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Files in this Data Supplement:
Fig. S1. Loss of Hai1 activity affects morphology of the olfactory epithelium, but not the morphology of the pronephric ducts and the gut. (A-C) Frontal views on head of live embryos at 24 hpf. Black arrows point to dissociating olfactory epithelium in hai1a mutant (with low penetrance and expressivity; B) and hai1a mutant injected with hai1b MO (with high penetrance and expressivity; C). (D-G) Eosin/hematoxylene stainings of cross-sections through posterior trunk region of hai1a mutants (E,G) and wild-type siblings (D,F) at 24 hpf (D,E) or 48 hpf (F,G). Pronephric ducts are indicated with white arrows, the gut in (F,G) with white asterisks. Black arrowhead in (E) points to dissociating epidermis on yolk extension.
Fig. S2. Concomitant inactivation of cMet and its related receptors Prgfr3 and Ron is insufficient to rescue the epidermal defects of hai1a mutants. (A-D) prgfr3 and ron MOs efficiently block the translation of their respective mRNAs encoding Prgfr3-GFP or Ron-GFP fusion proteins. Upper panels show bright-field images, lower panel GFP fluorescence of embryos at the 80% epiboly stage, after injection of prgfr3-gfp mRNA (A,B) or ron-gfp mRNA (C,D), and with (B,D) or without (A,C) the respective MOs. Co-injection of MOs leads to complete suppression of GFP fluorescence. (E-F) Lateral Nomarski images of 24 hpf un-injected wild-type embryo (E) and wild-type embryos co-injected with cmet MO, prgfr3 MO and ron MO (each at 0.16 mM) (F), revealing that all three receptors are dispensable for normal skin development. (G,H) Lateral Nomarski images of 24 hpf un-injected hai1a mutant (G) and hai1a mutant co-injected with cmet MO, prgfr3 MO and ron MO (each at 0.16 mM), revealing that Hai1 does not act by inactivating cMet, Prgfr3 and Ron. White arrows point to dissociating epidermis on yolk extension.
Movie 1. Confocal in vivo time-lapse recording of a cluster of mGFP-labelled wild-type basal epidermal cells in a ventral position above the anterior yolk-sac extension. The movie starts at 24 hpf and runs for 90 minutes. Cells are maintained in a rigid epithelium.
Movie 2. Confocal in vivo time-lapse recording of a cluster of mGFP-labelled hai1a morphant basal epidermal cells in a ventral position above the anterior yolk-sac extension, labeled mosaically with mGFP. The movie starts at 24 hpf and runs for 90 minutes. Cells display a fibroblastoid shape and migratory behavior.
Movie 3. Confocal in vivo time-lapse recording of a cluster of mGFP-labelled hai1a morphant basal epidermal cells in a dorsal position in the posterior trunk. The movie starts at 24 hpf and runs for 90 minutes. At this location morphant cells appear less motile than in more ventral positions (compare with Movie 2).
Movie 4. Confocal in vivo time-lapse recording of a cluster of mGFP-labelled hai1a, hai1b double-morphant basal epidermal cells. The movie starts at 20 hpf and runs for 90 minutes. Cell motility is evident earlier and in dorsal regions of the embryo, at a site where hai1a single-morphant keratinocytes show normal epithelial properties (compare with Movie 3).
Movie 5. Confocal in vivo time-lapse recording of a cluster of mGFP-labelled basal epidermal cells of embryos injected with both hai1a and hai1b morpholinos. The movie starts at 20 hpf and runs for 90 minutes. Notice how keratinocytes migrate on top of each other.
Movie 6. Confocal in vivo time-lapse recording of the posterior yolk sac and the anterior yolk extension of a tg(fli:egfp) embryo. The movie starts at 23 hpf and runs for 90 minutes.
Movie 7. Confocal in vivo time-lapse recording of the posterior yolk sac and the anterior yolk extension of a tg(fli:egfp), hai1a-/- embryo stained with acridine orange. The movie starts at 23 hpf and runs for 90 minutes. The embryo was only lightly stained with acridine orange to label apoptotic cells without interfering with the GFP signal. This acridine orange stain rapidly bleaches during the course of the movie. GFP-positive leukocytes are seen migrating over the yolk and towards the site of apoptosis.
Movie 8. Confocal in vivo time-lapse recording of a cluster of mGFP-labelled hai1a, mat1a double-morphant basal epidermal cells in a ventral position above the yolk-sac extension, at a site which showed aberrant mesenchymal-like behavior in regular hai1a morphants. Compare with Movie 2. The movie starts at 24 hpf and runs for 90 minutes. Epithelial properties are restored upon ablation of Matriptase1a.
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