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Research Article
Cooperative activity of noggin and gremlin 1 in axial skeleton development
David A. Stafford, Lisa J. Brunet, Mustafa K. Khokha, Aris N. Economides, Richard M. Harland
Development 2011 138: 1005-1014; doi: 10.1242/dev.051938

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Figures

  • Fig. 1.
    Fig. 1.

    Nog and Grem1 expression during somite pattern formation. Whole-mount in situ hybridization for mouse Nog (A,A′,B,C) and Grem1 (D,D′,E,F). (A) E8.5 Nog expression in the notochord (arrowheads) and dorsal neural tube. (A′) Transverse section through somite 6 showing E8.5 Nog expression in the dorsal neural tube (arrow) and notochord (arrowhead). (B) Nog is expressed at E9.0 in the dorsal neural tube (arrowheads) but has become restricted to the posterior notochord (bracket). (C) At E9.5, Nog expression is detectable in the tail notochord, dorsal neural tube and medial dermomyotome. (D) E8.5 Grem1 expression in the recently formed somites and posterior paraxial mesoderm. (D′) Transverse section through somite 6 showing E8.5 Grem1 expression in the dorsal neural tube, dorsal-lateral somite (arrows) and lateral plate mesoderm (arrowheads). (E) At E9.0, somitic Grem1 expression is restricted to the lateral dermomyotome. Expression also appears in the posterior dorsal neural tube and lateral mesoderm (arrow). (F) E9.5 Grem1 expression is concentrated in the lateral dermomyotome (arrowheads) and has initiated in the medial dermomyotome of anterior somites (arrow). Lateral views, facing left. Scale bars: 300 μm.

  • Fig. 2.
    Fig. 2.

    Nog mutants and Nog;Grem1 mutant mouse embryos have impaired somite formation. (A) E9.5 Nogfx/+;Grem1fx/+ embryo. This genetic class resembles the wild-type condition. (B) A Nogfx/+;Grem1fx/fx embryo. There are no evident developmental consequences of Grem1 mutation at E9.5. (C) A Nogfx/fx;Grem1fx/+ embryo. At E9.5, Nog mutants exhibit small posterior somites and kinked neural tubes. (D) A Nogfx/fx;Grem1fx/fx embryo. Double-mutant embryos have severe defects in neural tube, somite, head and tail development. (E-H) Whole-mount E9.5 in situ hybridizations for the somite marker Meox1; lateral views, facing left. A minimum of five embryos were examined for each genetic class. (E,F) Nogfx/+;Grem1fx/+ (E) and Nogfx/+;Grem1fx/fx (F) embryos appear as wild type. (G) A Nogfx/fx;Grem1fx/+ embryo showing reduced somite size from somites 7-15 (bracket), an absence of Meox1 expression in the lumbar region, and a restoration of signal in the tailbud (arrow). (H) A Nogfx/fx;Grem1fx/fx double-mutant embryo. Whereas Meox1 expression is similar to that in Nog mutants in anterior somites, no expression appears posterior to somite 15. (I-K) E9.0 in situ hybridizations for Meox1. Transverse sections through the trunk between somites 8-12. (I) Somites of Nogfx/+;Grem1fx/+ embryos have clear epithelial morphology and somitocoele (asterisk). (J,K) Nogfx/fx;Grem1fx/+ (J) and Nogfx/fx;Grem1fx/fx (K) somites are disorganized and reduced in size. (L-N) Transverse sections through the trunk between somites 8-12 of E9.0 embryos stained for F-actin (green) and atypical protein kinase C (PKC, red) to mark the apical (internal) surface of the epithelial somite. Three embryos of each genotype were examined. (L) Somites of Nogfx/+;Grem1fx/+ embryos show F-actin and PKC accumulation at the apical surface. The discontinuity in the stain medially (arrows) indicates the epithelial-to-mesenchymal transition associated with sclerotome formation. (M,N) Epithelium formation in Nogfx/fx;Grem1fx/+ (M) and Nogfx/fx;Grem1fx/fx (N) somites (arrowheads) is aberrant.

  • Fig. 3.
    Fig. 3.

    Nog;Grem1 double-mutant mouse embryos lack sclerotome. (A-D) Whole-mount E9.5 in situ hybridizations for the sclerotome marker Pax1; lateral views. (A,B) Nogfx/+;Grem1fx/+ (A) and Nogfx/+;Grem1fx/fx (B) embryos display normal Pax1 expression. (C) Pax1 expression in Nogfx/fx;Grem1fx/+ embryos is normal in anterior somites but is lost posterior to the thoracic region. (D) A Nogfx/fx;Grem1fx/fx double-mutant embryo lacking Pax1 expression in the sclerotome but retaining expression in the pharyngeal arches (arrowheads). (E-H) Whole-mount E9.5 in situ hybridizations for Pax3; lateral views. (E,F) Nogfx/+;Grem1fx/+ (E) and Nogfx/+;Grem1fx/fx (F) embryos display normal Pax3 expression in dermomyotome and dorsal neural tube. (G,H) Pax3 expression in Nogfx/fx;Grem1fx/+ (G) and Nogfx/fx;Grem1fx/fx (H) embryos is reduced in anterior somites (H, arrows). Somitic Pax3 expression does not appear posterior to somite 13 but is retained in the dorsal neural tube (bracket).

  • Fig. 4.
    Fig. 4.

    Grem1 heterozygosity enhances the Nog mutant skeletal phenotype. (A-D) Whole-mount E13.5 mouse embryos stained with Alcian Blue to detect cartilage; dorsal views. (A,B) Nogfx/+;Grem1fx/+ (A) and Nogfx/+;Grem1fx/fx (B) examples have normal axial skeletal morphology. (C) A Nogfx/fx mutant displaying thickened cervical and thoracic skeletal elements and reduced lumbar cartilage. (D) In Nogfx/fx;Grem1fx/+ animals, formation of the cartilage of the axial skeleton is dramatically impaired. (E-H) Whole-mount E9.5 in situ hybridizations for Meox1 (E,F) and Pax1 (G,H). (E,F) Although reduced relative to controls, this Nogfx/fx embryo (E) exhibits greater Meox1 expression than a Nogfx/fx;Grem1fx/+ embryo (F). (G,H) Pax1 expression in this Nogfx/fx embryo (G) appears in the lumbar region (arrow) to the level of the hindlimb buds, a region that does not express Pax1 in the Nogfx/fx;Grem1fx/+ embryo (H).

  • Fig. 5.
    Fig. 5.

    Neither aberrant cell death nor proliferation explains sclerotome agenesis. (A-D) Transverse sections between somites 8-12 of E9.0 mouse embryos. TUNEL-positive cells (red) indicate cell death, whereas phosphorylated histone H3 (PH3)-positive cells (green) reveal mitosis, and Hoechst (blue) labels nuclei. (A,B) Nogfx/+;Grem1fx/+ control (A) and Nogfx/+;Grem1fx/fx Grem1 mutant (B) examples exhibit few apoptotic cells. (C,D) Nogfx/fx;Grem1fx/+ Nog mutant (C) and Nogfx/fx;Grem1fx/fx double-mutant (D) examples exhibit apoptotic cells in the dorsal neural tube (arrows). No overt differences in somitic PH3-positive cells were observed among the different genotypes. Dashed lines indicate somite perimeters. (E) Bar chart showing mitotic cells within the somite as a percentage of all somitic nuclei. The three genetic classes analyzed – control Nogfx/+;Grem1fx/+ (left), Nog mutant Nogfx/fx;Grem1fx/− (center), and double-mutant Nogfx/fx;Grem1fx/fx (right) – exhibited no significant differences in somitic proliferation rates. A minimum of three embryos of each genotype were analyzed, and at least five sections from each embryo were counted. Error bars indicate 2× s.e.m.

  • Fig. 6.
    Fig. 6.

    Inhibition of BMP signaling does not enhance sclerotome formation. Whole-mount E9.5 in situ hybridizations for mouse Pax1(A-D) and Lbx1 (E-H); lateral views. (A,B,E,F) Conditional Bmpr1a mutants. Tamoxifen was administered via the mother at E7.5, 40 hours before dissection. Embryos were processed for β-galactosidase activity (pink) prior to in situ hybridization. Bmpr1afx/−;R26R controls displaying normal Pax1 (A, n=6) and Lbx1 (E, n=4) expression. Bmpr1afx/−;R26R;Tg(Cre/Esr1) embryos express Pax1 (B, n=5) normally but do not express Lbx1 (F, n=4). (C,D,G,H) Embryos cultured from E8.5 for 30 hours in DMSO vehicle alone (C,G) or 2.5 μM dorsomorphin (D,H). Dorsomorphin does not interfere with Pax1 expression (D) but does block Lbx1 induction (H). At least five embryos of each condition were analyzed. WT, wild type.

  • Fig. 7.
    Fig. 7.

    Elevated BMP signaling in mouse Nog;Grem1 double mutants interferes with Hh signaling. (A-M) Whole-mount in situ hybridizations for Pax1 (A-D), Ptc (E-G), Gli1 (H-J) and Gli2 (K-M); lateral views, facing left. (A-D) Nogfx/fx;Grem1fx/fx (A,B) and Smo−/− (C,D) embryos, cultured from E8.5 for 30 hours in DMSO vehicle alone (A,C) or 2.5 μM dorsomorphin (B,D). Dorsomorphin restores somitic Pax1 expression as well as some posterior somite formation in Nogfx/fx;Grem1fx/fx embryos (B) (arrowheads) but does not rescue Pax1 expression in the absence of Hh signaling (D). (E-M) Nogfx/+;Grem1fx/+ (E,H,K), Nogfx/fx;Grem1fx/+ (F,I,L) and Nogfx/fx;Grem1fx/fx (G,J,M) embryos. Ptc, Gli1 and Gli2 are expressed in all genetic classes. (E-G) Note reiterated Ptc expression in somites (arrowheads). Insets are midline lateral views of similarly staged embryos bisected at the level of the forelimb, revealing ventral medial Ptc expression. (J) Although reduced, Gli1 was detected in Nogfx/fx;Grem1fx/fx embryos (arrowhead). Insets are transverse sections through somite 10, revealing somite expression. (N) QPCR analysis of Ptc and Pax1 expression for control (black bars), Nog mutant (gray bars) or Nog;Grem1 double-mutant (white bars) embryos cultured with purmorphamine or vehicle alone. Ptc expression increases in response to smoothened activation in all genotypes. Pax1 expression is elevated in response to purmorphamine in controls but decreased in antagonist mutants. A minimum of three embryos were used for each condition. Expression for mock-treated Nogfx/+;Grem1fx/+ controls was set to 1. Error bars indicate 2× s.e.m. *, P<0.05 between purmorphamine- and control-treated embryos of that genotype.

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Research Article
Cooperative activity of noggin and gremlin 1 in axial skeleton development
David A. Stafford, Lisa J. Brunet, Mustafa K. Khokha, Aris N. Economides, Richard M. Harland
Development 2011 138: 1005-1014; doi: 10.1242/dev.051938
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