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Ectopic expression screen identifies genes affecting Drosophila mesoderm development including the HSPG Trol.

Trisnadi N, Stathopoulos A - G3 (Bethesda) (2014)

Bottom Line: These include the FGF ligand Pyramus, α-integrins, E-cadherin, Cueball, EGFR, JAK/STAT signaling components, as well as the heparan sulfate proteoglycan (HSPG) Terribly reduced optic lobes (Trol).Our data support the view that both HSPGs function to support FGF-dependent processes in the early embryo as they share phenotypes with FGF mutants: Trol in terms of effects on mesoderm migration and caudal visceral mesoderm (CVM) migration and Sdc in terms of dorsal mesoderm specification.The differential roles uncovered for these two HSPGs suggest that HSPG cofactor choice may modify FGF-signaling outputs.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard, MC 114-96, Pasadena, California 91125.

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Endogenous expression and mutant phenotypes of adhesion molecules and signaling components isolated from the screen. Cross-sectioned embryos are of stage 9–10 when mesoderm cells are at the end of their migration. (A–D) A comparison of wild-type with mild, moderate, and severe mesoderm spreading phenotypes. (A) Wild-type embryos have a monolayer of mesoderm cells. The arrowhead marks where the mesoderm cells have reached the dorsal region of the embryo, where cells receive additional differentiation signals. (B) pyr18/Df BSC25 trans-heterozygous mutant embryos have a mild phenotype marked by regions where mesoderm cells are multilayered (arrow). However, some cells intercalate into a single layer (arrowhead). (C) pyre02915/Df BSC25 embryos have a moderate phenotype where the mesoderm is uniformly multilayered. Df BSC25 is a deficiency that encompasses both Pyr and Ths, FGF ligands for the FGFR Htl. (D) htlAB42 mutants have severe defects such that the mesoderm forms lumps of cells. (E–T) Preliminary expression and mutant analysis of genes isolated in the screen. RNA expression patterns in wild-type embryos of stage 8–9 (lateral views: E–H, M–P) and cross-section of zygotic mutant embryos showing α-Twi expression to mark mesoderm (cross-sections: I–L, Q–T). Single mutants were assayed if available (I, J, K, L, Q, R) for genes isolated from the ectopic expression screen; otherwise, data for deficiencies are shown (aos: S). For assay of egfr, the dominant negative (DN) form of egfr was overexpressed using the Twi-Gal4 driver (T). In situ hybridization was performed using riboprobes specified for the indicated genes. Genes in red denote those isolated from this screen.
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fig2: Endogenous expression and mutant phenotypes of adhesion molecules and signaling components isolated from the screen. Cross-sectioned embryos are of stage 9–10 when mesoderm cells are at the end of their migration. (A–D) A comparison of wild-type with mild, moderate, and severe mesoderm spreading phenotypes. (A) Wild-type embryos have a monolayer of mesoderm cells. The arrowhead marks where the mesoderm cells have reached the dorsal region of the embryo, where cells receive additional differentiation signals. (B) pyr18/Df BSC25 trans-heterozygous mutant embryos have a mild phenotype marked by regions where mesoderm cells are multilayered (arrow). However, some cells intercalate into a single layer (arrowhead). (C) pyre02915/Df BSC25 embryos have a moderate phenotype where the mesoderm is uniformly multilayered. Df BSC25 is a deficiency that encompasses both Pyr and Ths, FGF ligands for the FGFR Htl. (D) htlAB42 mutants have severe defects such that the mesoderm forms lumps of cells. (E–T) Preliminary expression and mutant analysis of genes isolated in the screen. RNA expression patterns in wild-type embryos of stage 8–9 (lateral views: E–H, M–P) and cross-section of zygotic mutant embryos showing α-Twi expression to mark mesoderm (cross-sections: I–L, Q–T). Single mutants were assayed if available (I, J, K, L, Q, R) for genes isolated from the ectopic expression screen; otherwise, data for deficiencies are shown (aos: S). For assay of egfr, the dominant negative (DN) form of egfr was overexpressed using the Twi-Gal4 driver (T). In situ hybridization was performed using riboprobes specified for the indicated genes. Genes in red denote those isolated from this screen.

Mentions: These phenotypes were classed into three different levels of severity (Figure 2, A–D). Mild indicates only a few cells did not intercalate, creating an uneven layer. Mesoderm defects of moderate phenotype present multilayered cells, which nevertheless evenly spread on the ectoderm but fail to form a monolayer. Severe phenotypes include uneven spreading of cells on the ectoderm (i.e., not centered at the midline) as well as multilayered/clumping of cells, as in the case with htl mutants, which exhibit defects in mesoderm collapse, spreading, and intercalation (McMahon et al. 2008). Also, we have observed that htl phenotypes are variable, ranging from mild to severe (Table 2, see htl). To quantify phenotypes that could be variable, at least 7 and as many as 23 embryos were examined for mutants assayed. Furthermore, a score was calculated based on frequency of phenotypes observed (Table 2). In a recent study, cadherin mutants were found to exhibit nonmonolayer phenotypes, but a role for these molecules in supporting mesoderm formation was dismissed because germ layers were specified (Schafer et al. 2014). Because the goal of our screen was to uncover signals guiding proper mesoderm migration, lack of a monolayer is relevant and indicates defects in effective mesoderm migration. For this reason, we considered mutant phenotypes that span the range of mild to severe.


Ectopic expression screen identifies genes affecting Drosophila mesoderm development including the HSPG Trol.

Trisnadi N, Stathopoulos A - G3 (Bethesda) (2014)

Endogenous expression and mutant phenotypes of adhesion molecules and signaling components isolated from the screen. Cross-sectioned embryos are of stage 9–10 when mesoderm cells are at the end of their migration. (A–D) A comparison of wild-type with mild, moderate, and severe mesoderm spreading phenotypes. (A) Wild-type embryos have a monolayer of mesoderm cells. The arrowhead marks where the mesoderm cells have reached the dorsal region of the embryo, where cells receive additional differentiation signals. (B) pyr18/Df BSC25 trans-heterozygous mutant embryos have a mild phenotype marked by regions where mesoderm cells are multilayered (arrow). However, some cells intercalate into a single layer (arrowhead). (C) pyre02915/Df BSC25 embryos have a moderate phenotype where the mesoderm is uniformly multilayered. Df BSC25 is a deficiency that encompasses both Pyr and Ths, FGF ligands for the FGFR Htl. (D) htlAB42 mutants have severe defects such that the mesoderm forms lumps of cells. (E–T) Preliminary expression and mutant analysis of genes isolated in the screen. RNA expression patterns in wild-type embryos of stage 8–9 (lateral views: E–H, M–P) and cross-section of zygotic mutant embryos showing α-Twi expression to mark mesoderm (cross-sections: I–L, Q–T). Single mutants were assayed if available (I, J, K, L, Q, R) for genes isolated from the ectopic expression screen; otherwise, data for deficiencies are shown (aos: S). For assay of egfr, the dominant negative (DN) form of egfr was overexpressed using the Twi-Gal4 driver (T). In situ hybridization was performed using riboprobes specified for the indicated genes. Genes in red denote those isolated from this screen.
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fig2: Endogenous expression and mutant phenotypes of adhesion molecules and signaling components isolated from the screen. Cross-sectioned embryos are of stage 9–10 when mesoderm cells are at the end of their migration. (A–D) A comparison of wild-type with mild, moderate, and severe mesoderm spreading phenotypes. (A) Wild-type embryos have a monolayer of mesoderm cells. The arrowhead marks where the mesoderm cells have reached the dorsal region of the embryo, where cells receive additional differentiation signals. (B) pyr18/Df BSC25 trans-heterozygous mutant embryos have a mild phenotype marked by regions where mesoderm cells are multilayered (arrow). However, some cells intercalate into a single layer (arrowhead). (C) pyre02915/Df BSC25 embryos have a moderate phenotype where the mesoderm is uniformly multilayered. Df BSC25 is a deficiency that encompasses both Pyr and Ths, FGF ligands for the FGFR Htl. (D) htlAB42 mutants have severe defects such that the mesoderm forms lumps of cells. (E–T) Preliminary expression and mutant analysis of genes isolated in the screen. RNA expression patterns in wild-type embryos of stage 8–9 (lateral views: E–H, M–P) and cross-section of zygotic mutant embryos showing α-Twi expression to mark mesoderm (cross-sections: I–L, Q–T). Single mutants were assayed if available (I, J, K, L, Q, R) for genes isolated from the ectopic expression screen; otherwise, data for deficiencies are shown (aos: S). For assay of egfr, the dominant negative (DN) form of egfr was overexpressed using the Twi-Gal4 driver (T). In situ hybridization was performed using riboprobes specified for the indicated genes. Genes in red denote those isolated from this screen.
Mentions: These phenotypes were classed into three different levels of severity (Figure 2, A–D). Mild indicates only a few cells did not intercalate, creating an uneven layer. Mesoderm defects of moderate phenotype present multilayered cells, which nevertheless evenly spread on the ectoderm but fail to form a monolayer. Severe phenotypes include uneven spreading of cells on the ectoderm (i.e., not centered at the midline) as well as multilayered/clumping of cells, as in the case with htl mutants, which exhibit defects in mesoderm collapse, spreading, and intercalation (McMahon et al. 2008). Also, we have observed that htl phenotypes are variable, ranging from mild to severe (Table 2, see htl). To quantify phenotypes that could be variable, at least 7 and as many as 23 embryos were examined for mutants assayed. Furthermore, a score was calculated based on frequency of phenotypes observed (Table 2). In a recent study, cadherin mutants were found to exhibit nonmonolayer phenotypes, but a role for these molecules in supporting mesoderm formation was dismissed because germ layers were specified (Schafer et al. 2014). Because the goal of our screen was to uncover signals guiding proper mesoderm migration, lack of a monolayer is relevant and indicates defects in effective mesoderm migration. For this reason, we considered mutant phenotypes that span the range of mild to severe.

Bottom Line: These include the FGF ligand Pyramus, α-integrins, E-cadherin, Cueball, EGFR, JAK/STAT signaling components, as well as the heparan sulfate proteoglycan (HSPG) Terribly reduced optic lobes (Trol).Our data support the view that both HSPGs function to support FGF-dependent processes in the early embryo as they share phenotypes with FGF mutants: Trol in terms of effects on mesoderm migration and caudal visceral mesoderm (CVM) migration and Sdc in terms of dorsal mesoderm specification.The differential roles uncovered for these two HSPGs suggest that HSPG cofactor choice may modify FGF-signaling outputs.

View Article: PubMed Central - PubMed

Affiliation: Division of Biology and Biological Engineering, California Institute of Technology, 1200 East California Boulevard, MC 114-96, Pasadena, California 91125.

Show MeSH
Related in: MedlinePlus