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The dorsal root ganglion (DRG) is a component of the peripheral nervous system that contains the cell body of sensory neurons, which are divided mainly into three subtypes: mechanoceptive, proprioceptive and nociceptive. The development of these cells depends on the sequential expression of specific genes to progress from sensory lineage commitment to subtype differentiation. This transcriptomic evolution is mediated by a complex network of transcription factors (TFs) that can act jointly, antagonistically, or in feedback loops. This network is also known as a gene cascade. Here, we focused on the individual role and relationships between TFs that are expressed during the early stages of differentiation. Specifically, we centered our studies on SCRT2, expressed only in neural postmitotic cells. This study aims to define the transcriptomic profile of SCRT2-positive cells and its role in defining the next elements in the gene cascade. We first analyzed the transcriptome of SCRT2 positive cells with the publicly available mouse DRG scRNA-seq data1. SCRT2 is not specifically expressed in a cell population at all the stages that we analyzed. We then searched for co-expression with other TFs (NEUROG2, ISL1, and POU4F1) whose role in specific steps of DRG development is known. The evolution of the temporal expression pattern of SCRT2 and the late commitment genes ISL1 and POU4F1 suggest a positive covariance between the three. For instance, in advanced stages, the number of SCRT2 positive cells and average single-cell expression levels decrease. The same occurs with ISL1 and POU4F1. Further, cells that express SCRT2 do not express early sensory lineage commitment genes, such as NEUROG2. Together, these data suggest that SCRT2 participates in the gene cascade that is active during late stages of sensory lineage commitment. To confirm the above co-expression results in embryo, we analyzed the spatial expression pattern of SCRT2, ISL1, POU4F1, and NEUROG2 in chick embryonic DRGs. Our in situ hybridization data show that SCRT2 is indeed co-expressed with ISL1 and POU4F1 and its expression domain is complementary to NEUROG2. NEUROG2, ISL1, and POU4F1 have been shown by others to participate in a neuronal genetic regulatory network, with diverse feedback connections. Thus, our next step was to verify if SCRT2 is also part of this network and could regulate the expression of NEUROG2, ISL1, and POU4F1. We then used neural tube CUT&RUN and ATAC-seq data to identify SCRT2 target sites in the genome. We identified SCRT2-target sites in the genomic region of all three genes. These sites were all located in evolutionarily conserved non-coding regions. Finally, these regions were enriched for binding sites of other TFs, suggesting that these are potential regulatory regions. Thus, with this analysis, we conclude that SCRT2 can bind to those transcription factor binding sites and regulate the other genes present in this GRN. To confirm if SCRT2 regulates the expression of ISL1 we overexpressed SCRT2 in chick embryos and counted the number of ISL1 positive cells in the DRG. Exogenous SCRT2 increased the number of ISL1 positive cells, indicating that SCRT2 indeed regulates the expression of ISL1.

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#	Name
1	Vitória Botezelli(vitoriasamartin@icb.usp.br)
2	Carolina Goes(goes.cp@gmail.com)
3	Ana elisa Orsi(ana.orsi@usp.br)
4	Irene Yan(ireneyan@usp.br)

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1	SHARMA, N. et al. The emergence of transcriptional identity in somatosensory neurons. Nature, v. 577, n. 7790, p. 392–398, 2020.

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