Alterations in heart looping induced by overexpression of the tight junction protein Claudin-1 are dependent on its C-terminal cytoplasmic tail.
In vertebrates, the positioning of the internal organs relative to the midline is asymmetric and evolutionarily conserved. A number of molecules have been shown to play critical roles in left-right patterning. Using representational difference analysis to identify genes that are differentially expressed on the left and right sides of the chick embryo, we cloned chick Claudin-1, an integral component of epithelial tight junctions. Here, we demonstrate that retroviral overexpression of Claudin-1, but not Claudin-3, on the right side of the chick embryo between HH stages 4 and 7 randomizes the direction of heart looping. This effect was not observed when Claudin-1 was overexpressed on the left side of the embryo. A small, but reproducible, induction of Nodal expression in the perinodal region on the right side of the embryo was noted in embryos that were injected with Claudin-1 retroviral particles on their right sides. However, no changes in Lefty,Pitx2 or cSnR expression were observed. In addition, Flectin expression remained higher in the left dorsal mesocardial folds of embryos with leftwardly looped hearts resulting from Claudin-1 overexpression on the right side of the embryo. We demonstrated that Claudin-1's C-terminal cytoplasmic tail is essential for this effect: mutation of a PKC phosphorylation site in the Claudin-1 C-terminal cytoplasmic domain at threonine-206 eliminates Claudin-1's ability to randomize the direction of heart looping. Taken together, our data provide evidence that appropriate expression of the tight junction protein Claudin-1 is required for normal heart looping and suggest that phosphorylation of its cytoplasmic tail is responsible for mediating this function.
Simard
Annie
A
Departments of Pediatrics and Human Genetics, McGill University, Montréal, Que., Canada.
Di Pietro
Erminia
E
Young
Christopher R
CR
Plaza
Silvia
S
Ryan
Aimee K
AK
D000595:Amino Acid Sequence; D000818:Animals; D002642:Chick Embryo / Q000378:metabolism*; D062445:Claudin-1; D062465:Claudin-3; D003001:Cloning, Molecular; D003593:Cytoplasm / Q000378:metabolism; D047108:Embryonic Development; D005775:Gastrula / Q000502:physiology; D005786:Gene Expression Regulation; D006321:Heart / Q000196:embryology*; D018398:Homeodomain Proteins / Q000378:metabolism; D008565:Membrane Proteins / Q000235:genetics / Q000378:metabolism*; D008969:Molecular Sequence Data; D012190:Retroviridae / Q000235:genetics; D017386:Sequence Homology, Amino Acid; D019108:Tight Junctions / Q000378:metabolism*; D014157:Transcription Factors / Q000378:metabolism; D000097577:Homeobox Protein PITX2
D062445:Claudin-1; D062465:Claudin-3; D018398:Homeodomain Proteins; D008565:Membrane Proteins; D014157:Transcription Factors; D000097577:Homeobox Protein PITX2; C568108:CLDN1 protein, human; C568122:CLDN3 protein, human