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PubMed1978

Early changes in the mouse neuroepithelium preceding exencephaly induced by hypervitaminosis A.

Excess vitamin A orally administered to pregnant mice during neurulation causes most of the embryos to become exencephalic. The study presents light and electron microscopic observations that trace the origin and course of the early cellular and tissue alterations associated with the malformation. The main effects of the teratogen are seen in the neuroepithelial cells of the cephalic region of the presumptive nervous system. The degree to which each embryo is malformed varies but no matter how extensive the abnormalities in each embryo, the neuroepithelium is always affected and the lesions are similar. Mesodermal abnormalities appear only in the most seriously affected embryos. With the electron microscope, changes are apparent in the neuroepithelial cells within a few hours after maternal treatment. These changes include the formation of abnormally shaped nuclei with swollen nuclear envelopes, dilation of the rough endoplasmic reticulum, and formation of cytoplasmic inclusions that contain DNA and/or RNA. The inclusions are thought to arise as an autophagocytic response of the cells to the sub-lethal injury induced by the teratogen. In addition, a number of cells are more severely affected and degenerate. Light microscopic examination of serial sections reveals that by 20 hours after treatment, the neuroepithelium becomes disorganized: the cells are round and misaligned and intercellular connections are lost. We postulate that the early cellular alterations lead to a disruption of the architecture of the early neuroepithelium so that the neural folds fail to meet and close. The tissue may survive the initial insult, but continues to grow in the everted manner characteristic of exencephaly.

Animals↗

PubMed1983

Craniofacial malformation in Xenopus laevis tadpoles caused by the exposure of early embryos to ethanol.

The principal features of fetal alcohol syndrome (FAS) include microcephaly, reduced body size, and characteristic facial malformation, most of which are associated with hypoplasticity of structures around the mouth. A recent study using pregnant mice suggested that the malformations in FAS are caused by the effects of ethanol on early embryos during gastrulation and neurulation. Exposure of Xenopus laevis early embryos to 1-2% ethanol until they developed to the late neurula stage produced craniofacial malformations in tadpoles which have many similarities with the described features of FAS. They include reduced length of brain, reduced body size, and hypoplasticity of the anterior end of the body around the mouth. Size reduction from the control group was about 20% in the mouth region, and 10% in other head regions. Ethanol inhibited migration of mesodermal cells toward the animal pole during gastrulation, thus inducing a smaller neural plate with reduced anterior-posterior length. These very early effects of ethanol in development seem to produce various craniofacial malformations in tadpoles, and possibly also in human infants with FAS. In contrast to mammalian embryos, one can conveniently study in detail the effects of ethanol on morphogenetic movements in various in vitro experiments by using a large number of amphibian embryos. Thus, amphibian embryos represent a valuable animal model for the study of mechanisms of the teratogenic effects of ethanol.

Animals↗

PubMed1984

Effects of ethanol on the primitive streak stage mouse embryo.

Recent studies of mouse models have suggested that malformations associated with the fetal alcohol syndrome (FAS) are caused by the effects of ethanol on early embryos during gastrulation and neurulation. A study of Xenopus laevis embryos showed that exposure of gastrula stage amphibian embryos to ethanol inhibits migration of the mesodermal cells, causes formation of small neural plates, and subsequently causes hypoplastic craniofacial malformations in tadpoles. We now report effects of ethanol on the primitive streak stage mouse embryos. An ethanol solution (25%) was injected intraperitonealy twice into mice of 6.5-7.0 days of pregnancy at a dose of 0.015 ml/gm of body weight. Histological and morphometric examinations of 7.5-day embryos, 20 hr after the second injection, showed that the epiblast layer was disorganized and shrunk with formation of many blebs. In addition, formation of the mesodermal cell layer was retarded in the ethanol-treated embryos, suggesting that exposure of gastrula stage embryos to ethanol causes similar abnormalities in mouse and Xenopus embryos. These results suggest that the inhibition of the morphogenetic movements during gastrulation may be the primary effect of ethanol in causing major craniofacial malformations of FAS.

Animals↗

PubMed1986

Methacrylic acid as a teratogen in rat embryo culture.

Using day 10 rat embryos cultured in vitro, we have shown that methacrylic acid is teratogenic at concentrations ranging from 1.2 to 2.1 mM. At these concentrations, methacrylic acid produced concentration-dependent decreases in growth parameters, i.e., crown-rump length, number of somites, and embryo protein content. In addition, methacrylic acid exposure produced malformed embryos characterized primarily by abnormal neurulation. Less frequent abnormalities included hypoplasia of the prosencephalon, edema, malpositioned heart, abnormal flexion, and dilated otic vesicles. Accompanying these abnormalities was an underlying increase in methacrylic acid-induced cell death.

Acrylates↗

PubMed1987

D-(-)-beta-hydroxybutyrate-induced effects on mouse embryos in vitro.

The effects of the "physiologically occurring" D-isomer of beta-hydroxybutyrate (D-BOHB) were evaluated in neurulating mouse embryos by using the technique of whole embryo culture. Following a 24-hour culture period D-BOHB induced malformations and growth retardation in a concentration-dependent manner. At a 48 mM concentration essentially all embryos exhibited neural tube defects, and decreased rates of glucose metabolism by the pentose phosphate pathway (PPP) and Krebs cycle were observed when compared to controls. The relationship between an inhibition of the PPP and induction of malformations by DL-BOHB has been reported, and thereby suggests a similar mechanism may be operating for the D-isomer. In contrast, the effect of the D-isomer on the Krebs cycle may result from a replacement of glucose intermediates by those generated from metabolism of D-BOHB. Concentrations as high as 20 mM D-BOHB have been reported in the serum of uncontrolled diabetic patients, and since ketones rapidly equilibrate across extraembryonic membranes, embryos in vivo may be exposed to concentrations equivalent to those which induced malformations in vitro. However, the incidence of malformations induced by D-BOHB was less than that reported for the DL-racemic mixture at equivalent concentrations, thereby suggesting that the L-isomer is also teratogenic. Therefore, until the presence and concentration of L-BOHB in the serum of diabetics are documented, the assessment of the impact of maternal ketosis on the embryo remains incomplete.

3-Hydroxybutyric Acid↗

PubMed1988

Embryonic catch-up growth after exposure to the ketone body D,L,-beta-hydroxybutyrate in vitro.

The capability of rodent embryos to recover in growth and development subsequent to exposure to an insult was investigated employing whole embryo culture. Early somite stage mouse embryos were exposed to 32 mM D,L,-beta-hydroxybutyrate (D,L,-beta OHB) for 24 hr (Period I), and were then transferred and maintained in control medium for an additional 36 hr maximum (Period II). Growth of this recovery group (Group B) was monitored at various time points of Period II and the results were compared with an unexposed control group (Group A) and another continuously-exposed reference group (Group C). At the end of Period I, 100% of D,L,-beta OHB-exposed embryos exhibited neural tube closure defects and were growth retarded. At 36 hr of Period II, cranial and caudal neural tube defects of Group B embryos were reduced to 3-7% and 0%, respectively. These embryos also demonstrated an excess in growth velocity during recovery so that at the end of Period II, total protein content was comparable to control values. In contrast, Group C embryos remained growth retarded and showed a significant increase in cranial and caudal neural tube defects (20 and 75%, respectively). Thus, neurulating mouse embryos were capable of catch-up growth following an embryotoxic exposure to D,L,-beta OHB. The progression of development of total protein values and morphological features such as elimination of neural tube defects was intimately related to the amount of time allowed for recovery. Moreover, an elevation of growth rate over normality, which is characteristic of catch-up growth, was observed.

3-Hydroxybutyric Acid↗

PubMed1989

Effects of L-asparaginase on rat embryonic development and yolk sac membrane in vitro.

A comparative analysis of the teratogenic effects of L-asparaginase on 10.5- and 11.5-day rat embryos after 24 and 48 hours of exposure in vitro, respectively, were performed. Several medium concentrations of L-asparaginase (0.05, 0.25, and 1.5 IU/ml) were tested in both embryo series. Resulting embryos were submitted to morphological studies in a search for a specific route of pathogenesis. Morphological alterations of the visceral yolk sac were also studied to investigate its contribution to L-asparaginase teratogenicity in rats. Main embryonic malformations (open truncal neural tube, open encephalic vesicles, anophthalmia, lack of inversion, abnormal frontolateral protrusions, great vascular dilations at the cephalic level) and developmental retardation were already generated after the first 24 hours of culture (embryos of 10.5 days) and presented a dose-response relationship. Vascular dilations and neurulation disturbances seemed to be related to an early mesenchyme deficiency. Reduced number of mesenchymal cells was more evident in embryos of 10.5 days than those of 11.5 days, suggesting the existence of a later compensatory mechanism of cellular proliferation in the older embryo. Visceral yolk-sac endodermal cells at both embryonic stages were greatly deformed and enlarged by an increase of the high electron-dense vacuolar system. Therefore, both a blockage of the processes of lysosomal digestion and derived trophic deficiencies probably existed. A double teratogenic mechanism for L-asparaginase is postulated: a direct action mainly in younger embryos (before invagination of the embryo into the yolk sac) and a yolk sac-mediated one.

Analysis of Variance↗

PubMed1990

Delayed neural crest cell emigration from Sp and Spd mouse neural tube explants.

Splotch (Sp) and splotch-delayed (Spd) are allelic mutations on chromosome 1 of the mouse. Embryos homozygous for either allele have neural tube defects (NTDs) and deficiencies in neural crest cell (NCC) derived structures. The fact that Spd mouse mutants sometimes have deficiencies in NCC derivatives in the absence of an NTD led to the hypothesis that neurulation and the release of NCCs may depend on a regulatory event that is common to both processes. Therefore, it may be possible to understand the cause of NTDs in these mutants by examining the basis of aberrant NCC derivatives. Caudal neural tubes were excised from day 9 Sp and Spd embryos and placed into gelatin-coated tissue culture dishes, or 3-dimensional basement membrane matrigel, and cultured for 72 hours. A cytogenetic marker was used to genotype the embryos. In planar cultures, no morphological differences were observed between NCCs from neural tube explants of Spd mutants compared to those from heterozygous or wild-type embryos. However, there appeared to be a delay in the release of NCCs from the neural tube in both Sp and Spd mutants, which was particularly evident in Sp. After 24 hours in culture, the extent of NCC outgrowth, as well as the number of NCCs emigrating from explanted neural tubes, was significantly lower in Sp and Spd mutant cultures than in controls. No differences were observed in the mitotic indices among cells which had emigrated. By 72 hours, mutant cultures and their non-mutant counterparts were similar in terms of outgrowth, cell number, and migratory capability. After 24 hours in 3-dimensional basement membrane matrigel, cell outgrowth from Sp explants was also significantly less than controls. The pattern of NCC outgrowth in both types of culture conditions indicates a 24 hour delay in mutant cultures compared to controls. This stems from a delay in the release of NCCs from the neural tube, suggesting that the defect lies within the neuroepithelium with respect to the release of NCCs.

Alleles↗

PubMed1990

Expression of a new mutation (Axd) causing axial defects in mice correlates with maternal phenotype and age.

A new autosomal mutation, Axd (axial defects), is described. Axd segregates in a simple Mendelian fashion, and it is dominant with incomplete penetrance and variable expressivity. The phenotype of Axd heterozygotes ranges from a variety of tail anomalies to visibly normal tails. Approximately 12% of neonates from curly-tail (CT) F1 (Axd/+) x F1 (Axd/+) matings exhibit open neural tube defects (NTD) in the lumbosacral region and 16% have curly tails. Mean litter sizes and resorption rates comparable to wild type indicate that homozygosity for Axd is not obligately lethal. Genetic background plays a major role in Axd expression. Strains such as BALB/cByJ allow the highest penetrance of the mutation in single dose (46%), whereas, in CF-1 mice Axd is recessive. The tail phenotype of heterozygous Axd/+ dams, in part reflective of their genetic background, correlates with the incidence of NTD in F2 offspring: CT mothers produce significantly more neonates with frank NTD than normal tail mothers. At the one embryonic period examined for this study (D13/D14 post-coitus), an 85% higher incidence of total axial defects is observed than among the F2 at birth. Unchanging litter size and the relative increase in phenotypically normal offspring by birth suggest that Axd acts by delaying posterior neural tube closure. One of the most significant findings in this study is that maternal age influences the survival of Axd embryos in utero. Axd/+ dams older than 8 months yield fewer mean implants, higher resorption rates, and fewer viable embryos with axial defects than do Axd/+ dams younger than 8 months. Axd is not allelic to nor linked to the Sp (splotch) gene which also affects neurulation.

Animals↗

PubMed1990

Biochemical basis for D,L,-beta-hydroxybutyrate-induced teratogenesis.

Previous investigations have demonstrated that a potential mechanism for D,L,-beta-hydroxybutyrate (BOHB)-induced teratogenesis in neurulating mouse embryos (5-6 somite stage) after 24 hours of exposure in vitro is mediated by an inhibition of the pentose phosphate pathway (PPP) (Hunter, et al. '87). Employing conceptuses of an earlier stage (2-3 somite stage), the biochemistry of BOHB-induced abnormalities was examined further by exposing embryos to 32 mM BOHB for 24 hour and comparing results with controls with respect to the rate of metabolism via the PPP, de novo pyrimidine biosynthesis (PB), and BOHB utilization. Moreover, the capability of these BOHB-exposed embryos to recover from such an insult was also assessed by transferring them to fresh control medium and allowing them to grow for an additional 36 hours. Both controls and BOHB-exposed embryos showed a progressive increase in rate of BOHB utilization between days 9 and 11.5 of gestation in vitro. Exposure to ketone body produced a 100% rate of neural tube defects and a 25.2% decrease in total embryonic protein content. In contrast to results obtained at the 5-6 somite stage, no inhibition of the PPP in whole conceptuses, embryos, or visceral yolk sacs was observed in the group exposed to BOHB at the 2-3 somite stage. Furthermore, a 7.5 mM D-ribose supplement, an intermediate in the PPP, was unable to rescue the younger embryos from BOHB-induced abnormalities and growth retardation. On the other hand, BOHB produced a 34.3% decrease in pyrimidine biosynthesis in the 2-3 somite embryos, but not in the visceral yolk sac. In addition, embryos recovered biochemically after being transferred to control medium, demonstrating a 25.5% overshoot in pyrimidine biosynthesis. Therefore, the mechanism of BOHB-induced teratogenesis appears to differ depending on the stage of embryonic development at the time of initial exposure.

3-Hydroxybutyric Acid↗

PubMed1991

Altered visceral yolk sac function produced by a low-molecular-weight somatomedin inhibitor.

A fraction from diabetic rat serum containing a low-molecular-weight (800-1000) somatomedin inhibitor (SI) alters growth and development in both neurulation and early limb bud staged mouse embryos in vitro. Previous studies suggested that an accumulation of serum proteins and morphological changes of the visceral yolk sac (VYS) were produced following exposure to the SI in early limb bud staged conceptuses. The morphological changes, characterized by the presence of large endosomes in the endodermal cells, suggested that the SI altered histiotrophic nutrition, whereby proteins are pinocytosed by the endodermal VYS cells and degraded to constituent amino acids. Therefore, the effects of the SI on pinocytosis and protein degradation by the VYS were evaluated using the whole embryo culture system. Results showed that the SI reduced fluid phase pinocytosis as determined by the uptake of [U-14C]sucrose, but that accumulation of [3H]leucine-labeled hemoglobin ([3H]Hb) by the VYS was greater following exposure to the SI than in controls. In contrast, the accumulation of 3H-labeled amino acids in the embryo (produced from the degradation of [3H]Hb by the VYS) was reduced by the SI. The extent of amino acid reduction in embryonic accumulation is dependent upon the concentration of SI in the culture medium and correlates with the incidence of malformations produced by the SI, i.e., high rates of malformations occur with large reductions in embryonic 3H-labeled amino acid accumulation. The apparent paradox of high [3H]Hb accumulation in the presence of decreased pinocytosis appears to be the result of altered processing of the [3H]Hb in the endodermal cells. The altered processing decreases the "elimination" of the proteins from the VYS and results in the decrease in 3H-labeled amino acid present in the embryo proper. Therefore, the SI appears to alter two processes of VYS histiotrophic function. (1) decreased pinocytosis and (2) altered protein processing, ultimately resulting in a decreased availability of substrates for the embryo. During the early stages of embryogenesis in the human, the trophoblast cells of the placenta are responsible for the transport of nutrients from the maternal to embryonic systems. Since these cells show high phagocytic and pinocytotic activities, the SI may also disrupt these processes in the chorioallantoic placenta and contribute to diabetes-induced embryopathies.

Animals↗

PubMed1992

Maternal methionine supplementation promotes the remediation of axial defects in Axd mouse neural tube mutants.

The Axd (axial defects) mouse model system (Essien et al., Teratology 42:183-194, '90) is characterized by a dominant mutation which causes posterior open neural tube defects (NTD) and a variety of tail anomalies (curly tails, or CT). Repeated backcrosses to BALB/cByJ mice have resulted in a 50% increase in Axd penetrance among neonates of heterozygous matings and loss of a correlation with maternal tail phenotype. Analysis of D12-D18 embryos from Axd/+ x Axd/+ matings indicates that soft tissues can superficially heal over some lesions from open NTD and that some curly tails can straighten (macroscopically) as gestation proceeds. Similarly, in embryos of Axd/+ x BALB crosses, there is remediation of approximately 33% of the tail flexion defects by birth. Numerous studies show that maternal nutritional status can affect the development of the neural tube and related axial structures. One nutrient of special interest is methionine, which is required for neurulation in cultured rat embryos (Coelho et al., J. Nutr. 119:1716, '89). Thus, the major question addressed by this study was whether supplemental methionine administered to Axd/+ dams crossed to Axd/+ males would alter the prenatal expression of the gene. When given IP (70 mg/kg) on D8 and D9, methionine resulted in a 41% reduction (from 29% to 17%) in the incidence of NTD in D 14 embryos (P less than 0.01).

Animals↗

PubMed1992

Inositol deficiency increases the susceptibility to neural tube defects of genetically predisposed (curly tail) mouse embryos in vitro.

Curly tail (ct/ct) mouse embryos, which have a genetic predisposition for neural tube defects (NTD), were grown in culture from the 2-5 somite stage, before the initiation of neurulation, up to the 22-24 somite stage, when closure of the anterior neural tube is normally complete. The embryos were cultured in whole rat serum or in extensively dialysed serum supplemented with glucose, amino acids, and vitamins, with inositol omitted or added at concentrations of 2, 10, 20, and 50 mg/l. Two strains were used as controls; CBA mice, which are related to curly tails, and an unrelated PO stock. It was found that ct/ct embryos were particularly sensitive to inositol deficiency; both they and the CBA embryos showed a similar high incidence of cranial NTD after culture in inositol deficient medium (12/17 and 11/18, respectively). Furthermore, the lowest dose of inositol had no effect on the frequency of head defects in ct/ct mice, though it halved the incidence in CBA embryos. With higher inositol concentrations, the majority of ct/ct embryos completed head closure normally, and their development was generally similar to that obtained in whole serum. PO embryos showed a lower proportion (5/19) of cranial NTD in the inositol deficient medium than the other two strains, and this was further reduced by even the lowest inositol dose.

Animals↗

PubMed1993

The teratogenic effects of salicylic acid on the developing nervous system in rats in vitro.

Aspirin ingestion in humans and animals has been reported to lead to a range of undesirable outcomes, including fetal death, growth retardation, and congenital abnormalities. Rat embryos were cultured for 48 h in 100-300 micrograms/ml of salicylic acid, a metabolite of aspirin, days 9.5-11.5 of gestation. When compared to growth in control embryos, a significant dose-dependent decrease in crown-rump lengths, somite numbers, and yolk sac diameters was observed. There was also a significant increase in overall dysmorphology, including eye, brachial arch, and heart anomalies, and an absence of forelimb buds. The neural tube was especially vulnerable and had frequently failed to close. Cellular and ultrastructural examination revealed extensive cell death in the neuroepithelium, with a lesser effect on the mesenchymal cells. Large condensed blebs projected into the ventricular lumen, and cell membranes as well as the basal lamina were severely disrupted, with all cytoplasmic organelles affected in dying cells. It is likely that the extensive cell necrosis and blebbing in the developing neuroepithelium at the site of neural tube fusion may be involved in failed neurulation, while necrosis at other sites in the cranial neuroepithelium may be linked with previously reported intellectual and behavioural abnormalities.

Abnormalities, Multiple↗

PubMed1994

Effects of the biguanide class of oral hypoglycemic agents on mouse embryogenesis.

The incidence of birth defects among offspring of mothers with non-insulin dependent diabetes mellitus (NIDDM) is 2-3-fold higher than among infants of non diabetics. Since mothers with NIDDM are frequently given oral hypoglycemic agents, including sulphonylureas and biguanides, to control their disease and since these agents have been associated with the occurrence of congenital malformations in humans and animals, the embryotoxic effects of the most commonly employed biguanides, metformin and phenformin, were evaluated in whole embryo culture. Neurulating mouse embryos were exposed to therapeutic concentrations (metformin 500-2,550 mg per day; phenformin 50-400 mg per day, respectively) of the compounds for 24-48 h. Concentrations of metformin in culture ranged from 0.15 to 1.8 mg/ml and phenformin from 2.5 x 10(-5) to 0.4 mg/ml. Cultures were terminated and scored for gross morphological alterations and total protein content. Metformin produced no alterations in embryonic growth and no major malformations. Approximately 10% of all embryos exposed to metformin regardless of dose, exhibited open cranial neuropores after 24 h of culture. However, this anomaly appeared to represent a delay in closure as opposed to an overt defect, since no embryos exposed to the highest concentration of the drug and cultured for 48 h showed open neural tubes. In contrast, phenformin produced dose dependent changes in incidence of malformations, protein content, and embryolethality. Malformations included neural tube closure defects, craniofacial hypoplasia, and reduction in size of the first and second visceral arches. Doses above 0.1 mg/ml produced embryolethality and all embryos were killed at the 0.4 mg/ml concentration.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1994

Early sialylation on N-CAM in splotch neural tube defect mouse embryos.

The splotch (Sp) mutant mouse is a model for neural tube defects and Waardenburg syndrome type I. The neural tube defects that arise in Sp, which include spina bifida and exencephaly, are thought to be caused by a change in the timing of the cellular events which are taking place during neurulation. Cell adhesion molecules are strongly implicated in a variety of cell-cell interactions throughout development, thus the neural cell adhesion molecule (N-CAM) may play a role in neural tube formation and closure. The N-CAM in day 9 Sp embryos is altered showing a heavy 200 kD species rather than the 180 and 140 kD isoforms which are normally present at that developmental stage [Moase and Trasler (1991) Development 113:1049-1058]. These N-CAM isoforms normally become modified post-translationally by the addition of alpha-2,8 linked polysialosyl (PSA) units beginning at gestational day 11. Sp/Sp, Sp/+, and +/+ embryos were examined by Western blot analysis with an antibody (mAb 5A5) which specifically recognizes PSA residues on N-CAM. Mutant and heterozygote embryos display a sialylated N-CAM form at 20, 14, and 12 somite-stages which is absent in controls. Enzymatic removal of PSA on N-CAM resulted in a reduction of the 200 kD PSA-free N-CAM isoforms. These results in the observed 200 kD species, and suggest that the Sp gene is involved in the regulation of expression or the post-translational modification of N-CAM.

Animals↗

PubMed1995

Interactions of Wnt-1 and Wnt-3a are essential for neural tube patterning.

Wnt-1 and Wnt-3a have been postulated to share functional redundancy in spinal cord morphogenesis due to their homologies in protein structure and overlapping expression patterns. In this study, antisense oligonucleotides and a murine whole embryo culture system were used to examine functional interactions of Wnt-1 and Wnt-3a in late gastrulation and neurulation. Early somite mouse embryos were injected with combinations of Wnt-1 and Wnt-3a antisense oligonucleotides and then grown in vitro for up to 48 hr. Simultaneous inhibition of Wnt-1 and Wnt-3a expression resulted in pattern loss in the presumptive spinal cord, which was apparent within 4 hr following antisense treatment. The neural tube was wavy, there was a reduction in the number of nuclear layers in the walls of the neural tube, and evidence of decreased cell adhesion between neuroepithelial cells by 12 hr postinjection. In addition, notochord and primitive streak abnormalities accompanied neural tube abnormalities. The existence of regulatory interactions between Wnt-1, Wnt-3a, and engrailed genes was also examined in this study. Antisense inhibition of Wnt-1 or Wnt-3a expression resulted in reduction of engrailed protein levels in the brain, somites, and spinal cord. However, simultaneous inhibition of both Wnt genes resulted in more complete loss of engrailed protein in these regions. Herein, we present data suggesting functional redundancy of Wnt-1 and Wnt-3a in neural tube patterning and in regulation of engrailed expression.

Animals↗

PubMed1995

Antisense inhibition of engrailed genes in mouse embryos reveals roles for these genes in craniofacial and neural tube development.

The roles of engrailed-1 (En-1) and engrailed-2 (En-2) have been investigated during gastrulation and neurulation in mouse embryos. Using antisense oligonucleotides and murine whole embryo culture, early somite embryos were injected with antisense phosphorothioated oligonucleotides directed against En-1 or En-2 transcripts and then grown in vitro for 48 hr. Inhibition of En-1 by antisense targeting during this period resulted in embryos with craniofacial abnormalities, specifically loss of mid- and hindbrain tissue and hypoplasia in associated neural crest derived areas such as the face and first and second pharyngeal arches. In addition, En-1 appeared to be essential in early patterning of the neural tube. Embryos removed from culture as early as 8 hr following injection exhibited undulations in the presumptive spinal cord. Histological analysis of the affected neural tubes at 48 hr showed disrupted cytoarchitecture and in some cases, apparent dorsal-ventral duplication of the neural tube and underlying notochord. Using S100 beta as a notochord marker, embryos removed from culture at 8 or 12 hr following injection exhibited loss of S100 beta expression in the notochord region subjacent to affected neural tube segments, suggesting that S100 beta, or other notochordal genes, may be downstream components of the En-1 regulatory cascade. Furthermore, antisense inhibition of En-1 induced caudal dysgenesis, suggesting disruption in primitive streak function. Antisense targeting of En-2 expression of early somite stages produced few alterations in development, although approximately one third of the embryos exhibited a series of lateral folds in the spinal cord at the level of the forelimb-bud.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗