A mouse model for neural tube defects: the curtailed (Tc) mutation produces spina bifida occulta in Tc/+ animals and spina bifida with meningomyelocele in Tc/t

Curtailed (Tc), a dominant mutation on mouse chromosome 17, causes a tailless phenotype and occasional hindlimb paralysis in heterozygotes. Histologically, Tc/+ embryos show a variety of abnormalities including budding and ventral duplication of the developing spinal cord, duplication and intermittent absence of the notochord, and partial or complete absence of bony vertebrae, all posterior to midliver level. When Tc is heterozygous with t-haplotypes that contain the "tail interaction factor," tct, the phenotype is more severe, and a dorsal blood blister exists in the lumbosacral area. Our microscopic observations reveal that Tc/tw5 mice have a lumbosacral spina bifida with meningomyelocele. This results from the absence of bony vertebrae, extensive thinning of the dermis dorsally, and the rupturing of the previously closed neural tube, probably by increased cerebrospinal fluid (CSF) pressure on the necrotic, attenuated roof plate. Thinning of the roof plate, which facilitates the rupturing of the spinal cord, is not observed in Tc/+, which suggests that this phenomenon is associated with the interaction of Tc with the t-allele. Later in the development of Tc/tw5 embryos, adjacent blood vessels are ruptured, resulting in hemorrhage into the CSF space to give the external appearance of a blood blister. Tc/+ mice also show an absence of bony vertebrae dorsally in the lumbosacral region, but they lack the dorsal blood blister, and the dermal layer overlying the bony defect retains its normal thickness; these observations describe a spina bifida occulta.     

The forkhead gene FH1 is involved in evolutionary modification of the ascidian tadpole larva.

The forkhead gene FH1 encodes a HNF-3beta protein required for gastrulation and development of chordate features in the ascidian tadpole larva. Although most ascidian species develop via a tadpole larva, the conventional larva has regressed into an anural (tailless) larva in some species. Molgula oculata (the tailed species) exhibits a tadpole larva with chordate features (a dorsal neural sensory organ or otolith, a notochord, striated muscle cells, and a tail), whereas its sister species Molgula occulta (the tailless species) has evolved an anural larva, which has lost these features. Here we examine the role of FH1 in modifying the larval body plan in the tailless species. We also examine FH1 function in tailless speciesxtailed species hybrids, in which the otolith, notochord, and tail are restored. The FH1 gene is expressed primarily in the presumptive endoderm and notochord cells during gastrulation, neurulation, and larval axis formation in both species and hybrids. In the tailless species, FH1 expression is down-regulated after neurulation in concert with arrested otolith, notochord, and tail development. The FH1 expression pattern characteristic of the tailed species is restored in hybrid embryos prior to the development of chordate larval features. Antisense oligodeoxynucleotides (ODNs) shown previously to disrupt FH1 function were used to compare the developmental roles of this gene in both species and hybrids. As described previously, antisense FH1 ODNs inhibited endoderm invagination during gastrulation, notochord extension, and larval tail formation in the tailed species. Antisense FH1 ODNs also affected gastrulation in the tailless species, although the effects were less severe than in the tailed species, and an anural larva was formed. In hybrid embryos, antisense FH1 ODNs blocked restoration of the otolith, notochord, and tail, reverting the larva back to the anural state. The results suggest that changes in FH1 expression are involved in re-organizing the tadpole larva during the evolution of anural development.     

A tail length modifier gene discovered in the Japanese wild mice (Mus musculus molossinus)

The presence of the t haplotypes in strains derived from the Japanese wild mice (Mus musculus molossinus) was investigated. Crosses between the T/+ heterozygous short tailed mice and five normal tailed molossinus strains (MOL-ANJ, MOA, MOL-NEM, MOM and Mns) produced no tailless mice, indicating that these strains possess no t haplotype. In contrast, tailless mice were produced by a cross between the T/+ heterozygotes and a MOL-NIS strain. Mating experiments showed that the tailless character was due to an interaction between the T gene and an autosomal recessive gene carried by the MOL-NIS strain that expresses the short tail character under the homozygous condition. We have tentatively named this gene brachyury-interacting tail length modifier (btm). It remains to be investigated whether the btm gene is located in the t complex region or in the other locus.     

Homeotic transformations of murine vertebrae and concomitant alteration of Hox codes induced by retinoic acid.

Exposure of murine embryos to teratogenic doses of retinoic acid (RA) induced homeotic transformations of vertebrae. Posterior transformations occurred along the complete body axis after RA administration on day 7 of gestation and were accompanied by anterior shifts of Hox gene expression domains in embryos. Anterior transformations of vertebrae in the caudal half of the vertebral column were induced on day 8.5. We suggest that the identity of a vertebral segment is specified by a combination of functionally active Hox genes, a "Hox code." In this concept the sequential activation of Hox genes defines sequentially more posterior axial levels, while mesodermal cells leave the primitive streak. Exogenous RA interferes with the normal establishment of Hox codes and thus with axial specification.     

Evidence of a role for endogenous electrical fields in chick embryo development.

We have tested directly the hypothesis that the endogenous electrical field in the chick embryo plays a causal role in development. Conductive implants, which shunt currents out of the embryo and thus alter the internal field, were placed under the dorsal skin at the mid-trunk level of stage 11-15 embryos. Currents leaving the posterior intestinal portal (p.i.p.) of these embryos were reduced by an average of 30%. Control embryos receiving non-conductive implants showed no change in p.i.p. currents. In the group receiving current shunts, 92% of the embryos exhibited some developmental abnormality. Only 11% of the control group displayed defects. The most common defect in the experimental group (81%) was in tail development. Tail defects ranged from complete absence to the formation of a normal length, but morphologically abnormal tail. Internally, tail structures (neural tube, notochord and somites) were frequently absent or aberrantly formed. In 33% of the experimental embryos, the notochord continued lengthening in the absence of any other tail development. This led to the formation of ourenteric outgrowths from the hindgut. Defects in limb bud and head development were also found in experimentally treated embryos, but at a much lower frequency than tail defects. The abnormalities observed in experimental embryos were very similar to those produced naturally in rumpless mutant chicks. A vibrating probe analysis of these mutants (from both dominant and recessive strains) showed that currents leaving the p.i.p. were significantly lower in phenotypically abnormal mutants than in wild-type and phenotypically normal mutant embryos from both strains. There was no apparent correlation between the average transepithelial potential (TEP) of these mutants and the development of tail abnormalities. The possible role of endogenous electrical fields in chick tail development is discussed.     

The zygotic mutant tailless affects the anterior and posterior ectodermal regions of the Drosophila embryo

The recessive zygotic lethal mutation tailless maps to region 100A5,6-B1,2 at the tip of the right arm of chromosome 3, and results in shortened pharyngeal ridges in the head skeleton of the mature embryo and the elimination of the eighth abdominal segment and telson. Although they have a normal body length, tailless embryos have a smaller number of abdominal segments, some of which are larger than normal. The mutant phenotype is seen as early as 8 hr postfertilization, when tailless embryos are observed to have fewer tracheal pits than wildtype. At 9 hr, tailless embryos appear to be missing segments A8, A9, and A10 and have an abnormal clypeolabrum, optic lobes, and procephalic lobe. Segments A4, A5, A6, and A7 appear larger in tailless embryos than wildtype at this stage. The tailless mutation, although affecting anterior and posterior ectodermal structures in the mature embryo, does not affect the formation of pole cells, the posterior midgut, or the proctodeum, which arise from the most posterior region of the embryo. The mutation does result, however, in the failure of Malpighian tubule formation. Consistent with its effect on ectodermal segments, tailless leads to a reduction in the number of segmented, paired ganglia in the ventral nerve cord as well as to an abrupt alteration in the posterior region of the tracheal system. The role the tailless gene may play in the formation of the most anterior and posterior regions of the embryo's ectodermal body plan is discussed.     

Relationship between insecticide-induced short and wry neck and cervical defects visible histologically shortly after treatment of chick embryos

In order to clarify the anatomical precursor of short and wry neck, 48-hr chick embryos were injected with 6.25-200 micrograms of the organophosphate (OP) insecticide diazinon and recovered either at 96 hr for histological evaluation or at 19 days for gross observation. Among embryos recovered at 96 hr, all receiving a dose of 25-200 micrograms showed, in serial cross sections, the cervical notochord severely folded in the vertical, horizontal, and diagonal planes and the adjacent neural tube variously folded (often with branching of its canal), deformed by the notochord, rotated, and/or displaced from the midline. Virtually all embryos injected with 6.25 or 12.5 micrograms were fully free of such abnormalities. The coinjection of 2-pyridinealdoxime methochloride (2-PAM, which protects the embryo from certain OP insecticide-induced teratisms) along with 200 micrograms of diazinon markedly reduced the notochord and neural wry neck at 19 days paralleled the 96-hr cervical histology: pronounced in all embryos receiving greater than or equal to 25 micrograms, virtually nonexistent in those receiving 6.25 or 12.5 micrograms. Though more marked at higher doses, wry neck occurred to varying extents at all doses, 6.25-100 micrograms. We conclude that 1) the primary insecticide effect is upon the notochord rather than the neural tube, 2) short neck is a direct consequence of notochord folding, 3) wry neck is apparently not linked with notochord folding, and 4) vertebral fusion is not the consequence solely of muscle paralysis as argued elsewhere. We propose that the notochord folds because diazinon disrupts normal formation of its sheath.     

A central role for the notochord in vertebral patterning

The vertebrates are defined by their segmented vertebral column, and vertebral periodicity is thought to originate from embryonic segments, the somites. According to the widely accepted 'resegmentation' model, a single vertebra forms from the recombination of the anterior and posterior halves of two adjacent sclerotomes on both sides of the embryo. Although there is supporting evidence for this model in amniotes, it remains uncertain whether it applies to all vertebrates. To explore this, we have investigated vertebral patterning in the zebrafish. Surprisingly, we find that vertebral bodies (centra) arise by secretion of bone matrix from the notochord rather than somites; centra do not form via a cartilage intermediate stage, nor do they contain osteoblasts. Moreover, isolated, cultured notochords secrete bone matrix in vitro, and ablation of notochord cells at segmentally reiterated positions in vivo prevents the formation of centra. Analysis of fss mutant embryos, in which sclerotome segmentation is disrupted, shows that whereas neural arch segmentation is also disrupted, centrum development proceeds normally. These findings suggest that the notochord plays a key, perhaps ancient, role in the segmental patterning of vertebrae.     

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)     

Morphogenetic features in the tail region of the rat embryo.

The secondary (direct) body formation is a mechanism of development in which morphogenesis of various organs occurs directly from a mass of undifferentiated mesenchymal cells (blastema) without previous formation of germ layers. It is characteristic of the posterior end of the embryonic body, i.e. of the tail bud of tailless and the tail of tailed mammals. Development of the neural tube occurring by this mechanism (secondary neurulation) has been previously explained. We investigated the morphogenetic mechanism by which two other axial structures in the rat tail develop: the tail gut and the notochord. Both structures develop from an axial condensation of undifferentiated mesenchymal cells (tail cord) of tail bud origin. The tail gut forms in a similar way to the secondary neural tube: cells in the ventral part of the tail cord elongate, acquire an apicobasal polarity and form a rosette-like structure around a lumen in the centre. The notochord forms by detachment of a group of cells of the tail cord dorsally to the developing tail gut. The peculiarities of this morphogenetic mechanism in comparison with those in other parts of the embryo are discussed. Causal (including evolutionary) explanations of this mechanism are ruled out.     

Diplomyelia: caudal duplication of the neural tube in mice

Diplomyelia (duplication of the neural tube or spinal cord) was studied histologically in nine cases of T/+ mouse embryos at 10-15 days of gestation. Grüneberg investigated T/+ fetuses and interpreted the extra neural tube to be notochord, but a reexamination of this material demonstrated that the interpretation is incorrect. Diplomyelia is produced in the mutations Fused, Kinky, vestigial tail, homozygous Brachyury, and t-haplotype t9 and in the new mutation, NM 529, described here.     

Effect of mitomycin C on the neural tube defects of the curly-tail mouse

Around 60% of the mouse mutants called curly-tail, have tail aberrations in the form of a coil or a kink, with or without lumbosacral spina bifida, and rarely, exencephaly. These neural tube defects (NTD) are the result of an incompletely penetrant recessive gene. A single injection of various doses (1-6 mg/kg) of the DNA inhibitor mitomycin C was given to pregnant curly-tail mice on day 7, 8, or 9 of gestation, and its effect on the NTD of the embryos was noted. No dose used was lethal to the embryo. When given on day 7 or day 8, mitomycin C markedly increased the number of exencephalics, and additionally, on day 8, it reduced the number of posterior abnormalities. However, on day 9, no exencephaly was produced, and there was a drastic reduction in the number of tail and spinal defects, the overall incidence of NTD being as low as 15% with 2 mg/kg. A twofold effect of mitomycin C on the curly-tail embryos was thus observed--according to the time in development it was administered, firstly, a teratogenic effect, and later, a "remedial" or preventive effect.     

Wnt signaling maintains the notochord fate for progenitor cells and supports the posterior extension of the notochord

The notochord develops from notochord progenitor cells (NPCs) and functions as a major signaling center to regulate trunk and tail development. NPCs are initially specified in the node by Wnt and Nodal signals at the gastrula stage. However, the underlying mechanism that maintains the NPCs throughout embryogenesis to contribute to the posterior extension of the notochord remains unclear. Here, we demonstrate that Wnt signaling in the NPCs is essential for posterior extension of the notochord. Genetic labeling revealed that the Noto-expressing cells in the ventral node contribute the NPCs that reside in the tail bud. Robust Wnt signaling in the NPCs was observed during posterior notochord extension. Genetic attenuation of the Wnt signal via notochord-specific β-catenin gene ablation resulted in posterior truncation of the notochord. In the NPCs of such mutant embryos, the expression of notochord-specific genes was down-regulated, and an endodermal marker, E-cadherin, was observed. No significant alteration of cell proliferation or apoptosis of the NPCs was detected. Taken together, our data indicate that the NPCs are derived from Noto-positive node cells, and are not fully committed to a notochordal fate. Sustained Wnt signaling is required to maintain the NPCs’ notochordal fate.     

Characterization of a novel insertional mouse mutation, kkt: A closely linked modifier of Pax1

We describe a novel transgene insertional mouse mutant with skeletal abnormalities characterized by a kinked tail and severe curvature of the spine. The disrupted locus is designated kkt for "kyphoscoliosis kinked tail." Malformed vertebrae including bilateral ossification centers and premature fusion of the vertebral body to the pedicles are observed along the vertebral column, and the lower thoracic and lumbar vertebrae are the most affected. Some of the homozygous kkt neonates displayed two backward-pointing transverse processes in the sixth lumbar vertebra (L6) that resembled the first sacral vertebra, and some displayed one forward- and one backward-pointing transverse process in L6. The fourth and fifth sternebrae were also fused, and the acromion process of the scapula was missing in kkt mice. The skeletal abnormalities are similar to those observed in the mouse mutant undulated (un). The transgene is integrated at the distal end of chromosome 2 close to the Pax1 gene, as revealed by FISH analysis. However, mutation of the Pax1 gene is responsible for the un phenotype, but the Pax1 gene in the kkt mice is not rearranged or deleted. Pax1 is expressed normally in kkt embryos and in the thymus of mature animals, and there is no mutation in its coding sequence. Thus, the skeletal abnormalities observed in the kkt mutant are not due to a lack of functional Pax1. Mouse genomic sequences flanking the transgene and PAC clones spanning the wild-type kkt locus have been isolated, and reverse Northern analysis showed that the PACs contain transcribed sequence. Compound heterozygotes between un and kkt (un(+/-)/kkt(+/-)) display skeletal abnormalities similar to those of un or kkt homozygotes, but they have multiple lumbar vertebrae with a split vertebral body that is more severe than in homozygous un or kkt neonates. Furthermore, the sternebrae are not fused and no backward-pointing transverse processes are detected in L6. It is therefore apparent that these two mutations do not fully complement each other, and we propose that a gene in the kkt locus possesses a unique role that functions in concert with Pax1 during skeletal development.     

Interspecific hybridization between an anural and urodele ascidian: differential expression of urodele features suggests multiple mechanisms control anural development

Anural development in the ascidian Molgula occulta was examined using tissue-specific markers and interspecific hybridization. Unlike most ascidians, which develop into a swimming tadpole larva (urodele development), M. occulta eggs develop into a tailless slug-like larva (anural development) which metamorphoses into an adult. M. occulta embryos show conventional early cleavage patterns, gastrulation, and neurulation, but then diverge from the urodele developmental mode during larval morphogenesis. M. occulta larvae do not contain a pigmented sensory cell in their brain or form a tail with differentiated notochord and muscle cells. As shown by in situ hybridization with cloned probes and analysis of in vitro translation products, M. occulta embryos do not accumulate high levels of alpha actin or myosin heavy chain mRNA. In contrast, acetylcholinesterase is expressed in muscle lineage cells, indicating that various muscle cell features are differentially suppressed. M. occulta embryos also lack tyrosinase activity, suggesting that suppression of brain pigment cell differentiation occurs at an early step in development. M. occulta eggs fertilized with sperm from Molgula oculata (a closely related urodele species) develop into hybrid larvae exhibiting some of the missing urodele features. Some hybrid embryos develop tyrosinase activity and differentiate a brain pigment cell and a short row of notochord cells, and form a short tail. These urodele features appeared together or separately in different hybrid embryos suggesting that they develop by independent mechanisms. In contrast, alpha actin and myosin heavy chain mRNA accumulation was not enhanced in hybrid embryos. These results suggest that multiple mechanisms control anural development.     

Deformation of the notochord by pressure from the swim bladder may cause malformation of the vertebral column in cultured Atlantic cod Gadus morhua larvae: a case study

This study describes a malformation that frequently occurs in Atlantic cod Gadus morhua in intensive culture systems. The malformation is characterised by a slight upward tilt of the head and an indented dorsal body contour at the transition between the head and the trunk, and is first evident to the fish farmer when the cod reach the juvenile stage. These abnormalities are associated with malformations of the neurocranium, the cranial region of the vertebral column and the cranial part of the epaxial lateral muscles. The pathogenesis involves deformation of the notochord, which can be observed in larvae about 7 d post-hatch (dph) and onwards. The deformation consists of an increase in dorsal curvature of the notochord in the region above the swim bladder. In the same region, the notochord has an abnormal cross-sectional outline, characterised by a groove-shaped, longitudinal impression along the ventral surface of the sheath. In most cases, the swim bladder fills the impression, and in severely affected larvae it forms a hernia-like lesion in the notochord. The deformation of the notochord seems to be conveyed to the vertebral body anlagen (chordacentra), which in teleosts are formed by mineralisation within the notochordal sheath. The vertebral bodies adopt an abnormal wedge shape, with a ventral concavity, and the neural arches are most often S-shaped. A continuous range of degrees of the malformation can be observed. All these pathomorphological characteristics are compatible with the notion that the notochord has been subjected to an upward mechanical force, probably generated by a persistent increase in pressure between the swim bladder and the notochord during the period of development of the vertebral anlagen. Our results thus indicate that the critical time window with regard to development of the malformation is from 18 to 36 dph, when the initial formation of the vertebrae takes place. Chronic overinflation of the swim bladder or pathological dilatation of the digestive tract may cause the lesions, and aetiology may be related to factors that influence the function of these organs.     

Programmed cell death in the ascidian embryo: modulation by FoxA5 and Manx and roles in the evolution of larval development

Programmed cell death (PCD) has been discounted in the ascidian embryo because the descendants of every embryonic cell appear to be present in the tadpole larva. Here we show that apoptotic PCD is initiated in the epidermis and central nervous system (CNS) but not in the endoderm, mesenchyme, muscle, and notochord cells during embryogenesis in molgulid ascidians. However, the affected cells do not actually die until the beginning of metamorphosis. Although specific patterns of PCD were different in distantly related ascidian species, the results suggest that removal of CNS cells by apoptosis is a urchordate feature predating the origin of the vertebrates. Certain molgulid ascidian species have evolved an anural (tailless) larva in which notochord cells fail to undergo the morphogenetic movements culminating in tail development. These anural species include Molgula occulta, the sister species of the urodele (tailed) species Molgula oculata. We show that PCD in the notochord cell lineage precedes the arrest of tail development in M. occulta and other independently evolved anural species. The notochord cells are rescued from PCD and a tail develops in hybrid embryos produced by fertilizing M. occulta eggs with M. oculata sperm, implying that apoptosis is controlled zygotically. Antisense inhibition experiments show that zygotic expression of the FoxA5 and Manx genes is required to prevent notochord PCD in urodele species and hybrids with restored tails. The results provide the first indication of PCD in the ascidian embryo and suggest that apoptosis modulated by FoxA5 and Manx is involved in notochord and tail regression during anural development. Differences in PCD that occur between ascidian species suggest that diversity in programming apoptosis may explain differences in larval form.     

Expression pattern of the Brachyury gene in whole-mount TWis/TWis mutant embryos.

The murine Brachyury (T) gene is required in mesoderm formation. Mutants carrying different T alleles show a graded severity of defects correlated with gene dosage along the body axis. The phenotypes range from shortening of the tail to the malformation of sacral vertebrae in heterozygotes, and to disruption of trunk development and embryonic death in homozygotes. Defects include a severe disturbance of the primitive streak, an early cessation of mesoderm formation and absence of the allantois and notochord, the latter resulting in an abnormality of the neural tube and somites. The T gene is expressed in nascent mesoderm and in the notochord of wild-type embryos. Here the expression of T in whole-mount mutant embryos homozygous for the T allele TWis is described. The TWis gene product is altered, but the TWis/TWis phenotype is very similar to that of T/T embryos which lack T. In early TWis/TWis embryos T expression is normal, but ceases prematurely during early organogenesis coincident with a cessation of mesoderm formation. The archenteron/node region is disrupted and the extension of the notochord precursor comes to a halt, followed by a decrease and finally a complete loss of T gene expression in the primitive streak and the head process/notochord precursor. It appears that the primary defect of the mutant embryo is the disruption of the notochord precursor in the node region which is required for axis elongation. Thus the T gene product is directly or indirectly involved in the organization of axial development.