Additional Supporting Information may be found in the online version of this article.

DVDY_22685_sm_suppfig1.tif4774KSupporting Figure 1 Alcian blue staining indicates that ductin inhibition affects facial cartilage morphology at stage 45. A: Embryos injected with xduct-noTM4 and uninjected embryos were fixed, stained with alcian blue, and the soft tissues were dissected away from the cartilage. Arrowheads indicate branchial arches, arrows indicate jaws; in all images, red indicates abnormal and green indicates normal morphologies. Alcian blue staining clearly indicates that both the jaw and branchial arch cartilage were affected by ductin inhibition. B: An embryo injected with xduct-noTM4 was imaged at stage 45 (B.i), fixed, stained with alcian blue, embedded in paraffin, and sectioned. B.ii: Ventral sections show a normal-sized and shaped eye on the left side, and a small branchial arch on the right side. B.iii: More dorsal sections also show a small branchial arch on the right side. B.iv: The right eye is actually two fused eyes, which are joined to the olfactory epithelium. This is also apparent in the image of the whole animal (B.i)
DVDY_22685_sm_suppfig2.tif1646KSupporting Figure 2 Lineage tracers distinguish characteristics of ductin inhibition on placodal versus neural crest–derived organs. Embryos were co-injected with a β-gal lineage tracer plus xduct-noTM4, or they were injected with xduct-YFP. In both cases, one blastomere of 2-cell embryos was injected. A: Embryo co-injected with xduct-noTM4 and β-gal has an eye attached directly to the brain. The affected eye (blue arrow) is on the same side as the injection, as was observed in almost all cases. B: An embryo co-injected with xduct-noTM4 and β-gal with small, underdeveloped jaws and BA on both sides (red arrows). The β-gal signal indicates that only one side of this embryo actually received injected mRNA. C: To account for possible differences in the localization of β-gal lineage tracer and ductin mutant mRNA, we further verified these results using a fluorescently labeled xduct construct (xduct-YFP). C.i.: Brightfield image. Cii: Fluorescent signal from YFP. Again, eye phenotypes (blue arrow) were observed only on the injected side. This embryo also has abnormal growth of brain (white arrows), which is clearly labeled with YFP.
DVDY_22685_sm_suppfig3.tif8242KSupporting Figure 3 Craniofacial phenotypes induced by wildtype xduct and ductin-independent gain-of-function reagents. Ductin loss-of-function produced a range of craniofacial phenotypes (see Fig. 2). Reagents predicted to act as gain-of-function agents, including the sodium-hydrogen exchanger nhe3, and a yeast proton pump pma1.2, were also tested for their affects on morphology. We also examined the effect of wildtype ductin mRNA (xduct). Tadpoles were scored and imaged between stage 45 and 48. All three of these constructs produced a similar suite of phenotypes to each other and to xduct-noTM4 and concanamycin. In all images, the construct injected is indicated and arrowheads point to abnormal structures. All views are dorsal except where indicated by a V (ventral) or a P (profile). Anterior is up except in I, where anterior is left and dorsal is up. A: Tadpole illustrating unilateral small branchial arches (BA; red arrowhead). B: Tadpole with bilateral reduction in BA and jaw (red arrowheads). C: Tadpole with bilateral reduction in BA and jaw (red arrowheads), and eyes with thickened pigmented optic nerves (blue arrowhead) that are joined across the midline. The left otolith is missing (orange arrowhead). D: Tadpole with abnormal right BA and jaw (red arrowhead), an eye with thickened pigmented optic nerve (blue arrowhead), and a missing right otolith (orange arrowhead). E: Tadpole with pigmented optic nerves (blue arrowheads). F: Tadpole with missing left olfactory pit (yellow arrowhead), left eye fused to the brain (blue arrowhead), and malformed left otocyst (orange arrowhead). G: Tadpole with its right olfactory pit fused to the brain (yellow arrowhead), and a malformed right eye (blue arrowhead). H: Tadpole with a missing right otolith (red circle); normal left otolith circled in green. I: Profile of tadpole lacking jaws (red arrowhead). Inset: normal profile.
DVDY_22685_sm_suppmovie1.mov3108KSupporting Movie 1 Time-lapse movie of the dynamic patterns of regions distinguished by membrane voltage (see also Fig. 2). Embryos were stained using the ratiometric CC2-DiBAC double dye technique (see Experimental Procedures section). Brighter means the voltage is more negative inside the cell (more polarized), darker means less negative (less polarized). Two embryos are shown; the real length of the video is 18 hr. The embryo to the left is a few hours older than the embryo to the right. Three distinct types of dynamic pattern are illustrated. The earliest, course I, is best illustrated in the embryo on the right: a flash of hyperpolarization moves from the posterior to the anterior pole. When this flash is over, the embryo begins to rotate in its vitelline envelope, and the dorsal midline appears, revealing that the view is now of the left side of the embryo. The end of course I and the second pattern, course II, are clear on the left embryo. The interior of the closing neural tube appears at the upper edge of the embryo and moves towards the viewer. As the tube closes and its signal disappears from view, other temporary regions of brightness appear in regions delineating the future face. Course III is clear on both embryos. Randomly placed foci of brightness appear, spread, then disappear as the embryos change shape from spheres to flatter, elongated ovals.

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