinct clusters and identify two neuronal classes (NPF+ and AstA+) that both map to two closely related clusters. Our data support the hypothesis that each transcriptional cluster represents one or a few closely related neuron subtypes.

Abstract In both invertebrates such as Drosophila and vertebrates such as mouse or human, the brain contains the most diverse population of cell types of any tissue. It is generally accepted that transcriptional diversity is an early step in generating neuronal and glial diversity, followed by the establishment of a unique gene expression profile that determines morphology, connectivity, and function. In Drosophila, there are two types of neural stem cells, called Type 1 (T1) and Type 2 (T2) neuroblasts. In contrast to T1 neuroblasts, T2 neuroblasts generate intermediate neural progenitors (INPs) that expand the number and diversity of cell types. The diversity of T2-derived neurons contributes a large portion of the central complex (CX), a conserved brain region that plays a role in sensorimotor integration. Recent work has revealed much of the connectome of the CX, but how this connectome is assembled remains unclear. Mapping the transcriptional diversity of neurons derived from T2 neuroblasts is a necessary step in linking transcriptional profile to the assembly of the adult brain. Here we perform single nuclei RNA sequencing of T2 neuroblast-derived adult neurons and glia. We identify clusters containing all known classes of glia, clusters that are male/female enriched, and 161 neuron-specific clusters. We map neurotransmitter and neuropeptide expression and identify unique transcription factor combinatorial codes for each cluster (presumptive neuron subtype). This is a necessary step that directs functional studies to determine whether each transcription factor combinatorial code specifies a distinct neuron type within the CX. We map several columnar neuron subtypes to distinct clusters and identify two neuronal classes (NPF+ and AstA+) that both map to two closely related clusters. Our data support the hypothesis that each transcriptional cluster represents one or a few closely related neuron subtypes.

Drosophila nervous system development progresses through a series of well -characterized steps in which homeodomain transcription factors (HDTFs) play key roles during most, if not all, phases. Strikingly, although some HDTFs have only one role, many others are involved in multiple steps of the developmental process. Most Drosophila HDTFs engaged in nervous system development are conserved in vertebrates and often play similar roles during vertebrate development. In this Spotlight, we focus on the role of HDTFs during embryogenesis, where they were first characterized.

Notch signaling is an evolutionarily conserved pathway for specifying binary neuronal fates, yet how it specifies different fates in different contexts remains elusive. In our accompanying paper, using the Drosophila lamina neuron types (L1-L5) as a model, we show that the primary homeodomain transcription factor (HDTF) Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates. Here we test the hypothesis that Notch signaling enables Bsh to differentially specify L4 and L5 fates. We show asymmetric Notch signaling between newborn L4 and L5 neurons, but they are not siblings; rather, Notch signaling in L4 is due to Delta expression in adjacent L1 neurons. While Notch signaling and Bsh expression are mutually independent, Notch is necessary and sufficient for Bsh to specify L4 fate over L5. The Notch ON L4, compared to Notch OFF L5, has a distinct open chromatin landscape which allows Bsh to bind distinct genomic loci, leading to L4-specific identity gene transcription. We propose a novel model in which Notch signaling is integrated with the primary HDTF activity to diversify neuron types by directly or indirectly generating a distinct open chromatin landscape that constrains the pool of genes that a primary HDTF can activate.

How our brain generates diverse neuron types that assemble into precise neural circuits remains unclear. Using Drosophila lamina neuron types (L1-L5), we show that the primary homeodomain transcription factor (HDTF) brain-specific homeobox (Bsh) is initiated in progenitors and maintained in L4/L5 neurons to adulthood. Bsh activates secondary HDTFs Ap (L4) and Pdm3 (L5) and specifies L4/L5 neuronal fates while repressing the HDTF Zfh1 to prevent ectopic L1/L3 fates (control: L1-L5; Bsh-knockdown: L1-L3), thereby generating lamina neuronal diversity for normal visual sensitivity. Subsequently, in L4 neurons, Bsh and Ap function in a feed-forward loop to activate the synapse recognition molecule DIP-β, thereby bridging neuronal fate decision to synaptic connectivity. Expression of a Bsh:Dam, specifically in L4, reveals Bsh binding to the DIP-β locus and additional candidate L4 functional identity genes. We propose that HDTFs function hierarchically to coordinate neuronal molecular identity, circuit formation, and function. Hierarchical HDTFs may represent a conserved mechanism for linking neuronal diversity to circuit assembly and function.

Recent advances in single-cell transcriptional profiling have enabled us to map diverse neuron types across Drosophila, mice, and even human brains, yet we still lack a mechanistic understanding of how these neuron types arise in the brain and expand during evolution. We identified homeodomain transcription factor (HDTF) codes in newborn neurons that specify the five neuron types (L1-L5) in the Drosophila lamina, the first ganglion of the visual system. Specifically, we discovered that the pan-lamina HDTF Scro is required for lamina progenitors to exit proliferation and differentiate into lamina neurons. Furthermore, we showed that specific HDTFs are expressed in distinct newborn neurons and specify different lamina neuron types: Zfh1 for L1 and L3, Dve for L2, and Bsh for L4 and L5. Intriguingly, we found that the loss of both Dve and Bsh causes L2, L4, and L5 neurons to revert to L1 and L3 neuron types, potentially representing ancestral lamina neuron types. HDTFs are evolutionarily conserved and broadly expressed in brains across species. Based on these findings, we propose a model in which distinct HDTFs in newborn neurons specify different neuron types in the brain and have been utilized to expand neuron diversity during evolution. ### Competing Interest Statement The authors have declared no competing interest.

The generation of neuronal diversity is important for brain function, but how diversity is generated is incompletely understood. We used the development of the Drosophila central complex (CX) to address this question. The CX develops from eight bilateral Type 2 neuroblasts (T2NBs), which generate hundreds of different neuronal types. T2NBs express broad opposing temporal gradients of RNAbinding proteins. It remains unknown whether these protein gradients are sufficient to directly generate all known neuronal diversity, or whether there are temporal transcription factors (TTFs) with narrow expression windows that each specify a small subset of CX neuron identities. Multiple candidate TTFs have been identified, but their function remains uncharacterized. Here, we show that: (1) the adult E-PG neurons are born from early larval T2NBs; (2) the candidate TTF Castor is expressed transiently in early larval T2NBs when E-PG and P-EN neurons are born; and (3) Castor is required to specify early born E-PG and P-EN neuron identities. We conclude that Castor is a TTF in larval T2NB lineages that specifies multiple, early born CX neuron identities.

The generation of neuronal diversity remains incompletely understood. In Drosophila, the central brain is populated by neural stem cells derived from progenitors called neuroblasts (NBs). There are two types of NBs, type 1 and 2. T1NBs have a relatively simple lineage, whereas T2NBs expand and diversify the neural population with the generation of intermediate neural progenitors (INPs), contributing many neurons to the adult central complex, a brain region essential for navigation. However, it is not fully understood how neural diversity is created in T2NB and INP lineages. Imp, an RNA-binding protein, is expressed in T2NBs in a high-to-low temporal gradient, while the RNA-binding protein Syncrip forms an opposing gradient. It remains unknown if Imp expression is carried into INPs; whether it forms a gradient similar to NBs; and whether INP expression of Imp is required for generating neuronal identity or morphology. Here, we show that Imp/Syp are both present in INPs, but not always in opposing gradients. We find that newborn INPs adopt their Imp/Syp levels from their parental T2NBs; that Imp and Syp are expressed in stage-specific high-to-low gradients in INPs. In addition, there is a late INP pulse of Imp. We find that neurons born from old INPs (E-PG and PF-R neurons) have altered morphology following both Imp knock-down and Imp overexpression. We conclude that Imp functions in INPs and newborn neurons to determine proper neuronal morphology and central complex neuropil organization.

An unanswered question in neurobiology is how are diverse neuron cell types generated from a small number of neural stem cells? In the Drosophila larval central brain, there are eight bilateral Type 2 neuroblast (T2NB) lineages that express a suite of early temporal factors followed by a different set of late temporal factors and generate the majority of the central complex (CX) neurons. The early-to-late switch is triggered by the orphan nuclear hormone receptor Seven-up (Svp), yet little is known about how this Svp-dependent switch is involved in specifying CX neuron identities. Here, we: (1) birth date the CX neurons P-EN and P-FN (early and late, respectively); (2) show that Svp is transiently expressed in all early T2NBs; and (3) show that loss of Svp expands the population of early born P-EN neurons at the expense of late born P-FN neurons. Furthermore, in the absence of Svp, T2NBs fail decommissioning and abnormally extend their lineage into week-old adults. We conclude that Svp is required to specify CX neuron identity, as well as to initiate T2NB decommissioning.