UCLA

Craniofacial development is a highly coordinated biological process that depends on tightly regulated interactions between epithelial, mesenchymal, musculoskeletal, and connective tissues. These interactions are regulated by complex molecular signaling networks that regulate cell proliferation, differentiation, tissue patterning, and homeostasis throughout embryonic development and postnatal growth. Disruptions in these regulatory mechanisms can contribute to craniofacial abnormalities, impaired tissue regeneration, and developmental disorders affecting the jaws, teeth, and associated craniofacial structures. Understanding the signaling pathways that control craniofacial tissue development and renewal is therefore essential for advancing developmental biology, regenerative medicine, and translational applications in orthodontics and craniofacial therapeutics. This study investigates molecular mechanisms regulating two distinct but complementary aspects of craniofacial biology: dental epithelial renewal in the continuously growing murine incisor and embryonic mandibular osteochondral fate specification. Although these systems represent different developmental contexts, both rely on coordinated signaling interactions and transcriptional controls that regulate progenitor cell proliferation, differentiation, and tissue structural organization. However, the underlying mechanisms driving coordinated cell behaviors to generate spatiotemporally organized craniofacial tissues remain an important open question. The first component of this work focuses on the role of Hippo signaling in regulating dental epithelial cell proliferation and plasticity. The continuously erupting mouse incisor serves as an ideal model for studying epithelial stem cell maintenance due to its lifelong regenerative capacity. Within this system, the Hippo signaling pathway plays a central role in controlling tissue growth and cellular differentiation through regulation of the transcriptional co-activator Yes-associated protein (YAP). We therefore hypothesized that increased YAP activity can revert differentiated cells back to a progenitor-like state, informing strategies for human tooth regeneration. To investigate the effects of ectopic YAP activation in dental epithelial cells, conditional deletion of the Hippo pathway kinases Lats1 and Lats2 was done within the dental epithelium. Histological analysis, proliferation assays, and immunostaining were used to assess alterations in tissue organization, progenitor behavior, and cellular differentiation. Conditional activation of YAP resulted in expansion of proliferative dental epithelial progenitor populations within the apical bud and induced thickening of the suprabasal epithelial compartment. Increased proliferative activity was observed in epithelial progenitor regions, indicating that YAP signaling promotes maintenance of a proliferative progenitor state during incisor renewal. Despite this proliferative expansion, differentiated ameloblasts remained largely non-proliferative in vivo, suggesting that terminal differentiation imposes significant restrictions on proliferative re-entry within intact tissue environments. Additionally, disruption of normal tissue architecture and altered differentiation of pre-ameloblast populations were observed following ectopic YAP activation. To further investigate epithelial plasticity outside the native tissue environment, dissociated dental epithelial cells were cultured in three-dimensional spheroid conditions and treated with the LATS inhibitor TRULI. Under these conditions, a subset of differentiated ameloblasts demonstrated the capacity to form colonies, suggesting that cellular plasticity can be partially restored in vitro following tissue dissociation and pharmacologic modulation of Hippo signaling. Collectively, these findings demonstrate that YAP signaling promotes expansion of dental epithelial progenitors while revealing context-dependent limitations in the regenerative potential of differentiated ameloblasts. These studies provide insight into mechanisms governing epithelial stem cell behavior and suggest potential strategies for modulating dental epithelial regeneration in future stem cell-based therapies. The second component of this thesis investigates signaling interactions regulating embryonic mandibular development and Meckel’s cartilage formation. While these structures must form at precise times and locations, how different cell types emerge in a spatiotemporally regulated manner is still not fully understood. To understand how distinct cell types are specified within a relatively confined space, our lab has conducted single cell RNA-sequencing (scRNA-seq) to define the transcriptional signature of each cell population. This revealed many markers and my thesis aimed to characterize the spatial distribution of multiple molecular regulators involved in Meckel’s cartilage and mandibular development. Spatial gene expression analysis demonstrated distinct regional expression patterns corresponding to chondrogenic and osteogenic compartments within the developing mandible. Markers associated with cartilage differentiation and extracellular matrix organization, including Sox9, Col2a1, Matn4, and Col9a2, localized primarily within the chondrogenic regions of Meckel’s cartilage, whereas osteogenic regulators such as Runx2 and Sp7 were enriched in surrounding mandibular bone-forming regions. Additional signaling molecules, including Bmp4, Fgf8, Fgf9, Tgfb2, and Pdgfa, demonstrated region-specific expression patterns consistent with their potential roles in craniofacial patterning, mesenchymal proliferation, and tissue differentiation. To further characterize spatial gene expression during embryonic mandibular development, this work optimized and modified HCRTM Gold RNA-FISH protocol for three-dimensional whole- mount imaging of E12.5 embryonic mandibles. Complementary RNAscope analysis confirmed spatial localization and signal specificity at higher resolution. In addition, lineage tracing of Sp7- expressing cell populations using Sp7-tTA;tetO-Cre;R26mCherry mice provided further insight into the spatial organization of osteogenic cell populations during mandibular morphogenesis at E12.5 and E13.5. Combined with optical tissue clearing and two-photon microscopy, this approach enabled visualization of tissue-wide gene expression patterns within intact developing mandibular structures. Together, these approaches established imaging and molecular tools for examining three-dimensional signaling and lineage relationships during early craniofacial development. Among all the signaling pathways identified from the scRNA-seq, fibroblast growth factor (FGF) signaling is particularly interesting, as Fgf9 is specifically expressed in the mesoderm and its receptor Fgfr3 is specifically expressed in the cartilage. To further investigate the functional role of FGF signaling during mandibular development, pregnant mice were treated with the FGFR inhibitor infigratinib at E10.5 and E11.5. Embryos harvested at E12.5 were evaluated using proliferation assays and RNAscope spatial gene expression analysis. Pharmacologic inhibition of FGFR signaling did not significantly alter overall mesenchymal proliferation within the embryonic mandible. However, expression of Matn4 was reduced following FGFR inhibition, suggesting that FGFR signaling contributes to cartilage maturation and extracellular matrix organization rather than directly regulating cellular proliferation and specification during this early developmental stage. Together, the studies presented in this thesis highlight the importance of tightly regulated signaling pathways in controlling craniofacial tissue development, progenitor maintenance, and differentiation. By investigating the Hippo-YAP and FGFR signaling pathways in distinct biological systems, my studies demonstrate how modulation of developmental signaling networks influences tissue organization, cellular behavior, and regenerative potential within the craniofacial complex. These findings contribute to a broader understanding of craniofacial developmental biology and may provide foundational insight for future therapeutic approaches targeting tissue regeneration, developmental abnormalities, and craniofacial reconstruction.