Establishment of a neuroepithelial barrier by Claudin5a is essential for zebrafish brain ventricular lumen expansion

Abstract

Lumen expansion driven by hydrostatic pressure occurs during many morphogenetic processes. 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. 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. 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. Perfusion experiments with the electron-dense small molecule lanthanum nitrate reveal that paracellular tightness of the cerebral-ventricular barrier decreases upon loss of Claudin5a. 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 B rain morphogenesis in the zebrafish embryo involves a wellstudied ventricular lumen expansion process which occurs at early stages of development between 17 and 21 h after fertilization (hpf) (1). 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). However, the embryonic cerebral barrier expected to maintain luminal fluids and ions within the brain ventricles remains unknown. It is known that the bloodbrain barrier forms 2 days after brain ventricle expansion (2). 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). 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. 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). Claudins are characterized as either barrier-or pore-forming. In a recent study, Bagnat and colleagues demonstrated an involvement of the pore-forming Cldn15 in gut lumen expansion in the zebrafish embryo (7). 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). Together, these studies implied an important function of Claudins in brain ventricular lumen expansion.Her...

Mitotic cell rounding and epithelial thinning regulate lumen growth and shape

“…We explored the cellular mechanisms responsible for this anisotropic growth. While the relevance of fluid movement in increasing the size of the cavity has been demonstrated in several experimental models 5,10,11 , with the involvement of paracellular transportation and epithelial chloride secretion 9,23 , its role in shaping the lumen has still not been evaluated. It is widely assumed that the increased intraluminal concentration of chloride (and other ions) promotes the movement of water from outside the organ into the lumen (by paracellular or transcellular transport), similar to what occurs in secretory epithelial organs 12,13 .…”

“…Lumen growth requires coordination between fluid ingress and increase of the luminal epithelial surface area. Fluids ingress has been shown to rely on a functional Na þ /K þ ATPase pump, the CFTR chloride channel and the formation of both paracellular pores and barriers by claudin proteins 5,[9][10][11] . The present view of the process indicates that fluid ingress is mediated by transepithelial fluid transport, in which the epithelium is considered as a pump driving fluid movement from outside of the organ into the lumen [12][13][14] .…”

Luminal matrices: An inside view on organ morphogenesis

“…1A and 1B). Indeed, such hydrostatic pressure was proposed to play important roles in lumen formation and expansion in the gut [12], in Kupffer's vesicle [13] and in brain ventricles [14] in zebrafish, and in the excretory cell [15] and vulva [16] in C. elegans. Moreover, the cylindrical geometry of tubular structures brings along physical constraints that are distinct from those experienced by cells in planar epithelial sheets.…”

An Assay for Permeability of the Zebrafish Embryonic Neuroepithelium

“…Multiple processes are associated with brain ventricle formation, including formation of a neuroepithelium, tight junction formation that regulates permeability and CSF production. We showed that the Na,K-ATPase is required for brain ventricle inflation, impacting all these processes 7,8 , while claudin 5a is necessary for tight junction formation 9 . Additionally, we showed that "relaxation" of the embryonic neuroepithelium, via inhibition of myosin, is associated with brain ventricle inflation.…”