DEV027904 Supplementary Material

Files in this Data Supplement:

• Supplemental Figure S1 -

  Fig. S1. pyr and ths ectopic expression in the mesoderm results in ectopic dpERK activation. Cross-sections of stage 9/10 embryos stained with anti-dpERK antibody. (A) Wild type; (B) twist-Gal4 supporting UAS-bnl expression; (C) twist-Gal4 supporting UAS-pyr expression; (D) 69B-Gal4 supporting UAS-pyr expression; (E) twist-Gal4 supporting UAS-ths expression; 69B-Gal4 supporting UAS-ths expression. Red arrowhead marks the leading edge. (A) dpERK staining is observed only at the leading edge of migrating mesoderm in the wild-type embryo. (B) No change in the dpERK profile from that in wild-type embryos is observed when bnl is expressed (red arrowhead). (C,D) Ectopic expression of pyr in the mesoderm using twist-Gal4 and in the ectoderm using 69B-Gal4 results in expansion of dpERK staining throughout the mesoderm. Staining is no longer limited to the leading edge. (E,F) Ectopic expression of ths in the mesoderm using twist-Gal4 results in expansion of dpERK staining throughout the mesoderm (E). dpERK is observed only in the leading edge, as in the wild type, when ths is ectopically expressed in the ectoderm using 69B-Gal4. As in the wild type, we also see dpERK at the ventral part of mesoderm (yellow arrowhead) (F).

• Supplemental Figure S2 -

  Fig. S2. Ectopic expression of pyr and ths does not support tracheal branching. Embryos are oriented with anterior to the left and dorsal up, and stained using the 2A12 monoclonal antibody to identify trachea. (A) Wild type; (B) 69B-Gal4 supporting UAS-bnl expression; (C) 69B-Gal4 supporting UAS-pyr expression; (D) 69B-Gal4 supporting UAS-ths expression. (A) Typical branching pattern of the developing trachea is observed in wild-type embryos. (B) Ectopic expression of bnl throughout the entire ectoderm causes ectopic branching. (C,D) Ectopic expression of pyr or ths throughout the entire ectoderm using 69B-Gal4 has no detectable effect on branching of the trachea.

• Supplemental Figure S3 -

  Fig. S3. pyr and ths expression domains suggest that both ligands influence dorsal mesoderm specification. Embryos are oriented with anterior to the left and dorsal up. (A,C,D,E) Stage 9/10; (B) stage 10/11. (A,B) In situ hybridization using the specified riboprobes. (C-E) Double antibody fluorescence using anti-Eve and anti-β-galactosidase. (A,B) pyr (A) and ths (B) expression in wild-type embryos is present in the dorsal ectoderm, overlying the site of Eve expression within the dorsal mesoderm cells beneath. Within this domain, pyr is expressed slightly earlier than ths. (C) A wild-type embryo stained with anti-Eve antibody depicting expression in cells derived from the dorsal mesoderm as well as within cells of the CNS. (D,E) Additional Eve⁺ cells are supported by both pyr (D) and ths (E) ectopic expression through 69B-Gal4 in Df(2R)BSC25/CyO wg-lacZ embryos. β-galactosidase staining in apparent in these heterozygous embryos. The same results are obtained in a wild-type background.

• Supplemental Figure S4 -

  Fig. S4. Quantitative PCR analyses of ectopic pyr and ths expression demonstrates that the levels of ectopic expression supported by the respective transgenes are similar. Quantification of transcript levels in different lines using specific primers for pyr and ths. The data obtained are represented in arbitrary units in the table as the number of molecules of transcript for each primer combination. Ectopic expression of pyr in twist-Gal4>UASpyr1 and twist-Gal4>UASpyr2 results in a 3- to 4-fold increase in transcript levels as compared with the twist-Gal4>UASlacZ or the ths-expressing lines. Similarly, ths transcript levels show a similar increase in ectopically expressing ths lines (twist-Gal4>UASths1 and twist-Gal4>UASths2). See Materials and methods for details.

• Supplemental Figure S5 -

  Fig. S5. Clumping phenotypes. Embryos were scored based on the presence of a clump of mesoderm cells in at least one section of an embryo (clump), or complete absence of a clump (no clump); clumps occurred on either the top or bottom half of the embryo, or both. Seventy-two percent of deficiency mutant embryos exhibit a mesoderm clumping phenotype. Compared with the deficiency, ectopic expression of pyr using sim-Gal4 resulted in a significantly enhanced clumping phenotype (P<0.01, Fisher’s exact test). By comparison, the ths-expressing lines did not result in any significant increase in clumping (P=0.259). Rescue of the mesoderm spreading phenotype by ectopic expression of pyr using zenVRE.Kr-Gal4 resulted in a significant reduction as compared with deficiency lines (P<0.0001). The ectopic expression of ths did not result in a significant reduction of the clumping phenotype (P<0.04).

• Supplemental Figure S6 -

  Fig. S6. Multilayer formation at the dorsal mesoderm. The mesoderm was scored as multilayered if more than one layer of mesoderm cells was present near the dorsal-lateral boundary of the embryo in one or several sections of each embryo. The multilayered phenotype occurred on the bottom of the embryo (multilayered one side), on the top and bottom (multilayered both sides), or not at all (no multilayer). In the deficiency lines, 75% of embryos do not form multilayers. Similarly, ectopically expressed pyr or ths genes using sim-Gal4 do not form a multilayer phenotype. However, ectopic expression of pyr using zenVRE.Kr-Gal4 resulted in 22% of embryos having multilayer on one side (P<0.01, Fishers exact test) and 78% having multilayer on both sides (P<0.0001), as compared with the deficiency lines. Ectopic expression of ths using zenVRE.Kr-Gal4 resulted in 47% of embryos with multilayer on side (P<0.0001) and 53% embryos with multilayer on both sides (P<0.0001), respectively.