Expression of Sonic Hedgehog downstream genes is modified in rat embryos exposed in utero to a distal inhibitor of cholesterol biosynthesis.

Holoprosencephaly is a common developmental anomaly of the forebrain and midface, that has been associated with mutations in the Sonic Hedgehog gene, and with perturbations of cholesterol synthesis and metabolism in mammalian embryos. The study presented here was aimed to evaluate the functional relationship between these two causal agents in the genesis of the phenotype. Therefore, we used rat embryos exposed in utero to a distal inhibitor of cholesterol biosynthesis (AY9944) in which we analyzed different Shh-dependent processes, as evaluated by the expression of eight target genes. In addition, to delineate between the impact of cholesterol shortage and/or sterol precursors accumulation on the Shh signaling cascade we exposed rat embryos to AY9944 and we provided complementary diets rich in cholesterol and 7-DHC. At the early-somite stage we observed a reduction of Shh signaling in AY9944 treated embryos, resulting in the definition of a narrower ventral domain. Later in development this reduction of Shh signaling led to a complete interruption of the pathway in the rostral hindbrain and caudal midbrain. Other regions such as the forebrain and the spinal cord appeared less sensitive to the reduction of Shh signaling and interruption of the pathway was only observed in a subset of embryos. Finally, we did provide evidence that 7-DHC accumulation is compatible with normal activity of Shh, as long as cholesterol levels in embryonic tissue is sufficient.

Effects of the curly tail genotype on neuroepithelial integrity and cell proliferation during late stages of primary neurulation.

The curly tail (ct/ct) mouse mutant shows a high frequency of delay or failure of neural tube closure, and is a good model for human neural tube defects, particularly spina bifida. In a previous study we defined distinct domains of gene expression in the caudal region of non-mutant embryos during posterior (caudal) neuropore closure (Gofflot et al. Developmental Dynamics 210, 431-445, 1997). Here we use BrdU incorporation into S-phase nuclei to investigate the relationship between cell proliferation and the previously described gene expression domains in ct/ct mutant embryos. The BrdU-immunostained sections were also examined for abnormalities of tissue structure; immunohistochemical detection of perlecan (an extracellular heparan sulphate proteoglycan) was used as an indicator of neuroepithelial basement membrane structure and function. Quantitation of BrdU uptake revealed that at early stages of neurulation, cell proliferation was specifically reduced in the paraxial mesoderm of all ct/ct embryos compared with wild type controls, but at later stages (more cranial levels) it was increased. Those ct/ct embryos with enlarged posterior neuropore (indicating delay of closure) additionally showed an increased BrdU labelling index within the open neuroepithelium at all axial levels; however, this tissue was highly abnormal with respect to cell and nuclear morphology. It showed cell death and loss of cells from the apical surface, basement membrane defects including increased perlecan immunoreactivity, and increased separation from the underlying mesenchyme and notochord. These observations suggest that the mechanism of delay or failure of neuroepithelial curvature that leads to neural tube defects in curly tail embryos involves abnormalities of neuroepithelial-mesenchymal interactions that may be initiated by abnormal cellular function within the neuroepithelium. Minor histological and proliferation abnormalities are present in all ct/ct embryos, regardless of phenotype.

Absence of ventral cell populations in the developing brain in a rat model of the Smith-Lemli-Opitz syndrome.

The Smith-Lemli-Opitz syndrome (SLOS) is an autosomal recessive condition involving craniofacial and central nervous system malformations with occasional holoprosencephaly (HPE). It is caused by a defect in the 7-dehydrocholesterol (7-DHC) reductase, the enzyme catalyzing the last step of cholesterol biosynthesis. Treatment of pregnant rats with inhibitors of 7-DHC reductase, either AY9944 or BM15.766, has provided a valuable model to study the pathogenesis in SLOS. Recently, cholesterol has been shown to be involved in the post-translational activation of the signaling protein Sonic Hedgehog. To identify the early defects associated with HPE in a rat model of SLOS, and to compare the phenotype of the treated embryos with that of the Shh(-/-) mutants, we examined brain morphology and expression of three developmental genes (Shh, Otx2, and Pax6 ) in 23-somite stage embryos from AY9944-treated dams. We report clearly abnormal morphology of the developing brain, concerning primarily the ventral aspect of the neural tube. We observed a reduced or absent expression of Shh and Otx2 in their ventral domain associated with extended ventral expression of Pax6. The results suggest an absence of the midline ventral cell type at all levels of the cranial neural tube. They provide further evidence that cholesterol-deficiency-induced HPE originates from impaired Shh signaling activity in the ventral neural tube.

Genetic patterning of the posterior neuropore region of curly tail mouse embryos: deficiency of Wnt5a expression.

The mouse mutant curly tail (ct) develops tail flexion defects and spina bifida as the result of delayed or failed closure of the posterior neuropore (PNP). With the aim of identifying genes involved in the chain of events resulting in defective neurulation, which can be detected at day 10.5 of development, we examined the expression patterns of a number of genes implicated in patterning of axial structures, mesoderm and neuroepithelium. The genes analyzed were Shh, HNF3alpha, HNF3beta, Brachyury, Hoxb1, Evx1, Fgf8, Wnt5a and Wnt5b. No differences could be detected between non-mutant embryos and ct/ct embryos with normal PNP size for any of these genes. Comparisons between ct/ct embryos with enlarged PNP and phenotypically normal ct/ct or nonmutant embryos showed differences only for Wnt5a. Expression of this gene was greatly reduced in the ventral caudal mesoderm and hindgut endoderm. Analysis of younger embryos revealed that prior to the stage at which embryos at risk of developing neural tube defects can be detected, the same proportion of ct/ct embryos shows reduced Wnt5a expression. The proportion of embryos showing reduced expression and almost undetectable expression of Wnt5a reflects the proportions of tail defects and spina bifida seen at later stages. We suggest that deficiency of Wnt5a signaling in the ventral caudal region tissues is an important component of the mechanism of development of the defects in affected curly tail mutant mice, and that it is causally related to decreased cell proliferation within the ventral caudal region. A possible relationship between decreased Wnt5a expression and reduced levels of heparan sulphate proteoglycan is discussed.

Postimplantation mouse embryos cultured in vitro. Assessment with whole-mount immunostaining and in situ hybridization.

The postimplantation embryos of rodents have been particularly convenient to study in culture using the whole embryo culture (WEC) system developed by New. Two serious limitations of the method will be illustrated in the present paper and proposals will be made to improve the quality of the information. The first limitation is that the developmental period amenable to culture has not been significantly extended in recent years. In the present paper, we show that the culture of mouse presomitic stages for 48 h leads to poorly reproducible results and frequent dysmorphogenic embryos. We also show that early somite stages cultured for 54 h or less have a normal growth and differentiation. In contrast, the culture of these embryos for 72 h results in subtle abnormalities of the head and the first branchial arch. The second limitation is that the gross morphology and histology are often not informative enough to distinguish between overall toxicity and developmental toxicity. We suggest some improvements by the association of WEC with two specific techniques: 1) whole-mount immunostaining of sensory ganglia and nerves and 2) in situ hybridization on histological sections using molecular probes for some developmental genes. Embryos reaching about the 30 somite stage at the end of the culture were processed for whole-mount immunostaining of sensory ganglia and nerves. We show that these structures are very sensitive to the noxious effects of HgCl2 and valproate. Both developmental retardations and dysmorphogeneses of the cervical ganglia and nerves were observed. Embryos were also exposed in vitro to low concentrations of all-trans-retinoic acid (AT-RA) and processed for in situ hybridization with radiolabeled anti-sense RNA probes for the Hoxb-1 and Hoxb-2 developmental genes. Three-dimensional reconstructions of the expression domains were performed. The data show that AT-RA induces ectopic expression domains of Hoxb-1. Our experiments demonstrate that techniques such as immunostaining and in situ hybridization can significantly expand the information obtained from whole postimplantation embryo culture.

Genetic patterning of the developing mouse tail at the time of posterior neuropore closure.

Posterior neuropore (PNP) closure coincides with the end of gastrulation, marking the end of primary neurulation and primary body axis formation. Secondary neurulation and axis formation involve differentiation of the tail bud mesenchyme. Genetic control of the primary-secondary transition is not understood. We report a detailed analysis of gene expression in the caudal region of day 10 mouse embryos during primary neuropore closure. Embryos were collected at the 27-32 somite stage, fixed, processed for whole mount in situ hybridisation, and subsequently sectioned for a more detailed analysis. Genes selected for study include those involved in the key events of gastrulation and neurulation at earlier stages and more cranial levels. Patterns of expression within the tail bud, neural plate, recently closed neural tube, notochord, hindgut, mesoderm, and surface ectoderm are illustrated and described. Specifically, we report continuity of expression of the genes Wnt5a, Wnt5b, Evx1, Fgf8, RARgamma, Brachyury, and Hoxb1 from primitive streak and node into subpopulations of the tail bud and caudal axial structures. Within the caudal notochord, developing floorplate, and hindgut, HNF3alpha, HNF3beta, Shh, and Brachyury expression domains correlate directly with known genetic roles and predicted tissue interdependence during induction and differentiation of these structures. The patterns of expression of Wnt5a, Hoxb1, Brachyury, RARgamma, and Evx1, together with observations on proliferation, reveal that the caudal mesoderm is organised at a molecular level into distinct domains delineated by longitudinal and transverse borders before histological differentiation. Expression of Wnt5a in the ventral ectodermal ridge supports previous evidence that this structure is involved in epithelial-mesenchymal interaction. These results provide a foundation for understanding the mechanisms facilitating transition from primary to secondary body axis formation, as well as the factors involved in defective spinal neurulation.

Cranial nerves and ganglia are altered after in vitro treatment of mouse embryos with valproic acid (VPA) and 4-en-VPA.

Prenatal valproic acid (VPA) exposure results in neural tube defects and in the fetal valproate syndrome (FVS), associated with developmental delay. In the present study we investigate the alterations induced by VPA and one of its metabolite, 4-en-VPA, on specific neural structures: branchial nerves and ganglia. This study was performed on 8-9 pairs of somites mouse embryos exposed in vitro for 24 h to 0.75 mM of VPA or 1 mM of 4-en-VPA. After an additional culture period of 20 h without drug, the embryos were processed for whole mount immunostaining using the monoclonal antibody 2H3, directed against the 155 kDa neurofilament protein. This technique makes it possible to visualise the branchial nerves/ganglia. VPA and 4-en-VPA induced a delay in the development of the trigeminal (V), glossopharyngeal (IX) and vagus (X) nerves/ganglia. The development of the facial (VII) nerve was delayed to a lesser extend. These treatments also induced defects in the four ganglia. The main abnormalities were a reduced dorsal component of ganglion V, the absence of the dorsal root of ganglion IX, a disorganised dorsal part of ganglion X and diffuse ventral fibres in nerves VII-VIII. In addition, scattered fibres were observed around and between ganglia. In conclusion, VPA and 4-en-VPA deeply altered the differentiation of branchial nerves/ganglia. The dorsal part of the ganglia, arising from the rhombencephalic neural crest, was particularly sensitive. The disorganisation of fibres could possibly be explained by alteration of the extracellular matrix.

Defects in the development of branchial nerves and ganglia induced by in vitro exposure of mouse embryos to mercuric chloride.

The embryotoxic and dysmorphogenic effects of mercuric chloride (HgCl2) have been studied in mouse embryos cultured in vitro. In addition, the alterations induced in the developing branchial nerves and ganglia were analyzed. Mouse embryos with 6-8 pairs of somites were exposed for 26 hr to increasing concentrations (0, 12.5, 25, 50 microM) of HgCl2. After this period, a first set of embryos was removed and a second set of embryos transferred to culture medium without HgCl2 and remained in culture for an additional 22 hr. Both sets of embryos were examined for (1) survival, (2) presence of external dysmorphogenesis, (3) growth, and (4) differentiation. Dose-related alterations of these parameters were observed. The main target was the cephalic neural tube (mainly the forebrain), but several other systems were also affected (e.g., the turning of the embryos, the optic system). The 48-hr cultured embryos were immunostained using a monoclonal antineurofilament antibody to visualize defects in the development of branchial nerves and ganglia. HgCl2 induced a pronounced retardation in the differentiation of ganglion/nerve V and a slight retardation in the differentiation of ganglia/nerves VII and IX. The ganglia/nerves VIII and X were not retarded. In addition, hight percentages of abnormalities of ganglion/nerve V and fusions between ganglia/nerves IX and X were observed in these embryos. Disorganized fibers between ganglia/nerves VII-VIII and IX and between ganglia/nerves IX and X were also more frequently observed. At the highest concentration, asymmetric defects were induced by HgCl2 with a more pronounced effect observed on the right side of the embryos. These results demonstrate the usefulness of this approach in evaluating the susceptibility of the developing branchial nerves to the adverse effects of developmental toxicants.

In vitro neuroteratogenicity of valproic acid and 4-en-VPA.

Mouse embryos displaying 8 to 9 pairs of somites were cultured during 26 h in presence of 0.75 mM of VPA, or of 1 mM of 4-en-VPA. These concentrations induced approximately 50% of dysmorphogenic embryos. Irregular suture of caudal neural tube, abnormal head shape, cranial neural tube defects, and deformed optic vesicles were the most common defects observed with both compounds. The main differences in the types of dysmorphogeneses detected between the two compounds concerned the suture of the caudal neural tube and the telencephalic region. Other macroscopic effects induced by the two compounds were similar. Several of the observed abnormalities can be correlated with defects reported after in vivo exposure. The major alteration of the histological structure of the neural tube concerned a specific area in the hindbrain : VPA and 4-en-VPA induced an abnormal and irregular budding of the neuroepithelium at this level. Immunohistology with an antibody specific for radial glial fibers (RC-2) as well as SEM analysis showed a moderate effect on glial development, mainly after exposure to VPA.

Alterations of mouse embryonic branchial nerves and ganglia induced by ethanol.

An immunostaining technique using monoclonal antibodies to a neurofilament protein has allowed us to visualize defects in the development of cranial nerves and ganglia of 10 to 10.5 days mouse embryos following exposure to ethanol in whole embryo culture. Reference patterns for development of cranial nerves and ganglia of control mouse embryos explanted and examined when they had 25 to 34 pairs of somites were established. Additionally, control mouse embryos were grown in whole embryo culture for 48 h, with culture being initiated in embryos having 6 to 7 somite pairs. At the end of the culture period, only minor differences were observed between the control groups. An experimental group of embryos was cultured in the presence of increasing doses (1.6, 3.2, 4, and 4.8 g/l) of ethanol. Defects were observed in the development of the glossopharyngeal and vagus nerves. These abnormalities included absence of the dorsal root (superior ganglion) of IX, star-like shape of inferior ganglion IX, disorganization of the rootlets of nerve X and abnormal fibers between the two nerves and ganglia. These results suggest that the migration and patterning of neural crest cells derived from r6 and r7 may be particularly affected by ethanol. The results also demonstrate the usefulness of this approach in evaluating the susceptibility of the developing cranial nerves to toxicant exposure.

Whole embryo culture of presomitic mouse embryos.

Rat embryos explanted at the presomite stage and cultured through limb bud stages develop to well formed embryos and exhibit growth and differentiation which mimic those observed in vivo at corresponding stages. In contrast, the culture of presomite stage mouse embryos has proven to be much less successful. In the present study presomitic and 3-4 somite stage mouse embryos were cultured for 48 hr. Four stages of presomitic mouse embryos corresponding to late primitive streak, early neural plate, mid neural plate and late neural plate were cultured for 24 hr in a mixture of mouse serum (12.5%), rat serum (25%) and human serum (62.5%) and during a further period of 24 hr in a mixture of human (80%) and rat (20%) serum. Two explantation procedures were used. Embryos of 3-4 somites, cultured as a reference, developed in quite a similar way to embryos in vivo and in this group very few dysmorphogeneses were observed. In contrast, the development of earlier embryos was not reproducible and the two explantation procedures led to similar results.

Developmental toxicity of valproic acid assessed in a sequential culture of hepatocytes and embryos.

The absence of maternal metabolism in the whole rodent embryo culture (WEC) may partially be considered as a limitation when chemicals are tested for teratogenicity. In the present study, the possibility to combine incubation of rat hepatocytes and WEC in a sequential way was investigated, and valproic acid (VPA) was used as a model compound. Rat hepatocytes were incubated at a density of 2 x 10(6) cells/ml in a mixture of Waymouth medium and human and rat serum (5:4:1, by vol.). After 4 hr the culture medium was recovered and used to culture 8.5-day-old mouse embryos for 24 hr. When VPA (1 mm) was added at the beginning of embryo culture, the rates of mortality and dysmorphogenesis were 87 and 100%, respectively. When VPA was added at the beginning of the incubation of hepatocytes, these values were 18 and 78%, respectively. Moreover, the differentiation of embryos was less affected when VPA was added at the beginning of the hepatocyte culture. The concentration of VPA decreased during the incubation of hepatocytes and glucurono-VPA reached 56% at the end of the incubation. Five other unconjugated metabolites were also detected. It is concluded that addition of an exogenous metabolic activation system to embryo culture results in a decrease of the teratogenic potential of VPA.

Developmental table of the early mouse post-implantation embryo.

The developmental tables of early somite mouse embryos that are presently available from the literature give clear and useful descriptions of the differentiation at successive stages. However, they provide no easy access to the correlation between the growth of the embryo and its differentiation. In the present study, quantitative data concerning normal mouse embryonic development as well as the major developmental events occurring between 0 and 30 pairs of somites were established. Measurements of growth (crown-rump length, head length, absorbancy at 280 nm) and differentiation parameters (morphological score) of 168 to 310 explanted mouse embryos were recorded for each developmental stage (number of pairs of somites). A short description of the major events occurring at the corresponding stages is also presented. The table is more detailed than those presently available and provides a rapid and practical overview of the timing of the appearance of developmental events and differentiation in correlation to the progressive growth of the embryo. It could, therefore, be useful for embryologists and toxicologists. In addition, the development of 67 post-implantation mouse embryos cultured in vitro was compared with the reference table established from in vivo embryos. Our results confirm and extend previous reports showing that embryos cultured in vitro grow and differentiate at a pace very similar to that of embryos developed in vivo.

Effects of retinoic acid, auranofin and mercuric chloride on plasminogen activator activity in post-implantation cultured mouse embryos.

Plasminogen activator (PA) activity has been suggested to be an important determinant of cell migration and tissue modelling during organogenesis. We have postulated that in the developing embryo, any abnormal modulation of this enzymatic activity may lead to the production of teratogenic effects. In the present study, we investigated the effect of teratogenic doses (inducing about 50% of malformed embryos) of retinoic acid, auranofin and mercuric chloride on PA activity in post-implantation cultured mouse embryos. At the end of the culture period, PA activities of malformed and normal embryos in the same treatment group were compared. PA activities in compound-exposed embryos were also compared with those in untreated controls. The design of the present experiment allowed the identification of the effect of drugs on PA activity and its possible relation with induced malformations. An increased PA activity was observed in malformed embryos treated with mercuric chloride. PA activity was slightly increased in both groups (normal and malformed) exposed to retinoic acid. No effect of auranofin was observed on embryo PA activity. In conclusion, the data do not confirm but they also do not contradict the hypothesis that abnormal levels of PA activity lead to dysmorphogenesis.

Early neurogenesis and teratogenesis in whole mouse embryo cultures. Histochemical, immunocytological and ultrastructural study of the premigratory neuronal-glial units in normal mouse embryo and in mouse embryos influenced by cocaine and retinoic acid.

Yolk sacs of postimplantation mouse embryos were cultured in a mixture of human and rat sera. The central nervous system of these cultured normal embryos was studied from the stage of 5-9 somites (approximately 8.5 postcoital days) to 20-21 somites (approximately 9.5 postcoital days) and compared with in vivo embryos at the same stages. This developmental period covers most of the neural tube closure, the early premigratory differentiation of the neuroectodermal epithelium, and the glial commitment of a population of germinative cells. The neuronal and glial elements of the in vitro cultivated embryos were found to be identical to the corresponding neural tissue in in vivo embryos (light and electron microscopic comparisons); the morphological identity between the in vivo and in vitro embryos was confirmed by morphometry and by stainings revealing the differentiation of the glial elements and precursors. The study of the neuronal-glial units in this material revealed that the fascicular organization of the radial glial cells occurs before the stage of 20 somites. When submitted to a single low dose of retinoic acid at the 7-somite stage, the expression of the epitope recognized by radial cell 2 (RC2), a glial marker, is delayed in the in vitro embryos 12-16 hours, but the glycogen and the other glial parameters mature in time. The in vitro embryos exposed to cocaine at the 7-somite stage displayed a prosencephalon remaining deprived of almost all glial cytological features during the entire culture period, although the other developmental parameters evolved normally. This in vitro whole embryo model seems to be a powerful tool for studying early neurogenesis and teratogenesis.