A dual role of the extracellular domain of Drosophila Crumbs for morphogenesis of the embryonic neuroectoderm

Abstract

Epithelia are highly polarised tissues and several highly conserved polarity protein complexes serve to establish and maintain polarity. The transmembrane protein Crumbs (Crb), the central component of the Crb protein complex, is required, among others, for the maintenance of polarity in most epithelia in the Drosophila embryo. However, different epithelia exhibit different phenotypic severity upon loss of crb Using a transgenomic approach allowed us to more accurately define the role of crb in different epithelia. In particular, we provide evidence that the loss of epithelial tissue integrity in the ventral epidermis of crb mutant embryos is due to impaired actomyosin activity and an excess number of neuroblasts. We demonstrate that the intracellular domain of Crb could only partially rescue this phenotype, while it is able to completely restore tissue integrity in other epithelia. Based on these results we suggest a dual role of the extracellular domain of Crb in the ventral neuroectoderm. First, it is required for apical enrichment of the Crb protein, which in turn regulates actomyosin activity and thereby ensures tissue integrity; and second, the extracellular domain of Crb stabilises the Notch receptor and thereby ensures proper Notch signalling and specification of the correct number of neuroblasts.

Keywords: Actomyosin; Adhesion; Neurogenesis; Notch; Polarity.

Fig. 1.

The ICD of Crb restores epithelial integrity of crb mutant embryos. (A) Schematic representation of the fosmids. Depicted proteins are based on the Crb-PA isoform (2.146 amino acids). Adopted from Klose et al. (2013). (B) Analysis of embryonic lethality. The graph represents the percent of embryos that hatch. N>1000 embryos. The experiment was repeated 5 times. Error bars show standard error of the mean. (C-I) Classification of cuticle phenotypes of unhatched embryos. White arrowhead (D,E,F,G), intact head structure; yellow arrow (D,F), intact denticle belts; magenta arrowheads (D), fused or lost denticle belts; cyan arrowhead (E,F), dorsal/ventral hole, respectively; cyan arrowheads (G), ventral and dorsal hole. Scale bar: 50 μm. (J) Quantification of cuticle phenotypes. N>300 embryos. The experiment was repeated 3 times.

Fig. 2.

The Crb ICD is sufficient for tissue integrity of most embryonic epithelia. (A-F) Stage 12-13 embryos, stained for SAS. Dotted lines in E and F mark the disintegrated ventral epidermis. Dorsal is up, anterior left. Scale bar: 50 μm. The experiment was repeated 3 times. (G-N) Stage 12-13 foscrb; crb^GX24 control (G-J) and foscrb_ICD crb^11A22 (K-N) embryos, stained with anti-SAS. Polarity of epithelial tubes is restored in the hindgut (G,K), the Malpighian tubules (H,L), the salivary gland (I,M) and the trachea (J,N). Scale bar: 10 μm. (O-R″) Stills of time lapse movies of endogenously tagged DE-Cadherin-GFP lines. Dorsal (O-P″) and lateral (Q-R″) views of foscrb; crb^GX24 control and foscrb_ICD crb^11A22 embryos. Red dotted lines in P,P′ mark the disintegrated ventral epidermis. Yellow arrow in P, disintegrated head epidermis; cyan arrowhead in P″, recovered head epidermis; red arrowheads in R′,R″, ‘wounds’ in ventral epidermis. Anterior is to the left. Scale bar: 50 μm. The experiment was repeated 3 times.

Fig. 3.

Apico-basal polarity is restored in foscrb_ICD crb embryos. Stage 13 foscrb; crb^GX24 control (A-C′,J-L′), crb^11A22 (D-F′,M-O′) and foscrb_ICD crb^11A22 (G-I′,P-R′) embryos stained with anti-SAS (apical) and anti-FasIII (lateral). (A-I,J-R) Lateral view of the dorsal and ventral epidermis respectively. (A′-I′,J′-R′) XZ projections of images in A-I,J-R respectively. Magenta arrows in A point to filopodia. White arrows in R,R′, cyst-like structures with the apical membrane facing the lumen. Scale bar: 10 μm (A-R), 5 μm (A′-R′). The experiment was repeated twice.

Fig. 4.

The Crb ICD partially rescues apico-basal polarity of crb mutant embryos. (A-F″) Stage 12-13 foscrb; crb^GX24 control (A-A″,D-D″), crb^11A22 (B-B″,E-E″) and foscrb_ICD crb^11A22 (C-C″,F-F″) embryos, co-stained with anti-DPatj (magenta) and anti-Dlg (green) (A-C″) and anti-Par6 (magenta) and anti-Baz (green) (D-F″). Magenta arrows in C and C″, intracellular punctate accumulation of DPatj. Scale bar: 5 μm. The experiment was repeated 4 times. (G-J′) Stage 12-13 foscrb; crb^GX24 control (G-H′) and foscrb_ICD crb^11A22 (I-J′) embryos, co-stained with anti-Crb_ICD and anti-Crb_ECD. H-H′ and J-J′, magnifications of the epidermis shown in G-G′ and I-I′, respectively. Anterior is left, dorsal up. Cyan arrow in I′, hindgut. Scale bar: 50 μm (G,G′,I,I′) and 20 μm (H,H′,J and J′). The experiment was repeated 4 times. (K) Quantification of embryonic lethality. The graph shows the percentage of embryos that hatched. Control: foscrb; crb^GX24. Note that embryos represented by the third column have only one copy of foscrb_ICD. The experiment was repeated 3 times. Error bars show standard error of the mean. (L) Quantification of the cuticle phenotypes of crb mutant embryos with one or two copies of foscrb_ICD. The experiment was repeated 3 times.

Fig. 5.

crb and foscrb_ICD crb mutant embryos develop a neurogenic phenotype. (A-F) Stage 9 foscrb; crb^GX24 control (A,D), crb^11A22 (B,E) and foscrb_ICD crb^11A22 (C,F) embryos stained with anti-Hb (A-C) and anti-Dpn (D-F). Yellow dotted line marks the ventral midline. Scale bar: 25 μm. The experiment was repeated 4 times. (G-L′) Dorsal (G-I′) and ventral (J-L′) epidermis of stage 9 foscrb; crb^GX24 control (G,G′,J,J′), crb^11A22 (H,H′,K,K′) and foscrb_ICD crb^11A22 (I,I′,L,L′) embryos, stained with anti-Baz and anti-Notch-ICD. Scale bar: 20 μm. The experiment was repeated 3 times. (M-O′) Stage 9 foscrb; crb^GX24 control (M,M′), crb^11A22 (N,N′) and foscrb_ICD crb^11A22 (O,O′) embryos stained with anti-Achaete, anterior to the left. (M′,N′,O′) Close up of images shown in M-O, respectively. Magenta arrowheads in N,N′,O,O′ point to supernumerary neuroblasts. Scale bar: 50 μm (M,N,O) and 20 μm (M′,N′,O′). The experiment was repeated 3 times. (P-Q) Stage 9/10 embryos stained with anti-Bazooka. (P′,Q′) Close up of posterior ventral epidermis of embryos shown in P,Q. Anterior is left. Scale bar: 50 μm (P,Q) and 20 μm (P′-Q′). The experiment was repeated twice.

Fig. 6.

Defects in the ventral epidermis of foscrb_ICD crb embryos are rescued by overexpression of DE-cadherin or flapwing. Ventral views of stage 9/10 foscrb_ICD crb^11A22 embryos overexpressing HA-tagged flapwing (flw; B,B′) or DE-cadherin (C,C′), stained with an anti-phosphotyrosine antibody. Yellow lines mark the ventral midline. (A′,B′,C′) Close up of the ventral epidermis of embryos shown in A,B,C, respectively. Magenta arrows in A′ point to cells without proper junctional staining. Anterior to the left. Scale bar: 50 μm (A,B,C) and 20 μm (A′,B′,C′). The experiment was repeated twice.