Figure 6.1: Development of the zebrafish embryo
Stages are named after anatomical features of the embryos and the age corresponds to the embryonic development of the at 28ºC. The percentage of Epiboly describes the percentage to which the yolk cell is covered by embryonic cells. The embryos are illustrated in lateral view; germ ring and shield stage embryos are also shown from the top. Until the 25h stage, the dorsal side of the embryo is to the right. This figure is modified after camera lucida drawings of (Kimmel et al., 1995).
6.2 Expression pattern of wnt11, fz7a and fz7b in zebrafish
embryos
Figure 6.2 Expression pattern of wnt11, frizzled7a and b during early zebrafish development
Embryos were fixed at different stages of development and (16cell; 30%epiboly (5hpf); shield stage (6hpf); bud stage; (10hpf)) followed by in situ hybridisation with anti-sense mRNA probes against zebrafish wnt11 (upper panel); zebrafish homologue of frizzled7a (fz7a; middle panel) and zebrafish homologue of frizzled7b (fz7b; lower panel). Embryos are oriented onto the animal pole; the dorsal side is to the right. Wnt11 is not present at 16-cell stage and starts to be expressed in the germ ring margin at 30% epiboly. Note that wnt11 is absent from the animal pole at that stage. Fz7a is not present at 16-cell stage and starts to be weakly expressed at 30% epiboly. It becomes stronger expressed at shield stage in the germ ring margin. Fz7b is present at 16 –cell stage (maternally provided) and shows a strong ubiquitous expression throughout early stages and gastrulation. Note that Fz7b is strongly expressed in the germ ring margin (30% epiboly; shield stage). This data was kindly provided by Sylvia Schneider and Carl-Philipp Heisenberg.
6.3 Phenotype of Flamingo morphant embryos
Morpholino Construct Epiboly delay bud; % Unaffect ed 24hpf; % T a i l m o r p h o - genesis; 24hpf; % mild strong Narrow eyes (24hpf; %) total (n) Fmi1a/1b/2 - 91 24 69 7 21 331 Fmi1a/1b/2 fz7-YFP+wnt11 86 15 43 42 47 206 Morpho- lino Construct Convergent extension CE bud; % Unaffected 24hpf;% CE phenotype (24hpf;%) mild strong Narrow eyes (24hpf;%) total (n) - fz7-YFP + wnt11 75 43 34 23 57 35Table 6.1 Phenotype of Flamingo morphant embryos in the absence or presence of Fz7- YFP and Wnt11
For each Fz7+Wnt11+FmiMo time-lapse movie made, we have analyzed the phenotype of wt embryos which were either only injected with a mixture of Morpholinos against fmi1a, fmi1b and fmi2 (4ng/MO) or co-injected with 60pg of fz7-YFP and 10pg of wnt11 mRNA at the one-cell stage. The phenotype of the embryos was analyzed according to their morphology at bud stage (12hpf) and at 24hpf. The phenotypes were divided into ‘delayed epiboly’ at bud-stage, ‘abnormal tail morphogenesis’ at 24hpf (embryos with mild defects showed a slightly shorter and kinked tail and embryos with strong defects exhibited a very short and frequently curly tail), and ‘narrow eyes’ at 24hpf. Embryos with ‘narrow eyes’ phenotype also showed defects in tail morphogenesis. Over-expression of 60pg of fz7-YFP and 10pg of wnt11 mRNA led to convergent extension phenotypes at bud stage, and narrow eyes at 24hpf; in severe cases embryos showed narrow eyes and an very short and curly tail.
6.4 Supplementary videos
The corresponding videos can be found on the CD, which is supplemented with this thesis.
Supplementary video1: Separating cells in pre-gastrula stage embryos expressing Fz7-YFP and Wnt11
Embryos were injected at the one-cell-stage with 60pg of fz7-YFP and 10pg of wnt11 mRNA. 3D time- lapse analysis was performed by two-photon microscopy of blastodermal cells at the animal pole of pre- gastrula stage embryos (30% epiboly, 5hpf). The movie corresponds to the image sequence in Fig.12A and shows the separation behaviour of two cells exhibiting Wnt11-induced Fz7-YFP accumulation at their sites of cell-cell contact. Arrowheads indicate sites of cell-cell contacts. Time-intervals=130s. Scale bar=10µm.
Supplementary video2: Separating cells of gastrula stage embryos expressing Fz7-YFP and Wnt11
Embryos were injected at the one-cell-stage with 60pg of fz7-YFP and 10pg of wnt11 mRNA. 3D time- lapse analysis was performed by two-photon microscopy of ectodermal cells at the germ ring margin of gastrula stage embryos (starting at shield stage, 6hpf). The movie shows the separation behaviour of two epiblast cells exhibiting Wnt11-induced Fz7-YFP accumulation at their site of cell-cell contact. Arrowheads indicate sites of cell-cell contacts. Time-intervals=144s. Scale bar =10µm.
Supplementary video3: Separating cells in pre-gastrula stage embryos expressing Fz7-YFP only
Embryos were injected at the one-cell-stage with 60pg of fz7-YFP mRNA. 3D time-lapse analysis was performed by two-photon microscopy of blastodermal cells at the animal pole of pre-gastrula stage embryos (30% epiboly, 5hpf). The movie corresponds to the image sequence in Fig.12B and shows the separation behaviour of two cells expressing Fz7-YFP uniformly at their plasma membranes. Arrowheads indicate sites of cell-cell contacts. Time-intervals= 130s. Scale bar =10µm.
Supplementary video4: Separating cells in pre-gastrula stage embryos expressing Lyn-YFP only
Embryos were injected at the one-cell-stage with 25pg of lyn-YFP mRNA. 3D time-lapse analysis was performed by two-photon microscopy of blastodermal cells at the animal pole of pre-gastrula stage embryos (30% epiboly, 5hpf). The movie corresponds to the image sequence in Fig.12C and shows the separation behaviour of two cells expressing Lyn-YFP uniformly at their plasma membranes. Arrowheads indicate sites of cell-cell contacts. Time-intervals 123s. Scale bar =10µm.
Supplementary video5: Separating cells in pre-gastrula stage embryos expressing Fmi2-YFP only
Embryos were injected at the one-cell-stage with 25-50pg of fmi2-YFP pCS2-plasmid DNA. 3D time- lapse analysis was performed by two-photon microscopy of blastodermal cells at the animal pole of shield stage embryos (5-6hpf). The movie corresponds to the image sequence in Fig.16A and shows the separation behaviour of two cells containing Fmi2-YFP accumulations at their contact sites. Arrowheads indicate sites of cell-cell contacts. Time-intervals=172s. Scale bar =10µm.
Supplementary video6: Separating cells in pre-gastrula stage fmi morphant embryos expressing Fz7-YFP and Wnt11
Embryos were co-injected at the one-cell-stage with 60pg of fz7-YFP and 10pg of wnt11 m RNA and MOs targeted against fmi1a, fmi1b and fmi2 (4ng/MO). 3D time-lapse analysis was performed by two- photon microscopy of blastodermal cells at the animal pole of pre-gastrula stage embryos (30% epiboly, 5hpf). The movie serves as an example for the data analysis in Fig.16B-D and shows the separation behaviour of two blastodermal cells exhibiting Wnt11-induced Fz7-YFP accumulation at their site of cell-cell contact (Arrowheads); Time-intervals=137s; Scale bar=10µm.
Supplementary video7: Separating cells in gastrula stage rhoA morphant embryos expressing Fz7-YFP and Wnt11
One-cell stage embryos were injected with 8ng of rhoAa and rhoAd MO each, and a mix of 60pg of fz7-
YFP and 10pg of wnt11 mRNA. 3D time-lapse analysis was performed by two-photon microscopy of
gastrula stage embryos (starting at shield stage, 6hpf) mounted with the dorsal side to the right. Ectodermal cells in lateral regions of the germ ring were imaged (note that these cells show slower convergence movements due to reduced RhoA activity). After completion of the time-lapse analysis, imaged embryos were screened for their morphant phenotype at the end of gastrulation (Zhu et al., 2006). The movie corresponds to the image sequence in Fig.20A and shows the separation behaviour of cells containing Fz7 accumulations at their contact sites. Arrowheads indicate sites of cell contacts; Time-intervals=157s; Scale bar =10µm.
Supplementary video8: Separating cells in gastrula stage rhoA/rok2
morphant/inhibited embryos expressing Fz7-YFP and Wnt11
One-cell stage embryos were injected with 8ng of rhoAa and rhoAd MO each, and a mix of 60pg of fz7-
YFP and 10pg of wnt11 m RNA. Dechorionated embryos were incubated within 50µM of the Rok-
specific inhibitor Y-27632 from 30% epiboly onwards. 3D time-lapse analysis was performed by two- photon microscopy of gastrula stage embryos (starting at shield stage, 6hpf) mounted with the dorsal side to the right. Ectodermal cells in lateral regions of the germ ring margin were imaged (note that these cells show slower convergence movements due to reduced RhoA/Rok2 activity). After completion of the time-lapse analysis, imaged embryos were screened for their morphant phenotype at the end of gastrulation (Marlow et al., 2002; Zhu et al., 2006). The movie corresponds to the image sequence in Fig.20B and shows the separation behaviour of cells containing Fz7 accumulations at their contact sites. Arrowheads indicate sites of cell-cell contacts; Time-intervals=165s; Scale bar =10µm.
Supplementary video9: Separating epiblast cells in gastrula stage embryos expressing Wnt11-YFP only
Embryos were injected at the one-cell-stage with 80pg of wnt11-YFP and 80pg of GPI anchored-RFP (membrane RFP) mRNA. 3D time-lapse analysis was performed by confocal microscopy of epiblast and hypoblast cells at the germ ring of late shield stage embryos (7hpf). The movie shows the separation behaviour of two hypoblast cells, in which Wnt11-YFP localized in ‘puncta’ at sites of cell-cell contacts (arrowheads) and remained there until the cells were completely separated. Time-intervals=112s. Scale bar=10µm.
Supplementary video10: Separating hypoblast cells in gastrula stage embryos expressing Wnt11-YFP only
Embryos were injected at the one-cell-stage with 80pg of wnt11-YFP and 80pg of GPI anchored-RFP (membrane RFP) mRNA. 3D time-lapse analysis was performed by confocal microscopy of epiblast and hypoblast cells at the germ ring of late shield stage embryos (7hpf). The movie corresponds to the image sequence in Fig.24A and shows the separation behaviour of two hypoblast cells, in which Wnt11-YFP localized in ‘puncta’ at sites of cell-cell contacts (arrowheads) and remained there until the cells were completely separated. Time-intervals=112s; Scale bar=10µm.
Supplementary video11: Separating cells in pre-gastrula stage embryos expressing Wnt11-YFP only
Embryos were injected at the one-cell-stage with 80pg of wnt11-YFP and 80pg of GPI anchored-RFP (membrane RFP) m RNA. 3D time-lapse analysis was performed by confocal microscopy of blastodermal cells at the pre-gastrula stage embryos (5hpf). The movie serves as an example for the data analysis in Fig.24B-E and shows the separation of blastodermal cells, in which Wnt11-YFP localized in ‘puncta’ at sites of cell-cell contacts (arrowheads) and remained there until the cells were completely separated; Time-intervals=151s; Scale bar=10µm.