Functional Study Of β-Catenin In The Segmentation Of Zebrafish Pronephrons

Kidney is an essential organ to play roles in regulating the osmotic pressure and excreting biological waste in vertebrates. The segmentation is a cell differentiation process in kidney organogenesis. Despite some differences in organ morphology between the kidney of zebrafish and that of the human beings, many common elements exist at the cellular and molecular level that can be exploited to further our general understanding of renal development and biology. We took the zebrafish as an animal model to study the mechanism of kidney segmentation, which may be applicable for the development of mammalian kidney.The fluorescence immunostaining assay showed that β-Catenin started to express in the pronephrons of zebrafish embryos at ~20hpf(hour post fertilization), and β-Catenin was highly expressed in the posterior region of pronephrons with a decreasing gradient from 1dpf to 5dpf. These results indicated that β-Catenin may play an important role in pronephrons organogenesis in Zebrafish. Meanwhile, the TOP signal of Tg(TOP-dgfp) was found to be expressed in the posterior region of pronephrons in zebrafish embryos, which was consistent with the expression of β-Catenin. It suggested that β-Catenin was regulated by a canonical Wnt signaling pathway. The segmentation of pronephrons in Zebrafish is completed at 24 hpf, and the pronephrons contains 8 segments with specific markers: glomerulus(wt1a, wt1b); neck(rfx2); proximal convoluted tubule, PCT(slc20a1a); proximal straight tubule, PST(slc13a1/trpm7); distal early, DE(slc12a1); distal late, DL(slc12a3); corpuscle of Stannius, CS(stc1); pronephric duct, PD(gata3). Tg(hsp70:dkk1-GFP) embryos were chosen to perform the experiment of down-regulating β-Catenin. The heat treatment of these transgenic embryos at 24 hpf resulted in the expression change of slc12a3 and gata3 at 34 hpf. After heat treatment, the expression region of slc12a3 got longer in pronephrons while the expression region of gata3 became shorter and down-regulated, and this result was re-confirmed by of double-whole mount in-situ hybridization of slc12a3 and gata3.BIO was a chemical inhibitor of GSK3, which was a suppressor of β-Catenin, therefore, BIO treatment can elevate the expression of β-Catenin. After BIO treatment, slc12a3 expression got weaker while gata3 expression also became weaker and the expression region of gata3 got shorter. These results implied that an accurate concentration of β-Catenin is important to maintain the different differentiations of pronephrons cells, and cell fates of pronephrons may be changed with the fluctuation of the β-Catenin expression. The results of the recover assay, in which the expression of β-Catenin was decreased firstly followed by gradually increasing the expression of β-Catenin to the normal level, supported the former findings. The result of a shorter heat-treatment experiment in transgenic embryos(24hpf treatment, 31 hpf fix) was similar with that obtained in the former assay(24hpf treatment, 34 hpf fix). A similar result appeared when the heat treatment of transgenic embryos was shifted to an early stage(18hpf). Furthermore, the decreased expression of β-Catenin at 18 hpf influenced the development of other segments in pronephrons. A different result was obtained when embryos were treated before 18hpf(16hpf treatment, 24 hpf fix). After a heat treatment before 18 hpf, the expression region of slc12a3 got shorter, while the expression level of gata3 became weaker, and the expression region of gata3 got shorter; However, BIO treatment induced a longer expression region of slc12a3, but the influence of BIO treatment on the gata3 expression was the same as that of the heat treatment. Moreover, the development of other segments in pronephrons were affected by the expression change of β-Catenin before 18 hpf. This was similar to the result of the decreased expression of β-Catenin at 18 hpf. Combined together, it indicated that 18 hpf may be an important time point for pronephrons organogenesis in zebrafish, and β-Catenin may play dual roles before and after this point. The WISH results of these two markers(slc12a3 and gata3) displayed that the two differentiation markers began to be expressed from 18 hpf. This suggested that the segmentation of zebrafish pronephrons were completed at 18 hpf, and the β-Catenin played dual roles before and after segmentation.Our results clearly demonstrated that the function of β-Catenin was essential to maintain cell differentiation and cell fates in the segmentation of zebrafish pronephrons, and it provides a valuable reference for the study of the development of mammalian kidney.