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, 240 (7), 1745-55

Abelson Tyrosine Kinase Is Required for Drosophila Photoreceptor Morphogenesis and Retinal Epithelial Patterning

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Abelson Tyrosine Kinase Is Required for Drosophila Photoreceptor Morphogenesis and Retinal Epithelial Patterning

Wenjun Xiong et al. Dev Dyn.

Abstract

Coordinated differentiation and morphogenesis transform the Drosophila retina from a layer of epithelial cells into a complex three-dimensional organ. In this study we show that the Abelson (Abl) tyrosine kinase localizes to the dynamically remodeling apical-junctional membrane domains of the developing photoreceptor cells. Analyses of abl mutant clone phenotypes demonstrate that abl is required for enriched localization of adherens junction and apical polarity complex proteins at photoreceptor-photoreceptor cell junctions and apical membrane domains, respectively, for rhabdomere generation and for spatial organization of ommatidial cells along the apical-basal axis of the epithelium. Loss of abl does not alter expression or localization of Enabled (Ena) nor does heterozygosity for ena dominantly suppress the abl phenotypes, suggesting the downstream effector mechanisms used by Abl in the eye may differ from those used in the embryo. Together our results reveal a prominent role for Abl in coordinating multiple aspects of photoreceptor morphogenesis.

Figures

Figure 1
Figure 1. Abl protein localization in the developing eye disc
A) A schematic summarizing the major morphogenetic events during Drosophila ommatidial development. In the third instar larval eye disc the newly specified photoreceptor cells display a similar apical-basal polarity to that of other cells in the epithelium. By the early pupal stage, the apical membrane of each photoreceptor has turned 90° towards the center of its cluster and detached from the apical surface of the epithelium, with the four newly specified cone cells covering the apical space left by the involution of the photoreceptor apical membranes. During mid-pupal stages, rhabdomeres are assembled at the apex of the elongating apical membranes of the photoreceptors. Both the photoreceptor apical membranes and the rod-like rhabdomeres continue to extend basally as all cells in the epithelium elongate. By 90hr APF, shortly before eclosion to the adult, the final ommatidial organization has been achieved: eight photoreceptor cells with rod-like rhabdomeres occupy the center of each ommatidium, with four cone cells surrounding and capping them and eleven pigment cells forming two outer whorls. Figures are not drawn to scale and were adapted from (Bate and Arias, 1993, Chapter 22, Figure 2) and from (Tepass and Harris, 2007). B) A maximum confocal projection of all transverse sections crossing a wild type third instar larval eye disc stained with anti-Abl (red, B′), anti-Arm (green, B″), and anti-aPKC (blue, B‴). Abl expression initiates a few rows posterior to the furrow, coincident with the onset of aPKC enrichment at the constricting apical membrane of the developing photoreceptors (white arrowheads, B′&B‴). All third instar larval eye discs shown in this paper are oriented with anterior left, dorsal up, and position of the morphogenetic furrow marked with yellow arrows. Scale bars: 20 μm. C) An orthogonal section of the disc shown in (B) shows the extent of overlap of Abl with aPKC (C′) and Arm (C″) along the apical-basal axis. All orthogonal sections shown in this paper are oriented with apical up and basal down. Scale bars: 2 μm. D-E) Zoomed-in views of single transverse sections crossing the most apical (D) and subapical (E) regions of the disc shown in (B). Scale bars: 2 μm. D) Abl colocalizes with aPKC at the apical domain of the photoreceptors at the center of each developing cluster, but is absent from the apical membranes of the undifferentiated epithelial cells where aPKC is expressed. E) Abl overlaps with Arm at the AJs of the photoreceptor clusters. F) An orthogonal section of a wild-type third instar larval eye disc stained with anti-Abl (red) antibody and OG-Phalloidin (green) shows colocalization of Abl with actin filaments at the apical region. Scale bars: 2 μm. G-L) 48hr APF wild-type eye discs stained with anti-Abl (red) and OG-Phalloidin (green, G-H), anti-aPKC (green, I-J) or anti-Arm (green, K-L) antibodies. G, I, K) are the face views and H, J, L) are the orthogonal sections. Scale bars: 10 μm. G-H) A single middle section showing Abl colocalization with F-actin in the center of each ommatidium. I-J) A single middle section showing Abl partially overlaps with aPKC at the apical surface of the photoreceptor cells, but that most Abl protein localizes to the center of each ommatidium where no aPKC is observed. K-L) A single middle section showing Abl does not overlap with Arm at this stage.
Figure 2
Figure 2. Loss of abl delays and disrupts the organization of adherens junctions between the photoreceptors
Eye discs with abl clones were stained with anti-Arm (red) and anti-GFP (green) antibodies. Lack of GFP marks abl mutant clones in this and all other figures in this paper. Scale bars: 10 μm. A-C) are confocal projections of all transverse sections, and D-K) are zoomed-in single transverse (D, F-G, I-J) or orthogonal (E, H, K) sections of the two adjacent ommatidia (outlined by white boxes in A-C) with the wild-type ommatidium outlined by a solid yellow circle/box and the abl mutant ommatidium outlined by a dashed yellow circle/box. A, D-E) Arm expression appears reduced, but fairly normally patterned and localized in abl mutant ommatidia in a third instar disc. B, F-H) At 24hr APF, the adherens junctions between the cone cells in abl mutant ommatidia appear normal (F). Arm enrichment between the abl mutant photoreceptor cells is clearly evident at this stage, although still mildly weaker and less tightly organized than wild type (G). C, I-K) At 48hr APF, apical Arm enrichment is undetectable in abl mutant ommatidia (I) and has collapsed to the basal plane of the epithelium (J-K). Supplementary Fig. 2 shows analogous phenotypes in the hypomorphic abl1 allele.
Figure 3
Figure 3. Defects in organization of the apical membrane domains in abl mutant ommatidial clusters
Eye discs carrying abl mutant clones stained with anti-GFP (green) and either anti-aPKC (red, A-C, J-M), anti-Crumbs (red, D-F, N-Q), or anti-PATJ (red, G-I, R-U). Scale bars: 10 μm. A, D, G) are maximum projection confocal images of third instar discs, with zoomed-in views (B, E, H, outlined by white boxes in A, D and G) and orthogonal sections of the zoomed-in regions (C, F and I). The enrichment of all three apical membrane markers in the center of abl mutant ommatidium (open arrows in B-C, E-F, H-I) is reduced compared to that of wild-type ommatidia (solid arrows in B-C, E-F, H-I). J, N, R) are maximum projection confocal images of 48hr APF discs with zoomed-in single transverse (K-L, O-P, S-T, outlined by white boxes in J, N and R) or orthogonal (M, Q, U) sections of adjacent wild-type (solid circles/boxes) and abl mutant (dashed circles/boxes) ommatidia. Enrichment of all three apical markers in the center of abl mutant ommatidial clusters is evident although the structures are disorganized and have collapsed basally.
Figure 4
Figure 4. Loss of abl disrupts photoreceptor apicobasal positioning
A-E) Orthogonal sections through third instar (A), 24hr APF (B) and 48hr APF (C-E) eye discs. Scale bars: 5 μm. A-C) Eye discs with abl clones were stained with anti-Elav (red) and anti-GFP (green) antibodies. A) abl mutant photoreceptor cell nuclei (yellow arrow) reside slightly basal relative to wild type. B-C) At 24hr APF and 48hr APF, this phenotype becomes more apparent. D-E) Eye discs with wild-type (D) and abl mutant (E) clones were stained with anti-Elav (red) and anti-So (green) antibodies. Wild-type and abl mutant tissues were distinguished by the presence or absence of GFP (not shown). D) A wild-type clone showing the normal apical to basal arrangement of cone cell, photoreceptor cell and pigment cell nuclei. E) In abl mutant clones, the Elav positive photoreceptor nuclei are found basal to the pigment cell nuclei. F) A schematic diagram summarizing the disrupted apical-basal architecture of abl mutant ommatidial clusters. G) A single apical section through a 48hr APF eye disc stained with anti-Elav (red, G′), anti-Arm (white, G″) and anti-GFP (green) antibodies. The nuclei of the abl mutant cells (yellow arrowheads in G′) in the mosaic ommatidia reside normally at the apical plane of the epithelium, and the AJs between the abl mutant cells (white arrows) and those between the abl mutant cell and their wild-type neighbor (white arrowheads) appear normal. A mosaic ommatidium with three adjacent abl mutant photoreceptors with normally positioned nuclei is outlined by the yellow circle. Scale bars: 5 μm. Supplementary Fig. 3 shows apical-basal photoreceptor nuclear position is similarly disrupted in the hypomorphic abl1 allele.
Figure 5
Figure 5. Rhabdomeres fail to generate in abl clones
A-E) All eye imaginal discs were stained with Rhodamine-phalloidin (red) and anti-GFP (green). All panels are single transverse sections. Scale bars: 10 μm. A) In the third instar disc, F-actin appears normally distributed in abl clones. B) At 24hr APF, F-actin appears normal in abl clones, despite the disrupted organization of the mutant ommatidia. C) At 48hr APF, the central enrichment of F-actin at the microvillar extensions in wild-type tissue is absent in abl mutant tissue. D) At 72hr APF, the expanding rhabdomeres in each wild-type ommatidium are absent in abl mutant clones. E) At 96hr APF, no rhabdomeres are detected in abl clones. F) A histological section of an adult eye shows the lack of rhabdomeres and ommatidial organization in abl clones, which are marked by the absence of the product of the white gene. The few rhabdomeres seen in the center of the mutant clone actually belong to wild-type cells, as pigment granules can be detected in their cell bodies. Scale bar: 10 μm. Supplementary Fig. 5 shows analogous defects in F-actin and loss of rhabdomeres in the hypomorphic abl1 allele.
Figure 6
Figure 6. abl function during photoreceptor polarity establishment may be independent of enabled
A-E) Single transverse sections of eye discs with abl clones stained with anti-Enabled (red) and anti-GFP (green) antibodies. Scale bars: 10 μm. A) Enabled staining looks normal in the third instar abl clones. B-C) At 24hr APF, the enrichment of Enabled at the apical membrane (B) and its localization to the basal-lateral membrane (C) appears comparable between wild-type and abl mutant ommatidia. D-E) At 48hr APF, Enabled expression is detected basally in abl mutant clones, presumably reflecting the disrupted apical-basal positioning of the photoreceptor cells. F-G) Confocal projections of third instar eye discs with enaGC1/+; abl clones were stained with anti-GFP (green) and either anti-Arm (red, F) or anti-aPKC (red, G) antibodies. Arm and aPKC staining is weaker in the enaGC1/+; abl clones relative to wild-type, but comparable to that seen abl clones as shown in Figures 2A and 3A. Scale bars: 10 μm. H) An orthogonal section through a 48hr APF eye disc stained with anti-Elav (red) and anti-GFP antibodies. In the enaGC1/+; abl clones, the nuclei of the mutant photoreceptor cells reside at the basal plane of the epithelium, a phenotype similar to that of abl clones. I) Confocal projection of a 48hr APF eye disc stained with Rh-Phalloidin (red) and anti-GFP (green) antibody. F-actin accumulation at the center of the enaGC1/+; abl ommatidia remains greatly reduced relative to wild type.

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