TCF4 at the crest of development: a zebrafish model to explore craniofacial and gastrointestinal defects in Pitt-Hopkins syndrome.

BACKGROUND: Pitt-Hopkins Syndrome (PTHS) is a rare neurodevelopmental disorder caused by haploinsufficiency of the TCF4 gene. It is characterized by intellectual disability, distinctive facial features, breathing abnormalities, and gastrointestinal dysfunction. While the role of TCF4 in central nervous system development has been extensively investigated, the developmental basis underlying craniofacial and enteric alterations remains poorly understood. METHODS: We generated a zebrafish tcf4 mutant line using CRISPR/Cas9 genome editing to unveil the role of Tcf4 in neural crest-derived lineages that contribute to craniofacial and Enteric Nervous System development. The model was characterized by multiple integrated approaches to evaluate the morphological, cellular and functional alterations associated with tcf4 haploinsufficiency. As a proof-of-concept that the observed phenotypes resulted from Tcf4 loss of function, we reinstated human TCF4 expression in mutant larvae and assessed the rescue of the pathological phenotypes previously described. RESULTS: Heterozygous mutants exhibit key features of PTHS, including craniofacial skeletal abnormalities and impaired gastrointestinal motility. Functional analysis revealed a significant reduction of spontaneous peristaltic contractions and a delayed swallow-induced gut transit, consistently with human patients and PTHS mouse model. To further elucidate these developmental alterations, we showed that these defects are associated with a reduced number of Phox2b-positive enteric progenitors and HuC-positive enteric neurons, though early vagal neural crest migration appears unaffected. Reintroducing human TCF4 mRNA in tcf4 heterozygous mutant embryos rescues both craniofacial and gastrointestinal phenotypes, confirming the specificity of the observed phenotypes. Furthermore, we showed that the human TCF4 mRNA overexpression can alter craniofacial development in zebrafish embryos suggesting that TCF4 reinstatement dosage should be evaluated in gene therapy approaches to avoid a gain of function phenotype. CONCLUSION: Together, our results shed light on TCF4 as a key regulator of neural crest-derived lineages and provide a new perspective on two major clinical features of PTHS. The tcf4 mutant line represents a novel in vivo platform for investigating PTHS pathogenesis at cellular and molecular level and testing novel therapeutic strategies.