Crooked Tail(Cd) Models Human Folate-Responsive Neural Tube Defects

Genetic correlation of human neural tube defects(NTDs) with NTD genes identified in mouse mayunravel predisposing complex traits for assessment ofindividual risk and treatment in clinical settings. Folicacid (FA) can reduce the recurrence of NTDs in humanpopulations by as much as 50–70%, though themechanism of this rescue is unknown. We examinedwhether Crooked tail (Cd), a mouse strain prone toexencephaly, could provide a genetic animal model forfolate-responsive NTDs. The Cdlocus was localized toa 0.2 cM interval of the Mouse Genome Databasegenetic map, identifying tightly linked markers forgenotyping prior to phenotypic expression. In acontrolled diet study, Cdwas found to mimic closelythe clinical response to FA. FA supplementationreduced the recurrence risk of Cdexencephaly by asmuch as 55%. Thisrescue was dosedependent anddidnot require subjects to be inherently folate deficient.Like the female predominance of NTDs in humans,female Cd embryos were most likely to displayexencephaly and were more responsive than males totheFA rescue. Importantly, FA supplementation shiftedthe severity of Cd phenotypic expression from earlyembryonic lethality to longer survival, and reduced theincidence of NTDs. The Cdlocusisdistinctfromtheknown genes associated with neurulation defects, andisolation of this gene will assist identification ofbiochemical, genetic and gender-dependent factorscontributing to folate-responsive NTDs.INTRODUCTIONNeural tube defects (NTDs) are a leading cause of infantmortality and developmental disability, occurring in 0.5–8 per1000 live births (1), though underestimation is likely due togeographical variations in reporting of prenatal losses (2). Avariety of teratogens, folic acid (FA) status and genetic suscep-tibility factors are associated with NTDs (3,4). Human studieshave identified dietary FA as capable of reducing NTD recur-rence by 50–70% (5,6). However, the mechanism of thisrescue and the FA-responsive factors leading to NTDs areunknown, largely due to a paucity of appropriate animalmodels for study. Whether FA acts to promote neurulation ordecreases viability of NTD-susceptible embryos, or both, is notknown (7). We have begun to positionally clone theCrookedtail (Cd) mouse locus, a spontaneously arising mutation thatproduces a crooked tail in heterozygotes and an increased inci-dence of exencephaly among homozygotes (8). For mice andrats, exencephaly is the more common presentation of failedneural tube closure in the head region and is most closelyrelated to anencephaly in humans (9–11). In this report,linkage analysis of an intraspecies backcross placed Cd withina 0.2 cM interval on the Mouse Genome Database (MGD)(12,13) map of chromosome 6. Tightly linked markers identi-fying Cd homozygotes permitted an extensive dietary supple-mentation study to determine how closely Cd mimics humanNTDs in response to FA. The parallels between Cd and humanstudies were striking and suggest that Cd is an importantbiological model for understanding the implications of clinicalobservations in NTDs. Therefore, positional cloning ofCd andits modifier loci will help to elucidate the molecular pathwaysassociated with folate-responsive NTDs arising in humanpopulations.RESULTSFeatures of the Cd/Cd exencephalic fetus help to define appro-priate expression patterns for candidate genes. The mammalianneural tube closes in a zipper-like fashion, extending sequen-tially from four primary closure points (14). In Cd exen-cephalic embryos (Fig. 1), the neural folds in themesencephalon, closure point 2, remain separated in themidline and splay over the skull base. Cd exencephalicssurviving to the fetal period are of normal size, and the orbitsand lower cranial structures are preserved (Fig. 1B). In addi-tion, the digits are normally formed and, unlikeSplotch NTDmice, there is no hypoplasia of limb muscles. Vascular mark-ings outside of the exencephalic brain appear normal. Sagittalhistological sections are also consistent with failure primarilyof closure point 2 (Fig. 1D). TheCd/Cd mesencephalon is mostdistorted, pushed up and over the cortex, with markedlyatrophic tectum. Cerebellum is relatively well preserved.Therefore, genes expressed predominantly in the mesencephalon