Conditional switches for extracellular matrix patterning in Drosophila melanogaster

Abstract

An F(1) mutagenesis strategy was developed to identify conditional mutations affecting extracellular matrix (ECM) patterning. Tubulogenesis requires coordinated movement of epithelial cells and deposition of a multilayered ECM. In the Drosophila ovary, an epithelium of follicle cells creates the eggshells, including the paired tubular dorsal appendages (DAs) that act as breathing tubes for the embryo. A P-element mutagenesis strategy allowed for conditional overexpression of hundreds of genes in follicle cells. Conditional phenotypes were scored at the level of individual mutant (F(1)) female flies. ECM pattern regulators were readily identified including MAPK signaling gene ets domain lacking (fused DAs), Wnt pathway genes frizzled 3 and osa (long DAs), Hh pathway gene debra (branched DAs), and transcription factor genes sima/HIF-1alpha, ush, lilli, Tfb1, broad, and foxo. In moving cells the [Ca(2+)]/calcineurin pathway can regulate adhesion to ECM while adherens junctions link cells together. Accordingly, thin eggshell and DA phenotypes were identified for the calcineurin regulator calreticulin and the adherens junction component arc. Finally a tubulogenesis defect phenotype was identified for the gene pterodactyl, homologous to the mammalian serine/threonine receptor-associated protein (STRAP) that integrates the TGF-beta and PI3K/AKT signaling pathways. Because phenotypes can be scored in each mutant fly before and after gene induction, this F(1) conditional mutagenesis strategy should allow for increased scale in screens for mutations affecting repeated (reiterated) events in adult animals, including gametogenesis, movement, behavior, and learning.

Figure 1.—

Outline of signaling pathways affecting tubulogenesis. Canonical pathway components are indicated in black. Details of Drosophila homologs are indicated in blue. Genes identified in this study are indicated in red. The ETS domain is a DNA-binding domain that specifically interacts with sequences containing the common core trinucleotide GGA and is involved in protein–protein interactions with cofactors that help determine its biological activity. RBPs, receptor binding proteins; ROS, reactive oxygen species.

Figure 2.—

DOX-dependent gene overexpression in ovarian follicle cells. Ovaries and stage-10 egg chambers were photographed under a visible light source and fluorescent light source and merged pictures are shown. The scale is indicated in the lower left corner. (A) Ovaries dissected from female containing the GFP-reporter insertion and rtTA(3)E2 insertion cultured in absence of DOX. (B) Ovaries dissected from female containing the GFP reporter and rtTA(3)E2 cultured in the presence of DOX. (C) Stage-10 egg chamber dissected from female containing the GFP-reporter and rtTA(3)E2 cultured in the absence of DOX. (D) Stage-10 egg chamber dissected from female containing the GFP-reporter and rtTA(3)E2 cultured in the presence of DOX. Results are typical of multiple flies and experiments.

Figure 3.—

Identification of conditional, dominant mutations affecting the eggshell. (A) Scheme to identify conditional eggshell mutants. Crosses 1 and 2 were done en masse in bottles. F₁ females bearing new PdL insertions obtained from the same bottle in cross 2 could contain unique events or the same event and were named to reflect that information. The mini-white+ gene in the rtTA(3)E2 insertion yields only an orange eye color, and so new white+ PdL insertions could be identified in this background by a more red or wild-type eye color. (B–H) Eggshell phenotypes. The indicated PdL mutant strains were crossed to rtTA(3)E2 driver strain, and female progeny were cultured in the presence and absence of DOX. The −DOX control eggs (B and F) are from strain edl^4M127 (as in C), however they are characteristic of the normal morphology of control eggs for the other two lines shown (D and E) as well as most other PdL conditional eggshell mutations. (I) Recessive wing phenotype of pterodactyl^2-4 insertion and excision derivatives. Representative wings are presented from wild-type flies and from flies homozygous for the starting pterodactyl^2-4 insertion and homozygous for the indicated excision (exc) derivatives. The class of pterodactyl wing phenotype (mild, intermediate, severe) is indicated.

Figure 3.—

Identification of conditional, dominant mutations affecting the eggshell. (A) Scheme to identify conditional eggshell mutants. Crosses 1 and 2 were done en masse in bottles. F₁ females bearing new PdL insertions obtained from the same bottle in cross 2 could contain unique events or the same event and were named to reflect that information. The mini-white+ gene in the rtTA(3)E2 insertion yields only an orange eye color, and so new white+ PdL insertions could be identified in this background by a more red or wild-type eye color. (B–H) Eggshell phenotypes. The indicated PdL mutant strains were crossed to rtTA(3)E2 driver strain, and female progeny were cultured in the presence and absence of DOX. The −DOX control eggs (B and F) are from strain edl^4M127 (as in C), however they are characteristic of the normal morphology of control eggs for the other two lines shown (D and E) as well as most other PdL conditional eggshell mutations. (I) Recessive wing phenotype of pterodactyl^2-4 insertion and excision derivatives. Representative wings are presented from wild-type flies and from flies homozygous for the starting pterodactyl^2-4 insertion and homozygous for the indicated excision (exc) derivatives. The class of pterodactyl wing phenotype (mild, intermediate, severe) is indicated.

Figure 3.—

Identification of conditional, dominant mutations affecting the eggshell. (A) Scheme to identify conditional eggshell mutants. Crosses 1 and 2 were done en masse in bottles. F₁ females bearing new PdL insertions obtained from the same bottle in cross 2 could contain unique events or the same event and were named to reflect that information. The mini-white+ gene in the rtTA(3)E2 insertion yields only an orange eye color, and so new white+ PdL insertions could be identified in this background by a more red or wild-type eye color. (B–H) Eggshell phenotypes. The indicated PdL mutant strains were crossed to rtTA(3)E2 driver strain, and female progeny were cultured in the presence and absence of DOX. The −DOX control eggs (B and F) are from strain edl^4M127 (as in C), however they are characteristic of the normal morphology of control eggs for the other two lines shown (D and E) as well as most other PdL conditional eggshell mutations. (I) Recessive wing phenotype of pterodactyl^2-4 insertion and excision derivatives. Representative wings are presented from wild-type flies and from flies homozygous for the starting pterodactyl^2-4 insertion and homozygous for the indicated excision (exc) derivatives. The class of pterodactyl wing phenotype (mild, intermediate, severe) is indicated.

Figure 4.—

Molecular characterization of selected mutations. (A–D) Intron and exon boundaries of the mutated genes are indicated, with numbering according to DNA sequences obtained from the NCBI web site (http://www.ncbi.nlm.nih.gov/). Locations for transcriptional initiation are indicated by arrows. PdL inserts are indicated by triangles and each is oriented 5′ to 3′, as indicated by internal arrows. DNA fragments used as probes in Northern analyses are indicated at the top of each diagram. (A) edl (genomic scaffold sequence AE003798.3). (B) dbr (genomic scaffold sequence AE003590.3). (C) ush (genomic scaffold sequence AE003589.3; SD5608 cDNA sequence is from accession no. AI541608 in the NCBI database. (D) foxo. (E–G) Northern analysis of selected lines. Oregon R wild-type strain and the indicated PdL mutant strains were crossed to rtTA(3)E2 driver strain. Total RNA was isolated from adult progeny cultured 1 week in the presence and absence of DOX, transferred to Northerns blots, and hybridized with the gene-specific probes indicated. Ribosomal protein 49 gene Rp49 was used as control for loading. Two amounts of RNA were loaded for each sample (once and twice, as indicated), and signals were visualized by phosphoimager. (E) edl^4M127 mutant strains and controls. (F) dbr^3M1753 mutant strain and controls. (G) ush^3-38 mutant strains and controls. Galectin probe D downstream of gene dbr and lwr probe F downstream of gene ush did not yield any altered signal in the presence and absence of DOX, indicating that transcription did not go beyond genes dbr and ush. The predicted sizes of the edl^4M127, dbr^3M1753, and ush^3-38 PdL transcripts (A–C) matched the measured sizes of the corresponding +DOX transcripts (E–G).

Figure 4.—

Molecular characterization of selected mutations. (A–D) Intron and exon boundaries of the mutated genes are indicated, with numbering according to DNA sequences obtained from the NCBI web site (http://www.ncbi.nlm.nih.gov/). Locations for transcriptional initiation are indicated by arrows. PdL inserts are indicated by triangles and each is oriented 5′ to 3′, as indicated by internal arrows. DNA fragments used as probes in Northern analyses are indicated at the top of each diagram. (A) edl (genomic scaffold sequence AE003798.3). (B) dbr (genomic scaffold sequence AE003590.3). (C) ush (genomic scaffold sequence AE003589.3; SD5608 cDNA sequence is from accession no. AI541608 in the NCBI database. (D) foxo. (E–G) Northern analysis of selected lines. Oregon R wild-type strain and the indicated PdL mutant strains were crossed to rtTA(3)E2 driver strain. Total RNA was isolated from adult progeny cultured 1 week in the presence and absence of DOX, transferred to Northerns blots, and hybridized with the gene-specific probes indicated. Ribosomal protein 49 gene Rp49 was used as control for loading. Two amounts of RNA were loaded for each sample (once and twice, as indicated), and signals were visualized by phosphoimager. (E) edl^4M127 mutant strains and controls. (F) dbr^3M1753 mutant strain and controls. (G) ush^3-38 mutant strains and controls. Galectin probe D downstream of gene dbr and lwr probe F downstream of gene ush did not yield any altered signal in the presence and absence of DOX, indicating that transcription did not go beyond genes dbr and ush. The predicted sizes of the edl^4M127, dbr^3M1753, and ush^3-38 PdL transcripts (A–C) matched the measured sizes of the corresponding +DOX transcripts (E–G).

Figure 4.—

Molecular characterization of selected mutations. (A–D) Intron and exon boundaries of the mutated genes are indicated, with numbering according to DNA sequences obtained from the NCBI web site (http://www.ncbi.nlm.nih.gov/). Locations for transcriptional initiation are indicated by arrows. PdL inserts are indicated by triangles and each is oriented 5′ to 3′, as indicated by internal arrows. DNA fragments used as probes in Northern analyses are indicated at the top of each diagram. (A) edl (genomic scaffold sequence AE003798.3). (B) dbr (genomic scaffold sequence AE003590.3). (C) ush (genomic scaffold sequence AE003589.3; SD5608 cDNA sequence is from accession no. AI541608 in the NCBI database. (D) foxo. (E–G) Northern analysis of selected lines. Oregon R wild-type strain and the indicated PdL mutant strains were crossed to rtTA(3)E2 driver strain. Total RNA was isolated from adult progeny cultured 1 week in the presence and absence of DOX, transferred to Northerns blots, and hybridized with the gene-specific probes indicated. Ribosomal protein 49 gene Rp49 was used as control for loading. Two amounts of RNA were loaded for each sample (once and twice, as indicated), and signals were visualized by phosphoimager. (E) edl^4M127 mutant strains and controls. (F) dbr^3M1753 mutant strain and controls. (G) ush^3-38 mutant strains and controls. Galectin probe D downstream of gene dbr and lwr probe F downstream of gene ush did not yield any altered signal in the presence and absence of DOX, indicating that transcription did not go beyond genes dbr and ush. The predicted sizes of the edl^4M127, dbr^3M1753, and ush^3-38 PdL transcripts (A–C) matched the measured sizes of the corresponding +DOX transcripts (E–G).