The mechanisms underlying the generation of the immense diversity of neuronal cell types remain a fundamental question of developmental biology. In the spinal cord, different "cardinal classes" of neurons that share a common molecular identity are produced from spatially segregated progenitor domains. Within many such classes, a stereotyped sequence of divergent neuronal types of related function is generated over time, raising the question of the molecular mechanisms that control this process. Here, we show that the successive expression of mouse transcription factors Onecut2, Pou2f2, and Pou3f1 within the cardinal classes giving rise to motor and sensory circuits, correlates with the emergence of sequentially generated subpopulations of neurons within those domains. We demonstrate that the genetic loss of Pou2f2 results in impaired development of two early-born motor neuron columns and re-specification of anterolateral system projection neurons as a later-born subset. Similarly, we show that Pou3f1 expression is required for the normal development of later-born subsets of motor neurons and anterolateral system projection neurons. Together, our observations provide functional evidence that horologic diversification of output neurons of spinal motor and sensory circuits are driven by a conserved sequential order of expression of transcription factors.

Funding provided by: Canadian Institutes of Health ResearchROR ID: https://ror.org/01gavpb45Award Number: 494078 Funding provided by: Deutsche ForschungsgemeinschaftROR ID: https://ror.org/018mejw64Award Number: 455354162 Funding provided by: Friedrich-Alexander-Universität Erlangen-NürnbergROR ID: https://ror.org/00f7hpc57Award Number: IZKF-E36 Funding provided by: Friedrich-Alexander-Universität Erlangen-NürnbergROR ID: https://ror.org/00f7hpc57Award Number: IZKF-S1 Funding provided by: Canadian Institutes of Health ResearchROR ID: https://ror.org/01gavpb45Award Number: PJT-162225 Funding provided by: Canadian Institutes of Health ResearchROR ID: https://ror.org/01gavpb45Award Number: MOP-77556 Funding provided by: Canadian Institutes of Health ResearchROR ID: https://ror.org/01gavpb45Award Number: PJT-153053 Funding provided by: Canadian Institutes of Health ResearchROR ID: https://ror.org/01gavpb45Award Number: PJT-159839 Funding provided by: Canadian Institutes of Health ResearchROR ID: https://ror.org/01gavpb45Award Number: PJT-183824