REGULATION OF NEURAL CREST CELL MIGRATION BY SDF1-CXCR4 SIGNALING

University Of Louisville, Louisville KY

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Abstract

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. Project #1: The overall goal of the project is to examine the role of SDF1/CXCR4 signaling in the migration of trunk neural crest cells (NCCs) to the Dorsal Root Ganglion (DRG) and to identify factors that regulate this process. Defective migration of trunk NCCs can cause perinatal lethality and affect the development and formation of the peripheral nervous system. Towards this end, we constructed riboprobes for chicken SDF1 and CXCR4, and utilized them in in situ hybridization (ISH) analyses to determine the spatiotemporal expression of the two genes during embryonic development. The hypothesis being addressed is that trunk NCCs express CXCR4 and migrate towards mesodermal regions of high SDF-1 concentration where they differentiate into the DRG. Chicken embryos (Hamilton-Hamburger (HH) stages ~7-22) were used for the study as their developmental stages have been well defined and are more amenable to analysis and manipulation. Gene expression profiling of SDF1 and CXCR4 expression spanning these developmental stages in the chick embryo has largely been accomplished. Ongoing studies are determining if the expression patterns of CXCR4 and SDF-1 correspond to the developmental paths undertaken by migrating NCCs to become DRG using specific antibodies -- anti-HNK-1 for NCCs and anti-Islet-1 for the DRG. In a parallel study, PC12 cells (derived from the adrenal gland;neural crest origin) are being used in experiments involving transwells and chemotaxis chambers to address possible roles for upstream factors (e.g., TGF[unreadable]1 and BMP4) in regulating NCC migration via SDF1/ CXCR4 signaling, and the effect of this signaling on putative downstream targets, such as IP3 kinase. Project #2: (new): Orofacial clefts such as cleft palate (CP), and cleft lip with or without cleft palate (CL/P), are amongst the most prevalent birth defects in the United States. CP is caused by defects in the development of the secondary palate, which in mice occurs during gestational days (gd) 12-14. Genetic and environmental factors have been implicated in the etiology of CP. Environmentally-induced epigenetic alterations provide a mechanism by which environmental insults could trigger abnormal DNA methylation without a change in DNA sequence. Previous analyses of mRNA profiling during secondary palate development, using Affymetrix GeneChips, coupled with bioinformatic analyses, identified Sox4 as a gene that is potentially regulated by DNA methylation during palate development, and therefore, relevant to the study of orofacial clefting. Sox4 mRNA was found to decrease two-fold from gd12 to gd13/14, indicative of increased methylation during this period. An extensive CpG methylation profile was developed for mouse Sox4 in the 5'upstream region in gd12-14 secondary palates. The resulting analyses indicated a strong association between DNA methylation in the 5'flanking region and decreased mRNA levels seen during gd12-14. Specifically, two CpG residues residing in a Differentially Methylated Region (DMR), which are 60%-70% methylated at gd12, become fully methylated by gd13. Increased methylation of these residues correlates with the decrease in Sox4 mRNA levels during this developmental time-frame. Current studies seek to validate the functionality of these CpG residues using methylation-dependent luciferase expression vectors.