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INTRODUCTION Neurons are by far the most diverse of all cell types in animals, to the extent that “cell types” in mammalian brains are still mostly heterogeneous groups, and there is no consensus definition of the term. The Drosophila optic lobes, with approximately 200 well-defined cell types, provides a tractable system with which to address the genetic basis of neuronal type diversity. We previously characterized the distinct developmental gene expression program of each of these types using single-cell RNA sequencing (scRNA-seq), with one-to-one correspondence to the known morphological types. RATIONALE The identity of fly neurons is determined by temporal and spatial patterning mechanisms in stem cell progenitors, but it remained unclear how these cell fate decisions are implemented and maintained in postmitotic neurons. It was proposed in Caenorhabditis elegans that unique combinations of terminal selector transcription factors (TFs) that are continuously expressed in each neuron control nearly all of its type-specific gene expression. This model implies that it should be possible to engineer predictable and complete switches of identity between different neurons just by modifying these sustained TFs. We aimed to test this prediction in the Drosophila visual system. RESULTS Here, we used our developmental scRNA-seq atlases to identify the potential terminal selector genes in all optic lobe neurons. We found unique combinations of, on average, 10 differentially expressed and stably maintained (across all stages of development) TFs in each neuron. Through genetic gain- and loss-of-function experiments in postmitotic neurons, we showed that modifications of these selector codes are sufficient to induce predictable switches of identity between various cell types. Combinations of terminal selectors jointly control both developmental (e.g., morphology) and functional (e.g., neurotransmitters and their receptors) features of neurons. The closely related Transmedullary 1 (Tm1), Tm2, Tm4, and Tm6 neurons (see the figure) share a similar code of terminal selectors, but can be distinguished from each other by three TFs that are continuously and specifically expressed in one of these cell types: Drgx in Tm1, Pdm3 in Tm2, and SoxN in Tm6. We showed that the removal of each of these selectors in these cell types reprograms them to the default Tm4 fate. We validated these conversions using both morphological features and molecular markers. In addition, we performed scRNA-seq to show that ectopic expression of pdm3 in Tm4 and Tm6 neurons converts them to neurons with transcriptomes that are nearly indistinguishable from that of wild-type Tm2 neurons. We also show that Drgx expression in Tm1 neurons is regulated by Klumpfuss, a TF expressed in stem cells that instructs this fate in progenitors, establishing a link between the regulatory programs that specify neuronal fates and those that implement them. We identified an intronic enhancer in the Drgx locus whose chromatin is specifically accessible in Tm1 neurons and in which Klu motifs are enriched. Genomic deletion of this region knocked down Drgx expression specifically in Tm1 neurons, leaving it intact in the other cell types that normally express it. We further validated this concept by demonstrating that ectopic expression of Vsx (visual system homeobox) genes in Mi15 neurons not only converts them morphologically to Dm2 neurons, but also leads to the loss of their aminergic identity. Our results suggest that selector combinations can be further sculpted by receptor tyrosine kinase signaling after neurogenesis, providing a potential mechanism for postmitotic plasticity of neuronal fates. Finally, we combined our transcriptomic datasets with previously generated chromatin accessibility datasets to understand the mechanisms that control brain wiring downstream of terminal selectors. We built predictive computational models of gene regulatory networks using the Inferelator framework. Experimental validations of these networks revealed how selectors interact with ecdysone-responsive TFs to activate a large and specific repertoire of cell surface proteins and other effectors in each neuron at the onset of synapse formation. We showed that these network models can be used to identify downstream effectors that mediate specific cellular decisions during circuit formation. For instance, reduced levels of cut expression in Tm2 neurons, because of its negative regulation by pdm3 , controls the synaptic layer targeting of their axons. Knockdown of cut in Tm1 neurons is sufficient to redirect their axons to the Tm2 layer in the lobula neuropil without affecting other morphological features. CONCLUSION Our results support a model in which neuronal type identity is primarily determined by a relatively simple code of continuously expressed terminal selector TFs in each cell type throughout development. Our results provide a unified framework of how specific fates are initiated and maintained in postmitotic neurons and open new avenues to understanding synaptic specificity through gene regulatory networks. The conservation of this regulatory logic in both C. elegans and Drosophila makes it likely that the terminal selector concept will also be useful in understanding and manipulating the neuronal diversity of mammalian brains. Terminal selectors enable predictive cell fate reprogramming. Tm1, Tm2, Tm4, and Tm6 neurons of the Drosophila visual system share a core set of TFs continuously expressed by each cell type (simplified). The default Tm4 fate is overridden by the expression of a single additional terminal selector to generate Tm1 ( Drgx ), Tm2 ( pdm3 ), or Tm6 ( SoxN ) fates.
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Neuronal identity control at the resolution of a single transcription factor isoform
SUMMARY The brain exhibits remarkable neuronal diversity which is critical for its functional integrity. From the sheer number of cell types emerging from extensive transcriptional, morphological, and connectome datasets, the question arises of how the brain is capable of generating so many unique identities. ‘Terminal selectors’ are transcription factors hypothesized to determine the final identity characteristics in post-mitotic cells. Which transcription factors function as terminal selectors and the level of control they exert over different terminal characteristics are not well defined. Here, we establish a novel role for the transcription factorbroadas a terminal selector inDrosophila melanogaster. We capitalize on existing large sequencing and connectomics datasets and employ a comprehensive characterization of terminal characteristics including Perturb-seq and whole-cell electrophysiology. We find a single isoformbroad-z4serves as the switch between the identity of two visual projection neurons LPLC1 and LPLC2.Broad-z4is natively expressed in LPLC1, and is capable of transforming the transcriptome, morphology, and functional connectivity of LPLC2 cells into LPLC1 cells when perturbed. Our comprehensive work establishes a single isoform as the smallest unit underlying an identity switch, which may serve as a conserved strategy replicated across developmental programs.
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- Award ID(s):
- 1921065
- PAR ID:
- 10534910
- Publisher / Repository:
- bioRxiv
- Date Published:
- Format(s):
- Medium: X
- Institution:
- Drexel University
- Sponsoring Org:
- National Science Foundation
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- Posted Content:
- https://doi.org/10.1101/2024.06.14.598883
