heparitin-sulfate and Neural-Tube-Defects

Syndecan 4 (Sdc4) is a cell-surface heparan sulfate proteoglycan (HSPG) that regulates gastrulation, neural tube closure and directed neural crest migration in Xenopus development. To determine whether Sdc4 participates in Wnt/PCP signaling during mouse development, we evaluated a possible interaction between a null mutation of Sdc4 and the loop-tail allele of Vangl2. Sdc4 is expressed in multiple tissues, but particularly in the non-neural ectoderm, hindgut and otic vesicles. Sdc4;Vangl2(Lp) compound mutant mice have defective spinal neural tube closure, disrupted orientation of the stereocilia bundles in the cochlea and delayed wound healing, demonstrating a strong genetic interaction. In Xenopus, co-injection of suboptimal amounts of Sdc4 and Vangl2 morpholinos resulted in a significantly greater proportion of embryos with defective neural tube closure than each individual morpholino alone. To probe the mechanism of this interaction, we overexpressed or knocked down Vangl2 function in HEK293 cells. The Sdc4 and Vangl2 proteins colocalize, and Vangl2, particularly the Vangl2(Lp) mutant form, diminishes Sdc4 protein levels. Conversely, Vangl2 knockdown enhances Sdc4 protein levels. Overall HSPG steady-state levels were regulated by Vangl2, suggesting a molecular mechanism for the genetic interaction in which Vangl2(Lp/+) enhances the Sdc4-null phenotype. This could be mediated via heparan sulfate residues, as Vangl2(Lp/+) embryos fail to initiate neural tube closure and develop craniorachischisis (usually seen only in Vangl2(Lp/Lp)) when cultured in the presence of chlorate, a sulfation inhibitor. These results demonstrate that Sdc4 can participate in the Wnt/PCP pathway, unveiling its importance during neural tube closure in mammalian embryos.

Topics: Animals; Cell Polarity; Embryo, Mammalian; Female; Gene Knockdown Techniques; Hair Cells, Auditory; HEK293 Cells; Heparitin Sulfate; Humans; Mice; Nerve Tissue Proteins; Neural Tube; Neural Tube Defects; Syndecan-4; Wnt Signaling Pathway; Wound Healing; Xenopus

Perlecan is a heparan sulfate proteoglycan that is expressed in all basement membranes (BMs), in cartilage, and several other mesenchymal tissues during development. Perlecan binds growth factors and interacts with various extracellular matrix proteins and cell adhesion molecules. Homozygous mice with a null mutation in the perlecan gene exhibit normal formation of BMs. However, BMs deteriorate in regions with increased mechanical stress such as the contracting myocardium and the expanding brain vesicles showing that perlecan is crucial for maintaining BM integrity. As a consequence, small clefts are formed in the cardiac muscle leading to blood leakage into the pericardial cavity and an arrest of heart function. The defects in the BM separating the brain from the adjacent mesenchyme caused invasion of brain tissue into the overlaying ectoderm leading to abnormal expansion of neuroepithelium, neuronal ectopias, and exencephaly. Finally, homozygotes developed a severe defect in cartilage, a tissue that lacks BMs. The chondrodysplasia is characterized by a reduction of the fibrillar collagen network, shortened collagen fibers, and elevated expression of cartilage extracellular matrix genes, suggesting that perlecan protects cartilage extracellular matrix from degradation.

Topics: Animals; Basement Membrane; Calcification, Physiologic; Cartilage; Cells, Cultured; Collagen; Exostoses, Multiple Hereditary; Gene Targeting; Genes, Lethal; Heart Defects, Congenital; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Homozygote; Mice; Mice, Congenic; Mice, Mutant Strains; Mutagenesis, Insertional; Neural Tube Defects; Ossification, Heterotopic; Proteoglycans; Restriction Mapping

The splotch (Sp) mutation on mouse chromosome I is a genetic model for the neural tube defects spina bifida and exencephaly. Embryos carrying Sp or its allele splotch-delayed (Spd), have been shown to have delays in neural tube closure, and neural crest cell emigration, as well as a reduction in extracellular space around the neural tube. Pre-spina bifida Sp and Spd embryos have abnormalities of notochord, mesoderm and neuroepithelial development. Chondroitin sulphate proteoglycans (CSPG) and heparan sulfate proteoglycans (HSPG) have been shown to play essential roles during neural tube closure and neural crest cell emigration and migration and thus might well be affected by the splotch mutation. Therefore, the effects of Sp and Spd on the temporal and spatial distributions of CSPG and HSPG were studied in pre-spina bifida embryos cytogenetically identified as Sp/Sp (Spd/Spd), Sp/ + (Spd/ +) or +/+. Immunohistochemical localization of CSPG by means of the CS-56 monoclonal antibody showed that in Sp/Sp head sections, the neuroepithelial basement membranes stained more intensely at 5-, 10-, and 15-somite stages, whereas similar staining was observed at 16- and 19-somite stages compared with matched +/+ sections. In caudal sections Sp/Sp again showed a more intense stain for CSPG in the neuroepithelial basement membranes in all sections (except one comparison, in which staining was similar) from embryos of 14-, 15-, 16-, and 19-somite stages, compared to matched +/+ sections. Heterozygotes did not differ consistently from the mutant or the normal (+/+) embryos in CS-56 stain intensity.(ABSTRACT TRUNCATED AT 250 WORDS)

Topics: Animals; Antibodies, Monoclonal; Basement Membrane; Chondroitin Sulfate Proteoglycans; Gestational Age; Heparan Sulfate Proteoglycans; Heparitin Sulfate; Immunohistochemistry; Mice; Mice, Mutant Strains; Neural Tube Defects; Proteoglycans; Spinal Dysraphism

Regional patterns of deposition of laminin (LN), fibronectin (FN), type IV collagen (IV), and heparan sulfate proteoglycan (HSPG) were examined during the formation of the caudal neural tube in embryos homozygous for the delayed Splotch gene and in their normal littermates. Delayed Splotch embryos had neural tube closure defects which extended from the posterior neuropore into the region formed by secondary neurulation. During posterior neuropore closure these components were normally restricted to forming basal laminae, with FN and HSPG additionally deposited in the mesenchyme. Unlike control embryos in which medial regions of the neuroepithelial basal lamina contained greatest amounts of all four, the dorsolateral zone contained less LN and IV and more FN and HSPG, in affected embryos these components were less densely deposited medially, reflecting perhaps the poor structural organization of the notochord. The neuroepithelial basal lamina was often disorganized and wavy compared to the linear pattern typical of controls. By the 12th day, the posterior neuropore of controls had closed and secondary neurulation was underway; however in delayed Splotch embryos, the neural folds remained widely splayed and epithelium newly formed via secondary neurulation extended that abnormally open configuration to the tip of the tailbud. In controls, with mesenchymal cell aggregation FN and HSPG were displaced from between cells to the forming basal lamina. As a central lumen formed within the aggregate LN and IV were added to the basal lamina, and the newly formed epithelium merged with the anterior neural tube. In delayed Splotch embryos, FN and HSPG were incompletely removed from aggregating cell surfaces, the normal morphogenetic cell shaping changes failed to occur and in many embryos a central lumen did not form; the overgrown, aggregated cells merging with the abnormally splayed anterior neural folds. In addition, the critical enrichment of FN and HSPG present between newly formed and consolidated neuroepithelium was displaced in delayed Splotch embryos.

Topics: Animals; Basement Membrane; Central Nervous System; Extracellular Matrix; Fibronectins; Heparitin Sulfate; Heterozygote; Laminin; Mice; Neural Tube Defects