The Role Of Paraxial Protocadherin In Xenopus Otic Placode Development

Vertebrate inner ear develops from its rudiment, otic placode, which later forms otic vesicle and gives rise to tissues comprising the entire inner ear. Although several signaling molecules have been identified as candidate responsible for inner ear specification and patterning, many details remain elusive.Here, we report that Paraxial Protocadherin (PAPC) is required for otic vesicle formation in Xenopus embryos. PAPC is expressed strictly in presumptive otic placode and later in otic vesicle during inner ear morphogenesis. Knockdown of PAPC by dominant negative PAPC results in the failure of otic vesicle formation and the loss of early inner ear markers Sox9 and Tbx2 expression, suggesting the requirement of PAPC in the early stage of otic vesicle development. However, PAPC alone is not sufficient to induce an ectopic otic placode formation.In order to further investigate PAPC function,it is necessary to prepare PAPC antibody. GST expression system was used to express the fusion protein PAPC-GST. Rabbits were immunized with PAPC-GST fusion protein and the PAPC polyclonal antibody was obtained. In the dilution of 1: 3000 the anibody recognizes a specific band in western blot analysis of FL-PAPC transfected HEK 293T cell extract, which can be specifically blocked when pre-adsorbed with PAPC protein. Furthermore,the expression of PAPC protein in FL-PAPC injected Xenopus animal cap could be observed on cell membrane by immunofluorescence analysis using the antibody (1: 500). The detection of endogenous PAPC protein expression pattern using PAPC polyclonal antibody is being carried out.