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First published online 21 June 2006
doi: 10.1242/dev.02442

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Odd-skipped related 1 is required for development of the metanephric kidney and regulates formation and differentiation of kidney precursor cells

Richard G. James^1,2,*, Caramai N. Kamei^1,2,*, Qingru Wang³, Rulang Jiang³ and Thomas M. Schultheiss^1,2,

¹ Molecular and Vascular Medicine Unit, Beth Israel Deaconess Medical Center, 330 Brookline Ave, RW-663, Boston, MA 02215, USA.
² Program in Biological and Biomedical Sciences, Harvard Medical School, Boston, MA, USA.
³ Center for Oral Biology and Department of Biomedical Genetics, University of Rochester School of Medicine and Dentistry, Rochester, NY, USA.

View larger version (98K): [in a new window]   Fig. 1. Expression of Odd1 in the chick embryo. (A,B) Whole-mount in situ hybridization for Odd1 in HH Stage 5 (A) and Stage 9 (B) chick embryos. (C,D) In situ hybridization for Pax2 at Stage 9 (C) and Stage 11 (D). Note that Odd1 is expressed earlier than Pax2, and that it extends more broadly than Pax2 in lateral, anterior and posterior directions. Arrows in A and C indicate nascent Odd1 and Pax2 expression, respectively. (E-G) Sections of whole-mount in situ hybridization for Odd1. At Stage 11 (E) Odd1 is expressed in the intermediate mesoderm and extends into the lateral plate but is not found in the nephric duct. At Stage 13 (F), Odd1 is expressed in mesenchymal cells adjacent to mesonephric tubules and also in splanchnic mesoderm derivatives, including the dorsal mesentery, but not in epithelialized nephric duct or tubules. At embryonic day 5 (G), Odd1 is expressed in the coelomic lining (arrowhead) and in the rudimentary somatic gonad (arrow). (H-M) Expression of Odd1 and Pax2 protein during formation of the nephric duct rudiment. At Stage 9 (H-J), before the duct rudiment is morphologically detectable, most Pax2-expressing cells (arrow) also express Odd1. Inset in J shows merged detail from I and J. By Stage 10 (K-M), the nephric duct rudiment has begun to bulge dorsally (arrow) and continues to express Pax2 but no longer expresses Odd1. Inset in M shows merged detail from L and M. (N-P) In the developing mesonephros, Odd1 protein is expressed in mesenchymal cells adjacent to the tubules, but not in the Pax2-expressing tubules themselves. Inset in P shows merged detail from O and P. d, nephric duct; im, intermediate mesoderm; lp, lateral plate; n, Hensen's node; t, tubules.

View larger version (127K): [in a new window]   Fig. 2. Expression of Odd1 in the mouse embryo. LacZ staining of mouse embryos heterozygous for the ß-galactosidase gene inserted into the Odd1 locus. At E9.5, Odd1 is expressed in the intermediate and part of the lateral plate mesoderm (A). In a section through the mesonephros (B), lacZ staining is present in the coelomic lining and mesenchyme, but is absent from the nephric duct and largely absent from the mesonephric tubules. (C) Schematic of events in metanephros formation: (left) invasion of the ureteric bud (white) into the metanephric mesenchyme (blue); (center) initial branching of the ureteric bud; (right), continued branching of the ureteric bud and formation of epithelial vesicles (yellow). See text for details. (D) At E10.5, Odd1 is expressed in the metanephric mesenchyme but not in the ureteric bud. (E,F) At E11.5, Odd1 is expressed in the undifferentiated condensed mesenchyme of the metanephros and at lower levels in mesenchyme surrounding the ureteric stalk, but not in ureteric bud derivatives. Odd1 is also expressed in mesenchyme adjacent to the ventral aorta (arrow in E). At E13.5 (G-I), Odd1 continues to be expressed in the condensed mesenchyme, but not in pretubular aggregates (H, arrow) or comma-shaped bodies (I, arrow). The lacZ staining in the ureter epithelium in G is non-specific. a, aorta; cm, condensed mesenchyme; d, nephric duct; du, ureter epithelium; m, mesonephric mesenchyme; mm, metanephric mesenchyme; s, ureteric stalk; t, mesonephric tubules; u, ureteric bud.

View larger version (91K): [in a new window]   Fig. 3. The metanephric mesenchyme fails to form in Odd1-/- embryos. (A,B) Sections through the metanephric region of E11.5 heterozygous and homozygous null embryos stained with hematoxylin and eosin. In heterozygotes (A), a nephric duct, ureteric bud and condensed metanephric mesenchyme are seen. Homozygous null embryos (B) completely lack a ureteric bud and condensed mesenchyme (arrow). A duct is often present (on right side but not left in B), but smaller than normal. The region between the aorta and nephric duct is also significantly smaller in the Odd1 null embryos. (C-J) Sections through the metanephric region of mouse embryos stained by in situ whole-mount hybridization (C-F) or immunofluorescence (G-J) for the indicated gene products (Dapi nuclear stains in G and I correspond to sections in H and J, respectively). At E10.5, the metanephric mesenchyme of Odd1 heterozygous embryos expresses Six2 (C), Eya1 (E) and Pax2 (H). All three gene products are absent from the metanephric mesenchyme region of Odd1 null mutants (D,F,J). The mutant nephric duct does express Pax2 (J). The duct in mutant embryos is outlined with a dotted line (D,F,I). The fluorescence in the upper right corner in J is autofluoresc;ence from blood cells in the aorta. a, aorta; d, nephric duct; mm, metanephric mesenchyme; u, ureteric bud.

View larger version (82K): [in a new window]   Fig. 4. Effects of loss of Odd1 on nephric duct and mesonephric tubule development. Odd1 heterozygous (A,B,E) and homozygous null (C,D,F) mouse embryos stained by whole-mount in situ hybridization for Lim1 (A-D) or Pax2 (E,F). B and D show sections through the mesonephric region. In homozygous null embryos, the nephric duct is thinner and often interrupted (C, left arrowhead in D). A few mesonephric tubules form in the null mutants (F, arrowhead), but fewer than in the heterozygotes (E, arrowheads). In Odd1 mutants, the nephric duct also does not turn normally toward the midline at the posterior end (arrows in E,F). (G,H) Sections through the mesonephric region of E10.5 embryos stained with immunofluorescence against the indicated antigens. Tubules are smaller in mutant (H) compared with control (G) embryos. In both mutant and control embryos, Pax2 is expressed in the nephric duct and the tubules, while Wt1 is expressed in the proximalmost part of the tubule (arrow), as well as in the mesenchyme and coelomic lining. d, nephric duct; t, tubules.

View larger version (100K): [in a new window]   Fig. 5. Timing of apoptosis and loss of nephric gene expression in Odd1 mutant embryos. Odd1 heterozygous (A,C,G) and homozygous null (B,D,H) embryos were analyzed by TUNEL to detect apoptotic cells and stained with antibodies against Pax2. In the mesenchyme surrounding anterior mesonephric tubules, levels of apoptosis are significantly elevated in Odd1 mutant (B, asterisk) compared with control embryos (A). By contrast, levels of apoptosis in E9.5 metanephric mesenchyme precursor cells are low in both Odd1 mutants (D) and controls (C). At this stage and axial level, Pax2 (D) and Eya1 (F) expression in prospective metanephric mesenchyme are absent in mutant embryos, while dermamyotomal Eya1 expression is normal (E,F). By E10.5, Odd1 null mutants (H) have increased levels of apoptosis in the metanephric mesenchyme (asterisk) compared with heterozygotes (G). At E10.5, Wt1 is expressed in the mesenchyme of both mutant (J, arrowhead) and wild-type (I) embryos. Similarly, Odd1 mutant embryos (L) exhibit substantial levels of Odd1 promoter activity, as detected by lacZ expression driven by the Odd1 promoter (heterozygote shown in K). a, aorta; d, duct; m, nephric mesenchyme; mm, metanephric mesenchyme; t, tubule; u, ureter.

View larger version (78K): [in a new window]   Fig. 6. Odd1 can induce early nephric markers when ectopically expressed in the somites. Control chicken embryos (A,D) and embryos electroporated with Mes-Odd1 expression vector (B,C,E) were stained with immunofluorescence against Pax2 (A,B) or GFP (C), or by whole-mount in situ hybridization for Lim1 (D,E). Ectopic cells expressing Pax2 or Lim1 are found in the somite region (arrowheads in B,E). In B and C, note that GFP cells in the lateral plate (arrows) do not express ectopic Pax2. im, intermediate mesoderm; nt, neural tube; s, somite.

View larger version (101K): [in a new window]   Fig. 7. Expression of Odd1 inhibits kidney tubule formation. (A-C) Representative control chicken embryos infected with RCAS-Gfp at day 0 and harvested at day 5, showing extensive infection (A) and robust mesonephric tubule formation (A-C). (D-F) RCAS-Odd1 infected embryos analyzed by section in situ hybridization for Odd1 to document extent of infection (D), by Nomarski microscopy (E) and with immunofluorescence for Pax2 (F). The mesonephroi of RCAS-Odd1 infected embryos contain fewer epithelialized tubules (compare B,C with E,F), but do contain significant numbers of Pax2-expressing non-epithelialized cells (arrow in F). When epithelialized tubules are present in RCAS-Odd1 infected embryos, the tubules tend to be uninfected (D), whereas tubules are readily infected by control RCAS-Gfp virus (A) (in situ detection conditions were calibrated to detect only ectopic and not endogenous Odd1 message). The area of total tubular tissue (defined as Pax2-expressing cells arranged either as lumenized epithelia or as aggregates with polarized nuclei) in RCAS-Odd1 infected embryos is significantly smaller than in control embryos (G), but the nephric ducts are approximately the same size (H). Bar graphs show mean and standard error of 8 RCAS-Gfp and 10 RCAS-Odd1 samples. The y-axis is in arbitrary units. d, nephric duct; t, tubule.