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Control of photoreceptor cell morphology, planar polarity and epithelial integrity during Drosophila eye development

¹ Program in Developmental Biology, Research Institute, The Hospital for Sick Children, 555 University Avenue, Toronto, Ontario M5G 1X8, Canada
² Department of Molecular & Medical Genetics, University of Toronto, Toronto, Canada
³ Department of Biology, University of Virginia, Charlottesville, VA 22904, USA
⁴ Cornell Theory Center, Cornell University, Ithaca, NY 14853, USA
⁵ OSI Pharmaceuticals, Uniondale, NY 11553, USA

View larger version (62K): [in a new window]   Fig. 1. HNT is expressed in sensory neuron precursor cells in larval discs. Third instar larval and pupal discs stained with -HNT antibody: (A-D) larval eye discs; (E) pupal eye disc; (F) wing disc; (G,H) leg discs. (A) In wild type, HNT accumulates posterior to the morphogenetic furrow of the eye disc (arrow) in the nuclei of all R cells before their differentiation as neurons, in all sensory neuron precursor cells in the antennal disc (black arrowhead points to a subset of these cells) and in the ocellar precursor cells (gray arrowhead). (B) Eye disc from a larva carrying a dpp-lacZ enhancer trap (see Materials and Methods) double-stained with -HNT antibody (green) and an -ß-gal antibody to visualize dpp-reporter expression (red). The most anterior boundary of HNT expression is in the middle of the furrow, partially overlapping with dpp-reporter expression. (C) HNT expression in the furrow is restricted to clusters of cells, with non-staining cells in between (the arrow marks the furrow). (D) More posteriorly, HNT is found in the nuclei of all R cells in the developing clusters (the arrow marks the furrow). Inset: there is higher accumulation of HNT in the R3 and R4 precursor cells (white arrowheads). (E) HNT accumulates in the bristle precursor cell nuclei in the basal plane of a pupal eye disc. (F,G) HNT accumulates in sensory precursor cells in third instar wing (F) and leg (G) discs. (H) In a leg disc from a Nts1/Y fly pulsed at the restrictive temperature, HNT accumulates in the additional cells that are now destined to become neurons.

View larger version (94K): [in a new window]   Fig. 2. hntpeb is a viable allele that causes eye roughening. Scanning electron micrographs of adult eyes. (A) Wild-type eye. Note the regular arrangement of facets. (B) Eye from a hntpeb/hntpeb fly raised at the restrictive temperature. Note the disorganized facets, causing roughening. (C) Eye from a hntpeb/hntXE81 fly. Note the similar phenotype to that shown in B. This failure to complement hntpeb was observed for three other alleles of hnt (hntEH704a, hntXO01 and hnt1142; data not shown). In all cases, anterior is towards the left and dorsal is towards the top of the page.

View larger version (71K): [in a new window]   Fig. 3. dpp expression, neuronal determination and precluster formation in hnt mutant eye discs. (A-D) Expression of a dpp-lacZ reporter in wild-type (A) and hnt mutant (B-D) eye tissue. (A) In a late third instar disc from a dpp-lacZ/+ larva, reporter expression occurs along the furrow and is abruptly downregulated posterior to the furrow. The arrow marks the posterior border of the furrow. The reporter was visualized by X-Gal staining. (B) A disc with a hntXE81 clone visualized by the lack of -HNT antibody staining (brown) behind the furrow. The dpp-lacZ reporter expression, visualized by X-Gal staining, looks normal. The same result was obtained with hntEH704a clones (not shown). (C) A disc from a hntpeb/Y larva raised at the restrictive temperature, showing that the dpp-reporter expression is no longer tightly downregulated at the posterior edge of the furrow but persists in some of the emerging clusters in the eye field (reporter was visualized by X-Gal staining). (D) A disc with a hntXO01 clone, showing that the dpp-reporter expression (blue-black staining), again persists posterior to the furrow, when it would normally be shut off. In this case, reporter expression was visualized with -ß-gal antibody. (E) Eye disc containing a hntXE81 clone visualized by lack of immunostaining with -HNT antibody (HNT-expressing cells are green). -HRP staining (red) labels clusters of neuronally determined R cell precursors. Within the hnt tissue, the neuronal determination in the clusters is delayed relative to the adjacent wild-type clusters. The white arrowheads mark the first neuronal cells in the mutant patch versus in the surrounding wild-type tissue; note that the arrowhead is two rows closer to the furrow in the wild-type tissue. (F) Eye disc containing a hntXE81 clone visualized by lack of immunostaining with -HNT antibody (HNT-expressing cells are red). The outlines of emerging R cell clusters are highlighted using -PY antibody (green). Inside the mutant patch, the arc stage of development looks normal (arrow), but defects in cell arrangement and number can be observed in the preclusters (arrowheads). (G) Eye disc from a wild-type larva stained with the -Glass antibody, which labels all of the presumptive R cells. Cells of the definitive precluster are labeled with red arrowheads. (H) Eye disc from a hntpeb larva, raised at the restrictive temperature and stained with the -Glass antibody. Many of the preclusters (yellow arrowheads) have defects, including missing or extra Glass-positive cells and displaced nuclei within a cluster. Red arrowheads point to preclusters with a normal complement of cells.

View larger version (99K): [in a new window]   Fig. 4. R-cell specification in preclusters in hnt mutants. Eye discs containing clones of hnt tissue are shown. In all cases, the clone is marked by a lack of brown -HNT antibody staining. The location of the furrow together with the direction of the equator is indicated by the arrows. (A) Apical membrane expression of the R8 marker, Boss, visualized by -Boss antibody immunostaining in blue-black. Within the hnt patch, there is always a determined R8 cell, although occasionally there is a lower level of Boss accumulation than normal (red arrowheads). (B,C) Nuclear expression of the rhoPX81 enhancer trap, which labels determined R8, R2 and R5 precursor cells, visualized by -ß-gal antibody staining in blue-black and outlined in pink in the patch or in blue in surrounding wild-type clusters (C). Within the hnt patch, 47% of clusters have one or two non-staining cells, 74% of the clusters have misarrangements of their cells and 79% of the triads show misrotation of the cluster relative to its wild-type counterparts. (D,E) Nuclear expression of Spalt, which labels the R3 and R4 precursor cells, is shown by -Spalt antibody staining in blue-black. The nuclei of the R3 and R4 cells are outlined in blue in wild-type clusters or in yellow in cells in the hnt patch (E). Pale yellow indicates nuclei with reduced -Spalt staining. Inside the hnt patch, 57% of clusters have a non-staining cell, 84% have misarrangements of their cells and 67% show misrotation.

View larger version (69K): [in a new window]   Fig. 5. Mosaic analysis of hnt clones in the eye. (A) The results of a clonal analysis of mosaic ommatidia along the borders of 94 hnt patches are schematized. Each data set represents ommatidia with different phenotypic organization: I, wild type; II, chiral flip; III, 45°rotation; IV, 90° rotation; V, chiral flip and 45° rotation; VI, chiral flip and 90° rotation; VII, symmetrical; VIII, symmetrical and chiral flip. Black R cells are wild type with respect to hnt; white R cells are mutant for hnt. The number of ommatidia with that particular phenotype is shown in red. (B) Apical tangential section of a hntXO01 clone (mutant patch is to the left of the red line). The polarity of each ommatidium is labeled with a pink arrow as shown on the schematic in A. The yellow asterisk marks the hnt single mutant cell (R6) in a mosaic ommatidium that is an example of class V (i.e. misrotated by 45° and chirally flipped with respect to the adjacent wild-type ommatidia). (C) More basal tangential section of a hntXE81 clone. A class VII symmetrical ommatidium with a mutant R4 cell (asterisk) is labeled with a yellow arrow. The R8 projection (between R1 and R2) is marked with an orange arrowhead. A chirally flipped and misrotated (class V) ommatidium can be seen on the bottom left of the mutant patch. In this case, there is a mutant R3 cell (asterisk).

View larger version (152K): [in a new window]   Fig. 6. Adult phenotypes of hnt mutants. (A) Scanning electron micrograph (SEM) of part of an eye from a hntpeb/hntpeb fly. Some of the facets do not have the stereotypical hexagonal shape, others are fused. The sensory bristles are sometimes mispositioned and show a variation in size never seen in wild type. (B) Apical tangential section through a wild-type eye, showing each ommatidium with a trapezoidal arrangement of the seven photoreceptor (R) cells. All the ommatidia in this half of the eye have the same polarity. (C) In an eye from a hntpeb/Y fly raised at the restrictive temperature, 40% of the ommatidia are missing outer or central R cells (black arrowheads), 12% have extra R cells (white arrowhead) and some of the ommatidia are fused (yellow arrows point to a pair of fused ommatidia). The ommatidia also have variable polarity. (D) SEM of an eye with a scar caused by a clone of hntXO01 tissue. Clones mutant for three different hnt alleles (hntXE81, hntEH704a and hntXO01) form scars. (E) An apical tangential section through a hntXE81 scar (to right of red dots) reveals that the interior of the clone contains no R cells but that pigment cells are still present. (F) A cross section through another hntXE81 scar, shows that some of the R cells (arrowheads) have fallen out of the retinal epithelium. The R cells come to reside on the optic lobe of the brain and the basal, fenestrated membrane of the retina, which is assembled after the R cells fall out of the epithelium (see Fig. 7 and Results), separates these R cells from the retina.

View larger version (72K): [in a new window]   Fig. 7. Rhabdomere structure and integrity in hnt mutants. (A) Schematic of a longitudinal section through an adult ommatidium, showing the rhabdomeres (black) extending along the apicobasal axis of the eye epithelium. Gray arrows indicate the level of transverse cross-sections through two distinct mosaic ommatidia, shown in the blue panels either side of the schematic. The mosaic ommatidium on the left has an R5 cell that is mutant for hnt. The R5 rhabdomere is seen in an apical plane of section (yellow asterisk), but does not extend all the way basally, unlike the rhabdomeres belonging to the other, hnt+, outer R cells in this ommatidium. The mosaic ommatidium on the right has an R3 cell that is mutant for hnt (yellow asterisk). At an apical plane of section the rhabdomeres have a wild-type appearance, whereas, at more basal levels, the R3 and R4 rhabdomeres have failed to separate. (B) Third instar larval eye disc stained with an -HNT antibody (red), which is absent from the hntXO01 patch, and phalloidin-FITC (green), which stains the F-actin concentrated at the apical tips of the developing photoreceptor cell clusters. The F-actin is depleted in the more posterior area of the hnt patch compared with its levels in the adjacent wild-type tissue. (C) Apical section of a late pupal disc stained with -HNT antibody (red) and phalloidin-FITC (green), and containing a clone of hntXE81 tissue. In the area where the R cells are all mutant, there is no F-actin accumulation. HNT-positive R cells within two mosaic ommatidia along the clone border (arrows) have less F-actin apically than their genetically wild-type neighbors within the cluster. (D) More basal view of the same clone as in C, showing that there are low levels of F-actin in the more basal regions of these cells (arrows).