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Interaction between EGFR signaling and DE-cadherin during nervous system morphogenesis

Karin Dumstrei^1,*, Fay Wang^1,*, Diana Shy¹, Ulrich Tepass² and Volker Hartenstein^1,

¹ Department of Molecular Cell and Developmental Biology, University of California Los Angeles, Los Angeles, CA 90095, USA
² Department of Zoology, University of Toronto, Toronto, Ontario M5S3G5, Canada
^* These authors contributed equally to this work

View larger version (80K): [in a new window]   Fig. 1. Morphogenesis of the Drosophila embryonic visual system. (A-D) SEM photos of embryonic head at stage 7 (gastrulation; A), stage 9 (early extended germ band; B); stage 11 (late extended germ band; C) and stage 12 (germ band retraction; D). In all SEM anterior is to the left. Orientation in A is dorsal, whereas B,C and D are lateral views with dorsal up. The migrating eye field (eyf in A,B) and optic placode (opl in C,D) are distinguishable by surface morphology. (E) Fate map of the eye field. Schematic representation of stage 7 embryonic head, lateral view. Approximate position of anlagen are indicated (Bo, larval eye; ed, adult eye disc; ola, anterior lip of optic lobe, which gives rise to inner optic anlage; olp posterior lip of optic lobe, which gives rise to outer optic anlage). (F) Morphogenesis of the optic placode. Schematic representation of the invaginating optic placode at three successive timepoints. (G) Visual system of late embryo. Other abbreviations: as, amnioserosa; Bn, Bolwig’s nerve; cf, cephalic furrow; fg, foregut; prn, protocerebral neurectoderm.

View larger version (77K): [in a new window]   Fig. 2. Pattern of expression of DE-cadherin and EGFR in the embryonic head. (A) Anti-DE-cadherin labeling of stage 11 embryo, dorsal view. Confocal section of head epidermis close to apical surface, showing concentration of DE-cadherin in zonula adherens of epithelial cells. (B) Anti-EGFR (red) and anti-Armadillo (green) labeling of stage 11 embryo, dorsal view. Note diffuse expression of EGFR in membrane of epithelial cells and neuroblasts, and apical concentration of Armadillo (arrows). Merged EGFR and Armadillo staining is shown in yellow. (C) In situ probe showing expression of rho, an activator of the EGFR signal, in two stripes (as, anterior stripe; ps, posterior stripe) in stage 7 embryo, dorsal view. (D-F) Pattern of EGFR activity, visualized by antibody against phosphorylated MAPK (dpERK; red). At stage 7 (D; dorsal view), EGFR is active in two stripes, corresponding to the anterior (as) and posterior stripe (ps) of rho expression (see C). The preparation was double labeled by GFP expressed in the sine oculis pattern by an so-Gal4 driver (green). Sine oculis is the earliest marker for the eye field (ef). Note partial overlap between eye field and posterior stripe of ERK activity (yellow). During later stages (E, stage 9; F, stage 11) posterior stripe of ERK activity stretches laterally and stays in spatial overlap with the optic lobe placode (opl). (G) Anti-Crumbs staining of stage-15 wild-type embryo, lateral view. Anti-Crumbs labels the apical membrane of the optic lobe (indicated by dashed outline). (H) Anti-Coracle labeling of wild-type embryo, same stage and view as in G. Anti-Coracle is a marker for the septate junctions. In the optic lobe (outlined by dashed lines) septate junctions are absent. Insert in H shows anti-Coracle-labeled septate junctions in the dorsal tracheae.

View larger version (121K): [in a new window]   Fig. 3. Defects in the optic placode following DE-cadherin loss of function and overexpression. (A,B) Anti-Crumbs, labeling apical membrane of ectodermal cells. Stage 15, lateral view of wild-type embryo (A) and shgIH mutant (B). Note invaginated optic lobe (ol) attached to basal brain surface (br) in wild-type. In the mutant, the optic placode does not invaginate and remains at the surface. Placode cells lose contact and dissociate, as evidenced by reduced and patchy Crumbs expression. (C,D) Anti-FasII, labeling Bolwig’s organ (Bo) and posterior lip of optic lobe (ol). Stage 15, lateral view of wild-type (C) and shgIH mutant (D). In wild type, Bolwig’s organ has separated from optic lobe and differentiated as photoreceptor neurons with axons staying attached to optic lobe. In shg mutant, cells of optic lobe and Bolwig’s organ still express the marker FasII but do not differentiate structurally. (E,F) Anti-FasII, stage 15, lateral view of embryo in which (E) heatshock mouse E-cadherin (hs-ME-cadherin4b) and (F) Drosophila full length DE-cadherin (UAS-DE-cadherin5,9) construct is expressed by da-Gal4. Cells of optic placode maintain their epithelial shape but do not invaginate. Bolwig’s organ does not separate from optic lobe.

View larger version (128K): [in a new window]   Fig. 4. Role of ß-catenin in optic placode morphogenesis. (A) Invaginating optic placode of stage-13 wild-type embryo, labeled with anti-Crumbs. (B,C) Rudimentary optic placode of arm1; arms14 embryo (B) stage 13 labeled with anti-Crumbs and (C) stage 16 using anti-Fasll to label optic lobe (ol) and Bolwig’s nerve (Bn). The arms14 is a deletion construct of ß-catenin, which lack the -catenin binding domain. Arms14 is able to carry out Wg signaling function but not DE-cadherin-related function. Optic placode cells have dissociated into small, disjunct vesicles that fail to invaginate. (D) Anti-Fasll, stage 16, showing lack of separation of optic lobe and Bolwig’s organ in embryos expressing the fusion protein UAS-DE-cad--catenin under the control of da-Gal4. Other abbreviations: bo, Bolwig’s organ; br, brain.

View larger version (114K): [in a new window]   Fig. 5. Cell death in the neurectoderm of EGFR loss-of-function embryos. (A) SEM of head of stage 11 Egfrf5 mutant, dorsal view, anterior to the left. Note domains of cell death in anterior head (pa) and posterior head (pc). The posterior domain covers the optic placode. (B) In stage-14 Egfrf5 mutant the brain (br) is exposed and only a small rudiment of optic placode is left (olp; labeled with anti-FasII; compare to Fig. 3C). (C,D) SEM of stage-11 embryos, ventral view, showing cell death in ventral neurectoderm of Egfrf5 mutant embryos. Cell death, indicated by light, round cell fragments (arrows in D), occurs scattered throughout ventral neurectoderm of stage 10/11 embryo. (E) Wild-type neurectoderm for comparison. (F,G) Comparison of EGFR activation with ‘pre-apoptotic domains’ in dorsal head of stage 10 embryo. (F) Domains of EGFR activation are visualized by anti-dpERK antibody (black staining), embryo is also labeled with Anti-phosphohistone H3 (brown staining). (G) Pre-apoptotic domains are labeled by probe against reaper. Note presence of anterior and posterior band of labeling (pa, pc). Other abbreviations: cf, cephalic furrow; ml, midline; tp, tracheal pit.

View larger version (126K): [in a new window]   Fig. 6. Hyperadhesive phenotype in optic placode following reduction of EGFR function. (A) Anti-FasII labeling of stage-15 wild-type embryo, lateral view. Optic placode has invaginated and split into Bolwig’s organ (Bo) and optic lobe (ol), the latter attached to the brain (br). (B) In embryo lacking EGFR (Egfrf5) and carrying deficiency H99, which inhibits cell death, optic placode fails to invaginate and Bolwig’s organ remains attached to optic lobe (‘non-disjunction’). (C) Labeling of Bolwig’s organ with mab22C10 demonstrates that photoreceptor neurons remain arranged in a layered array, rather than transforming into spindle shaped cells as in wild type (compare with A). (D-F) Defects in visual system following application of 2-hour heat pulses (3°C) to EGFR temperature sensitive allele (Egfrf1). All panels show heads of stage-15 embryos, labeled with anti-FasII antibody. Heat pulses between 3 and 6 hours (D) cause defects in head development, but leave the visual system intact. (E) Heat pulses around the time of optic placode invagination and disjunction (6-8 hours) result in non-disjunction phenotype of Bolwig’s organ and optic lobe (arrowhead), although usually milder than phenotype observed in Egfr null. Frequently phenotype is asymmetric: in the embryo shown in dorsal view, non-disjunction is prominent on right side, but absent from left side (arrow). (F) Heat pulses from 8-10 hours do not affect optic placode morphogenesis, but causes reduction in Bolwig’s neurons. Note small size of Bolwig’s organ, compared with wild type shown in G.

View larger version (102K): [in a new window]   Fig. 7. Genetic interaction between components of EGFR signaling pathway and DE-cadherin. (A-D) Cuticle preparations from wild-type and experimental embryos, all ventral views. (A) Wild-type embryo. (B) ShgP34-1 homozygous embryos, head cuticle is missing but ventral trunk cuticle has relatively minor defects such as small holes in the cuticle (arrows). 12% of these cuticles have a more severe phenotype in which the entire ventral cuticle is missing, n=117. C: Da-GAL4; UAS-Activated EGFR; shgP34–1/shgP34–1, 27% of these embryos show lack of ventral cuticle, P=0.001, n=117, (arrows). (D) shgP34–1/shgP34–1;rhoM3/rhoM3. Among these double mutants, no embryos were observed that lacked the entire ventral cuticle. 8% of shgP34–1/shgP34–1 sibling embryos from this cross, lacked ventral cuticle, P=0.01, n=83. (E) Histogram of data.

View larger version (33K): [in a new window]   Fig. 8. EGFR is co-immunoprecipitated with DE-cadherin and Armadillo in the Drosophila embryo. (A-C) Co-immunoprecipitation of CCC-associated proteins using anti-DE-cadherin (DE-cad) antibody (A), anti-Armadillo (Arm) antibody (B), and anti-EGFR (C). (Left column) Blots were probed with anti-Armadillo antibody. (Center) Blots were probed with anti-EGFR antibody. (Right) Blots were probed with anti-DE-cadherin antibody. Owing to the different sources of the antibodies, it was not possible to normalize the amount of antibody used in conducting the CoIP experiments. Thus, the quantity of proteins pulled down using one antibody differs from that obtained with another antibody. (D) Western blot probed with anti-neurotactin (BP106), documenting the absence of neurotactin in anti-DE-cad and anti-Arm IP products. Western blot contains anti-Arm and Anti-DE-cad IP products (left two lanes) and embryo lysate (right two lanes; A: lysate used for anti-Arm IP; B: lysate used for anti-DE-cad IP). In the lanes loaded with embryo lysate, a band labeled by anti-neurotactin antibody proves that neurotactin is present in the lysate.