The Temporal Neurogenesis Patterning of Spinal p3-V3 Interneurons into Divergent Subpopulation Assemblies

Neuronal diversity provides the spinal cord with the functional flexibility required to perform complex motor tasks. Spinal neurons arise during early embryonic development with the establishment of spatially and molecularly discrete progenitor domains that give rise to distinct, but highly heterogeneous, postmitotic interneuron (IN) populations. Our previous studies have shown that Sim1-expressing V3 INs, originating from the p3 progenitor domain, are anatomically and physiologically divergent. However, the developmental logic guiding V3 subpopulation diversity remains elusive. In specific cases of other IN classes, neurogenesis timing can play a role in determining the ultimate fates and unique characteristics of distinctive subpopulations. To examine whether neurogenesis timing contributes to V3 diversity, we systematically investigated the temporal neurogenesis profiles of V3 INs in the mouse spinal cord. Our work uncovered that V3 INs were organized into either early-born [embryonic day 9.5 (E9.5) to E10.5] or late-born (E11.5-E12.5) neurogenic waves. Early-born V3 INs displayed both ascending and descending commissural projections and clustered into subgroups across dorsoventral spinal laminae. In contrast, late-born V3 INs became fate-restricted to ventral laminae and displayed mostly descending and local commissural projections and uniform membrane properties. Furthermore, we found that the postmitotic transcription factor, Sim1, although expressed in all V3 INs, exclusively regulated the dorsal clustering and electrophysiological diversification of early-born, but not late-born, V3 INs, which indicates that neurogenesis timing may enable newborn V3 INs to interact with different postmitotic differentiation pathways. Thus, our work demonstrates neurogenesis timing as a developmental mechanism underlying the postmitotic differentiation of V3 INs into distinct subpopulation assemblies.SIGNIFICANCE STATEMENT Interneuron (IN) diversity empowers the spinal cord with the computation flexibility required to perform appropriate sensorimotor control. As such, uncovering the developmental logic guiding spinal IN diversity is fundamental to understanding the development of movement. In our current work, through a focus on the cardinal spinal V3 IN population, we investigated the role of neurogenesis timing on IN diversity. We uncovered that V3 INs are organized into early-born [embryonic day 9.5 (E9.5) to E10.5] or late-born (E11.5-E12.5) neurogenic waves, where late-born V3 INs display increasingly restricted subpopulation fates. Next, to better understand the consequences of V3 neurogenesis timing, we investigated the time-dependent functions of the Sim1 transcription factor, which is expressed in postmitotic V3 INs. Interestingly, Sim1 exclusively regulated the diversification of early-born, but not late-born, V3 INs. Thus, our current work indicates neurogenesis timing can modulate the functions of early postmitotic transcription factors and, thus, subpopulation fate specifications.