In vivo functional validation of transcriptional regulators involved in vertebrate skeletal cell fate convergence

The vertebrate skeleton was a major evolutionary innovation that appeared sequentially in different parts of the body. Developmentally, cells forming this endoskeleton originate from three different embryonic lineages. The ectoderm-derived cranial neural crest gives rise to the craniofacial skeleton while the mesoderm-derived somite and lateral plate give rise to the axial and appendicular skeleton, respectively. Despite these distinct developmental origins, the three lineages differentiate into skeletal cell populations displaying not only functional but also transcriptional similarities. However, the exact molecular mechanisms underlying the convergent specification and differentiation of these tissues remain unclear.
This PhD thesis aims at identifying origin-specific regulators involved in this process of skeletogenic cell fate convergence, as well as investigating their regulatory interactions, to provide insights into the underlying gene regulatory networks in each skeletal lineage.
To do so, single-cell transcriptional and chromatin-profiling analyses were conducted on chicken-derived, differentiating skeletal progenitors from each embryonic lineage, revealing a set of shared and embryonic-specific transcription factors (TFs) with a potential role in skeletal cell fate specification. Additionally, predicted cis-regulatory elements were identified and validated for their activity and origin-specificity.
Furthermore, we developed and tested an optimized in vivo clustered regularly interspaced short palindromic repeats (CRISPR) screening procedure with single-cell transcriptomic readouts, to functionally validate TFs in developing chicken forelimbs, specifically targeting the tissue giving rise to skeletal progenitors. To do so, we adapted CRISPR constructs to enable the detection of guides during single-cell RNA sequencing while still maintaining CRISPR perturbation efficiencies. We conducted bioinformatic analyses on CRISPR perturbed cells and identified potential perturbation hits for a series of TFs with effects in genes related to cell proliferation, transcriptional regulation and skeletal development.
Collectively, this present work contributes to our understanding of the gene regulatory mechanisms underlying the convergent skeletal cell fate specification from three distinct precursor lineages during vertebrate embryogenesis.