Independent control of neurogenesis and dorsoventral patterning by NKX2-2
Human neurogenesis is disproportionately protracted, lasting >10 times longer than in mouse, allowing neural progenitors to undergo more rounds of self-renewing cell divisions and generate larger neuronal populations. In the human spinal cord, expansion of the motor neuron lineage is achieved through a newly evolved progenitor domain called vpMN (ventral motor neuron progenitor) that uniquely delays and expands motor neurogenesis. This behavior of vpMNs is controlled by transcription factor NKX2-2, which in vpMNs is co-expressed with classical motor neuron progenitor (pMN) marker OLIG2. In this study, we sought to determine the molecular basis of NKX2-2-mediated extension and expansion of motor neurogenesis. We found that, unlike in mouse or chick, NKX2-2 in the human spinal cord does not repress dorsoventral patterning genes like OLIG2. However, it retains its ability to repress NEUROG2, a proneural gene that promotes exit from cell cycle and motor neurogenesis. Interestingly, we found that ectopic expression of Tinman-mutant Nkx2-2 in mouse pMNs phenocopies human vpMNs, repressing Neurog2 but not Olig2, resulting in delayed motor neurogenesis. Thus, our studies reveal that the classical patterning function of NKX2-2 that depends on its Tinman repressive domain is dissociated from NKX2-2's ability to repress NEUROG2 to control the onset and duration of motor neurogenesis in human ventral motor neuron progenitors. Overall design: Mouse ES cell- or human iPS cell-derived motor neuron organoids were used to generate high-throughput sequencing data. Human organoids were dissociated on day 11 of motor neuron differentiation following established protocols (Maury et al., Nat. Biotech.), followed by fixation, immunolabeling for NKX2-2 and OLIG2, then FACS-purified for NKX2-2+/OLIG2+ or NKX2-2-/OLIG2+ populations. RNA was extracted from purified populations and sequenced. Mouse cultures (following established motor neuron differentiation conditions; Wichterle et al., Cell) were exposed to doxycycline on day 3.5 to overexpress WT, TN domain mutant or SD mutant Nkx2-2 for 24 hours (control population was cultured in the absence of doxycycline). These populations were then used for RNA-seq and ChIP-seq against NKX2-2 or H3K27 acetylation.