Deciphering primary-cilia-mediated signaling networks during periocular neural crest morphogenesis, specification and function

Abstract

Purpose : The mechanisms that control the morphogenesis, differentiation, and function of the periocular neural crest cells (pNC), the precursors for most structures of the anterior segment of the eye (AS), are poorly understood. Our previous studies and clinical evidence indicate a major role for primary cilia in these processes, but little is known about the signaling networks involved. The purpose of this study is to identify cilia-mediated signaling networks that may impact the behavior of pNC during AS morphogenesis

Methods : We performed single-cell (sc)RNA-Seq on isolated pNC from cornea and sclera of control and cilia mutant mice. We then profiled FACS-isolated mGFP+ pNC from Wn1-Cre;Ift88^fl/+;mT/mG (control) and Wn1-Cre;Ift88^fx/fx;mT/mG (cilia mutant) E18.5 embryos using droplet-based scRNA-Seq. The resulting data were plotted using a Uniform Manifold Approximation and Projection (UMAP) for analysis. We are currently validating scRNA-Seq results using an immunolocalization approach and assessing the function of selected differentially expressed genes by generating NC-specific conditional knockout mice

Results : The UMAPs from control and cilia-deficient pNC at E18.5 showed a similar distribution of clusters for both genotypes. However, the number of cells in clusters identified as sclera, iridocorneal angle and sensory nerves was lower in the mutant when compared to the control. In contrast, the number of cells in clusters identified as corneal stromal stem cells and Schwann cells and 4 clusters not yet identified was higher in the mutant. Interestingly, in control mice, the most highly expressed genes in the cluster identified as sclera were genes involved in cartilage production, including the transcription factor Sox9. Notably, cartilage-specific collagen genes were only downregulated in cells of the sclera cluster of the mutant. Thus, a cartilage synthesis program is present in the periocular mesenchyme of the control but is missing in the cilia mutant. The Sox9 function in the development of the sclera and other structures of the AS is under investigation

Conclusions : We have identified a cilia-dependent cartilage production pathway that takes place in a subpopulation of Sox9-expressing pNC. Our data suggest a putative role for the Sox-9-dependent signaling network and cartilage production in the development of the sclera

This abstract was presented at the 2024 ARVO Annual Meeting, held in Seattle, WA, May 5-9, 2024.