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Epithelial cell adhesion molecule EpCAM is a transmembrane glycoprotein that is dynamically expressed in human and murine renal epithelia during development. The levels of EpCAM in the renal epithelium are upregulated both during regeneration after ischemia/reperfusion injury and in renal-derived carcinomas. The role of EpCAM in early kidney development, however, has remained unclear. The zebrafish pronephros shows a similar segmentation pattern to the mammalian metanephric nephron, and has recently emerged as a tractable model to study the regulatory programs governing early nephrogenesis. Since EpCAM shows persistent expression in the pronephros throughout early development, we developed a method to study the global changes in gene expression in specific pronephric segments of wild type and EpCAM-deficient zebrafish embryos. In epcam mutants, we found 379 differentially expressed genes. Gene ontology analysis revealed that EpCAM controls various developmental programs, including uretric bud development, morphogenesis of branching epithelium, regulation of cell differentiation and cilium morphogenesis.

Eph/ephrin molecules are widely expressed during embryonic development, and function in a variety of developmental processes. Here we studied the roles of the Eph receptor EphA7 and its soluble form in Xenopus pronephros development. EphA7 is specifically expressed in pronephric tubules at tadpole stages and knockdown of EphA7 by a translation blocking morpholino led to defects in tubule cell differentiation and morphogenesis. A soluble form of EphA7 (sEphA7) was also identified. Interestingly, the membrane level of claudin6 (CLDN6), a tetraspan transmembrane tight junction protein, was dramatically reduced in the translation blocking morpholino injected embryos, but not when a splicing morpholino was used, which blocks only the full length EphA7. In cultured cells, EphA7 binds and phosphorylates CLDN6, and reduces its distribution at the cell surface. Our work suggests a role of EphA7 in the regulation of cell adhesion during pronephros development, whereas sEphA7 works as an antagonist.

Claudins are tetratransmembrane tight junction proteins and play important roles in regulating paracellular permeability of different nephron segments of the kidney. However, the roles of claudins in kidney development remain largely unknown. Here we studied the expression and functions of claudin-6 in Xenopus pronephros development. Xenopus claudin-6 is expressed in the developing pronephric tubule and duct but not glomus. Knockdown of claudin-6 by specific morpholino led to severe defects in pronephros tubular morphogenesis and blocked the terminal differentiation of the tubule cells. The claudin-6 morpholino targeted tubule cells showed failure of apical accumulation of actin and reduced lateral expression of tight junction protein Na/K-ATPase, suggesting an incomplete epithelization likely due to defected cell adhesions and apical-lateral polarity. Our work uncovered a novel role for claudin-6 in embryonic kidney development.

NDRG1 is a member of the N-myc downstream-regulated gene (NDRG) family and is involved in cellular differentiation, activation of p53, cell cycle arrest, metastasis, and hypoxia. Expression of NDRG1 is repressed by the proto-oncogene, N-myc during mouse development, although the exact functional role of NDRG1 in development remains unknown. Here, we report the characterization of Xenopus laevis NDRG1 (xNDRG1) during X. laevis development. Expression of xNDRG1 transcript was first detected at stage 15, and was localized to the presumptive pronephric anlagen at stage 26 and to pronephros, eye, branchial arches, and tail-bud at stage 32. Overexpression of xNDRG1 results in a reduced pronephros and disorganized somites. Depletion of xNDRG1, using morpholinos, causes failure of pronephros development. These results suggest that xNDRG1 is required for pronephros development in X. laevis.

In zebrafish embryos, bilateral symmetry is broken by asymmetric nodal flow generated in Kupffer's vesicle (KV), the transient cilia-rich organ, analogous to the mouse node. Asymmetric nodal flow induces the asymmetric expression of several genes, which are critical for the determination of correct LR body patterning. seson encoding three consecutive C2H2 zinc finger protein is predominantly expressed in the cilia-rich organs including KV. Inhibition of its function by the injection of a seson-specific MO inhibited the left-side biased expression of spaw, and resulted in randomization of the heart, gut looping and brain laterality. Disruption of the LR patterning in seson morphants appeared to be due to severe cilia defects in KV. Seson function was also required for ciliogenesis in other tissues such as the pronephros and olfactory organs. Collectively, our data suggest that Seson has critical roles in ciliogenesis and LR body axis patterning.

Despite the known importance of long-chained polyunsaturated fatty acids (LC-PUFA) during development, very little is known about their utilization and biosynthesis during embryogenesis. Combining the advantages of the existence of a complete range of enzymes required for LC-PUFA biosynthesis and the well established developmental biology tools in zebrafish, we examined the expression patterns of three LC-PUFA biosynthesis genes, Elovl2-like elongase (elovl2), Elovl5-like elongase (elovl5) and fatty acyl desaturase (fad) in different zebrafish developmental stages. The presence of all three genes in the brain as early as 24 hours post fertilization (hpf) implies LC-PUFA synthesis activity in the embryonic brain. This expression eventually subsides from 72 hpf onwards, coinciding with the initiation of elovl2 and fad expression in the liver and intestine, 2 organs known to be involved in adult fish LC-PUFA biosynthesis. Collectively, these patterns strongly suggest the necessity for localized production of LC-PUFA in the brain during in early stage embryos prior to the maturation of the liver and intestine. Interestingly, we also showed a specific expression of elovl5 in the proximal convoluted tubule (PCT) of the zebrafish pronephros, suggesting a possible new role for LC-PUFA in kidney development and function.

Vertebrate gap junctions are constituted of connexin (Cx) proteins. In Xenopus laevis, only seven different Cxs have been described so far. Here, we identify two new Cxs from X. laevis. Cx28.6 displays >60% amino acid identity with human Cx25, Cx29 displays strong homology with mouse Cx26 and Cx30. Cx29 is expressed throughout embryonic development. Cx28.6 mRNA is only transiently found from stage 22 to 26 of development. While no Cx28.6 expression could be detected by whole mount in situ hybridization, expression of Cx29 was found in the developing endoderm, lateral mesoderm, liver anlage, pronephros, and proctodeum. Ectopic expression of Cx28.6 failed to produce functional gap-junctions. In contrast, ectopic expression of full-length Cx29 in HEK293 and COS-7 cells resulted in the formation of gap junction-like structures at the cell–cell interfaces. Ectopic expression of Cx29 in communication deficient N2A cell pairs led to functional electrical coupling.

Vertebrates use two or three forms of kidney successively during development and the nephric duct is essential for this succession of kidney induction. While transcripts of many Wnt ligands and Wnt receptor Frizzled genes have been localized in developing kidney, the relationship between Wnt signaling and nephric duct development remains unknown. This study investigated the role of Xenopus frizzled-8 (Xfz8) in pronephros development. Translational inhibition of Xfz8 caused a significant reduction in the staining of a duct-specific antibody, but did not affect the expression of early pronephric maker genes in the duct region. Defects in pronephric tubule branching were also observed following inhibition of Xfz8. Histological analysis revealed that the Xfz8-inhibited cells failed to form a normal epithelium structure. These results suggest that Xfz8 is involved in the process of normal epithelium formation in the developing pronephric duct and tubules after specification.

We describe the isolation and characterization of the RNA-binding protein XC3H-3b that is expressed during pronephros development. XC3H-3b is a member of the TTP/TIS family of CCCH tandem zinc-finger proteins, which are physiological stimulators of instability for the mRNA encoding tumor necrosis factor-alpha in certain cell types. XC3H-3b is localized primarily to the mesodermal tissues around the pronephros. Overexpression of XC3H-3b markedly and specifically inhibits kidney development. Morpholino-mediated knockdown of XC3H-3b also results in defects in nephrogenesis. In both cases, the expression of numerous pronephric marker genes, such as Xlim-1, Xpax-2, Xpax-8, Xwnt-4, and XWT1, is decreased and morphological development of the pronephric tubules is abrogated. We conclude that XC3H-3b plays an important role in the regulation of pronephros differentiation. This is the first report of a gene localized around the pronephros that regulates pronephros development.

In a screen for genes expressed in neural tissues and pronephroi, we isolated a novel gene, named dullard. Dullard protein contains the C-terminal conserved domain of NLI-IF (Nuclear LIM Interactor-Interacting Factor), a protein whose function is not yet characterized. Dullard mRNA was maternally derived and localized to the animal hemisphere. At neurula stages, the expression was in neural regions and subsequently localized to neural tissues, branchial arches, and pronephroi. Using antisense morpholino oligonucleotide-mediated inhibition, we showed that dullard was required for neural development. The translational knock-down of dullard resulted in failure of neural tube development and the embryos consequently showed a reduction of head development. Expression of neural marker genes in dullard-inhibited embryos was also suppressed. These results suggest that dullard is necessary for neural development.

ARNT factors are a cluster of bHLH-PAS factors that heterodimerize with other specific bHLH-PAS factors to mediate a wide range of biological responses. Previously, we obtained a truncated form of ARNT2-like factor, ARNT2A, from zebrafish, which encompasses the basic-helix-loop-helix and PAS A/B domains, but lacks a transactivation domain at its carboxyl end. Herein, we report another truncated ARNT2-like factor, ARNT2X, in zebrafish, which differs from ARNT2A at its N-terminal region. In cultured ZLE cells, transiently expressed ARNT2X and ARNT2A inhibited 2,3,7,8-TCDD-activated cyp1a1 transcription with different efficiencies. In the developing embryo, arnt2X mRNA was consistently expressed in the retinal and neural tube regions until the hatching stages, but it exhibited a more specific pattern at larval stages, including expression in the brain, eyes, hypothalamus, pharyngeal skeleton, heart, liver, pronephros duct, pectoral fin, and epithelial cells of the swim bladder. In contrast, arnt2A transcription diminished after hatching. Microinjecting a recombinant arnt2X-expression vector into fertilized eggs before cleavage stages caused severe defects in brain, eyes, pectoral fin, heart, and gut development. This suggests that the ARNT-mediated signal transduction pathways play important roles in fish tissue development.