Early morphological abnormalities in splotch mouse embryos and predisposition to gene- and retinoic acid-induced neural tube defects

Genetic and environmental factors contribute to an individual's neural tube defect liability. In the mouse, the gene mutation Splotch (Sp) causes a pigmentation defect in heterozygotes while homozygotes have spina bifida +/- exencephaly. Splotch homozygotes, heterozygotes, and wild-type embryos were examined for somite number, anterior neuropore closure, and posterior neuropore length. The aim was to distinguish potentially affected homozygotes early in pathogenesis and find a morphological basis for increased teratogen susceptibility in heterozygotes. Posterior neuropore closure as well as anterior neuropore closure was significantly delayed in potentially affected Sp as compared to wild-type litter embryos exceeding the incidence found in day-10-diagnosed homozygotes. Part of this excess was attributed to a transient delay in heterozygotes which in turn might predispose to retinoic acid-induced neural tube defects. This idea was supported by an outcross of Sp heterozygote males by inbred SWV females and wild-type males by SWV where a significant increase in retinoic acid-induced neural tube defects was found in Sp carrier litters.     

Studies of the effect of retinoic acid on anterior neural tube closure in mice genetically liable to exencephaly

Previously we have shown that all SELH/Bc mouse embryos close their anterior neural tubes by an abnormal mechanism and that 10-20% of SELH/Bc embryos are exencephalic. The purposes of these studies were (1) to observe the effects of retinoic acid on the frequency of exencephaly in SELH/Bc embryos; (2) to compare the SELH/Bc response with those of normal strains and of other neural tube mutants; and (3) to compare, between SELH/Bc and a normal strain (SWV/Bc), the effects of retinoic acid on morphology of the closing anterior neural tube. SELH/Bc was more liable to retinoic acid-induced exencephaly than were normal strains. After maternal treatment with 5 mg/kg retinoic acid on day 8.5 of gestation, 53% of SELH/Bc embryos had exencephaly, compared with 22% in ICR/Bc and 14% in SWV/Bc. When these results were transformed according to the assumptions of the developmental threshold model, the effects of genotype and retinoic acid appeared to be additive. Similar treatment on day 9 or 10 of gestation had little or no effect on the frequency of exencephaly in SELH/Bc mice. These results are similar to the reported responses of the curly-tail and Splotch mutants, where frequencies of spina bifida but not exencephaly were decreased. This pattern suggests that studies of effects of periconceptional vitamin treatment on risk of human neural tube defects should consider anencephaly and spina bifida separately. The study comparing the morphology of anterior neural tube closure in SELH/Bc and normal SWV/Bc embryos showed that retinoic acid delays the elevation of the mesencephalic neural folds. This results in a "stalling" of many embryos in the first steps of neural tube closure, with their neural folds remaining convex and splayed wide apart. The delay in fold elevation was superimposed on the different closure patterns of the two strains. The overall conclusion is that there is no nonadditive interaction in the parameters studied between retinoic acid treatment and the SELH/Bc genotype.     

Mini-review: toward understanding mechanisms of genetic neural tube defects in mice.

We review the data from studies of mouse mutants that lend insight to the mechanisms that lead to neural tube defects (NTDs). Most of the 50 single-gene mutations that cause neural tube defects (NTDs) in mice also cause severe embryonic-lethal syndromes, in which exencephaly is a nonspecific feature. In a few mutants (e.g., Trp53, Macs, Mlp or Sp), other defects may be present, but affected fetuses can survive to birth. Multifactorial genetic causes, as are present in the curly tail stock (15-20% spina bifida), or the SELH/Bc strain (15-20% exencephaly), lead to nonsyndromic NTDs. The mutations indicate that "spina bifida occulta," a dorsal gap in the vertebral arches over an intact neural tube, is usually genetically and developmentally unrelated to exencephaly or "spina bifida" (aperta). Almost all exencephaly or spina bifida aperta of genetic origin is caused by failure of neural fold elevation. The developmental mechanisms in genetic NTDs are considered in terms of distinct rostro-caudal zones along the neural folds that likely differ in mechanism of elevation. Failure of elevation leads to: split face (zone A), exencephaly (zone B), rachischisis (all of zone D), or spina bifida (caudal zone D). The developmental mechanisms leading to these genetic NTDs are heterogeneous, even within one zone. At the tissue level, the mutants show that the mechanism of failure of elevation can involve, e.g., (1) slow growth of adjacent tethered tissue (curly tail), (2) defective forebrain mesenchyme (Cart1 or twist), (3) defective basal lamina in surface ectoderm (Lama5), (4) excessive breadth of floorplate and notochord (Lp), (5) abnormal neuroepithelium (Apob, Sp, Tcfap2a), (6) morphological deformation of neural folds (jmj), (7) abnormal neuroepithelial and neural crest cell gap-junction communication (Gja1), or (8) incomplete compensation for a defective step in the elevation sequence (SELH/Bc). At the biochemical level, mutants suggest involvement of: (1) faulty regulation of apoptosis (Trp53 or p300), (2) premature differentiation (Hes1), (3) disruption of actin function (Macs or Mlp), (4) abnormal telomerase complex (Terc), or (5) faulty pyrimidine synthesis (Sp). The NTD preventative effect of maternal dietary supplementation is also heterogeneous, as demonstrated by: (1) methionine (Axd), (2) folic acid or thymidine (Sp), or (3) inositol (curly tail). The heterogeneity of mechanism of mouse NTDs suggests that human NTDs, including the common nonsyndromic anencephaly or spina bifida, may also reflect a variety of genetically caused defects in developmental mechanisms normally responsible for elevation of the neural folds.     

<Emphasis Type="Italic">Pax1/E2a</Emphasis> Double-Mutant Mice Develop Non-Lethal Neural Tube Defects that Resemble Human Malformations

Many mouse models exist for neural tube defects (NTDs), but only few of them are relevant for human patients that are born alive with spina bifida aperta. NTDs in humans show a complex inheritance, which most likely result from the involvement of a variety of predisposing genetic and environmental factors. Hints toward the identity of predisposing genetic factors for human NTDs could come from mouse studies on the development of the neural tube and spinal cord, as well as from studies on associated features of this type of diseases. Among such features is the observation that pregnancies affected by a neural tube defect frequently show changes in thymus morphology, and in both neonatal and maternal T-cell repertoire. The genes for E2a and Pax1 have both been implicated in not only paraxial mesodermal development, but also in that of the immune system. Moreover, Pax1 mutant mice have been shown to display NTDs in digenic mouse models. In the present study we have investigated the phenotype of E2a null mutant mice that are also heterozygous for the so-called undulated mutation in Pax1. Here we report that such double-mutant mice develop a non-lethal NTD that strongly resembles the classic human NTD: spina bifida aperta, associated with defects of the axial skeleton, immune system and urinary tract.     

Transgenic mice expressing PAX3-FKHR have multiple defects in muscle development, including ectopic skeletal myogenesis in the developing neural tube

The t(2;13) chromosomal translocation is found in the majority of human alveolar rhabdomyosarcomas (RMS). The resulting PAX3-FKHR fusion protein contains PAX3 DNA-binding domains fused to the potent transactivation domain of FKHR, suggesting that PAX3-FKHR functions to deregulate PAX3-specific target genes and signaling pathways. We previously developed transgenic mice expressing PAX3-FKHR under the control of mouse Pax3 regulatory sequences to test this hypothesis. We reported that PAX3-FKHR interferes with normal Pax3 developmental functions, with mice exhibiting neural tube and neural crest abnormalities that mimic those found in Pax3-deficient Splotch mice. Here we expanded those studies to show that developmental expression of PAX3-FKHR results in aberrant myogenesis in the developing somites and neural tube, leading to ectopic skeletal muscle formation in the mature spinal cord. Gene expression profiling indicated that PAX3-FKHR expression in the developing neural tube induces a myogenic pattern of gene expression at the expense of the normal neurogenic program. Somite defects in PAX3-FKHR transgenic animals resulted in skeletal malformations that included rib fusions and mis-attachments. As opposed to the neural tube defects, the severity of the rib phenotype was rescued by reducing Pax3 levels through mating with Splotch mice. Embryos from the transgenic line expressing the highest levels of PAX3-FKHR had severe neural tube defects, including exencephaly, and almost half of the embryos died between gestational ages E13.5-E15.5. Nearly all of the embryos that survived to term died after birth due to severe spina bifida, rather than the absence of a muscular diaphragm. These studies reveal a prominent role for PAX3-FKHR in disrupting Pax3 functions and in deregulating skeletal muscle development, suggesting that this fusion protein plays a critical role in the pathogenesis of␣alveolar RMS by influencing the commitment␣and differentiation of the myogenic cell lineage.Electronic Supplementary Material Supplementary material is available to authorised users in the online version of this article at .     

Immunohistochemical localization of chondroitin and heparan sulfate proteoglycans in pre-spina bifida splotch mouse embryos.

The splotch (Sp) mutation on mouse chromosome I is a genetic model for the neural tube defects spina bifida and exencephaly. Embryos carrying Sp or its allele splotch-delayed (Spd), have been shown to have delays in neural tube closure, and neural crest cell emigration, as well as a reduction in extracellular space around the neural tube. Pre-spina bifida Sp and Spd embryos have abnormalities of notochord, mesoderm and neuroepithelial development. Chondroitin sulphate proteoglycans (CSPG) and heparan sulfate proteoglycans (HSPG) have been shown to play essential roles during neural tube closure and neural crest cell emigration and migration and thus might well be affected by the splotch mutation. Therefore, the effects of Sp and Spd on the temporal and spatial distributions of CSPG and HSPG were studied in pre-spina bifida embryos cytogenetically identified as Sp/Sp (Spd/Spd), Sp/ + (Spd/ +) or +/+. Immunohistochemical localization of CSPG by means of the CS-56 monoclonal antibody showed that in Sp/Sp head sections, the neuroepithelial basement membranes stained more intensely at 5-, 10-, and 15-somite stages, whereas similar staining was observed at 16- and 19-somite stages compared with matched +/+ sections. In caudal sections Sp/Sp again showed a more intense stain for CSPG in the neuroepithelial basement membranes in all sections (except one comparison, in which staining was similar) from embryos of 14-, 15-, 16-, and 19-somite stages, compared to matched +/+ sections. Heterozygotes did not differ consistently from the mutant or the normal (+/+) embryos in CS-56 stain intensity.(ABSTRACT TRUNCATED AT 250 WORDS)     

Effect of mitomycin C on the neural tube defects of the curly-tail mouse

Around 60% of the mouse mutants called curly-tail, have tail aberrations in the form of a coil or a kink, with or without lumbosacral spina bifida, and rarely, exencephaly. These neural tube defects (NTD) are the result of an incompletely penetrant recessive gene. A single injection of various doses (1-6 mg/kg) of the DNA inhibitor mitomycin C was given to pregnant curly-tail mice on day 7, 8, or 9 of gestation, and its effect on the NTD of the embryos was noted. No dose used was lethal to the embryo. When given on day 7 or day 8, mitomycin C markedly increased the number of exencephalics, and additionally, on day 8, it reduced the number of posterior abnormalities. However, on day 9, no exencephaly was produced, and there was a drastic reduction in the number of tail and spinal defects, the overall incidence of NTD being as low as 15% with 2 mg/kg. A twofold effect of mitomycin C on the curly-tail embryos was thus observed--according to the time in development it was administered, firstly, a teratogenic effect, and later, a "remedial" or preventive effect.     

The BMP antagonist Noggin promotes cranial and spinal neurulation by distinct mechanisms

Here we characterize the consequences of elevated bone morphogenetic protein (BMP) signaling on neural tube morphogenesis by analyzing mice lacking the BMP antagonist, Noggin. Noggin is expressed dorsally in the closing neural folds and ventrally in the notochord and somites. All Noggin-/- pups are born with lumbar spina bifida; depending on genetic background, they may also have exencephaly. The exencephaly is due to a primary failure of neurulation, resulting from a lack of mid/hindbrain dorsolateral hinge point (DLHP) formation. Thus, as previously shown for Shh signaling at spinal levels, BMP activity may inhibit cranial DLHP morphogenesis. However, the increased BMP signaling observed in the Noggin-/- dorsal neural tube is not sufficient to cause exencephaly; it appears to also depend on the action of a genetic modifier, which may act to increase dorsal Shh signaling. The spinal neural tube defect results from a different mechanism: increased BMP signaling in the mesoderm between the limb buds leads to abnormal somite differentiation and axial skeletal malformation. The resulting lack of mechanical support for the neural tube causes spina bifida. We show that this defect is due to elevated BMP4 signaling. Thus, Noggin is required for mammalian neurulation in two contexts, dependent on position along the rostrocaudal axis.     

Pax3 functions in cell survival and in pax7 regulation

In developing vertebrate embryos, Pax3 is expressed in the neural tube and in the paraxial mesoderm that gives rise to skeletal muscles. Pax3 mutants develop muscular and neural tube defects; furthermore, Pax3 is essential for the proper activation of the myogenic determination factor gene, MyoD, during early muscle development and PAX3 chromosomal translocations result in muscle tumors, providing evidence that Pax3 has diverse functions in myogenesis. To investigate the specific functions of Pax3 in development, we have examined cell survival and gene expression in presomitic mesoderm, somites and neural tube of developing wild-type and Pax3 mutant (Splotch) mouse embryos. Disruption of Pax3 expression by antisense oligonucleotides significantly impairs MyoD activation by signals from neural tube/notochord and surface ectoderm in cultured presomitic mesoderm (PSM), and is accompanied by a marked increase in programmed cell death. In Pax3 mutant (Splotch) embryos, MyoD is activated normally in the hypaxial somite, but MyoD-expressing cells are disorganized and apoptosis is prevalent in newly formed somites, but not in the neural tube or mature somites. In neural tube and somite regions where cell survival is maintained, the closely related Pax7 gene is upregulated, and its expression becomes expanded into the dorsal neural tube and somites, where Pax3 would normally be expressed. These results establish that Pax3 has complementary functions in MyoD activation and inhibition of apoptosis in the somitic mesoderm and in repression of Pax7 during neural tube and somite development.     

Effect of hydroxyurea on neural tube defects in the curly-tail mouse

Around 60% of curly-tail mice spontaneously develop neural tube defects (NTD), that is, exencephaly, and/or spina bifida (open lesions), or a curly tail (closed lesion), due to an incompletely penetrant recessive gene. Various doses of hydroxyurea, a teratogen to the rodent central nervous system, were administered to curly-tail mice on either day 8 or day 9 of pregnancy in an attempt to increase the number of NTD in the embryos. No dose used on either day achieved this. However, on day 8, the proportion of affected mice with open lesions increased from around 30% in control mice to 78% with 400 mg/kg hydroxyurea, and this was accounted for specifically by the production of exencephaly. When administered on day 9, 400, 500, and 600 mg/kg hydroxyurea (the latter two doses being lethal to embryos on day 8) actually reduced the incidence of total NTD, to around 30%. Among these affected mice, even though reduced in number, there was still a slight tendency for an increase in the number of exencephalics. Hydroxyurea also produced gastroschisis in a small percentage of embryos; the greatest incidence was 36% with 400 mg/kg on day 8.     

Inositol- and folate-resistant neural tube defects in mice lacking the epithelial-specific factor Grhl-3

The neural tube defects (NTDs) spina bifida and anencephaly are widely prevalent severe birth defects. The mouse mutant curly tail (ct/ct) has served as a model of NTDs for 50 years, even though the responsible genetic defect remained unrecognized. Here we show by gene targeting, mapping and genetic complementation studies that a mouse homolog of the Drosophila grainyhead (grh) gene, grainyhead-like-3 (Grhl3), is a compelling candidate for the gene underlying the curly tail phenotype. The NTDs in Grhl3-null mice are more severe than those in the curly tail strain, as the Grhl3 alleles in ct/ct mice are hypomorphic. Spina bifida in ct/ct mice is folate resistant, but its incidence can be markedly reduced by maternal inositol supplementation periconceptually. The NTDs in Grhl3-/- embryos are also folate resistant, but unlike those in ct/ct mice, they are resistant to inositol. These findings suggest that residual Grhl3 expression in ct/ct mice may be required for inositol rescue of folate-resistant NTDs.     

Quantification and localization of expression of the retinoic acid receptor-beta and -gamma mRNA isoforms during neurulation in mouse embryos with or without spina bifida

BACKGROUND: Previous studies observed that retinoic acid receptor-gamma (RARgamma) is expressed in the open caudal neuroepithelium but that RARbeta is expressed in the closed neural tube. Furthermore, retinoic acid (RA) induces RARbeta expression, a molecular event associated with neural tube closure, but treatment with RA at the appropriate gestation time causes failure of neural tube closure. Since there are four isoforms of RARbeta, perhaps the isoforms expressed in the closed neural tube and induced by RA are different. To investigate the hypothesis that the switch from RARgamma to RARbeta is mechanistically linked to neural tube closure, this study determined the concentrations and distributions of RARbeta and RARgamma isoforms in mouse embryos with RA-induced neural tube defects and in splotch (Sp) mutant embryos with spina bifida. METHODS: Absolute concentrations of RARbeta and RARgamma isoforms were determined throughout primary neurulation (gestational day 8.5-10.0) in treated or untreated C57BL/6J mouse whole embryos by ribonuclease protection analysis. Treatment consisted of an oral dose of 100 mg/kg of all-trans-RA on gestational day 8.5. Spatial distributions of RARbeta and RARgamma were examined in RA-treated and Sp mutant embryos by in situ hybridization. RESULTS: RARbeta2, gamma1, and gamma2 were expressed in untreated embryos and were induced 4.5-, 1.6-, and 4.0-fold, respectively, 4 hr after treatment with RA. In embryos with RA-induced spina bifida, RARbeta2 was expressed in the closed neural tube while RARgamma1 and RARgamma2 were expressed in the open caudal neuroepithelium. In splotch mice with spina bifida, the boundary between RARbeta and RARgamma did not correspond to the site of neural tube closure. CONCLUSIONS: In RA-treated embryos, the relationship between RARbeta expression in the closed and RARgamma in the open caudal neuroepithelium was not altered. However, in splotch embryos with spina bifida, the juncture between RARbeta and RARgamma expression remained in the same anatomical position in the neuroepithelium irrespective of the neural tube closure status and suggests that the switch from RARgamma to RARbeta expression in the closing caudal neuroepithelium may not be causally linked to neural tube closure in the splotch mutant.     

Characterization of a novel missense mutation on murine Pax3 through ENU mutagenesis

N-ethyl-N-nitrosourea (ENU) mutagenesis has led to the elucidation of several regulator genes for melanocyte and skin development.Here we characterized a mutant from ENU mutagenesis with similar phenotype as that of Splotch mutant,including exencephaly,spina bifida and abnormal limbs in homozygotes as well as white belly spotting and occasionally loop-tail in heterozygotes.This novel mutant was named as SpxG..Through genome-wide linkage analysis in backcross progenies with microsatellite markers,the SpxG was confined to a region between D1MIT415 and D1M IT7 on chromosome 1,where notable Pax3 gene was located.Direct sequencing revealed that SpxG carried a nucleotide A894G missense transition in exon 6 of Pax3 gene that resulted in Asn to Asp substitution at amino acid 269 within the highly-conserved homeodomain (HD) DNA recognition module,which was the first point mutation found in this domain in mice.This N269D mutation impaired the transactivation capacity of Pax3 protein,but exerted no effect on Pax3 protein translation.The characterization of the new mutation expanded our understanding the transactivation and DNA-binding structure of Pax3 protein.     

Neural tube development requires the cooperation of p53- and Gadd45a-associated pathways

BACKGROUND: Numerous genetically engineered mouse models for neural tube defects (NTDs) exist, and some of the implicated proteins are functionally related. For example, the growth arrest and DNA damage-inducible protein Gadd45a and tumor suppressor p53 are functionally similar, and both are involved in neural tube development (Gadd45a- and Trp53-null embryos show low levels of exencephaly). To assess their roles in neural tube development, we generated double-null mice from Gadd45a- and Trp53-null mice, as well as from cyclin-dependent kinase inhibitor (Cdkn1a) (p21)-null and xeroderma pigmentosum group C (XPC)-null mice that do not show spontaneous exencephaly. METHODS: Gadd45a-, Trp53-, Cdkn1a-, and XPC-null mice were crossed to generate several double-null mouse models. Embryos (embryonic day [ED] 16-18) from the single- and double-null crosses were scored for NTDs. RESULTS: Deletion of both Gadd45a and Trp53 in mice increased exencephaly frequencies compared to the deletion of either single gene (34.0% in Gadd45a/Trp53-null compared to 8.4% and 9.1% in the Gadd45a- and Trp53-null embryos, respectively). Furthermore, although deletion of another p53-regulated gene, Cdkn1a, is not associated with exencephaly, in conjunction with Gadd45a deletion, the exencephaly frequencies are increased (30.5% in the Gadd45a/Cdkn1a-null embryos) and are similar to those in the Gadd45a/Trp53-null embryos. Although XPC deletion increased exencephaly frequencies in Trp53-null embryos, XPC deletion did not increase the exencephaly frequencies in Gadd45a-null embryos. CONCLUSIONS: The increased genetic liability to exencephaly in the Gadd45a/Trp53- and Gadd45a/Cdkn1a-null embryos may be related to the disruption of multiple cellular pathways associated with Gadd45a and p53.     

Valproic acid-induced spina bifida: a mouse model.

Prenatal exposure to the antiepileptic drug valproic acid (VPA) has been associated with the formation of spina bifida aperta, meningocele, and meningomyelocele in the human. Until now, a direct relationship between VPA application and spina bifida has not been experimentally demonstrated. VPA was known only to induce exencephaly in mice, a defect of the anterior neural tube. Maximal sensitivity toward production of this defect was on day 8 of gestation (plug day = day 0). The closure of the posterior neuropore occurs later in the development of mice than the closure of the anterior neuropore. To investigate whether there is a direct relationship between VPA application during pregnancy and induction of spina bifida in mice, we administered various doses of the drug on day 9 of gestation, at three time intervals (at 0, 6, and 12 hr). This administration of VPA produced spina bifida aperta and spina bifida occulta in mice. High doses of VPA (3 x 450 and 3 x 500 mg/kg) induced a low rate of spina bifida aperta in the lumbosacral region. High incidences of spina bifida occulta, a less serious form of spina bifida, were induced with lower doses. This malformation was demonstrated in double-stained fetal skeletons by measurements of the distance between the cartilaginous ends of each vertebral arch. The occurrence of this defect and its localization was dose-dependent. The lumbar region was affected by all doses investigated (3 x 300, 3 x 350, 3 x 400, 3 x 450, and 3 x 500 mg/kg). The sacral/coccygeal region was affected additionally, but with higher doses (3 x 400, 3 x 450, and 3 x 500 mg/kg). A comparison of the results obtained with day 16 and 17 control fetuses showed that the pattern of gaps present in the lumbar and sacral region of the spinal cord in treated groups was drug-specific and not related to a developmental delay. Our results indicate that multiple administrations of VPA on day 9 of gestation in mice result in a low incidence of spina bifida aperta and a high incidence of spina bifida occulta, and provides a relevant model for the study of human spina bifida defects.