Dual PAX3 and PAX7 transcriptional activities spatially encode spinal cell fates through distinct gene networks
Understanding how transcription factors regulate organized cellular diversity in developing tissues remains a major challenge due to their pleiotropic functions. We addressed this by monitoring and genetically modulating the activity of PAX3 and PAX7 during the specification of neural progenitor pools in the embryonic spinal cord. Using mouse models, we show that the balance between the transcriptional activating and repressing functions of these factors is modulated along the dorsoventral axis and is instructive to the patterning of spinal progenitor pools. By combining loss-of-function experiments with functional genomics in spinal organoids, we demonstrate that PAX-mediated repression and activation rely on distinct cis-regulatory genomic modules. This enables both the coexistence of their dual activity in dorsal cell progenitors and the specific control of two major differentiation programs. PAX promotes H3K27me3 deposition at silencers to repress ventral identities, while at enhancers, they act as pioneer factors, opening and activating cis-regulatory modules to specify dorsal-most identities. Finally, we show that this pioneer activity is restricted to cells exposed to BMP morphogens, ensuring spatial specificity. These findings reveal how PAX proteins, modulated by morphogen gradients, orchestrate neuronal diversity in the spinal cord, providing a robust framework for neural subtype specification. Overall design: Profiling of PAX3 and PAX7 recruitment by CUT&Tag in spinal neuronal progenitors differenciated from HM1 ES cells in presence or absence of BMP4. RNA-Seq, ATAC-Seq, and CUT&Tag profiling (H3K4me2 and H3K27me3 marks) in spinal neuronal progenitors differenciated in presence or absence of BMP4 from wildtype HM1 ES cells, as well as the knock-out cell lines (Pax3-/-, Pax7-/-, or Pax3-/-; Pax7-/- (DKO)).