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First published online 17 December 2008
doi: 10.1242/dev.026542

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Flamingo regulates epiboly and convergence/extension movements through cell cohesive and signalling functions during zebrafish gastrulation

¹ Department of Cell and Developmental Biology, University College London, Gower Street, London WC1E 6BT, UK.
² MRC Laboratory for Molecular Cell Biology & Cell Biology Unit, University College London, Gower Street, London WC1E 6BT, UK.
³ Department of Genetics, University of Cambridge, Cambridge CB2 3EH, UK.
⁴ Laboratory for Developmental Gene Regulation, Brain Science Institute, The Institute of Physical and Chemical Research (RIKEN), Saitama 351-0198, Japan.

View larger version (92K): [in this window] [in a new window]   Fig. 1. Injection of celsr1a and celsr1b morpholinos in MZord embryos leads to a defective epiboly phenotype during zebrafish gastrulation. MZord embryos (A,B,C,G,H) or MZord embryos injected with 0.4 pmoles each of celsr1a and celsr1b morpholinos (D,E,F,I,J) were visualised at tail-bud stage with markers, as indicated in the bottom right-hand corner, by in situ hybridisation. hgg1 was used to indicate the prechordal plate (pcp), ntl for the prospective notochord (n) and germ ring blastopore margin, and dlx3 for the anterior edge of the neural plate (A-F). Embryos were also visualised at 90% epiboly with phalloidin for actin (G,I) or anti--catenin antibody (H,J). An arrowhead represents the leading edge of the EVL with the actin cable being formed (I). An arrow indicates the leading edge of deep cells (J). Note that the deep cells are delayed from the leading edge of the enveloping layer in the mutant/morphant embryos. y, yolk. (K) Cell elongation of the EVL along the animal-vegetal (AV) axis and the mediolateral (ML) axis at 90% epiboly is quantified as AV/ML ratio (see the inset of G), and the measurement is made using ImageJ from 80 cells (four embryos of each group). Cells that are attached with the actin cable are expressed as `Row1', whereas cells that are not attached to that as `Row2/3'. Means and standard deviations (s.d.) are shown, and an asterisk indicates a statistically significant difference (P<0.05; Student's t-test).

View larger version (136K): [in this window] [in a new window]   Fig. 2. A C-terminal truncated form of Celsr2 (Celsr-C) is dominant negative and Celrs regulates epiboly independently of the PCP. Wild-type (A,B,D,E) or MZord embryos (C,F) were injected with 300 pg (H, high) (A,D) or 150 pg (L, low) (B,C,E,F) RNA encoding Celsr-C-HA were visualised at 90% epiboly with phalloidin for actin (A-C) or anti--catenin (D-F). Arrowheads indicate the leading edge of deep cells, showing epiboly defects with deep cells retracted from the leading edge of the EVL. (G-I) MZord embryos (G) or MZord embryos injected with 0.4 pmoles stbmMO (H) or 0.75 pmoles dvl2MO (I) were visualised at 90% epiboly with anti--catenin. Dorso-posterior views of 90% epiboly embryos. Anterior is upwards and the antibodies used are indicated in the bottom right-hand corner. MZord embryos injected with stbmMO or dvl2MO show normal epiboly movements, as assessed by closure of the germ ring blastopore.

View larger version (62K): [in this window] [in a new window]   Fig. 3. Celsr/Flamingo has a potential pro-region mediating dimer formation. (A) The comparison of the region N-terminal to the Cadherin repeats indicates a Furin-like protease cleavage site (arrowhead) conserved amongst Drosophila Fmi and vertebrate Celsr. (B) The design for Celsr-Activin fusion proteins. A potential pro-region of zebrafish Celsr2 (blue), including the Furin-like cleavage site, is fused to the mature region of mouse Activin A (pink) to generate FmiP-Act (see Materials and methods for details). The potential cleavage site is mutated in alanine. (C-N) Wild-type embryos were injected with 5 pg mouse activin RNA (F-H), 50 pg FmiP-Act RNA (I-K), 100 pg FmiP-Act-RA RNA(L-N) or left uninjected (C-E), and fixed at 50% epiboly to examine expression of the mesoderm or endoderm markers ntl (C,F,I,L), gsc (D,G,J,M) or bon (E,H,K,N).

View larger version (44K): [in this window] [in a new window]   Fig. 4. Celsr-C is retained in the golgi and inhibits membrane presentation of Celsr. (A-F) Wild-type embryos were injected with 30 pg Celsr-C-HA DNA alone (A-C) or together with 30 pg Rab5-CFP RNA (D-F) and fixed at 40% epiboly to visualise with an HA antibody (A,D) or either a GM130 antibody for the golgi (B) or a GFP antibody for early endosomes (E). The merge images are shown in C and F. (G-L) Wild-type embryos were co-injected with 30 pg Celsr-Venus DNA and 30 pg Celsr-C-HA DNA, and fixed at 40% epiboly to visualise with a GFP antibody (G,J) or an HA antibody (H,K). The merge images are shown in I and L. When the level of Celsr-C is low, Celsr-Venus is presented at the membrane (shown by arrowhead) (62 cells out of 15 embryos examined but the level of Celsr-C is high, Celsr-Venus is prevented from presenting at the membrane (15 cells out of 15 embryos examined). (M) Non-covalent dimer formation of Celsr. Celsr-Venus and/or Celsr-deltaC-HA were transiently expressed in HEK293 cells. Cell lysates were immunoprecipitated with anti-GFP antibody, and immunoprecipitated proteins were further incubated in Laemmli's buffer with or without 2-mercaptoethanol (2-ME), followed by western blotting with anti-GFP or anti-HA antibody.

View larger version (70K): [in this window] [in a new window]   Fig. 5. Overexpression of a membrane-targeted intracellular domain of Celsr (Lyn-Celsr) causes a convergence extension defect during zebrafish gastrulation. (A-H) Wild-type (WT) embryos (A-D) or wild-type embryos injected with 100 pg RNA encoding Lyn-Celsr (E-H). (A,E) Lateral views of pharyngula stage living WT (A) and Lyn-Fmi-expressing (E) embryos. The Lyn-Fmi-expressing embryo shows a shorter body axis in this case associated with cyclopia. Dorsal views (B,C,F,G) of tail-bud stage wild-type embryos (B,C) and Lyn-Celsr-expressing embryos (F,G). Lateral views of 80% epiboly WT (D) and Lyn-Celsr-expressing (H) embryos. Anterior is upwards and genes analysed are indicated in the bottom right-hand corner. (I-R) Analysis of axial and lateral mesendermal cells using a photo-conversion strategy. Labelled axial mesendermal cells at shield stage (6 hpf) in control (I) and Lyn-Celsr embryos (K) were analysed at tailbud stage (10 hpf) (J,L, respectively). Labelled lateral mesendermal cells at shield stage (6 hpf) in control (M) and Lyn-Celsr embryos (O) were analysed at tailbud stage (10 hpf) (N,P, respectively). Lateral views (I-P). Quantification of anterior migration of axial cells (Q) and dorsal migration of lateral cells (R). Blue, Lyn-Celsr-expressing embryos; red, WT embryos. Means and s.d. are shown. Asterisks indicate statistically significant differences (P<0.05; Student's t-test). Note that Lyn-Celsr-expressing embryos show both convergence and extension defects.

View larger version (72K): [in this window] [in a new window]   Fig. 6. Lyn-Celsr inhibits Frizzled-induced membrane localisation of Dishevelled through a highly conserved SE/D domain. (A) Schematic illustration of the intracellular region of Fmi/Celsr. Two domains are conserved among vertebrates as highlighted in blue. Only one of these, the SD/E domain is conserved between vertebrates and Drosophila. The partial sequence of a construct lacking this domain is shown. (B-M) Wild-type embryos injected with 150 pg Dsh-GFP RNA in the absence (B-D) or presence (E-M) of 100 pg Fz7 RNA, together with 100 pg membrane-RFP (E-G), Lyn-Celsr-RFP (B-D,H-J) or Lyn-Celsr--RFP (K-M), as indicated and analysed at 40% epiboly by confocal microscopy. Scanning for GFP and RFP was carried out simultaneously and merged (D,G,J,M). Fz7-mediated membrane localisation of Dsh is inhibited by Lyn-Celsr (H,J) but not by Lyn-Celsr- (K,M), which lacks the SD/E domain conserved between vertebrates and Drosophila (A).

View larger version (40K): [in this window] [in a new window]   Fig. 7. Differential cell cohesive properties of wild-type cells compared with cells from celsr mutant/morphants and cells from embryos expressing Celsr-C. Wild-type and MZord embryos were injected with RNA/morpholinos together with fluorescein-dextran (FDX, green) or rhodamine-dextran (RDX, red), as indicated. (A) 30-40% epiboly embryos were dissociated and dissociated cells from different populations were mixed in a minimal volume of a hanging drop and kept overnight to analyse aggregates by confocal microscopy. (B) Cells from wild-type embryos (green) and wild-type embryos (red). (C) Cells from MZord embryos injected with 0.4 pmoles each of celsr1a and celsr1b morpholinos (green) and wild-type embryos (red). (D) Cells from wild-type embryos injected with 300 pg Celsr-C-HA RNA (green) and wild-type embryos (red). (E) Cells from wild-type embryos injected with 100 pg Lyn-Celsr RNA (green) and wild-type embryos (red). Celsr mutant/morphant cells (C) or Celsr-C-expressing cells (D) are strongly segregated from wild-type cells, whereas Lyn-Celsr-expressing cells are only weakly segregated from wild-type cells (E).

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