Date Published: May 29, 2012
Publisher: Wiley Subscription Services, Inc., A Wiley Company
Author(s): Scott W Rogers, Petr Tvrdik, Mario R Capecchi, Lorise C Gahring.
Lumbosacral spina bifida is a common debilitating birth defect whose multiple causes are poorly understood. Here, we provide the first genetic delineation of cholinergic nicotinic receptor alpha7 (Chrna7) expression and link the ablation of the Chrna7 cell lineage to this condition in the mouse. Using homologous recombination, an IRES-Cre bi-cistronic cassette was introduced into the 3′ noncoding region of Chrna7 (Chrna7:Cre) for identifying cell lineages expressing this gene. This lineage first appears at embryonic day E9.0 in rhombomeres 3 and 5 of the neural tube and extends to cell subsets in most tissues by E14.5. Ablation of the Chrna7:Cre cell lineage in embryos from crosses with conditionally expressed attenuated diphtheria toxin results in precise developmental defects including omphalocele (89%) and open spina bifida (SB; 80%). We hypothesized that like humans, this defect would be modified by environmental compounds not only folic acid or choline but also nicotine. Prenatal chronic oral nicotine administration substantially worsened the defect to often include the rostral neural tube. In contrast, supplementation of the maternal diet with 2% choline decreased SB prevalence to 38% and dramatically reduced the defect severity. Folic acid supplementation only trended towards a reduced SB frequency. The omphalocele was unaffected by these interventions. These studies identify the Chrna7 cell lineage as participating in posterior neuropore closure and present a novel model of lower SB that can be substantially modified by the prenatal environment. © 2012 Wiley Periodicals, Inc.
Neural tube closure in higher vertebrates involves several complex developmental sequences that, if disrupted, can lead to open neural tube defects (NTD). In humans these are relatively common disorders broadly grouped as rostral or cranial NTD (exencephaly or anencephaly) or caudal NTD (spina bifida; SB). Definition of the genetics and the specific mechanisms leading to these defects is complicated by the interaction between the predisposing genes and environmental processes such as maternal diet or toxin exposure.
We began the examination of how α7 participates in prenatal development by crossing the Chrna7:Cre male mice (Methods and Fig. 1) with females harboring the conditional attenuated diphtheria toxin (DTA) construct, ROSA26-loxP(DTA). Pups from these crosses were stillborn (Fig. 2A) with several developmental abnormalities. Overall they had a grossly normal body but they often exhibited a hunched posture, flat head and abnormally proportioned limbs (especially lengthened hind limbs). Most evident was protrusion of abdominal organs (liver and intestines) suggestive of an omphalocele (Fig. 2A). Also prominent was the defect of open spina bifida (SB) in nine of 13 pups from three litters (Fig. 2A–C). This defect was always accompanied by a tail deformity reminiscent of curly tail [van Straaten and Copp, 2001; Ting et al., 2003]. We next examined prenatal litters at embryonic day 16.5 (E16.5). The open SB defect and omphalocele were evident (Fig. 2C,D) although neither defect was fully penetrant (Table I; see van Straaten and Copp ; and Harris and Juriloff, ). Also, careful removal of embryos at this stage reveals the abdominal wall defect to include peritoneal membrane enclosing the extruded liver and intestines that is characteristic of omphalocele (Fig. 2E). Additional developmental deficiencies included irregularities in the retinal pigmented epithelium and a severe reduction in dorsal root ganglion sensory neurons (not shown). Craniofacial malformations were rare and generally restricted to the mandible, which was shortened. Hematopoiesis was present as indicated by red blood cells although as many as 30% of the embryos in some litters could be anemic. RNA levels for other nAChR subunits (α2–α6 and β2–β4, respectively) were not altered relative to controls as might be expected if there were significant compensatory mechanisms or if other receptor subtypes contributed to this phenotype (not shown). The location of the embryos in the placental order also failed to correspond with the defect penetrance or severity (not shown).
We have used homologous recombination to modify the nicotinic acetylcholine receptor alpha7 (Chrna7:Cre) transcript to provide a flexible approach for examining the temporal and spatial expression of this receptor in the developing mouse. The expression of Chrna7:Cre is detected first in rhombomeres 3 and 5 of the E9.0 mouse embryo and thereafter it extends to include subsets of cells located in tissues throughout the embryo. A notable developmental defect resulting from the conditional DTA ablation of the Chrna7:Cre cell lineage is a precise caudal NTD consistent with open spina bifida (SB). The prevalence and severity of this defect is responsive to dietary supplementation with agents that also affect SB frequency in humans. Also evident is the prominent omphalocele, possibly resulting from disproportionately large organs. Thus processes in this mouse leading to the SB and omphalocele defects are mechanistically separable as demonstrated by the differing response of SB to oral nicotine and choline treatment in the absence of a change in the frequency of omphalocele (Fig. 5 and Table I).