Database searches ( ) combined with whole-mount in situ hybridization analyses revealed that two zebrafish clnd5 genes encoding the predicted orthologs of the human four-transmembrane pass TJ protein Cldn5 are expressed in the zebrafish embryo ( cldn5a: gi|47086108| cldn5b: gi|53733860|) ( Fig. There are at least 20 Claudin family members in teleosts ( 9, 10). Together, these studies implied an important function of Claudins in brain ventricular lumen expansion. Moreover, the C-terminal half of Clostridium perfringens enterotoxin (C-CPE), a polypeptide with inhibitory activity to several barrier-forming Claudins including Cldn3, Cldn4, and Cldn6, affected murine blastocoel cavity expansion, which is another lumen expansion process that requires hydrostatic pressure ( 8).
In a recent study, Bagnat and colleagues demonstrated an involvement of the pore-forming Cldn15 in gut lumen expansion in the zebrafish embryo ( 7). Claudins are characterized as either barrier- or pore-forming. We hypothesized that Claudins (Cldn) may contribute to the cerebral-ventricular barrier based on their established roles in the regulation of tight junction (TJ) barriers ( 5, 6). To elucidate the nature of the cerebral-ventricular barrier system required for initial lumen expansion, we focused our attention on the neuroepithelium lining the cerebral cavities. Also, the choroid plexus, which develops from the ependymal layer lining the ventral floor of the cerebral ventricle, is not functional at these stages and forms the blood-cerebrospinal fluid barrier only at later stages and in the adult fish ( 3, 4). It is known that the blood–brain barrier forms 2 days after brain ventricle expansion ( 2). However, the embryonic cerebral barrier expected to maintain luminal fluids and ions within the brain ventricles remains unknown. Genetic analyses showed that the osmoregulatory ion pump ATPase, Na +/K + transporting, alpha 1 polypeptide (Atp1a1) is critically important for lumen expansion which suggests a role in the generation of hydrostatic pressure ( 1). These data establish an essential role of a barrier-forming Claudin in ventricular lumen expansion, thereby contributing to brain morphogenesis.īrain morphogenesis in the zebrafish embryo involves a well-studied ventricular lumen expansion process which occurs at early stages of development between 17 and 21 h after fertilization (hpf) ( 1). Genetic analyses show that the apical neuroepithelial localization of Claudin5a depends on epithelial cell polarity and provide evidence for concerted activities between Claudin5a and Na +,K +-ATPase during luminal expansion of brain ventricles. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. Loss of Claudin5a or expression of a tight junction-opening Claudin5a mutant reduces brain ventricular volume expansion without disrupting the polarized organization of the neuroepithelium.
Here, we identify Claudin5a as a core component of an early cerebral-ventricular barrier system that is required for ventricular lumen expansion in the zebrafish embryonic brain before the establishment of the embryonic blood–brain barrier. Although it is well established that members of the Claudin family of transmembrane tight junction proteins determine paracellular tightness within epithelial/endothelial barrier systems, functional evidence for their role in the morphogenesis of lumenized organs has been scarce. Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes.
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