Delayed neural crest cell emigration from Sp and Sp^d mouse neural tube explants

Abstract

Splotch (Sp) and splotch-delayed (Spd) are allelic mutations on chromosome 1 of the mouse. Embryos homozygous for either allele have neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derived structures. The fact that Spd mouse mutants sometimes have deficiencies in NCC derivatives in the absence of an NTD led to the hypothesis that neurulation and the release of NCCs may depend on a regulatory event that is common to both processes. Therefore, it may be possible to understand the cause of NTDs in these mutants by examining the basis of aberrant NCC derivatives. Caudal neural tubes were excised from day 9 Sp and Spd embryos and placed into gelatin-coated tissue culture dishes, or 3-dimensional basement membrane matrigel, and cultured for 72 hours. A cytogenetic marker was used to genotype the embryos. In planar cultures, no morphological differences were observed between NCCs from neural tube explants of Spd mutants compared to those from heterozygous or wild-type embryos. However, there appeared to be a delay in the release of NCCs from the neural tube in both Sp and Spd mutants, which was particularly evident in Sp. After 24 hours in culture, the extent of NCC outgrowth, as well as the number of NCCs emigrating from explanted neural tubes, was significantly lower in Sp and Spd mutant cultures than in controls. No differences were observed in the mitotic indices among cells which had emigrated. By 72 hours, mutant cultures and their non-mutant counterparts were similar in terms of outgrowth, cell number, and migratory capability. After 24 hours in 3-dimensional basement membrane matrigel, cell outgrowth from Sp explants was also significantly less than controls. The pattern of NCC outgrowth in both types of culture conditions indicates a 24 hour delay in mutant cultures compared to controls. This stems from a delay in the release of NCCs from the neural tube, suggesting that the defect lies within the neuroepithelium with respect to the release of NCCs.

Signals from the notochord and floor plate regulate the region-specific expression of two Pax genes in the developing spinal cord

“…However, it is unclear whether changes to DV patterning underly these defects in the spinal cord. The differences in the migration of neural crest cells in vitro that have been described may be in part responsible for the loss of melanocytes and other neural crest-derived cell types in Pax gene expression in the developing chick spinal cord splotch mice (Moase and Trasler, 1990). Since little is known of the changes that occur at the cellular level in either mutant, further analysis of both mutants will be required to ascertain the role, if any, that both genes play in the DV patterning of the spinal cord.…”

Pax genes: regulators of lineage specification and progenitor cell maintenance

“…As PSM cells migrate along established β-neuregulin 1-secreting axon tracts, their maintenance is regulated by Sox2, Sox10 (Britsch et al, 2001;Le et al, 2005) and Pax3 (Blanchard et al, 1996;Kioussi et al, 1995). Pax3 is a known survival factor for PSM cells during this mitotic and chemotactic stage; when homozygous Splotch mice perish at E13.5, PSM cells cannot be detected (Franz, 1990) and mice homozygous for the Splotch-delayed allele (Table 2) (which survive until E18.5) contain few PSM cells, all of which perish by E15.5 (Moase and Trasler, 1990).…”

Endogenous bone morphogenetic protein antagonists regulate mammalian neural crest generation and survival

“…Explants were photographed and measured daily, for 4 days. The migration rate of cultured NCCs was gauged by calculation of a "migratory index" for each sample (Moase and Trasler, 1990;Huang et al, 1998). Here, overall outgrowth area per unit of neural explant perimeter was calculated daily for 3 days and was normalized at the start of differential protein treatment (day 2).…”