Hedgehog Signaling Is a Principal Inducer of Myosin-II-Driven Cell Ingression in Drosophila Epithelia

Developmental Cell

Article

Hedgehog Signaling Is a Principal Inducerof Myosin-II-Driven Cell Ingressionin Drosophila EpitheliaDouglas Corrigall,1 Rhian F. Walther,1 Lilia Rodriguez,1 Pierre Fichelson,1 and Franck Pichaud1,*1MRC Laboratory for Molecular Cell Biology and Cell Biology Unit, Department of Anatomy and Developmental Biology,

University College London, Gower Street, WC1E 6BT, London, United Kingdom

*Correspondence: [email protected] 10.1016/j.devcel.2007.09.015

SUMMARY

Cell constriction promotes epithelial sheet in-vagination during embryogenesis across phyla.However, how this cell response is linked toglobal patterning information during organo-genesis remains unclear. To address this issue,we have used the Drosophila eye and studiedthe formation of the morphogenetic furrow (MF),which is characterized by cells undergoing asynchronous apical constriction and apicobasalcontraction. We show that this cell responserelies on microtubules and F-actin enrichmentwithin the apical domain of the constricting cellas well as on the activation of nonmuscle myo-sin. In the MF, Hedgehog (Hh) signaling is re-quired to promote cell constriction downstreamof cubitus interruptus (ci), and, in this context,Ci155 functions redundantly with mad, themain effector of dpp/BMP signaling. Further-more, ectopically activating Hh signaling in flyepithelia reveals a direct relationship betweenthe duration of exposure to this signaling path-way, the accumulation of activated Myosin II,and the degree of tissue invagination.

INTRODUCTION

Organogenesis requires the precise regulation of cell

proliferation, movement, and apoptosis to achieve proper

organ shape and size. In addition, the folding of epithelial

cell sheets plays a crucial role in shaping organs such as

the heart, lung, and kidney. In many organisms, epithe-

lial-cell sheet invagination is promoted when a few epithe-

lial cells adopt a bottle- or wedge-like shape resulting from

a striking constriction of their apical domain. Such cell-

shape changes create a local mechanical stress within

the epithelium that is instrumental for promoting tissue

invagination (Kimberly and Hardin, 1998). This is likely to

be a conserved feature in tissue patterning because the

formation of bottle-shaped cells associated with tissue

invagination has been identified in the sea urchin during

730 Developmental Cell 13, 730–742, November 2007 ª2007 E

gastrulation (Davidson et al., 1995), during neural tube clo-

sure in the vertebrate notochord (Schroeder, 1970), and in

the developing fly embryo (Kiehart et al., 1990), to name

but a few examples.

Apical cell constriction is crucial for mesoderm invagina-

tion during Drosophila development, and, in the past de-

cade, key molecular players involved in this process

have been identified. In the fly embryo, cell constriction is

characterized by an accumulation of apical F-actin and

activation of hexameric, nonmuscle MyoII through the

RhoA-Rok signaling pathway (Dawes-Hoang et al., 2005;

Hacker and Perrimon, 1998; Nikolaidou and Barrett,

2004; Seher et al., 2006). Cell constriction is achieved

through the assembly of MyoII into bipolar filaments and

the assembly of short bundles of unbranched F-actin that

act as a substrate for the motile activity of MyoII. Rok ap-

pears to be the principle kinase that activates MyoII via

phosphorylation of a conserved serine at position 19 of

the myosin regulatory light chain (MRLC) (encoded by spa-

ghetti squash [sqh]) (Amano et al., 1996). Activated MyoII is

then thought to be involved in pulling the adherens junc-

tions (AJs) toward the apical pole of the cell, thereby ‘‘eat-

ing-up’’ the apical surface (Dawes-Hoang et al., 2005;

Kolsch et al., 2007). In addition, a recent report revealed

that transient AJ disassembly also takes place during cell

constriction (Kolsch et al., 2007). In the fly embryo, ventral

furrow formation involves the transcription factor Twist

upstream of the G protein Concertina pathway (Parks

and Wieschaus, 1991; Seher et al., 2006) as well as the

recently identified transmembrane protein T48 (Kolsch

et al., 2007). In this context, the putative secreted protein

Folded gastrulation induces the cytoskeletal changes

required to promote tissue invagination (Costa et al., 1994;

Morize et al., 1998). However, knowledge about the up-

stream signaling pathways that provide the patterning

information that governs cell constriction and eventually

cell ingression during organogenesis remains scant.

A situation strongly reminiscent of cell constriction is

cell cytokinesis. Specifically, the assembly of the contrac-

tile ring necessary for the function of the cleavage furrow

and cell division also requires the assembly of parallel

F-actin and motile forces provided by MyoII (Dean et al.,

2005; Hickson et al., 2006). Additionally, the MyoII-driven

motile force required to achieve cell cleavage involves

the RhoA-Rok signaling pathway (Glotzer, 2001). In this

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Hh Signaling and Cell Constriction

context, the microtubule-associated and F-actin-nucleat-

ing formin Diaphanous (Dia) is required for stabilizing

MyoII at the cleavage furrow. This is achieved through the

actin-nucleating activity of the formin homology (FH) 2

domain present in Dia and is presumably combined with

the ability of this protein to directly interact with Profilin,

another F-actin effector (Chang et al., 1997). However,

a role for dia in cell constriction remains to be examined.

To gain insight into how cell constriction and tissue

invagination are regulated during development, we turned

to the genetically amenable Drosophila eye imaginal disc.

Patterning of this columnar pseudostratified epithelium

depends on the formation and movement of the morpho-

genetic furrow (MF) along the anterior-posterior axis. The

MF is characterized by cells undergoing a synchronous

apical constriction and apicobasal contraction together

with a cell-cycle arrest in G1 (Ready et al., 1976; Wolff

and Ready, 1991). Hh and Dpp signaling are instrumental

for propagating the MF; Hh is produced by the developing

neurons and then diffuses more anteriorly (Figure 1A). The

two signaling pathways rely on transcriptional regulation

of genes downstream of Ci and Mad, respectively (Lum

and Beachy, 2004).

We show that the cell constriction and mild ingression

observed in the MF are transcriptionally controlled down-

stream of Hh. This signaling pathway requires the small

GTPase RhoA and its effector, Rok, to control the activa-

tion of nonmuscle MyoII, whereas Dia, coupled with the

regulated activity of Cofilin and Profilin, is required for

proper F-actin enrichment within the apical cortex of the

constricting cells. Our data are consistent with a model

in which the stabilization of apical MTs in the MF is cou-

pled to the assembly of parallel F-actin within the apical

cell cortex and in which the motile force necessary for

apical constriction is provided by MyoII. Importantly, our

work suggests a dose-dependent relationship between

the level of phosphorylated MyoII within the apical mem-

brane and the types of structures produced in a columnar

epithelium. Short-term exposure to Hh signaling leads to

cell constriction and mild ingression, whereas longer

exposure to this pathway is accompanied by higher levels

of activated MyoII and leads to major tissue invagination.

RESULTS

Cell Constriction in the Morphogenetic FurrowRequires RhoA/Rok and MyoIICells in the MF are characterized by a striking apical cell

constriction (Figures 1A–1C) and apico-basal contraction

(Figures 1D–1F), which can be viewed as a mild case of

tissue invagination (Ready et al., 1976; Wolff and Ready,

1991). This is accompanied by an increase in F-actin

and E-cadherin (encoded by shotgun [shg]) within the api-

cal cortex of the constricting cells (Figures 1A and 1H). In-

terestingly, we also observed a substantial stabilization of

the apical MT network (Figures 1E and 1F) in the constrict-

ing cells in a region spanning the apical domain and sep-

tate junctions (SJs), which are located basal to the AJs.

These cytoskeletal changes are accompanied by a dra-

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matic reduction of the apical cell surface area (Figures

1A–1C) also apparent at the level of the basal SJs, as

seen by labeling with an antibody against Discs-large

(Dlg) (not shown). Whereas the MF is characterized by

an acute decrease in the apical cell surface of over �10

cell diameters, the whole anterior compartment of the

eye disc is characterized by a graded decrease of the api-

cal cell surfaces along the anterior-posterior axis (Figures

1A and 1B); smaller apical surfaces are measured closer

to the MF. This suggests that the MF response is distrib-

uted over the entire anterior compartment of the develop-

ing eye. In addition, optical sagittal sections (Figure 1D)

revealed that, in the anterior compartment, cells become

increasingly taller along the anterior-posterior axis; cells

in the anterior-most part of the epithelium are more cuboi-

dal than the columnar cells close to the MF.

Consistent with the hypothesis that MyoII could be

required for cell constriction in the MF, we observed

MRLC enrichment at the apical cortex of the constricting

cells in the MF (Figure 1G), where it is activated through

phosphorylation of Ser19 (Figures 1H–1K). We next used

the FLP/FRT system (Xu and Rubin, 1993) in the develop-

ing eye to examine the effects of removing the function of

genes previously shown to activate and modulate MyoII

activity. Removing rok function in the MF caused a marked

decrease in apical F-actin quantity and a statistically sig-

nificant (p < 0.05) impairment in apical cell constriction

(WT-MF cell surface/rok cell surface: 0.539) (Figures 2A

and 2B; Tables S1 and S2, see the Supplemental Data

available with this article online for quantification) and

apico-basal contraction (Figures 2C and 2D). This was ac-

companied by a statistically significant (p < 0.05) decrease

in Ser19 MRLC phosphorylation (Figures 2E–2H; Table S3

for quantification). Nevertheless, we could detect apical

Myosin Heavy Chain (Zipper [Zip]) in these mutant cells

(Figures 2A and 2D and data not shown). Importantly, ep-

ithelial cell polarity was maintained in these mutant cells

(not shown), indicating that the impaired MF cell response

was not caused by a loss of polarity. Although Rok func-

tion is key for cell constriction, expressing an activated

form of this kinase (RokCAT) (Winter et al., 2001) in clones

was not sufficient to promote cell constriction in the ante-

rior compartment of the eye disc (Figures S1A–S1C).

We next examined mutant cells deficient for MRLC (sqh).

We were only able to recover small mutant clones by using

a hypomorphic allele of MRLC, probably because MRLC is

required for normal cytokinesis (Edwards and Kiehart,

1996). In these mutant clones, we observed a failure in

the MF cell response in that both constriction and apico-

basal contraction failed to occur (Figures 2I–2L and data

not shown). Similarly, cell constriction and apico-basal cell

contraction failed to occur when a dominant-negative ver-

sion of MRLC lacking the F-actin-binding domain (Dawes-

Hoang et al., 2005) was expressed in clones (not shown).

Interestingly, removal of the Myosin phosphatase-binding

subunit (mbs) in mosaic eye discs led to groove formation

in the case of clones located in the MF (Figures 2M–2P0).

This finding suggests that different levels of MRLC phos-

phorylation might promote different degrees of tissue

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Figure 1. Epithelial Morphogenesis and Drosophila Eye Organogenesis

All of the tissue samples are orientated along the anterior-posterior axis. The MF is marked with a solid, white line.

(A) Apical view of a developing eye labeled for the AJ marker DECadherin:GFP. The anterior compartment is indicated by a yellow line, whereas the

proneuronal compartment and MF are indicated by turquoise and blue lines, respectively. The posterior compartment is indicated by a purple line.

(B) Representation of the apical cell surface measured along the anterior-posterior axis of the developing eye (n = 5 discs). A red line marks the MF.

(C) Schematic representation of a columnar epithelial cell found in the anterior compartment ahead of the MF (left) and a constricting cell found in the

middle of the MF (right).

(D) Sagittal section of a developing eye labeled for F-actin (red) and a marker of the SJs, Dlg (green). ppm, peripodial membrane.

(E) High magnification of the eye primordium centered on the MF. The F-actin is labeled in red, whereas the microtubules (MTs) are labeled with

a-tubulin (green).

(F) Same as (E), but showing the MTs only.

(G) A fusion protein between MLRC and GFP is used to report MyoII localization (green). PATJ (blue) labels the apical domain, and Dlg (red) labels the SJs.

(H–K) Phosphorylation at Ser19 of MLRC (green) in the MF. Arm marks the AJs (blue); phalloidin labels F-actin (red).

invagination, with higher levels associated with tube for-

mation. Importantly, high levels of expression of an acti-

vated form of MRLC (SqhE20E21) in the anterior compart-

732 Developmental Cell 13, 730–742, November 2007 ª2007

ment of the developing eye disc proved to be sufficient

for inducing reproducible cases of minor cell constriction

accompanied with tissue ingression (Figures 2Q–2T).

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Figure 2. Patterning of the Eye Imaginal

Disc: MF and MyoII-Driven Cell Constric-

tion

In all panels, the MF is indicated by a white line,

and the clones, marked by the absence of blue

(b-galactosidase or GFP staining), are circled

with a dashed, white line.

(A and B) Cells mutant for the null allele rok2

showing a strong reduction in apical F-actin

staining (red) correlated with a failure to prop-

erly achieve cell constriction. The dashed lines

labeled (C) and (D) mark the position corre-

sponding to the sagittal sections seen in (C)

and (D), respectively.

(C and D) Sagittal sections showing a failure of

the (D) rok2 mutant cells to undergo apicobasal

contraction, compared to the (C) wild-type tis-

sue shown in the same preparation.

(E–H) Cells mutant for the null allele rok2. F-

actin staining is shown in red, and Phospho-

Ser19 MRLC staining is shown in green. The

cell nuclei are labeled with DAPI (white) in (H).

(I–L) Cells mutant for MRLC (sqh1). F-actin

staining is shown in red, and Dlg staining is

shown in green.

(M–P0) Eye disc presenting a clone mutant for

mbs791. (M)–(P) show an optical section at the

level of the apical membrane of the constricting

cells in the MF, and the position of the mutant

clone is indicated with the dashed line. (M0–

P0) show a basal view of the same region of

the eye disc. This reveals that the apical mem-

branes of the mbs791 mutant cells are located

at the level of the basal nuclei (F-actin staining

is shown in red; the AJ marker Arm staining is

shown in green).

(Q–T) Activated MRLC (SqhE20E21) induces

mild cell ingression (lack of green staining).

F-actin staining is shown in red; Nuclei are

stained blue. An arrowhead points to the ec-

topic cell ingression.

(U–X) Eye disc mutant for RhoA72M1 induced by

using the Minute technique. MRLC phosphory-

lation at Ser19 is shown in green.

Finally, in the absence of RhoA function, we observed

a dramatic loss of cell constriction and apicobasal con-

traction in the MF, associated with weak F-actin staining

and a diffuse pattern of MRLC phosphorylation at Ser19

when compared to wild-type (Figures 2U–2X). Taken to-

gether, our findings demonstrate that cell constriction

and ingression in the MF depend upon the action of

RhoA, Rok, and MyoII. Our observation of residual phos-

phorylation of MRLC at Ser19 in rok mutant cells (Table

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S3) confirms a previous report (Lee and Treisman, 2004)

and suggests that another kinase acts in parallel with

Rok to phosphorylate this residue.

The Formin Dia Is Required for Apical CellConstrictionOne major effect of removing RhoA and rok function in the

MF is a significant reduction in F-actin accumulation in the

apical domain of the cell (Figures 2B and 2V; Table S2 for

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quantification). Previous work on the regulation of cytoki-

nesis has revealed that RhoA acts upstream of the formin

Dia (Dean et al., 2005; Hickson et al., 2006). In addition, to-

gether with Dia, both profilin (encoded by chickadee [chic])

and ADF/cofilin (encoded by twin star [tsr]) have been

shown to be important for cytokinesis through promoting

F-actin reorganization at the contractile ring and thus

providing a substrate for MyoII-mediated motility (Ishizaki

et al., 2001; Palazzo et al., 2001). This prompted us to

test whether dia could function in apical constriction. First,

we showed that Dia is localized in the apical domain of

constricting cells (Figures 3A–3C). We then showed that

the removal of dia completely inhibited apical constriction

in the MF (Figures 3D–3G) and largely prevented F-actin

enrichment in the mutant cells (Figures 3E and 3G). To-

gether, these observations suggest that cell constriction

is dependent upon Dia-driven enrichment of actin fila-

ments in the apical domain of these cells. However,

ectopic expression of a constitutively activated form of

Dia, Dia-CA (Somogyi and Rorth, 2004), failed to trigger

cell constriction or MT stabilization in the corresponding

expressing cells in the eye and wing imaginal discs (not

shown).

We then examined clones mutant for profilin. As previ-

ously reported in the developing eye (Lee and Treisman,

2004), a clear loss of apical F-actin is observed in the cor-

responding mutant cells (Figures 3H–3K). However, we

also noted that this was accompanied by an alteration of

cell shape and a substantial loss of apico-basal polarity,

including a strong decrease in the SJ marker Dlg (Fig-

ure 3J). We next assayed a role for ADF/cofilin and, as

previously reported (Lee and Treisman, 2004), we noted

a strong statistically significant (p < 0.05) increase in cor-

tical F-actin in the corresponding mutant cells (Figures

3L–3O; Tables S1 and S2). The corresponding apical cell

surface areas were significantly (p < 0.05) enlarged (Ta-

bles S1 and S2). A similar situation was obtained when re-

moving the cofilin phosphatase slingshot (ssh) in clones

(Figures 3P–3S; Tables S1 and S2). The increase in cell

surface area measured in cofilin and ssh mutant cells in

the MF is likely to be the result of these cells being filled

with a large excess of F-actin that spreads basally toward

the SJs (not shown). This set of data indicates that Dia,

Profilin, and Cofilin act in concert to promote apical F-

actin enrichment in parallel with MyoII, which is needed

for cell constriction. Our data are compatible with the

possibility that both MyoII and dia act downstream of rok

and RhoA, a situation strongly reminiscent of that ob-

served during cytokinesis (Dean et al., 2005).

Cell Constriction and Actin EffectorsA recent study has revealed a role for the nonreceptor and

actin-binding Abelson tyrosine kinase (Abl) and the actin

effector Enabled (Ena) in promoting cell constriction in

the fly mesoderm (Fox and Peifer, 2007). However, we

could not detect any defect in cell constriction in the MF

when inducing mutant clones for either Abl or ena by using

null alleles for these two genes (Abl4 and ena23, respec-

tively) (Figures 3T–3W and 3X–3A0). This might be due to


some redundancy in this cell response, and, in that

respect, it is interesting to note that in the mesoderm, cells

undergoing apical constriction could be detected in the

absence of Abl function (Fox and Peifer, 2007). Similarly,

we failed to detect defects in apical constriction in the

MF when generating loss-of-function mutant cells for

DWave (using a null allele, scarD37) (Figures 3B0–3E0) and

Wasp (using a combination of independent alleles, wsp1

and wsp3; data not shown), both key effectors of F-actin

and regulators of the Arp2/3-actin-nucleating complex

(Machesky et al., 1994; Miki and Takenawa, 2003; Naka-

gawa et al., 2001). It is also interesting to note that, despite

our efforts with various allelic combinations and loss-of-

function mutant clones, we failed to detect defects in

cell constriction in mutant cells for Rho-GEF2 (Figures

S1E–S1G), a conserved Guanosine Exchange Factor

(GEF) responsible for loading GTP onto Rho1/A. When

generating whole mutant eye discs for Rho-GEF2 we

could readily detect defects in the gross morphology of

the disc, including the folding of the corresponding disc

onto itself (data not shown). These data suggest that, in

the MF, another GEF might function in parallel with Rho-

GEF2, or that Rho-GEF2 is not involved in cell constriction

in the MF. Consistent with an important role for the AJs

during cell constriction, mutant clones for arm induced

in the MF led to a failure in this cell response (Figures

S1H–S1K).

Hh Signaling Is a Principal Inducer of Apical CellConstrictionWe next investigated the potential upstream regulator(s)

responsible for orchestrating the MF cell response. It

has previously been reported that both the Dpp and Hh

pathways provide spatial and temporal patterning infor-

mation in the eye imaginal disc (Chanut and Heberlein,

1997; Borod and Heberlein, 1998). Hh and Dpp signaling

are involved in MF induction and propagation, but it is

not clear if both of these pathways are required for cell

constriction in the MF. To address this issue, we induced

loss-of-function clones in the developing eye disc for

mothers against dpp (mad; vertebrate smad5/7) (Figures

4A–4D; Figure S1D), the Hh coreceptor smoothened (smo)

(Figures 4E–4H and 4M–4P), or both mad and smo (Fig-

ures 4I–4L, 4Q–4T, and 4U–4X). As previously reported

in the wing disc (Gibson and Perrimon, 2005; Shen and

Dahmann, 2005), removing mad function in the eye by

using a null allele led to clones producing cysts that some-

times could be recovered in the basal part of the epithe-

lium (Figure S1D). However, we could also recover a num-

ber of clones in the MF that were relatively small and did

not produce cysts. In these clones, we did not detect sig-

nificant changes (p > 0.05; Table S1) in cell constriction

(cell surfaces WT-MF/mad: 0.82), F-actin accumulation,

or MRLC-Ser19 phosphorylation at the apical cortex (Fig-

ures 4A–4C; Tables S1–S3). The fraction of mad mutant

clones that we were able to recover in the MF did not

affect the characteristic apico-basal contraction of the cor-

responding cells (Figure 4D). Conversely, in smo mutant

clones, we consistently observed a strong and statistically

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Figure 3. Cell Constriction Depends on

the Activity of Dia, Profilin, and Cofilin

(A–C) Sagittal section of an eye imaginal disc.

Dia (blue) colocalizes with MLRC:GFP (green)

and the AJ marker Arm (red).

(D–G) Cells mutant for dia5. F-actin staining is

shown in red, and Dlg marks the SJs (green).

(H–K) Cells mutant for profilin (chic221).

(L–O) Cells mutant for cofilin (tsr1).

(P–S) Cells mutant for ssh221.

(T–W) Cells mutant for Abl4. F-actin is labeled in

red, whereas the AJ marker Arm is in green.

(X–A0) Cells mutant for ena23. F-actin is labeled

in red, whereas the neuronal marker Elav is in

green.

(B0–E0) Cells mutant for SCARD37. F-actin is la-

beled in red, whereas the SJ marker Dlg is in

green.

significant (p < 0.05) impairment in apical cell constriction

in the MF (cell surfaces WT-MF/smo: 0.43; Figures 4E–4H

and Table S1), which was accompanied by a dramatic

Developme

decrease in F-actin accumulation (Figure 4F; Table S2)

and a marked decrease in b-catenin (Armadillo [Arm]) ac-

cumulation at the AJs (data not shown). In addition, we


Developmental Cell


Figure 4. Hh Signaling Regulates MyoII-

Driven Cell Constriction

(A–C) Cells mutant for mad12. F-actin is labeled

in red.

(D) Sagittal section across a mad12 mutant

clone showing that cells still achieve proper

apicobasal contraction (marked with a white

line).

(E–H) Cells mutant for smoD16 exhibiting a fail-

ure in apical F-actin enrichment (red). Dlg

marks the SJs (green).

(I–L) Double mutant clone for mad1-2 and smo3

exhibiting a failure in apical F-actin enrichment

(red). Dlg marks the SJs (green).

(M–P) smoD16 mutant cells showing a total loss

of MRLC phosphorylation at Ser19 (green).

(Q–T) Double smo3, mad1-2 mutant cells exhib-

iting failure to achieve apical constriction

(F-actin, red) and a total loss of MRLC phos-

phorylation at Ser19 (green).

(U–X) Loss of Dia staining (green) at the apical

cortex in the MF in the absence of smo3 and

mad1-2 function. F-actin is labeled in red, and

the mutant cells are indicated with a white

asterisk.

measured a significant decrease (p < 0.05) in the levels of

MRLC phosphorylation at Ser19 at the apical cortex of

these mutant cells (Figure 4Q; Table S3), implying that

Hh signaling plays a major role in inducing cell constriction

and apico-basal contraction in the MF. However, by using

Dlg to label the SJs, we could still observe a remnant of the

MF (Figure 4G), suggesting that Dpp and Hh might act in

a partially redundant manner in the control of the MF cell

response. To test this hypothesis, we removed both of

these two signaling pathways in clones mutant for both

mad and smo. The mutant cells completely failed to

undergo apical constriction (cell surfaces WT-MF/mad,

smo: 0.30; Figures 4I–4L and Table S1) and apico-basal

contraction. Furthermore, there was no F-actin accumula-

tion at the apical cortex (Table S2) or MRLC phosphoryla-


tion at Ser19 (Figures 4Q–4T; Table S3). In these mutant

cells, we also observed a substantial decrease in apical

Dia staining (Figures 4U–4X). Nonetheless, cell polarity

was preserved, as assayed by using marginal zone, AJ,

and SJ markers (Figure 4K and data not shown).

Apical Cell Constriction in the MF IsTranscriptionally Regulated Downstream of HhTo further test the function of Hh signaling in cell constric-

tion, we removed the ci locus (i.e., both Ci155-transcrip-

tional activator and Ci75-transcriptional repressor), the

zinc finger transcription factor responsible for mediating

Hh signaling downstream of Smo (for a review, see [Lum

and Beachy, 2004]). In ci mutant clones induced in the

anterior compartment of the eye imaginal disc, we

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Figure 5. Hh Signaling Transcriptionally

Regulates Cell Constriction and Ingres-

sion

(A–D) Cells mutant for ci showing ectopic api-

cal constriction immediately anterior to the

endogenous MF. F-actin (red) and Dlg labeling

the SJs (green).

(E) Sagittal section of a cullin116E clone.

(F–H) Double mutant clone for the transcription

factor ci and mad1-2 fail to show ectopic apical

constriction.

(I–M) Sagittal section across a clone mutant

for ci (loss of green staining, dashed lines).

(I) A phospho-specific antibody labels P-Mad

(blue); (K) F-actin staining is shown in red,

and the (I) cell nuclei are labeled with DAPI

(white).

(N–R) (N) Sagittal section across an eye disc

expressing spastin:GFP. (O) P-Ser19 staining

(blue). (P) MT staining in red. (R) Nuclei are

stained with DAPI (white).

consistently observed ectopic MF-like cell responses,

including apical MT stabilization and MyoII activation

(Figures 5A–5D and not shown). Similarly, we prevented

Ci75-transcriptional repressor formation by removing

cullin1, a ubiquitin-ligase required to generate the tran-

scriptional repressor Ci75 from the transcriptional activa-

tor Ci155 (Ou et al., 2002). cullin1 loss-of-function clones

led to ectopic MF cell responses in the anterior compart-

ment immediately ahead of the MF (Figures 5E). These

experiments suggest that alleviating Ci75-mediated tran-

scriptional repression is sufficient to induce cell constric-

tion. Surprisingly, while mad is not absolutely required

for a cell to constrict in the MF, loss of mad function in ci

mutant cells (in double ci and mad mutant cells) led to

a lack of ectopic cell constriction in the corresponding

cells (Figures 5F–5H). This indicates that, in the absence

of Ci155-mediated transcriptional activation, ectopic cell

constriction requires Dpp signaling to proceed. Although,

all of the cells located in the anterior compartment of the

eye disc receive the Dpp signal, as reported by a phos-

pho-specific antibody raised against active P-Mad (Fig-

ures 5I–5M), we did not observe increased P-Mad staining

in ci mutant clones. Altogether, this suggests that mad

function is required for cell constriction to occur in ci

mutant cells, whereas in the MF, Ci-155 and Mad might

act redundantly to promote this cell response.

In ci mutant cells and in the endogenous MF, cell con-

striction is accompanied by MT stabilization within the

apical cell domain. To assay their contribution during cell

Developmen

constriction, we used the MT-severing factor Spastin

(Sherwood et al., 2004) to depolymerize MTs in vivo (Fig-

ures 5N–5R). When expressed in the MF (Figure 5N),

Spastin led to a near total depletion of the MT network,

accompanied by a loss of apical F-actin accumulation

and a loss of Ser19-P MyoII staining. In addition, the

basally located nuclei normally found in the MF appeared

randomly distributed and had a tendency to be close to

the apical cell surfaces (Figure 5Q).

Hh Signaling Induces a Range of TissueInvaginationWe next tested whether Hh signaling was sufficient to trig-

ger cell constriction in fly epithelia. To this end, we ectop-

ically activated the Hh signaling pathway in the eye,

antenna, and wing imaginal discs by generating mutant

clones in which the inhibitory coreceptor patched (ptc)

was removed (Hooper and Scott, 1989). In ptc mutant

clones, Hh signaling is constitutively activated at its max-

imal strength. Interestingly, these clones led to a range of

responses, depending on the levels of activation of MyoII

measured through quantifying the Ser19-P MRLC staining

(Table S3). In the case of ptc clones dissected 24 hr

after clone induction, we invariably observed MF-like

responses (Figures 6A–6D; Tables S1 and S2) together

with levels of Ser19-P MRLC comparable to those mea-

sured in the endogenous MF cells (p > 0.005; Table S3).

This was accompanied by a pronounced bundling of

the apical MTs (Figures 6E–6H) and a striking apical

tal Cell 13, 730–742, November 2007 ª2007 Elsevier Inc. 737

Developmental Cell


Figure 6. Hh Signaling Promotes MyoII-

Driven Tissue Ingression

(A–D) Sagittal sections showing a mild case of

cell ingression for ptcS2 mutant cells in the eye

disc. F-actin staining is shown in red, and

Ser19-P-MRLC staining is shown in green.

The endogenous MF is indicated by a white

line in (B) and (C) and by a red line in (D).

(E–H) ptcS2 mutant clones induced in the ante-

rior compartment of the eye showing F-actin

(red) and MT stabilization (green).

(I–L) Sagittal section of a ptcS2 mutant clone

induced in the wing disc and leading to tissue

invagination. F-actin staining is shown in red,

and Ser19-P-MRLC staining is shown in green.

(M) Sagittal section of a ptcS2 mutant clone

induced in the eye disc and leading to groove

formation. F-actin staining is shown in red,

and Dlg staining is shown in blue.

(N) Schematic representation of the endoge-

nous MF cell response. Blue labels the dpp-ex-

pressing cells, whereas orange labels the Hh-

producing cells.

(O) Schematic representation of the situation

described in (L) upon removal of patched

(ptcS2). This leads in the wing to alleviation of

Ci75-mediated transcriptional repression, and

it allows for Ci155-mediated transcriptional ac-

tivation as well as the induction of dpp expres-

sion (Methot and Basler, 1999).

enrichment of F-actin (Figures 6F; Table S2). This was not

limited to the eye imaginal disc because removing ptc func-

tion in the antennal or wing discs also resulted in ectopic

activation of MyoII and enrichment of apical F-actin and

MT stabilization, leading to a striking cell constriction in

the affected tissue (Figures 6I–6L and not shown).

Interestingly, when ptc mutant clones were dissected

48–72 hr after their induction, we primarily observed an

ingression of the ptc mutant cells, with the cells dropping

down within the columnar epithelium, without loss of

polarity or loss of adhesion. This was the case in all imag-

inal discs, including the wing (Figures 6I–6L) and the eye

(Figure 6M). As expected, the ptc mutant cells showed

an increase in apical F-actin and MRLC phosphorylation

at Ser19 (Figure 6J, 6K, and 6M). Quantification of Ser19

phosphorylation of MRLC in the eye revealed that cells

located at the bottom of such ptc-induced tubes exhibit

a significantly higher level (p < 0.05) of activated MyoII

when compared to the endogenous MF cells (Table S3).

These observations suggest that Hh signaling can induce

cell ingression and groove formation in a MyoII-dependent

manner (Figures 6N–6O; Table S3) provided there are

a minimum number of Hh signaling cells and adequate

levels of activated MyoII.


DISCUSSION

Hh Signaling and Tissue PatterningThe Hh and Dpp families of ligands are of crucial impor-

tance for patterning a wide variety of tissues across spe-

cies. Our work on Hh signaling and that of others on

Dpp (Gibson and Perrimon, 2005; Shen and Dahmann,

2005) suggest that these major signaling pathways also

function in modulating MTs, F-actin nucleation, and myo-

sin activity during organogenesis. When removing ci (both

Ci155-act and Ci75-rep), we consistently observed ec-

topic MF-like responses in the eye imaginal disc, even

when generating relatively large patches of mutant cells.

The failure to induce apical constriction in double ci,

mad clones demonstrates that transcription downstream

of Dpp signaling is required for ectopic cell constriction

to proceed in the absence of Ci155. As mad function is

dispensable in the MF, this also suggests that, in this con-

text, Ci155 and Mad might function redundantly.

We found that a number of mad mutant clones form

cysts in the eye disc and, as previously reported (Gibson

and Perrimon, 2005; Shen and Dahmann, 2005), this is ac-

companied by defects in apical MT organization. It is not

clear why some of the mad mutant clones lead to cyst

lsevier Inc.

Developmental Cell


formation while others remain within the epithelium, and it

is possible that the timing of mutant-clone induction is

a key parameter. Interestingly, cells in the MF present

the highest levels of P-Mad, and this correlates with our

observation that, in these cells, the apical MTs appear

more bundled and abundant.

Linking MT and F-Actin during Cell ConstrictionThe Formin-Homology (FH) protein family has been impli-

cated in a number of actin- and MT-dependent cellular

processes, including the alignment of the MTs and F-actin

during stress-fiber formation (Tsuji et al., 2002), cell polar-

ity, and cytokinesis (Wasserman, 1998). In the fly embryo,

dia loss of function leads to defects in pseudocleavage-

furrow invagination during cellularization (Afshar et al.,

2000) and contractile-ring morphogensis (Giansanti et al.,

1998). Consistent with a role for dia during cytokinesis,

we observed that a high proportion of dia mutant cells

were located at the apical surface of the epithelium and

arrested in telophase. Nevertheless, we could recover

small dia mutant clones in the MF that retained their overall

apico-basal polarity, thus enabling us to reveal a function

for this factor for F-actin enrichment during cell constric-

tion in the MF. However, we were not able to induce

ectopic cell constriction by using an activated form of

dia (Somogyi and Rorth, 2004).

FH proteins such as Dia contain a central proline-rich

FH1 domain that, in yeast, has been shown to mediate

a direct interaction with the actin effector Profilin (Chang

et al., 1997), whereas the amino-terminal part of several

FH proteins is able to interact with the GTP-bound Rho-

GTPases such as Rho1 (Watanabe et al., 1997, 1999).

Our data suggest that a RhoA-Dia-Profilin biochemical

module might be evolutionarily conserved (Chang et al.,

1997). Interestingly, mdia has been shown to be trans-

ported on MT and act as a MT (+) end stabilizer in 3T3

fibroblasts (Palazzo et al., 2001). Whereas MTs remain

largely unaffected in the absence of dia in the MF (not

shown), depolymerizing MTs (Sherwood et al., 2004) led

to the absence of cell constriction in the MF, accompanied

by a loss of apical F-actin and Ser19-P MyoII staining. In

this context, we favor a model in which dia is involved in

maintaining F-actin at the apical cell cortex in the MF,

but is not required for the apparent tight bundling and sta-

bilization of the apical MTs in these cells. Finally, it is inter-

esting to note that, in the fly syncytial blastoderm embryo,

F-actin and MyoII are transported toward the MT (+) end

(Foe et al., 2000). The failure in apical F-actin and activated

MyoII enrichment when MTs are depolymerized in the MF

is consistent with a role for MTs in targeting MyoII to the

apical cell surface.

MyoII Activation and Cell ConstrictionIn the MF, we note that loss of rok function does not lead to

a total loss of phosphorylation of MRLC at the conserved

Ser19. This is consistent with a previous report in the

developing eye disc (Lee and Treisman, 2004) and sug-

gests that other kinases act in parallel with Rok in the MF.

Potential candidates are the P21-activated kinases (PAK),

Developme

encoded by three loci in Drosophila (PAK1/PAK, DPAK2/

mbt, and PAK3). We used the null allele mbtP1 as well as

various PAK1 alleles (including PAK16, which encodes

a truncated protein) to examine the role of these kinases

in apical cell constriction in the MF, and, in both cases,

we failed to detect defects in apical cell constriction (not

shown). In addition, expressing the auto-inhibitory domain

(PAK-AID) found in the group-I PAKs (including PAK1 and

PAK3) in clones, and thus interfering with these two

kinases’ function (Conder et al., 2004), did not lead to

loss of cell constriction in the MF (not shown). It is possible

that residual DPAK2/mbt activity could compensate for

the lack of PAK1 and PAK3 function, and further work will

be required to analyze the effects of removing all three of

these loci in the MF. Another kinase potentially responsible

for phosphorylating MRLC in the MF is myosin light chain

kinase (MLCK), a large, complex locus for which loss-

of-function mutations have not been identified.

MyoII Activation and Tissue InvaginationIn ptc mutant clones, we are ectopically inducing maxi-

mum levels of Hh signaling. In this context, MF-like ingres-

sion (or indentation) is associated with lower levels of

Ser19-P-MyoII than those we measured in cases of

groove formation. Interestingly, the situation in the endo-

genous MF involves a very transient stimulation of the Hh

pathway due to the activity of the ubiquitin ligases Cullin1

and Cullin3, which process Ci155 in the anterior and pos-

terior edge of the MF, respectively (Ou et al., 2002). Our

data are consistent with a model in which, upon Hh signal-

ing, activated MyoII accumulates at the apical cortex of

the constricting cell over time. The degree of activated

MyoII accumulation is linked to the duration of Hh signal-

ing perceived by the corresponding constricting cells and

appears to be the main parameter involved in groove

formation in columnar epithelia.

It is likely that the Hh-induced MyoII-driven pathway we

report here also plays a role in tissue patterning in higher

organisms. For example, cell constriction and apico-basal

contraction occur during the invagination of the neural

plate to form the neural tube in vertebrates. In this case,

the notochord and the floor plate are a potent source of

sonic hedgehog (shh) (Jessell, 2000), and shh knockout

mice exhibit a failure in neural tube closure. In these

mice, the neural plate lacks characteristic bottle cells,

pointing to the possibility that neural tube closure is driven

by MyoII following a similar pathway to the one defined in

the present study.

EXPERIMENTAL PROCEDURES

Genotypes Used in This Study

The functional Ubi-Sqh:GFP was expressed in a sqhAX3 null mutant

background (Royou et al., 2002). w, hsflp122; Tub>w[+]>Gal4/UAS-

mYFP:myosinDN (Dawes-Hoang et al., 2005). y,w,FRT101 sqh1/UbiGFP

FRT101; eyflp/+ (Karess et al., 1991). sqh1 is a hypomorphic mutation in

the MRLC. rok2 FRT19A/FRT19A arm-LacZ; eyflp/+ (Winter et al.,

2001). arm8 FRT19A/FRT19A arm-LacZ; eyflp/+ (Uemura et al.,

1996). w, eyflp; madB1 FRT40A/FRT40A arm-LacZ and w, eyflp;

mad12 FRT40A/FRT40A arm-LacZ (Wiersdorff et al., 1996). mad12 is


Developmental Cell


an insertion in the mad locus that prevents most Dpp signaling. w,

eyflp; smoD16 FRT40A/FRT40A arm-LacZ (van den Heuvel and Ing-

ham, 1996). smoD16 is a null allele (Chen and Struhl, 1996). w, eyflp;

smo3, mad1-2 FRT40A/FRT40A arm-LacZ (Curtiss and Mlodzik,

2000). mad1-2 is a hypomorphic allele, and smo3 is an amorphic allele.

y, w, eyflp; [Ci+] FRT40A/FRT40A arm-LacZ;;Ci94/Ci94 (Zhang and

Kalderon, 2001). y, w, eyflp; [Ci+], mad1-2 FRT40A/FRT40A arm-

LacZ;;Ci94/Ci94 (Fu and Baker, 2003). w, eyflp; dia5 FRT40A/FRT40A

arm-LacZ (Afshar et al., 2000). w, eyflp; chic221 FRT40A/FRT40A

arm-LacZ (Verheyen and Cooley, 1994). The chic221 allele is an amor-

phic allele. w, eyflp; RhoA720M1 FRT40A/FRT40A arm-LacZ, Minutes

(Strutt et al., 1997). w, eyflp; scarD37 FRT40A/FRT40A arm-LacZ (Zallen

et al., 2002). w, hsflp122; ptcS2 FRT42D/FRT42D arm-LacZ (Jiang and

Struhl, 1995). ptcS2 is an amorphic allele. w, hsflp122; tsr1 FRT42D/

FRT42D arm-LacZ (Gunsalus et al., 1995). w, hsflp122; ena23

FRT42D/FRT42D arm-LacZ (Ahern-Djamali et al., 1998). w, eyflp;

FRT80A mbsT541/FRT80A arm-LacZ and w, eyflp; FRT80 mbsT791/

FRT80 arm-LacZ (Lee and Treisman, 2004). w, eyflp; FRT80A

cul116E/FRT80A arm-LacZ and w, eyflp; FRT80A cul1107D/FRT80A

arm-LacZ (Ou et al., 2002). w, eyflp; FRT80A Abl4/FRT80A arm-LacZ

(Bennett and Hoffmann, 1992). w, eyflp; FRT82B ssh26-1/FRT82B

arm-LacZ as well as w, eyflp; FRT82B ssh1-11/FRT82B arm-LacZ and

w, eyflp; FRT82B sshP01207/FRT82B arm-LacZ (Niwa et al., 2002). w,

eyflp; FRT82B wsp1/FRT82B arm-LacZ and w, eyflp; FRT82B wsp3/

FRT82B arm-LacZ (Ben-Yaacov et al., 2001). w, eyflp; FRT82B

pak16/FRT82B arm-LacZ (Hing et al., 1999). pak16 is an amorphic

allele. Homozygous RhoGEF24.1/RhoGEF2PX6 animals (Barrett et al.,

1997). RhoGEF24.1 is a null allele, and RhoGEF2PX6 is a hypomorph.

w, UAS-GFP, eyflp; TubGal4; FRT82B, TubGal80/FRT82B UAS-Pak-

AID (Conder et al., 2004). w, UAS-GFP, eyflp; TubGal4; FRT82B, Tub-

Gal80/FRT82B, UAS-ROKCAT (Winter et al., 2001).

y,w, hflp122; Tub>GFP,y+>Gal4; UAS-sqh-E20E21. UAS-sqh-E20E21

contains the sqh transgene with two activating phosphomimetic

mutations (Winter et al., 2001) under the control of the UAS sequence.

y,w, hflp122; Tub>GFP,y+>Gal4; UAS-spastin:GFP (Sherwood et al.,

2004).

Fly cultures and crosses were carried out at 25�C. Overexpression of

SqhE20E21 was performed at 29�C.

Immunostaining

Immunostainings were performed with the following antibodies: anti-

Armadillo (Riggleman et al., 1990) (mouse, 1/50); anti-MyoII (Kiehart

and Feghali, 1986) (rabbit, 1/500); anti-PSer19-MRLC (rabbit, 1/10;

Cell Signaling Technology, Beverly, MA); anti-Diaphanous (Afshar

et al., 2000) (rabbit, 1/200); anti-P-Mad (Gift from G. Morata) (rabbit,

1/500); anti-b-galactosidase (rabbit, 1/5000; Cappel Laboratories,

Durham, NC); anti-b-galactosidase (goat, 1/500; Cappel Laboratories);

anti-a-tubulin (Sigma, mouse, 1/2000); anti-Discs large (mouse, 1/50);

phalloidin-Texas red (Sigma, 20 mM); anti-mouse, -rabbit, and -goat

secondary antibodies (Jackson) (used at 1/200).

Spastin:GFP-expressing cells were generated by heat shocking

third-instar larvae for 30 minutes at 37�C, and the corresponding ani-

mals were dissected 5 hr later. ptc-mutant clones were induced in

early second-instar larvae by performing a 1 hr heat shock at 37�C.

The corresponding animals were dissected 24, 48, 72, or 96 hr after

heat shock.

Imaginal discs were dissected in 13 PBS and fixed in 4% formalde-

hyde for 20 min at room temperature. Samples were permeabilized in

PBS and 0.3% Triton and then blocked in 5% FCS. Antibodies were

incubated in PBS, Triton (0.3%). Imaging was performed by using a

Leica SP2, SP5 or Biorad Radiance 1040 confocal microscope.

Quantification

Images were analyzed by using IMAGEJ v1.33. To avoid complications

with variations from preparation to preparation, we chose to compare

wild-type and mutant tissue from the same disc. Surface areas of wild-

type and mutant patches of cells in the MF (or anterior to the MF in the

case of ptc-mutant clones) were measured. For F-actin quantity, aver-


age pixel intensity over a transect in the MF (or in clones in the anterior

compartment for ptc) was calculated. The data were compared by

using a 2-tailed Student’s paired-t test to compare the mutant and

wild-type cell responses.

Supplemental Data

Supplemental Data include statistical analyses and Figure S1 and are

available at http://www.developmentalcell.com/cgi/content/full/13/5/

730/DC1/.

ACKNOWLEDGMENTS

This work was funded by the Medical Research Council (MRC). We

particularly acknowledge L. Alphey for the kind gift of the UAS-

sqhE20E21 flies and Matthew Freeman for sharing unpublished data

concerning this transgene. The authors would also like to acknowl-

edge Buzz Baum, Nick Baker, Kathy Barrett, Andrea Brand, Roger

Karess, Gines Morata, and Jessica Treisman for kindly providing us

with fly stocks and reagents; the Bloomington Stock Center for fly

stocks; and the Developmental Study Hybridoma Bank (DSHB) for

antibodies. We are grateful to the Pichaud lab for their input during

this work. R.F.W. is a recipient of an MRC career development fellow-

ship. P.F. is a recipient of a European Molecular Biology Organization

long-term fellowship.

Received: February 23, 2007

Revised: July 17, 2007

Accepted: September 25, 2007

Published: November 5, 2007

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Hedgehog Signaling Is a Principal Inducer of Myosin-II-Driven Cell Ingression in Drosophila Epithelia

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