Normal mouse strains differ in the site of initiation of closure of the cranial neural tube

The scanning electron microscopic study of day 9 embryos reported here documents differences among normal mouse strains in morphology of cranial neural tube closure. The site of initiation of contact and fusion of the cranial neural folds, previously defined as Closure 2 (Macdonald et al., '89), is located in the region of the junction between the forebrain (prosencephalon) and midbrain (mesencephalon) in three normal strains: LM/Bc, AEJ/RkBc, and ICR/Bc. However in a fourth normal strain, SWV/Bc, Closure 2 is initiated much further rostral, in the prosencephalic region. In addition, the anterior neuropore, rostral to Closure 2, closes late in ICR/Bc embryos, relative to the posterior progress of development of the Closure 2 seam. Initiation of closure from the most rostral end of the neural tube (Closure 3) appears to be relatively delayed in ICR/Bc embryos. We hypothesize that the observed genetic polymorphism in location of the first site of fusion between the cranial neural folds in normal mouse embryos may be one basis for differences among normal strains in liability to exencephaly induced by teratogens.     

Observations on closure of the neuropores in the chick embryo.

Neuropore closure was studied in chick embryos by light and electron microscopy. Surface ectoderm reflects over the crests of the neural folds at all craniocaudal levels, merging with the neural ectoderm lining the neural groove. Apices of surface ectodermal cells have an essentially identical morphology prior to approximation of folds, both within the presumptive fusion sites and more laterally. Cells of these areas have slightly convex profiles exhibiting few cellular protrusions. Each neural fold contains a superficial half, composed of neural ectoderm covered by surface ectoderm, and a deep half consisting entirely of neural ectoderm. Initial contact between folds usually occurs near the junction between these halves in cranial regions, but is restricted primarily to surface ectoderm at caudal levels. Subsequent fusion of folds at all levels involves both ectodermal layers. Cellular protrusions and small, morphologically unspecialized intercellular junctions often interconnect cells of apposed folds in areas undergoing fusion. The anterior neuropore closes at stages 10-11, but fusion of folds in this region is not completed until stages 13-14. Fusion occurs dorsoventrally in this area and is more advanced internally than externally. Numerous pleomorphic inclusions and a few apparently necrotic cells are present in areas bordering the anterior neuropore. The posterior neuropore closes at stages 12-13 and fusion is completed in this region during stages 13-14. The caudal end of the posterior neuropore closes dorsal to the developing tail bud. Several morphological features of this closure may at least partially account for the high susceptibility to myeloschisis localized specifically at caudal spinal cord levels.     

The two sites of fusion of the neural folds and the two neuropores in the human embryo

BACKGROUND: Since reports on a pattern of multiple sites of fusion of the neural folds in the mouse appeared, it has been widely assumed that a similar pattern must be valid for the human. In the absence of embryological evidence, claims have been made that such a pattern can be discerned by classifying neural tube defects. METHODS: The neural folds and tube, as well as the neuropores, were reassessed in 98 human embryos of Stages 8-13; 61 were controlled by precise graphic reconstructions. RESULTS: Careful study of an extensive series of staged human embryos shows that two de novo sites of fusion of the neural folds appear in succession: alpha in the rhombencephalic region and beta in the prosencephalic region, adjacent to the chiasmatic plate. Fusion from Site alpha proceeds bidirectionally (rostrad and caudad), whereas that from beta is unidirectional (caudad only). The fusions terminate in neuropores, of which there are two: rostral and caudal. Highly variable accessory loci of fusion, without positional stability and of unknown frequency, may be encountered in Stage 10 but seemingly not later, and their existence has been known for more than half a century. CONCLUSIONS: Two sites of fusion (a term preferred to closure) of the neural folds and two neuropores are found in the human embryo. No convincing embryological evidence of a pattern of multiple sites of fusion, such as has been described in the mouse, is available for the human. The construction of embryological details from information derived from other species or from the examination of later anomalies is liable to error. Neural tube defects are reviewed and although they have been considered on the basis of five, four, or three sites of fusion, interpretations based on two sites can as readily be envisaged.     

Developmental study of neural tube closure in a mouse stock with a high incidence of exencephaly

About 17% of embryos and fetuses in the SELH/Bc mouse stock have the anterior neural tube defect, exencephaly. No other malformations are seen. The genetic liability to exencephaly was shown to be probably genetically fixed in the SELH/Bc stock. This means that SELH/Bc embryos with successful neural tube closure are genetically the same as exencephalics. Females were significantly more likely to be affected than males (66% females). The pattern of morphological developmental events during anterior neural tube closure on days 8 and 9 of gestation was compared among 322 ICR/Bc (normal), 304 SWV/Bc (normal), and 265 SELH/Bc embryos. Anterior neural tube closure was found to follow a strikingly different pattern in almost all SELH/Bc embryos than in either of the normal strains or in previous published studies. SELH/Bc embryos lack the initial contact between the anterior folds in the posterior prosencephalon/anterior mesencephalon region (Closure 2). In spite of this, all but 17% manage to close the anterior neural tube by extending caudally the later occurring normal anterior zone of contact and fusion at the most rostral aspect of the prosencephalon (Closure 3) through the region of Closure 2 to meet the zone of closure of the rhombencephalon, Closure 4. Anterior neural tube closure was completed late, and in some SELH/Bc embryos, elevation and fusion in the mesencephalon did not occur at all. In histological sections of six- and eight-somite embryos, elevated numbers of pyknotic cells in the neuroepithelium and mesenchyme, and elevated numbers of unstained inclusions in the neuroepithelium were found; but their relationship, if any, to the abnormal pattern of neural tube closure is not clear.     

The initial development of the human brain.

An account of the early development of the human brain has been prepared from the data available for the Carnegie Collection, as well as from published information from other sources. Although the site of the neural plate can be discerned at stage 7, the first visible indication of the nervous system is the neural groove in certain embryos of stage 8, in which the embryonic disc measures more than 1 mm and the notochordal process at least 0.3 mm. The progressive fusion of the neural folds during stage 10, and the closure of the rostral and caudal neuropores at stages 11 and 12, respectively, are detailed with further precision than hitherto. It is emphasized that the major subdivisions of the human brain do not begin as vesicles, but as enlargements of the open neural folds at stage 9. The relationships of the neuromeres to the otic region, the somites, and the neural crest are clarified and illustrated. The early appearance of the telencephalon medium (before cerebral vesicles have formed) is stressed, and the terminological implications for the subdivisions of the brain are discussed.     

Retinoic acid-induced spina bifida: evidence for a pathogenetic mechanism

Treatment of C57Bl/6J mice with three successive doses of all-trans retinoic acid (28 mg kg-1 body weight) on 8 day, 6 h (8d,6h), 8d,12h, and 8d,18h of gestation resulted in a high incidence (79%, 31/39 fetuses) of spina bifida with myeloschisis (spina bifida aperta) in near term fetuses. Twelve hours following the last maternal dose (9d,6h), the caudal aspects of treated embryos, were abnormal, with eversion of the neural plate at the posterior neuropore, as compared to its normal concavity in comparably staged control specimens. This eversion persisted in affected embryos through the time that the posterior neuropore should normally close. The distribution of cell death in control and experimental embryos was determined using vital staining with Nile blue sulphate and with routine histological techniques. Twelve hours following the maternal dosing regimen, experimental embryos showed evidence of excessive cell death, predominantly in the mesenchyme associated with the primitive streak and in the endoderm of the tail gut, both of which are readily identifiable sites of physiological cell death at this stage of development. In addition, the presumptive trunk neural crest cells located in the dorsal midline, cranial to the posterior neuropore, exhibited a marked amount of cell death in the experimental embryos. We propose that the major factor in the generation of spina bifida in this model is excessive cell death in the tail gut and mesenchyme ventral to the neuroepithelium of the posterior neuropore.(ABSTRACT TRUNCATED AT 250 WORDS)     

Cephalic neurulation and optic vesicle formation in the early mouse embryo.

The overall pattern of cephalic neurulation and the concomitant early development of the optic vesicles in mouse embryos were examined by scanning electron microscopy. Paraffin-sectioned specimens were also examined. The overall pattern of closure of the cephalic neural folds accords well with earlier observations of this process. The earliest indication of optic placode formation was seen in histological sections of embryos at the 4-somite stage, while optic pit formation was first observed at the 5- to 6-somite stage. The upper halves of the optic vesicles were formed in 10- to 15-somite embryos by the fusion of the neural folds at the junction between the mesencephalon and prosencephalon, while closure of the lower halves was associated with the closure of the rostral neuropore, and was usually completed by about the 20-somite stage. By the 25- to 30-somite stage, a rapid increase in the volume of the forebrain was observed, so that the optic vesicles were displaced laterally. An overall increase in the volume of the optic vesicles and decrease in the diameter of the optic stalks were also observed at this time. This account of cephalic neurulation and optic organogenesis provides useful baseline data relevant to the study of the normal early development of the mouse. A comparison is made between similar events in the rat, the hamster, and the human embryo.     

Morphogenesis of the cranial segments and distribution of neural crest in the embryos of the snapping turtle,Chelydra serpentina

Recent studies of the heads of vertebrates have shown a primitive pattern of segmentation in the mesoderm and neural plate not previously recognized. The role of this pattern in the subsequent distribution of cranial crest and the development of branchial arches and cranial nerves, may resolve century-old arguments about the evolution of vertebrate segmentation. In this study, we examine the early embryonic development of the cranium of a primitive amniote, the snapping turtle, with the SEM. We show that the paraxial mesoderm cranial to the first-formed somites is segmented and that this pattern is based on somitomeres, similar to those described in the embryos of chick and mouse. Seven contiguous pairs of somitomeres comprise the “head mesoderm”; the first pair of somites actually arise from the eighth pair of somitomeres added to the axis. Cranial somitomeres are associated with specific brain regions, in that the first pair lie adjacent to prosencephalon, the second and third pair are adjacent to the mesencephalon, and the fourth, fifth, sixth, and seventh pair of somitomeres lie adjacent to individual neuromeres of the rhombencephalon. Prior to the closure of the anterior neuropore, cranial neural crest cells first emerge from the mesencephalon and migrate onto the second and third somitomeres. Shortly thereafter, neural crest cells emerge at more caudal levels of the rhombencephalon, beginning at the juncture of the fifth and sixth somitomeres. Eventually, neural crest originating from the mesencephalon spreads caudally as far as the fourth somitomere, leaving a gap in crest emigration adjacent to the fifth somitomere. The otic placode develops from the surface ectoderm covering the sixth and seventh somitomeres, and the adjacent rhombencephalic neural crest moves around the cranial and caudal edge of the placode. At more caudal levels, rhombencephalic crest cells merge with cervical crest populations to form a continuous sheet over the somites. By the time the anterior neuropore closes, some of the mesencephalic crest cells return from the paraxial mesoderm to spread onto the rostral wall of the optic vesicle and future telencephalon. The segmentation of the mesoderm and patterned distribution of cranial neural crest seen in snapping turtle embryos, further strengthens the argument that the heads of amniotes are derived from a common metameric pattern established early during gastrulation.     

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.     

Neural Tube Closure in Mouse Whole Embryo Culture

Genetic mouse models are an important tool in the study of mammalian neural tube closure (Gray & Ross, 2009; Ross, 2010). However, the study of mouse embryos in utero is limited by our inability to directly pharmacologically manipulate the embryos in isolation from the effects of maternal metabolism on the reagent of interest. Whether using a small molecule, recombinant protein, or siRNA, delivery of these substances to the mother, through the diet or by injection will subject these unstable compounds to a variety of bodily defenses that could prevent them from reaching the embryo. Investigations in cultures of whole embryos can be used to separate maternal from intrinsic fetal effects on development.Here, we present a method for culturing mouse embryos using highly enriched media in a roller incubator apparatus that allows for normal neural tube closure after dissection (Crockett, 1990). Once in culture, embryos can be manipulated using conventional in vitro techniques that would not otherwise be possible if the embryos were still in utero. Embryo siblings can be collected at various time points to study different aspects of neurulation, occurring from E7-7.5 (neural plate formation, just prior to the initiation of neurulation) to E9.5-10 (at the conclusion of cranial fold and caudal neuropore closure, Kaufman, 1992). In this protocol, we demonstrate our method for dissecting embryos at timepoints that are optimal for the study of cranial neurulation. Embryos will be dissected at E8.5 (approx. 10-12 somities), after the initiation of neural tube closure but prior to embryo turning and cranial neural fold closure, and maintained in culture till E10 (26-28 somities), when cranial neurulation should be complete.     

Expression of protocadherin 18 in the CNS and pharyngeal arches of zebrafish embryos

Here, we report the results of molecular cloning and expression analyses of a non-clustered protocadherin (pcdh), pcdh18 in zebrafish embryos. The predicted zebrafish pcdh18 protein shows 6566% identity and 7879% homology with its mammalian and Xenopus counterparts. It has a Disabled-1 binding motif in its cytoplasmic domain, which is characteristic of pcdh18. Zebrafish embryos expressed pcdh18 by the early gastrula stage, 6 h post-fertilization (hpf), in their animal cap but not in the germ ring or the shield. pcdh18 was expressed in the neural tube and the central nervous system (CNS) from 12 hpf. Some populations of cells in the lateral neural tube and spinal cord of 1218 hpf embryos expressed pcdh18, but expression in these cells disappeared by 24 hpf. The hindbrain of embryos at 2456 hpf expressed pcdh18 in cells closely adjacent to the rostral and caudal rhombomeric boundaries in a thread-like pattern running in the dorsoventral direction. The pcdh18-positive cells were localized in the ventral part of the hindbrain at 24 hpf and in the dorsal part from 36 hpf. pcdh18 was also expressed in the telencephalon, diencephalon, tectum, upper rhombic lip, retina and otic vesicle. Expression in the CNS decreased markedly before hatching. Pharyngeal arch primordia, arches, jaws and gills expressed pcdh18, and the molecule was also expressed in some endodermal cells in late embryos.     

Differential deposition of basement membrane components during formation of the caudal neural tube in the mouse embryo

The distribution of basement membrane and extracellular matrix components laminin, fibronectin, type IV collagen and heparan sulphate proteoglycan was examined during posterior neuropore closure and secondary neurulation in the mouse embryo. During posterior neuropore closure, these components were densely deposited in basement membranes of neuroepithelium, blood vessels, gut and notochord; although deposition was sparse in the midline of the regressing primitive streak. During secondary neurulation, mesenchymal cells formed an initial aggregate near the dorsal surface, which canalized and merged with the anterior neuroepithelium. With aggregation, fibronectin and heparan sulphate proteoglycan were first detected at the base of a 3- to 4-layer zone of radially organized cells. With formation of a lumen within the aggregate, laminin and type IV collagen were also deposited in the forming basement membrane. During both posterior neuropore closure and secondary neurulation, fibronectin and heparan sulphate proteoglycan were associated with the most caudal portion of the neuroepithelium, the region where newly formed epithelium merges with the consolidated neuroepithelium. In regions of neural crest migration, the deposition of basement membrane components was altered, lacking laminin and type IV collagen, with increased deposition of fibronectin and heparan sulphate proteoglycan.     

Early morphological abnormalities in splotch mouse embryos and predisposition to gene- and retinoic acid-induced neural tube defects

Genetic and environmental factors contribute to an individual's neural tube defect liability. In the mouse, the gene mutation Splotch (Sp) causes a pigmentation defect in heterozygotes while homozygotes have spina bifida +/- exencephaly. Splotch homozygotes, heterozygotes, and wild-type embryos were examined for somite number, anterior neuropore closure, and posterior neuropore length. The aim was to distinguish potentially affected homozygotes early in pathogenesis and find a morphological basis for increased teratogen susceptibility in heterozygotes. Posterior neuropore closure as well as anterior neuropore closure was significantly delayed in potentially affected Sp as compared to wild-type litter embryos exceeding the incidence found in day-10-diagnosed homozygotes. Part of this excess was attributed to a transient delay in heterozygotes which in turn might predispose to retinoic acid-induced neural tube defects. This idea was supported by an outcross of Sp heterozygote males by inbred SWV females and wild-type males by SWV where a significant increase in retinoic acid-induced neural tube defects was found in Sp carrier litters.     

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)     

Do Peanut Agglutinin Receptors on Somites Control the Behavior of Neural Cells?

Peanut agglutinin (PNA) receptors are expressed in the caudal halves of sclerotomes in chick embryos after 3 days of incubation (stages 19–20 of Hamburger & Hamilton). The neural crest cells forming dorsal root ganglia (DRG) and motor nerves appear to avoid PNA positive regions and concentrate into rostral halves of sclerotomes. To investigate the role of PNA receptors in gangliogenesis and nerve growth, we examined PNA binding ability in quail sclerotomes and in chick-quail chimeric embryos made by transplanting quail somites to chick embryos, comparing the development of DRG, motor nerves and sclerotomes. PNA did not bind to any part of the somites of 4.5-day quail embryos, although dorsal root ganglia and motor nerves appeared only in the rostral halves of sclerotomes as in chick embryos. Moreover, in spite of no PNA binding ability of the transplanted quail somite in 4.5-day chick-quail chimeric embryos, DRG and motor nerves derived from chick tissues appeared only in the rostral halves of the sclerotomes derived from these somites. Thus, both quail and chick neural crest cells and motor nerves recognized the difference between the rostral and caudal halves of sclerotomes of quail embryos in the absence of PNA binding ability, indicating that PNA binding site on somite cells does not support the selective neural crest migration and nerve growth.     

Glycosaminoglycans vary in accumulation along the neuraxis during spinal neurulation in the mouse embryo

We have utilized the method of whole embryo culture for metabolic labeling of mouse embryos with [3H]glucosamine during closure of neural folds at the posterior neuropore (27- to 29-somite stage). Accumulations of newly synthesized glycopeptides, lactosaminoglycans, hyaluronate, and sulfated glycosaminoglycans (GAG) were assessed by ion-exchange chromatography of glycoconjugates isolated from labeled embryos. Accumulation of hyaluronate and sulfated GAG was greatest in the posterior neuropore and decreased progressively toward the hindbrain where neurulation was already complete. Hyaluronate comprised a progressively smaller proportion of total newly synthesized glycoconjugate from the posterior neuropore toward the cranial region and glycopeptides showed the opposite trend. Sulfated GAG and lactosaminoglycans showed no consistent differences in relative abundance along the neuraxis. Autoradiographic analysis of newly synthesized glycoconjugates revealed especially heavy incorporation into developing basement membranes, beneath the neuroepithelium and around the notochord, in the posterior neuropore and recently closed neural tube regions, but not at more cranial levels of the neuraxis. Predigestion of sections with a specific hyaluronidase showed a significant quantity of this glycoconjugate to be hyaluronate. These results are consistent with a role for neuroepithelial and notochordal basement membrane hyaluronate in spinal neurulation.     

Cerebral dysraphia (future anencephaly) in a human twin embryo at stage 13

Cerebral dysraphia was studied histologically and by graphic reconstruction in a twin at stage 13, and comparisons were made with the normal (discordant) twin. The normal, bidirectional closure of the rostral neuropore was investigated in several embryos, from which it was concluded that the situs neuroporicus is represented by the future commissural plate rather than by the (adult) lamina terminalis. In the abnormal twin the neural tube was open over part of the midbrain and forebrain, although the situs neuroporicus was closed. The experimental production of anencephaly by Giroud and co-workers was reviewed, and comparisons between embryonic staging systems in the rat, mouse, and human were made. Three corresponding phases are found in the human: 1) cerebral dysraphia, occurring before or during Carnegie stage 11 (approximately 23-25 days); 2) exposure of a highly developing and well-differentiated brain during the remainder of the embryonic period; and 3) degeneration of the exposed brain throughout the fetal period, resulting in anencephaly. Hence the abnormal twin described here is believed to represent a precursor of typical anencephaly, and is the earliest example of purely cerebral dysraphia so far recorded.