Ectopic x-chromosome inactivation is critical for limb patterning

The National Institute of Child Health and Human Development (NICHD) awarded the Regents of the University of Michigan a $386,100 Project Grant under the Child Health and Human Development Extramural Research program (CFDA 93.865) beginning August 4, 2025, with completion targeted for July 31, 2027. This award supports investigator-initiated biomedical research designed to advance understanding of human development and improve child health outcomes. The research will be conducted at the University of Michigan's Ann Arbor campus in Michigan.

The funded research addresses limb patterning and development by investigating the role of ectopic X-chromosome inactivation in mesenchymal condensation and skeletogenesis. The project will utilize mutant mouse lines and bioinformatics approaches to characterize how bone morphogenetic protein (BMP) signaling regulates X-inactivation and gene suppression during early limb development, with the goal of identifying novel mechanisms essential for appropriate morphological and functional limb development. The research encompasses two primary aims: mapping the landscape of genes differentially suppressed by ectopic X-chromosome inactivation in a spatial-temporal manner, and identifying the functional role of X-chromosome inactivation during limb development through ex vivo culture systems and genetic models.

Abstract

Morphologically and functionally appropriate limb development is critical for quality of life. There is compelling evidence that growth factors, including bone morphogenetic proteins (bmps), regulate limb development. However, the mechanism of how growth factor signaling is involved in early patterning of mesenchymal condensation and subsequent cell fate specification in mesenchymal stem/progenitor cell population remains unclear. We recently found a formation of massive ectopic cartilage in the craniofacial region of a mutant mouse line engineered to augment BMP-smad signaling in a neural crest-specific manner. During preliminary characterization, we made three interesting findings: 1. Augmented BMP signaling in cranial neural crest cells (nccs) results in an increase of xist expression, a non-coding RNA critical for x chromosome inactivation, (XCI) leading to ectopic XCI in cranial NCC-specific manner, 2. The ectopic XCI is functionally essential for ectopic cartilage formation, and 3. Transient upregulation of xist expression and ectopic XCI are found in wildtype limb development. Based on these experimental data, we hypothesize that transient ectopic XCI is an unappreciated unique mechanism essential for limb development during early mesenchymal condensation and subsequent skeletogenesis. We will fully utilize mutant mouse lines generated in our laboratories and ones from our collaborators, and unbiased bioinformatics approaches to test the hypothesis. In aim 1, we will uncover the landscape of how differentially suppressed genes by ectopic XCI in a spatial-temporal manner. In aim 2, we will identify the functions of ectopic XCI during limb development. Our preliminary data using limb BUD EX vivo culture suggest that ectopic XCI is involved in early mesenchymal patterning. Our unique experimental tools and approaches will allow US to determine the role of BMP signaling in mesenchymal patterning and subsequent cell fate specification in limb development. Completing the proposed study will generate a novel knowledge informative to understand unique mechanisms of limb patterning and thus provide new insight for better treatment options for congenital limb abnormalities and directions toward limb regeneration.