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During spinal cord development, Sonic hedgehog (Shh), secreted from the floor plate, plays an important role in the production of motor neurons by patterning the ventral neural tube, which establishes MN progenitor identity. It remains unknown, however, if Shh signaling plays a role in generating columnar diversity of MNs that connect distinct target muscles. Here, we report that Shh, expressed in MNs, is essential for the formation of lateral motor column (LMC) neurons in vertebrate spinal cord. This novel activity of Shh is mediated by its downstream effector ARHGAP36, whose expression is directly induced by the MN-specific transcription factor complex Isl1-Lhx3. Furthermore, we found that AKT stimulates the Shh activity to induce LMC MNs through the stabilization of ARHGAP36 proteins. Taken together, our data reveal that Shh, secreted from MNs, plays a crucial role in generating MN diversity via a regulatory axis of Shh-AKT-ARHGAP36 in the developing mouse spinal cord.

During spinal cord development, motor neuron (MN) axons exit the spinal cord ventrally, although the molecular basis for this process remains poorly understood. STAM1 and Hrs form a complex involved with endosomal targeting of cargo proteins, including the chemokine receptor CXCR4. Interestingly, the absence of CXCR4 signaling in spinal MNs is known to enforce improper extension of the axons into the dorsal side of the spinal cord. Here we report that the MN-specific Isl1-Lhx3 complex directly transactivates the Stam1 gene and STAM1 functions in determining the ventral spinal MN axonal projections. STAM1 is co-expressed with Hrs in embryonic spinal MNs, and knock-down of STAM1 in the developing chick spinal cord results in down-regulation of the expression of CXCR4, accompanied by dorsally projecting motor axons. Interestingly, overexpression of STAM1 or CXCR4 also results in dorsal projection of motor axons, suggesting that proper CXCR4 protein level is critical for the ventral motor axon trajectory. Our results reveal a critical regulatory axis for the ventral axonal trajectory of developing spinal MNs, consisting of the Isl1-Lhx3 complex, STAM1 and CXCR4.

While microRNAs have emerged as an important component of gene regulatory networks, it remains unclear how microRNAs collaborate with transcription factors in the gene networks that determines neuronal cell fate. Here we show that in the developing spinal cord, the expression of miR-218 is directly upregulated by the Isl1–Lhx3 complex, which drives motor neuron fate. Inhibition of miR-218 suppresses the generation of motor neurons in both chick neural tube and mouse embryonic stem cells, suggesting that miR-218 plays a crucial role in motor neuron differentiation. Results from unbiased RISC-trap screens, in vivo reporter assays and overexpression studies indicated that miR-218 directly represses transcripts that promote developmental programs for interneurons. In addition, we found that miR-218 activity is required for Isl1–Lhx3 to effectively induce motor neurons and suppress interneuron fates. Together our results reveal an essential role of miR-218 as a downstream effector of the Isl1–Lhx3 complex in establishing motor neuron identity. microRNAs have emerged as important components of numerous gene regulatory networks. Here the authors demonstrate that miR-218 is an essential component of the gene regulatory network that controls motor neuron fate specification in the developing spinal cord.