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First published online 30 November 2005
doi: 10.1242/dev.02160


Development 133, 21-32 (2006)
Published by The Company of Biologists 2006


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p38 MAPK is essential for secondary axis specification and patterning in sea urchin embryos

Cynthia A. Bradham and David R. McClay*

DCBM Group, Biology Department, Duke University, Durham, NC 27708, USA



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Fig. 1. Lvp38 inhibition specifically blocks oral specification. (A) Alignment of Lvp38 (Lv) and human p38 (Hs). Residues that are identical or 90% similar (Corpet, 1988Go) are indicated in red. The phosphorylation motif TGY is labeled blue. Specific SB-interacting residues are labeled green, and non-specific SB-interacting structural elements are labeled gray. (B) Brightfield images are shown of control (B1), KI p38-expressing (1.4 pg/pl, B2) and SB-treated (20 µM, B3) embryos. (C) Control (top) and SB-treated (bottom) embryos shown at early gastrula (C1,C2), late gastrula (C3,C4) and pluteus stages (C5,C6). (D) D1 and D2 show immunostaining of PMCs with 1d5 at the mesenchyme blastula stage in control (D1) and SB-treated (D2) embryos. Images are full projections of confocal z-series. (D3,D4) Whole-mount in situ analysis of FoxA in control (D3, late gastrula) and SB-treated (D4, pluteus stage) embryos, shown in lateral views. (E) Control (E1), KI p38-expressing (E2) and SB-treated (E3) embryos were immunolabeled with the aboral marker 1c10 at pluteus stage. Full projections are shown.

 


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Fig. 2. p38 is required for the expression of Nodal (Nod) and Goosecoid (Gsc). (A) Brightfield or DIC images of control (A1), SB-treated (A2), LvNod-overexpressing (A3) and LvGsc-overexpressing (A4) embryos. Doses were 0.2 pg/pl for LvNod and 0.05 pg/pl for LvGsc mRNA. (B) Whole-mount in situ analysis in control (B1,B3) and SB-treated (B2,B4) embryos for LvNod (B1,B2, late gastrula) and LvGsc (B3,B4, prism), shown in vegetal views. (C,D) Whole-mount in situ analysis for Nodal (C) and Gsc (D) in control (top) and SB-treated (bottom) embryos at early blastula (4h, left), late blastula (5.5h, middle) and hatched blastula (7h, right).

 


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Fig. 3. p38 is required for oral specification at late blastula stage. (A) Schematic depicting the experiment in B. Corresponding hours post-fertilization (h) are indicated. (B) Embryos were treated with SB at the indicated stages (B1-B4), and then cultured to the pluteus stage. Morphological (top) and skeletal (bottom, illuminated with plane-polarized light) images are shown. (C-E) Whole-mount in situ analysis for Nodal (C), Gsc (D) and Tbx2/3 (E) in embryos treated with SB at 5 (1), 6 (2,3) and 7 (4) hpf. Vegetal views at late gastrula stage are shown. EB, early blastula; HB, hatched blastula; TVP, thickened vegetal plate; MB, mesenchyme blastula; Plut, pluteus larvae.

 


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Fig. 4. SB removal reveals OA patterning defects. (A) Schematic depicting the experiment in B. (B) In B1, data are expressed as the percentage of embryos with abnormal patterning ±s.e.m. following SB-wash at various time points. Corresponding stages are indicated above the graph. (B2,B3) Phenotype of SB-wash embryos, illuminated with brightfield (B2) and plane-polarized light (B3). (C) QPCR analysis for Nodal (C1) and Gsc (C2) relative expression levels in control, SB-treated and SB-wash embryos. Data are expressed as net {Delta}CT±s.e.m., with controls at hatched blastula (HB) arbitrarily set to zero. Dashed lines indicate the threshold for significance. EG, early gastrula; LG, late gastrula.

 


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Fig. 5. p38 is transiently inactivated on the future aboral side of the embryo. (A) Western blot analysis for phospho-p38 at the indicated stages. P-p38 is indicated by an arrowhead. 8, 8 cell; 60, 60 cell. (B) Embryos stained with anti-P-p38 antibody (B1-B3) and Hoechst's dye (B4-B6) at the indicated timepoints post-fertilization. The cleared region is indicated by arrowheads. Images are internal projections of 10 sections from a confocal z-series. (C) A single embryo expressing Lvp38-GFP (0.015 pg/pl), imaged using a live confocal timecourse, is shown at the indicated timepoints post-fertilization (C1-C3). The cleared region is indicated by arrowheads. Images are internal projections of 20 sections. (D) Schematic depicting the experimental approach used in E. (E) A similar embryo to the one in C, but also labeled with DiI (red). (E1-E3) Internal projections of 25 sections at the indicated hpf. (E4-E6) The same embryo at late gastrula stage, with morphology (E4), skeleton (E5) and DiI labels shown (E6). The oral (O) and aboral (A) poles are indicated.

 


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Fig. 6. Nodal expression oralizes embryos downstream from p38. (A) A1 and A2 show DIC images of a control embryo (A1) and an embryo injected with Nod MASO (0.7 mM, A2) at the pluteus stage. (A3) Embryo co-injected with Nod MASO and Lvp38-GFP, then imaged with live confocal microscopy at late blastula stage, as in Fig. 5C and 5E. (B-E) Whole-mount in situ analysis of Gsc (B), Dri (C), Tbx2/3 (D) and Bra (E) expression shown in control (1), SB-treated (2), Nod-injected (3), and Nod-injected plus SB-treated (4) embryos. Vegetal views are shown at prism stage, except for D (lateral views; D1 at late gastrula and D2-D4 at pluteus stage).

 


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Fig. 7. LvGsc expression oralizes embryos downstream from p38. (A-C) Whole-mount in situ analysis of Nodal (A), Dri (B) and Tbx2/3 (C) expression in control (1), SB-treated (2), Gsc-injected (3), and Gsc-injected plus SB-treated (4) embryos. Vegetal views at prism stage are shown, except for A (lateral views at late gastrula). (D) Control (D1), SB-treated (D2), and Gsc-injected (D3) embryos stained with 1c10 at the pluteus stage. In D4, embryos were injected with Gsc in one blastomere at the 2-cell stage and then SB-treated and stained as in D1-D3. Full confocal projections are shown.

 


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Fig. 8. Asymmetric expression of Gsc or Nodal restores oral-aboral patterning downstream from p38. The left column shows schematics representing the experiments for each panel. (A) SB-wash (SB W) treatment with a control 2-cell (2c) injection. (B) 2c Gsc injections without SB treatment. (C) 2c Gsc injections with SB-wash treatment. (D) 2c Nod injections with SB-wash treatment. Brightfield or DIC (1), skeletal (2) and fluorescence (3) images are shown.

 


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Fig. 9. Model summarizing the data presented. p38 is asymmetrically active on the future oral side of the embryo at late blastula stage, and initiates the expression of Nodal through an unknown intermediate transcription factor (T) that is a direct target of p38. The relationships between Nodal, Gsc, Dri and Tbx2/3 observed in L. variegatus are consistent with those observed in other species (Amore et al., 2003Go; Croce et al., 2003Go; Duboc et al., 2004Go). The induction of Dri is positioned downstream from Gsc via an intermediate repressor R, in keeping with the relationship observed in S. purpuratus embryos (Amore et al., 2003Go). Both Dri and Gsc are upstream from the oral differentiation genes, first because SpDri is necessary but not sufficient to induce oral-specific genes (Amore et al., 2003Go), and second because Gsc is sufficient for oralization downstream from p38 (Fig. 8C). Gsc is depicted as relieving the repression of the oral differentiation genes. A single repressor (R) is shown, although multiple repressors might exist. A single ubiquitous activator of aboral genes is shown in the aboral compartment for simplicity; there may of course be several. This activator may also induce the repressor/s of oral differentiation genes that is/are in turn repressed by Gsc. In the aboral ectoderm, the transcription factor Tbx2/3 might contribute to the induction of the aboral differentiation genes (Croce et al., 2003Go; Gross et al., 2003Go); the 1c10 antigen is expressed later in development and is thus likely to be an aboral differentiation gene.

 





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