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Fig. S3. Characterizing the morpholino phenotype. (A, parts a,b) Whole embryo phenotype. (a) frzb-1/crescent morphants at stage 23-24 compared with control morpholino-injected embryo. Note the overall similarity in the embryos, suggesting that the positioning and specification of many of the anterior structures are normal. (b) frzb-1/crescent morphants at stage 40-41 compared with control morpholino-injected embryo. Note the curved axis and reduced head structures. Scale bars: 1000 (c,d) Alcian Blue staining of cartilage at stage 45 in (c) control embryos, and (d) frzb-1 and crescent morphants. Note the almost complete lack of Alcian Blue staining in the morphants. Scale bars: 340 . (e,f) Axon tracts of the CNS at stage 40 were visualized by acetylated tubulin immunohistochemistry in (e) controls and (f) frzb-1/crescent morphants. Note the unorganized axon tracts in frzb-1/crescent morphants. Scale bars: 370 . (g,h) Phalloidin labeling of somites at stage 45 in (g) controls and (h) frzb-1/crescent morphants, using F-actin labeling with Phalloidin conjugated to Alexa Fluor 488. Scale bar: 1200 . Insets show a 100magnification of the somites, note the unorganized muscle fibers in frzb-1/crescent morphants. Scale bar: 1200 . (B, parts a,b) Embryos and tadpoles were processed for terminal deoxynucleotidyl transferase-mediated dTP nick-end labeling (TUNEL) as described (Dickinson and Sive, 2006), using the Apoptag kit (Chemicon). TUNEL labeling (green) in the primary mouth region of (a) control and (b) frzb-1/crescent morphants; embryos were counterstained with propidium iodide (red). Note that there are few TUNEL-positive cells in the presumptive primary mouth at stage 24-26, and no observable difference in frzb-1/crescent morphants (n=5; two experiments, not quantified). Scale bars: 100 . (c,d) Phosho-histone3 (pH3) immunohistochemistry (green) using a polyclonal anti-phosphohistone3 antibody diluted 1:1000 and a secondary goat anti-rabbit Alexa Fluor-conjugated antibody diluted 1:500, counterstained with propidium iodide (1:1000). (c) Control. (d) frzb-1/crescent morphants have similar amounts of pH3-positive cells to controls (6.33%, no significant difference) in the presumptive primary mouth regions (four stages examined, n=5 at each stage, one stage in one experiment quantified). Arrows indicate the presumptive primary mouth. cg, cement gland. Scale bars: 100 . |
