Spinal level of myelomeningocele lesion as a contributing factor in posterior fossa volume, intracranial cerebellar volume, and cerebellar ectopia

Object McLone and Knepper's unified theory of Chiari malformation Type II (CM-II) describes how the loss of CSF via the open posterior neuropore fails to create adequate distending pressure for the developing rhomboencephalic vesicle. The authors of the present article describe the relationship between the posterior fossa volume and intracranial cerebellar volume as being related to the distance from the obex of the fourth ventricle to the myelomeningocele lesion using a common mathematical model, the Hagen-Poiseuille law. Methods All newborns who required closure of a myelomeningocele at the authors' institution between 2008 and 2011 and who were between 4 weeks premature and 2 months, corrected gestational age, at the time of MRI were included in this study. Volumes and measurements were obtained from axial and sagittal T2-weighted MR images of the brain and spine. Results: A total of 56 newborn infants met the inclusion criteria. There was a direct linear relationship between both posterior fossa volume and cerebellar volume and the spinal level of the myelomeningocele lesion (p = 0.0012 and p = 0.0041, respectively). There was a negative linear relationship between the cerebellar descent, the spinal level of the lesion, and posterior fossa volume and cerebellar volume. These relationships strengthen in patients with no syringomyelia and are not significant in those groups with syringomyelia. The results of a 1-way ANOVA for the 3 groups did not reach significance. Conclusions Using a linear equation derived from the Hagen-Poiseuille law that describes pressure in the fourth ventricle as being directly related to the length of the central canal from the obex to the myelomeningocele lesion, the authors were able to explain the directly observed linear relation between posterior fossa volume, intracranial cerebellar volume, and cerebellar descent to the level of the spinal lesion. As this model assumes a uniform radius of the central canal they were able to validate this model when they observed a strengthening in relationships in the no syringomyelia group and statistically insignificant correlations in the groups with syringomyelia. They therefore propose that the spinal level of the lesion is one of the major determinants of posterior fossa volume, intracranial cerebellar volume, and cerebellar ectopia.

Heparan sulphate proteoglycans and spinal neurulation in the mouse embryo

Heparan sulphate proteoglycans have been implicated in the binding and presentation of several growth factors to their receptors, thereby regulating cellular growth and differentiation. To investigate the role of heparan sulphate proteoglycans in mouse spinal neurulation, we administered chlorate, a competitive inhibitor of glycosaminoglycan sulphation, to cultured E8.5 embryos. Treated embryos exhibit accelerated posterior neuropore closure, accompanied by suppression of neuroepithelial bending at the median hinge point and accentuated bending at the paired dorsolateral hinge points of the posterior neuropore. These effects appear specific, as they can be prevented by addition of heparan sulphate to the culture medium, whereas heparitinase-treated heparan sulphate and chondroitin sulphate are ineffective. Both N- and O-sulphate groups appear to be necessary for the action of heparan sulphate. In situ hybridisation analysis demonstrates a normal distribution of sonic hedgehog mRNA in chlorate-treated embryos. By contrast, patched 1 transcripts are abnormally abundant in the notochord, and diminished in the overlying neuroepithelium, suggesting that sonic hedgehog signalling from the notochord may be perturbed by inhibition of heparan sulphation. Together, these results demonstrate a regulatory role for heparan sulphate in mouse spinal neurulation.

The T gene is necessary for normal mesodermal morphogenetic cell movements during gastrulation

The T (Brachyury) deletion in mouse is responsible for defective primitive streak and notochord morphogenesis, leading to a failure of the axis to elongate properly posterior to the forelimb bud. T/T embryonic stem (ES) cells colonise wild-type embryos, but in chimeras at 10.5 days post coitum (dpc) onwards they are found predominantly in the distal tail, while trunk paraxial and lateral mesoderm are deficient in T/T cells (Wilson, V., Rashbass, P. and Beddington, R. S. P. (1992) Development 117, 1321–1331). To determine the origin of this abnormal tissue distribution, we have isolated T/T and control T/+ ES cell clones which express lacZ constitutively using a gene trap strategy. Visualisation of T/T cell distribution in chimeric embryos throughout gastrulation up to 10.5 dpc shows that a progressive buildup of T/T cells in the primitive streak during gastrulation leads to their incorporation into the tailbud. These observations make it likely that one role of the T gene product is to act during gastrulation to alter cell surface (probably adhesion) properties as cells pass through the primitive streak. As the chimeric tail elongates at 10.5 dpc, abnormal morphology in the most distal portion becomes apparent. Comparison of T expression in the developing tailbud with the sites of accumulation of T/T cells in chimeras shows that T/T cells collect in sites where T would normally be expressed. T expression becomes internalised in the tailbud following posterior neuropore closure while, in abnormal chimeric tails, T/T cells remain on the surface of the distal tail. We conclude that prevention of posterior neuropore closure by the wedge of T/T cells remaining in the primitive streak after gastrulation is one source of the abnormal tail phenotypes observed. Accumulation of T/T cells in the node and anterior streak during gastrulation results in the preferential incorporation of T/T cells into the ventral portion of the neural tube and axial mesoderm. The latter forms compact blocks which are often fused with the ventral neural tube, reminiscent of the notochordal defects seen in intact mutants. Such fusions may be attributed to cell-autonomous changes in cell adhesion, possibly related to those observed at earlier stages in the primitive streak.

Genesis and prevention of spinal neural tube defects in the curly tail mutant mouse: involvement of retinoic acid and its nuclear receptors RAR-beta and RAR-gamma

A role for all-trans-retinoic acid in spinal neurulation is suggested by: (1) the reciprocal domains of expression of the retinoic acid receptors RAR-beta and RAR-gamma in the region of the closed neural tube and open posterior neuropore, respectively, and (2) the preventive effect of maternally administered retinoic acid (5 mg/kg) on spinal neural tube defects in curly tail (ct/ct) mice. Using in situ hybridisation and computerised image analysis we show here that in ct/ct embryos, RAR-beta transcripts are deficient in the hindgut endoderm, a tissue whose proliferation rate is abnormal in the ct mutant, and RAR-gamma transcripts are deficient in the tail bud and posterior neuropore region. The degree of deficiency of RAR-gamma transcripts is correlated with the severity of delay of posterior neuropore closure. As early as 2 hours following RA treatment at 10 days 8 hours post coitum, i.e. well before any morphogenetic effects are detectable, RAR-beta expression is specifically upregulated in the hindgut endoderm, and the abnormal expression pattern of RAR-gamma is also altered. These results suggest that the spinal neural tube defects which characterise the curly tail phenotype may be due to interaction between the ct gene product and one or more aspects of the retinoic acid signalling pathway.

Interaction between splotch (Sp) and curly tail (ct) mouse mutants in the embryonic development of neural tube defects

The mouse mutations splotch (Sp) and curly tail (ct) both produce spinal neural tube defects with closely similar morphology, but achieve this by different embryonic mechanisms. To determine whether the mutants may interact during development, we constructed mice carrying both mutations. Double heterozygotes exhibited tail defects in 10% of cases, although the single heterozygotes do not express this phenotype. Backcrosses of double heterozygotes to ct/ct produced offspring with an elevated incidence of neural tube defects, both spina bifida and tail defects, compared with a control backcross in which Sp was not involved. Use of the deletion allele Sp2H permitted embryos carrying a splotch mutation to be recognised by polymerase chain reaction assay. This experiment showed that only embryos carrying Sp2H develop spina bifida in the backcross with ct/ct, suggesting that the genotype Sp2H/+, ct/ct is usually lethal around the time of birth as a result of severe disturbance of neurulation. The interaction between Sp and ct was investigated further by examining embryos in the backcross for developmental markers of the Sp/Sp and ct/ct genotypes. Sp/Sp embryos characteristically lack neural crest derivatives, such as dorsal root ganglia, and die on day 13 of gestation. Double mutant embryos from the backcross did not exhibit either of these characteristics suggesting that homozygosity for ct does not cause Sp/+ embryos to develop as if they were of genotype Sp/Sp. The angle of ventral curvature of the posterior neuropore region is enhanced in affected ct/ct embryos whereas it was found to be reduced in Sp/Sp embryos compared with their normal littermates.(ABSTRACT TRUNCATED AT 250 WORDS)

Intrinsic and extrinsic factors in the mechanism of neurulation: effect of curvature of the body axis on closure of the posterior neuropore

Neurulation has been suggested to involve both factors intrinsic and extrinsic to the neuroepithelium. In the curly tail (ct) mutant mouse embryo, final closure of the posterior neuropore is delayed to varying extents resulting in neural tube defects. Evidence was presented recently (Brook et al., 1991 Development 113, 671–678) to suggest that enhanced ventral curvature of the caudal region is responsible for the neurulation defect, which probably originates from an abnormally reduced rate of cell proliferation affecting the hindgut endoderm and notochord, but not the neuroepithelium (Copp et al., 1988, Development 104, 285–295). This axial curvature probably generates a mechanical stress on the posterior neuropore, opposing normal closure. We predicted, therefore, that the ct/ct posterior neuropore should be capable of normal closure if the neuropore should be capable of normal closure if the neuroepithelium is isolated from its adjacent tissues. This prediction was tested by in vitro culture of ct/ct posterior neuropore regions, isolated by a cut caudal to the 5th from last somite. In experimental explants, the neuroepithelium of the posterior neuropore, together with the contiguous portion of the neural tube, were separated mechanically from all adjacent non-neural tissues. The posterior neuropore closed in these explants at a similar rate to isolated posterior neuropore regions of non-mutant embryos. By contrast, control ct/ct explants, in which the caudal region was isolated but the neuroepithelium was left attached to adjacent tissues, showed delayed neurulation. To examine further the idea that axial curvature may be a general mechanism regulating neurulation, we cultured chick embryos on curved substrata in vitro. Slight curvature of the body axis (maximally 1 degree per mm axial length), of either concave or convex nature, resulted in delay of posterior neuropore closure in the chick embryo. Both incidence and extent of closure delay correlated with the degree of curvature that was imposed. We propose that during normal embryogenesis the rate of neurulation is related to the angle of axial curvature, such that experimental alterations in curvature will have differing effects (either enhancement or delay of closure) depending on the angle of curvature at which neurulation normally occurs in a given species, or at a given level of the body axis.

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.

The induction of tail malformations in trisomy 16 mouse fetuses heterozygous for the curly tail recessive gene

SummaryThe mouse mutant curly tail is thought to be inherited as an autosomal recessive (ct/ct) with incomplete penetrance so that approximately 60% of ct/ct individuals exhibit the curly tail (CT) phenotype. By outcrossing ct/ct with mouse stock carrying specific heterozygous combinations of Robertsonian (Rb) chromosomes, trisomy 16 (Ts16) and Ts19 mouse fetuses (and their chromosomally balanced littermates) were derived which were heterozygous for the ct gene. All of the Ts16 (ct/Rb;Rb) fetuses, studied between days 14–19 gestation had tail malformations, 86% of which were tail flexion defects (TFD) apparently very similar to the curly tail phenotype. Neither Ts19 nor any of the chromosomally balanced (ct/Rb) littermates from both experimental crosses showed any type of tail or other spinal malformation. At the 27–29 somite stage of development, Ts16 (ct/Rb;Rb) fetuses did not show any significant delay in the closure of the posterior neuropore (PNP) compared with their littermate controls, suggesting that the tail malformation observed in Ts16 (ct/Rb;Rb) occur as a result of mechanisms which differ significantly from those thought to be responsible to causing the curly tail malformation.