During embryonic development, the dorsal spinal cord generates numerous interneuron populations eventually involved in motor circuits or in sensory networks that integrate and transmit sensory inputs from the periphery. The molecular mechanisms that regulate the specification of these multiple dorsal neuronal populations have been extensively characterized. In contrast, the factors that contribute to their diversification into smaller specialized subsets and those that control the specific distribution of each population in the developing spinal cord remain unknown. Here, we demonstrate that the Onecut transcription factors, namely Hepatocyte Nuclear Factor-6 (HNF-6) (or OC-1), OC-2 and OC-3, regulate the diversification and the distribution of spinal dorsal interneuron (dINs). Onecut proteins are dynamically and differentially distributed in spinal dINs during differentiation and migration. Analyzes of mutant embryos devoid of Onecut factors in the developing spinal cord evidenced a requirement in Onecut proteins for proper production of a specific subset of dI5 interneurons. In addition, the distribution of dI3, dI5 and dI6 interneuron populations was altered. Hence, Onecut transcription factors control genetic programs that contribute to the regulation of spinal dIN diversification and distribution during embryonic development.

Acquisition of proper neuronal identity and position is critical for the formation of neural circuits. In the embryonic spinal cord, cardinal populations of interneurons diversify into specialized subsets and migrate to defined locations within the spinal parenchyma. However, the factors that control interneuron diversification and migration remain poorly characterized. Here, we show that the Onecut transcription factors are necessary for proper diversification and distribution of the V2 interneurons in the developing spinal cord. Furthermore, we uncover that these proteins restrict and moderate the expression of spinal isoforms of Pou2f2 , a transcription factor known to regulate B-cell differentiation. By gain- or loss-of-function experiments, we show that Pou2f2 contribute to regulate the position of V2 populations in the developing spinal cord. Thus, we uncovered a genetic pathway that regulates the diversification and the distribution of V2 interneurons during embryonic development.

Spinal dorsal interneurons, which are generated during embryonic development, relay and process sensory inputs from the periphery to the central nervous system. Proper integration of these cells into neuronal circuitry depends on their correct positioning within the spinal parenchyma. Molecular cues that control neuronal migration have been extensively characterized but the genetic programs that regulate their production remain poorly investigated. Onecut (OC) transcription factors have been shown to control the migration of the dorsal interneurons (dINs) during spinal cord development. Here, we report that the OC factors moderate the expression of Pou2f2, a transcription factor essential for B-cell differentiation, in spinal dINs. Overexpression or inactivation of Pou2f2 leads to alterations in the differentiation of dI2, dI3 and Phox2a-positive dI5 populations and to defects in the distribution of dI2-dI6 interneurons. Thus, an OC-Pou2f2 genetic cascade regulates adequate diversification and distribution of dINs during embryonic development.

32 Acquisition of proper neuronal identity and position is critical for the formation of neural 33 circuits. In the embryonic spinal cord, cardinal populations of interneurons diversify into 34 specialized subsets and migrate to defined locations within the spinal parenchyma. 35 However, the factors that control interneuron diversification and migration remain poorly 36 characterized. Here, we show that the Onecut transcription factors are necessary for proper 37 diversification and distribution of the V2 interneurons in the developing spinal cord. 38 Furthermore, we uncover that these proteins restrict and moderate the expression of spinal 39 isoforms of Pou2f2, a transcription factor known to regulate B-cell differentiation. By gain-40 or loss-of-function experiments, we show that Pou2f2 contribute to regulate the position of 41 V2 populations in the developing spinal cord. Thus, we uncovered a genetic pathway that 42 regulates the diversification and the distribution of V2 interneurons during embryonic 43 development. 44 45 46 Significance statement 47 In this study, we identify the Onecut and Pou2f2 transcription factors as regulators of spinal 48 interneuron diversification and migration, two events that are critical for proper CNS 49 development. 50 51