Neural crest and mesoderm lineage-dependent gene expression in orofacial development.

The present study utilizes a combination of genetic labeling/selective isolation of pluripotent embryonic progenitor cells, and oligonucleotide-based microarray technology, to delineate and compare the "molecular fingerprint" of two mesenchymal cell populations from distinct lineages in the developing embryonic orofacial region. The first branchial arches-bi-lateral tissue primordia that flank the primitive oral cavity-are populated by pluripotent mesenchymal cells from two different lineages: neural crest (neuroectoderm)- and mesoderm-derived mesenchymal cells. These cells give rise to all of the connective tissue elements (bone, cartilage, smooth and skeletal muscle, dentin) of the orofacial region (maxillary and mandibular portion), as well as neurons and glia associated with the cranial ganglia, among other tissues. In the present study, neural crest- and mesoderm-derived mesenchymal cells were selectively isolated from the first branchial arch of gestational day 9.5 mouse embryos using laser capture microdissection (LCM). The two different embryonic cell lineages were distinguished through utilization of a novel two component transgenic mouse model (Wnt1Cre/ZEG) in which the neural crest cells and their derivatives are indelibly marked (i.e., expressing enhanced green fluorescent protein, EGFP) throughout the pre- and post-natal lifespan of the organism. EGFP-labeled neural crest-derived, and non-fluorescent mesoderm-derived mesenchymal cells from the first branchial arch were visualized in frozen tissue sections from gestational day 9.5 mouse embryos and independently isolated by LCM under epifluorescence optics. RNA was extracted from the two populations of LCM-procured cells, and amplified by double-stranded cDNA synthesis and in vitro transcription. Gene expression profiles of the two progenitor cell populations were generated via hybridization of the cell-type specific cRNA samples to oligo-based GeneChip microarrays. Comparison of gene expression profiles of neural crest- and mesoderm-derived mesenchymal cells from the first branchial arch revealed over 140 genes that exhibited statistically significant differential levels of expression. The gene products of many of these differentially expressed genes have previously been linked to the development of mesoderm- or neural crest-derived tissues in the embryo. Interestingly, however, hitherto uncharacterized coding sequences with highly significant differences in expression between the two embryonic progenitor cell types were also identified. These lineage-dependent mesenchymal cell molecular fingerprints offer the opportunity to elucidate additional mechanisms governing cellular growth, differentiation, and morphogenesis of the embryonic orofacial region. The chemokine stromal cell-derived factor 1, (SDF-1), was found to exhibit greater expression in mesoderm-derived mesenchyme in the branchial arch when compared with neurectoderm, suggesting a possible chemotactic role for SDF-1 in guiding the migratory neural crest cells to their destination. The novel combination of genetic labeling of the neural crest cell population by EGFP coupled with isolation of cells by LCM for gene expression analysis has enabled, for the first time, the generation of gene expression profiles of distinct embryonic cell lineages.

Fluorescence-activated cell sorting of EGFP-labeled neural crest cells from murine embryonic craniofacial tissue.

During the early stages of embryogenesis, pluripotent neural crest cells (NCC) are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt 1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt 1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR). The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development.