Ripply3 overdosage induces mid-face shortening through Tbx1 downregulation in Down syndrome models

The most frequent and unique features of Down syndrome (DS) are learning disability and craniofacial (CF) dysmorphism. The DS-specific CF features are an overall reduction in head dimensions (microcephaly), relatively wide and broad neurocranium (brachycephaly), reduced mediolaterally orbital region, reduced bizygomatic breadth, small maxilla, small mandible, and increased individual variability. Until now, the cellular and molecular mechanisms underlying the specific craniofacial phenotype have remained poorly understood. Investigating a new panel of DS mouse models with different segmental duplications on mouse chromosome 16 in the region homologous to human chromosome 21, we identified new regions and the role of two candidate genes for DS-specific CF phenotypes. First, we confirmed the role of Dyrk1a in the neurocranium brachycephaly. Then, we identified the role of the transcription factor Ripply3 overdosage in midface shortening through the downregulation of Tbx1, another transcription factor involved in the CF midface phenotype encountered in DiGeorge syndrome. This last effect occurs during the development of branchial arches through a reduction in cell proliferation. Our findings define a new dosage-sensitive gene responsible for the DS craniofacial features and propose new models for rescuing all aspects of DS CF phenotypes. This data may also provide insights into specific brain, immune and cardiovascular phenotypes observed in DiGeorge and DS models, opening avenues for potential targeted treatment to soften craniofacial dysmorphism in Down syndrome.Author summary: An extra copy of chromosome 21 causes Down syndrome (DS) and leads to intellectual disability and distinct facial features. To understand which genes drive these traits, we have engineered mouse models that mimic the DS genetic changes. By studying these mice, we identified regions of mouse chromosome 16, equivalent to human chromosome 21, that are associated with the head and face differences in Down syndrome (DS). We pinpointed two genes, Dyrk1a and Ripply3, whose increased activity disrupts normal facial development by altering the growth of specific cells during embryonic development. Notably, reducing Ripply3 activity in these mice corrected some facial abnormalities, highlighting its crucial role. These findings clarify how extra genetic material leads to DS features and suggest new therapeutic targets. The study also highlights the importance of animal models in elucidating the genetic and developmental basis of human conditions, such as DS. Additionally, the mechanism involving Ripply3 connects DS with DiGeorge syndrome, another disorder with similar facial changes, suggesting shared pathways behind some features of both conditions.