The objective of this proposal is to delineate the role of the transcriptional repressor ZEB2 during human neural crest cell formation, craniofacial differentiation, and Mowat-Wilson Syndrome. The neural crest is a multipotent embryonic cell population unique to vertebrates which migrates extensively and differentiates into a variety of derivatives, including most of the craniofacial bone, cartilage, and dental organ tissues. Numerous human pathologies are associated with abnormal neural crest cell development, including the recently delineated neurocristopathy Mowat-Wilson Syndrome (MWS). MWS is a rare syndrome caused by de novo heterozygous mutations in ZEB2 leading to a distinct facial phenotype, dental abnormalities, intellectual disability, and other anomalies including Hirschprung disease which are indicative of neural crest cell defects at multiple axial levels. However, significant genotypic and phenotypic variability has been observed among MWS patients, and the underlying mechanisms that contribute to this disease are not well understood. Furthermore, while ZEB2 has been implicated as a major developmental regulator of the nervous system as well as in neural crest cell epithelial-to-mesenchymal transition and migration, recent evidence in our human neural crest cell model indicates a crucial role for ZEB2 in the early formation of the neural plate border and neural crest. ZEB2 is known to negatively regulate TGFb superfamily signaling, whose modulation is essential for proper neural crest formation and differentiation. Therefore, it is hypothesized that ZEB2 functions reiteratively to establish the repressive epigenetic landscape throughout neural crest cell formation and craniofacial development by modulating proper TGFb superfamily signaling, and that the failure of this regulation underscores the MWS pathology. To test this hypothesis, three specific aims are proposed.
Aim 1 will examine the modular functionality of ZEB2 in regulating the proper levels of TGFb signaling throughout wild-type human cranial neural crest cell formation. In particular, the cis-regulatory interactions between ZEB2 and TGFb receptor activated Smad proteins and the functional requirement of co-repressors leading to changes in chromatin accessibility will be delineated.
In Aim 2, the MWS pathology will be addressed through the establishment of induced pluripotent stem cells from MWS patients of diverse genotypes. Using these tools, the precise transcriptomic and epigenetic misregulation during MWS-based neural crest cell formation will be interrogated, providing much needed molecular insight into this understudied pathology.
In Aim 3 of this proposal, the role of ZEB2 in wild-type and MWS human tooth formation will be interrogated through the establishment of a human neural crest cell-based tooth organoid model. These results will reveal for the first time the molecular role of this transcriptional repressor throughout multiple stages of human neural crest cell formation, differentiation, and pathology.
The multipotent neural crest cell population contributes to a wide range of derivatives including most of the craniofacial skeleton and dental tissue, and their aberrant development results in numerous human congenital defects including Mowat-Wilson Syndrome. These experiments will demonstrate precisely how ZEB2 regulates human neural crest cell formation, differentiation, and the Mowat-Wilson Syndrome pathology by coupling pluripotent stem cells with the development of a novel organoid model of human tooth formation. This project is relevant to the mission of NIDCR because the expected results will advance our understanding of the molecular underpinnings of this understudied pathology, and provide a unique platform from which to further explore molecular and environmental influences on human craniofacial and dental development.
