Study of pronephric-glomerular morphogenesis in zebrafish
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Midline convergence of organ primordia is an important mechanism for shaping the vertebrate body-plan at various stages of development, such as the morphogenesis of the heart and endoderm. Down regulation of wnt or noncanonical wnt signalling components, such as dishelleved (Dvl) or RhoA GTPase (RhoA), impairs midline convergence of the heart primordia and endoderm in zebrafish. This suggests that wnt signaling plays an important role in regulating midline convergence. At the early patterning stage of the zebrafish kidney, the two pronephric-glomerular primodia (PGP), which derive from intermediate mesoderm, converge towards the midline and fuse to form a functional pronephros. In contrast, during development of the mammalian kidney, the pronephros degenerates as the mesonephros develops without midline convergence. The hypothesis is thus that there is/are mechanisms underlying midline convergence of PGP in zebrafish, which is/are in addition to the control of the non-canonical wnt/Dvl/RhoA pathway and specific to kidney morphogenesis. In this study, the aim was to identify genetic factors that are specifically involved in the mechanism of PGP midline convergence by establishing a cell lineage tracing transgenic system with Cre-loxP, followed by the analysis of selected mutant embryos using the cell lineage tracing system, whole-mount in situ hybridization (WISH) and immunostaining. The cell lineage tracing system was generated, and tested. Constructs, in which β-actin promoter drives the transcription of the reporter genes, were microinjected into zebrafish embryos at 1- to 4-cell stages. The mRNAs in microinjected embryos (5 dpf) were analyzed by RT-PCR. The results show that the constructs induced indiscriminate alternative splicing. RNA splicing mechanisms were not affected by transcription termination when the polyA signal was located in introns. To provide an alternative approach, three mutants were selected after screening of available ENU zebrafish mutants. These mutants were chosen not only because of their genetic importance in cell adhesion and motility but also because of their respective developmental defects in tissues surrounding the PGP, such as the notochord (no tail, ntl), somite (spadetail, spt), and endodermal tissues (zygotic oneeyed pinhead, Zoep). Spt and ntl are key targets of fibroblast growth factor (FGF) signalling in the trunk and tail respectively. EGP-CFC gene oep is a Nodal signalling cofactor. Firstly, a rapid genotyping technique was developed, which was applied in identifying the mutant alleles. Since the tools for tracing PGP using transgenes were unavailable, the three mutants were analyzed by WISH and immunostaining. Zygotic mutation of Zoep causes a PGP midline convergence phenotype of variable severity due to maternal Oep effects. In more than 90% of Zoep-/- embryos, PGP midline convergence was impaired. Even though the abnormality could be observed as early as 15 hpf, the differentiation of the PGP was not affected. Heart primordial phenotypes were also observed but they did not correlate with that of the PGP phenotypes. Embryos homozygous for mutations in T-box transcription factors, ntl or spt had normal heart midline convergence phenotypes. PGP midline convergence abnormality was observed in spt-/- but not in ntl-/- prior to 36 hpf. In addition the extracellular matrix (ECM) might play a key role in the mechanisms of PGP midline convergence. Furthermore, PGP midline convergence proceeds from 10 hpf (the specification of intermediate mesoderm) to 48 hpf (fused pronephric glomerulus) in wild type zebrafish embryos. The process was quantified by 2D image analysis of the PGP distance. Prior to 18 hpf, PGP midline convergence is closely correlated with the midline convergence of mesoderm but not at later stages in Zoep-/-. Spt is essential for PGP but not for cardiac primordium midline convergence. Data from this research suggests that there is not one universal mechanism, which controls all the midline convergence of organ primordia. Indeed, specific factors, which depend on tissues and development stages, are also required.