Craniofacial dysmorphogenesis in transgenic mice.

While using transgenic mice to study the regulation of alpha-fetoprotein (AFP) it was noted that two different alpha-fetoprotein-chloramphenicol acetyl transferase (CAT) transgenes resulted in the appearance of craniofacial anomalies in 11% of the offspring derived from crosses between transgenic mice and nontransgenic mates. A total of 13 fetuses exhibited abnormalities; two are described in detail. Ninety-two percent of the affected fetuses had some form of mandibular abnormality while zygomatic and ossicular defects appeared in more than 40% of the specimens. Aglossia and aberrant musculature were also present in the most severely involved specimen. Eight of the affected fetuses were screened for the presence of the AFP-CAT plasmid and all were found to be heterozygous for the transgene. Since the probability that all 8 of the abnormal fetuses known to carry the CAT gene would have done so by chance was only 1 in 256, it may be assumed that these anomalies did not appear spontaneously, but were somehow created by the transgenic procedure. It is not known how the transgenic material led to the observed dysmorphogenetic pattern, but theoretically introduction of the AFP-CAT plasmid could have disrupted morphogenesis through the presence of the "foreign" CAT protein or a decrease in the availability of AFP. Since AFP levels were found to be normal in both the liver and the yolk sac of transgenic fetuses, it appears that the presence of CAT was responsible for the craniofacial anomalies described here.     

Congenital malformations in rats with the semilethal mutation fused pulmonary lobes

It has been demonstrated that an autosomal recessive gene, fused pulmonary lobes (fpl), causes fusion of the right pulmonary lobes with several associated malformations and a high incidence of death in homozygous newborns (Aoyama et al. Teratology 1988; 37:159-166). The aim of the present study was to investigate whether the deaths of fpl/fpl newborns were caused by functional abnormalities of the malformed lung or other associated malformations. Day-20 fpl/fpl and fpl/+ fetuses were weighed and examined for gross abnormalities. The lungs of selected fetuses were further examined for histological abnormalities. A wide variety of associated external, visceral, and skeletal anomalies as well as relatively lower body weights than those of phenotypically normal fpl/+ littermates were observed in the fpl/fpl fetuses. The associated anomalies consisted of hematomas and/or subcutaneous hemorrhages in the head, truncus and limbs, eyelid anomalies, CNS defects, lobation anomalies of the liver, hypoplasia of the spleen, partial absence of the skull bones, and dorsi- or ventriflexion of the phalanges of the limbs. Among them, CNS defects and partial absence of the skull bones were considered to be possible causes of newborn deaths. However, the incidence of these malformations was approximately 10% and was lower than the neonatal mortality, which had been estimated to be approximately 50% in the previous study (Aoyama et al. Teratology 1988; 37:159-166). The lungs of fpl/fpl fetuses consistently had hypoplasia of the intermediate lobe and fusion of the right pulmonary lobes. No histological changes suggesting postnatal respiratory insufficiency were found in the lungs of day-20 fpl/fpl fetuses, and the cause of newborn death remains unclear.     

Homozygous WNT3 mutation causes tetra-amelia in a large consanguineous family

Tetra-amelia is a rare human genetic disorder characterized by complete absence of all four limbs and other anomalies. We studied a consanguineous family with four affected fetuses displaying autosomal recessive tetra-amelia and craniofacial and urogenital defects. By homozygosity mapping, the disease locus was assigned to chromosome 17q21, with a maximum multipoint LOD score of 2.9 at markers D17S931, D17S1785, D17SS1827, and D17S1868. Further fine mapping defined a critical interval of approximately 8.9 Mb between D17S1299 and D17S797. We identified a homozygous nonsense mutation (Q83X) in the WNT3 gene in affected fetuses of the family. WNT3, a human homologue of the Drosophila wingless gene, encodes a member of the WNT family known to play key roles in embryonic development. The Q83X mutation truncates WNT3 at its amino terminus, suggesting that loss of function is the most likely cause of the disorder. Our findings contrast with the observation of early lethality in mice homozygous for null alleles of Wnt3. To our knowledge, this is the first report of a mutation in a WNT gene associated with a Mendelian disorder. The identification of a WNT3 mutation in tetra-amelia indicates that WNT3 is required at the earliest stages of human limb formation and for craniofacial and urogenital development.     

Preliminary observations on isotretinoin-induced ear malformations and pattern formation of the external ear.

Retinoic acid is a morphogenic substance capable of inducing a variety of limb malformations, including duplications and reduction-type defects. Whether retinoic acid plays a similar role in controlling pattern formation of other vertebrate structures is unclear. Many fetuses and infants exposed to isotretinoin (13-cis-retinoic acid) in utero have a characteristic pattern of anomalies, chiefly involving brain, craniofacial, and thymic morphogenesis. Among the craniofacial anomalies, external ear malformations are common and the specific types of auricular malformations include partial duplications, and tissue reductions and displacements. These similarities to the types of limb malformations that retinoic acid can induce suggest that retinoic acid may play an important role in controlling pattern formation of facial structures.     

Spontaneous malformations in JW-NIBS rabbits

The frequency of spontaneous anomalies among JW-NIBS rabbits in our laboratory is reported. The study was based on 1217 live fetuses obtained from 185 of an origin sample of 195 pregnant females; the remaining 10 (5.1%) aborted. Seven (0.57%) of the fetuses had the following external anomalies: multiple anomalies with craniofacial anomalies and thoraco-, gastroschisis (3 cases), microcephaly with open eyelids and microstomia (1), microphthalmia (1), anury (1) and brachyury (1). Among 1213 fetuses, 2 (0.16%) had abdominal visceral anomalies: agenesis of gall bladder and hypoplasia of the ovary were each found in one animal. Head and thoracic visceral anomalies were found in 6 (1.73%) of 347 fetuses, and skeletal anomalies in 6 (0.69%) of 867 fetuses. 4.03% of fetuses had 13 ribs.     

ENU induced mutations causing congenital cardiovascular anomalies

We used non-invasive high frequency ultrasound to screen N-ethyl-N-nitrosourea mutagenized mouse fetuses for congenital cardiovascular anomalies. We ultrasound scanned 7546 mouse fetuses from 262 mutagenized families, and identified 124 families with cardiovascular defects. Represented were most of the major congenital cardiovascular anomalies seen clinically. The ENU-induced mutations in several families were mapped using polymorphic microsatellite DNA markers. One family with forelimb anomalies and ventricular septal defects, phenotypes similar to Holt-Oram syndrome, and one family with transposition of the great arteries and heart situs anomalies were mapped to different regions of mouse chromosome 4. A third mutation causing persistent truncus arteriosus and craniofacial defects, phenotypes reminiscent of DiGeorge syndrome, was mapped to mouse chromosome 2. We note that mouse chromosomes 4 and 2 do not contain Tbx5 or Tbx1, genes previously linked to Holt-Oram and DiGeorge syndromes, respectively. In two other families, the ENU-induced mutation was identified--Sema3CL605P was associated with persistent truncus arteriosus with interrupted aortic arch, and the Gja1W45X connexin43 mutation caused conotruncal malformation and coronary aneurysms. Although our screen was designed as a recessive screen, a number of the mutations showed cardiovascular phenotypes in both heterozygote and homozygote animals. These studies show the efficacy of ENU mutagenesis and high-throughput ultrasound phenotyping in recovering mutations causing a wide spectrum of congenital heart defects. These ENU-induced mutations hold promise in yielding new insights into the genetic basis for human congenital heart disease.     

Limb reduction defects in humans associated with prenatal isotretinoin exposure.

Retinoic acid has long been used to induce limb reductions defects in experimental animal studies. No limb malformations, however, have been reported among malformed retinoic acid-exposed human fetuses from case reports or epidemiologic studies. We report a child and a fetus with limb reduction malformations following maternal use of isotretinoin (13-cis-retinoic acid) during the first trimester of pregnancy. The child had a unilaterally absent clavicle and nearly absent scapula, with a short humerus and short, synostotic forearm bones. He also had ventriculomegaly and developmental delay, minor dysmorphic facial features, and a short sternum with a sterno-umbilical raphe. The fetus had a unilaterally absent thumb with normal proximal bony structures. Other findings included hydrocephalus, craniofacial anomalies, thymic agenesis, supracristal ventricular septal defect, single umbilical artery, anal and vaginal atresia, and urethral agenesis with dysplastic, multicystic kidneys. Although the limb malformations were quite dissimilar, a number of anomalies that are frequently found among isotretinoin-exposed fetuses/infants were present in both cases. This increases the probability that retinoic acid caused these limb defects, but a causal association cannot be conclusively drawn on the basis of these two retrospective case reports.     

Teratologic studies on rat perinates and offspring from dams treated with ethylnitrosourea (ENU).

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues, limbs and male reproductive organs. Recently we clarified that excess cell death caused by apoptosis occurred in these organs and tissues of rat fetuses from dams treated with ENU at day 13 of gestation (GD13). In this study, we examined fetuses at GD21 and offspring at 10 weeks of age after ENU administration to pregnant rats at GD13 in order to clarify the relationship between ENU-induced apoptosis in the fetal tissues and teratogenicity of ENU. Severe intrauterine growth retardation was observed in the ENU group, and the body weight of the offspring in the ENU group was significantly lower than that of the control group throughout the experiment. In addition, a high incidence of microencephaly, ectrodactyly and curved caudal vertebrae was observed in the offspring from dams treated with ENU at GD13. Judging from the results of our previous and present studies, it was strongly suggested that ENU-induced apoptosis in rat fetal tissues may play an important role in the induction of anomalies in the corresponding tissues.     

The power of zebrafish models for understanding the co‐occurrence of craniofacial and limb disorders

Craniofacial and limb defects are two of the most common congenital anomalies in the general population. Interestingly, these defects are not mutually exclusive. Many patients with craniofacial phenotypes, such as orofacial clefting and craniosynostosis, also present with limb defects, including polydactyly, syndactyly, brachydactyly, or ectrodactyly. The gene regulatory networks governing craniofacial and limb development initially seem distinct from one another, and yet these birth defects frequently occur together. Both developmental processes are highly conserved among vertebrates, and zebrafish have emerged as an advantageous model due to their high fecundity, relative ease of genetic manipulation, and transparency during development. Here we summarize studies that have used zebrafish models to study human syndromes that present with both craniofacial and limb phenotypes. We discuss the highly conserved processes of craniofacial and limb/fin development and describe recent zebrafish studies that have explored the function of genes associated with human syndromes with phenotypes in both structures. We attempt to identify commonalities between the two to help explain why craniofacial and limb anomalies often occur together.     

Teratogenic effects of dosage levels and time of administration of carbamazepine, sodium valproate, and diphenylhydantoin on craniofacial development in the CD-1 mouse fetus

The objective of this investigation was to study the teratogenic effects of dosage levels and time of administration of three anticonvulsant drugs (carbamazepine [CMZ], sodium valproate [NaV], and diphenylhydantoin [DPH]) on craniofacial development in the CD-1 mouse fetus. Pregnant females were intubated on each of days 8-10, 11-13, 14-16, and 8-16 of gestation with the following dose levels for each drug: 375, 563, 938 mg/kg CMZ; 225, 338, 563 mg/kg NaV; 50, 75, 125 mg/kg DPH. Appropriate control groups were maintained for each drug. On gestation day 17, pregnant females were killed and implantation sites were recorded as live, dead, or resorbed. All live fetuses were examined for craniofacial defects. Results of examination of 1,398 fetuses indicated that CMZ, NaV, and DPH were teratogenic and embryotoxic at all dose levels. This study indicated that the observed decrease in mean fetal weight was drug-, dose-, and time-dependent. There was a drug-, dose-, and time-dependent increase observed in the number of dead fetuses, whereas the number of resorbed fetuses was observed to be only time-dependent. The observed frequencies of hydrocephalies, secondary palatal clefts, and submucous palatal clefts were significant for all three factors (drug, dose, and time) whereas the observed frequencies of hematomas and exencephalies were significant only for drug and time. Cleft lips were observed only in the highest dose level of DPH. Uterine horn distribution of defects indicated that fetuses located at the proximal end of the horns were less subject to major defects than those fetuses located at the distal end of the uterine horns. Fetuses with craniofacial hematomas were found in the proximal one-third of the uterine horn, resorbed fetuses, and fetuses with submucous palatal clefts in the middle one-third of the uterine horns and dead fetuses and fetuses with exencephalies, cleft lips, and secondary palatal clefts were localized in the distal one-third of the uterine horns. In comparing the effect of drug, dosage, and time on the development of craniofacial malformations in the CD-1 mouse fetus, CMZ was the least teratogenic and embryotoxic of the three anticonvulsant drugs employed in this study.     

Face off against ROS: Tcof1/Treacle safeguards neuroepithelial cells and progenitor neural crest cells from oxidative stress during craniofacial development

One‐third of all congenital birth defects affect the head and face, and most craniofacial anomalies are considered to arise through defects in the development of cranial neural crest cells. Cranial neural crest cells give rise to the majority of craniofacial bones, cartilages and connective tissues. Therefore, understanding the events that control normal cranial neural crest and subsequent craniofacial development is important for elucidating the pathogenetic mechanisms of craniofacial anomalies and for the exploring potential therapeutic avenues for their prevention. Treacher Collins syndrome (TCS) is a congenital disorder characterized by severe craniofacial anomalies. An animal model of TCS, generated through mutation of Tcof1, the mouse (Mus musculus) homologue of the gene primarily mutated in association with TCS in humans, has recently revealed significant insights into the pathogenesis of TCS. Apoptotic elimination of neuroepithelial cells including neural crest cells is the primary cause of craniofacial defects in Tcof1 mutant embryos. However, our understanding of the mechanisms that induce tissue‐specific apoptosis remains incomplete. In this review, we describe recent advances in our understanding of the pathogenesis TCS. Furthermore, we discuss the role of Tcof1 in normal embryonic development, the correlation between genetic and environmental factors on the severity of craniofacial abnormalities, and the prospect for prenatal prevention of craniofacial anomalies.     

Morphometric analysis of the craniofacial development of the CD-1 mouse fetus exposed to alcohol on gestational day eight

Fetal alcohol syndrome (FAS) describes a pattern of dysmorphogenesis observed in some offspring of women who consumed alcohol during pregnancy; partial expression of this pattern are fetal alcohol effects (FAE). The purpose of this investigation was to measure selected craniofacial parameters in the CD-1 mouse fetus following exposure to alcohol on gestational day (D) 8. CD-1 mice were mated for 1 hr; D0.0 designated by the presence of a vaginal plug. On D8, 0 hr, and D8, 4 hr, 33 dams were injected intraperitoneally (IP) with 25% (v/v) alcohol in physiological saline solution (0.015 ml/gm maternal body weight). Appropriate controls were maintained. The animals were sacrificed every 12 hr from D12.0 through D17.0. Implantation sites were examined and recorded as live, dead, or resorbed fetuses. All live fetuses were weighed, examined for gross defects, and fixed in Bouin's solution. Twenty-three bilateral parameters were recorded for linear dimensions defining face and cranium. The fetal weights were statistically lower in treated as compared to control fetuses only on D16.0 through D17.0. Statistical analysis of the morphometrics identified five distinct growth patterns in treated mice as compared to controls. The anomalies induced in the CD-1 mouse fetus following exposure to alcohol on D8.0 resembled FAE rather than FAS. Morphometric analysis of the craniofacial region may be an important clinical tool for the quantitative identification of alcohol-related effects in the offspring of women who consumed alcohol while pregnant.     

Disorganization is a completely dominant gain-of-function mouse mutation causing sporadic developmental defects.

Disorganization (Ds) is an exceptional mutation because of its diverse and profound developmental effects. Although other mouse mutations produce similar congenital defects, extreme pleiotropism, random occurrence, developmental independence of multiple defects, and type of anomaly make Ds unique. Examples of developmental defects include cranioschisis, rachischisis, thoracoschisis, exencephaly, hamartomas, and anomalies of appendages, digestive, genital and urinary tracts, sense organs, limbs and girdles, tail and pharynx. No other mutation in the mouse has such broad effects. Ds is therefore an important model for studying not only the genetic control of lineage determination and pattern formation, but also the occurrence of sporadic congenital defects. To characterize the effects of gene dosage, we examined the viability and phenotype of Ds homozygotes and the phenotype of +/+/Ds trisomic fetuses. Occurrence of homozygotes was tested by intercrossing Ds/+ heterozygotes, typing genetic markers that flank Ds, and examining homozygotes for morphological abnormalities. Not only were Ds homozygotes found in their expected frequency, homozygotes were not more severely affected than heterozygotes. Trisomies provide a direct test for determining whether Ds is a gain-of-function mutation. Trisomic fetuses were derived by crossing Ds/Ds homozygous mice to hybrid mice that were heterozygous for two related Robertsonian translocations. Two trisomic fetuses had developmental defects characteristic of Ds mice. Together these results demonstrate that Ds is a completely dominant, gain-of-function mutation.     

Fetal pathology produced by ethylene oxide treatment of the murine zygote

Exposure of female mice to ethylene oxide by inhalation 1 or 6 h after mating produced not only multitemporal death of conceptuses but also high rates of abnormalities among surviving fetuses. In contrast, only marginal effects were observed when females were exposed 9 or 25 h after mating. The abnormalities found among 17 day gestation live fetuses were predominated by hydrops and eye defects, which, together, constitute 54% of all anomalies. Most of the remaining anomalies were distributed among 5 other types: small size, cleft palate, and cardiac, abdominal wall, or extremity and/or tail defects. In a follow-up study, the fetuses of females treated 6 h postmating were examined at 11-15 days gestation and the progression of fetal death and of malformations was studied. Results indicate that the expression of most fetal anomalies does not become apparent until late in gestation. Several of these induced anomalies are similar to common human sporadic birth defects. This new class of experimentally induced fetal anomalies provides a new avenue for investigating zygotic biology and a system for studying the progression of aberrant development.     

Lectin teratogenesis: defects produced by concanavalin A in fetal rabbits.

Concanavalin A (con A) is teratogenic to rabbit embryos during gestational days 12--15. Intracoelomic injections of 40 microliter con A solution (4 microgram/microliter) were performed on rabbit embryos during gestational days 10--15. Control embryos received either 40 microliter of saline, sham injection or no treatment. Con A caused increased fetal resorptions on days 10 and 11, but malformation levels did not differ from controls. On days 12--15, con A produced craniofacial, trunk and limb anomalies. The highest percentage of malformation occurred on day 14. The defects were classified into four groups: (1) malformations of limbs including paw and digital dysplasias as well as fusions of the limbs to the head or body wall; (2) "closure" defects such as umbilical hernia, encephalocoele, exencephaly or ectopia cordis; (3) "contracture" defects such as club paws, extended knees, or clenched digits, which exhibited normal osseous and cartilaginous skeletons; and (4) miscellaneous, non-specific anomalies including fused or dysplastic sternebrae or ribs. Histologic analysis of selected 12-day embryos 4 to 18 hours post-injection was performed to ascertain potential sites of teratogenic action. At 12 hours ectodermal necrosis was observed in the limb buds adjacent to the apical ectodermal ridge. By 18 hours, the ectoderm had eroded, exposing the basal lamina to the amniotic fluid. Focal areas of mesenchymal necrosis were observed in association with the ectodermal erosion. The potential roles of amniocentesis and limb bud repair in the genesis of the malformations are discussed.     

Compensatory defects associated with mutations in Hoxa1 restore normal palatogenesis to Hoxa2 mutants.

The rhombencephalic neural crest play several roles in craniofacial development. They give rise to the cranial sensory ganglia and much of the craniofacial skeleton, and are vital for patterning of the craniofacial muscles. The loss of Hoxa1 or Hoxa2 function affects the development of multiple neural crest-derived structures. To understand how these two genes function together in craniofacial development, an allele was generated that disrupts both of these linked genes. Some of the craniofacial defects observed in the double mutants were additive combinations of those that exist in each of the single mutants, indicating that each gene functions independently in the formation of these structures. Other defects were found only in the double mutants demonstrating overlapping or synergistic functions. We also uncovered multiple defects in the attachments and trajectories of the extrinsic tongue and hyoid muscles in Hoxa2 mutants. Interestingly, the abnormal trajectory of two of these muscles, the styloglossus and the stylohyoideus, blocked the attachment of the hyoglossus to the greater horn of the hyoid, which in turn correlated exactly with the presence of cleft palate in Hoxa2 mutants. We suggest that the hyoglossus, whose function is to depress the lateral edges of the tongue, when unable to make its proper attachment to the greater horn of the hyoid, forces the tongue to adopt an abnormal posture which blocks closure of the palatal shelves. Unexpectedly, in Hoxa1/Hoxa2 double mutants, the penetrance of cleft palate is dramatically reduced. We show that two compensatory defects, associated with the loss of Hoxa1 function, restore normal attachment of the hyoglossus to the greater horn thereby allowing the palatal shelves to lift and fuse above the flattened tongue.     

Ethylnitrosourea (ENU)-induced apoptosis in the rat fetal tissues

Ethylnitrosourea (ENU), a well known DNA alkylating agent, induces anomalies in the central nervous system (CNS), craniofacial tissues and male reproductive organs. In this study, pregnant rats were treated with 60 mg/kg ENU at day 13 of gestation, and their fetuses were examined from 1 to 48 hours after treatment (HAT) to find a clue for clarifying the mechanisms of the ENU fetotoxicity and teratogenicity. From 3 to 12 HAT, the moderate to marked increase in the number of pyknotic cells was detected in the fetal CNS, craniofacial mesenchymal tissues, gonads and so on. These pyknotic cells had nuclei positively stained by the TUNEL method, which is widely used for the detection of apoptotic nuclei, and they also showed electron microscopic characteristics identical to those of apoptotic cells. The present results strongly suggest that excess cell death by apoptosis in the fetal CNS, craniofacial tissues and gonads may have a close relation to the later occurrence of anomalies reported in these tissues following ENU-administration.     

Craniofacial anomalies of the amniotic band syndrome in serial clinical cases

Amniotic band syndrome has been proposed as a sequela of intrauterine rupture of the amnion, resulting in oligohydramnios and passage of the fetus into the chorionic cavity. Amnion disruption with loss of amniotic fluid, causing fetal compression and localized fetal ischemia, possibly results in a pathogenic mechanism of extremely variable malformations. The prominence of the nasal processes and the adjacent stomodeal orifice facilitates free band attachment and adherence, resulting in a spectrum of similarly oriented facial defects. The authors present six consecutive cases of amniotic band syndrome with cleft lip and palate (facial cleft 3, 5, 7, and 10, isolated or combined) that were associated with other craniofacial anomalies, such as craniosynostosis and hypertelorbitism. They also present limb malformations and discuss the proposed pathogenesis and the surgical challenges in functional and aesthetic restoration.     

Arachidonic acid reversal of phenytoin-induced neural tube and craniofacial defects in vitro in mice

Diphenylhydantoin (DPH), a common anticonvulsant drug, is known to produce anomalies in the craniofacial region of animals and humans. We hypothesize that phenytoin disrupts craniofacial morphogenesis by inhibiting the arachidonic acid cascade; a pathogenesis already demonstrated for glucocorticoids and hyperglycemia. This hypothesis was tested in vitro by administering DPH, with and without the addition of exogenous arachidonic acid (AA), to murine embryos. Forty-five 8.7-day-old embryos were randomly assigned to one of three groups: control, DPH, or DPH plus AA. After 48 hours in culture, all specimens were examined at 6x magnification for defects in the facial arches, head fold, and neural tube fusion. The DPH-treated specimens had a significantly greater (P less than or equal to .05) number of anomalies in each of the three anatomical areas than did the controls. Specimens cultured in DPH plus AA had significantly fewer defects in each of the three features than those treated with DPH alone. These data support the hypothesis that phenytoin disrupts normal embryonic development through interruption of the arachidonic acid cascade. Furthermore, removal of the visceral yolk sac from 50% of the specimens in control and treatment groups provided evidence that the drug had a direct effect on the embryos rather than secondary to yolk sac involvement as has been suggested in the literature.