Neural crest cells (NCCs) are a population of highly migratory stem-like cells that have unique characteristics including the ability to undergo an epithelial-to-mesenchymal transition (EMT), migrate extensively throughout the embryo and differentiate into specific derivatives. Defects in neural crest migration can be one of the underlying causes of several human congenital birth defects, termed neurocristopathies including several craniofacial syndromes including DiGeorge and Treacher Collins syndromes. We have identified a new role for the zebrafish (Danio rerio) cell adhesion protein, protocadherin10a (pcdh10a) in which knockdown of pcdh10a causes specific defects in NCC migration. My hypothesis is that Pcdh10a functions in a subset of NCCs to regulate cell migration by the regulation of F-actin distribution. To test this, I will complete the following specific aims:. 1) Determine the cellular localization in the subpopulations of NCCs that require protocadherin10a. I will use immunofluorescence and fluorescent in-situ hybridization to identify exactly which populations of NCCs are expressing pcdh10a and what part of the cell Pcdh10a is localized to in order to determine how it may be regulating cell migration; 2) Investigate pcdh10a requirement in migratory NCCs in regulating F-actin mediated cell motility and cell adhesion. I will determine how F-actin is regulated by pcdh10a by a gain-of-function and loss-of-function approaches. For this experiment I will image fixed samples with the phalloidin, along with live cell imaging using actin binding proteins LifeAct and UtrCH fused to RFP or GFP. This will allow me to determine how F-actin is regulated dynamically during NCC migration in live zebrafish embryos; and 3) Determine if zebrafish Pcdh10a directly interacts with Nap1 and WAVE to regulate cell motility and cell adhesion. Here, I will determine if Pcdh10a binds with any homologues of known proteins that interact with Pcdh10 in mouse, such as proteins of the Nap-1/WAVE complex, which regulates actin. Determining what proteins Pcdh10a is interacting with will allow me to make more specific hypotheses about its function and role in neural crest migration. Together these data will determine a novel role for protocadherins mediating NC cell migration.
Neural crest defects are implicated in multiple human syndromes resulting in cleft lip and palate in approximately 6800 live births in the United States alone (March of Dimes, 2010). Understanding the genes involved in neural crest migration during embryological development is critical to the understanding of both normal and defective craniofacial development, as well as for expanding potential therapies and treatments for craniofacial disorders.
| Williams, Jason S; Hsu, Jessica Y; Rossi, Christy Cortez et al. (2018) Requirement of zebrafish pcdh10a and pcdh10b in melanocyte precursor migration. Dev Biol : |
