Supplementary MaterialsS1 Fig: terminal cells exhibit multiple tube defects. S2 Fig: EnR-IchDBD-FLAG, but not full-length Ich, induced apical membrane discontinuities. (A-B) terminal cell clones stained for GFP, the apical membrane using Wkd antiserum (A), as well as the Flag epitope (B,B). terminal cell clones show cystic, discontinuous lumens (A,A). EnR-IchDBD-FLAG localizes towards the nucleus of terminal cells (B, B). (C-D) Control (C,C) and (D,D) terminal cell clones overexpressing Ich. BAY-598 As opposed to wild-type settings (C,C), terminal cells overexpressing full-length Ich (D,D) show serious pruning with rudimentary lumens (D). (E-E) terminal cell clones stained to label cortical f-actin (E), cortical acetylated tubulin (E), and aPKC (E). Unlike EnR-IchDBD, full-length Ich overexpression in terminal cells will not perturb lumen patency but will disrupt localization of particular apical membrane markers, such as for example aPKC. (Scale Bar: A-B, E-E 5m; C-D, 50 m).(TIF) pgen.1007146.s002.tif (5.7M) GUID:?4B0AF599-54F9-4B78-8839-2ADEA2FAD7FC S3 Fig: An transcriptional reporter is expressed in cuticle-secreting epithelia. Embryos heterozygous for the P element enhancer trap insertion were immunostained for nuclear LacZ (nLacZ, green) and the tracheal specific transcripton factor Trachealess (Trh, red). (A,A) Rabbit polyclonal to Receptor Estrogen alpha.ER-alpha is a nuclear hormone receptor and transcription factor.Regulates gene expression and affects cellular proliferation and differentiation in target tissues.Two splice-variant isoforms have been described. LacZ signal is first detected in Stage 10 embryos in broad BAY-598 epidermal stripes (A). During germband retraction (B, B), epidermal expression is strongest in the T2, T3, and A8 epidermal parasegments (arrowheads in A-B). LacZ reporter expression is not detected during primary branching (B-C). Pan-tracheal LacZ expression is first detected at St. 14 (D, D) and continues during later stages (St. 15: E, E), coinciding with lumen growth and cuticle deposition. In addition to tracheal expression, LacZ is also expressed in the epidermis (arrowhead in E), foregut (F, G), and hindgut (arrowhead in H). All are ectodermally-derived epithelia that secrete chitin-based cuticles. (Scale Bars: 20 m).(TIF) pgen.1007146.s003.tif (5.7M) GUID:?2186E9E0-E662-4BD4-A057-36900BC1F381 S4 Fig: is dispensable for the formation and modification of the tracheal chitin cable. (A-B) Wild type (WT) (A,A) and (B,B) embryos immunostained for Trachealess (white) and chitin-binding probe (CBP, red). embryos deposit a wild-type chitin filament and exhibit neither cystic nor convoluted lumens. (C, C) embryos stained for Gasp, showing is dispensable for lumenal accumulation of Gasp. (F-F) hemizygotes stained for Trh and DE-cadherin (red) and the Chitin Deacetylase Verm (magenta). is dispensable for luminal accumulation of Verm. (G,J) Maternal-zygotic mutant embryos (H) exhibit wild-type lumen morphogenesis in the embryonic trachea. Restoring zygotic expression in maternally-deficient embryos (G) has no effect on tracheal lumen morphogenesis. (Scale Bars: 10 m).(TIF) pgen.1007146.s004.tif (3.4M) GUID:?CB2ED165-7146-43DA-9D24-59E16F90B439 S5 Fig: Characterization of alleles. (A-C) GFP-labeled MARCM clones in wholemount heat-killed third instar larvae. Unlike wild-type control terminal cells (asterisk in A), terminal cell clones exhibit a cell-autonomous gas-filling defect (arrow in A). Isolated clones in the dorsal trunk (B, B) causes cell-autonomous divots (arrows in B) in the gas-filled lumen. Cell-autonomous loss of in autocellular branches (C,C) causes a cell-autonomous gas-filling defect (arrow in C). (D-G) mutant embryos and heterozygous control siblings stained for GFP (green) and chitin-binding probe (red). mutants fail to form the transient chitin filament and exhibit cystic lumens in the dorsal trunk. (H-L) Analysis of lumen morphology in wild-type (H), hemizygous mutants (I and K), and homoallelic mutants (J and L) using mAb2A12. The cystic dorsal trunks of homoallelic mutants (J). However, homozygotes (L) can exhibit a severe reduction of luminal 2A12 staining not observed in hemizygotes (K). (Scale Bars: A-C 50 m; D-L, 10 m).(TIF) pgen.1007146.s005.tif (4.5M) GUID:?D9F66CD6-5F7F-4115-AFE7-7840F89E41EC S6 Fig: regulates ectodermal expression in the foregut and epidermis. (A, B, G) Wild-type (WT, is expressed in the foregut primordium (brackets in A) and posterior spiracles (black arrowhead within a). By Stage 16, is certainly expressed in every cuticle-secreting epithelia, like the foregut BAY-598 (bracket BAY-598 in B), epidermis (arrow in B), trachea (dark arrowhead in B), and hindgut (white arrowhead in B). This sign is certainly particular to transcript as the corresponding feeling probe provides no such design (G). (C, D) Control.