Retinoic acid induces down-regulation of Wnt-3a, apoptosis and diversion of tail bud cells to a neural fate in the mouse embryo.

The tail bud comprises the caudal extremity of the vertebrate embryo, containing a pool of pluripotent mesenchymal stem cells that gives rise to almost all the tissues of the sacro-caudal region. Treatment of pregnant mice with 100 mg/kg all-trans retinoic acid at 9.5 days post coitum induces severe truncation of the body axis, providing a model system for studying the mechanisms underlying development of caudal agenesis. In the present study, we find that retinoic acid treatment causes extensive apoptosis of tail bud cells 24 h after treatment. Once the apoptotic cells have been removed, the remaining mesenchymal cells differentiate into an extensive network of ectopic tubules, radially arranged around the notochord. These tubules express Pax-3 and Pax-6 in a regionally-restricted pattern that closely resembles expression in the definitive neural tube. Neurofilament-positive neurons subsequently grow out from the ectopic tubules. Thus, the tail bud cells remaining after retinoic acid-induced apoptosis appear to adopt a neural fate. Wnt-3a, a gene that has been shown to be essential for tail bud formation, is specifically down-regulated in the tail bud of retinoic acid-treated embryos, as early as 2 h after retinoic acid treatment and Wnt-3a transcripts become undetectable by 10 h. In contrast, Wnt-5a and RAR-gamma are still detectable in the tail bud at that time. Extensive cell death also occurs in the tail bud of embryos homozygous for the vestigial tail mutation, in which there is a marked reduction in Wnt-3a expression. These embryos go on to develop multiple neural tubes in their truncated caudal region. These results suggest that retinoic acid induces down-regulation of Wnt-3a which may play an important role in the pathogenesis of axial truncation, involving induction of widespread apoptosis, followed by an alteration of tail bud cell fate to form multiple ectopic neural tubes.

Differential expression of glial fibrillary acidic protein (GFAP) in the retinae and visual cortices of rats with experimental renal hypertension.

To examine the expression of the GFAP protein in the retina and visual cortex under normal and pathological conditions, hypertension was induced in adult male Sprague-Dawley rats by applying silver clips onto renal arteries and the change in GFAP expression was followed by Western blotting and immunocytochemical staining. One week after operation when the induced hypertension was at the initial stage, GFAP expression in the retina was reduced to half of the sham control. By 4 weeks, when consistent hypertension was developed, a further decrease in the level of GFAP expression in the retina to one third of the sham control was observed. Immunocytochemical staining showed that the number of GFAP-positive cells in the nerve fiber layer of the retina of the hypertensive rat was reduced to less than one third of the sham control. However, similar changes in GFAP expression in the visual cortex of hypertensive rats were not observed. This study represents the first report to date on GFAP expression in the retina and visual cortex and includes discussion of the possible mechanisms through which GFAP expression is mediated.

Regional differences in morphogenesis of the neuroepithelium suggest multiple mechanisms of spinal neurulation in the mouse.

A study of neuroepithelial morphogenesis in the mouse embryo has identified three modes of neural tube formation that occur consecutively as neurulation progresses along the spinal region. The three modes of neurulation differ in the extent to which the neuroepithelium exhibits formation of "hinge points', i.e. localised bending owing to reduction in apical surface area. In Mode 1, bending occurs only in the neuroepithelium overlying the notochord, creating a median hinge point. The neural folds remain straight along both apical and basal surfaces, resulting in a neural tube with a slitshaped lumen. In Mode 2, the neuroepithelium forms paired dorsolateral hinge points, as well as a median hinge point, whereas the remaining portions of the neuroepithelium do not bend. This produces a neural tube with a diamond-shaped lumen. In Mode 3 neurulation, the entire neuroepithelium exhibits bending, so that the cells specific hinge points are not discernible; the resulting neural tube has a circular lumen. The three modes of neurulation are present in all three strains of mice studied: C57BL/6, CBA/Ca and curly tail, a mutant predisposed to neural tube defects. However, curly tail embryos exhibit a delay in transition from Mode 2 to Mode 3, preceding faulty closure of the posterior neuropore. This heterogeneity of neurulation morphogenesis in the mouse embryo indicates that the underlying mechanisms may vary along the body axis. Specifically, we suggest that Mode 1 neurulation is driven largely by forces generated extrinsic to the neuroepithelium, in adjacent tissues, whereas Mode 3 neurulation is dependent primarily on forces generated intrinsic to the neuroepithelium. Down the body axis, there is a gradual decrease in the area of ectoderm involved in neural induction and, as neurulation reaches lower spinal levels, the newly induced neural plate exhibits marked indentation from the time of its first appearance. The transition from primary neurulation (neural folding of Mode 3 type) to secondary neurulation (neural tube formation by cavitation) appears to be a smooth continuation of this trend, with loss of contact between the newly induced neuroepithelium and the outside of the embryo.

Relationship between altered axial curvature and neural tube closure in normal and mutant (curly tail) mouse embryos.

Neural tube defects, including spina bifida, develop in the curly tail mutant mouse as a result of delayed closure of the posterior neuropore at 10.5 days of gestation. Affected embryos are characterized by increased ventral curvature of the caudal region. To determine whether closure of the neuropore could be affected by this angle of curvature, we experimentally enhanced the curvature of non-mutant embryos. The amnion was opened in 9.5 day embryos; after 20 h of culture, a proportion of the embryos exhibited a tightly wrapped amnion with enhanced curvature of the caudal region compared with the control embryos in which the opened amnion remained inflated. Enhanced curvature correlated with a higher frequency of embryos with an open posterior neuropore, irrespective of developmental stage within the range, 27-32 somites. Thus, within this somite range, caudal curvature is a more accurate determinant for normal spinal neurulation than the exact somite stage. Enhanced ventral curvature of the curly tail embryo correlates with an abnormal growth difference between the neuroepithelium and ventral structures (the notochord and hindgut). We experimentally corrected this imbalance by culturing under conditions of mild hyperthermia and subsequently determined whether the angle of curvature would also be corrected. The mean angle of curvature and length of the posterior neuropore were both reduced in embryos cultured at 40.5 degrees C by comparison with control embryos cultured at 38 degrees C. We conclude that the sequence of morphogenetic events leading to spinal neural tube defects in curly tail embryos involves an imbalance of growth rates, which leads to enhanced ventral curvature that, in turn, leads to delayed closure of the posterior neuropore.

Analysis of neural tube defects in a mouse mutant using whole embryo culture.

The method of whole embryo culture permits a variety of experimental manipulations to be performed on the mammalian embryo. When used in conjunction with mouse mutants, this technique can provide information on the pathogenetic mechanisms underlying the development of birth defects. To illustrate this approach, we review in vitro studies on the development of embryos homozygous for the mutation curly tail (ct). These studies have involved making repeated observations on individual embryos, performing surgical manipulations, applying environmental influences and metabolic labelling. As a result of this work, we have now partially elucidated the developmental sequence of events that precedes the appearance of spina bifida in the ct mutant.

Curvature of the caudal region is responsible for failure of neural tube closure in the curly tail (ct) mouse embryo.

Delayed closure of the posterior neuropore (PNP) occurs to a variable extent in homozygous mutant curly tail (ct) mouse embryos, and results in the development of spinal neural tube defects (NTD) in 60% of embryos. Previous studies have suggested that curvature of the body axis may delay neural tube closure in the cranial region of the mouse embryo. In order to investigate the relationship between curvature and delayed PNP closure, we measured the extent of ventral curvature of the neuropore region in ct/ct embryos with normal or delayed PNP closure. The results show significantly greater curvature in ct/ct embryos with delayed PNP closure in vivo than in their normal littermates. Reopening of the posterior neuropore in non-mutant mouse embryos, to delay neuropore closure experimentally, did not increase ventral curvature, suggesting that increased curvature in ct/ct embryos is not likely to be a secondary effect of delayed PNP closure. Experimental prevention of ventral curvature in ct/ct embryos, brought about by implantation of an eyelash tip longitudinally into the hindgut lumen, ameliorated the delay in PNP closure. We propose, therefore, that increased ventral curvature of the neuropore region of ct/ct embryos imposes a mechanical stress, which opposes neurulation and thus delays closure of the PNP. Increased ventral curvature may arise as a result of a cell proliferation imbalance, which we demonstrated previously in affected ct/ct embryos.

Hyperglycaemia potentiates the teratogenicity of retinoic acid in diabetic pregnancy in mice.

AIMS/HYPOTHESIS: We recently showed in mice that maternal diabetes increases embryonic susceptibility to caudal regression induced by vitamin A metabolite retinoic acid. Here we tested whether in the maternal diabetic milieu hyperglycaemia is the critical factor responsible for mediating this increased susceptibility. METHODS: Non-diabetic pregnant mice were made hyperglycaemic by subcutaneous injections of glucose at regular intervals. Conversely, diabetic pregnant mice were treated with phlorizin to induce renal glucosuria and thus reduce blood glucose concentrations. Pregnant mice were treated with retinoic acid and the extent of caudal regression in mouse embryos, measured in terms of the ratio of tail length to crown-rump length was assessed. Embryos were also examined for Wnt-3a expression and cell death. RESULTS: Embryos of mice treated with glucose had a greater extent of caudal regression induced by retinoic acid than saline-treated controls, with enhanced down-regulation of Wnt-3a expression and exacerbated cell death specifically at the caudal end of the embryo. Embryos of diabetic mice treated with phlorizin had a similar extent of caudal regression to embryos of non-diabetic mice after treatment with retinoic acid. CONCLUSIONS/INTERPRETATION: Hyperglycaemia increases embryonic susceptibility to caudal regression induced by retinoic acid, with the underlying cellular and molecular changes closely mimicking those that occur in maternal diabetes. Reduction of blood glucose concentrations in diabetic mice completely abolishes this increased susceptibility to retinoic acid. These results suggest that in maternal diabetes hyperglycaemia is the critical factor responsible for potentiating the teratogenic effect of retinoic acid.

Temporal and spatial regulation of H19 imprinting in normal and uniparental mouse embryos.

The mouse H19 gene is imprinted so that the paternal copy is both methylated and repressed during fetal development. However, the CpG-rich promoter region encompassing the transcription start is not methylated in sperm; this region must therefore become methylated postzygotically. We first examined the timing of DNA methylation of this region and the corresponding expression of H19. Both parental copies are initially undermethylated in blastocysts and the paternal copy then becomes fully methylated in the embryo around implantation; this methylation is more protracted in the extraembryonic lineages, especially in the trophoblast. By contrast to the lineage-dependent methylation, we observed exclusive expression of the maternal copy in preimplantation embryos and in all the lineages of early postimplantation embryos although variability may exist in cultured embryos. This indicates that methylation of the CpG-rich promoter is not a prerequisite for the paternal repression. We then examined whether methylation and expression occurs appropriately in the absence of a maternal or a paternal genome. Both H19 copies in androgenetic embryos are fully methylated while they are unmethylated in parthenogenetic embryos. This correlates with the lack of expression in androgenetic embryos but expression in parthenogenetic embryos. However, the androgenetic trophoblast was exceptional as it shows reduced methylation and expresses H19. These results suggest that promoter methylation is not the primary inactivation mechanism but is a stabilizing factor. Differential methylation in the more upstream region, which is established in the gametes, is a likely candidate for the gametic signal and may directly control H19 activity.