Lmo4 Establishes Rostral Motor Cortex Projection Neuron Subtype Diversity

The mammalian neocortex is parcellated into anatomically and functionally distinct areas. The establishment of area-specific neuronal diversity and circuit connectivity enables distinct neocortical regions to control diverse and specialized functional outputs, yet underlying molecular controls remain largely unknown. Here, we identify a central role for the transcriptional regulator Lim-only 4 (Lmo4) in establishing the diversity of neuronal subtypes within rostral mouse motor cortex, where projection neurons have particularly diverse and multi-projection connectivity compared with caudal motor cortex. In rostral motor cortex, we report that both subcerebral projection neurons (SCPN), which send projections away from the cerebrum, and callosal projection neurons (CPN), which send projections to contralateral cortex, express Lmo4, whereas more caudal SCPN and CPN do not. Lmo4-expressing SCPN and CPN populations are comprised of multiple hodologically distinct subtypes. SCPN in rostral layer Va project largely to brainstem, whereas SCPN in layer Vb project largely to spinal cord, and a subset of both rostral SCPN and CPN sends second ipsilateral caudal (backward) projections in addition to primary projections. Without Lmo4 function, the molecular identity of neurons in rostral motor cortex is disrupted and more homogenous, rostral layer Va SCPN aberrantly project to the spinal cord, and many dual-projection SCPN and CPN fail to send a second backward projection. These molecular and hodological disruptions result in greater overall homogeneity of motor cortex output. Together, these results identify Lmo4 as a central developmental control over the diversity of motor cortex projection neuron subpopulations, establishing their area-specific identity and specialized connectivity.