The remarkable diversity of neurons in the nervous system is generated during development, when properties such as cell morphology, receptor profiles and neurotransmitter identities are specified. In order to gain a greater understanding of neurotransmitter specification we profiled the transcription state of cholinergic, GABAergic and glutamatergic neurons in vivo at three developmental time points. We identified 86 differentially expressed transcription factors that are uniquely enriched, or uniquely depleted, in a specific neurotransmitter type. Some transcription factors show a similar profile across development, others only show enrichment or depletion at specific developmental stages. Profiling of Acj6 (cholinergic enriched) and Ets65A (cholinergic depleted) binding sites in vivo reveals that they both directly bind the ChAT locus, in addition to a wide spectrum of other key neuronal differentiation genes. We also show that cholinergic enriched transcription factors are expressed in mostly non-overlapping populations in the adult brain, implying the absence of combinatorial regulation of neurotransmitter fate in this context. Furthermore, our data underlines that, similar to Caenorhabditis elegans, there are no simple transcription factor codes for neurotransmitter type specification. This article has an associated First Person interview with the first author of the paper.

Condensin complexes are essential for mitotic chromosome assembly and segregation during cell divisions, however, little is known about their functions in post-mitotic cells. Here we report a role for the condensin I subunit Cap-G in Drosophila neurons. We show that, despite not requiring condensin for mitotic chromosome compaction, post-mitotic neurons express Cap-G. Knockdown of Cap-G specifically in neurons (from their birth onwards) results in developmental arrest, behavioural defects, and dramatic gene expression changes, including reduced expression of a subset of neuronal genes and aberrant expression of genes that are not normally expressed in the developing brain. Knockdown of Cap-G in mature neurons results in similar phenotypes but to a lesser degree. Furthermore, we see dynamic binding of Cap-G at distinct loci in progenitor cells and differentiated neurons. Therefore, Cap-G is essential for proper gene expression in neurons and plays an important role during the early stages of neuronal development.

8The remarkable diversity of neurons in the nervous system is generated during development, when 9properties such as cell morphology, receptor profiles and neurotransmitter identities are specified. 10Neurotransmitters are essential for neuronal communication, and transcription factors are the key 11 determinants of neurotransmitter fate. In order to gain a greater understanding of neurotransmitter 12 specification we profiled the transcription state of cholinergic, GABAergic and glutamatergic neurons 13 in vivo at multiple developmental time points. We identified 86 differentially expressed transcription 14 factors that are uniquely enriched, or uniquely depleted, in a specific neurotransmitter subtype. Some 15 transcription factors show a similar profile across development, others only show enrichment or 16 depletion at specific developmental stages. Profiling of acj6 (cholinergic enriched) and Ets65A 17 (cholinergic depleted) binding sites in vivo reveals that they both directly bind the ChAT locus, in 18 addition to a wide spectrum of other key neuronal differentiation genes. Our data underlines that, 19 similar to C. elegans, there are no simple transcription factor codes for neurotransmitter type 20 specification, and provides a useful resource for studying the developmental basis of neurotransmitter 21 specification in Drosophila and other model systems. 22 enrichment of the GAL4-trapped genes (ChAT, Gad1 and VGlut) ( Figure S1) that provide subtype 126 specific expression for the TaDa experiments. 127 CATaDa profiling of chromatin accessibility (Aughey et al., 2018) reveals that, similar to RNA Pol II, 128 global chromatin accessibility does not vary greatly between cell types (Figure 2) but shows more 129 differences between developmental stages. Correspondingly, when examining regions of the genome 130 that display robust changes in chromatin accessibility (>10 RPM differences across 3 consecutive 131 GATC fragments) during embryo development, only 37 GATC fragments (13 individual peaks) are 132 identified, with 62% mapping to the loci of the three neurotransmitter synthesis genes (ChAT, Gad1 133

AbstractThe condensin complex is essential for mitotic chromosome assembly and segregation during cell divisions, however, little is known about its function in post-mitotic, differentiated cells. Here we report a novel role for the condensin I subunit Cap-G in Drosophila neurons. We show that, despite not requiring condensin for mitotic chromosome compaction, post-mitotic neurons express Cap-G and that knockdown of Cap-G specifically in neurons (from their birth onwards) results in developmental arrest, behavioural defects, and dramatic gene expression changes. These include reduced expression of a subset of neuronal genes and aberrant expression of genes that are not normally expressed in the developing brain. Knockdown of Cap-G in more mature neurons also results in similar phenotypes but to a lesser degree. Furthermore, we see dynamic binding of Cap-G to chromatin at distinct loci in neural stem cells and differentiated neurons. Therefore, Cap-G is essential for proper gene expression in neurons and plays an important role during the early stages of neuronal development.

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