Search

The human EYA1 gene is an ortholog of the fruit fly eyes absent gene (eya), which was identified as a regulator of compound eye development. In contrast to the single eya gene found in Drosophila melanogaster, mammals possess four Eya paralogs, designated Eya1-4. The respective gene products are characterized by a C-terminal domain called the Eya domain, which is conserved in both length (271 to 274 amino acids [aa]) and sequence. The Eya domain has an intrinsic phosphatase activity (24, 39, 53) and is required for protein-protein interactions (38). Recent studies provided evidence that the phosphatase function of Eya is involved in the innate immune system and the regulation of DNA damage response (8, 35). Interestingly, all Eya interaction partners that have been identified so far, for example, the Six and Dach proteins or inhibitory G protein α (Gα) subunits, bind to the Eya domain (5, 10, 34). It has been demonstrated that the cooperative action of Eya and Six is essential for the development of several tissues and organs in a variety of species throughout evolution (reviewed in reference 6). In vertebrates, Six induces nuclear translocation of Eya and recruitment to the DNA, and Eya enhances Six-mediated activation of target gene expression (34). The N termini of Eya proteins, which are highly divergent between Eya family members, harbor a proline-serine-threonine (PST)-rich transactivation domain, which is indispensable for their function as coactivators of transcription (31, 45, 50, 58). Natural target genes of the vertebrate Eya-Six complex are, for example, Six2, Sall1, and Myogenin. Activation of Six2 and Sall1 expression has been shown to be essential for proper kidney development in the mouse, whereas activation of Myogenin is required for muscle development (3, 4, 46). Mutations in the human EYA1 gene are associated with several congenital disorders, like BOR (branchio-oto-renal) and BO (branchio-oto) syndrome, as well as ocular defects. BOR syndrome patients suffer from severe malformations of the ears, the branchial arch derivatives, and the kidneys, while in BO syndrome patients, the kidneys are not affected (11, 12, 30). The physiological importance of Eya1 was underlined by knockout studies in mice. It was shown that the phenotype of Eya1-heterozygous mice resembles the characteristic symptoms of human patients suffering from BOR syndrome with renal abnormalities, including hypoplasia and unilateral agenesis, and conductive hearing loss. In contrast, Eya1 homozygote mice die at birth, showing severe craniofacial and skeletal defects and the complete absence of the thymus, parathyroid glands, ears, and kidneys due to defective inductive tissue interactions (57, 59). Interestingly, similar to EYA1, the paralogous EYA4 gene has also been demonstrated to be associated with human disease. EYA4 is most closely related to EYA1 rather than EYA2 and EYA3. Mutations in EYA4 have been linked to cardiomyopathy and hearing loss (37, 42, 55). In contrast, there is no evidence for the existence of disease-associated mutations in EYA2 and EYA3 up to now. The identification and characterization of an Eya1 ortholog in zebrafish revealed a remarkable conservation in both the structure and expression pattern between Eya1 genes in fish and humans. In zebrafish, eya1 expression was detected in several organs during embryogenesis, such as the ears, the lateral line organ, the branchial arches, and the somites (41). As in mammals, eya1 in zebrafish is essential for the development of several tissues and organs. A quite well-studied example is the zebrafish ear, whose proper formation depends on eya1 expression, as shown by morpholino-mediated knockdown analysis (23). Since Eya1 is involved in the development of many different organs, we proposed that additional factors bind to it and, thereby, modulate its function. Using a yeast two-hybrid-based approach, we identified the related proteins Sipl1 (Shank-interacting protein-like 1) and Rbck1 (RBCC protein interacting with PKC1) as so-far-unknown Eya1 interaction partners. We characterized the interactions and demonstrated that both Sipl1 and Rbck1 enhance the function of Eya proteins to act as coactivators for the Six transcription factors. Furthermore, we showed that Sipl1 and Rbck1 are coexpressed with Eya1 in several organs during embryogenesis of both the mouse and zebrafish. Importantly, the morpholino-mediated knockdown of Sipl1 and Rbck1 orthologs in zebrafish led to a BOR syndrome-like phenotype, indicating the relevance of the Eya1-Sipl1/Rbck1 interaction in vivo.

The venom of marine cone snails contains a variety of conformationally constrained peptides utilized by the animal to capture prey. Besides numerous conotoxins, which are characterized by complex disulfide patterns, other peptides with only a single disulfide bridge were isolated from different conus species. Here, we report the synthesis, structure elucidation and biological evaluation of the novel C-terminally amidated decapeptide CCAP-vil, PFc[CNSFGC]YN-NH 2 , from Conus villepinii . The linear precursor peptide was generated by standard solid phase synthesis. Oxidation of the cysteine residues to yield the disulfide-bridged peptide was investigated under different conditions, including several ionic liquids (ILs) as new biocompatible reaction media. Among the examined ILs, 1-ethyl-3-methylimidazolium tosylate ([C 2 mim][OTs]) was most efficient for CCAP-vil oxidative folding, since oxidation occurred without any byproduct formation. The structure of CCAP-vil was determined by NMR methods in aqueous solution and revealed a loop structure adopting a type(I) β-turn between residues 4–7 imposed by the flanking disulfide bridge. The amino acid side chains of Pro 1 , Phe 2 , Phe 6 and Tyr 9 point in three directions away from the cyclic core into the solvent creating a rather hydrophobic surface of the molecule. Based on sequence homology to cardioactive peptides (CAPs) from gastropods and arthropods, such as PFc[CNAFTGC]-NH 2 (CCAP), the influence of CCAP-vil on heart rate using zebrafish embryos was investigated. CCAP-vil reduced the heart rate immediately upon injection into the heart as well as upon indirect application indicating an opposite effect to the cardioaccelerating CCAP.

Background: Kcnh1 is a voltage-gated potassium channel that is primarily expressed in brain. Results: Knockdown of kcnh1 in zebrafish delays neural development and causes embryonic lethality. Conclusion: Kcnh1 is involved in cell proliferation during early zebrafish development. Significance: The finding that Kcnh1 has basic functions beyond neural signaling will help to elucidate its roles in physiology and cancer formation., The Kcnh1 gene encodes a voltage-gated potassium channel highly expressed in neurons and involved in tumor cell proliferation, yet its physiological roles remain unclear. We have used the zebrafish as a model to analyze Kcnh1 function in vitro and in vivo. We found that the kcnh1 gene is duplicated in teleost fish (i.e. kcnh1a and kcnh1b) and that both genes are maternally expressed during early development. In adult zebrafish, kcnh1a and kcnh1b have distinct expression patterns but share expression in brain and testis. Heterologous expression of both genes in Xenopus oocytes revealed a strong conservation of characteristic functional properties between human and fish channels, including a unique sensitivity to intracellular Ca2+/calmodulin and modulation of voltage-dependent gating by extracellular Mg2+. Using a morpholino antisense approach, we demonstrate a strong kcnh1 loss-of-function phenotype in developing zebrafish, characterized by growth retardation, delayed hindbrain formation, and embryonic lethality. This late phenotype was preceded by transcriptional up-regulation of known cell-cycle inhibitors (p21, p27, cdh2) and down-regulation of pro-proliferative factors, including cyclin D1, at 70% epiboly. These results reveal an unanticipated basic activity of kcnh1 that is crucial for early embryonic development and patterning.

The physiological role of KCNH1 channels has not been clarified in detail yet, but the predominant neuronal expression in mammals indicates a role in electrical signaling. The only described physiological function of KCNH1 so far is the promotion of myoblast fusion. Moreover, KCNH1 channels apparently enhance proliferation of cancer cells, thus exhibiting oncogenic potential. Many genes involved in cancerogenesis play a physiological role during embryonic development. To study KCNH1 channel function in this respect, we surveyed genomic databases of Danio rerio, a widely used vertebrate-development model organism, and found two putative kcnh1 paralogs located on chromosomes 17 (kcnh1a) and 22 (kcnh1b). The corresponding amino acid sequences show >80% identity to that of human KCNH1. We observed both kcnh1 genes to be endogenously expressed in different organs of adult fish and at early developmental stages, most prominently in neuronal tissues. We electrophysiologically analyzed the encoded channels by two-electrode voltage-clamp and patch-clamp techniques on Xenopus oocytes, showing the gene products to form functional potassium channels. The Danio rerio channels exhibited typical characteristics known from other species, such as dependence of activation kinetics on prepulse potential and extracellular Mg2+ concentration, as well as current inhibition by intracellular Ca2+/calmodulin. After morpholino-mediated knockdown of the individual kcnh1 paralogs in zebrafish embryos we observed severe effects on development. The morphants were retarded in growth, showed abnormal body curvature, exhibited complete body malformation or even died within less than 24 hrs after fertilization, depending on the degree of knockdown. The most remarkable phenotypes were alterations of brain structures like head edemas, incomplete brain growth and necrotic degeneration. Given the electrophysiological similarity of Kcnh1 channels to the human homolog, we suggest that human KCNH1 potassium channels play a similar role during early embryonic development in humans.

The Wilms' tumor suppressor gene Wt1 encodes a zinc-finger transcription factor that plays an essential role in organ development, most notably of the kidney. Despite its importance for organogenesis, knowledge of the regulation of Wt1 expression is scarce. Here, we have used transgenesis in zebrafish harboring two wt1 genes, wt1a and wt1b, in order to define regulatory elements that drive wt1expression in the kidney. Stable transgenic lines with approximately 30 kb of the upstream genomic regions of wt1a or wt1b almost exactly recapitulated endogenous expression of the wt1 paralogs. In the case of wt1b, we have identified an enhancer that is located in the far upstream region that is necessary and sufficient for reporter gene expression in the pronephric glomeruli. Regarding wt1a, we could also identify an enhancer that is located approximately 4 kb upstream of the transcriptional start site that is required for expression in the intermediate mesoderm. Interestingly, this intermediate mesoderm enhancer is highly conserved between fish and mammals, is bound by members of the retinoic acid receptor family of transcription factors in gel shift experiments and mediates responsiveness to retinoic acid both in vivo and in cell culture. To our knowledge, this is the first functional demonstration of defined regulatory elements controlling Wt1 expression in vivo. The identification of kidney-specific enhancer elements will help us to better understand the integration of extracellular signals into intracellular networks in nephrogenesis.