Holoprosencephaly: clinical, anatomic, and molecular dimensions

Holoprosencephaly is addressed under the following headings: alobar, semilobar, and lobar holoprosencephaly; arrhinencephaly; agenesis of the corpus callosum; pituitary abnormalities; hindbrain abnormalities; syntelencephaly; aprosencephaly/atelencephaly; neural tube defects; facial anomalies; median cleft lip; minor facial anomalies; single maxillary central incisor; holoprosencephaly-like phenotype; epidemiology; genetic causes of holoprosencephaly; teratogenic causes of holoprosencephaly; SHH mutations; ZIC2 mutations; SIX3 mutations; TGIF mutations; PTCH mutations; GLI2 mutations; FAST1 mutations; TDGF1 mutations; and DHCR7 mutations.     

Correlation between mandibular retrognathia and induction of cleft palate with 6-aminonicotinamide in the rat.

A single injection of the niacin antimetabolite 6-aminonicotinamide (6-AN) late in gestation produces cleft palate in the rat. In order to achieve an understanding of the mechanism of induction of cleft palate, craniofacial growth and palate development were studied in Sprague-Dawley rats after treatment with 6-AN on day 15 of gestation. The rats were maintained on a high niacin diet (95 ppm) and subjected to three different teratogenic levels of 6-AN. The first group was injected with 8 mg/kg, the second was fasted and injected with 8 mg/kg and the third was treated with 16 mg/kg. The lowest teratogenic dose, 8 mg/kg, produced mild mandibular retrognathia on day 16, delayed shelf elevation a few hours and resulted in small rostral and small caudal clefts of the secondary palate. The moderate dose, 8 mg/kg with fasting, produced more severe mandibular retrognathia, delayed shelf elevation about 24 hours and resulted in 37% full clefts and 63% partial clefts of the palate. The highest teratogenic dose, 16 mg/kg, produced severe mandibular retrognathia, delayed shelf elevation by more than 24 hours and resulted in 100% full clefts of the palate. In each 6-AN group, the most severe mandibular retrognathia was present between days 16 and 17, the critical time for palate closure in the rat. Treatment with 6-AN also produced abnormality of the epithelial cells of the palate, the toothbuds and the nasal septum. Molar and incisor toothbuds were small and malformed, and the epithelial surfaces of the palate and the soft tissue nasal septum did not fuse.     

The effects of 5-bromodeoxyuridine on fusion of the cranial neural folds in the mouse embryo

The effects of 500 and 300 mg/kg bromodeoxyuridine (BUdR) on the process of fusion of the neural folds were tested after injection into pregnant mice on day 8 of gestation (192 hours postcoitum). Various doses of the natural nucleoside, thymidine (TdR), were also tested. Both doses of BUdR retarded growth to the same extent, but only the larger dose caused neural tube defects in 28.8% of embryos. Treatment with the larger dose also caused extensive cell necrosis to appear in the neuroepithelium of the neural folds between 12 and 15 hours after treatment. No changes were detectable with the light microscope up to this time. Measurement of the cell generation time in treated and control embryos indicated that the BUdR prolonged the cycle by about 2 hours and that the dying cells were in the second DNA synthetic phase following incorporation of the analog. Treatment with the smaller dose of BUdR caused minimal cell necrosis. This was taken as evidence for the importance of cell necrosis in the pathogenesis of BUdR-induced neural tube defects. Treatment with excess TdR did not cause either neural tube defects or cell necrosis, and a dose of TdR equimolar with the large dose of BUdR (400 mg/kg TdR) did not retard growth. Doses of 800 and 1,200 mg/kg TdR retarded growth to the same extent as BUdR. The administration of an equimolar amount of TdR, along with the teratogenic dose of BUdR, prevented the occurrence of cell necrosis and neural tube defects. When treatments were given on day 9 of gestation, 500 mg/kg BUdR caused cell necrosis in the neuroepithelium about 15 hours after treatment but no neural tube defects were produced by day 9 after treatment. It is suggested that in this case cell necrosis occurred too late to interfere with neural fold fusion. It was concluded that the ability of BUdR to cause exencephaly in mouse embryos was due to cell necrosis in the neuroepithelium.     

Common hierarchies of susceptibility to the induction of neural tube defects in mouse embryos by valproic acid and its 4-propyl-4-pentenoic acid metabolite

The teratogenic effects of valproic acid and its 4-propyl-4-pentenoic acid (4-en) metabolite were investigated in three inbred mouse strains that were known to possess differing sensitivity to heat-induced neural tube defects. In the heat-resistant DBA/2J strain, administration of either valproic acid or the metabolite during the critical period of neural tube development failed to produce any abnormal offspring. Similar treatment in the moderately heat-sensitive LM/Bc strain resulted in up to 19.8% exencephalic fetuses. The highly heat-sensitive SWV strain was also very susceptible to the induction of neural tube defects by either valproic acid or its 4-en metabolite. When administered on gestational day 8 plus 12 hours, the parent compound produced 35% exencephalic fetuses, while the metabolite had a response frequency of 32.4%. Thus, the hierarchy of susceptibility for the induction of neural tube defects in these inbred mouse strains was exactly the same whether the teratogen was a physical agent such as hyperthermia or a chemical compound such as valproic acid. If such diverse agents as these should interact to produce malformations, then it is possible that a wide variety of other agents might interact in a similar manner to produce neural tube defects.     

Birth Prevalence of Neural Tube Defects and Orofacial Clefts in India: A Systematic Review and Meta-Analysis

Background

In the last two decades, India has witnessed a substantial decrease in infant mortality attributed to infectious disease and malnutrition. However, the mortality attributed to birth defects remains constant. Studies on the prevalence of birth defects such as neural tube defects and orofacial clefts in India have reported inconsistent results. Therefore, we conducted a systematic review of observational studies to document the birth prevalence of neural tube defects and orofacial clefts.

Methods

A comprehensive literature search for observational studies was conducted in MEDLINE and EMBASE databases using key MeSH terms (neural tube defects OR cleft lip OR cleft palate AND Prevalence AND India). Two reviewers independently reviewed the retrieved studies, and studies satisfying the eligibility were included. The quality of included studies was assessed using selected criteria from STROBE statement.

Results

The overall pooled birth prevalence (random effect) of neural tube defects in India is 4.5 per 1000 total births (95% CI 4.2 to 4.9). The overall pooled birth prevalence (random effect) of orofacial clefts is 1.3 per 1000 total births (95% CI 1.1 to 1.5). Subgroup analyses were performed by region, time period, consanguinity, and gender of newborn.

Conclusion

The overall prevalence of neural tube defects from India is high compared to other regions of the world, while that of orofacial clefts is similar to other countries. The majority of studies included in the review were hospital based. The quality of these studies ranged from low to moderate. Further well-designed, high quality community-based observational studies are needed to accurately estimate the burden of neural tube defects and orofacial clefts in India.     

Differential and stage dependent effects of retinoic acid on chondrogenesis and synthesis of extracellular matrix macromolecules in chick craniofacial mesenchyme in vitro

Retinoic acid (RA) is well known to be a potent teratogen and induces a variety of facial defects in vivo, but at concentration levels lower than those that cause facial defects, RA seems to play an important role in normal facial development. In a previous study, we demonstrated the ability of RA to stimulate chondrogenesis in vitro in HH stage 23/24 chick mandibular (MND) but not frontonasal (FNP) mesenchyme cultured in a serum-free medium. The present study furthers these results by examining the effects of RA on chondrogenesis of chick facial mesenchyme at earlier embryonic stages and the effects on cell proliferation and synthesis of specific extracellular matrix macromolecules at stage 23/24. MND and FNP cells were cultured as micromasses for 4 days in defined media. As described previously, chondrogenesis in stage 23/24 MND cells was significantly enhanced by concentrations of RA of 0.1-1 ng/ml; however, at all earlier stages examined (18 to 22) RA at these concentrations had no significant effect. Higher concentrations of the retinoid inhibited chondrogenesis in MND cultures from all stages tested. Cells of the FNP from all stages displayed no significant change in chondrogenesis below 1 ng/ml RA and a dose dependent inhibition at higher concentrations. Thus RA's promotional effects in the face are not only tissue specific (MND), but also stage-dependent (HH 23/24). The specific effects of RA on matrix production and cell proliferation of stage 23/24 MND and FNP cells was examined by analysis of 35S sulfate, 3H thymidine and 3H proline incorporation. Analysis of 35S sulfate incorporation into sulfated proteoglycans confirmed that concentrations of RA of 0.1-1 ng/ml stimulated cartilage matrix production in MND but not FNP cultures. Above this level of RA, 35S sulfate incorporation was reduced in both. Likewise, 3H proline incorporation into collagenous protein, and to a lesser extent non-collagenous proteins, was stimulated by low levels of RA in MND, but not FNP cultures. Higher concentrations of the retinoid in either MND or FNP cultures did not lower collagen production, undoubtedly due to stimulation of non-chondrogenic cells within the population. This indicates that levels of RA as high as 100 ng/ml cause phenotypic change rather than cell death. This last point is corroborated by the analysis of 3H thymidine uptake in the cultures which was only transiently modified in most. The data indicate that cell proliferation occurred even in the presence of high RA levels.     

Changes in the mouse neuroepithelium associated with cadmium-induced neural tube defects

The aim of this study was to investigate the teratogenic action of cadmium (Cd) on the developing mouse CNS. Pregnant mice were injected with 4 mg/kg CdCl2 on day 7, 8, 9, or 10 of gestation. These animals and saline injected controls were sacrificed either on the day before birth or at various times up to 48 hours after injection and the embryos examined grossly and histologically. Exencephaly occurred after Cd treatment on day 7 or 8 and its development was examined in day 8 embryos. Eight hours after Cd injection many cells of the closing neural plate contained dense-staining inclusions, thought to be autophagic vacuoles. After 24 hours this damage had almost disappeared, but the anterior neural folds, although looking histologically normal, were more open than in controls. Forty-eight hours after injection it was apparent that this part of the neural tube was not going to close and would result in exencephaly. Cd exposure on day 9 or 10 did not cause gross CNS defects such as exencephaly. On both days, twelve hours after Cd injection, similar dark-staining inclusions were seen in many cells throughout the CNS. After twenty-four hours there were variable amounts of cell death, resulting in some embryos in breakdown of parts of the wall of the neural tube. Forty-eight hours after treatment all inclusions and cellular debris had disappeared, indicating repair had taken place, but in some embryos, treated on day 9, severe lasting damage was seen as dorsal openings in the previously closed neural tube.     

Oblique facial clefts: pathology, etiology, and reconstruction

Modern views on embryology have increased our understanding of the nature of oblique facial clefts. The anomalies that have their origin at the junction of facial processes, such as the nasomaxillary dysplasias, may be named primary clefts or transformation. The maxillary clefts that are due to a developmental arrest of the skeleton are in fact secondary defects of differentiation defects. The teratology of these malformations is discussed, and attention is drawn to the amniotic rupture syndrome as a possible cause. All these clefts are rare, their incidence ranging from 0.75 to 5.4 per 1000 common clefts. This author has been involved in the treatment of nine of these patients. Four had their malformation reconstructed with one of the conventional procedures described in the literature, but the results, although initially acceptable, soon deteriorated. A more aggressive approach was therefore chosen. Rotation and advancement of the cheek proved to be extremely effective and is now advocated as the procedure of choice. The transposition of a median forehead flap is considered an excellent alternative. Use of these procedures in five patients is reported. There were no complications.     

Ultrastructural analysis of malformations of the embryonic neural axis induced by in vitro hyperglycemic conditions

Neural tube defects are the most common malformations associated with diabetic pregnancies. Although the teratogenic effects of excess glucose have been investigated in in vivo and in vivo studies, a cellular basis for neural tube defects has not been elucidated. We used rat embryo culture to study the organogenesis period of development, with excess d-glucose added to the serum medium to induce neural tube anomalies. Light and electron microscopic examination of control 12-day-old embryos grown 48 hours in culture revealed blastlike cells with few organelles or cellular processes. Twelve-day-old embryos cultured in excess d-glucose had advanced cellular maturation with differentiation, including the presence of free polysomes and copious cell processes, regardless of whether they had an open neural tube. Cytoarchitectural changes such as decreased numbers of mitotic figures with mitotic cells in the mantle layer were focally distributed throughout the neural epithelium but with predominance at the site of failed closure. In vivo studies failed to demonstrate neural processes in day 12 normal embryos. Fourteen-day-old embryos grown in utero also had foci of cell processes in the neural tube but to a much lesser degree than that observed in the in vitro day 12 glucose-exposed embryos. The cellular aberrations in the excess d-glucose-treated embryos are characteristic of a premature maturational change. Since they are present in excess d-glucose-exposed embryos with or without failure of neural tube closure, these maturational and cytoarchitectural changes may contribute to the cellular basis for neural tube defects.     

Maternal treatment with teratogen causes congenital malformations in mouse embryos

Heterozygotes for the tail-short (Ts) mutant gene in the Balb/c strain have minor skeletal defects and a short, kinky tail. If heterozygote Ts/+ mothers are mated with normal-tail +/+ males and are treated with teratogenic doses of trypan blue on the eighth day of pregnancy, the mutant F1 heterozygotes develop exencephaly, folded neural tube and spina bifida significantly more often than non-mutants. This is indicative of gene-teratogen interaction, with the Ts gene increasing the embryo's susceptibility to trypan blue-induced neural tube defects.     

Gene Expression Profiling Within the Developing Neural Tube

The developing mammalian nervous system is subject to devastating congenital malformations with clinical significance that extends into the billions of health care dollars annually worldwide. Neural tube defects (NTDs) are among the most common of all human congenital defects, yet their etiology remains poorly understood. This is largely due to the complexity of the genetic factors regulating the intricate events involved in neurulation. Using mouse model systems and the application of modern molecular biological technologies, we have recently gained a greater appreciation for the factors that not only regulate normal neural tube closure (NTC), but those genetic factors that predispose an embryo to significant birth defects such as anencephaly or spina bifida. We have selected prominent murine mutants, both spontaneous and genetically modified, as well as the use of teratogenic agents, to examine the impact of altering the normal pattern of gene expression in the developing neural tube.     

Lack of concordance between heat shock proteins and the development of tolerance to teratogen-induced neural tube defects

The present study was undertaken to examine the role of heat shock response in the development of tolerance and cross-tolerance in an in vivo murine model of teratogen-induced neural tube defects. The experimental paradigm designed to address this question was to utilize inbred mouse strains that differed in their sensitivity to hyperthermia and valproic acid induced neural tube defects, subjecting the dams to subteratogenic pretreatments with either heat or valproic acid at two different timepoints during development prior to the administration of the teratogenic insult. A statistically significant reduction in the frequency of neural tube defects and/or embryolethality following a pretreatment in dams subsequently exposed to a teratogenic treatment was considered evidence for the induction of tolerance. This was observed in the SWV embryos exposed to the 38°C pretreatment at 8:06 and to embryos exposed to either pretreatment temperature at 8:10 priorto a teratogenic heat shock at 8:12. In the LM/Bc embryos, only the 41°C pretreatment at 8:06 induced thermotolerance. There was no evidence of tolerance induced in either mouse strain using valproic acid. On the other hand, cross-tolerance was clearly demonstrated in this study, with a low temperature (41°C) pretreatment successfully protecting SWV fetuses from a subsequent teratogenic treatment with valproic acid, while valproic acid (200 mg/kg) was effective in reducing the risk of hyperthermia-induced neural tube defects in the LM/Bc fetuses. In all instances, tolerance was induced in the absence of significant induction of hsp synthesis. The lack ofconcordance between hsps and thermotolerance suggests that some other factor(s) is involved in conferring thermotolerance on developing murine embryos. © 1993 Wiley-Liss, Inc.     

Teratogenic interaction of ethanol and hyperthermia in mice

Alcohol and maternal hyperthermia have been implicated in human birth defects. Both ethanol and heat can induce neural tube defects (NTDs) and other developmental abnormalities in mice when large doses are given during pregnancy. To explore the teratogenic interaction of both agents, pregnant ICR mice were injected with a single dose of 25% ethanol and/or were heat-stressed in a water bath at 42 degrees C on the morning of Day 8 of gestation. Combined treatment with ethanol (0.01-0.02 ml/g) and heat (10 min), when they were given concurrently or 1 hr apart, resulted in a significant increase of resorptions and externally malformed fetuses. Skeletal malformations and visceral variations also increased significantly following a concurrent exposure to both agents. These results indicate that ethanol and heat can be synergistically teratogenic in mice when the doses of each agent are below the teratogenic threshold. It was also suggested that pretreatment with a small dose of ethanol may not enhance the teratogenicity of heat when the hyperthermic stress is strong enough and teratogenic by itself.     

Inhibition of histone deacetylase activity on specific embryonic tissues as a new mechanism for teratogenicity

BACKGROUND: the inhibition of histone deacetylase (HDAC) has been reported as an effective mechanism on therapy in neoplastic diseases. Among HDAC inhibitors, Trichostatin A (TSA) and Valproic Acid (VPA) prevent the tumorigenesis in rodent and human models. Malformations as neural tube and axial skeletal defects are well-known VPA side effects. Recent hypotheses suggest the HDAC inhibitor activity as the teratogenic mechanism of VPA. The teratogenic potency of TSA is, at the moment, unknown. The aim of the present work is to investigate the HDAC inhibition on embryos exposed in utero to TSA or VPA and to compare the teratogenic potential of these two molecules on the axial skeleton morphogenesis. METHODS: Pregnant CD mice were i.p. treated on day 8 post coitum (9.00 a.m.) with 400 mg/kg VPA or with 0, 2, 4, 8, 16 mg/kg TSA. Embryos explanted 1 hr after the treatment from some females exposed to 400 mg/kg VPA or to 16 mg/kg TSA were processed for Western blotting and immunohistochemical analysis, in order to evaluate the histone hyperacetylation in the total embryo homogenates and to visualize the hyperacetylated tissues. Foetuses at term were processed for skeletal examination. RESULTS: Both VPA and TSA were able to induce hyperacetylation on embryos, specifically at the level of the caudal neural tube and of somites. At term, TSA showed teratogenic effects at the axial skeleton, quite similar to those observed after VPA exposure. CONCLUSIONS: In conclusion, both VPA and TSA are teratogenic in mice. A direct correlation between somite hyperacetylation and axial abnormalities could suggest the HDAC inhibition as the mechanism of the teratogenic effects.     

Identification of early-responsive genes correlated to valproic acid-induced neural tube defects in mice

BACKGROUND: Valproic acid (VPA) causes the failure of neural tube closure in newborn mice. However, the molecular mechanism of its teratogenesis is unknown. This study was conducted to investigate the genomewide effects of VPA disruption of normal neural tube development in mice. METHODS: Microarray analysis was performed on the head part of NMRI mouse embryos treated for 1 hr with VPA on gestational day (GD) 8. Subsequently, we attempted to isolate genes that changed in correlation with the teratogenic action of VPA by employing reduced teratogenic VPA analogs, valpromide (VPD) and valnoctamide (VCD), in a real-time PCR study. RESULTS: Microarray results demonstrated that during neurulation, many genes, some of whose functions are known and some unknown, were either increased or decreased after VPA injection. Some genes were affected by VPD or VCD in the same way as VPA, but others were not changed by the analogs. In this way, our system identified 11 increased and 20 decreased genes. Annotation analysis revealed that the increased genes included gadd45b, ier5, per1, phfl3, pou3f1, and sox4, and the decreased genes included ccne2, ccnl, gas5, egr2, sirt1, and zfp105. CONCLUSIONS: These findings demonstrate that expression changes in genes having roles in the cell cycle and apoptosis pathways of neural tube cells were strongly expected to relate to the teratogenic, but not antiepileptic, activity of VPA. Our approach has allowed the expansion of the catalog of molecules immediately affected by VPA in the developing neural tube.     

The role of sonic hedgehog in normal and abnormal craniofacial morphogenesis.

There is growing evidence that implicates a role for Sonic hedgehog (SHH) in morphogenesis of the craniofacial complex. Mutations in human and murine SHH cause midline patterning defects that are manifested in the head as holoprosencephaly and cyclopia. In addition, teratogens such as jervine, which inhibit the response of tissues to SHH, also produce cyclopia. Thus, the loss of SHH signaling during early stages of neural plate patterning has a profound influence of craniofacial morphogenesis. However, the severity of these defects precludes analyses of SHH function during later stages of craniofacial development. We have used an embryonic chick system to study the role of SHH during these later stages of craniofacial development. Using a combination of surgical and molecular experiments, we show here that SHH is essential for morphogenesis of the frontonasal and maxillary processes (FNP and MXPs), which give rise to the mid- and upper face. Transient loss of SHH signaling in the embryonic face inhibits growth of the primordia and results in defects analogous to hypotelorism and cleft lip/palate, characteristics of the mild forms of holoprosencephaly. In contrast, excess SHH leads to a mediolateral widening of the FNP and a widening between the eyes, a condition known as hypertelorism. In severe cases, this widening is accompanied by facial duplications. Collectively, these experiments demonstrate that SHH has multiple and profound effects on the entire spectrum of craniofacial development, and perturbations in SHH signaling are likely to underlie a number of human craniofacial anomalies.     

The embryolethality and teratogenicity of acrolein in cultured rat embryos

Acrolein, a three-carbon unsaturated aldehyde, is teratogenic to rats in vivo following intraamniotic administration but has been reported not to be teratogenic in vitro in the rat whole embryo culture system. In this study the effects of acrolein on rat embryos cultured in the standard medium consisting of rat serum were assessed over a narrow-concentration range. Additionally, a comparison was done of the effects of culture in a serum medium vs. a serum-free medium. In the serum medium acrolein caused 100% embryolethality at 140 microM and was found to be teratogenic in the concentration range of 80-120 microM. In the serum-free medium acrolein was 100% embryolethal at 20 microM and was teratogenic in the range of 5-15 microM. The EC50 for malformations in the serum medium was 137 microM, whereas that for embryolethality was 115 microM; the EC50s for malformations and embryolethality in the serum-free medium were 2.8 microM and 8.3 microM, respectively. Malformations were observed in the brain, facial area, and heart in addition to blebs and twisted or kinked bodies. Decreases in yolk sac diameter, crown-rump length, head length, number of somites, morphological score, and protein content were observed within the teratogenic ranges in each type of medium. Thus acrolein is teratogenic and embryolethal in vitro as well as in vivo. Dissociation between embryolethality and teratogenicity was seen in the serum-free medium. The slope of the acrolein log concentration-response curve in the serum-free medium was twice that in the serum medium, indicating that acrolein may have a different mechanism of action in this medium.     

Transplacental exposure to methylene blue initiates teratogenesis in the mouse: preliminary evidence for a mechanistic implication of cyclic GMP pathway disruption

BACKGROUND: The vital dye methylene blue (MB) has been shown to be teratogenic when injected into the amnion in the second trimester. On the other hand, the teratogenic potential of transplacental exposure to MB has not been determined. METHODS: MB was administered subcutaneously to ICR (CD-1) mice at 0, 35, 50, 60, or 70 mg/kg on gestation day 8 (plug day = day 0). Teratological assessments were carried out at term gestation, on gestation day 18. Since MB inhibits soluble guanylate cyclase enzyme activity, zaprinast (ZPN), a selective cGMP-phosphodiesterase type V inhibitor, was administered to prevent developmental disorders initiated by MB at 50 mg/kg. RESULTS: There was a dose-dependent increment of embryolethality. MB treatment also produced axial skeleton and neural tube defects. Coadministration of ZPN (20 mg/kg per three times) abolished completely MB-induced neural tube defects and reduced by one-half the incidence of fetuses exhibiting axial skeletal defects. ZPN did not provide protection against the embryocidal effects of MB. CONCLUSIONS: This study showed that transplacental exposure to MB is teratogenic in the mouse. Coadministration of ZPN prevented partly MB-induced teratogenesis, which supports the hypothesis that imbalance of cGMP pathway accounts, in part, for the teratogenicity of MB.     

Teratogenic potential of two neurotropic drugs, haloperidol and dextromoramide, tested on mouse embryos

Potential teratogenic activity of two neurotropic drugs, haloperidol and dextromoramide tartrate, was tested by using the mouse embryo experimental model. Like numerous other drugs of this class these two are also embryotoxic. After treatment 1 hour into the 9th gestation day they induce the neurotropic syndrome of malformations comprising exencephaly, craniorachischisis, kinking of the spinal cord, brachyury, and dilation of the fourth brain ventricle. In addition, dextromoramide tartrate was found to induce one more, so far unknown, neural tube defect, namely the ectopia of the neural tube. Delay of treatment by 1 or 2 hours tends to displace the location of the neural tube defects along the length of the neural axis towards the anterior and posterior directions. Even after accumulating these results at relatively high doses, it is difficult to estimate human reproductive risks from this animal data for human therapeutic doses.