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First published online 6 February 2008
doi: 10.1242/dev.015321

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Wnt3a-mediated chemorepulsion controls movement patterns of cardiac progenitors and requires RhoA function

Qiaoyun Yue¹, Laura Wagstaff^1,*, Xuesong Yang^2,, Cornelis Weijer² and Andrea Münsterberg^1,

¹ School of Biological Sciences, University of East Anglia, Norwich NR4 7TJ, UK.
² College of Life Sciences Biocentre, University of Dundee, Dundee DD1 5EH, UK.

View larger version (75K): [in this window] [in a new window]   Fig. 1. GFP labelling of cardiac progenitors in HH3 primitive streak. (A) Schematic illustration of the electroporation and grafting procedure; embryos were placed in EC culture with their ventral side up. (B) HH3 donor embryo (left) after electroporation of primitive streak with pCS2+-GFP, and HH3 host embryo (right). Green fluorescence identifies labelled cells; small grafts were dissected and transplanted into a stage-matched host. The white dotted semi-circles show the anterior margin of the primitive streak; the white dotted oval indicates the graft. (C) Transverse cryosection (15 µm) through an HH5 embryo shows GFP-positive cells in the mesoderm of the lateral plate. (D) HH7 embryo with GFP-positive cells in the area of the bilateral heart fields, indicated by a white line (Stalsberg and DeHaan, 1969). (E) GFP-positive cells in the heart tube at HH10. (F) Transverse cryosection (30 µm) of a similar embryo, stained with anti-GFP (green) and MF20 (red) antibodies, showing that GFP-positive cells were present in endocardium and myocardium. White line in E indicates the approximate level of the section shown in F. ao, area opaca; ec, ectoderm; en, endoderm; end, endocardium; fg, foregut; h, heart; hf, heart field; my, myocardium; nt, neural tube; ps, primitive streak.

View larger version (81K): [in this window] [in a new window]   Fig. 2. Cardiac progenitors move on directed trajectories, which are controlled by Wnt3a. (A) Long-term video microscopy followed by image processing revealed movement trajectories; still images at the time points indicated in hours (h) are shown. (B) Corresponding bright-field images. The last panel shows the location of GFP cells in the bilateral heart fields, indicated by white outline (for the corresponding movie, see Movie 1 in the supplementary material). (C) vMHC in situ hybridisation of an HH11 embryo after live imaging shows normal heart morphogenesis and cardiac specific gene expression. (D) Paraffin transverse section of the embryo shown in C. (E,F) In situ hybridisation shows (E) Fgf8 and (F) Wnt3a transcripts are expressed in the HH3 primitive streak. (G) A weak signal for Wnt11 was detected in the node of an HH4 embryo (arrowhead). The contrast was enhanced and saturation of the magenta colour was increased to visualise this signal. (H) Illustration of Wnt3a-expressing cells (yellow dot) grafted into HH3 embryos to challenge the movement of GFP-labelled cardiac progenitors. (I) Long-term video microscopy followed by image processing revealed the movement trajectories of GFP-labelled cardiac progenitors in the presence of Wnt3a-expressing cells (yellow dot). Still images at the time points indicated show a wider trajectory. (J) Corresponding bright-field images show that most cells failed to reach the midline and remained in the area opaca and lateral mesoderm (see Movie 2 in the supplementary material). (K,L) Many embryos developed cardia bifida as shown by vMHC in situ hybridisation: whole mount (K) and section (L) of an embryo after imaging. (M,N) Paraffin sections of an HH10 embryo implanted with a control cell pellet (M) or with a Wnt3a cell pellet to induce cardia bifida (N). Black arrowheads indicate the endoderm and red lines indicate the foregut pocket; h, heart. (O) Graph showing the percentage of embryos with cardia bifida under different conditions. The embryo treatment is indicated below each bar. LNCX ccp, control cell pellet; ep, electoporation; cp, cell pellet. (P) HH5 embryo with Wnt3a-expressing cells (arrowhead) implanted at HH3 (as shown in H) and hybridised with a probe detecting Fgf8 transcripts.

View larger version (75K): [in this window] [in a new window]   Fig. 3. Wnt3a-expressing cells repell cardiac progenitors on the area opaca. (A) Illustration of GFP-positive primitive streak explants cultured in the presence of a cell pellet on the area opaca of an HH4 host embryo. (B) Merged bright-field and fluorescent images of a representative streak explant and cell pellet on the area opaca. Still images from the start (0h) and end points (16h) of the experiments are shown. (C) Image tracks of movies showed that HH3 primitive streak cells migrated in all directions away from a graft cultured on the area opaca and ignored the presence of a control cell pellet (yellow asterisk). (D) HH3 primitive streak cells electroporated with GFP (on the right of the cell pellet) or with a dominant-negative form of Fgf receptor 1, -Fgfr1-YFP (on the left of the cell pellet) migrated away from Wnt3a-expressing cells (see Movie 3 in the supplementary material). (E) HH4 primitive streak cells did not respond to Wnt3a-expressing cells. (F) HH3 cardiac progenitors were repelled by a bead soaked in Fgf8. (G) HH3 cardiac progenitors electroporated with DN-Wnt3a-GFP no longer responded to Wnt3a-expressing cells. (H) Wnt11-expressing cells repelled HH3 prospective cardiac cells. Yellow asterisks indicate the position of cell pellets or beads. 0h, start of imaging; 16h, end of imaging; ao, area opaca; ps, primitive streak.

View larger version (87K): [in this window] [in a new window]   Fig. 4. DN-Wnt3a can rescue the effects of Wnt3a on cell movement patterns in vivo. (A) DN-Wnt3a-GFP-(green) and RFP-(red) expressing HH3 streak cells grafted into a stage-matched host displayed normal migration patterns in control embryos. (B) A graft of Wnt3a-expressing cells affected GFP- and RFP-labelled cells at different anterior-posterior levels and caused cells to take a wider path. (C) The movement trajectories of cells expressing DN-Wnt3a-GFP were restored to normal in the presence of Wnt3a-expressing cells but RFP-transfected cells still displayed wider migration trajectories. (A-C) Panels on the left show GFP and RFP fluorescence with bright field at the start point of imaging. The two central panels show dark-field images at the time points indicated. Panels on the right show fluorescence with bright field at the end of imaging. Vertical white lines indicate the axial midline; angled white lines indicate the main trajectory of cells leaving the streak. Heart-forming regions at HH8 [based on Stalsberg and DeHaan (Stalsberg and DeHaan, 1969)] are outlined in white. Scale bar in A: 500 µm.

View larger version (85K): [in this window] [in a new window]   Fig. 5. Wnt3a-mediated repulsion requires RhoA function, and dominant-negative or constitutively active forms of RhoA cause cardia bifida. (A) HH3 primitive streak cells electroporated with a constitutively active form of RhoA, RhoA-V14, were repelled by Wnt3a-expressing cells (yellow asterisk) in area opaca explants. (B) HH3 primitive streak cells electroporated with a dominant-negative form of RhoA, RhoA-N19, ignored the source of Wnt3a and behaved like unchallenged explants or explants transfected with DN-Wnt3a (see Fig. 3C,G), migrating in all directions away from the graft. (C) RhoA transcripts were expressed in the primitive streak at HH4. (D) vMHC in situ hybridisation showed that embryos electroporated with RhoA-V14-IRES-GFP developed cardia bifida. (E) Graph showing the percentage of embryos with cardia bifida. Embryo treatment is indicated below each bar. ccp, control cell pellet; ep, electoporation. (F) Long-term video microscopy followed by image processing revealed the movement trajectories of RhoA-V14-IRES-GFP labelled cardiac progenitors. Still images at the time points indicated are shown. (G) Corresponding bright-field images show the location of GFP cells within the embryo. The bilateral heart fields are indicated by a white outline in HH8 embryos.

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