Blast software (version 2.1.1) and were assembled with the Consed package ... Cap1 morpholino-injected (E), and Quo morpholino-injected (F) em- bryos ...
Supplemental Data
S1
Developmentally Restricted Actin-Regulatory Molecules Control Morphogenetic Cell Movements in the Zebrafish Gastrula David F. Daggett, Catherine A. Boyd, Philippe Gautier, Robert J. Bryson-Richardson, Christine Thisse, Bernard Thisse, Sharon L. Amacher, and Peter D. Currie Supplemental Experimental Procedures Microscopy and Time-Lapse Imaging Living wild-type and morpholino-injected embryos were mounted in 3% methyl cellulose, and anterior mesodermal regions were observed under 20–40⫻ magnification on a Zeiss AxioSkop equipped with DIC optics. Images were collected with IPLab software, and frames were exported to QuickTime movie format. cap1 and quo Cloning and Bioinformatics A cDNA with homology to human and Drosophila cap1 genes was obtained from an expressed sequence tag (EST) database (www. genetics.wustl.edu). Sequencing of the complete open reading frame revealed a zebrafish cap1 ortholog (GenBank AY162326). A partial quo cDNA was originally identified by its expression profile in an in-situ hybridization screen, and a larger incomplete fragment was subsequently obtained by cDNA library screening. Creating a composite of the sequenced partial cDNAs allowed identification of a complete open reading frame for Danio rerio quo, shotgun genomic reads assembled into a “contig,” and a deposited EST (GenBank AI588674). This yielded a complete cDNA sequence (GenBank AY654285) consistent with an ortholog predicted from the Fugu genome (GenBank BK000661). The zebrafish reads were isolated in the Trace Server database (http://trace.ensembl.org/) with Blast software (version 2.1.1) and were assembled with the Consed package (version 11.0). Multiple alignments were made with CLUSTALW (version 1.82). Positions in alignments containing gaps were omitted from subsequent analyses. All phylogenetic trees were constructed by the neighbor-joining method [S1] with MEGA (version 2.1; http://www.megasoftware.net/). The radiating tree was made from a multiple alignment of all the RhoGEF domain sequences belonging to the Pfam RhoGEF family (http://www.sanger.ac.uk/ cgi-bin/Pfam/getacc?PF00621).
In situ Hybridization and Immunocytochemistry Whole mount in situ hybridization was performed essentially as described [S2]. For Fkd/phalloidin staining, embryos were fixed in 4% paraformaldehyde, washed in PBS, permeabilized in 2% TritonX-100, washed, and incubated with fluorescently conjugated phalloidin (Molecular Probes). Embryos were then treated with AntiFkd antibodies (kind gift of R. Warga) as described [S3] in conjunction with fluorescently conjugated secondary antibodies. Morpholino Design and Injections Two unique antisense morpholino sequences each were designed against the 5⬘ untranslated and start codon regions of both cap1 and quo predicted mRNAs (CapA/B, QuoA/B; sequences are available on request) along with 5–6 base pair CapB and QuoB mismatch morpholinos. Embryos were injected at the 1–2 cell stage as described [S4]. For both genes, initial studies revealed that injection of 3–5 nl of either the A or B morpholino resulted in comparable, specific phenotypes at 0.25–1.0 mM, whereas CapB and QuoB mismatch morpholinos, and a morpholino sequence effective in knocking down GFP expression in GFP transgenic fish, had no effect on polster or embryo morphology. Subsequent experiments were performed with 0.5 mM injections except for transplant experiments, in which some donors received a mixture of 0.35 mM CapB and 0.35 mM QuoB morpholinos. Cell Transplantation Donor embryos were injected with a mixture of fluorescent and biotinylated dextrans in the presence or absence of morpholino as above. Cells were removed by aspiration from the dorsal or lateral marginal zone of shield stage donor embryos with a polished micropipette attached to a microsyringe pump filled with mineral oil and transplanted into the dorsal or lateral marginal zone of wild-type hosts of the same stage. Further analysis was performed on embryos
Figure S1. N Terminus of Quo Is a Novel Conserved Domain in Multiple Predicted Proteins from ESTs and Genomic Sequences across Species
S2
for which morphant cell donors consequently developed polster and convergent extension defects, confirming morpholino efficacy (data not shown). Transplanted cells were visualized by fluorescence in live embryos and, subsequently, biotin was revealed with the Vectastain kit. Supplemental References S1. Saitou, N., and Nei, M. (1987). The neighbor-joining method: A new method for reconstructing phylogenetic trees. Mol. Biol. Evol. 4, 406–425. S2. Broadbent, J., and Read, E.M. (1999). Wholemount in situ hybridization of Xenopus and zebrafish embryos. Methods Mol. Biol. 127, 57–67. S3. Warga, R.M., and Nusslein-Volhard, C. (1999). Origin and development of the zebrafish endoderm. Development 126, 827–838. S4. Nasevicius, A., and Ekker, S.C. (2000). Effective targeted gene ‘knockdown’ in zebrafish. Nat. Genet. 26, 216–220.
Figure S2. Coordinated Cell Movements during Polster Development Require Cap1 and Quo Embryos were examined at 75% epiboly (A, D, and G), 90% epiboly (B, E, and H), and tailbud (C, F, and I) stages. In wild-type embryos, individual hgg1-expressing cells consolidate and coordinately move anterior to the anterior edge of the neural plate (dlx3 expression) as the tissue advances toward and beyond the animal pole (A–C). Analysis of Cap1 morphants (D–F) shows a general consolidation of polster cells but their failure to move anterior to the neural plate. In the case of Quo morphants (G–I), polster precursor cells fail to properly aggregate in the anterior-most mesoderm or to advance beyond the neural plate.
Figure S3. Early Specification and Patterning Events Are Unaffected in Cap1 and Quo Morpholino-Injected Embryos (A–C), gata2 in situ hybridization staining at the shield stage in wildtype (A), Cap1 morpholino-injected (B), and Quo morpholino-injected (C) embryos. (A–C), animal view; A⬘–C⬘, lateral view; * denotes dorsal shield. (D–F), goosecoid staining at 65% epiboly in wild-type (D), Cap1 morpholino-injected (E), and Quo morpholino-injected (F) embryos; dorsal view.
