|
Fig. 2. Embryonic expression pattern of Evi1 and MEL1. (A) Evi1 in Xenopus. Top panels: RNAase protection analysis of Evi1 transcripts during oogenesis and embryogenesis (left) and in adult organs (right). FGFR or EF1-alpha are used as internal controls. Bottom panels: whole-mount in situ hybridization analysis of Evi1 expression. Embryos were fixed at the indicated Nieuwkoop-Faber stages and analyzed with the probes as indicated. (1–5) Expression of Evi1 at late neurula stage. (1) Initial expression of Evi1 is observed in the pronephric mesoderm. (2) Transversal section of the embryo shown in (1) showing restricted Evi1 expression in the somatic layer. (3) Dorsal view of a slightly later stage embryo showing Evi1 expression in selected regions of the developing brain. (4) Comparison of Evi1 (purple) and Pax8 (light blue) expression in the pronephros; note that Evi1 expression is restricted to the ventral posterior part of the Pax8 expression domain. (5) Transversal section at the level of the pronephros. (6–11) Evi1 expression at tailbud stages. (6) Lateral view of an embryo; note Evi1 expression outside the pronephros in the forebrain, midbrain and hindbrain and in cranial neural crest cells. (7) Transversal section of the embryo shown in (6) at the level indicated; note that Evi1 expression can be seen only in the ventral part of the pronephros anlagen. (8) Horizontal section showing Evi1 expression in the dorsal telencephalon, ventral diencephalons and in the olfactory placodes. (9) Transversal section at the level of r4 in the hindbrain; note that only the dorsal part of the neural tube is labeled by the Evi1 probe. (10) High-magnification view of the hindbrain with anterior towards the top double stained with Evi1 (red) and XKrox20 (purple) probes indicating that Evi1 is expressed in r4. (11) Embryo double stained with Evi1 (light blue) and XKrox-20 (purple); note that the Evi1 strongest staining in the migrating neural crest cells is found anteriorly to the XKrox-20 expressing cells. (12–16) Evi1 expression at tadpole stages. (12) High-magnification view of the head; note that Evi1 expression is restricted to the posterior part of the pronephros and that in the hindbrain, its expression initially restricted to r4 has expanded anteriorly and posteriorly. (13) Transversal section at the level of the otic vesicles; note that Evi1 expression can be seen in addition to the dorsal neural tube in a subset of differentiated neurons. (14) Horizontal section through the visceral arches, with anterior towards the right; note Evi1 expression in the neural crest component of the branchial arches. (15) Lateral view of a late tadpole; note that within the pronephros, Evi1 expression is restricted to the distal tubule and duct compartments. (16) Transversal section at the level of the trunk showing Evi1 expressing pronephric duct cells.
Abbreviations: a1–a4, arch 1–4; ba, branchial arches; bcs, branchial crest segment; cnc, cranial neural crest; dd, dorsal diencephalon; dt, dorsal telencephalon; e, eye; fb, forebrain; hb, hindbrain; hcs, hyoid crest segment; h, heart; hm, head mesenchyme; im, intermediate mesoderm; lb, limb buds; mcs, mandibular crest segment; mb, midbrain; nc, notochord; nd, nephric duct; nt, neural tube; olp, olfactory placodes; ov, otic vesicle; pf, pectoral fins; pn, pronephros; pnd, pronephric duct; pndt, pronephric distal tubule; ppnd, posterior pronephric duct; rpe, retinal pigment epithelium; r3–5, rhombomere 3–5; sc, spinal cord; som, somatic lateral plate mesoderm; spm, splanchnic lateral plate mesoderm; tel, telencephalon; tg, tegmentum; va, visceral arches; vd, ventral diencephalon.
|
|
Fig. 3. Comparison of ClC-K, ret, Sat1, Evi1 and PDZK1 expression in Xenopus and zebrafish pronephros. The stages and probes analyzed are indicated in each figure. Anterior is toward the right. (A–D,G–J) Single in situ hybridization of the indicated markers. Note that in zebrafish ClC-K and ret1 expression is restricted to the posterior part of the duct while the proximal tubule specific markers Sat1 and PDZK1 are expressed in its anterior portion. (E) Comparison by fluorescent in situ hybridization of Evi1 (green) and ret1 (red) expression in a 24-hpf zebrafish embryo. Note that Evi1 expression extends further anteriorly than that of ret1. (F) Comparison by double in situ hybridization of ClC-K (red) and ret1 (purple) expression in a 24-hpf zebrafish embryo. Note that ClC-K expression extends further anteriorly than that of ret1. Abbreviations: apnd, anterior pronephric duct; pnd, pronephric duct; pndt, pronephric distal tubule; ppnd, posterior pronephric duct; ppt, proximal pronephric tubules.
|
|
Fig. 4. Evi1 expression in the pronephros is upregulated by retinoid signaling and repressed by xWT1 and Notch signaling. Embryos were injected with the indicated mRNA together with β-galactosidase mRNA and processed at tailbud stage by whole-mount in situ hybridization to visualize Evi1 expression. In all panels, anterior is to the right. Control and injected sides revealed with X-gal are shown. (A–H) Evi1 expression is elevated in embryos by RA treatment or by injection of mRNA encoding XRALDH2 in the presence of its substrate, all-trans retinal. Conversely, it is decreased in embryos where RA signaling is inhibited by injection of xCYP26 mRNA or the dominant negative RARα receptor xRARα1405*. (I–L) Evi1 expression is increased in embryos where Notch signaling is inhibited by injection of mRNA encoding the dominant negative form of Su(H), Su(H)DBM. Conversely, a reduction of Evi1 expression is observed in embryos overexpressing the activated form of Notch, NICD. (M, N) Evi1 expression is downregulated by xWT1 mRNA injection.
|
|
Fig. 5. Overexpression of Evi1 down-regulates Xbra and chordin expression but not other genes in the early Xenopus embryo. (A) Embryos injected with Evi1 mRNA failed to complete gastrulation. (B–F) Whole-mount in situ analysis of the indicated markers in embryos coinjected with Evi1 and β-galactosidase mRNA. In all cases, β-galactosidase activity is revealed with X-gal. (B, C) Vegetal views of injected embryos at early gastrula stage. A reduction of the expression of the mesodermal markers Xbra and chordin is seen in the injected area. (D–F) Anterior view (D) and dorsal views (E, F) of neurula stage injected embryos analyzed for the expression of the ectodermal EpK, the neural crest Slug and the neural Sox2 marker. Note that Evi1 mRNA injection has no effect on the expression of these markers.
|
|
Fig. 6. Overexpression of Evi1-hGR in the pronephros with DEX addition from stage 18 specifically suppresses the expression of proximal but not distal markers. (A,B, E–L, O–P) Lateral views and (C, D, M, N and Q, R) sections through the pronephros of tailbud or early tadpole stage embryos coinjected with Evi1-hGR mRNA and β-galactosidase mRNA and stained with the indicated markers. Control and injected sides marked with Red-gal are shown. Note the reduction of the expression of the X-Delta-1 (A–D), EphA4 (E,F) and XSMP-30 (G, H) proximal tubule and xWT1 (K–N) and nephrin (O, R) glomus markers (arrowheads) while the expression of the Gremlin (G, H) and xClC-K (I, J) distal tubule and duct markers is unaffected (arrow). Abbreviations: pn, pronephros; spm, splanchnic layer of the mesoderm.
|
|
Fig 2D. Whole-mount in situ analysis of MEL1 in Xenopus. (1, 2) Lateral views of early tailbud stage embryos. Note MEL1 expression in cranial crest cells, in the head mesenchyme and in the otic vesicle. (3, 5) High magnification view of the head and transversal sections of early tadpoles. Note that MEL1 staining is now also apparent in various parts of the brain and in the retinal pigment epithelium. (6) Lateral view and horizontal sections of late tadpoles. Note that additional MEL1 staining is seen in the heart and in the pronephric distal tubule and duct.
Abbreviations: a14, arch 1; ba, branchial arches; bcs, branchial crest segment; cnc, cranial neural crest; dd, dorsal diencephalon; dt, dorsal telencephalon; e, eye; fb, forebrain; hb, hindbrain; hcs, hyoid crest segment; h, heart; hm, head mesenchyme; im, intermediate mesoderm; lb, limb buds; mcs, mandibular crest segment; mb, midbrain; nc, notochord; nd, nephric duct; nt, neural tube; olp, olfactory placodes; ov, otic vesicle; pf, pectoral fins; pn, pronephros; pnd, pronephric duct; pndt, pronephric distal tubule; ppnd, posterior pronephric duct; rpe, retinal pigment epithelium; r3, rhombomere 3; sc, spinal cord; som, somatic lateral plate mesoderm; spm, splanchnic lateral plate mesoderm; tel, telencephalon; tg, tegmentum; va, visceral arches; vd, ventral diencephalon.
|
|
Fig. 7. Test of Evi1 ZF1, ZF2 and CtBP mutants for their ability to inhibit XSMP-30 expression. Embryos overexpressing the indicated inducible Evi1 mutants were fixed at tadpole stage (stage 36) and the expression of the XSMP-30 proximal tubule marker was analyzed. Control and injected sides marked with Red-gal are shown. Note that while Evi1δZF2-hGR is as effective as the wild type protein to repress XSMP-30 expression (A, B), the deletion of ZF1 or the CtBP interaction motifs abolishes Evi1 activity (C).
|
|
Fig. 8. Overexpression of Evi1-VP16-hGR disrupts distal and expands early proximal markers. (A, I, O) Lateral views and (G, H, M, N and Q, R) sections through the pronephros of tailbud or early tadpole stage embryos coinjected with Evi1-VP16-hGR mRNA and β-galactosidase mRNA and stained with the indicated markers. Control and injected sides marked with Red-gal are shown. Note the inhibition of the expression of the expression of the XClC-K (A, B) and Gremlin (C, D) distal tubule and duct markers (arrowheads), the expansion of the xWT1 (E) and nephrin (I, J) glomus and X-Delta-1 (K) and EphA4 (O, P) early proximal markers (arrows). (Q) Example of injected embryos with reduced proximal tubules. (R) Example of an embryo with a pronephros containing additional tubule branches and ectopic xPDZK1 staining (arrow). Abbreviations: pn, pronephros; spm, splanchnic layer of the mesoderm; pt, proximal tubules.
|
