Claudin-3 in the non-neural ectoderm is essential for neural fold fusion in chicken embryos.

The neural tube, the embryonic precursor to the brain and spinal cord, begins as a flat sheet of epithelial cells, divided into non-neural and neural ectoderm. Proper neural tube closure requires that the edges of the neural ectoderm, the neural folds, to elevate upwards and fuse along the dorsal midline of the embryo. We have previously shown that members of the claudin protein family are required for the early phases of chick neural tube closure. Claudins are transmembrane proteins, localized in apical tight junctions within epithelial cells where they are essential for regulation of paracellular permeability, strongly involved in apical-basal polarity, cell-cell adhesion, and bridging the tight junction to cytoplasmic proteins. Here we explored the role of Claudin-3 (Cldn3), which is specifically expressed in the non-neural ectoderm. We discovered that depletion of Cldn3 causes folic acid-insensitive primarily spinal neural tube defects due to a failure in neural fold fusion. Apical cell surface morphology of Cldn3-depleted non-neural ectodermal cells exhibited increased membrane blebbing and smaller apical surfaces. Although apical-basal polarity was retained, we observed altered Par3 and Pals1 protein localization patterns within the apical domain of the non-neural ectodermal cells in Cldn3-depleted embryos. Furthermore, F-actin signal was reduced at apical junctions. Our data presents a model of spina bifida, and the role that Cldn3 is playing in regulating essential apical cell processes in the non-neural ectoderm required for neural fold fusion.

Functional Validation of CLDN Variants Identified in a Neural Tube Defect Cohort Demonstrates Their Contribution to Neural Tube Defects.

Neural tube defects (NTDs) are severe malformations of the central nervous system that affect 1-2 individuals per 2,000 births. Their etiology is complex and involves both genetic and environmental factors. Our recent discovery that simultaneous removal of Cldn3, -4, and -8 from tight junctions results in cranial and spinal NTDs in both chick and mouse embryos suggests that claudins play a conserved role in neural tube closure in vertebrates. To determine if claudins were associated with NTDs in humans, we used a Fluidigm next generation sequencing approach to identify genetic variants in CLDN loci in 152 patients with spinal NTDs. We identified eleven rare and four novel missense mutations in ten CLDN genes. In vivo validation of variant pathogenicity using a chick embryo model system revealed that overexpression of four variants caused a significant increase in NTDs: CLDN3 A128T, CLDN8 P216L, CLDN19 I22T, and E209G. Our data implicate rare missense variants in CLDN genes as risk factors for spinal NTDs and suggest a new family of proteins involved in the pathogenesis of these malformations.

Developing a link between toxicants, claudins and neural tube defects.

The cause and effect relationship between environmental factors, including toxins (naturally occuring) and toxicants (man-made environmental contaminants), and neural tube defects is well-established. More recent evidence has demonstrated a requirement for the claudin family of tight junction proteins in regulating epithelial remodelling events that transform the plate neural plate into a closed tube. At the molecular level, toxicants are known to disrupt claudin expression and tight junction barrier function. In this review we consider the evidence leading to the hypothesis that toxins and toxicants affect neural tube closure due to their effects on the claudin family of tight junction proteins.

Claudins in morphogenesis: Forming an epithelial tube.

The claudin family of tetraspan transmembrane proteins is essential for tight junction formation and regulation of paracellular transport between epithelial cells. Claudins also play a role in apical-basal cell polarity, cell adhesion and link the tight junction to the actin cytoskeleton to exert effects on cell shape. The function of claudins in paracellular transport has been extensively studied through loss-of-function and gain-of-function studies in cell lines and in animal models, however, their role in morphogenesis has been less appreciated. In this review, we will highlight the importance of claudins during morphogenesis by specifically focusing on their critical functions in generating epithelial tubes, lumens, and tubular networks during organ formation.

Claudins are essential for cell shape changes and convergent extension movements during neural tube closure.

During neural tube closure, regulated changes at the level of individual cells are translated into large-scale morphogenetic movements to facilitate conversion of the flat neural plate into a closed tube. Throughout this process, the integrity of the neural epithelium is maintained via cell interactions through intercellular junctions, including apical tight junctions. Members of the claudin family of tight junction proteins regulate paracellular permeability, apical-basal cell polarity and link the tight junction to the actin cytoskeleton. Here, we show that claudins are essential for neural tube closure: the simultaneous removal of Cldn3, -4 and -8 from tight junctions caused folate-resistant open neural tube defects. Their removal did not affect cell type differentiation, neural ectoderm patterning nor overall apical-basal polarity. However, apical accumulation of Vangl2, RhoA, and pMLC were reduced, and Par3 and Cdc42 were mislocalized at the apical cell surface. Our data showed that claudins act upstream of planar cell polarity and RhoA/ROCK signaling to regulate cell intercalation and actin-myosin contraction, which are required for convergent extension and apical constriction during neural tube closure, respectively.

Claudin-10 is required for relay of left-right patterning cues from Hensen's node to the lateral plate mesoderm.

Species-specific symmetry-breaking events at the left-right organizer (LRO) drive an evolutionarily-conserved cascade of gene expression in the lateral plate mesoderm that is required for the asymmetric positioning of organs within the body cavity. The mechanisms underlying the transfer of the left and right laterality information from the LRO to the lateral plate mesoderm are poorly understood. Here, we investigate the role of Claudin-10, a tight junction protein, in facilitating the transfer of left-right identity from the LRO to the lateral plate mesoderm. Claudin-10 is asymmetrically expressed on the right side of the chick LRO, Hensen's node. Gain- and loss-of-function studies demonstrated that right-sided expression of Claudin-10 is essential for normal rightward heart tube looping, the first morphological asymmetry during organogenesis. Manipulation of Claudin-10 expression did not perturb asymmetric gene expression at Hensen's node, but did disrupt asymmetric gene expression in the lateral plate mesoderm. Bilateral expression of Claudin-10 at Hensen's node prevented expression of Nodal, Lefty-2 and Pitx2c in the left lateral plate mesoderm, while morpholino knockdown of Claudin-10 inhibited expression of Snail1 in the right lateral plate mesoderm. We also determined that amino acids that are predicted to affect ion selectivity and protein interactions that bridge Claudin-10 to the actin cytoskeleton were essential for its left-right patterning function. Collectively, our data demonstrate a novel role for Claudin-10 during the transmission of laterality information from Hensen's node to both the left and right sides of the embryo and demonstrate that tight junctions have a critical role during the relay of left-right patterning cues from Hensen's node to the lateral plate mesoderm.

Claudin family members exhibit unique temporal and spatial expression boundaries in the chick embryo.

The claudin family of proteins are integral components of tight junctions and are responsible for determining the ion specificity and permeability of paracellular transport within epithelial and endothelial cell layers. Several members of the claudin family have been shown to be important during embryonic development and morphogenesis. However, detailed embryonic expression patterns have been described for only a few claudins. Here, we provide a phylogenetic analysis of the chicken claudins and a comprehensive analysis of their mRNA expression profiles. We found that claudin family members exhibit both overlapping and unique expression patterns throughout development. Especially striking were the distinct expression boundaries observed between neural and non-neural ectoderm, as well as within ectodermal derivatives. Claudins were also expressed in endodermally-derived tissues, including the anterior intestinal portal, pharynx, lung and pancreas and in mesodermally derived tissues such as the kidney, gonad and heart. The overlapping zones of claudin expression observed in the chick embryo may confer distinct domains of ion permeability within the early epiblast and in epithelial, mesodermal and endothelial derivatives that may ultimately influence embryonic patterning and morphogenesis during development.

Claudin-5 expression in the vasculature of the developing chick embryo.

The claudin family of proteins are integral components of tight junctions and are responsible for determining the ion specificity and permeability of paracellular transport within epithelial and endothelial cell layers. Studies in human, mouse, Xenopus, and zebrafish have shown that only a limited number of claudins are expressed in endothelial cells. Here, we report the expression pattern of Claudin-5 during chick development. Between HH stage 4 and 6 Claudin-5 expression was observed exclusively in extraembryonic tissue. Claudin-5 expression was not observed in the embryo until HH stage 8, coincident with the onset of embryonic vascularization. Claudin-5 expression was maintained in the developing vasculature in the embryonic and extraembryonic tissue throughout organogenesis (HH stage 19-35), including the vasculature of the ectoderm and of organs derived from the mesoderm and endoderm lineages. These data describe a conserved expression pattern for Claudin-5 in the endothelial tight junction barrier and is the first report of the onset of Claudin-5 expression in a vertebrate embryo.