Figure 1.
Embryos with either global or local loss of Egfr signaling have defects in epithelial morphogenesis.
(A–F, I, J) Cuticle preparations. Black arrows, posterior spiracles; black arrowheads, puckers in cuticle; white arrowheads, dorsal hole or scab. (G, H, K, K´) Confocal micrographs of embryos during dorsal closure (DC). (A) Dorsolateral view of wild-type embryo showing intact dorsal surface. (B) Egfrf2/Egfr2C82 embryo, selected by absence of GFP balancer chromosome, showing “curled up” phenotype. (C) Egfrf2/Egfr2C82 embryo showing large dorsal hole. (D) Embryo from temperature-sensitive Egfr1F26 stock that had been allowed to develop at 18°C before shifting to restrictive temperature of 29°C at about stage 10/11. Embryo has a bowed appearance characterized by pulling in of tail (marked by posterior spiracles) and puckering of cuticle. There is a small dorsal hole in the cuticle. (E) Embryo in which EgfrDN had been expressed in the epidermis using the 69B-Gal4 driver showing dorsal scab and mild bowing. (F) Embryo in which EgfrDN had been expressed in the epidermis using the ptc-Gal4 driver showing bowed appearance and dorsal hole, similar to the embryo in panel D. (G, H) Still images from Movie S1 showing restricted expression pattern of LE-Gal4 driver, revealed using a UAS-GFP-NLS reporter. Cell outlines were revealed through expression of a Ubi-DEcadherin-GFP transgene. (G) LE-Gal4 is not expressed at beginning of DC. (H) Midway through DC, GFP-NLS is expressed in the first two rows of cells flanking the amnioserosa (AS), visualized as GFP signal in nuclei. (I) Embryo in which EgfrDN had been expressed using the LE-Gal4 driver showing mild bowing and dorsal scab. (J) Embryo in which EgfrDN had been expressed in the AS using the Gal4c381 driver showing small dorsal hole. (K) Anti-phospho-MAPK staining of a wild-type embryo showing strong immunoreactivity in the center of the amnioserosa and lateral epidermis but little staining in dorsal epidermis and cells at periphery of the AS. (K´) Same embryo as in K with phospho-MAPK in red and cell outlines revealed with anti-phosphotyrosine (PY, green). Scale bars: 50 µm (A–J)(K, K´).
Figure 2.
Impairment of Egfr signaling affects morphogenesis of the AS and dorsal epidermis.
(A, B) Still images from Movie S2 showing unfolding of the AS as germband retraction proceeds in a Ubi-DEcadherin-GFP-expressing embryo. (C, D) Still images from Movie S3 showing delayed germband retraction and disintegration of AS in Egfrf2 mutant embryo expressing Ubi-DEcadherin-GFP. (E, F) Still images from Movie S4 showing delayed germband retraction and disintegration of AS in Egfrf2/EgfrH25 mutant embryo expressing Ubi-DEcadherin-GFP. (G, H) Still images from Movie S5 showing bowing of Egfr1a15/EgfrH25 mutant, Ubi-DEcadherin-GFP-expressing embryo. Note bunching of segments (arrowhead in panel H). (I–L) Still images from Movie S6 showing morphogenesis of the AS in a Ubi-DEcadherin-GFP-expressing embryo. (M–P) Close-ups of dorsal surface in still images from Movie S5 showing defective AS morphogenesis in Egfr mutant embryo. Note that AS has fewer cells than wild-type and constricts perpendicular to the normal anterior-posterior direction. Note that the posterior end of the embryo moves anteriorly in progression from panel N to panel P as the embryo undergoes bowing. (Q-S) Confocal micrographs of dorsal epidermis of embryos stained with anti-phosphotyrosine. (Q) Wild-type embryo showing uniform shape of DME cells and fairly smooth leading edge. (R) Egfr1a15/EgfrH25 embryo showing considerable variation in shape of DME cells and jagged leading edge. Arrow marks a cluster of very constricted DME cells and arrowhead a cluster of cuboidal DME cells. (S) Embryo in which EgfrDN had been expressed using the LE-Gal4 driver showing bunching of segments. Scale bars: 50 µm (A–H)(I–P)(Q–S).
Figure 3.
dpp transcription is repressed by Egfr signaling during DC.
Panels A, C, E and F are digoxigenin in situ hybridizations and panels B, D and H–H´ ´ are FISH, with all embryos at beginning of DC. (A, B) Wild-type embryos showing horizontal dorsal and ventrolateral stripes of dpp expression. The dorsal stripe is dpp expression in the DME cells. (C, D) Egfrf2 embryo (C) and Egfrf2/Egfr2C82 embryo (D) showing ectopic dpp expression ventral to the DME cells (arrowheads). Arrow in (C) shows ventrolateral stripe visible on other side of embryo due to decreased distance between stripes compared to wild-type. (E) Embryo in which EgfrDN had been expressed in the epidermis using the 69B-Gal4 driver showing ectopic dpp expression (arrowhead). Arrow shows ventrolateral stripe visible on other side of embryo. (F) Embryo in which EgfrDN had been expressed using the LE-Gal4 driver showing elevated dpp expression in the dorsal epidermis (arrowhead). (G, H–H´ ´) Increasing EGFR signaling by expression of sSpi (G) or Egfr-EGFP (H–H´ ´) in vertical stripes using the ptc-Gal4 driver causes breaks in the dorsal and ventrolateral dpp stripes. Anti-GFP staining (H´, H´ ´) reveals the expression pattern of Egfr-EGFP. Note that remnants of dpp expression (arrowheads in H–H´ ´) are seen where Egfr-EGFP was not expressed. Scale bar: 50 µm.
Figure 4.
zip transcription is repressed by Egfr signaling during DC.
zip FISH on embryos at beginning of DC. (A) Wild-type embryo showing high levels of zip transcription in DME cells and absence of zip expression in the AS. Prior to completion of germband retraction there are high levels of zip in the AS of wild-type embryos (see Fig. 6A). (B) Egfrf2 embryo showing intense zip signal in DME cells and ectopic zip expression (arrowheads). (C) Mildly affected Egfrf2 embryo showing modest retention of zip in AS. (D, E) Embryos in which Egfr signaling had been impaired in the AS by expression of either EgfrDN (D) or RasN17 (E) showing significant retention of zip in AS, modest elevation of zip expression in the DME cells and ectopic zip transcripts in the head. (F) Elevation of Egfr signaling in the AS through expression of Egfr-EGFP causes down-regulation of zip expression in DME cells. Scale bar: 50 µm.
Figure 5.
Egfr inhibits apoptosis and morphogenesis in the AS.
(A–F´ ´) Apoliner signals in the AS. Apoliner reporter had been expressed either globally with the tub-Gal4 driver or in the AS using the LP1-Gal4 driver. For each embryo RFP, EGFP signals and merge are shown. On the right side of each panel is a higher power view of AS cells. In the absence of caspase activity, RFP and EGFP co-localize at various membranes and there is little EGFP signal in the nucleus. In the presence of caspase activity, EGFP is cleaved away from RFP and moves into the nucleus. (A–A´ ´) AS of wild-type embryo prior to germband retraction showing co-localization of RFP and EGFP signals and weak EGFP signals in the nucleus. (B–B´ ´) AS of Egfr mutant embryo prior to germband retraction showing strong EGFP signals in the nucleus. (C–C´ ´) AS of wild-type embryo during DC showing strong EGFP signals in the nucleus. (D–D´ ´) AS of p35-expressing embryo during DC showing weak EGFP signals in the nucleus. (E–E´ ´) AS of sSpi-expressing-expressing embryo during DC showing weak EGFP signals in the nucleus. (F–F´ ´) AS of RasV12-expressing embryo during DC showing weak EGFP signals in the nucleus. (G) Still from Movie S7 showing AS of stage 15 wild-type embryo in which GFP had been expressed with the Gal4NP3312 AS driver, showing narrow, tube-like AS. (H, I) Stills from Movies S8 (H) and S9 (I) showing AS of stage 15 embryos in which Egfr-EGFP and GFP-NLS had been expressed with the double driver combination Gal4NP3312+ Gal4NP5328 showing failure of AS morphogenesis. The AS in panel H has failed to narrow throughout while that in panel I has failed to narrow at the anterior end. Scale bars: 50 µm (A–B´ ´); 10 µm (C–I).
Figure 6.
Evidence that Egfr signaling is negatively regulated by endocytosis in the AS.
(A–D) zip FISH on embryos late in germband retraction. (A) Wild-type embryo showing zip expression in AS. (B) Expression of Ack in the AS using the Gal4c381 driver causes an increase in zip levels in this tissue relative to wild-type. (C) Ack fails to elevate zip levels when co-expressed with Egfr–EGFP. (D) zip levels are elevated when Ack is co-expressed with control lacZ gene. (E–É ´´) AS in which Ack had been over-expressed in prd stripes, triple-stained with anti-phosphotyrosine (anti-PY) (E), anti-Egfr (É) and anti-Rab5 (É ´). (E) Cells over-expressing Ack are marked by high levels of anti-PY (outlined with dotted lines). (É) Egfr shows strong cortical localization in wild-type AS cells but a more cytoplasmic distribution in Ack-over-expressing cells. (É ´) There is an increase in Rab5-positive early endosomes in Ack-over-expressing cells. (É ´´) Merge of panels É and É ´. Arrowheads and arrows mark Egfr-positive early endosomes in wild-type cells and Ack-over-expressing cells, respectively. (F) Egfr-EGFP expressed in the AS using the Gal4NP3312 driver shows vesicular accumulation in addition to being at the plasma membrane. (G) AS cells in embryo in which Apoliner has been expressed with LP1-Gal4 driver showing localization of Apoliner-RFP signal to membranes. (H) AS cells in embryo in which Apoliner and kinase-dead Ack have been co-expressed with LP1-Gal4 driver showing punctate localization of Apoliner-RFP signal. Scale bars: 50 µm in A-D; 5 µm in E-H.
Figure 7.
Model for Egfr acting as a brake on DC.
Egfr negatively regulates the production and/or secretion of a diffusible signal “X” in the AS (AS) and is itself negatively regulated by Ack through endocytosis. “X” signals into both the AS and the DME cells where it activates a pathway promoting transcription of myosin from the zip locus. Previous work from our group and others, and unpublished results from our group, suggest that Dpp from the DME cells diffuses to the AS where it regulates production of a second diffusible signal “Y” providing a parallel input into zip transcription. Myosin produced through the cooperation of the two pathways then drives morphogenesis of the AS and DME cells. Egfr additionally regulates this signaling network by negatively regulating dpp transcription in the epidermis, including the DME cells. Egfr further regulates AS morphogenesis by inhibiting apoptosis in this tissue.