Cellular and Molecular Mechanisms of Dermal Condensate Formation

About This Grant

Project Summary Dermal papilla (DP) cells and their progenitors in dermal condensates (DC) during embryonic hair follicle (HF) development are tightly clustered, postmitotic cells with remarkable hair-inductive properties together with skin epithelial cells. The hair-inductive capacity, however, is unfortunately lost during cell culture expansion of dispersed cells, thus rendering cell-based regenerative therapy of hair loss with competent cells still elusive. During embryonic HF morphogenesis, epidermal placodes induce dispersed upper dermal fibroblasts to become the specialized DC clusters that aggregate via directional cell migration. Despite the importance of the DC cell cluster as a signaling center for epidermal cells for HF formation, the molecular controls that regulate cell migration and DC cluster formation remain unknown. We have recently discovered an unclustered DC precursor state (pre-DC) as fibroblasts (Fb) exit proliferation and directionally migrate to form DC clusters of nascent HFs, and reported the dynamic gene expression changes during the FbDC cell fate transitions. Exploring these “time-lapse” gene signatures suggested an intriguing role of axon guidance signaling (AGS) in regulating pre- DC cell migration and DC clustering. With many new preliminary insights throughout HF morphogenesis from bulk and single-cell transcriptomics; immunofluorescence; single molecule in situ hybridization; in vitro and ex vivo migration; and in vivo functional ablation assays, we have now established the conditions for exploring the dynamic expression of AGS ligands-receptors during early HF morphogenesis; elucidating their functional roles in pre-DC migration and clustering; and dissecting the downstream molecular mechanism(s) of these axon guidance pathways. Overall, we will rigorously test the hypothesis that axon guidance signaling is at the center of regulating pre-DC cell migration and DC formation. We will precisely map the spatiotemporal expression of these key AGS pathway ligands-receptors towards the DC fate by immunofluorescence analyses and multiplexed fluorescence in situ hybridization at subcellular resolution. We will further dissect the global expression dynamics along the Fbpre-DCDC axis with single-cell spatial transcriptomics. Finally, we will map the FbDC trajectory in human developing skin and share again all novel data in an easily accessible online platform. We will define the functional role of AGS pathways in DC aggregation and HF formation in gene knockouts of AGS pathways and carefully dissect the cellular and molecular effects resulting from loss of AGS. Finally, we will determine how DC migration and aggregation is perturbed upon individual and combined deletions of AGS pathway members and determine the potential of AGS ligands to regulate directional pre-DC cell migration. Finally, we will define with biochemical, molecular biological, and super-resolution imaging assays the role of small GTPase activation of actin cytoskeleton dynamics in AGS-mediated DC cell migration and aggregation. This work will identify AGS as a key regulatory mechanism that controls mesenchymal cell migration and clustering to orchestrate DC formation, a critical requirement for the hair inducing function.