A Nuclear Magnetic Resonance (NMR)- and Mass Spectrometry (MS)-Based Saturation Kinetics Model of a Bryophyllum pinnatum Decoction as a Treatment for Kidney Stones.

Bryophyllum pinnatum (BP) is a medicinal plant used to treat many conditions when taken as a leaf juice, leaves in capsules, as an ethanolic extract, and as herbal tea. These preparations have been chemically analyzed except for decoctions derived from boiled green leaves. In preparation for a clinical trial to validate BP tea as a treatment for kidney stones, we used NMR and MS analyses to characterize the saturation kinetics of the release of metabolites. During boiling of the leaves, (a) the pH decreased to 4.8 within 14 min and then stabilized; (b) regarding organic acids, citric and malic acid were released with maximum release time (tmax) = 35 min; (c) for glycoflavonoids, quercetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (Q-3O-ArRh), myricetin 3-O-α-L-arabinopyranosyl-(1 → 2)-α-L-rhamnopyranoside (M-3O-ArRh), kappinatoside, myricitrin, and quercitrin were released with tmax = 5-10 min; and (d) the total phenolic content (TPC) and the total antioxidant capacity (TAC) reached a tmax at 55 min and 61 min, respectively. In summary, 24 g of leaves boiled in 250 mL of water for 61 min ensures a maximal release of key water-soluble metabolites, including organic acids and flavonoids. These metabolites are beneficial for treating kidney stones because they target oxidative stress and inflammation and inhibit stone formation.

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.

Claudin-3 regulates luminal fluid accumulation in the developing chick lung.

Claudins are a family of tight junction proteins expressed in epithelial tissues during development and in postnatal life. We hypothesized that claudins are required for branching morphogenesis in the developing chick lung. To test this hypothesis, we exposed cultured chick lung explants at embryonic day 5 to a truncated non-toxic form of the Clostridium perfringens enterotoxin known as C-CPE that removes C-CPE-sensitive claudins from tight junctions. Using in situ hybridization and immunofluorescence studies, we established that only one C-CPE-sensitive claudin, Claudin-3, was expressed in the chick lung at this stage. C-CPE treated lung explants did not exhibit any defect in lung branching compared to controls. However, they did exhibit a significantly smaller lumen area, suggesting that paracellular permeability was perturbed. The decrease in lumen area was associated with a loss of Claudin-3 expression within tight junctions of the respiratory epithelium and an increase in permeability of the respiratory epithelium. When C-CPE-treated lung explants were treated with forskolin, lumen area was restored. In summary, removal of a sealing claudin, Claudin-3, from tight junctions in embryonic lung epithelium results in a decrease in lumen area and in hydrostatic pressure needed for lung development.

Role of Claudins in Renal Branching Morphogenesis.

Claudins are a family of tight junction proteins that are expressed during mouse kidney development. They regulate paracellular transport of solutes along the nephron and contribute to the final composition of the urinary filtrate. To understand their roles during development, we used a protein reagent, a truncated version of the Clostridium perfringens enterotoxin (C-CPE), to specifically remove a subset of claudin family members from mouse embryonic kidney explants at embryonic day 12. We observed that treatment with C-CPE decreased the number and the complexity of ureteric bud tips that formed: there were more single and less bifid ureteric bud tips when compared to control-treated explants. In addition, C-CPE-treated explants exhibited ureteric bud tips with larger lumens when compared to control explants (p < .05). Immunofluorescent analysis revealed decreased expression and localization of Claudin-3, -4, -6, and -8 to tight junctions of ureteric bud tips following treatment with C-CPE. Interestingly, Claudin-7 showed higher expression in the basolateral membrane of the ureteric bud lineage and poor localization to the tight junctions of the ureteric bud lineage both in controls and in C-CPE-treated explants. Taken together, it appears that claudin proteins may play a role in ureteric bud branching morphogenesis through changes in lumen formation that may affect the efficiency by which ureteric buds emerge and branch.

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.

Temporal Effects of Quercetin on Tight Junction Barrier Properties and Claudin Expression and Localization in MDCK II Cells.

: Kidney stones affect 10% of the population. Yet, there is relatively little known about how they form or how to prevent and treat them. The claudin family of tight junction proteins has been linked to the formation of kidney stones. The flavonoid quercetin has been shown to prevent kidney stone formation and to modify claudin expression in different models. Here we investigate the effect of quercetin on claudin expression and localization in MDCK II cells, a cation-selective cell line, derived from the proximal tubule. For this study, we focused our analyses on claudin family members that confer different tight junction properties: barrier-sealing (Cldn1, -3, and -7), cation-selective (Cldn2) or anion-selective (Cldn4). Our data revealed that quercetin's effects on the expression and localization of different claudins over time corresponded with changes in transepithelial resistance, which was measured continuously throughout the treatment. In addition, these effects appear to be independent of PI3K/AKT signaling, one of the pathways that is known to act downstream of quercetin. In conclusion, our data suggest that quercetin's effects on claudins result in a tighter epithelial barrier, which may reduce the reabsorption of sodium, calcium and water, thereby preventing the formation of a kidney stone.

Regulatory interaction between the ZPBP2-ORMDL3/Zpbp2-Ormdl3 region and the circadian clock.

Genome-wide association study (GWAS) loci for several immunity-mediated diseases (early onset asthma, inflammatory bowel disease (IBD), primary biliary cholangitis, and rheumatoid arthritis) map to chromosomal region 17q12-q21. The predominant view is that association between 17q12-q21 alleles and increased risk of developing asthma or IBD is due to regulatory variants. ORM sphingolipid biosynthesis regulator (ORMDL3) residing in this region is the most promising gene candidate for explaining association with disease. However, the relationship between 17q12-q21 alleles and disease is complex suggesting contributions from other factors, such as trans-acting genetic and environmental modifiers or circadian rhythms. Circadian rhythms regulate expression levels of thousands of genes and their dysregulation is implicated in the etiology of several common chronic inflammatory diseases. However, their role in the regulation of the 17q12-q21 genes has not been investigated. Moreover, the core clock gene nuclear receptor subfamily 1, group D, member 1 (NR1D1) resides about 200 kb distal to the GWAS region. We hypothesized that circadian rhythms influenced gene expression levels in 17q12-q21 region and conversely, regulatory elements in this region influenced transcription of the core clock gene NR1D1 in cis. To test these hypotheses, we examined the diurnal expression profiles of zona pellucida binding protein 2 (ZPBP2/Zpbp2), gasdermin B (GSDMB), and ORMDL3/Ormdl3 in human and mouse tissues and analyzed the impact of genetic variation in the ZPBP2/Zpbp2 region on NR1D1/Nr1d1 expression. We found that Ormdl3 and Zpbp2 were controlled by the circadian clock in a tissue-specific fashion. We also report that deletion of the Zpbp2 region altered the expression profile of Nr1d1 in lungs and ileum in a time-dependent manner. In liver, the deletion was associated with enhanced expression of Ormdl3. We provide the first evidence that disease-associated genes Zpbp2 and Ormdl3 are regulated by circadian rhythms and the Zpbp2 region influences expression of the core clock gene Nr1d1.

Microsaccades and covert attention: Evidence from a continuous, divided attention task.

A substantial question in understanding expert behavior is isolating where experts look, and which aspects of their environment they process. While tracking the position of gaze provides some insight into this process, our ability to attend covertly to regions of space other than the current point of fixation, severely limits the diagnostic power of such data. Over the past decade, evidence has emerged suggesting that microscopic eye movements present during periods of fixation may be linked to the spatial distribution of covert attention, potentially offering a powerful tool for studying expert behavior. To date, the majority of studies in this field have tested the link under the constraints of a trial by trial, forced-response task. In the current study we sought to examine the effect when participants performed a continuous, divided-attention task, with the hope of bridging the gap to a range of more ecological, real-world tasks. We report various aspects of the eye movement and response data including (i) the relationship between microsaccades and drift correction, (ii) response behavior in brief time periods immediately following a microsaccade, (iii) response behavior briefly preceding a microsaccade. Analysis failed to reveal a link between task accuracy and the direction of a microsaccade. Most striking however, we found evidence for a timelocked relationship between the side of space responded to and the direction of the most recent microsaccade. The paper hence provides preliminary evidence that microsaccades may indeed be used to track the ongoing allocation of spatial attention.

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.

Are there conserved roles for the extracellular matrix, cilia, and junctional complexes in left-right patterning?

Many different types of molecules have essential roles in patterning the left-right axis and directing asymmetric morphogenesis. In particular, the relationship between signaling molecules and transcription factors has been explored extensively. Another group of proteins implicated in left-right patterning are components of the extracellular matrix, apical junctions, and cilia. These structural molecules have the potential to participate in the conversion of morphogenetic cues from the extracellular environment into morphogenetic patterning via their interactions with the actin cytoskeleton. Although it has been relatively easy to temporally position these proteins within the hierarchy of the left-right patterning pathway, it has been more difficult to define how they mechanistically fit into these pathways. Consequently, our understanding of how these factors impart patterning information to influence the establishment of the left-right axis remains limited. In this review, we will discuss those structural molecules that have been implicated in early phases of left-right axis development.

Claudin-7, -16, and -19 during mouse kidney development.

Members of the claudin family of tight junction proteins are critical for establishing epithelial barriers and for the regulation of paracellular transport. To understand their roles during kidney development, we first performed RT-PCR analyses and determined that 23 claudin family members were expressed in embryonic day (E) 13.5 mouse kidneys. Based on their developmental expression and phenotypes in mouse models, we hypothesized that 3 claudin members could affect nephron formation during kidney development. Using whole mount in situ hybridization and immunohistochemistry, we demonstrated that Claudin-7 (Cldn7) was expressed in the nephric duct, the emerging ureteric bud, and in tubules derived from ureteric bud branching morphogenesis. In contrast, Claudin-16 (Cldn16) and Claudin-19 (Cldn19) were expressed at later stages of kidney development in immature renal tubules that become the Loop of Henle. To determine if a loss of these claudins would perturb kidney development, we examined newborn kidneys from mutant mouse models lacking Cldn7 or Cldn16. In both models, we noted no evidence for any congenital renal malformation and quantification of nephron number did not reveal a decrease in nephron number when compared to wildtype littermates. In summary, Cldn7, Cldn16, and Cldn19 are expressed in different epithelial lineages during kidney development. Mice lacking Cldn7 or Cldn16 do not have defects in de novo nephron formation, and this suggests that these claudins primarily function to regulate paracellular transport in the mature nephron.

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.

Otic mesenchyme cells regulate spiral ganglion axon fasciculation through a Pou3f4/EphA4 signaling pathway.

Peripheral axons from auditory spiral ganglion neurons (SGNs) form an elaborate series of radially and spirally oriented projections that interpret complex aspects of the auditory environment. However, the developmental processes that shape these axon tracts are largely unknown. Radial bundles are comprised of dense SGN fascicles that project through otic mesenchyme to form synapses within the cochlea. Here, we show that radial bundle fasciculation and synapse formation are disrupted when Pou3f4 (DFNX2) is deleted from otic mesenchyme. Further, we demonstrate that Pou3f4 binds to and directly regulates expression of Epha4, Epha4⁻/⁻ mice present similar SGN defects, and exogenous EphA4 promotes SGN fasciculation in the absence of Pou3f4. Finally, Efnb2 deletion in SGNs leads to similar fasciculation defects, suggesting that ephrin-B2/EphA4 interactions are critical during this process. These results indicate a model whereby Pou3f4 in the otic mesenchyme establishes an Eph/ephrin-mediated fasciculation signal that promotes inner radial bundle formation.

Manipulating claudin expression in avian embryos.

Since the discovery of Claudin-1 and -2 by Tsukita and colleagues in the late 1990s [Furuse et al. J Cell Biol 141:1539-50,1998], claudin family members have been found to have critical roles in maintaining the integrity of epithelial and endothelial tight junctions [Furuse and Moriwaki Ann N Y Acad Sci 1165:58-61, 2009; Morita et al. Proc Natl Acad Sci USA 96:511-6, 1999; Tsukita and Furuse Ann N Y Acad Sci 915:129-35, 2000; Turksen and Troy J Cell Sci 117:2435-47, 2004]. The properties of distinct claudin family members in tight junction permeability and specificity have been extensively studied in vitro using cell culture models. In vivo, claudin family members are dynamically regulated during embryogenesis and alterations in their expression patterns can have detrimental effects on the formation and physiological function of the tissues in which they are expressed. The chick embryo provides an excellent system to dissect the roles of specific family members in vivo and to explore the effects of modulating claudin expression during the epithelial-to-mesenchymal and mesenchymal-to-epithelial transitions that are associated with tissue morphogenesis and differentiation. We are using the chick embryo to understand the roles of the claudin family of tight junction proteins during gastrulation and left-right patterning during embryogenesis. Here, we describe methodologies for manipulating claudin gene expression in specific target tissues during chick embryogenesis.

The tight junction protein claudin-3 shows conserved expression in the nephric duct and ureteric bud and promotes tubulogenesis in vitro.

The claudin family of proteins is required for the formation of tight junctions that are contact points between epithelial cells. Although little is known of the cellular events by which epithelial cells of the ureteric bud form tubules and branch, tubule formation is critical for kidney development. We hypothesize that if claudin-3 (Cldn3) is expressed within tight junctions of the ureteric bud, this will affect ureteric bud cell shape and tubule formation. Using transmission electron microscopy, we identified tight junctions within epithelial cells of the ureteric bud. Whole mount in situ hybridization and immunoassays were performed in the mouse and chick and demonstrated that Cldn3 transcript and protein were expressed in the nephric duct, the ureteric bud, and its derivatives at critical time points during tubule formation and branching. Mouse inner medullary collecting duct cells (mIMCD-3) form tubules when seeded in a type I collagen matrix and were found to coexpress CLDN3 and the tight junction marker zonula occludens-1 in the cell membrane. When these cells were stably transfected with Cldn3 fused to the enhanced green fluorescent protein reporter, multiple clones showed a significant increase in tubule formation compared with controls (P < 0.05) due in part to an increase in cell proliferation (P < 0.01). Cldn3 may therefore promote tubule formation and expansion of the ureteric bud epithelium.

Expression patterns of hormones, signaling molecules, and transcription factors during adenohypophysis development in the chick embryo.

The chick embryo is an ideal model to study pituitary cell-type differentiation. Previous studies describing the temporal appearance of differentiated pituitary cell types in the chick embryo are contradictory. To resolve these controversies, we used RT-PCR to define the temporal onset and in situ hybridization and immunohistochemistry to define the spatial localization of hormone expression within the pituitary. RT-PCR detected low levels of Fshbeta (gonadotropes) and Pomc (corticotropes, melanotropes) mRNA at E4 and Gh (somatotropes), Prl (lactotropes), and Tshbeta (thyrotropes) mRNA at E8. For all hormones, sufficient accumulation of mRNA and/or protein to permit detection by in situ hybridization or immunohistochemistry was observed approximately 3 days later and in all cases corresponded to a notable increase in RT-PCR product. We also describe the expression patterns of signaling (Bmp2, Bmp4, Fgf8, Fgf10, Shh) and transcription factors (Pitx1, Pitx2, cLim3) known to be important for pituitary organogenesis in other model organisms.