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First published online 25 April 2006
doi: 10.1242/jcs.02928

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Identification of regulators of germ layer morphogenesis using proteomics in zebrafish

Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr.108, 01307 Dresden, Germany

View larger version (24K): [in a new window]   Fig. 1. Experimental layout. Ectodermal and mesendodermal protein extracts were labeled with Cy3 or Cy5 fluorescent dyes, combined and subsequently separated by 2D gel electrophoresis (DIGE). A common standard labeled with Cy2 was used for normalization. The scanned images were analyzed with Proteomweaver software to identify spots that displayed statistically significant changes. Differential spots were cut, digested with trypsin and analyzed by LC-MS/MS. Database searches were performed in Ensembl and TIGR databases using MASCOT.

View larger version (74K): [in a new window]   Fig. 2. 2D gel electrophoresis. Three Cy-dye labeled extracts (50 µg protein each) were combined and separated by 2D gel electrophoresis. First dimension: 24 cm strips pI 4-7 (A) and pI 7-11 (B). Second dimension, 25.5 x 20.5 cm 10% SDS polyacrylamide gels. Spots differentially regulated between ectodermal and mesendodermal cells are outlined in red, numbered and listed in Table 1.

View larger version (35K): [in a new window]   Fig. 3. Gene expression analysis. Gene expression changes between ectodermal and mesendodermal cells. Wild-type embryos consisting of both ectodermal and mesendodermal cells were compared with samples of ectodermal or mesendodermal cells using Affymetrix zebrafish gene arrays in triplicate. (A) The 220 most significantly regulated genes were selected based on an expected false discovery rate (FDR) of 5%. After centering and normalizing, the genes were clustered according to their regulation pattern. Color coding: up-regulated, red; unchanged, black; downregulated, green. (B) The regulation factor comparing mesendodermal with ectodermal cells on a transcriptional level was plotted against the regulation factor of 2D gel spot intensities. Areas of regulation in the same direction are shaded gray. The overall correlation between gene expression and spot intensity is weak.

View larger version (36K): [in a new window]   Fig. 4. Zebrafish ezrin2. (A) Un-rooted tree of human (Hs), mouse (Mm), zebrafish (Dr) and Drosophila (Dm) ERM-family proteins. Corresponding NCBI accession numbers: Dr Ezrin2 NP_001025456, Hs Ezrin NP_003370, Mm Ezrin P26040, Dr Ezrin1 XP_699992, Hs Radixin AAH47109, Mm Radixin NP_033067, Mm Moesin NP_034963, Hs Moesin P26038, Dr Moesin-like NP_001004296, Dr Moesin XP_700327, Dm Moesin P46150, Dm Merlin AAF49005, Dr Merlin-like NP_998116, Mm Merlin P46662, Hs Merlin P35240, Dr Merlin XP_689682. Scale bar indicates point mutations per site. (B) In situ hybridization of shield-stage (6 hpf), 70% epiboly-stage (7 hpf) and bud-stage (10 hpf) sectioned and whole-mount wild-type embryos using an ezrin2-antisense probe. Lateral views with dorsal side to the right. (C) Close-up view of the dorsal side of a sectioned 70% epiboly-stage (7 hpf) embryo after in situ hybridization using ezrin2 antisense probe.

View larger version (53K): [in a new window]   Fig. 5. Ezrin2 regulation analyzed by 2D gels, western blotting and immunostaining of sections. (A) Close-up view of three out of seven gels (Cy3 and Cy5 labeled) in the region of the ezrin2 spot (circled red). (B) Quantification of ezrin2 spot intensities of the DIGE 2D gel experiment. (C) Western blot analysis of wild-type, mz-oep (ectoderm) and cyclops mRNA injected (mesendoderm) embryos at 8 hpf (corresponding to 80% epiboly stage in wild-type embryos). After analysis with phospho-ERM (p-ERM) antibody, blots were stripped and re-probed with ERM antibody. The lower part of the blot was probed for tubulin as a loading control. (D) Wild-type embryos were sectioned at the shield stage (6 hpf; sagittal section through the shield region) and at 80% epiboly (8 hpf; transverse section through the emerging body axis, paraxial region) and stained with phospho-ERM (p-ERM) antibody. In mesendodermal cells the level of phosphorylated ERM proteins is increased. Insets show section planes.

View larger version (43K): [in a new window]   Fig. 6. Ezrin2 morpholino antisense oligonucleotides efficiently reduce ezrin2 protein level and impair gastrulation. (A) Placement of the ezrin2 MOs used in this study (MO1 and MO2) on the ezrin2 mRNA. MO1 targets the translation initiation site and MO2 is placed in the upstream 5'UTR of ezrin2 mRNA. (B) Injection of ezrin2 MO1 efficiently reduces ezrin2 protein as detected by western blotting with ERM antibody (tubulin antibody was used as loading control). Co-injection of 100 pg ezrin2 mRNA with 8 ng of ezrin2 MO1 restores ezrin2 protein expression. (C) Quantification of the early loss-of-function phenotypes and partial rescue by ezrin2 mRNA injection. One-cell-stage embryos were injected with morpholino MO1 or MO2 and scored for blastoderm detachment or epiboly defects (wt 0/122, 2 ng MO1 37/91, 4 ng MO1 53/81, 4 ng MO2 38/109, 8 ng MO2 42/89). To rescue ezrin2 morpholino-induced phenotypes 4 ng ezrin2 MO2 followed by 100 pg ezrin2 mRNA were injected. Embryos were scored for blastoderm detachment or epiboly defects (MO2 20/107; MO2 + ezrin2 mRNA 9/90).

View larger version (70K): [in a new window]   Fig. 7. Phenotypic characterization of ezrin2 morphant embryos. (A) Images of wild-type (wt) and ezrin2-MO1 injected embryos at 50% epiboly (5 hpf) and 60% epiboly (6 hpf). Dorsal side is to the right and the animal pole is up. (B) Face-on views of blastodermal cells within the animal pole of 50% epiboly (5 hpf) wild-type (wt) and ezrin2 morphant (MO1) embryos as DIC images and stained for F-actin with phalloidin. Bar, 20 µm. (C) Images of wild-type (wt) and ezrin2-MO2 injected embryos at 80% epiboly (8 hpf) and bud stage (10 hpf). Dorsal side is to the right and the animal pole is up. (D) In situ hybridization of wild-type (wt) and ezrin2-MO2 injected embryos at the bud stage (10 hpf) with markers delineating convergent extension of the forming body axis. Upon morpholino injection, embryos developed a shortened notochord and a broadened neural plate. Animal views (upper panel) and dorsal views (lower panel), anterior to the left. Markers: notochord, notail (ntl); anterior edge of neural plate, distal-less homeobox 3 (dlx3); prechordal plate, hatching gland gene-1 (hgg1). (E) Analysis of cellular rearrangements within the axial germ ring (shield) of wild-type and ezrin2 morphant embryos (MO2) starting at shield stage (6 hpf) by confocal time-lapse microscopy. Images correspond to timepoints 0 minutes and 60 minutes of Movies 1 and 2 in supplementary material. One exemplary mesendodermal progenitor cell was labeled in red to mark its position at 0 and 60 minutes of the time-lapse movie. Note that during this time interval the labeled cell has moved further away from the germ ring margin in the wild-type embryo compared with the morphant embryo, suggesting that mesendodermal cell migration is reduced within the shield of ezrin2 morphant embryos. Lateral view, dorsal to the right. Bar, 40 µm.