Structure and sequential gene expression of in vitro-induced pronephros (kidney) in the amphibian development

We have successfully induced the formation of 20 different organs in vitro from animal caps (undifferentiated or multipotent cells) of Xenopus blastulae and six different tissues from mice embryonic stem cells. For the Xenopus embryo, we subsequently have attempted to transplant these in vitro-generated organs into embryos to determine their functional capacity. In the experiment we induced the pronephros (nephron) consisting of renal tubule, glomerulus, and renal duct from animal caps treated with activin and retinoic acid. Then we transplanted these cell mass treated with activin and retinoic acid into an embryo in which the renal rudiment had been removed. We found that the transplant developed into pronephros normally and function in vivo. Using this system, we have cloned and analyzed the pronephros-specific genes in Xenopus development. Sequential gene expressions of pronephros in vivo and in vitro resemble the mesonephros formation in mammals. In the screening for genes expressed in pronephroi, we isolated a novel gene, named dullard. Dullard protein contains the C-terminal conserved domain of nuclear LIM interactor-interacting factor (NLI-IF), a protein whose function is unknown. The spatial expression was in neural regions at neurula stages, and localized to neural tissues, branchial arches, and pronephroi. The translational knockdown of dullard resulted in failure of neural tube development and structural deformation of the pronephric tubules, and the embryos consequently showed a reduction of head development. In the same screening method for the pronephros-specific genes, we isolated the XTRAP-gamma gene that codes translucent-associated protein, one of the subunit of the translocon-associated protein complex. XTRAP is one of the proteins proposed to aid in the translocation of nascent polypeptides into the lumen of the endothelium reticulum. XTRAP-gamma is expressed in pronephros tubules during pronephros development. The translational knockdown of XTRAP-gamma caused the suppression of tubulogenesis, the increase of Xlim-1 expression, and the decrease of Xpax-2 and Xwnt-4 expression. This translational knockdown of XTRAP-gamma also inhibited differentiation of the pronephros in animal caps by the treatment with activin and retinoic acid. These results showed that XTRAP-gamma plays an important role in the pronephric development. As another approach to clarify the molecular mechanisms of renal development, we investigated the role of Xenopus frizzled-8 (Xfz8) in pronephros development to study the relation between Wnt signaling and nephric development. Translational inhibition of Xfz8 caused reduction in the staining of a duct-specific antibody and defects in pronephric tubule branching, while there was no effect on the expression of early pronephric maker genes in the duct region. Histologic analysis showed that the Xfz8-inhibited cells failed to form a normal epithelial structure. These results suggest that Xfz8 is involved in the formation of epithelium in the developing pronephric duct and tubules