Preferential adhesion maintains separation of ommatidia in the Drosophila eye

Abstract

In the Drosophila eye, neighboring ommatidia are separated by inter-ommatidial cells (IOCs). How this ommatidial spacing emerges during eye development is not clear. Here we demonstrate that four adhesion molecules of the Irre cell recognition module (IRM) family play a redundant role in maintaining separation of ommatidia. The four IRM proteins are divided into two groups: Kirre and Rst are expressed in IOCs, and Hbs and Sns in primary pigment cells (1 degrees s). Kirre binds Hbs and Sns in vivo and in vitro. Reducing activity of either Rst or Kirre alone had minimal effects on ommatidial spacing, but reducing both together led to direct ommatidium:ommatidium contact. A similar phenotype was also observed when reducing both Hbs and Sns. Consistent with the role of these factors in sorting ommatidia, mis-expression of Hbs plus Sns within a single IOC led to complete separation of the cell from neighboring ommatidia. Our results indicate mutual preferential adhesion between ommatidia and IOCs mediated by four IRM proteins is both necessary and sufficient to maintain separation of ommatidia.

Figure 1

Kirre and Rst act redundantly in patterning ommatidia. Eyes at 42 h APF were stained with an antibody against either Armadillo (A–D) or E-cadherin (E–G). (A-A’) A wild type eye. Tracing of an ommatidium is shown in (A’); inter-ommatidial cells (IOCs) are pseudo-colored in green. IR1 and IR2 indicate expression of one and two copies of indicated RNAi transgenes, respectively. IR3 refers to expression of a single copy of a transgene together with Dicer-2. Expression of all transgenes is controlled by GMR-Gal4 in this figure. (B) Strong reduction of Kirre by kirre-RNAi (kirre-IR3). Single cells failed to be selected in vertices (arrows). Occasionally bristle groups were mis-positioned (arrowhead). (C) Mild reduction of Rst by expressing a single copy of rst-RNAi (rst-IR1). An extra cell is highlighted by an arrow. (D) Expression of two copies of rst-RNAi (rst-IR2). A single cell was not selected in the vertex (arrows). Cells were often found surrounding a bristle group (arrowheads). (E) Strong reduction of Rst by rst-RNAi (rst-IR3). Defects in cone cells (arrowheads) and 1°s (asterisks) are indicated. Two ommatidia in direct contact are highlighted by double asterisks. (F) Strong reduction of both Rst and Kirre. Defects in cone cells (arrowheads) and 1°s (asterisks) are indicted. Contacting ommatidia are highlighted by double asterisks. IOCs completely failed to sort into single line. (G) Ommatidial patterning in the pupal eye does not require cell death. In hid mutants, cell death was strongly blocked and cells failed to sort into single line (arrows). However, ommatidia were separated as in wild type.

Figure 2

Kirre is expressed in IOCs. (A-A”) Kirre (red) co-localizes with Rst (green) on the surface. Kirre also co-localizes with Rst in all vesicles (open arrowheads). (B-B”) The Gal4–54; UAS-lacZ (54>lacZ) eye at 27 h APF was co-stained with anti-lacZ (red, left) and anti-Armadillo (green, middle) antibodies. Merged view is shown in the right panel. LacZ was detected exclusively in IOCs (asterisks). Arrowheads point to a bristle group from which lacZ staining was absent. (C-C”) kirre is transcribed in IOCs as revealed by an enhancer trap rP298-lacZ. The eye was stained with an anti-LacZ antibody (red) and marked by 54>GFP (green). Space for an ommatidium is indicated by an asterisk.

Figure 3

Sns and Hbs act redundantly in patterning ommatidia. Eyes at 42 h APF were stained with an antibody against either Armadillo (B–C), E-cadherin (A, D–F) or Echinoid (G). (A) Strong reduction of Sns by sns-RNAi (sns-IR3). Frequently, single cells failed to be selected at vertices (arrows). Occasionally, a cluster of cells was found surrounding a bristle group (arrowheads). (B) Mild reduction of Hbs by expressing a single copy of hbs-RNAi (hbs-IR1). A mis-positioned cell is highlighted by an arrow and a cluster of cells surrounding a bristle group indicated by an arrowhead. (C) Expression of two copies of hbs-RNAi (hbs-IR2). Single cells were not selected in vertices (arrows) and bristle groups misplaced (arrowheads). (D) Strong reduction of Hbs by hbs-RNAi (hbs-IR3). Defects in cone cells (arrowheads) and 1°s (asterisks) are highlighted. (E) Strong reduction of both Hbs and Sns. Ommatidia in direct contact are indicated by double asterisks. Defects in cone cells (arrowheads) and 1°s (asterisks) are highlighted. IOCs completely failed to sort into single file. (F) Strong reduction of both Kirre and Sns. Single cells failed to be selected within vertices (arrows). Extra cells (‘cone contact cells’) were commonly found in direct contact with cone cell quartets (arrowheads). (G) Strong reduction of both Kirre and Hbs. Frequently cone cells formed abnormal configurations (arrowheads). Occasionally IOCs failed to sort into single rows (arrows).

Figure 4

IRM proteins are expressed in complementary cell types. Eyes at 27 h APF are shown in A–B and an eye at 42 h APF in C. (A-A”) Hbs (red) and Rst (green) co-localize on the surface. Hbs also co-localizes with Rst in vesicles in 1°s (arrows). Rst is also found in vesicles in IOCs (arrowheads), where it does not co-localize with Hbs. A merged view is shown in A”. (B-B”) Sns (red) co-localizes with Kirre (green) on the cell surface. Sns and Kirre vesicles largely co-localize (arrows). Sns and Kirre were also found at the borders between IOCs and bristle groups (arrowheads). (C-C’) sns-RNAi (sns-IR3) was targeted to single cells (green, C’) and the eye was stained with an anti-Sns antibody (red). Sns is reduced at the border when sns-IR is targeted to 1°s (arrowheads). D) Schematic representation of expression domains of the IRM proteins. Hbs and Sns of the Nephrin group (magenta) are expressed 1°s and cone cells. Kirre and Rst of the Neph1 group are expressed in IOCs (green).

Figure 5

Kirre binds both Sns and Hbs. (A-A’) When Hbs (green) is over-expressed in a single 1°, ectopic Kirre (red) is recruited to the border (arrow). (B-B’) Upon over-expression of two copies of a sns transgene (green) in IOCs, ectopic Kirre (red) is attracted to the borders (arrow). (C-C’) When Kirre (green) is over-expressed in an IOC, ectopic Hbs is recruited to the border (arrowheads). Note ectopic Hbs is not found in between IOCs (open arrowheads). An arrow points to a bristle group. (D-D’) When Kirre (green) is over-expressed in IOCs, ectopic Sns is recruited to the border (arrowheads). Note ectopic Sns is not found in between IOCs (open arrowheads). An arrow points to a bristle group. (E) Both Sns and Hbs are co-immunoprecipitated with Kirre. Lane 1, Hbs+Kirre; Lane 2, Sns+Kirre; Lane 3, Rst+Kirre. Immunoprecitation was performed using an anti-Flag antibody.

Figure 6

Sns and Hbs drive separation of cells from ommatidia. Eyes at 42 h APF were stained with an antibody against either Armadillo (Arm) or E-cadherin (E-cad) as indicated. Merged views in A–D are shown in A’-D’. (A-A’) When a single copy of sns (green) was expressed in a single cell, the cell retained its normal position. (B-B’) Upon expression of hbs (green), two cells were separated from one but retained contact with another ommatidium. Wild type IOCs maintained small interfaces with each other (open arrowhead) while target IOCs established larger interfaces with IOCs (arrowheads). (C-C’) When both hbs and sns were expressed in a single cell (green), the cell was fully separated from ommatidia. Wild type IOCs maintained small interfaces with each other (open arrowheads) but larger interfaces with the target IOC (arrowhead). (D-D’) When two copies of a sns transgene were expressed in a single cell (green), the cell was fully separated from ommatidia. Wild type IOCs maintained small interfaces with each other (open arrowheads) but larger interfaces with the target IOC (arrowhead). (E-E’) Pupal eye at 20 h APF. The eye was stained with an anti-Armadillo antibody. Emerging 1°s are outlined (E’). Most IOCs (white asterisks) are found in touch with at least one 1°. In the same area, an IOC (gold asterisk) not in touch with any 1° is highlighted.

Figure 7

Hbs and Sns mediate preferential adhesion of ommatidia to IOCs. Ommatidia (o) are shaded in gold and IOCs (i) in light blue. A single IOC targeted with ectopic Hbs/Sns expression (O’ cell) is highlighted in green. If Hbs and Sns make the O’ cell more adhesive to ommatidia than to IOCs (W(o’-o) > W(o’-i)), upon mis-expression of Hbs/Sns, the O’ cell should remain attached to ommatidia (Case I). Conversely, if Hbs/Sns render the O’ cell less adhesive to ommatidia than to IOCs (W(o’-i) > W(o’-o)), the O’ cell should be detached from all ommatidia upon ectopic expression of Hbs/Sns (Case II).