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First published online 16 September 2003
doi: 10.1242/dev.00758

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Slb/Wnt11 controls hypoblast cell migration and morphogenesis at the onset of zebrafish gastrulation

Florian Ulrich^1,*, Miguel L. Concha^2,3,*, Paul J. Heid⁴, Ed Voss⁴, Sabine Witzel¹, Henry Roehl⁵, Masazumi Tada², Stephen W. Wilson², Richard J. Adams⁶, David R. Soll⁴ and Carl-Philipp Heisenberg^1,

¹ Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstrasse 108, 01307 Dresden, Germany
² University College London, Department of Anatomy and Developmental Biology, Gower Street, London WC1E 6BT, UK
³ Programa de Morfologia, Instituto de Ciencias Biomedicas, Facultad de Medicina, Universidad de Chile, PO Box 70079, Santiago de Chile, Chile
⁴ University of Iowa, Department of Biological Sciences, Iowa City, IA 52242, USA
⁵ Max-Planck-Institut für Entwicklungsbiologie, Spemannstrasse 35 / III, 72076 Tübingen, Germany
⁶ University of Cambridge, Department of Anatomy, Downing Street, Cambridge CB2 3DY, UK

View larger version (97K): [in a new window]   Fig. 1. slb/wnt11 is expressed in epiblast cells (ectoderm) overlying the first ingressing hypoblast cells (mesendoderm) at the germ ring during early stages of gastrulation. (A) Dorsal view of slb/wnt11 expression in the germ ring of a wild-type embryo at shield stage. The vertical line indicates the section plane shown in B-D. (B-D) Medial section of the embryo in A showing slb/wnt11 expression in epiblast cells (B). The boxed regions are shown at higher magnification in C (ventral germ ring) and D (shield). e, epiblast; h, hypoblast. Scale bars: 100 µm.

View larger version (81K): [in a new window]   Fig. 2. Extension of axial mesendodermal tissues (prechordal plate and notochord) is reduced in slb embryos throughout the early stages of gastrulation. Embryos were fixed at various times after shield stage (sh) and stained for the expression of hatching gland gene 1 (hgg1) and floating head (flh) to mark the positions of the prechordal plate (ppl) and the notochord (no), respectively. (A-L) Shape and position of prechordal plate and notochord in wild-type embryos (wt, A-D), slb mutants (slb, EH) and slb mutants overexpressing 5 pg slb/wnt11 mRNA (slb*, I-L) at the indicated time intervals. For each experiment and timepoint, 20 embryos were analysed. Anterior to the top, dorsal views.

View larger version (59K): [in a new window]   Fig. 3. Movement of axial hypoblast (presumptive prechordal plate) cells in dorsal regions of the germ ring (shield) is disturbed in slb mutant embryos at the onset of gastrulation. Embryos expressing GFP (green) under the control of the goosecoid (gsc) promotor in prechordal plate precursor cells were scatter labelled with rhodamine (red) in epiblast cells overlying the presumptive prechordal plate and followed in 3D over time by dual channel confocal microscopy. (A-D,F-I) Prechordal plate precursor cells (green) and overlying epiblast cells (red) in wild-type (A-D) and slb (F-I) gscGFP embryos at shield stage (A,C,F,H) and 45 minutes (45') later (B,D,G,I). Ventral views (A,B,F,G) and lateral views with ventral to the right and dorsal to the left (C,D,H,I). In all pictures, anterior is to the top and posterior to the bottom. The axes of orientation are shown in panels A and C. (E,E',J,J') Track diagrams showing the movements of prechordal plate precursor cells (green) and epiblast cells (red) in wild type (E,E') and slb gscGFP embryos (J,J') along the x (anterior-posterior) and y (mediolateral) axes (E,J) and along the x and z (dorsal-ventral) axes (E',J') of the embryo. The positions of the geometric centre of the cell (the centroid) were measured in 3D at 4-minute intervals and plotted as a single dot. Each line represents the track of one cell, with the first timepoint depicted in white. The net movement of the epiblast cells is along the +x (posterior) direction and that of prechordal plate precursor cells along the –x (anterior) direction. Note that the x, y and z axes depict the global coordinates within the gastrula, whereas in Figs 4, 5 and 7, these axes show the coordinates relative to the movement direction of individual cells. (K) Schematic diagram showing the orientation of the region analyzed in wild-type and slb embryos and the net movement direction of the cells (arrows) with respect to the x and z axes. The y axis is perpendicular to x and z and is not depicted in this diagram. Epiblast cells in red, hypoblast cells in green. Note that a left-handed coordinate system has been used. sh, shield; yol, yolk. Scale bar in A: 50 µm.

View larger version (55K): [in a new window]   Fig. 4. Distribution of pseudopodial processes in Wild-type and slb prechordal plate precursor cells at the onset of gastrulation. Prechordal plate precursor cells were labelled with a mixture of cytosolic and membrane-bound GFP and visualised in 3D over time by two-photon confocal microscopy. (A,D) 3D images of prechordal-plate-precursor cells in a wild-type (A) and slb mutant embryo (D) moving from bottom (germ ring margin) to top (animal pole) at shield stage. Arrowheads point to thick, pseudopod-like processes and the red asterisk (D) marks a pseudopod projecting into the dorsal direction. (B,E) Spherical plots showing the distribution of the outgrowth positions of pseudopods (blue dots) relative to the cell centroid and normalized to the movement direction of the cells (black dot) in wild-type (B) and slb mutant (E) embryos at shield stage. For these spherical diagrams, the 3D distribution of the positions where pseudopods emerged on the surface of each cell (blue dots) was measured relative to the centroid of the cell body. The position of the centroid for the succeeding timepoint was also measured. The distances between the positions of the pseudopod and the cell centroid were then calibrated to a constant value, leaving the orientation of the pseudopod positions unchanged. Plotted in 3D with the cell centroid at the origin, the pseudopod positions were, thus, placed at the surface of a sphere centred around the origin. These spherical graphs were then turned so that the positions of each cell centroid for the following timepoint (black dot) were placed onto the x-axis. The pseudopod positions in 20 cells from five wild-type and five slb embryos at four consecutive timepoints (0, 2.5, 5 and 7.5 minutes) were plotted into one diagram. To enhance the 3D appearance of the plots, an artificial transparent sphere centred at the origin was added to each diagram. Note that the x, y and z axes in these diagrams show the coordinates relative to the movement direction of individual cells (+x axis), whereas in Fig. 3, these axes depict the global coordinates within the gastrula. (B',E') Distribution of pseudopod lengths from B and E, respectively, along the x-axis. (B',E') – the individual movement axis of the cells – or the z-axis (B'',E''). Each diagram shows pseudopod lengths relative to the body length of the corresponding cell (in %); the numbers on the ordinate axis correspond to arbitrary units, with x=10 being the radius of the spheres in (B) and (E). (C,F) Distribution of the outgrowth positions of pseudopods in wild-type (C) and slb mutant (F) embryos. The columns show the relative distribution of pseudopods along (+x versus –x) or perpendicular (+z versus –z) to the individual movement direction of the cells, averaged over four consecutive timepoints (0, 2.5, 5 and 7.5 minutes), with the cell centroid at x=0 and z=0. The insets show examples of wild-type (a-d) and slb (e-h) prechordal plate cells after 3D reconstruction with the software used to quantify the data, with cell body and pseudopods in light blue and red, respectively. The corresponding timepoints are indicated. *, P<0.05, paired Student's t-test. Scale bar in A: 10 µm.

View larger version (50K): [in a new window]   Fig. 5. Distribution of processes in wild-type and slb epiblast cells overlying the presumptive prechordal plate at the onset of gastrulation. Cells were labelled with a mixture of cytosolic and membrane-bound GFP and visualised in 3D over time by two-photon confocal microscopy. (A,D) 3D images of typical epiblast cells in a wild-type (A) and slb mutant (D) embryo moving from top (animal pole) to bottom (germ ring margin) at shield stage. Arrowheads point to thick, pseudopod-like processes. Arrows mark smaller, filopod-like processes. In A, the pseudopod emerging from the upper cell is branched. (B,E) Spherical plots showing the distribution of the outgrowth positions of pseudopods (blue dots) relative to the cell centroid and normalized to the movement direction of the cells (black dot) in wild-type (B) and slb mutant (E) embryos at shield stage (see Fig. 4 for further information on spherical plots). The process positions of 20 cells (from four timepoints in wild type and the first three timepoints in slb) or from 11 cells (last timepoint in slb) from five Wild-type and eight slb embryos at four consecutive timepoints (0, 2.5, 5 and 7.5 minutes) were plotted into one diagram. Note that the x, y and z axes in these diagrams show the coordinates relative to the movement direction of individual cells (+x axis), whereas in Fig. 3, these axes depict the global coordinates within the gastrula. (B',B'',E',E'') Distribution of pseudopod lengths from B and E, respectively, along the x-axis (B',E') – the individual movement axis of the cells – or the z-axis (B'',E''). Each diagram shows pseudopod lengths relative to the body length of the corresponding cell (in %); the numbers on the ordinate axis correspond to arbitrary units, with x=10 being the radius of the spheres in (B) and (E). (C,F) Distribution of the outgrowth positions of pseudopods in wild-type (C) and slb embryos (F). The columns show the relative distribution of pseudopods along (+x versus –x) and perpendicular (+z versus –z) to the individual movement direction of the cells, averaged over four consecutive timepoints (0, 2.5, 5 and 7.5 minutes), with the cell centroid at x=0 and z=0. The insets show examples of wild-type (a-d) and slb (e-h) epiblast cells, with cell bodies in light blue and pseudopods in red. The corresponding timepoints are indicated. The dark blue structures are thin, filopod-like processes. *, P<0.05, paired Student's t-test. Scale bar in A: 10 µm.

View larger version (35K): [in a new window]   Fig. 6. Scatter plot of the relationship between the orientation of hypoblast cell processes towards their individual movement directions (y-axis with % of processes in movement direction) in relation to the degree of persistence of their movements (x-axis), determined for five consecutive timepoints (0, 2.5, 5, 7.5 and 10 minutes) in 20 wild type (blue-dots) and 20 slb cells (red dots) from five embryos each.

View larger version (65K): [in a new window]   Fig. 7. Two-dimensional distribution of pseudopodial processes in wild-type, slb and slb/wnt11 injected slb prechordal plate precursor cells at midgastrulation stages. As in Figs 4 and 5, cells were labelled with a mixture of cytosolic and membrane-bound GFP and visualised over time by two-photon confocal microscopy. (A-C) Polar plots of the distribution of the outgrowth positions of pseudopods (blue dots) relative to the cell centroid and normalized to the movement direction of the cells (black arrows) in wild-type (A), slb (B) and slb embryos injected with 5 pg slb/wnt11 mRNA (C) at 75% epiboly. These polar plots were made similar to the spherical plots shown in Figs 4 and 5 (see above), except that the outgrowth positions of the pseudopods and the cell centroids were originally determined in two dimensions. For each genotype, the process positions in 5-10 cells over 5-15 consecutive timepoints (4-minute time intervals) from two embryos were analysed and plotted into one diagram. Note that the x and y axes show the coordinates relative to the movement direction of individual cells (+x direction) and do not resemble the global coordinates within the gastrula. (D) Relative distribution of the outgrowth positions of pseudopods (in %) along the individual movement direction of the cells (+x and –x) in wild-type and slb embryos and slb embryos injected with 5 pg slb/wnt11 mRNA, averaged over all timepoints analysed.