Expression and phylogeny of claudins in vertebrate primordia

Abstract

Claudins, the major transmembrane proteins of tight junctions, are members of the tetraspanin superfamily of proteins that mediate cellular adhesion and migration. Their functional importance is demonstrated by mutations in claudin genes that eliminate tight junctions in myelin and the testis, abolish Mg(2+) resorption in the kidney, and cause autosomal recessive deafness. Here we report that two paralogs among 15 claudin genes in the zebrafish, Danio rerio, are expressed in the otic and lateral-line placodes at their earliest stages of development. Related claudins in amphibians and mammals are expressed in a similar manner in vertebrate primordia such as sensory placodes, branchial arches, and limb buds. We also show that the claudin gene family may have expanded along the chordate stem lineage from urochordates to gnathostomes, in parallel with the elaboration of vertebrate characters. We propose that tight junctions not only form barriers in mature epithelia, but also participate in vertebrate morphogenesis.

Figure 1

Zebrafish cldna and cldnb mark the earliest developmental stages of the acoustico-lateralis system. (a) Material dissected for the subtractive cDNA hybridization: ear region in yellow; head and tail regions in mauve. [Reprinted with permission from ref. (Copyright 1995, Wiley-Liss, a subsidiary of John Wiley & Sons, Inc.)]. (b and f–h) Whole-mount in situ hybridizations with antisense probes for cldna, which labels the developing ear, and krox20, which marks hindbrain rhombomeres 3 and 5. (c–e) Immunofluorescence detection of Cldna protein throughout the cell membranes of the otic vesicle after acetone permeabilization (c and d), but only at the apical surface of the sensory epithelium, the location of tight junctions, after detergent extraction (e; Cldna in red, nuclei in green). Each panel shows the combination of several adjacent confocal sections; in d, the vesicle was sectioned tangentially. (i–k) Whole-mount in situ hybridizations with antisense probes for cldnb, which labels placodal structures, including those of the ear and lateral-line organ, and for krox20. (k) Migrating placode of the posterior lateral line, with brown pigment cells and boundaries between somites 19 and 22 marked by slanted lines. Views are lateral (a, c–e, and k), dorsolateral (b), or dorsal (f–j), all with anterior to the left, and of whole embryos (a, b, and f–i) or details (c–e, j, and k); in i and j, the specimens have been flattened. L, lumen of the otic vesicle; A, apical surface of the otic vesicle's epithelium; HC, hair-cell nuclei; SC, supporting-cell nuclei; r3 and r5, hindbrain rhombomeres 3 and 5; OF, otic field; Olf, olfactory placode; OP, otic placode; PD, pronephric duct; ALL, anterior lateral-line placode; OV, otic vesicle; PLL, posterior lateral-line placode.

Figure 2

Zebrafish paralogs cldna and cldnb are orthologs of mammalian claudins 3–6 and 9. This 50% majority consensus tree with compatible groupings was reconstructed from the alignment in Fig. 5 by using maximum-likelihood distances and a heuristic tree-search algorithm (41) and rooted for display purposes with HrCldna from Halocynthia roretzi as the outgroup. Numbers indicate the percentage of 1,000 bootstrap replicates that support the adjacent node. Zebrafish proteins are shown in red, human and urochordate in black; introns in the coding region (Table 1) and the linkage group or chromosome of the corresponding gene (Table 2) are listed in parentheses. Claudins whose genes contain multiple introns in their coding regions are shaded in gray. CLDN12 and Cldn12 appear to be tetraspanins, but only distant relatives of the other claudins; their common branch (dashed line) was shortened to two-fifths to conserve space. Mouse Cldn13 was omitted from this tree because its placement, either with CLDN3 and CLDN4 or with CLDN12 and Cldn12, was the least consistent among bootstrap replicates and its omission markedly increased the tree's likelihood.

Figure 3

Orthologs of zebrafish cldna and cldnb are expressed in vertebrate primordia. (a) Conserved syntenies between zebrafish linkage groups 15 and 21, which harbor paralogous clusters of claudin genes that include cldna and cldnb, and human chromosome 7, which contains CLDN3 and CLDN4. We mapped putative zebrafish orthologs of six genes from the vicinity of CLDN3 and CLDN4 (45); two of them, hip1 and wbscr1, occur in LG15 near cldna (Table 2). In addition, zebrafish LG21 harbors the ortholog of the human CCT6A gene on 7p11 (46). (b) In situ hybridization of a stage 30 X. laevis embryo with an antisense probe for frog cldna. (c–e) In situ hybridizations of mouse embryos between embryonic days 9.0 and 9.5 with antisense probes for Cldn3, Cldn4, and Cldn6. All views are lateral. BA, branchial arches; OV, otic vesicle; PD, pronephric duct; TB, tail bud; Olf, olfactory placode; FB, forelimb bud.

Figure 4

Concurrent evolution of the claudin family and the vertebrate body plan. The number of distinct claudin genes found to date in each clade is given in brackets. The appearance of those vertebrate characters that we found to be associated with claudin expression is indicated beside the stem lineage (13). Arrows indicate two presumptive genome duplications (16); a third genome duplication may have taken place in ray-finned fishes alone (15, 47).