The PAX-FOXO1s trigger fast trans-differentiation of chick embryonic neural cells into alveolar rhabdomyosarcoma with tissue invasive properties limited by S phase entry inhibition.

The chromosome translocations generating PAX3-FOXO1 and PAX7-FOXO1 chimeric proteins are the primary hallmarks of the paediatric fusion-positive alveolar subtype of Rhabdomyosarcoma (FP-RMS). Despite the ability of these transcription factors to remodel chromatin landscapes and promote the expression of tumour driver genes, they only inefficiently promote malignant transformation in vivo. The reason for this is unclear. To address this, we developed an in ovo model to follow the response of spinal cord progenitors to PAX-FOXO1s. Our data demonstrate that PAX-FOXO1s, but not wild-type PAX3 or PAX7, trigger the trans-differentiation of neural cells into FP-RMS-like cells with myogenic characteristics. In parallel, PAX-FOXO1s remodel the neural pseudo-stratified epithelium into a cohesive mesenchyme capable of tissue invasion. Surprisingly, expression of PAX-FOXO1s, similar to wild-type PAX3/7, reduce the levels of CDK-CYCLIN activity and increase the fraction of cells in G1. Introduction of CYCLIN D1 or MYCN overcomes this PAX-FOXO1-mediated cell cycle inhibition and promotes tumour growth. Together, our findings reveal a mechanism that can explain the apparent limited oncogenicity of PAX-FOXO1 fusion transcription factors. They are also consistent with certain clinical reports indicative of a neural origin of FP-RMS.

BMP4 patterns Smad activity and generates stereotyped cell fate organization in spinal organoids.

Bone morphogenetic proteins (BMPs) are secreted regulators of cell fate in several developing tissues. In the embryonic spinal cord, they control the emergence of the neural crest, roof plate and distinct subsets of dorsal interneurons. Although a gradient of BMP activity has been proposed to determine cell type identity in vivo, whether this is sufficient for pattern formation in vitro is unclear. Here, we demonstrate that exposure to BMP4 initiates distinct spatial dynamics of BMP signalling within the self-emerging epithelia of both mouse and human pluripotent stem cell-derived spinal organoids. The pattern of BMP signalling results in the stereotyped spatial arrangement of dorsal neural tube cell types, and concentration, timing and duration of BMP4 exposure modulate these patterns. Moreover, differences in the duration of competence time-windows between mouse and human account for the species-specific tempo of neural differentiation. Together, this study describes efficient methods for generating patterned subsets of dorsal interneurons in spinal organoids and supports the conclusion that graded BMP activity orchestrates the spatial organization of the dorsal neural tube cellular diversity in mouse and human.

Pax3- and Pax7-mediated Dbx1 regulation orchestrates the patterning of intermediate spinal interneurons.

Transcription factors are key orchestrators of the emergence of neuronal diversity within the developing spinal cord. As such, the two paralogous proteins Pax3 and Pax7 regulate the specification of progenitor cells within the intermediate neural tube, by defining a neat segregation between those fated to form motor circuits and those involved in the integration of sensory inputs. To attain insights into the molecular means by which they control this process, we have performed detailed phenotypic analyses of the intermediate spinal interneurons (IN), namely the dI6, V0D, V0VCG and V1 populations in compound null mutants for Pax3 and Pax7. This has revealed that the levels of Pax3/7 proteins determine both the dorso-ventral extent and the number of cells produced in each subpopulation; with increasing levels leading to the dorsalisation of their fate. Furthermore, thanks to the examination of mutants in which Pax3 transcriptional activity is skewed either towards repression or activation, we demonstrate that this cell diversification process is mainly dictated by Pax3/7 ability to repress gene expression. Consistently, we show that Pax3 and Pax7 inhibit the expression of Dbx1 and of its repressor Prdm12, fate determinants of the V0 and V1 interneurons, respectively. Notably, we provide evidence for the activity of several cis-regulatory modules of Dbx1 to be sensitive to Pax3 and Pax7 transcriptional activity levels. Altogether, our study provides insights into how the redundancy within a TF family, together with discrete dynamics of expression profiles of each member, are exploited to generate cellular diversity. Furthermore, our data supports the model whereby cell fate choices in the neural tube do not rely on binary decisions but rather on inhibition of multiple alternative fates.