Characterization of chromosomal inversion of the mouse hairy ears (Eh) mutation associated with cleft palate

Abstract

The hairy ears (Eh) mutation in the mouse originated from neutron irradiation experiments and is associated with chromosomal inversion on chromosome 15. Eh/+ mice have small pinna and extra hairs on the pinna but the phenotypic features of Eh/Eh mice are unclear. In this study we found that Eh/Eh mice died shortly after birth and had a cleft palate caused by impaired growth of palate shelves. Because genes located on the breakpoints of inversion are likely to be responsible for the defects associated with chromosomal inversions, we determined the breakpoints of the Eh inversion. We used a new genetic method that uses recombinant chromosomes resulting from crossing over between two overlapping inversions to determine the breakpoints. Koa is a mouse mutation associated with inversion of chromosome 15, which partially overlaps with the Eh inversion. We made Eh +/+ Koa double heterozygotes and obtained the recombinant chromosomes possessing deletion and duplication of the regions flanked by the breakpoints of both inversions, which were generated by crossing over within the overlapped region of these inversions. By defining the deleted regions we identified the breakpoints of the Eh inversion. We then examined the expression of genes in the vicinities of the breakpoints and found ectopic expression of the Hoxc5 gene and a transcript with unknown function in the developing palate of Eh/Eh mice, which is likely to be responsible for the cleft palate.

A spontaneous mutation of the Wwox gene and audiogenic seizures in rats with lethal dwarfism and epilepsy

“…The products of the third nested PCR were cloned into pGEM‐T Easy vector (Promega, Madison, WI, USA) and sequenced using an ABI Prism 310 Genetic Analyzer and BigDye terminator v3.1 (Applied Biosystems, Foster City, CA, USA). For 3’‐RACE analysis, cDNA was synthesized using a 3'RACE oligo‐dT primer (Katayama et al 2007). The first PCR was performed with a 3'RACE adaptor and Wwox‐3 F, and the second PCR with a 3'RACE adaptor and 3'RACE TSP 1.…”

Homeobox family Hoxc localization during murine palate formation

“…Moreover, Hoxc5 positive cells were noticeably increased at E14.5 ( P < 0.001) in Eh / Eh mice. Previously it has been demonstrated that strong expression of the Hoxc5 gene is detected in the palates of Eh / Eh mice, while no Hoxc5 expression is detected in +/+ mice at this stage, suggesting that the elevated expression of Hoxc5 is likely to be responsible for the cleft palate in Eh / Eh mice (Katayama et al ). Our data showed that the distribution of Hoxc5 tend to localize in epithelial cells during palate formation in the palatal shelves of +/+.…”

“…Additionally, the palatal shelves do not elevate in the Hoxa2 knockout mouse, suggesting that the absence of Hoxa2 induces an increase in mesenchymal cell proliferation, which alters the palate growth pattern and affects palatal elevation (Smith et al ). However, in Eh / Eh mice, no significant differences in cellular proliferation or apoptosis (Katayama et al ) in the palatal shelves were observed; therefore, the cleft in Eh / Eh is not likely due to decreased cell proliferation or increased cell death (Katayama et al ). In this study, no difference was observed in the PCNA‐positive cells; moreover, it appeared that PCNA distribution patterns were similar to those of Hoxc5, though it remains unclear whether Hoxc5 could be involved in proliferation and differentiation of the cells of the palatal shelves.…”

“…The distal breakpoint is located at 8 kb telomeric to the HoxC gene cluster and does not result in gene disruption, suggesting that the developmental defects of Eh / Eh mice, including cleft palate, are caused by altered expression of genes in the vicinities of the breakpoints. It is also reported that strong expression of the Hoxc5 gene in the developing palate is likely to be responsible for the cleft palate observed in Eh / Eh mice (Katayama et al ). However, the distribution pattern of Hoxc5, Hoxc4, and Hoxc6 during palate formation has been so far reported.…”

“…Eh heterozygotes ( Eh /+) have small pinna with extra hair on the pinna; in contrast, Eh homozygotes ( Eh / Eh ) are reported to be embryonic lethal (Bangham, ). Recently, the breakpoints of the Eh inversion were determined using a genetic method that uses recombinant chromosomes resulting from crossing over between two overlapping inversions (Katayama et al ). The distal breakpoint is located at 8 kb telomeric to the HoxC gene cluster and does not result in gene disruption, suggesting that the developmental defects of Eh / Eh mice, including cleft palate, are caused by altered expression of genes in the vicinities of the breakpoints.…”