Double gene deletion reveals lack of cooperation between claudin 11 and claudin 14 tight junction proteins

Abstract

Members of the claudin family of proteins are the main components of tight junctions (TJs), the major selective barrier of the paracellular pathway between epithelial cells. The selectivity and specificity of TJ strands are determined by the type of claudins present. An understanding of the cooperation between different claudins in various tissues is thus important. To study the possible cooperation between claudin 11 and claudin 14, we have generated claudin 11/claudin 14 double-deficient mice, which exhibit a combination of the phenotypes found in each of the singly deficient mutants, including deafness, neurological deficits, and male sterility. These two claudins have distinct and partially overlapping expression patterns in the kidney. Claudin 11 is located in both the proximal and distal convoluted tubules, whereas claudin 14 occurs in both the thin descending and thick ascending limbs of the loop of Henle and in the proximal convoluted tubules. Although daily urinary excretion of Mg(++), and to a lesser extent of Ca(++), tends to be higher in claudin 11/claudin 14 double mutants, these changes do not reach statistical significance compared with wild-type animals. Thus, under normal conditions, co-deletion of claudin 11 and claudin 14 does not affect kidney function or ion balance. Our data demonstrate that, despite the importance of each of these claudins, there is probably no functional cooperation between them. Generation of additional mouse models in which different claudins are abolished should provide further insight into the complex interactions between claudin proteins in various physiological systems.

Fig. 1

Generation of Cldn11/Cldn14 double knockout mice. a Shown are the Cldn14 and Cldn11 genotypes of mice used in each set of matings and the genotypes of the offspring. The numbers above the genotypes indicate the proportion of mice with this genotype among all offspring in a given mating. b For specific amplification of the wt and the knockout (ko) alleles, if present, from each DNA sample, three different PCR-primers were used in each case (a common primer, a wt primer, and a mutant primer). For Cldn11 a single multiplexed PCR reaction with all three different PCR-primers is performed. PCR product sizes are 390 bp for the wt allele and 290 bp for the ko allele. For Cldn14 two separate PCR reactions with two sets of primers (common and wt primers or common and mutant primers) are performed. PCR product sizes are 340 bp for the wt allele and 275 bp for the ko allele. c Brain cDNA was analyzed by RT-PCR for Cldn11 and Cldn14 expression (512 and 664 bp products, respectively) in mice of various genotypes. As a control, the Actb gene (β-actin) (465 bp product) was equally amplified in all samples.

Fig. 2

Cochlear pathology in Cldn11^−/−, Cldn14^−/− and Cldn11^−/−/Cldn14^−/− mice. a–d Hematoxylin and eosin staining of paraffin embedded sections (8 μm thick) of the organ of Corti of two months old wt, Cldn11^−/−, Cldn14^−/− and Cldn11^−/−/Cldn14^−/− mice (10× magnification). In comparison with the wt mice (a), edema is detected in the St.V of Cldn11^−/− (b) and Cldn11^−/−/Cldn14^−/− mice (d) (asterisks). OHCs of Cldn14^−/− (c) and Cldn11^−/−/Cldn14^−/− (d) mice have degenerated (arrowheads). OHC, outer hair cells; IHC, inner hair cells; TM, tectorial membrane; RM, Reissner’s membrane; St.V, stria vascularis. e-l Whole mount organ of Corti of wt, Cldn11^−/−, Cldn14^−/− and Cldn11^−/−/Cldn14^−/− mice at P16 were processed for immunostaining with an anti-prestin antibody (green), which served as a marker for OHC bodies. Rhodamine-phalloidin staining (red) was used to observe the actin-rich stereocilia and cuticular plate of cochlear hair cells. Shown are confocal cross-sections from the apical region, taken at the level of stereocilia (e-h) and the OHC body (i-l). The single row of IHCs is marked by an arrow. The three rows of OHCs are marked by arrowheads. Scale bar = 5 μm.

Fig. 3

Segment-specific expression pattern of claudin 11 and claudin 14 in kidney nephrons. Kidney frozen sections of wt mice at the age of three months were stained with pAb for claudin 11 or claudin 14 (green), and for nephron segment-specific markers (red). Claudin 11 is partially co-localized with AQP1 in the proximal convoluted tubule (a-c) and fully co-localized with CK8 in the distal convoluted tubule (cortex) (j-l). Claudin 14 is co-localized with AQP1 in both proximal convoluted tubule (cortex) (d-f) and thin descending limb of the loop of Henle (medulla) (g-i), and with THP in the thick ascending limb of the loop of Henle (m-o). Scale bar = 10 μm.

Fig. 4

Relative expression levels of claudin genes in inner ears (a) and kidneys (b) of Cldn11/Cldn14 double knockout mice, as compared to wt mice.