A cell-type-specific abnormality of cell proliferation in mutant (curly tail) mouse embryos developing spinal neural tube defects

The mouse mutant curly tail (ct) provides a model system for studies of neurulation mechanisms. 60% of ct/ct embryos develop spinal neural tube defects (NTD) as a result of delayed neurulation at the posterior neuropore whereas the remaining 40% of embryos develop normally. In order to investigate the role of cell proliferation during mouse neurulation, cell cycle parameters were studied in curly tail embryos developing spinal NTD and in their normally developing litter-mates. Measurements were made of mitotic index, median length of S-phase and percent reduction of labelling index during a [3H]thymidine pulse-chase experiment. These independent measures of cell proliferation rate indicate a reduced rate of proliferation of gut endoderm and notochord cells in the neuropore region of embryos developing spinal NTD compared with normally developing controls. The incidence of cell death and the relative frequency of mitotic spindle orientations does not differ consistently between normal and abnormal embryos. These results suggest a mechanism of spinal NTD pathogenesis in curly tail embryos based on failure of normal cell proliferation in gut endoderm and notochord.     

Discontinuity of primary and secondary neural tube in spina bifida induced by retinoic acid in mice

This report shows by light microscopy the appearance of secondary neurulation separated from primary neurulation and its developmental fate in the spinal cord of mice exposed to retinoic acid in utero. The embryos and fetuses were derived from pregnant mice (ICR strain) given 60, 40, or 0 mg/kg of retinoic acid in olive oil on day 8 of gestation orally and killed 1, 2, or 10 days later. Separation of the primary neural fold from the secondary neural tube was seen in 9- and 10-day-old embryos: the caudal part of the neuroepithelium of the primary neural fold was disarranged with non-closed posterior neuropore, and underneath it the secondary neural tissue extended caudally with abnormal notochord. At term, fetuses showed spina bifida, including myeloschisis, myelocele, and diplomyelia (diastematomyelia) with abnormal distribution of ganglionic cells. These cord lesions were located between the third lumbar and second coccygeal levels. The former two cord anomalies were associated with diplomyelia and split the dorsal and ventral portions of the spinal cord with an overlapping zone between the third lumbar and third sacral levels. These findings suggest that the separation from primary neurulation is due to the lesions in both primary neural folds and notochord induced by retinoic acid and that the spinal cord caudal to the third lumbar level originates from both neuroectoderm and mesenchyme-like cells while that caudal to the third sacral level originates from mesenchyme-like cells only.     

Computer modelling of neural tube defects

Neurulation, the curling of the neuroepithelium to form the neural tube, is an essential component of the development of animal embryos. Defects of neural tube formation, which occur with an overall frequency of one in 500 human births, are the cause of severe and distressing congenital abnormalities. However, despite the fact that there is increasing information from animal experiments about the mechanisms which effect neural tube formation, much less is known about the fundamental causes of neural tube defects (NTD). The use of computer models provides one way of gaining clues about the ways in which neurulation may be compromised. Here we employ one computer model to examine the robustness of different cellular mechanisms which are thought to contribute to neurulation. The model, modified from that of Odell et al (Odell, G.M., Oster, G., Alberch, P. and Burnside, B., (1981)) mimics neurulation by laterally propagating a wave of apical contraction along an active zone within a ring of cells. We link the results to experimental evidence gained from studies of embryos in which neurulation has been perturbed. The results indicate that alteration of one of the properties of non-neural tissue can delay or inhibit neurulation, supporting the idea, gained from observation of embryos bearing genes which predispose to NTD, that the tissue underlying the neuroepithelium may contribute to the elevation of the neural folds. The results also show that reduction of the contractile properties of a small proportion of the neuroepithelial cell population may have a profound effect on overall tissue profiling. The results suggest that the elevation of the neural folds, and hence successful neurulation, may be vulnerable to relatively minor deficiencies in cell properties.     

Developmental potential of defined neural progenitors derived from mouse embryonic stem cells

The developmental potential of a uniform population of neural progenitors was tested by implanting them into chick embryos. These cells were generated from retinoic acid-treated mouse embryonic stem (ES) cells, and were used to replace a segment of the neural tube. At the time of implantation, the progenitors expressed markers defining them as Pax6-positive radial glial (RG) cells, which have recently been shown to generate most pyramidal neurons in the developing cerebral cortex. Six days after implantation, the progenitors generated large numbers of neurons in the spinal cord, and differentiated into interneurons and motoneurons at appropriate locations. They also colonized the host dorsal root ganglia (DRG) and differentiated into neurons, but, unlike stem cell-derived motoneurons, they failed to elongate axons out of the DRG. In addition, they neither expressed the DRG marker Brn3a nor the Trk neurotrophin receptors. Control experiments with untreated ES cells indicated that when colonizing the DRG, these cells did elongate axons and expressed Brn3a, as well as Trk receptors. Our results thus indicate that ES cell-derived progenitors with RG characteristics generate neurons in the spinal cord and the DRG. They are able to respond appropriately to local cues in the spinal cord, but not in the DRG, indicating that they are restricted in their developmental potential.     

Prevention of spinal neural tube defects in the mouse embryo by growth retardation during neurulation

Homozygous mutant curly tail mouse embryos developing spinal neural tube defects (NTD) exhibit a cell-type-specific abnormality of cell proliferation that affects the gut endoderm and notochord but not the neuroepithelium. We suggested that spinal NTD in these embryos may result from the imbalance of cell proliferation rates between affected and unaffected cell types. In order to test this hypothesis, curly tail embryos were subjected to influences that retard growth in vivo and in vitro. The expectation was that growth of unaffected rapidly growing cell types would be reduced to a greater extent than affected slowly growing cell types, thus counteracting the genetically determined imbalance of cell proliferation rates and leading to normalization of spinal neurulation. Food deprivation of pregnant females for 48 h prior to the stage of posterior neuropore closure reduced the overall incidence of spinal NTD and almost completely prevented open spina bifida, the most severe form of spinal NTD in curly tail mice. Analysis of embryos earlier in gestation showed that growth retardation acts by reducing the incidence of delayed neuropore closure. Culture of embryos at 40.5 degrees C for 15-23 h from day 10 of gestation, like food deprivation in vivo, also produced growth retardation and led to normalization of posterior neuropore closure. Labelling of embryos in vitro with [3H]thymidine for 1 h at the end of the culture period showed that the labelling index is reduced to a greater extent in the neuroepithelium than in other cell types in growth-retarded embryos compared with controls cultured at 38 degrees C.(ABSTRACT TRUNCATED AT 250 WORDS)     

Accumulation of basement membrane-associated hyaluronate is reduced in the posterior neuropore region of mutant (curly tail) mouse embryos developing spinal neural tube defects

We investigated the accumulation of newly synthesized glycoconjugates during spinal neurulation in mutant curly tail mouse embryos, a proportion of which develop lower spinal neural-tube defects (NTD). Embryos undergoing closure of the posterior neuropore (27- to 29-somite stage) were labeled in vitro with [3H]glucosamine, and [3H]glycoconjugates were analyzed by ion-exchange chromatography. Mutant embryos undergoing normal spinal neurulation exhibited a pattern of glycoconjugate accumulation closely similar to that observed for nonmutant embryos (Copp and Bernfield, 1988, Dev. Biol. 130, 573-582). Mutant embryos developing spinal NTD accumulated reduced amounts of [3H]hyaluronate specifically in the posterior neuropore region. Other embryonic regions and other glycoconjugates appeared unaffected by the developmental abnormality. Autoradiographic analysis of labeled curly tail embryos confirmed that [3H]hyaluronate accumulates in reduced amounts in the posterior neuropore region and indicated that this reduction is mainly localized to the site of developing basement membranes, beneath the neuroepithelium and around the notochord. Accumulation of [3H]hyaluronate in the interstitial mesenchymal matrix of the posterior neuropore region is not consistently affected in embryos developing spinal NTD. These results provide support for a role for basement-membrane hyaluronate in lower spinal neurulation.     

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).     

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.     

Spinal ganglia reduction in the splotch-delayed mouse neural tube defect mutant

Splotch and splotch-delayed mutants have anomalies in certain neural crest cell derivatives as well as neural tube defects. A genetic marker was used to identify mutant, heterozygote, and wild-type embryos within a litter, which enabled us to make intergenotypic comparisons. Histological studies of the lumbosacral region of day 15 and day 16 embryos indicated that the splotch-delayed mutant had similar but less severe defects in spinal ganglion development than those reported for splotch (Auerbach: Journal of Experimental Zoology 127:305-329, 1954). The ganglia were extensively reduced in size, residual, or missing in the splotch-delayed mutant, whereas in the splotch mutant, they were virtually nonexistent. Paired comparison analyses showed that all mutant embryos had a significant reduction in their volume of lumbosacral spinal ganglia when compared to their heterozygous and/or wild-type littermates. Also, some heterozygotes were found to have spinal ganglia volumes that were significantly reduced when compared to wild-type embryos. The volume of spinal ganglia was not related to the severity of the neural tube defect. In fact, three mutant embryos, which did not exhibit a neural tube defect, had spinal ganglia volumes comparable to or less than those mutants with open neural tube lesions or curly tails. This shows that the formation of abnormal neural crest cell derivatives is not a result of the neural tube closure defect. We hypothesize that the two anomalies observed in these mutants have a common etiological basis.     

Distribution of arginine-vasopressin in the trigeminal,dorsal root ganglia and spinal cord of the rat; depletion by capsaicin

We have measured arginine vasopressin in the neural lobe, the trigeminal ganglion (TG), dorsal root ganglia (DRG), spinal cord, trigeminal and sciatic nerves of the rat by radioimmunoassay. In control rats, the neural lobe contained 1600 pg/mg, the ganglia 52.5, 21.0, 8.5, 4.28, 3.85 pg/mg in the lumbar, sacral, cervical, thoracic, and trigeminal ganglion, respectively, the spinal cord contained 5.1, 4.3, 4.2 and 2.6 pg/mg in the lumbar, thoracic, sacral and cervical cord, respectively and the trigeminal and sciatic nerves contained 3.8 and 13 pg/mg. Neonatal capsaicin treatment depleted about 38–67% of AVP in the ganglia. Residual AVP amounted to 526.8, 30.55, 20.75, 12.88, 4.95, 2.74, 2.14, 7.94 and 2.53 pg/mg in the neural lobe, lumbar, thoracic, sacral, cervical DRG, lumbar, thoracic spinal cord, the sciatic and trigeminal nerves respectively. Capsaicin destroyed about 40.5% of total cells and 52% of AVP-immunoreactive neurons.     

Rescue of the neural tube defect of loop-tail mice by a BAC clone containing the Ltap gene

The mouse mutant loop-tail (Lp) is an accepted model for the study of neural tube defects (NTDs) in humans. Whereas Lp/+ heterozygotes show a mild tail defect (looped), homozygous Lp/Lp embryos show a very severe form of NTD, with a completely open neural tube from the hindbrain region to the caudal portion of the spinal cord (craniorachischisis). We have recently identified a positional candidate for Lp on chromosome 1, designated as Ltap. Here, we have used an in vivo complementation approach in transgenic mice to attempt to correct the looped-tail phenotype with a bacterial artificial chromosome clone (BAC280A23) that harbors a full-length copy of the Ltap gene. Genotype:phenotype correlations in Lp/+ heterozygotes carrying BAC280A23 show that this clone can rescue the looped-tail phenotype in two independent founder lines (P < 0.05 and P < 0.0001). Importantly, BAC280A23 is also observed to rescue the lethal NTD of Lp/Lp homozygotes, because several viable transgenic Lp/Lp mice could be identified and appeared normal (P < 0.05). Results from these gain-of-function transgenic animals strongly suggest that the positional candidate Ltap present in this BAC is indeed the gene that is defective in loop-tail.     

Regulation of Cell Number in Formation of the Dorsal Root Ganglion Revealed by Transplantation of Quail Neural Crest Cells into Chick Embryos

Neural crest cells appear transiently in early embryogenesis on the dorsal surface of the neural tube and subsequently migrate along specific pathways. Some migrate to between the neural tube and somites, aggregating to form the rudiments of dorsal root ganglia (DRG). The size of DRG at a given somite level is almost constant in all chick embryos. To determine the mechanisms controlling the size of DRG, we transplanted neural crest cells of 2.5-day-old quail embryos into 2.5-day-old chick embryos between the neural tube and the somites, and examined the size of DRG in these chimeric embryos with extra neural crest cells 2 days after the operation, when natural cell death in DRG had not yet occurred. The DRG on the operated side were composed of both chick and quail cells in various proportions. The cell numbers of these chimeric DRG were almost the same as those of the normal DRG on the opposite side. That is, there were significantly fewer chick cells in chimeric DRG than in DRG composed of only chick cells on the opposite unoperated side. This finding indicates that the size of DRG is not determined in migrating neural crest cells but is regulated by the circumstances.     

Effects of folic acid and homocysteine on spinal cord morphology of the chicken embryo

Maternal ingestion of folic acid (FA) reduces neural tube defects, which are associated with high homocysteine levels. Present study evaluated the effects of FA and homocysteine on cell proliferation and cell adhesion, as well as on apoptosis, throughout the development of the spinal cord and mesenchyme of chicken embryos. Normal closure of the neural tube and a regular distribution of the mesenchymal cells were observed in control and FA-treated embryos. All homocysteine-treated embryos and also 6 of 10 embryos treated with FA + homocysteine showed failure of closure of the neural tube. Homocysteine decreased the thickness of the mantle and marginal layers of the spinal cord, and FA did not prevent this effect. FA treatment reversed the decrease of proliferating cells in the spinal cord induced by homocysteine. FA-treated embryos showed the highest numerical density of apoptotic cells. Homocysteine treatment reduced NCAM expression in both spinal cord and mesenchymal tissue, and FA prevents this effect. These results are important because they demonstrate in situ that the imbalance between FA and homocysteine levels can lead to disruptions in spinal cord development, changing proliferation, apoptosis, and cell adhesion and consequently changing the arrangement of the spinal cord layers.     

Requirement of a neural tube signal for the differentiation of neural crest cells into dorsal root ganglia

The influence of the neural tube on early development of neural crest cells into sensory ganglia was studied in the chick embryo. Silastic membranes were implanted between the neural tube and the somites in 30-somite-stage embryos at the level of somites 21-24, thus separating the early migrated population of neural crest cells from the neural tube. Neural crest cells and peripheral ganglia were visualized by immunofluorescence using the HNK-1 monoclonal antibody and several histochemical techniques. Separation of crest cells from the neural tube caused the selective death of the neural crest cells from which dorsal root ganglia (DRG) would have developed. Complete disappearance of HNK-1 positive cells was evident already 10 hr after silastic implantation, before early differentiation sensory neurons could have reached their peripheral targets. In older embryos, DRG were absent at the level of implantation. In contrast, the development of ventral roots, sympathetic ganglia and adrenal gland was normal, and so was somitic differentiation into cartilage and muscle, while morphogenesis of the vertebrae was perturbed. To overcome the experimentally induced crest cell death, the silastic membranes were impregnated with a 3-day-old embryonic chick neural tube extract. Under these conditions, crest cells which were separated from the tube survived for a period of 30 hr after operation, compared to less than 10 hr in respective controls. The extract of another tissue, the liver, did not protract survival of DRG progenitor cells. Among the cells which survived with neural tube extract, some even succeeded in extending neurites; nevertheless, in absence of normal connections with the central nervous system (CNS) they finally died. Treatment of silastic implanted embryos with nerve growth factor (NGF) did not prevent the experimentally induced crest cell death. These results demonstrate that DRG develop from a population of neural crest cells which depends for its survival and probably for its differentiation upon a signal arising from the CNS, needed as early as the first hours after initiation of migration. Recovery experiments suggest that the subpopulation of crest cells which will develop along the sensory pathway probably depends for its survival and/or differentiation upon a factor contained in the neural tube, which is different from NGF.     

Therapeutic potential of in utero mesenchymal stem cell (MSCs) transplantation in rat foetuses with spina bifida aperta

Neural tube defects (NTDs) are complex congenital malformations resulting from incomplete neurulation in embryo. Despite surgical repair of the defect, most of the patients who survive with NTDs have a multiple system handicap due to neuron deficiency of the defective spinal cord. In this study, we successfully devised a prenatal surgical approach and transplanted mesenchymal stem cells (MSCs) to foetal rat spinal column to treat retinoic acid induced NTDs in rat. Transplanted MSCs survived, grew and expressed markers of neurons, glia and myoblasts in the defective spinal cord. MSCs expressed and perhaps induced the surrounding spinal tissue to express neurotrophic factors. In addition, MSC reduced spinal tissue apoptosis in NTD. Our results suggested that prenatal MSC transplantation could treat spinal neuron deficiency in NTDs by the regeneration of neurons and reduced spinal neuron death in the defective spinal cord.     

E-cadherin expression in a particular subset of sensory neurons.

We found that the dorsal root ganglia (DRG) and trigeminal ganglia of mouse embryos express the E-cadherin cell-cell adhesion molecule and analyzed its expression profile. E-cadherin expression began around Embryonic Day 12 (E12) in these ganglia, thereafter increased, and persisted to the adult stage. This cadherin was expressed by 10 and 30% of DRG neurons in E17 and postnatal animals, respectively, as well as by satellite cells and some Schwann cells. E-cadherin-positive primary sensory fibers terminated only in a narrow region of the dorsal horn of the spinal cord, which was identified as part of lamina II by double-staining for E-cadherin and substance P or somatostatin. This E-cadherin expressing area of the spinal cord extended to part of the trigeminal nucleus in the medulla. These results showed that E-cadherin is expressed in a particular subset of primary sensory neurons which may have specific functional properties. We suggest that this adhesion molecule may play a role in the selective adhesion of sensory neuronal fibers.     

The effect of back-transplants of the embryonic gut wall on growth of the neural tube

Experiments in which the developing gut of avian embryos was back-transplanted to permit the bowel to interact with the developing neural tube were undertaken. Segments of intestine from 4-day quail embryos were implanted between the somites and neural tubes of chick embryos of 7 to 24 somites. The spinal cord responded to the presence of the bowel by enlarging unilaterally on the side of the graft. This effect encompassed both gray and white matter and was accompanied by the extension of neuritic projections from the spinal cord into the enteric grafts. The growth-promoting effect of enteric transplants was manifest at all levels of the neural tube where the grafts were made and led to enlargement of the brain as well as the spinal cord; however, truncal neural crest derivatives in the region of the grafts, such as developing sympathetic and spinal ganglia, were unaffected. Neither sham operations nor grafts of ciliary ganglion, lung, pancreas, mesonephros, or rudiment of the eye mimicked the action of the gut. The effect of the bowel was manifest as early as 24 hr following back-transplantation and was found to be due to an increase in the number of cells in the neuroepithelium. The cell responsible for the ability of the gut wall to enhance neuroepithelial proliferation was not identified, but the effect lacked species specificity and could be elicited in the absence of endoderm or neural crest derivatives in the explant. We propose that the musculoconnective tissue of the gut produces a short-range diffusible factor that induces mitogenic activity in the neuroepithelial cells of the neural tube, but not in the crest cells that form sympathetic or sensory ganglia. Since the gut is not normally in apposition to the neural tube, we suggest that the physiological targets of this factor are the specialized crest cells that colonize the bowel and give rise to the enteric nervous system.     

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.