QUICK SEARCH:   [advanced] Author: Keyword(s):
Home     Help     Feedback     Subscriptions     Archive     Search     Table of Contents

This Article Summary Full Text Full Text (PDF) Alert me when this article is cited Alert me if a correction is posted Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Citing Articles Citing Articles via HighWire Citing Articles via Google Scholar Google Scholar Articles by Erter, C. E. Articles by Solnica-Krezel, L. Search for Related Content PubMed PubMed Citation Articles by Erter, C. E. Articles by Solnica-Krezel, L. Social Bookmarking                         What's this?

Wnt8 is required in lateral mesendodermal precursors for neural posteriorization in vivo

¹ Department of Cell Biology, Vanderbilt University School of Medicine, 1161 21st Avenue South, Nashville, TN 37232-2175, USA
² Department of Biological Sciences, Vanderbilt University, VU Station B 351634, Nashville, TN 37235, USA

View larger version (83K): [in a new window]   Fig. 1. Mesoderm induction in Nodal signaling-deficient embryos. Animal view. In situ hybridization of ntl expression in wild type (A,B), cyc;sqt (D,E) and cyc;sqt enh (G,H) embryos at 30% (A,D,G) and 60% (B,E,H) epiboly. Arrowheads indicate ntl boundaries. Bright field images (C,F,I) show posterior tail somite formation (arrows) in wild type (C) and cyc;sqt (F), but not cyc;sqt enh (I).

View larger version (94K): [in a new window]   Fig. 2. Proper AP brain regionalization correlates with wnt8 expression. Dorsal view; anterior is upwards. In situ hybridization for six3, pax2.1 and krox20 expression in the brain anlagen of wild type (A), cyc;sqt (B), atv RNA-injected (C) and cyc;sqt enh (D) embryos at the four-somite stage. In situ hybridization for wnt8 at 50% (E-H) and 70% (I-L) epiboly. Dorsal view. Black lines denote dorsal gap in wnt8 expression (F,G,I-K). Embryos were co-hybridized with gsc probe (I-L) to phenotypically distinguish double mutant embryos from wild-type or single mutant embryos before genotyping, and to monitor the dose of atv RNA injected. fb, forebrain; mhb, midbrain-hindbrain boundary; hb, hindbrain; 4s, four-somite stage.

View larger version (61K): [in a new window]   Fig. 3. Loss of wnt8 expression is associated with expansion of anterior neural fates during gastrulation. Lateral view; dorsal is towards the right. Wild-type (A,E), cyc;sqt (B,F), atv RNA-injected (C,G) and cyc;sqt enh (D,H) embryos at 60-65% epiboly (A-D) and YPC (E-H). ntl (red); otx1 (blue). Black arrowheads indicate the posterior otx1 boundary. Red arrowheads indicate the margin.

View larger version (80K): [in a new window]   Fig. 4. (A-H) Neuroectoderm of Nodal-deficient embryos is posteriorized between 80% epiboly and YPC. atv RNA/caged-fluorescein co-injected embryos at indicated stages. Dorsal is towards the right in A,C,E,G. B,D,F,H are dorsal views. Black brackets indicate in situ probe signal. Red brackets indicate uncaged cells. (I) Cartoon depicting the co-injection experiment in (A-H). (J) atv RNA-injected embryos were collected over a gastrulation timecourse (60%, 70%, 80%, 90% and 100% (YPC) epiboly), and analyzed for the number of embryos showing anterior displacement of the otx1 expression domain (n=2/55 (4%); n=4/42 (10%); n=2/23 (9%); n=23/39 (59%); and n=27/27 (100%), respectively).

View larger version (83K): [in a new window]   Fig. 5. Downregulation of Wnt8 function results in loss of spinal cord primary neurons. (A,B) Expression of ntl at 60% epiboly. (C-F) Expression of krx20/huC and ephA4/isl1 at six-somite stage. (A,C,E) Untreated wild-type embryos; (B,D,F) MOwnt8-injected wild-type embryos (class I). (A,B) Dorsal view, animal pole is upwards (asterisks mark animal pole position). (C-F) Dorsal view, anterior is upwards. The number of huC- or isl1-expressing Rohon-Beard neurons (rb) and motoneurons (mn) of the developing spinal cord was dramatically reduced after injection of MOwnt8 (146/219 (66.7%); arrowheads in D,F). By contrast, expression of the mesodermal marker ntl (B) and of ephA4 in the notochord (E,F; nc, not in focus) was not affected. General dorsalization of injected embryos is manifested by mediolateral enlargement of rhombomeres (marked by krx20 and ephA4, D) and notochord (F). Embryonic shield (es), rhombomere 3/5 (r3/5); Scale bar: 100 µm.

View larger version (46K): [in a new window]   Fig. 6. (A-D) MOwnt8 blocks neural posteriorization. In situ analysis of goosecoid (gsc) expression at 50% epiboly. Animal view, dorsal right. Black lines show lateral gsc borders. (E-L) six3, pax2.1, krox20 and papC expression, four-somite stage. Lateral view, anterior is towards the left and upwards, posterior is downwards. *, six3 posterior border; red arrowhead, pax2.1 posterior border; black arrowhead, anterior papC border. (M) Graph shows phenotypic distribution of injected embryos, recorded in the table below. Class I, loss of spinal cord huC expression and partial loss of rhombomere 5 (r5) krox20 expression; Class II, loss of r5 and partial loss of rhombomere 3 (r3) krox20 expression; Class III, loss of all krox20 expression; Class IV, loss of krox20 and pax2.1, altered embryonic morphology.

View larger version (110K): [in a new window]   Fig. 7. MOwnt8 blocks effects of Wnt8 in a highly specific manner. Dorsal view, anterior is upwards. (A) wnt8 RNA-injected, (B) wnt8 RNA + MOwnt8-injected, (C) wnt8mut RNA-injected and (D) wnt8mut + MOwnt8-injected embryos. In situ analysis of four-somite stage embryos with markers six3, pax2.1, krox20 and papC.

View larger version (100K): [in a new window]   Fig. 8. boz and Nodal signaling have opposing roles in regulation of wnt8 expression and forebrain specification. Expression of six3, pax2.1, krx20 and ntl in boz mutant embryos at the six-somite stage. (A) Untreated, (B) after MOwnt8 injection; dorsal views, anterior is upwards. Expression of wnt8 is upregulated in boz mutant embryos (D), but downregulated or completely lost in cyc;sqt (E) and boz;cyc;sqt (F) mutant embryos. (C) Wild type. Embryos (D-F) have been genotyped by PCR after in situ hybridization (for details, see text). The mesodermal marker gsc was used for better identification of phenotypic classes in triple mutant background (C-F). (G) Model illustrating the opposing roles of boz, cyc and sqt in wnt8 regulation. Scale bar: 100 µm.

CiteULike

Complore

Connotea

Del.icio.us

Digg

Reddit

Technorati

Twitter