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???displayArticle.abstract??? Pdzrn3, a multidomain protein with E3-ubiquitin ligase activity, has been reported to play a role in myoblast and osteoblast differentiation and, more recently, in neuronal and endothelial cell development. The expression of the pdzrn3 gene is developmentally regulated in various vertebrate tissues, including muscular, neural and vascular system. Little is known about its expression during kidney development, although genetic polymorphisms and alterations around the human pdzrn3 chromosomal region have been found to be associated with renal cell carcinomas and other kidney diseases. We investigated the pdzrn3 spatio-temporal expression pattern in Xenopus laevis embryos by in situ hybridization. We focused our study on the development of the pronephros, which is the embryonic amphibian kidney, functionally similar to the most primitive nephric structures of human kidney. To explore the role of pdzrn3 during renal morphogenesis, we performed loss-of-function experiments, through antisense morpholino injections and analysed the morphants using specific pronephric markers. Dynamic pdzrn3 expression was observed in embryonic tissues, such as somites, brain, eye, blood islands, heart, liver and pronephros. Loss of function experiments resulted in specific alterations of pronephros development. In particular, at early stages, pdzrn3 depletion was associated with a reduction of the pronephros anlagen and later, with perturbations of the tubulogenesis, including deformation of the proximal tubules. Rescue experiments, in which mRNA of the zebrafish pdzrn3 orthologue was injected together with the morpholino, allowed recovery of the kidney phenotypes. These results underline the importance of pdzrn3 expression for correct nephrogenesis.
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1. Whole-mount in situ hybridization analysis of pdzrn3 during Xenopus laevis development. Nieuwkoop-Faber stages (st) of embryogenesis are indicated at the bottom right corner of each panel. The analysed genes are indicated at the top right corner of each panel. Abbreviations: branchial arches (ba), cement gland (ce), central nervous system (CNS), cloaca (cl), cranial ganglia (cg), eye vesicle (e), heart (h), otic vesicle (ov), pronephric anlagen (p), pronephric duct (pd), pronephric proximal tubules (pt), somites (s), spinal cord (sc), trigeminal anlagen (tr). (A,D) Dorsal views of pdzrn3 hybridized embryos during neurulation; black arrows indicate the specific pdzrn3 signal. (E-E’’) Comparison of embryos hybridized with pdzrn3, hnf1b and pax8 (lateral views, anteriorleft); black arrowhead in (E) underlines the pdzrn3 signal below the somites; black arrow indicates the specific pdzrn3 signal in the ventral region. (F,F’) Comparison of embryos hybridized with pdzrn3 and hnf1b. Somites numbers 2, 3, 4 are indicated and the ventral region is pointed by the arrow. (G-G’’) Ventral views of embryos hybridized with pdzrn3, hnf1b and Nkx2.5. In (G) the white bracket indicates the ventral region and white arrowheads point to a region below the ce gland. The ce is dashed in (G’). (H) Lateral view, anterior to the left and (I) ventral view (head to the top) showing the strong signal in the ventral region of embryos (white bracket); open arrowheads point to specific regions of the CNS. (J,K) Lateral views of the developing pronephros at st. 33-34 and 37-38. The dashed box in (K) corresponds to the enlarged view in panel (N). (L) Complete view of the embryo at st. 37-38. (M-Q) Details of specific domains of expression. White brackets in (L), (M) and (M’) indicate the liver anlagen. (N) Enlargement of (K) dashed box. (O) Particular of the embryohead; open arrowheads: the regions of the CNS already detectable at st. 28. (P) Particular of the dorsal fin cells are highlighted in dashed box. (R) Transversal section of embryo, showing specific pdzrn3 signal in glomus (red arrows) and tubules (blue arrows). (S) Schematic representation adapted from Vize et al., (1997). Scale bars: A-E’, 150 mm; F-G’’,R, 250 mm; I,M,M’, 300 mm; H,J,K,L,O-Q, 500 mm.
Fig. 2. Analysis of pdzrn3 depletion effects using pax8 and hnf1b pronephric markers. Mo-pdzrn3 was injected into the marginal zone of one blastomere of Xenopus embryos at 2-cell stage; lacZ mRNA was used as lineage tracer. Control and injected sides of the same embryo are shown, respectively. Stages of embryos are indicated at the bottom right corner of each panel, while the analyzed gene is indicated to left of each row. Abbreviations: otic vesicles (ov), pronephric proximal tubules (pt). (A-F) Impairment of pdzrn3 gene function does affect early expression domains of pax8 and later the morphogenesis of nephrostomes, indicated by arrows. (G-L) In situ hybridization with hnf1b reveals reduction of the pronephron anlagen (black brackets) and pt alterations. The three nephrostomes, clearly detectable in the controls (arrowheads) are indistinct in the morphants. Embryos are shown in lateral views. Scale bars: A,B,G,H, 150 mm; C-F,I-L, 250 mm.
Fig. 3. Analysis of pdzrn3 depletion effects using Na+/K+ATPase and nephrin pronephric markers. Xenopus embryos were injected with Mo-pdzrn3 at 2-cell stage into the marginal zone of one blastomere or at 16-cell stage into V1.1 blastomere, as indicated at the top of the injected side panels. In situ hybridization with Na+/K+ATPase probe on embryos at st. 37-38 (A-H) reveals that impairment of pdzrn3 gene function does affect the formation of pronephric proximal tubules (pt) but not the formation of the duct (A,C,E,G), or the formation of the glomus, revealed by in situ hybridization with nephrin probe (I,J). Scale bars: A,J, 300 mm.
Fig. 4. Coinjections of Mo-pdzrn3 + full-length pdzrn3RNA of Danio rerio allow to significantly recover wild type phenotypes. The coinjections were carried out into one blastomere at 2-cell stage. Comparison of the injected and control side of Xenopus embryos reveals the rescue of the expression domains of pax8 (C,D) and hnf1b (G,H) at stage 22, and correct formation of the pronephric tubules and of the nephrostomes (arrows) at stage 37-38, as revealed using Na+/K+ATPase probe (A,B), pax8 probe (E,F), and hnf1b (I,J). Scale bars: A,B,E,F,I,J, 300 mm; C,D,G,H, 600 mm.
pdzrn3 (PDZ domain containing ring finger 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 13, dorsal view, anterior up.
pdzrn3 (PDZ domain containing ring finger 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, dorsal view, anterior up.
pdzrn3 (PDZ domain containing ring finger 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 25, lateral view, anteriorleft, dorsal up.
pdzrn3 (PDZ domain containing ring finger 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
