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The hallmarks of the alveolar subclass of rhabdomyosarcoma are chromosomal translocations that generate chimeric PAX3-FOXO1 or PAX7-FOXO1 transcription factors. Both PAX-FOXO1s result in related cell transformation in animal models, but both mutations are associated with distinct pathological manifestations in patients. To assess the mechanisms underlying these differences, we generated isogenic fibroblast lines expressing either PAX-FOXO1 paralog. Mapping of their genomic recruitment using CUT&Tag revealed that the two chimeric proteins have distinct DNA binding preferences. In addition, PAX7-FOXO1 causes stronger de novo transactivation of its bound regions than PAX3-FOXO1, resulting in greater transcriptomic dynamics involving genes regulating cell shape and cycle. Consistently, PAX3-FOXO1 accentuates fibroblast cellular traits associated with contractility and surface adhesion and limits entry into M phase. In contrast, PAX7-FOXO1 drives cells to adopt an amoeboid shape, reduces entry into S phase, and causes more genomic instabilities. Altogether, our results argue that the diversity of rhabdomyosarcoma manifestation arises, in part, from the divergence between the transcriptional activities of PAX3-FOXO1 and PAX7-FOXO1. Furthermore, the identified pronounced deleterious effects of PAX7-FOXO1 provide an explanation for the low frequency of the translocation generating this factor in patients with rhabdomyosarcoma.

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., Author summary The fusion-positive subtype of rhabdomyosarcoma (FP-RMS) is a rare malignant paediatric cancer, whose induction and evolution still remain to be deciphered. Out of the gross genetic aberrations found in these cancers, t(2:13) and t(1,13) chromosome translocations are the first to appear and lead to the expression of fusion proteins made of the DNA binding domains of either PAX3 or PAX7 and the transactivation domain of FOXO1. Both PAX3-FOXO1 and PAX7-FOXO1 have a strong impact on gene transcription, yet they only inefficiently promote the transformation of healthy cells into tumorigenic cells. To address this issue, we have used chick embryos to monitor in vivo the early response of cells to PAX-FOXO1 chimeric proteins. We showed that both proteins, but not the normal PAX3 and PAX7, transform neural cells into cells with FP-RMS molecular features. The PAX-FOXO1s also force polarized epithelial neural cells to adopt a mesenchymal phenotype with tissue invasive properties. However, the PAX-FOXO1s inhibit cell division and hence tumour growth. Genetically re-activating core cell cycle regulators rescues PAX-FOXO1 mediated cell cycle inhibition. Together, our findings bring further support to the idea that the PAX-FOXO1s are stricto sensu oncoproteins, whose oncogenicity is limited by negative effects on cell cycle.

Zebrafish are an emerging model in behavioral neuroscience. They display a wide range of measurable behaviors such as locomotion, aggression, anxiety, learning and memory, and social behavior. In addition, the relative ease of genetic manipulation and the increasing availability of disease models mean that zebrafish have gained in popularity as an animal model for various neurological and psychiatric diseases including autism spectrum disorder (ASD). In order to better characterize social behavior and behavioral abnormalities in zebrafish, we have developed the visually mediated social preference (VMSP) test, a novel assay to measure social preference and social novelty in two consecutive 5-min sessions. Using recording and video tracking, the time spent in different areas of the tank, the time spent immobile, swimming speed, and distance moved can be easily measured and analyzed. Untreated experimentally naive AB WT zebrafish typically show a strong preference for spending time near and interacting with a compartment containing unfamiliar conspecifics over the empty compartments during session 1 and a stronger preference for a group of unfamiliar zebrafish over familiar conspecifics from session 1, during session 2 of the test. Research in our lab has shown that the VMSP is suitable to measure the social behavior of individual zebrafish, to uncover social phenotypes of mutant strains, and to better understand animal models of disease that include impaired sociability such as ASD. The current paper provides a step-by-step guide on how to implement and perform this test and highlights important considerations for data acquisition, analysis, and interpretation.