UDP-Glucose Dehydrogenase Required for Cardiac Valve Formation in Zebrafish Emily C. Walsh and Didier Y. R. Stainier

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Supplemental Figure 2. Expression of ugdh during zebrafish development. (A) Wholemount in situ hybridization (39) of a 4-cell stage embryo shows that ugdh mRNA is maternally provided and initially ubiquitous. (B) At the 6-somite stage, ugdh is expressed in many cells at a low level, but appears more highly expressed in the cells of the hatching gland rudiment (arrow). (C) At the same stage, a dorsal view reveals heightened expression in the adaxial cells of the developing somites (arrow). (D) At 30 hpf, the developing otic vesicle (arrowhead) and branchial arches (arrow) express ugdh (dorsal view, anterior to the left). (E) From 37 hpf onward, a low level of ugdh expression can be detected in the developing heart (39 hpf shown, ventricle (V) and atrium (A) are indicated). (F) 48 hpf embryos exhibit ugdh expression in developing jaw elements (arrows), as well as continued expression in the otic vesicle (O) and fin bud (F) (lateral view, dorsal to the right). Bars, 100 m except (E) 20 m.

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Supplemental Figure 3. Atrial and ventricular myocardial differentiation appears normal in jekyll mutant embryos. Immunofluorescence staining with MF20 (red) and S46 antibodies (green) at 48 hpf. MF20 recognizes a myosin heavy chain present throughout the heart, and S46 recognizes an atrial-specific isotype of myosin heavy chain. Co-staining allows visualization of both chambers: ventricle appears red and atrium yellow as a result of overlap. Although jekyll mutant hearts are somewhat enlarged (B) compared to wild-type (A), the staining pattern appears normal. Staining performed as described previously (41). Bars, 20 m.

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Supplemental Figure 4. Comparison of branchial arch morphology between jekyll and pipetail/wnt5a mutant embryos. (A-C) Alcian green staining of wildtype (A), pipetail/wnt5a mutant (B), and jekyll mutant (C) embryos at 4 dpf (42). As noted previously (16), jekyll branchial arches are devoid of alcian staining which detects negatively charged sulfated proteoglycans deposited around developing chondrocytes. Closer examination reveals additional morphological defects in the jekyll mutant arches, similar to those seen in pipetail mutants. The first branchial arch, which is only 1-2 cells in diameter in wild-type embryos, is 3-4 and 5-6 cells wide in pipetail and jekyll mutants, respectively. Typical cells are outlined in white next to arrow (ventral view, anterior to the left). (D-F) Methylene blue staining of JB4 plastic parasagittal sections shows additional defects in the arches at 4 dpf (anterior to the left). In wildtype embryos, branchial arches exhibit a dorsoanterior-ventroposterior alignment (indicated by the arrow in D). By contrast, in pipetail and jekyll mutants the arches exhibit a ventroanterior-dorsoposterior alignment (arrows in E and F respectively). In addition, as methylene blue undergoes a metachromatic shift from blue to purple in the presence of negatively charged moieties like sulfated proteoglycans, jekyll chondrocytes fail to exhibit a surrounding purple calyx, in contrast to both wildtype and pipetail mutant embryos. Bars, 50 m.

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References

41. Antibody staining performed as described in J. Alexander, D. Y. Stainier, D. Yelon, Dev. Genet. 22, 288 (1998).

42. Alcian staining performed as described in (16). Methylene blue staining performed on parasagittal sections of wild-type and mutant embryos embedded in JB4 plastic as described in (6).