2024.01.17.575959v1.full-html.html

bioRxiv preprint

Summary

The biconvex shape of the

Drosophila

corneal lens, which enables it to focus light onto the

retina, arises by organized assembly of chitin and other apical extracellular matrix components.

We show here that the Zona Pellucida domain-containing protein Dusky-like is essential for

normal corneal lens morphogenesis. Dusky-like transiently localizes to the expanded apical

surfaces of the corneal lens-secreting cells, and in its absence, these cells undergo apical

constriction and apicobasal contraction. Dusky-like also controls the arrangement of two other

Zona Pellucida-domain proteins, Dumpy and Piopio, external to the developing corneal lens.

Loss of either

dusky-like

dumpy

delays chitin accumulation and disrupts the outer surface of

the corneal lens. Artificially inducing apical constriction with constitutively active Myosin light

chain kinase is sufficient to similarly alter chitin deposition and corneal lens morphology. These

results demonstrate the importance of cell shape for the morphogenesis of overlying apical

extracellular matrix structures.

Key words: Dusky-like,

Drosophila,

corneal lens, Zona Pellucida domain, apical constriction,

chitin, Dumpy, myosin

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Introduction

The extracellular matrix (ECM) is a complex array of proteins and polysaccharides that

provides structural and biochemical support to tissues. Mutations affecting the ECM can cause

numerous human genetic disorders including cancer, muscular dystrophy, hematuria, and

retinopathy

1-3

. Basement membrane ECM, which forms a sheet underlying epithelial cells, is

made up of conserved proteins such as collagen, nidogen, perlecan, and laminins. It can mediate

adhesion between tissue layers, insulate cells from the extracellular fluid, transmit mechanical

forces, influence the distribution of signaling molecules, and act as a substrate for cell migration

4,5

. Less is known about the apical ECM (aECM), which has diverse forms and functions.

External aECMs such as invertebrate cuticles can act as permeability barriers and protect against

desiccation, and the shapes of tubular organs such as the

Drosophila

trachea and

C. elegans

excretory system depend on luminal scaffolds composed of aECM

6,7

. In vertebrates, aECM

forms structures such as the tectorial membrane of the inner ear, the vascular glycocalyx,

pulmonary surfactant, and the mucin-rich lining of the gut

8-13

. The morphogenesis of aECM

assemblies is not fully understood, although in a few cases it has been linked to cytoskeletal

organization in the underlying cells

14,15

One of the most striking structures composed of aECM is the

Drosophila

corneal lens,

which consists of fibers of the polysaccharide chitin and associated proteins arranged to form a

precisely curved biconvex shape

16,17

. The corneal lens of each ommatidium of the compound

eye is secreted during the second half of pupal development by the underlying non-neuronal cells

. Major corneal lens constituents such as Crystallin are produced by the central cone and

primary pigment cells, while additional components are derived from the secondary and tertiary

pigment cells attached to the corneal lens periphery

16,19

. Although it is not known how these

cells specify the shape of the corneal lens, we have previously shown that the transcription factor

Blimp-1 is essential to generate its outer curvature

. Many Blimp-1 target genes encode aECM

components, including members of the most conserved family of aECM proteins, which are

characterized by a Zona Pellucida (ZP) domain

20,21

The ZP domain was initially identified in constituents of the extracellular coat

surrounding the mammalian oocyte

22,23

, but has since been found in proteins implicated in many

developmental processes

21,24

. Mutations affecting human ZP-domain proteins are associated

with disease conditions that include infertility, deafness, vascular disorders, inflammatory bowel

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disease, kidney disease, and cancer

22,25-30

. The 260 amino acid ZP domain can mediate

homomeric or heteromeric polymerization into filaments; it consists of N- and C-terminal

subdomains with structures related to immunoglobulin domains that are stabilized by conserved

disulfide bonds

31-33

. Some ZP-domain proteins are tethered to the plasma membrane by a

transmembrane domain or GPI linkage, while others undergo proteolytic cleavage and are

secreted into the extracellular space

34-37

ZP-domain proteins have been found to play a crucial role in shaping the aECM and

attaching it to the apical plasma membrane

7,21

. For instance, the

-tectorin ZP-domain protein is

essential for attachment of the tectorial membrane to the cochlear epithelium

. Filaments of the

giant

Drosophila

ZP-domain protein Dumpy (Dpy) anchor pupal appendages and tendon cells to

the external cuticle

39-41

, and the dendrites of sensory organs also require ZP-domain proteins to

connect them to the cuticle

37,42

. The lumen of tubular structures in

Drosophila,

C. elegans

, and

the mammalian kidney is organized by ZP-domain proteins

29,43-45

. Interestingly, mutations

affecting eight

Drosophila

ZP-domain proteins each have distinct effects on the morphology of

embryonic denticles, and each protein occupies a specific spatial location in the denticle

indicating that ZP-domain proteins are specialized to assemble aECM into diverse shapes.

Here, we show that the ZP-domain protein Dusky-like (Dyl)

46-48

is essential for normal

morphogenesis of the

Drosophila

corneal lens. During pupal development,

dyl

mutant ommatidia

undergo abnormal apical constriction and apicobasal contraction accompanied by changes in the

distribution of cytoskeletal proteins. Loss of

dyl

also results in changes in the organization of

other ZP-domain proteins such as Dpy and Piopio (Pio)

36,43,49

, and a delay in the accumulation

of chitin. Interestingly, many of these changes can be phenocopied by activating myosin to

artificially induce constriction of the corneal lens-secreting cells. These observations suggest that

Dyl acts as a mechanical anchor that transiently attaches the cone and primary pigment cells to

the aECM to maintain their expanded apical surface area, enabling the generation of a ZP-

domain protein scaffold that shapes the curved corneal lens surface.

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Results

Dyl is necessary for normal corneal lens morphology

The

Drosophila

corneal lens is a biconvex aECM structure that focuses light onto the

underlying photoreceptors

. The transcription factor Blimp-1 is necessary for normal corneal

lens curvature, and its target genes include several that encode ZP-domain proteins

. ZP-

domain proteins have been reported to connect aECM to the plasma membrane of epithelial

cells, and mutations affecting these proteins can alter the shape of cuticular structures such as

embryonic denticles

and bristles

47,48

. We therefore used existing mutants and RNAi lines to

test whether any ZP-domain targets of Blimp-1 were necessary for the normal shape of the

corneal lens.

We observed a dramatic effect on the external appearance of the adult eye in clones

mutant for one such gene,

dyl

. Scanning electron micrographs showed that in ommatidia

homozygous for

dyl

, a deletion of the entire gene

, the corneal lenses lacked external

curvature and had gaps in their outer surfaces (Fig. 1A). The same flat shape and abnormal

surface structure were visible in transmission electron micrographs (Fig. S1A, B). Cryosections

of eyes stained for chitin with Calcofluor White revealed that the flatter external surfaces of the

dyl

mutant corneal lenses were accompanied by more deeply curved internal surfaces than those

of the adjacent wild-type biconvex corneal lenses (Fig. 1C). Quantification of the outer and inner

angles between adjacent corneal lenses (Fig. 1E) confirmed that the outer angle was significantly

increased and the inner angle significantly decreased in

dyl

mutant regions (Fig. 1F, G). The

changes in corneal lens shape observed in

dyl

mutants, which included increased height and

reduced width, were rescued by expression of a

UAS-dyl

transgene

in all retinal cells with

lGMR-GAL4

, confirming that the defects were due to loss of

dyl

(Fig. 1B, D, F, G, Fig. S1E,

F). In transmission electron micrographs, we observed that the deeper inner curvature of the

corneal lens correlated with an increase in its area of contact with the peripheral secondary and

tertiary pigment cells (Fig. S1A-D, G). Taken together, our results show that the ZP-domain

protein Dyl is necessary for the normal biconvex shape and external morphology of the corneal

lens.

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Figure 1.

dyl

is necessary for normal corneal lens curvature.

(

A, B

) Scanning electron

micrographs of adult eyes. (

) in

dyl

∆42

mutant clones, the corneal lens surfaces are flat rather

than convex, and many appear to be missing parts of the surface layer. (

)

dyl

∆42

mutant clones

expressing a

UAS-dyl

transgene with

lGMR-GAL4

cannot be distinguished from wild-type eye

tissue. The regions in the yellow boxes are enlarged in (

A’, B’

), and the boundaries of the

dyl

mutant clone are marked with a yellow dotted line in (

A’

). (

C, D

) Horizontal frozen sections of

adult eyes in which

dyl

∆42

mutant clones (

) or

dyl

∆42

mutant clones expressing

UAS-dyl

with

lGMR-GAL4

(

) are positively marked with GFP (green). The corneal lenses are stained with

Calcofluor White (

C’, D’,

blue in

C, D

) and photoreceptors are marked with anti-Chaoptin (Chp,

red).

dyl

mutant ommatidia have externally flat corneal lenses (yellow arrows,

) while adjacent

wild-type ommatidia have biconvex corneal lenses (blue arrows,

). Rescue with

UAS-dyl

restores the normal corneal lens shape (yellow arrows,

). (

) Schematic illustration showing

how the outer and inner angles between adjacent corneal lenses and the height and width of a
corneal lens were defined. (

F, G

) Graphs showing the outer (

) and inner (

) angles between

adjacent corneal lenses in adult eye sections for wild type control regions,

dyl

∆42

mutant clones,

and

dyl

∆42

mutant clones in which

UAS-dyl

is driven with

lGMR-GAL4

. For both graphs, error

bars show mean ± SEM. n values are given as number of ommatidia/number of retinas for this
and subsequent graphs. Wild type n=70/20,

dyl

mutant n=103/20,

lGMR>dyl

;

dyl

mutant n=37/6.

****p < 0.0001, unpaired two-tailed t test with Welch’s correction. Scale bars: 200

m (

2.5

m (

A’

B’

), 20 µm (

C, D

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Loss of

dyl

causes apical constriction and apico-basal contraction of ommatidia

In order to understand how loss of

dyl

affects corneal lens shape, we traced the mutant

phenotype earlier in development. Corneal lens secretion initiates at 50 h APF and continues

through the late pupal stages

. Major components of the corneal lens such as Crystallin and

Retinin are produced by the underlying cone and primary pigment cells, and additional

constituents are secreted by the secondary and tertiary pigment cells (also called lattice cells, Fig.

2G)

. To determine the site of Dyl expression in the retina, we used an antibody against Dyl

and identified specific labeling by its absence in

dyl

mutant clones (Fig. 2A, B, Fig. S2A, B). We

observed that Dyl was present on the apical surface of the cone and primary pigment cells at 48 h

APF (Fig. 2A) and 50 h APF (Fig. S2A). Dyl protein levels were reduced at 52 h APF (Fig. S2B)

and became very low at 54 h APF (Fig. 2B), consistent with the transient 48 h APF peak of

dyl

mRNA reported by modENCODE

dyl

mutant

ommatidia showed a striking apical constriction starting at 48 h APF (Fig. 2A)

that became quite pronounced at 54 h APF (Fig. 2B, Fig. S2F). The change was driven by a large

decrease in the apical surface area of the cone and primary pigment cells (central cells) (Fig.

2D); in contrast, the apical width of the lattice cells expanded (Fig. 2E). The apical constriction

dyl

mutant ommatidia was accompanied by apical-basal contraction, placing their apical

surfaces in a deeper plane than the wild-type ommatidia at 54 h APF (Fig. 2B). This change

could also be visualized in cryosections of 60 h APF pupal retinas, in which

dyl

∆42

ommatidia

were shorter than their wild-type neighbors (Fig. 2C, F). In these sections, the wild-type corneal

lenses labeled with a fluorescent chitin-binding domain (CBD) had convex outer surfaces,

whereas corneal lenses corresponding to

dyl

clones were flat or convoluted (Fig. 2C). Expression

of the wild-type UAS-

dyl

transgene rescued both the apical constriction and apicobasal

contraction displayed by

dyl

∆42

mutants (Fig. S2C-F). These data suggest that Dyl acts at the

apical surfaces of cone and primary pigment cells to maintain their apical expansion (Fig. 2G).

The apicobasal contraction of

dyl

mutant ommatidia correlates with a change in the shape of the

developing corneal lens (Fig. 2H).

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Figure 2.

Loss of

dyl

causes apical constriction and apico-basal contraction of ommatidia.

(

A, B

) Retinas containing

dyl

∆42

mutant clones marked with GFP (green) and stained with anti-

Dyl (

A’

B’

, red in

) and anti-Armadillo (Arm) to mark apical junctions (

A’’

B’’

, blue in

) at (

) 48 h APF and (

) 54 h APF. The

dyl

∆42

mutant clones show loss of Dyl staining in cone

and primary pigment cells (there is non-specific staining of the lattice cells), and a marked
reduction in their apical surface area (yellow arrows,

A”

; yellow outline,

B’’

) as well as

apicobasal contraction; the apical plane of the

dyl

mutant ommatidium outlined in yellow is in

the same confocal section as a more basal plane of the wild-type ommatidium outlined in blue
(

B”

). (

) Horizontal cryosection of a 60h APF retina showing apicobasal contraction in

dyl

∆42

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mutant clones marked with GFP (green, yellow arrows) in comparison to wild type ommatidia
(blue arrows). An Alexa647-labeled chitin binding domain (CBD,

C’,

blue in

) marks the

corneal lenses, which are flattened and distorted in

dyl

mutant clones, and photoreceptor

rhabdomeres are stained with anti-Chp (red). Dotted lines show how ommatidial height was
measured. Scale bars: 10 µm (

A, B

), 20 µm (

). (

D, E

) Graphs depicting (

) apical surface area

of the central cone and primary pigment cells and (

) lattice cell width in

dyl

∆42

mutant clones

and control wild-type ommatidia in the same retinas, at 48 h APF (n=28/18 for control and 32/18
for

dyl

, n=69/7 for control and 61/7 for

dyl

) and 54 h APF (n=44/27 for control and

52/12 for

dyl

, n=37/7 for control and 57/7 for

dyl

). (

) graph showing ommatidial

height in

dyl

∆42

mutant clones and control wild-type ommatidia at 60 h APF (n=21/5 for control

and 35/5 for

dyl

). For all graphs, error bars show mean ± SEM. ****p < 0.0001, *p = 0.0221,

unpaired two-tailed t test with Welch’s correction. (

) Schematic representations of ommatidia at

54 h APF. Cone cells (CC, green) and primary pigment cells (1°, yellow), together known as the
central cells, are surrounded by a lattice of secondary pigment cells (2°, cyan), tertiary pigment
cells (3°, indigo), and bristles (B, red). In controls, we hypothesize that Dyl attaches the central
cells to the aECM to maintain their apical surfaces in an expanded state. In

dyl

mutants

the

central cells would lose their apical attachments and contract. (

) Schematic of horizontal views

of control and

dyl

mutant adult ommatidia. The corneal lens (pink) may maintain its attachment

to the lattice cells as the ommatidium contracts basally, flattening its external curvature and
deepening its internal curvature.

Loss of

dyl

causes cytoskeletal reorganization

We next investigated how the organization of the cytoskeleton was affected in

dyl

mutant

clones. Using the actin-binding domain of the ERM protein Moesin (Moe) tagged with mCherry

to label actin filaments, we found that at 60 h APF more Moe::mcherry accumulated at the

apical surface of

dyl

mutant ommatidia than in neighboring wild-type ommatidia, implying that

the actin cytoskeleton is condensed in

dyl

mutant cells (Fig. 3A). Actomyosin contraction can be

induced by phosphorylation of the non-muscle myosin II regulatory light chain, known in

Drosophila

as Spaghetti squash (Sqh)

. We stained

dyl

clones in the pupal retina

with an

antibody against phospho-Sqh (pSqh)

and found it to be enriched in lattice cells at 50 h APF in

both wild-type and

dyl

mutant regions (Fig. S3A). At 54 h APF pSqh was present in discrete foci

at the apical surfaces of wild-type cone and primary pigment cells, whereas in

dyl

∆42

clones,

pSqh was more uniformly distributed (Fig. 3B). Loss of

dyl

had a similar effect on the

distribution of beta-heavy spectrin (β

-spectrin) (Fig. 3C, S3B), a component of the spectrin-

based membrane cytoskeleton encoded by the gene

karst

(

kst

) that has been reported to control

apical cell surface area

. Similar foci of phospho-Sqh and β

-spectrin have been observed in

cells undergoing pulsatile apical constriction and ratcheting, in the embryonic mesoderm and

other contexts

56,57

. Myosin foci are also associated with fluctuations in the area of cone and

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primary pigment cells as they expand earlier in pupal development

. The lack of such foci in

dyl

mutant cells may indicate that their constriction is a less organized process.

Figure 3.

Loss of

dyl

alters the distribution of cytoskeletal proteins.

(

) Cryosection of a 60 h

APF retina showing Moe::mCherry (red) accumulation at the apical surface in

dyl

∆42

mutant

clones (yellow arrows) marked with GFP (green). Blue arrows mark wild type ommatidia. CBD
(blue) labels the corneal lens. (

B, C

) 54 h APF retinas containing

dyl

∆42

mutant clones marked

with GFP (green), stained with anti-Ecad and anti-Ncad (blue), anti-pSqh (

B’

, red in

), or anti-

-spectrin (

C’

, red in

). In wild-type ommatidia at this stage pSqh and β

-spectrin form foci,

but in

dyl

∆42

mutant clones they are diffusely localized. Confocal sections showing the apical

surfaces of individual wild-type (

B’’, C’’

) or

dyl

mutant (

B’’’, C’’’

) ommatidia labeled with anti-

Ecad and anti-Ncad (green) and anti-pSqh (magenta,

B’’, B’’’

) or anti-β

-spectrin (magenta,

C’’,

C’’’

) show that the differences are not due to the more basal position of

dyl

mutant ommatidia.

(

) A 54 h APF retina with

dyl

∆42

mutant clones expressing

UAS-Mlck RNAi

with

lGMR-GAL4

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marked with GFP (green). Apical cell junctions are stained with anti-Ecad and anti-Ncad (

D’

magenta in

). (

) Graph showing the apical surface area of central cells in

dyl

∆42

mutant clones

and internal wild type control ommatidia (data from

Fig. 2D

), and in

dyl

∆42

mutant clones

expressing

Mlck RNAi

(n=19/8) at 54 h APF. Knocking down

Mlck

partially rescues the apical

constriction phenotype. (

) Horizontal cryosection of an adult eye with

dyl

∆42

mutant clones

expressing

Mlck RNAi

labeled with GFP (green, yellow arrows), stained with anti-Chp (blue) and

CBD (red). Scale bars: 10 µm (

A-D

), 5

m (

B’’, B’’’, C’’, C’’’

), 20

m (

). (

) Graph showing

the outer angle between adjacent corneal lenses in control,

dyl

∆42

mutant clones (data from

Fig.

), and

dyl

∆42

mutant clones expressing

Mlck RNAi

(n=41/8)

Knocking down

Mlck

is not

sufficient to restore normal corneal lens curvature in

dyl

∆42

mutant clones. Error bars show mean

+/-SEM. ****p < 0.0001, unpaired two-tailed t test with Welch’s correction.

Artificially induced apical constriction alters corneal lens shape

We attempted to test whether apical constriction was necessary for the effect of

dyl

corneal lens shape by knocking down

Myosin light chain kinase

(

Mlck)

, which encodes a kinase

that phosphorylates Sqh to induce actomyosin contraction

, in

dyl

∆42

clones. A significant

change in corneal lens shape was still observed, but because the apical constriction was not fully

rescued, we could not determine whether

dyl

has an effect on corneal lens shape that is

independent of apical constriction (Fig. 3D-G). Knocking down

kst

also partially rescued the

apical constriction of

dyl

mutant ommatidia, but did not restore normal corneal lens curvature

(Fig. S3C-F). These knockdowns may have been incomplete, or the constriction may be partially

independent of Mlck and β

-spectrin activity. If Dyl-mediated attachments to the apical ECM

exert tension on the apical surfaces of the cone and primary pigment cells to maintain their

expansion, loss of these attachments could result in passive constriction of the apical surfaces.

We next tested whether apical constriction was sufficient to explain the effect of

dyl

corneal lens shape. We artificially induced apical constriction by expressing a constitutively

active form of Mlck (

UAS-Mlck

)

with

lGMR-GAL4

in clones in the retina. We confirmed

that

Mlck

-expressing clones exhibited strong apical constriction in the mid-pupal retina (Fig.

4A). At the adult stage, the overlying corneal lenses were externally flat with deep and distorted

internal curvature (Fig. 4B-D). Apical constriction in

dyl

∆42

clones is limited to the cone and

primary pigment cells (Fig. 2D, E). Expressing

UAS-Mlck

specifically in cone and primary

pigment cells with

sparkling-GAL4

(

spa-GAL4

)

resulted in a change in pSqh distribution and

corneal lens shape similar to

dyl

mutant clones, indicating that constriction of these cells is

sufficient to alter corneal lens curvature (Fig. 4E-G).

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Figure 4. Apical constriction of central cells is sufficient to alter corneal lens shape.

(

A, B

)

Expression of a constitutively active form of

Mlck

(UAS-

Mlck

) in clones marked with GFP

(green) in a 48 h APF pupal retina (

) and adult eye section (

) stained for Arm (

A’

, magenta in

), Chp (red in

) and Calcofluor White (B’, blue in B). UAS-

Mlck

clones display strong

apical constriction of central cells (yellow asterisk,

A’

), and in the adult eye, they show distorted

corneal lenses with flatter external surfaces and deeper internal curvature (yellow arrows in

B’

)

than controls (blue arrow). (

C, D

) Graphs showing (

) outer angle and (

) inner angle between

adjacent corneal lenses in control,

dyl

∆42

mutant clones (data from

Fig. 1F, G

), and

Mlck

overexpressing clones (n=31/5). Error bars show mean +/-SEM. ****p < 0.0001, ns p=0.369,
unpaired two-tailed t test with Welch’s correction. (

E, F

) UAS-

Mlck

is expressed only in cone

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and primary pigment cells with

spa-GAL4

in clones marked with GFP (green) at 48 h APF (

) or

54 h APF (

). Retinas are stained with anti-E-Cad and anti-N-Cad (

E’

, magenta in

blue in

)

and anti-pSqh (F’, red in F)

The clones show apical constriction of the central cells and

expansion of the lattice cells (asterisks in

E’

) and accumulation of disorganized pSqh. (

)

Horizontal cryosection of an adult eye in which

spa-GAL4

drives

UAS-MLCK

in all

ommatidia, stained for Chp (red), CBD (green) and the neuronal nuclear marker Elav (blue).
CBD staining shows external flattening and deep internal curvature of the corneal lenses. Scale
bars: 10 µm (

A, E, F

), 20 µm (

Dyl affects the organization of other ZP-domain proteins

The ZP domain is known to mediate homodimerization or heterodimerization, allowing

ZP-domain proteins to form extended filaments. Previous studies have shown that the ZP-

domain protein Dpy

interacts with another ZP-domain protein, Piopio (Pio), in the epidermis

and the lumen of the tracheal system

36,43

. Additionally, the ZP-domain protein Quasimodo

(Qsm) modifies the strength of the Dpy filaments that connect tendon cells to the external pupal

cuticle

. Since Dyl is only transiently expressed in the developing pupal retina, we wondered

whether it might interact with other ZP-domain proteins to maintain corneal lens structure. To

test this hypothesis, we examined the effect of loss of

dyl

on the organization of Dpy and Pio,

which we found to colocalize in the pupal retina (Fig. S5A). At 50 h APF, Dpy and Pio were

uniformly localized above the cone and primary pigment cells in both wild type and

dyl

∆42

ommatidia (Fig. 5A, Fig. S5B). By 54 h APF, Dpy and Pio were organized into a variety of linear

structures above the central region of wild-type ommatidia, but maintained their uniform

distribution in

dyl

∆42

clones (Fig. 5B, G). Loss of

dyl

had a similar effect on another ZP-domain

protein, Trynity (Tyn)

46,62

(Fig. S5C). These observations suggest that other ZP-domain proteins

depend on Dyl for their normal organization.

Tyn, like Dyl, was only detected at mid-pupal stages, but Dpy and Pio were maintained

into adulthood. At 60 h and 72 h APF, we found that Dpy and Pio were apical to chitin in the

developing corneal lens, and had a less convex shape in

dyl

∆42

clones than in wild-type

ommatidia (Fig. 5C, D, H). However, when pseudocone secretion begins at 84 h APF and in the

adult eye, Dpy and Pio were lost from the outer surface of the eye and only present in the

pseudocone below the corneal lens in both wild-type and

dyl

mutant ommatidia (Fig. 5E, F, I).

Dpy and Pio are thus in a position to provide outer and inner boundaries for the corneal lens as it

is being secreted.

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Figure 5. Loss of

dyl

affects the organization of Dpy and Pio at mid-pupal stages.

(

A, B

)

Pupal retinas at 50 h APF (

) and 54 h APF (

)

with

dyl

clones marked by myristoylated

Tomato (red), stained for Dpy-YFP (

A’

B’

, green in

) and Arm (blue). Dpy-YFP takes on a

linear organization that becomes more pronounced at later stages in wild-type but not

dyl

mutant

ommatidia.

(C-F)

Horizontal sections of retinas containing

dyl

clones marked by

myristoylated Tomato (red) at 60 h APF (

), 72 h APF (

), 84 h APF (

) and adult stage (

stained for Dpy-YFP (green) and CBD (blue,

) or Calcofluor White (blue,

). From 60-

72 h APF Dpy-YFP is present external to the chitin layer of the corneal lenses and is less convex
in

dyl

mutant clones (yellow arrows) than wild-type (blue arrows), but at 84 h APF it is lost from

the external surface and begins to be secreted into the pseudocone under the corneal lens, where
it remains in the adult. (

G-I

)

dyl

∆42

mutant clones marked with GFP (green) in a 54 h APF retina

(G)

, and in horizontal cryosections of 60 h APF

(H)

and adult

(I)

retinas, stained for Pio (

G’

, red

G-I

), Ecad and Ncad (blue in

), or CBD (blue in

). Pio localizes in a similar pattern to

Dpy-YFP in pupal retinas, but is restricted to the basal part of the pseudocone in adults. Dpy and
Pio are also visible in mechanosensory bristles in (

C, D, H

). Scale bars: 10 µm (

A, B, G

), 20 µm

(

C-F, H-I

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bioRxiv preprint

Dpy and Pio are required for normal corneal lens shape

The Dpy protein, the largest isoforms of which have molecular weights close to 2.5 MDa,

is known to promote membrane attachment to the cuticle in the embryonic trachea, the pupal

wing, and pupal tendon cells, where it forms long filamentous structures

39,40,43,49

. We

hypothesized that Dpy might also contribute to attaching the corneal lens cuticle to the

underlying cells. To test this idea, we generated clones homozygous for the lethal allele

dpy

lv1

in the retina and examined them at pupal and adult stages. In adults, the overlying corneal lenses

showed abnormal and irregular shapes (Fig. 6B, F, G), comparable to

dyl

∆42

clones. However,

dpy

mutant ommatidia were not shorter on the apical-basal axis, consistent with the lack of

significant apical constriction in

dpy

clones earlier in development (Fig. 6A, E). Clones mutant

for the protein null allele

pio

V132

(Fig. S6A) had no effect on apical constriction (Fig. 6C, Fig.

S6A) and did not alter the external curvature of the corneal lens (Fig. 6D, F), but showed a deep

and distorted inner corneal lens curvature (Fig. 6D, G). The protein null allele

tyn

did not

affect corneal lens shape (Fig. S6B-D). These results indicate that Dpy and Pio organized by Dyl

have critical roles in determining corneal lens shape independent of apical cell size.

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bioRxiv preprint

Figure 6. Loss of

dpy

pio

alters corneal lens shape.

(

A, B

) 50 h APF retina (

) and adult eye

section (

) containing

dpy

lv1

mutant clones labeled with GFP (green), stained for Ecad and Ncad

(

A’

, magenta in

), Chp (blue in

) and CBD (

B’

, red in

). Although

dpy

lv1

mutant clones in the

pupal retina show little apical constriction, corneal lenses in the adult eye have distorted shapes
including changes in the inner and outer curvature (yellow arrows) in comparison to adjacent
control corneal lenses (blue arrows). (

C, D

) 54 h APF retina (

) and adult eye section (

)

containing

pio

V132

mutant clones labeled with GFP (green), stained for Ecad and Ncad (

C’

magenta in

), Chp (red in

) and CBD (

B’

, blue in

pio

mutant clones do not show apical

constriction at 54 h APF. In adults,

pio

mutant corneal lenses have normal outer curvature, but

increased and distorted inner curvature (yellow arrows). (

) Graph showing the apical surface

area of central cells in

dpy

lv1

mutant clones (n=22/10) compared to internal control wild-type

ommatidia (n=27/10) and

dyl

mutant clones (n=73/23) at 50 h APF. Although

dyl

mutant

ommatidia are significantly constricted at this stage,

dpy

mutant ommatidia are not. (

F, G

)

Graphs illustrating the outer angle (

) and inner angle (

) between neighboring corneal lenses in

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bioRxiv preprint

control,

dyl

∆42

mutant clones (data from

Fig. 1F, G

dpy

lv1

mutant clones (n=30/5) and

pio

V132

mutant clones (n=58/8) in the adult eye. Error bars show mean +/-SEM. ****p < 0.0001, ***p =
0.0004, ns p = 0.3965 (

), 0.1538 (

), 0.6419 (

dyl

dpy

lv1

), 0.6217 (

dyl

pio

V132

0.4525 (

dpy

lv1

pio

V132

), unpaired two-tailed t test with Welch’s correction. Scale bars: 10

µm (

A, C

), 20 µm (

B, D

ZP-domain proteins act as a scaffold to retain chitin

A major component of the corneal lens is chitin, a polymer of N-acetylglucosamine.

Chitin assembles into strong fibrillar structures that provide structural integrity

. These chitin

polymers are associated with numerous chitin-binding proteins, such as Gasp

, which is highly

expressed in the pupal retina

. To examine whether the secretion or arrangement of chitin was

affected in

dyl

mutants, we stained

dyl

∆42

clones in the pupal retina with a fluorescently labeled

CBD probe

. We observed that at 50 h APF, chitin fibrils were located on the apical surface of

wild-type ommatidia, but in

dyl

∆42

clones chitin accumulation was sparse (Fig. S7A). At 54 h

APF high levels of chitin were localized above wild-type ommatidia, but chitin was absent above

dyl

clones and did not appear to be stuck in the secretory pathway within the mutant cells (Fig.

7A, B). This phenotype could be rescued by expressing a

UAS-dyl

transgene in all retinal cells

(Fig. S7H). Similarly, Gasp was not observed above

dyl

∆42

clones at either 50 h (Fig. S7B) or 54

h APF (Fig. 7D).

dpy

mutant clones also showed a loss of chitin accumulation (Fig. 7E), despite

normal Dyl localization (Fig. S7C), but

pio

was not required for chitin retention at this stage

(Fig. S7D). We first detected chitin above

dyl

clones at 57 h APF, but at a lower level than in

wild-type regions and without the appropriate curvature (Fig. 7C). All these data suggest that ZP-

domain proteins are necessary for the timely assembly of chitin in the corneal lens. We

hypothesize that an external scaffold that includes Dpy and Pio is crucial to retain the nascent

chitin layer and mold it into a biconvex shape. Changes in the arrangement of these proteins may

allow chitin to diffuse away at the stage when it would normally be deposited (Fig. 7H). This

external scaffold is lost, perhaps through proteolytic degradation

65,66

, later in development when

the chitinous corneal lens becomes a strong, rigid structure (Fig. 5E, F, I). The delay in chitin

assembly in

dyl

mutant clones may explain the defects in the surface structure of the adult

corneal lens (Fig. 1A).

Interestingly, we observed that chitin was also absent and Dpy was disorganized at 54 h

APF above clones in which apical constriction of cone and primary pigment cells was induced

by expressing activated Mlck (Fig. 7F, Fig. S7G). This suggests that the cell shape changes

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bioRxiv preprint

caused by loss of

dyl

may be sufficient to explain the altered ZP-domain scaffold and the lack of

chitin retention. It is also possible that direct interactions with Dpy and Pio contribute to the

effect of Dyl on their organization, as partially rescuing the constriction defect of

dyl

mutant

clones by knocking down

Mlck

failed to rescue Pio organization or chitin accumulation (Fig.

S7E, F). In addition, scanning electron micrographs confirmed the flat external surfaces of

ommatidia expressing Mlck

, but did not show the extensive surface gaps that were seen in

dyl

mutant clones (Fig. 7G). These results suggest that Dyl assembles a ZP-domain protein structure

that prevents dispersal of the components of the developing corneal lens primarily by

maintaining apical expansion of cone and primary pigment cells, but potentially also through an

independent mechanism.

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bioRxiv preprint

Figure 7. ZP-domain proteins and apical expansion are necessary for chitin accumulation.

(

A-D

) Pupal retinas with

dyl

clones marked with GFP (green), stained with CBD (

A’-C’

, blue

A-C

) or anti-Gasp (

D’

, blue in

) and anti-Ecad and Ncad (red,

A, B, D

) or anti-Pio (red,

)

at (

B, D

) 54 h APF and (

) a horizontal cryosection at 57 h APF. (

) is a more basal plane of

the retina shown in (

). At 54 h APF chitin and Gasp are completely absent above

dyl

mutant

cells. This decrease in accumulation is not due to a block in secretion, as no intracellular chitin is
detected in the

dyl

∆42

mutant clones. Chitin begins to appear above the

dyl

∆42

mutant clones at 57

h APF, internal to a layer of Pio (yellow arrows in

and

C’

). (

) 50 h APF pupal retina with

dpy

lv1

mutant clones marked by GFP (green) and (

) 54 h APF retina with clones overexpressing

Mlck

with

spa-GAL4

marked by GFP (green) stained with anti-Ecad and Ncad (red) and CBD

(

E’

F’

, blue in

). Loss of

dpy

and apical constriction induced by

Mlck

result in a similar

loss of chitin. (

) Scanning electron micrograph of an eye containing clones expressing

UAS-

Mlck

with

lGMR-GAL4

. The boxed area is enlarged in (

G’

), and the border of the clone is

indicated with a dashed yellow line. The corneal lenses in

Mlck

-expressing ommatidia have

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Discussion

We show here that forming the precisely curved architecture of the

Drosophila

corneal

lens from apical ECM requires the ZP-domain protein Dyl. Dyl acts transiently at a critical point

in development to maintain the apical expansion of corneal lens-secreting cells and to assemble a

scaffold containing ZP-domain proteins that acts as a convex outer boundary within which chitin

and other corneal lens components are retained. Our results add to a growing body of evidence

that the shapes of rigid structures composed of aECM rely on specific sites of attachment to the

underlying cells that are mediated by ZP-domain proteins

39,45,46,67

Cell shape determines apical ECM shape

Our data show that the dramatic apical expansion of primary pigment cells in the pupal

retina

18,68

is essential to build a foundation for corneal lens assembly. Reversing this expansion

either by removing Dyl or by activating myosin contraction results in severe corneal lens defects.

The apicobasal contraction that occurs in

dyl

mutant or

Mlck

-expressing ommatidia places the

apical surfaces of cone and primary pigment cells in a more basal position than their wild-type

neighbors. If secondary and tertiary pigment cells retain their attachments to the aECM, the

ommatidial surface would take on a concave shape, potentially explaining the deeper inner

curvature of the overlying corneal lenses (Fig. 2H). The smaller surface area of the central cells

could also directly alter the shape of the corneal lens if this structure is assembled one layer at a

time, like the tectorial membrane

. Such a pattern of assembly would be consistent with

labeling of the outer surface of the early corneal lens and the inner surface of the adult corneal

lens by our chitin-binding probe (Fig. 5H, I), if it preferentially binds to newly deposited chitin.

Constriction or folding of the apical cell surface has been shown to produce ridges of aECM in

the

C. elegans

cuticle and the

Drosophila

trachea

14,15

; our results suggest that apical expansion

can also drive the morphogenesis of specific aECM structures.

The striking apical constriction of cone and primary pigment cells that we observed in

dyl

mutants implies that Dyl is required to maintain their expanded shape. Dyl is located on the

apical surfaces of these cells, and it and other ZP-domain proteins have been shown to attach the

plasma membrane to the apical ECM

36,37,42,46,70,71

. These attachments can alter cell shape; for

instance, apical expansion of cells in the pupal wing is dependent on the ZP-domain proteins

Miniature and Dusky

, NOAH-1 and NOAH-2 are required for the concerted cell shape

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bioRxiv preprint

changes that drive elongation of the

C. elegans

embryo

, and Hensin is needed to convert flat

- to columnar

-intercalated cells in the kidney

. The NOAH-1, NOAH-2 and FBN-1 ZP-

domain proteins in the embryonic sheath resist deformation of the

C. elegans

epidermis by

mechanical forces

70,73

. We suggest that attachment to the aECM by Dyl enables cone and

primary pigment cells to resist mechanical tension exerted by the actomyosin cytoskeleton.

Active myosin and

-spectrin accumulate in

dyl

mutant cells, and reducing the level of these

components partially rescues apical constriction. However, these proteins appear disorganized

and do not form foci like those seen in the wild-type cells. Similar foci are associated with

changes in apical cell area driven by pulsatile constriction and ratcheting

56-58

, but continuous

constriction can occur with a diffuse apical actomyosin distribution, for instance in boundary

larval epithelial cells

53,75

. The loss of apical anchorage may cause

dyl

mutant cells to constrict in

a continuous, passive manner that does not involve controlled ratcheting.

ZP-domain proteins act as a scaffold for chitin retention

Our data also reveal a role for other ZP-domain proteins, including Dpy and Pio, in

establishing an external scaffold that helps to shape the corneal lens. Although

dpy

mutant

ommatidia do not show significant constriction, indicating that

dpy

acts downstream of or in

parallel to the cell shape changes, they fail to accumulate chitin at the normal time and produce

deformed corneal lenses. As chitin is not found trapped inside the mutant cells, it is likely that it

diffuses away in the absence of the scaffold. Although Pio is not necessary to retain chitin at this

stage, it appears to have an analogous role later in development, when it is present in the

pseudocone, to maintain the normal morphology of chitin at the inner surface of the corneal lens

(Fig. 6D). Dpy and Pio have a similar function in the trachea, where they provide an elastic

structural element of the lumen that controls its shape and size and maintains the chitin matrix

66,76,77

. Removal of this structure by proteolysis is necessary for subsequent gas filling of the

airway

65,66

, and it is possible that the external corneal lens scaffold is likewise proteolytically

degraded in late pupal stages. Transient aECM structures containing ZP-domain proteins also

shape many cuticular and tubular structures in

C. elegans

, and

-tectorin on the surface of

supporting cells templates the assembly of collagen fibrils in each layer of the tectorial

membrane

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We find that the arrangement of Dpy and Pio is dependent on Dyl; in wild-type

ommatidia, they form linear structures, but this organization is not apparent in

dyl

mutants. We

do not know the precise nature of these structures, nor what additional components they may

contain. ZP-domain proteins are capable of forming heteropolymeric filaments

31-33

, and another

ZP protein, Qsm, is known to promote the secretion and remodeling of Dpy filaments that link

tendon cells to the pupal cuticle, increasing their tensile strength

. Since Dyl is only present in

the retina for a short time, it may be required to initiate the assembly of a scaffold structure that

then becomes self-sustaining. Alternatively, since apical constriction is sufficient to alter Dpy

organization and delay chitin accumulation, the role of Dyl may be limited to maintaining apical

expansion.

Nevertheless, the requirement for

dyl

for normal chitin deposition in bristles and wing

hairs

47,48

suggests that promoting chitin assembly, directly or indirectly, is one of its primary

functions.

Other components of the corneal lens may also be affected by loss of

dyl

. Although chitin

levels appear normal in sections of adult retinas, scanning electron micrographs show that the

external surface of the corneal lens is incomplete. Induced apical constriction does not fully

reproduce this loss of surface structure, suggesting that it reflects an independent function of Dyl.

In the pupal wing,

dyl

belongs to a cluster of genes with peak expression at 42 h APF, when the

outer envelope layer of the cuticle is being deposited, but it influences the structure of inner

layers as well

. Another gene expressed at this time point,

tyn

, is required for the barrier

function of the embryonic envelope layer

. It is not clear whether the corneal lens has a typical

cuticular envelope, but its outermost layer forms nanostructures composed of the Retinin protein

and waxes

. Although

retinin

mRNA is not strongly expressed until very late in pupal

development

, its expression is initiated at mid-pupal stages when Dyl is present

. It is

possible that Dyl and the Dpy-Pio scaffold also control the retention of Retinin and other

components of the outer layer.

In summary, we show here that the development of a biconvex corneal lens depends on

both its upper and lower boundaries. The upper boundary, a convex shell of aECM that contains

the ZP-domain proteins Dpy and Pio, encloses chitin and its associated proteins as they are

secreted by cone and pigment cells. The shape of this shell may be defined by its attachment to

the peripheral lattice cells and the pressure exerted on its center by secreted corneal lens

components. The lower boundary is the apical plasma membrane of the cone and primary

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bioRxiv preprint

pigment cells. This membrane is flat until pseudocone secretion initiates late in pupal

development, and the phenotype of

dyl

mutants suggests that it is maintained taut and expanded

by attachment to the aECM. Loss of this expansion or of the pseudocone components Dpy or Pio

gives the inner surface of the corneal lens a deeper curvature. It would be interesting to

investigate whether mechanical forces exerted on the stromal extracellular matrix of the human

cornea, perhaps through the ZP-domain protein ZP4 present in the underlying corneal

endothelium

, affect its shape and refractive power

82,83

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bioRxiv preprint

STAR Methods

Key resource table

REAGENT or RESOURCE

SOURCE

IDENTIFIER

Antibodies

Chicken polyclonal anti-GFP

Thermo Fisher

Scientific

Cat#A-10262

RRID:AB_2534023

Rabbit polyclonal anti-DsRed

Takara Bio

Cat#

632496

RRID:AB_10013483

Mouse monoclonal anti-DsRed

Invitrogen

Cat# MA-15257

RRID:AB_10999796

Mouse monoclonal anti-Armadillo

Developmental Studies

Hybridoma Bank

(DSHB)

#N2 7A1

RRID:AB_528089

Rat monoclonal anti-E-cadherin

DSHB

#DCAD2

RRID:AB_528120

Rat monoclonal anti-N-cadherin

DSHB

#MNCD2

RRID:AB_528119

Rat monoclonal anti-Elav

DSHB

#7E8A10

RRID:AB_528218

Mouse monoclonal anti-Chaoptin

DSHB

#24B10

RRID:AB_528161

Mouse monoclonal anti-Gasp

DSHB

#2A12

RRID:AB_528492

Rabbit polyclonal anti-ß

spectrin

N/A

Guinea pig polyclonal anti-phospho-Squash

N/A

Rabbit polyclonal anti-Dyl

N/A

Rat polyclonal anti-Tyn

N/A

Rabbit polyclonal anti-Pio

N/A

Chemicals, peptides, and recombinant proteins

Calcofluor White ( Fluorescent Brightener 28 disodium
salt solution)

Sigma Aldrich

Cat# 910090

10% Formaldehyde

Polysciences

Cat#

04018-1

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this version posted January 20, 2024.

bioRxiv preprint

16% Paraformaldehyde

Fisher Scientific

Cat# 50-980-487

Chitin Binding Domain-Alexa Fluor 488

Chitin Binding Domain-Alexa Fluor 546

Chitin Binding Domain -Alexa Fluor 647

Experimental models: Organisms/strains

D. melanogaster: w

1118

Bloomington

Drosophila

Stock

Center (BDSC)

BDSC:3605; FlyBase:

FBst0003605

D. melanogaster: dyl

∆42

N/A

D. melanogaster: UAS-dyl

N/A

FlyBase:

FBal0244668

D. melanogaster: Moe::mCherry P{sChMCA}22

BDSC

BDSC:35520; FlyBase:

FBst0035520

D. melanogaster:UAS-kst RNAi

P{TRiP.GLC01654}attP40

BDSC

BDSC:50536; FlyBase:

FBst0050536

D. melanogaster: UAS-Mlck RNAi

P{KK113175}VIE-

260B

VDRC

VDRC:v109937;

FBst0481578

D. melanogaster: UAS-Mlck

BDSC

BDSC:37527; FlyBase:

FBst0037527

D. melanogaster: dpy

lv1

BDSC

BDSC:278; FlyBase:

FBst0000278

D. melanogaster: Dpy-YFP

PBac{681.P.FSVS-

1}dpy

CPTI001769

Kyoto Stock Center

DGGR:115238;

FlyBase: FBti0143891

D. melanogaster: tyn

FlyBase:

FBal0345589

D. melanogaster: FRT42D, pio

V132

BDSC: 99937; FlyBase:

FBst0099937

D. melanogaster: FRT80B, dyl

∆42

This study

N/A

D. melanogaster: UAS-dyl; FRT80B,dyl

∆42

This study

N/A

D. melanogaster: Moe::mCherry; FRT80B, dyl

∆42

This study

N/A

D. melanogaster: UAS-kst-RNAi; FRT80B, dyl

∆42

This study

N/A

D. melanogaster: UAS-Mlck-RNAi; FRT80B, dyl

∆42

This study

N/A

D. melanogaster: FRT42D; UAS-Mlck

This study

N/A

D. melanogaster: Dpy-YFP; FRT80B, dyl

∆42

This study

N/A

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bioRxiv preprint

D. melanogaster: FRT40A, dpy

lv1

This study

N/A

D. melanogaster: FRT19A, tyn

This study

N/A

D. melanogaster: ey3.5-FLP, spa-GAL4; FRT42D, tub-

GAL80; UAS-CD8-GFP

This study

N/A

D. melanogaster: spa-GAL4

BDSC

BDSC:26656; FlyBase:

FBti0114943

Software and algorithms

Image J

National Institutes of

Health;

http://imagej.nih.gov.ij/

Adobe Photoshop

Adobe

GraphPad Prism 10.0

GraphPad Software

https://www.graphpad.c

om/

MS-Office (MS-Word, MS-Powerpoint, MS-Excel)

MS-Office

Other

Confocal Microscope

Leica

SP8

Resource availability

Lead contact

Further information and requests for resources and reagents should be directed to and will

be fulfilled by the lead contact, Jessica E. Treisman (Jessica.Treisman@nyulangone.org).

Materials availability

All flies and custom reagents created for this study are available upon request to the lead

contact.

Experimental model details

Fly stocks and genetics

Drosophila melanogaster

strains were maintained on standard yeast-cornmeal-agar media

and raised at 25°C. For analysis of the pupal retina, white prepupae (0 h APF) were collected

with a soft wetted brush and cultured at 25°C till the appropriate developmental stage. Both

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bioRxiv preprint

sexes were used interchangeably for all the experiments, as no sex-specific differences were

observed.

Drosophila melanogaster

stocks used to generate

dyl

∆42

mutant clones were: (1)

UAS-

CD8-GFP, ey3.5-FLP; lGMR-GAL4; FRT80B, tub-GAL80/TM6B

; (2)

FRT80B

dyl

∆42

/TM6B

;

(3)

Moe::mCherry;

FRT80B

dyl

∆42

/SM6.TM6B; (4) Dpy-YFP; FRT80B

dyl

∆42

/SM6.TM6B

; (5)

eyFLP; lGMR-GAL4, UAS-myrTomato;

FRT80B, tub-GAL80/TM6B;

(6)

UAS-dyl; FRT80B

dyl

∆42

/TM6B;

(7)

UAS-kst RNAi; FRT80B

dyl

∆42

/SM6.TM6B

(8)

UAS-Mlck RNAi; FRT80B,

dyl

∆42

/SM6.TM6B

. Stocks used to generate

Mlck

overexpression clones were: (1)

UAS-CD8-

GFP, ey3.5-FLP; lGMR-GAL4; FRT42D, tub-GAL80/TM6B

; (2)

ey3.5-FLP, spa-GAL4;

FRT42D, tub-GAL80; UAS-CD8-GFP; (3) FRT42D; UAS- Mlck

/SM6.TM6B

. Stocks that were

used to generate

dpy

lv1

mutant clones were: (1)

UAS-CD8-GFP, ey3.5-FLP; FRT40A, tub-

GAL80; lGMR-GAL4 /TM6B

; (2)

FRT40A

dpy

lv1

/SM6.TM6B.

Stocks that were used to generate

pio

V132

mutant clones were: (1

UAS-CD8-GFP, ey3.5-FLP; lGMR-GAL4; FRT42D, tub-

GAL80/TM6B

; (2)

FRT42D

pio

V132

/ SM5.

Stocks that were used to generate

tyn

mutant clones

were: (1)

ey3.5-FLP, FRT19A, tub-GAL80; lGMR-GAL4, UAS-CD8-GFP /SM6-TM6B

; (2)

FRT19A

tyn

/ FM7, Tb, Ubi-RFP.

Immunohistochemistry

For cryosectioning, adult or pupal heads with the proboscis removed were glued onto

glass rods using nail polish and fixed for 4 h in 4% formaldehyde in 0.2 M sodium phosphate

buffer (pH 7.2) (PB) at 4°C. The heads were then incubated through an increasing gradient of

sucrose in PB (5%, 10%, 25%, and 30% sucrose) for 20 min each, transferred to plastic molds

containing OCT compound and frozen on dry ice. Cryosections of 12 µm were cut at −21°C,

transferred onto positively charged slides and postfixed in 0.5% formaldehyde in PB at room

temperature (RT) for 30 mins. The slides were then washed in PBS with 0.3% Triton X-100

(PBT) three times for 10 min each, blocked for 1 h at RT in 1% bovine serum albumin (BSA) in

PBT and incubated in primary antibodies overnight at 4°C in 1% BSA in PBT. After three 20-

minute washes in PBT, slides were incubated in secondary antibodies in 1% BSA in PBT for 2 h

at RT and mounted in Fluoromount-G (Southern Biotech). A 1:5 dilution of Calcofluor White

solution (25% in water; Sigma Aldrich, 910090) was included with the secondary antibodies

where indicated.

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bioRxiv preprint

Pupal retinas attached to the brain were dissected from staged pupae and collected in ice-

cold PBS in a glass plate. These samples were fixed on ice in 4% formaldehyde in PBS for 30

min. The samples were washed three times for 10 min each in PBT and incubated overnight at

4°C in primary antibodies in 10% donkey serum in PBT. After three 20-min washes in PBT, the

samples were incubated for 2 h in secondary antibodies in PBT/10% serum at RT, and washed

again three times for 20 min in PBT. Finally, the retinas were separated from the brain and

mounted in 80% glycerol in PBS.

The primary antibodies used were: mouse anti-Chp (1:50; Developmental Studies

Hybridoma Bank (DSHB), 24B10), chicken anti-GFP (1:400; Thermo Fisher, A-10262), rat anti-

Elav (1:100; DSHB, Rat-Elav-7E8A10), rat anti-Ecad (1:10, DSHB, DCAD2), mouse anti-Gasp

(1:20, DSHB, 2A12), rabbit anti-Dyl (1:300)

, rat anti-Tyn (1:100)

, rabbit anti-Pio (1:300)

guinea pig anti-pSqh (1:1000)

, and rabbit anti-ß

-spectrin (1:5000)

. All antibodies were

validated either using mutant or knockdown conditions as shown or by verifying that the staining

pattern matched previously published descriptions. The secondary antibodies used were from

either Jackson ImmunoResearch (Cy3 or Cy5 conjugates used at 1:200) or Invitrogen (Alexa488

conjugates used at 1:1000).

Fluorescently labeled SNAP-CBD-probes (1:200)

were included

with the secondary antibodies.

Images were acquired on a Leica SP8 confocal microscope with a

63X oil immersion lens and processed using ImageJ and Adobe Photoshop.

Electron microscopy

For transmission electron microscopy, adult heads were cut in half and incubated in

freshly made fixative containing 2.5% glutaraldehyde, 2% paraformaldehyde, and 0.05% Triton

X-100 in 0.1 M sodium cacodylate buffer pH 7.2 (CB) on a rotator for at least 4 h until all heads

had sunk to the bottom of the tube, and then in the same fixative without Triton X-100 overnight

at 4

C on a rotator. After washing three times for 10 min in CB, the heads were post-fixed in 1%

OsO

in CB for 1.5 h, washed three times for 10 min in water, dehydrated in an ethanol series

(30%, 50%, 70%, 85%, 95%, 100%), rinsed twice with propylene oxide and embedded in

EMbed812 epoxy resin (Electron Microscopy Sciences). 70 nm ultrathin sections were cut and

mounted on formvar-coated slot grids and stained with uranyl acetate and lead citrate

. Electron

microscopy imaging was performed on a Talos120C transmission electron microscope (Thermo

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this version posted January 20, 2024.

bioRxiv preprint

Fisher Scientific) and recorded using a Gatan (4k×4k) OneView Camera with Digital

Micrograph software (Gatan).

For scanning electron microscopy, whole flies were fixed for 2 h at room temperature and

then overnight at 4

C in 1% glutaraldehyde/1% formaldehyde/0.2% Triton X-100/0.1 M sodium

cacodylate pH 7.2. After three 10 min washes in PBS, the flies were postfixed in 1% OsO4 in

CB for 1 h and rinsed three times for 10 min in water. They were dehydrated in an ethanol series

(30%, 50%, 70%, 85%, 95%, 3X 100%) and stored in 100% ethanol overnight. The eyes were

critical point dried using a Tousimis Autosamdri®-931 critical point dryer (Tousimis, Rockville,

MD), mounted on SEM stabs covered with double sided electron-conductive tape, coated with

gold/palladium by a Safematic CCU-010 SEM coating system (Rave Scientific, Somerset, NJ),

and imaged on a Zeiss Gemini300 FESEM (Carl Zeiss Microscopy, Oberkochen, Germany)

using a secondary electron detector (SE

) at 5 kV with working distance (WD) between 15.2 mm

and 19.4 mm.

Quantification and statistical analysis

The outer and inner angles between adjacent corneal lenses were measured according to

the schematic in Fig. 1E, using the angle tool in ImageJ. To measure the apical surface areas of

ommatidia and central cells, ROI were drawn and measured in ImageJ using the freehand

selection tool in Z-projections that included the apical surface of the retina. To measure

ommatidial height, freehand straight lines were drawn from the corneal lens surface to the base

of the rhabdomere using the line tool in ImageJ and measured. Values were plotted in GraphPad

Prism v10. Significance was calculated using Welch's two-tailed unpaired

-tests. Sample

numbers and definitions of error bars are given in the figure legends. Sample sizes for

quantifications were not predefined and no samples were excluded.

Data and code availability

This study did not generate or analyze any datasets/codes. Any additional information

required to reanalyze the data reported in this paper is available from the Lead Contact upon

request.

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this version posted January 20, 2024.

bioRxiv preprint

Acknowledgments

We thank Markus Affolter, Shigeo Hayashi, François Payre, Robert Ward, Christian

Klämbt, the Bloomington

Drosophila

stock center, the Vienna

Drosophila

resource center, the

Kyoto stock center, and the Developmental Studies Hybridoma Bank for fly stocks and reagents.

Information available on FlyBase was invaluable for this work. We thank NYULH DART

Microscopy Laboratory members Alice F.-X. Liang, Joseph Sall, Jason Liang and Chris Petzold

for consultation and assistance with electron microscopy. This core is partially funded by NYU

Cancer Center Support Grant NIH/NCI P30CA016087, and the Gemini300 FESEM was

supported by NIH S10 OD019974. We thank Dhaval Gandhi, Sharukh Khan and Genie Jang for

technical assistance. The manuscript was improved by the critical comments of Gira Bhabha,

Maria Bustillo, Holger Knaut, Sudershana Nair, and Hongsu Wang. This work was funded by the

National Institutes of Health (grant R01EY032896 to J.E.T.).

Author contributions

Conceptualization, N.G. and J.E.T.; investigation, N.G.; data curation, N.G.; formal

analysis, N.G. and J.E.T.; writing- original draft, N.G.; writing – review and editing, J.E.T.;

funding acquisition, J.E.T.; supervision, J.E.T.

Declaration of interests

The authors declare no competing interests.

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bioRxiv preprint

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