Ultrastructural analysis of the midaxial extracellular matrix in spinal dysraphism

Ultrastructural aspects of the extracellular matrix (ECM) in the midaxial region of dysraphic embryos of the loop-tail (Lp) mutant mouse were analyzed by means of electron microscopy. In 17–23 somite embryos, ultrastructural differences in the ECM occurred with respect to the presence of a pair of long trailing basal laminar strands extending continuously from the ventral notochordal cells to the gut in abnormal (Lp/Lp) embryos, in contrast to short, ragged, discontinuous strands in normal (+ /+;Lpj +) embryos. The ultrastructural localization and configuration of fibronectin (FN) and laminin (L) associated with these strands, however, were similar in normals and abnormals. In addition, FN occurred over interstitial bodies, fibrils, and sporadically along the basal laminae of the neural tube (or folds), notochord, gut, and vessels, whereas L was largely confined to the basal laminae. The results indicate that although the ultrastructural pattern of FN and L reactivity are similar in normal and abnormal embryos, a disturbance in the manner whereby the notochord detaches from the gut in dysraphic embryos may be of causal significance in the etiology of dysraphism in this mutant.     

Abnormal elevation of the neural folds in the loop-tail mutant mouse.

The processes of elevation and convergence of the spinal neural folds were analyzed in normal (+/+; Lp/+) and abnormal (Lp/Lp) embryos of the loop-tail mutant mouse in order to determine possible mechanisms underlying the dysraphic defect characterized by a failure of the neural fold to close in this mutant. The results indicate that the neural folds are already defective during very early phases of elevation, with greater distances between the apical points of the paired walls of the neural groove, larger ventral angles and higher ratios of luminal/basal linear distances occurring in the abnormal embryos relative to those in normal embryos. The cross-sectional area of the neuroepithelium is also greater in abnormals, suggesting that faulty elongation of the neuraxis may contribute to the dysraphic condition.     

Deposition of laminin and fibronectin in dysraphic mutant mice: an immunofluorescent study

Abnormal loop-tail (Lp/Lp) mutant mouse embryos exhibiting severe exencephaly and myeloschisis were analyzed and compared with their normal (+/+; Lp/+) littermates by means of immunofluorescence histochemistry to determine regional differences in the distribution of laminin (L) and fibronectin (FN). In the neural basement membrane and adjacent mesenchymal cell matrix of the abnormal embryos, regional differences in the deposition of L and FN were similar to those in normal littermates. Moreover, most of the putative neural crest (NC) cells appeared to emigrate normally in terms of their site of detachment and migration pathways, despite the severe topographic distortions and loss of neuroepithelial integrity. However, some putative NC cells projected incorrectly from the 'luminal' surface of the neuroepithelium, suggesting that some of the NC may be abnormal or sequestered and prevented from appropriate detachment and emigration from the neural tube.     

Histochemical analysis of the neural basal lamina in the hindbrain region of exencephalic mutant mice

The neural basal lamina in hindbrain regions of exencephalic loop-tail (Lp/Lp) mice and of their normal (+/+; Lp/+) littermates was analyzed histochemically at the electron microscopic level by means of enzyme digestion and alcian blue staining with critical electrolyte concentrations (CEC) of MgCl2. At 9 days of gestation, the normal and abnormal embryos showed a similar pattern of alcian blue staining with a CEC of 0.00 M or 0.05 M MgCl2. However, with a CEC of 0.30 M MgCl2, the basal lamina in the abnormals stained more prominently, particularly the lamina rara externa, suggesting the presence of more sulfated glycosaminoglycans (GAG) in the abnormals. Moreover, predigestion of the tissues with Streptomyces hyaluronidase, which removes hyaluronic acid (HA), indicated that the abnormal basal lamina contained relatively less HA than in the normal embryos. By 10 days of gestation the normal basal lamina contained relatively more sulfated GAG and less HA and was thus more similar in appearance to that in the abnormal embryos. This apparently premature shift from HA predominance to sulfated GAG predominance in the abnormal basal lamina may be of significance in the etiology of dysraphism in this mutant.     

Aberrant convergence of the neural folds in the mouse mutant vl.

Progressive changes in the dorsolateral angles (DA) and ventral angle (VA) during elevation and convergence of the caudal neural folds were morphometrically analyzed in normal and dysraphic abnormal embryos of the mouse mutant vacuolated lens (vl), and correlations with the configuration of microfilaments in the apices of neuroepithelial cells were made by means of ultrastructural cytochemistry. In 22-28 somite stage abnormal (vl/vl) embryos, the DA and VA are larger than those in their normal counterparts at each comparable level of the caudal neural folds, suggesting that defective convergence involves both the DA and VA in this mutant. In 30-35 somite stage abnormal embryos, the VA is likewise larger than that in normal embryos in which the neural folds have converged and closed; however, the DAs are much smaller, indicating that a medial collapse of the dorsal ends of the neural folds may occur secondary to the closure failure. At the DA, the ultrastructural configuration of microfilaments is similar in abnormal and normal embryos in terms of their circumferential arrangement around the perimeters of the neuroepithelial cell apices. In abnormal embryos, however, the bundles of microfilaments are more delicate and less prominent than in normal embryos; thus it is possible that a quantitative and/or functional deficiency in these elements may be involved in the failure of the abnormal neuroepithelium to bend properly during convergence of the neural folds.     

Spatial expression of the alternatively spliced EIIIB and EIIIA segments of fibronectin in the early chicken embryo

Using domain-specific antibodies, we have analyzed the tissue distribution of fibronectins (FNs) containing the alternatively spliced EIIIB and EIIIA segments relative to total FN in early chicken embryos. The results show a selective loss of EIIIA+ FN staining in the notochordal sheath and in cartilaginous structures between 4.5 and 7.0 days of development. In other regions, EIIIB+ and EIIIA+ FNs are extensively codistributed in and around mesoderm-derived structures (somites, notochord, heart, and blood vessels), in basal laminae of endoderm and ectoderm-derived structures, as well as within the vicinity of neural crest formation and migration. We also noted that EIIIA staining overlaps with spatial patterns of distribution that have previously been described for the alpha4 integrin subunit, a component of the EIIIA receptor alpha4beta1.     

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.     

Ultrastructural analysis of basal neuroepithelial cells in dysraphic mice

Ultrastructural aspects of the cellular pathology in the basal neuroepithelium of the hindbrain and spinal cord were analyzed in dysraphic loop-tail mice at nine days of gestation. Whereas the basal cytoplasm of the neuroepithelium in normal littermates showed a consistent electron density, the neuroepithelium in abnormal embryos was characterized by "light" and "dark" cells scattered randomly along the basal aspect of the hindbrain and spinal cord. In the abnormals, gaps occurred in the neural basal lamina, and the neuroepithelial cells often were in direct contact with cytoplasmic processes from mesenchymal cells and from notochordal cells; in normal littermates, contact was observed only between the intact and continuous neural basal lamina and mesenchymal cells and notochordal cells. Thus, it is possible that the pathological features observed ultrastructurally in the basal neuroepithelium in dysraphic embryos may represent faulty tissue interaction with adjacent notochordal and mesenchymal cells.     

Immunogold localization of procollagen III,fibronectin and heparan sulfate proteoglycan on ultrathin frozen sections of the normal rat liver

Summary In the present study, we have localized by immunocytochemistry at the LM and EM level, procollagen type III (PIIIP), fibronectin (FN) and heparan sulfate proteoglycan (HSPG). Intracellularly, PIIIP was observed in both parenchymal and endothelial cells. In parenchymal cells, PIIIP was found in Golgi derived vesicles. This observation suggests that PIIIP synthesis is a normal function liver parenchymal cells. In endothelial cells, vesicles, which could not be identified, were seen to contain PIIIP. This result does not allow to conclude, whether sinusoidal endothelial cells secrete or take up PIIIP. Extracellularly, PIIIP was present around portal and central veins, in the space of Disse and between adjacent parenchymal cells. In the space of Disse, almost all interstitial collagen fibrils reacted with the anti PIIIP antibodies. This observation leads to the conclusion that most fibrils of the space of Disse contain type III in addition to type I collagen molecules.By immunofluorescence, FN was seen mainly along the sinusoids in discrete dots. By EM, FN was found to be present in diffuse material closely associated with the sinusoidal membrane of the parenchymal cells and in strands connecting adjacent parenchymal cells, parenchymal and endothelial cells or parenchymal cells and collagen fibrils. FN was also present in vascular and ductular basal laminae.Strong HSPG reaction was observed around bile ducts. Moderate reaction was seen around blood vessels and in the space of Disse. In the latter location, the ultrastructural distribution of HSPG resembles that of FN, i.e. HSPG is present in diffuse material and in strands.In honour of Prof. P. van Duijn     

Immunogold localization of procollagen III, fibronectin and heparan sulfate proteoglycan on ultrathin frozen sections of the normal rat liver

In the present study, we have localized by immunocytochemistry at the LM and EM level, procollagen type III (PIIIP), fibronectin (FN) and heparan sulfate proteoglycan (HSPG). Intracellularly, PIIIP was observed in both parenchymal and endothelial cells. In parenchymal cells, PIIIP was found in Golgi derived vesicles. This observation suggests that PIIIP synthesis is a normal function of liver parenchymal cells. In endothelial cells, vesicles, which could not be identified, were seen to contain PIIIP. This result does not allow to conclude, whether sinusoidal endothelial cells secrete or take up PIIIP. Extracellularly, PIIIP was present around portal and central veins, in the space of Disse and between adjacent parenchymal cells. In the space of Disse, almost all interstitial collagen fibrils reacted with the anti PIIIP antibodies. This observation leads to the conclusion that most fibrils of the space of Disse contain type III in addition to type I collagen molecules. By immunofluorescence, FN was seen mainly along the sinusoids in discrete dots. By EM, FN was found to be present in diffuse material closely associated with the sinusoidal membrane of the parenchymal cells and in strands connecting adjacent parenchymal cells, parenchymal and endothelial cells or parenchymal cells and collagen fibrils. FN was also present in vascular and ductular basal laminae. Strong HSPG reaction was observed around bile ducts. Moderate reaction was seen around blood vessels and in the space of Disse. In the latter location, the ultrastructural distribution of HSPG resembles that of FN, i.e. HSPG is present in diffuse material and in strands.     

Ultrastructure of the neural basal lamina in loop-tail mice

Ultrastructural features of the neural basal lamina were studied by means of the tannic acid and ruthenium red techniques in normal and abnormal dysraphic loop-tail mice at 9-11 days of gestation. With ruthenium red, the configuration of the neural basal lamina is similar in both normal and abnormal embryos at 9-11 days. However, differences were detected in the abnormal 9-day embryos processed with tannic acid, as compared with normal littermates. These include irregularities in the lamina rara externa, as well as differences in the staining pattern of the neuroepithelial cell plasma membrane. By 11 days of gestation, the lamina rara externa of the normal embryos shows features similar to those observed in the 9-day abnormal embryos.     

The distribution of fibronectin and tenascin along migratory pathways of the neural crest in the trunk of amphibian embryos

It is generally assumed that in amphibian embryos neural crest cells migrate dorsally, where they form the mesenchyme of the dorsal fin, laterally (between somites and epidermis), where they give rise to pigment cells, and ventromedially (between somites and neural tube), where they form the elements of the peripheral nervous system. While there is agreement about the crest migratory routes in the axolotl (Ambystoma mexicanum), different opinions exist about the lateral pathway in Xenopus. We investigated neural crest cell migration in Xenopus (stages 23, 32, 35/36 and 41) using the X. laevis-X. borealis nuclear marker system and could not find evidence for cells migrating laterally. We have also used immunohistochemistry to study the distribution of the extracellular matrix (ECM) glycoproteins fibronectin (FN) and tenascin (TN), which have been implicated in directing neural crest cells during their migrations in avian and mammalian embryos, in the neural crest migratory pathways of Xenopus and the axolotl. In premigratory stages of the crest, both in Xenopus (stage 22) and the axolotl (stage 25), FN was found subepidermally and in extracellular spaces around the neural tube, notochord and somites. The staining was particularly intense in the dorsal part of the embryo, but it was also present along the visceral and parietal layers of the lateral plate mesoderm. TN, in contrast, was found only in the anterior trunk mesoderm in Xenopus; in the axolotl, it was absent. During neural crest cell migration in Xenopus (stages 25-33) and the axolotl (stages 28-35), anti-FN stained the ECM throughout the embryo, whereas anti-TN staining was limited to dorsal regions. There it was particularly intense medially, i.e. in the dorsal fin, around the neural tube, notochord, dorsal aorta and at the medial surface of the somites (stage 35 in both species). During postmigratory stages in Xenopus (stage 40), anti-FN staining was less intense than anti-TN staining. In culture, axolotl neural crest cells spread differently on FN- and TN-coated substrata. On TN, the onset of cellular outgrowth was delayed for about 1 day, but after 3 days the extent of outgrowth was indistinguishable from cultures grown on FN. However, neural crest cells in 3-day-old cultures were much more flattened on FN than on TN. We conclude that both FN and TN are present in the ECM that lines the neural crest migratory pathways of amphibian embryos at the time when the neural crest cells are actively migrating. FN is present in the embryonic ECM before the onset of neural crest migration.(ABSTRACT TRUNCATED AT 400 WORDS)     

Ultrastructure of the basal lamina and its relationship to extracellular matrix of embryos of the starfish Pisaster ochraceus as revealed by anionic dyes

The ultrastructure of the 51/2–6-day-old embryonic asteroid basal lamina (BL) was studied by transmission electron microscopy (TEM) and scanning electron microscopy (SEM) and after treatment with anionic dyes. Conventional fixation in glutaraldehyde and osmium reveals a BL consisting of a lamina densa separated from the basal cell surface by a lamina lucida. Little or no reticular lamina is present. Material similar in appearance to the basal lamina extends into the blastocoel, forming an extracellular matrix (ECM). Following fixation in the presence of the dye ruthenium red, proteoglycan (PG) granules are visible in the lamina lucida and immediately beneath the lamina densa. The ECM consists of granules of a similar appearance, which are associated with fibers of an intermediate electron density resembling invertebrate collagen. After fixation in the presence of alcian blue under polyanionic conditions, all aspects of the basal lamina and the ECM stain very densely. The use of alcian blue in 0.3 M MgCl₂ (monoanionic condition) or in low concentrations reveals a lamina densa consisting of a fine feltwork and tubule-like structures. A meshwork composed of thick, densely stained and thinner, intermediately stained strands is embedded in the inner aspect (that adjacent to the blastocoel) of the ectodermal lamina densa. Similar elements are present in the endodermal BL, but the dense material is represented by short regions that do not form a meshwork. The dense and intermediate strands of both basal laminae also extend into the blastocoel as ECM. The tubule-like structures extend from the dense material of the inner meshwork into the lamina densa. They also cross both the lamina densa and lucida to associatee with the basal cell membranes. The fact that the basal cell surfaces are often puckered outward at the points of contact suggests that this configuration might be providing a means whereby forces can be transferred from the ECM through the basal lamina to the cells.     

The fine structure of ventricular cells in the brains of mouse embryos homozygous for the loop-tail gene

The neural tube in normal (+/+), heterozygous (Lp/+), and abnormal (Lp/Lp) mutant mouse embryos ranging in age from 10 to 12 days of gestation was studied by means of transmission electron microscopy. In the abnormal embryos, ventricular cells in defective regions of the brain show distortions and crowding together of internal cellular processes and a decrease in blebs and bulbous projections, as compared with their normal counterparts. At 12 days' gestation the abnormal brains show a scarcity of the T-shaped internal cellular processes characteristic of normal brains. The abnormal brains also show increased amounts of intercellular space and extensive gaps between the cells, particularly in basal regions. There are no striking differences between the normal and abnormal brains at 10 to 12 days' gestation with respect to the appearance and distribution of cilia, microfilaments, microtubules, tight junctions, and ribosomes.     

Fine Structural Observations on the Turbellarians Stenostomum sp. and Microstomum lineare with Special Reference to the Extracellular Matrix and Connective Tissue Systems

Employing transmission electron microscopy, observations were made on epidermis, muscle cells and connective tissue systems, with special emphasis on extracellular matrix components (ECM), in two rather primitive turbellarians: Stenostomum sp. (Catenulida) and Microstomum lineare (Macrostomida). In Stenostomum the only ECM components found are basal laminae, predominantly situated subepidermally. In Microstomum ECM is well developed and connective tissue filaments abundant in conspicuous extracellular spaces. It is uncertain whether basal laminae exist. The finding of basal lamina structures as the only ECM component present in Stenostomum makes it now possible to establish a complete ECM and connective tissue hierarchy in turbellarians, ranging from a purely cellular type with no ECM present to systems dominated by ECM and very similar to loose connective tissue in vertebrates. Comparative aspects of ECM and connective tissue systems in turbellarians are discussed in addition to the difficulties and ambiguities regarding definition and nomenclature of basal matrices as basal laminae and subepidermal membranes.     

Cranial effects of retinoic acid in the loop-tail (Lp) mutant mouse

The effects of retinoic acid (RA) on the manifestation and nature of neural tube defects (NTD) in heterozygous embryos of mutant mice carrying the gene loop-tail (Lp) and in normal (+/+) littermates and embryos from normal homozygous matings were compared with NTD that occur in untreated abnormal homozygous (Lp/Lp) embryos. A single intraperitoneal dose (5 mg/kg) of RA administered at 9 AM or 3 PM on day 8 of gestation induced NTD in +/+ as well as Lp/+ embryos removed on day 12 of gestation. All of the NTD were confined to the brain and consisted of exencephaly involving the diencephalon, mesencephalon, and metencephalon. In neither phenotype (Lp/+; +/+) was the massive exencephaly and myeloschisis characteristic of untreated Lp/Lp embryos produced; thus, it is possible that the teratogenic mechanisms of RA-induced defects and of Lp-induced defects may differ.     

Essential roles of fibronectin in the development of the left-right embryonic body plan

Studies in Xenopus laevis suggested that cell-extracellular matrix (ECM) interactions regulate the development of the left–right axis of asymmetry; however, the identities of ECM components and their receptors important for this process have remained unknown. We discovered that FN is required for the establishment of the asymmetric gene expression pattern in early mouse embryos by regulating morphogenesis of the node, while cellular fates of the nodal cells, canonical Wnt and Shh signaling within the node were not perturbed by the absence of FN. FN is also required for the expression of Lefty 1/2 and activation of SMADs 2 and 3 at the floor plate, while cell fate specification of the notochord and the floor plate, as well as signaling within and between these two embryonic organizing centers remained intact in FN-null mutants. Furthermore, our experiments indicate that a major cell surface receptor for FN, integrin α5β1, is also required for the development of the left–right asymmetry, and that this requirement is evolutionarily conserved in fish and mice. Taken together, our studies demonstrate the requisite role for a structural ECM protein and its integrin receptor in the development of the left–right axis of asymmetry in vertebrates.     

Effects of Extracellular Matrix Molecules on Subepidermal Neural Crest Cell Migration in Wild Type and White Mutant (dd) Axolotl Embryos

Migration of neural crest (NC) derived pigment cells is restricted in the white mutant (dd) axolotl embryo (Ambystoma mexicanum). Transplantations between mutant and wild type embryos show that the extracellular matrix (ECM) of the white mutant is unable to support the migration of prospective pigment cells in wild type embryos (Löfberg et al., 1989, Dev. Biol. 131:168–181). In the present study, we test the effects of various purified ECM molecules on NC cell migration in the subepidermal migratory pathway of wild type (D/-) and white mutant (dd) axolotl embryos. We adsorbed the ECM molecules onto membrane microcarriers, which were then implanted under the epidermis. Fibronectin (FN), tenascin (TN), collagens I and VI, and a chick aggrecan stimulated migration in both types of embryos. Laminin-nidogen, rat chondrosarcoma aggrecan, and shark aggrecan stimulated migration in dd embryos but did not affect migration in D/- embryos. Collagen III, fibromodulin and bovine aggrecan had no effect on migration in either type of embryo. NC cells did not migrate on control micro-carriers, which lacked ECM molecules. Some cells observed contacting, and presumably migrating on, coated microcarriers could be identified as pigment cells by their ultra-structure. Enzymatic digestion in vivo with chondroitinase ABC had no effect on NC cell migration. The neutral or stimulatory effect of the aggrecans is surprising; when tested in vitro they inhibited NC cell migration. The effect of three-dimensionality and other molecules present either in the embryonic ECM or in solution may overcome the inhibitory effect of aggrecans.     

Expression of the sonic hedgehog gene in human embryos with neural tube defects

BACKGROUND: To estimate the rate of malformations observed during early human development, a series of 38,913 first-trimester abortions were studied. Neural tube defects (NTD) were found in 57 cases. METHODS: A histological study of serial sections performed in 25 embryos revealed a spectrum of axial structure abnormalities. Expression of the SHH gene was studied by in situ hybridization in one case of CRS and in two cases of SB. RESULTS: A cervical notochord duplication was always found in craniorachischisis (CRS, n = 8), but not in spina bifida (SB, n = 10) or diplomyelia (split cord malformation, n = 3). In the embryo with CRS, expression of SHH was found in both domains, corresponding to the duplicated part of the notochord, whereas a single signal was observed in the nonduplicated part. This expression was associated at the cervical level of the open neural tube with a broad SHH expression domain and with two or even three domains in its lumbar region, suggesting multiple functional floor plates. Similarly, in two embryos with SB, two domains of SHH expression were found in the ventral neural tube. CONCLUSIONS: Our findings suggest that notochord splitting in the cervical region might be involved in the pathogenesis of CRS. Interestingly, similar notochord abnormality and altered expression of the shh gene are observed in Lp mice with NTD. This suggests that the Lp gene could be a candidate gene for human CRS. Further studies are needed to establish the primary event responsible for the notochord splitting and for the abnormal expression of the SHH gene in the floor plate in embryos with CRS and SB.