Laser capture microdissection of fluorescently labeled embryonic cranial neural crest cells

Abstract

This study is the first to report a unique genetic strategy to permanently label mammalian neural crest cells (NCC) with a fluorescent marker, selectively isolate the labeled NCC or their derivatives during murine ontogenesis by laser capture microdissection (LCM), and prepare molecular components, such as RNA, for selective gene expression analyses. Through utilization of a Cre recombinase/loxP system, a genetic strategy that has been used repeatedly to achieve tissue-specific activation of reporter transgenes in mice, a novel two-component mouse model was created in which neural crest cells (and their progeny) are indelibly marked throughout the pre- and postnatal lifespan of the organism. To generate this mouse model, a Wnt1-Cre transgenic line was crossed with a mouse line expressing a conditional reporter transgene ("floxed" enhanced green fluorescent protein). Resulting offspring, expressing both the Wnt1-Cre and "floxed" EGFP alleles, demonstrated EGFP expression in the NCC and all of their derivatives throughout embryonic, postnatal, and adult stages. In the present study, EGFP-labeled cranial NCC from the first branchial arch of gestational day 9.5 murine embryos were visualized in frozen tissue sections and isolated by LCM under epifluorescence optics. RNA was extracted from "captured" cells and amplified by double-stranded cDNA synthesis and in vitro transcription. Amplified mRNA samples from "captured" cells were evaluated by TaqMan quantitative, real-time PCR for the expression of a panel of NCC gene markers. The molecular genetic strategy delineated in this report will facilitate future embryo-genomic and -proteomic analyses of mammalian NCC that will serve to further our understanding of these pluripotent embryonic progenitor cells.

Molecular profiles of mitogen activated protein kinase signaling pathways in orofacial development

“…These may modulate the proliferative response of MEMM cells to EGF , which stimulates MAPK phosphorylation and activity in these cells (Bhattacherjee et al, 2004). Thus, it is reasonable to surmise that a convergence between the TGFb, cAMP, and/or EGF signaling pathways exists in embryonic orofacial tissue.…”

Customizing microarrays for neuroscience drug discovery

“…Laser microdissection (LCM) [75] , which combines advanced light microscopy with laser excision, provides an exceptionally high level of resolution and precision in obtaining tissue or cells from particular layers, cells, single cells or subcellular regions [76] . When combined with prior immunohistochemistry [77] or fluorescent marker transgenic animals [78] , LCM can enable gene expression studies in specific cellular phenotypes of interest. Low yields of RNA from single cells can be overcome by RNA amplification [79] .…”

Laser‐assisted microdissection (LAM) in developmental biology

“…An elegant two-component mouse model in combination with LCM has been described by Bhattacherjee et al (2004) which allowed in situ lineage analysis of the mammalian neural crest cells (NCC). A Wnt1-Cre transgenic line was crossed with a mouse line expressing a conditional reporter transgene enhanced green fluorescent protein (EGFP).…”