Cart1 Knockout

Gene targeting has been used to generate mice with a null mutation in Cartl, a homeobox-containing gene that encodes the transcription factor, cartilage homeoprotein 1 (16,17). Homozygous Cartl mutant mice develop cranial NTD and die shortly after birth. The penetrance is influenced by genetic background with a maximum NTD incidence of 100% on a 129/ SvEv strain background (17). Cranial NTD result from failure of closure at the prospective forebrain/midbrain boundary, so called Closure 2. Failure of neural tube closure is thought to be the result of a reduction in the number of mesenchymal cells in the forebrain of homozygous mutant embryos at embryonic day 9 (E9). This deficit appears to result from an increase in cell death and correlates with the forebrain-specific expression of Cart1 in the cranial mesenchyme. Cart1 expression is absent from the E9 midbrain mesenchyme, which is histologically normal. Interestingly, however, the neural folds do not close in the midbrain, raising the possibility either of undetec ted, low-level Cartl expression in the midbrain or of an influence from neighboring brain regions.

The deleterious effect of the Cartl mutation is greatly reduced by folic acid supplementation. Treatment by intraperitoneal injection during the first half of gestation reduced the incidence of NTD by approx 60% (17), although all the rescued mice died shortly after birth, indicating that there are residual defects that are not prevented by folic acid. Because treated litters were not examined during development, it is unclear whether folic acid treatment corrects the deficiency in forebrain mesenchyme. Folic acid may act to correct an abnormality of folate metabolism caused by loss of Cartl function. Alternatively, the protective effect may be unrelated to the underlying defect. For example, if Cartl deficiency causes mesenchymal cells to die, NTD could be prevented if folic acid serves to stimulate proliferation of the remaining mesenchymal cells.

Crooked Tail

Heterozygous crooked tail (Cd) mutant mice exhibit characteristic tail defects. Homozygotes display a range of phenotypes that include early embryonic lethality and exencephaly, whereas remaining mice are small in size and have vertebral skeletal defects (18,19). The risk of exencephaly is approximately twice as high in females as in males, mimicking the female preponderance among anencephalics seen in humans (20). The Cd gene has been mapped to mouse chromosome 6 but remains to be identified (19).

Prenatal dietary supplementation with folic acid causes a reduction in the percentage of affected Cd embryos and provides the first model for a dietary response to folic acid (19). A reduction in the risk of exencephaly has been reported for folate-controlled diets containing 0 mg/kg (folate free) and 10 mg/kg compared with a baseline 4 mg/kg folic acid treatment. One possible explanation for this surprising result is that at 0 mg/kg, the majority of exencephalic embryos die early in gestation, yielding a low NTD frequency, whereas at 4 mg/kg, most embryos are rescued from early lethality but still develop exencephaly, yielding an apparently higher NTD frequency. Because parallel data were not reported for control embryos, it is not clear whether Cd homozygotes are particularly prone to die in folic-acid-defi-cient conditions. Cd may provide a useful model for NTD in humans and it will be of interest to determine whether there is an underlying defect in folate uptake or metabolism. In addition, because embryos in the initial study were collected at E12.5-14.5, it is important to test whether "rescued" embryos are viable postnatally and whether less severe defects may persist after folate treatment.

Folic-Acid-Binding Protein Knockout

Knockout mice have been generated for folic-acid-binding proteins, folbp1 and folbp2, the murine homologs of human folate receptors a and p, respectively (9,21,22). Folbp1 is a high-affinity integral membrane receptor responsible for transport of folate into the cytoplasm, whereas folbp2 is a low-affinity, glycosylphosphatidylinositol (GPI)-anchored receptor (23). The genes encoding both proteins are expressed embryonically, suggesting that they could be involved in mediating the effect of folic acid during development (24).

Homozygous null embryos for folbp1 are severely growth retarded, fail to complete axial rotation, and die in utero, whereas folbp2 null mice are viable (22). The specific effect of folbp1 absence on neural tube closure cannot be determined from this study, as null embryos do not develop to a morphological stage at which the neural tube should have closed. It is clear, however, that folbp1 is essential for normal development, probably to maintain sufficient cytoplasmic folate levels to meet metabolic requirements. The uptake of folate may also be suboptimal, but sufficient for normal development, in folbp2 mutants and folbp1 and folbp2 heterozygotes. For example, although mice of these genotypes are apparently phenotypically normal, nonpregnant mice do show an abnormal response to folate deficiency. A folic-acid-deficient diet causes an increase in plasma homocysteine in wildtype mice, but this increase is significantly greater in mutants. Because elevated homocysteine is a risk factor for NTD, the offspring of such mice could be susceptible to develop NTD, perhaps in the presence of additional genetic or environmental factors. Therefore, it may be revealing to generate compound mutants of folbp knockouts with other folate-related mouse mutants.

Oral supplementation of heterozygous folbp1 females with folinic acid prior to and during pregnancy led to the survival to late gestation of some homozygous offspring (22). Litters were not collected until E18, when non-viable embryos would have been resorbed, so the proportion of rescued embryos could not be measured. Neither was the viability of rescued mice determined. Although the surviving folbp1 null homozygotes in this study do not exhibit NTD, it is not clear whether this represents the prevention of NTD by folinic acid or prolongation of survival, enabling otherwise normal neural tube closure to progress to completion.


Splotch mutant mice (Sp) are so called owing to the characteristic white belly spot of heterozygotes, which results from a neural-crest-related pig mentation defect (25). Homozygous mutants exhibit a range of neural-crest-related abnormalities, limb muscle defects, and neural tube defects, the latter comprising both exencephaly and spina bifida.

A proportion of homozygous embryos die around E14 as a result of heart defects (25,26). The Sp and Sp2H mutant alleles cause similar phenotypes and encode defective copies of the Pax3 gene (27-29). Pax3 is a transcription factor containing both paired-box and homeobox DNA-binding motifs. It is expressed in the dorsal neural tube, migrating neural crest, and der-momyotomal cells (30-32).

Mutations in the human PAX3 gene are found in Waardenburg syndromes types I and III in which there is a characteristic pigmentation defect (33,34). The occurrence of isolated NTD in heterozygous Waardenburg patients and in a suspected homozygous case shows that the splotch mouse may provide a useful model for NTD (35,36). However, PAX3 mutations do not appear to contribute directly to a large proportion of human NTD (37,38). Interestingly however, it has recently been shown that the PAX3 protein may be implicated in human DiGeorge syndrome, through binding to the candidate HIRA protein (39). This emphasizes the general principle that genes may be involved in human developmental defects in ways other than by direct mutation. In this case, misregulation of PAX3 binding may contribute to the neural crest phenotype of DiGeorge syndrome.

Splotch provides the first mouse model in which NTD are preventable by folic acid in association with a demonstrable abnormality of embryonic folate metabolism (15). Abnormal folate metabolism was detected in whole-embryo culture using the deoxyuridine (dU) suppression test. Incorporation of [3H]thymidine into DNA is suppressed by exogenous dUMP owing to the activation of thymidylate synthase that catalyzes the de novo synthesis of dTMP (Fig. 1, reaction 2) from dUMP and 5,10-methylene tetrahydrofolate (5,10-MeTHF). The degree of suppression by dUMP is diminished if folate metabolism is compromised, because the supply of 5,10-MeTHF then becomes limiting. For instance, the application of folate cycle inhibitors leads to diminished dU suppression in mouse embryos (15). Two abnormalities of the dU test are observed in homozygous splotch embryos (15). First, in the absence of exogenous dUMP there is an increased incorporation of [3H]thymidine in splotch homozygotes compared with wild-type embryos. Heterozygotes exhibit an intermediate level of [3H]thymidine incorporation. Second, the extent of suppression is significantly diminished in homozygotes compared with heterozygotes and wild-type embryos. These observations indicate that the supply of 5,10-MeTHF is insufficient to meet the developmental requirements of splotch embryos.

Treatment with folic acid reduces the incidence of cranial and spinal NTD both in embryo culture and following maternal treatment by intraperitoneal injection (15). There is also a corresponding normalisation of the excessive [3H]thymidine incorporation in splotch embryos following culture in the presence of folic acid. Therefore, in this model, prevention of NTD by folic acid appears to be associated with the correction of an underlying abnormality of folate metabolism (15,40).

A proportion of heterozygous splotch embryos, that normally complete cranial tube closure successfully, exhibits cranial NTD following methion-ine treatment in embryo culture. This apparent increase in the penetrance of the splotch defect is also associated with a further increase in the incorporation of thymidine, suggesting that methionine exacerbates the underlying folate abnormality (15). As described earlier, Axd mutants also exhibit occasional exencephaly following maternal methionine treatment, although methionine was curative with respect to the spinal defect (13). Methionine may, therefore, have a general inhibitory effect on cranial neural tube closure, which then causes NTD in predisposed embryos. Such an effect could be mediated through suppression of the folate cycle. For example, the presence of excess methionine inhibits synthesis of thymidylate (41), which is required for DNA synthesis.

Getting Back Into Shape After The Pregnancy

Getting Back Into Shape After The Pregnancy

Once your pregnancy is over and done with, your baby is happily in your arms, and youre headed back home from the hospital, youll begin to realize that things have only just begun. Over the next few days, weeks, and months, youre going to increasingly notice that your entire life has changed in more ways than you could ever imagine.

Get My Free Ebook

Post a comment