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First published online 11 April 2007
doi: 10.1242/dev.002972
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1 Department of Molecular Biology and Pharmacology, Washington University School
of Medicine, 660 South Euclid Avenue, Saint Louis, MO 63110, USA.
2 Division of Biological Sciences, University of California, San Diego, CA
92093, USA.
3 University of Kansas, Department of Molecular Biosciences, 7031 Haworth,
Lawrence, KS 66045, USA.
* Author for correspondence (e-mail: cagan{at}wustl.edu)
Accepted 11 March 2007
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Adhesion, BMP, Dpp, Epithelia, Patterning
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Hogan, 1996;
Ozdamar et al., 2005).
Although much is known of the basic signaling pathway, the role of BMPs in
cell morphogenesis remains poorly understood. In this paper we use the
Drosophila pupal retina to explore how temporal and cell
type-specific regulation of BMP signaling regulates the positioning of cells
within developing epithelia.
TGF-ß-family proteins activate signaling by binding Type I and Type II
serine-threonine kinase receptors, which in turn recruit and phosphorylate
receptor SMADs to regulate transcription of target genes
(Shi and Massague, 2003). Dpp
is the Drosophila ortholog of vertebrate BMP2/4. Its potential
receptors include the Type II receptors Punt (Put) and Wishful Thinking (Wit)
and the Type I receptors Thickveins (Tkv) and Saxophone (Sax), which activate
the downstream target Mad (Letsou et al.,
1995; Marques et al.,
2002; Newfeld et al.,
1997; Penton et al.,
1994; Xie et al.,
1994). The Dpp signaling pathway regulates multiple developmental
processes including dorsoventral patterning of the embryo, gut morphogenesis,
growth, patterning and differentiation of the imaginal discs, and epithelial
morphogenetic processes such as dorsal closure and imaginal disc spreading
(Ferguson and Anderson, 1992;
Firth and Baker, 2005;
Greenwood and Struhl, 1999;
Neumann and Cohen, 1997;
Panganiban et al., 1990;
Rogulja and Irvine, 2005;
Affolter et al., 1994;
Martin-Blanco et al.,
2000).
Recently, strong loss of Dpp signaling in the wing has been demonstrated to
cause the release of cells from the epithelium and the establishment of a
basal cyst (Gibson and Perrimon,
2005; Shen and Dahmann,
2005). This suggests that Dpp pathway activity is required to
maintain epithelial integrity. Epithelial integrity and tissue morphogenesis
are mediated through dynamic regulation of the apical junctions
(Schock and Perrimon, 2002).
Dpp signaling is also precisely regulated during development, and one
possibility is that it regulates epithelial patterning or maturation through
an association with apical junctions.
The Drosophila pupal retina has proven a useful system for
studying epithelial patterning. Its precise pattern emerges through a series
of morphogenetic processes that include changes in cell shape, cell position
and programmed cell death (Cagan and
Ready, 1989b). Formation of correct cell contacts and selective
cell adhesion - collectively known as cell sorting - are also key events
during patterning of the pupal retina (Bao
and Cagan, 2005; Grzeschik and
Knust, 2005; Hayashi and
Carthew, 2004; Reiter et al.,
1996). The adhesion molecule Roughest (Rst) is the ortholog of
vertebrate NEPH1 (also known as KIRREL1) and a member of the immunoglobulin
superfamily. Mutations in the rst gene result in impaired cell
sorting and subsequent blockade in programmed cell death during pupal retinal
development (Reiter et al.,
1996; Wolff and Ready,
1991). Rst regulates patterning of the pupal retina through
selective heterophilic adhesion with Hibris (Hbs) and formation of cell
junctions (Bao and Cagan,
2005). Additionally, the adhesion molecule DE-cadherin has been
proposed to regulate Rst during stages of maximal cell rearrangements in the
pupal retina (Grzeschik and Knust,
2005), although the precise relationship between these two
adhesion molecules remains to be elucidated.
In this paper, we demonstrate an essential role for the Dpp pathway in regulating epithelial cell shape and patterning in the pupal retina. We provide evidence that Dpp pathway activity is regulated dynamically across time and that it acts as a new component and functional link between two adhesion systems, Hbs-Rst and DE-cadherin. Our data support a novel role for temporal and cell-type specific Dpp/BMP signaling to direct shape and positioning of individual cells into an emerging epithelial pattern.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). We constructed two independent UAS-tkv-IR
(tkv-IR) lines by subcloning inverted repeats into pGEM-WIZ
(Bao and Cagan, 2006):
tkv-IR1, which targets part of the 5' half; and
tkv-IR2, directed towards the 3' half of the tkv
mRNA.
RNA extraction from pupal retinas and RT-PCR
Retinal-brain complexes from 10-15 pupae per genotype were dissected under
RNAse-free conditions. Retinas were separated from the brains using a sterile
surgical razor blade and subject to RNA extraction using TRIzol (Invitrogen).
The RNA was then used to detect tkv transcript levels by RT-PCR.
Temperature-sensitive experiments and clonal analysis
To examine the role of dpp in pupal patterning,
dppe90 homozygous flies were kept at 18°C (permissive
temperature), pupae were selected at 42 hours [equivalent to approximately 20
hours after puparium formation (APF)] at 25°C, and then switched to
25°C (restrictive temperature) for 22 hours and dissected. As a control,
dppe90 pupae were held at the permissive temperature until
dissection.
Whole eye mutants for puntP were generated using the
EGUF system (Stowers and Schwarz,
1999) using pupae with the following genotypes: (1) control:
ey-Gal4, UAS-FLP; FRT82B, cl, GMR-hid/FRT82B lacZ; (2) experimental:
ey-Gal4, UAS-FLP; FRT82B, cl, GMR-hid/FRT82B puntP.
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; Xu and Rubin,
1993). Recombinant clones were induced by heat shocking larvae 72
hours after egg laying for 1 hour at 37°C. For MARCM
tkv8 clones (Lee and
Luo, 1999), third instar larvae were heat shocked for 30 minutes
at 37°C. Clonal analysis was performed in pupae with the following
genotypes: (1) tkv8 clones: hs-FLP; tkv8
FRT40/ub-nGFP FRT40; (2) tkv4 clones: hs-FLP;
tkv4 FRT40/ub-nGFP FRT40; (3) Mad12
clones: hs-FLP; Mad12 FRT40/ub-nGFP FRT40; (4)
tkv8 single-cell clones: UAS-CD4:GFP, hs-FLP; Gal80,
FRT40/tkv8 FRT40; tubulin-Gal4/+.
Immunostaining and imaging
Pupal retinas and wings were processed as described previously
(Bao and Cagan, 2005;
Blair and Ralston, 1997).
Antibodies used were: mouse anti-Armadillo and rat anti-DE-cadherin (1:3 and
1:10, respectively, from the Developmental Studies Hybridoma Bank at the
University of Iowa); mouse anti-Rst (1:50, from Karl Fischbach); rabbit
anti-ß-galactosidase (1:2000, Cappel); rabbit anti-luminal Tkv (1:10,
from Marcos Gonzalez-Gaitán, Max Planck Institute, Dresden, Germany);
rabbit anti-GFP (1:2000, from Pam Silver, Harvard Medical School, Boston, MA);
mouse anti-Rho1 and mouse anti-Tubulin (E7) (1:10, from the Developmental
Studies Hybridoma Bank at the University of Iowa); mouse anti-Srf (1:50, from
M. Gilman, Cold Spring Harbor Laboratory, NY); and rabbit anti-p-Mad (1:5000,
from Tetsuya Tabata, University of Tokyo, Tokyo, Japan). Alexa488- and
Alexa568-conjugated secondary antibodies were used (1:1000, Molecular
Probes).
Whole-mount in situ hybridization was carried out as previously described
(Bao and Cagan, 2005). Cell
surface-associated Tkv was visualized with an antibody directed against the
extracellular domain of Tkv (Kruse et al.,
2004); dissected tissue was incubated with the antibody at 4°C
prior to fixation (Strigini and Cohen,
2000). The antibody did not work when added after fixation and
permeabilization.
Images were captured with a Zeiss Axiophot microscope equipped with a Quantix CCD camera (Photometrics) and Image Pro Plus software. Images were processed with Photoshop (Adobe). Confocal xzy projections were taken on a Leica confocal microscope using the Leica confocal software. For scanning electron microscopy, flies were prepared by ethanol fixation followed by critical-point drying. Images were captured using a Hitachi S-2600H scanning electron microscope.
In vivo visualization
In vivo imaging was performed in pupae with the following genotypes: (1)
experimental: GMR-gal4/+; UAS-
Catenin-GFP/tkv8;
UAS-tkv-IR1(2X)/UAS-tkv-IR1(2X) (Fig.
3; see Movie 2 in the supplementary material); (2) control:
GMR-gal4/+; UAS-Catenin-GFP/+ (see Movie 1 in the
supplementary material). Larvae were allowed to pupate at 29°C (0 hours
APF). Pupae were then collected and staged at 25°C until 25 hours APF. The
pupal case was removed in the head area and the animal was placed with the eye
region pressed against a coverslip. Temperature and humidity were controlled
and images were captured every 15 minutes.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) with three sensory bristles at alternating vertices. We
will collectively refer to mature interommatidial cells as `2°/3°s'.
This precise interommatidial pattern emerges 18-31 hours after puparium
formation (APF) (Fig. 1A,C,D).
`Interommatidial precursor cells' (IPCs) are precursors to the 2°/3°s
(Fig. 1A,B). They undergo
dynamic cell rearrangements that are necessary to direct them into a precise
2°/3° hexagonal array (Fig.
1E,F). In an early-stage pupal retina, IPCs are initially arranged
in double and triple rows between ommatidia
(Fig. 1A,B). These cells then
rearrange into single-cell rows (Fig.
1C-E). Throughout this patterning process, excess IPCs are
eliminated by programmed cell death
(Rusconi et al., 2000).
At least two adhesion systems are involved in directing IPC patterning:
Hbs-Rst and DE-cadherin. Reducing the activity of Rst or Hbs led to a failure
of IPC cell movement (Bao and Cagan,
2005; Reiter et al.,
1996). Rst is found primarily at the junction between IPCs and
1°s and is excluded from IPC:IPC junctions
(Reiter et al., 1996;
Bao and Cagan, 2005)
(Fig. 1G,H). Rst regulates
patterning of the pupal retina through selective heterophilic adhesion with
Hbs and formation of DE-cadherin-rich adherens junctions
(Bao and Cagan, 2005).
The relationship between these adhesion systems is complex. Experiments
that altered the activity or expression of DE-cadherin suggested that
DE-cadherin is required to drive Rst to the adherens junctions
(Grzeschik and Knust, 2005).
Conversely, overexpression of Rst led to an increase in DE-cadherin
(Bao and Cagan, 2005).
Additionally, we observed that mutations in rst disrupted the dynamic
localization of IPC:IPC adherens junctions
(Fig. 1I,J). Normally, the
adherens junctions between IPCs are strongly reduced by 31 hours APF
(Bao and Cagan, 2005;
Grzeschik and Knust, 2005)
(Fig. 1, compare D with A,C,E).
Retinas from rst mutants failed to clear these junctions
(Fig. 1I,J). Consistent with
the previous result, taking away one functional copy of shotgun
(shg), the gene encoding DE-cadherin, significantly suppressed the
rough eye phenotype of rst mutants
(Fig. 1K-P). Together, these
data emphasize the complexity of the relationship and epistatic order between
Hbs-Rst and components of the adherens junctions. To better understand this
relationship, we examined other potential regulators of IPC patterning.
Dpp is required for patterning the pupal retina
To explore the role of Dpp specifically in the pupa, we utilized the
temperature-sensitive allele dppe90
(Fig. 2A,B). Genotypically
dppe90 animals were kept either at 18°C
(Fig. 2A) or switched to the
non-permissive temperature at 20 hours APF just prior to the stage of cell
rearrangements in the pupal retina (Fig.
2B). Downregulation of dpp resulted in an abnormal
hexagonal pattern due to disruption in the shape and patterning of
2°/3°s as assessed with the junctional marker Armadillo
(Fig. 2B). The main
2°/3° defects we observed included: (1) abnormal IPC:IPC contacts; (2)
a failure of 3°s to establish a correct position within the vertex of the
hexagon; (3) 2°/3°s that were abnormally arranged around sensory
bristles; and (4) misplaced bristle organs. As previously reported
(Wharton et al., 1996), there
was some variability in the penetrance and expressivity of the phenotype (for
quantification of defects, see Table S1 in the supplementary material).
|
). A
dpp-lacZ reporter line indicated that Dpp was expressed exclusively
in all primary pigment cells at the time of active IPC morphogenesis
(Fig. 2C,D). Together, these
results suggest a model in which Dpp is provided by primaries to regulate
patterning of neighboring IPCs.
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;
Xu and Rubin, 1993) to
generate clonal patches bearing reduced activity of the Type I receptor Tkv,
or the Type II receptor Punt, in the pupal retina. Clones of the null allele
tkv8 (Nellen et al.,
1994) (Fig. 2E,F)
or of the hypomorphic allele tkv4
(Penton et al., 1994)
(Fig. 2G,H), led to defects in
the shape and patterning of 2°/3°s similar to those observed when Dpp
activity was reduced (Fig. 2B).
Additionally, we observed cases of ectopic 2°/3°s. Whole eye clones of
genotypically puntP tissue showed patterning defects that
were similar to, but milder than, those seen in dpp and tkv
mutants, and discrete puntP clones showed infrequent
defects (data not shown). Weaker phenotypes of punt versus
tkv clones have previously been documented in the wing disc
(Burke and Basler, 1996),
perhaps reflecting the hypomorphic nature of available punt alleles.
Clones of null alleles of wit or sax, which encode
alternative Type II and Type I receptors, respectively, gave no mutant
phenotype (data not shown).
In situ hybridization experiments and a tkv-lacZ reporter
indicated that tkv and punt transcripts were expressed in
IPCs and cone cells during stages of IPC patterning (data not shown). Antibody
staining (Kruse et al., 2004)
detected surface-exposed Tkv in puncta along the surface of IPCs, cone cells
and sensory bristles (Fig.
2I-K). Therefore, Dpp and its receptors Tkv and Punt are mainly
expressed in complementary cell types, supporting a model in which Dpp from
primary pigment cells binds to Tkv and Punt in the IPCs to regulate their
shape and positioning.
Tkv regulates cell shape autonomously in the pupal retina
To more closely explore Dpp pathway activity, we generated single-cell
clones of tkv8 using the MARCM system
(Lee and Luo, 1999). Each cell
within the pupal retina has a stereotyped apical profile
(Fig. 1E,F), and deviations are
readily observed. The apical profiles of isolated, genotypically
tkv8 cells failed to stretch out and fill their proper
niche within the hexagon (65%, n=30;
Fig. 2L-N). Instead, their
shortened profile was typically compensated for by a wild-type neighbor, which
expanded to fill the unoccupied space (Fig.
2L-N). Interestingly, when two tkv8 cells were
juxtaposed they typically exhibited normal apical profiles (88%,
n=40; Fig. 2N),
indicating that relative levels of Dpp signaling between neighboring cells
determine cell shape.
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IPC patterning defects are due to a failure in proper cell movement and morphogenesis
To better assess the role of Dpp signaling we used in vivo imaging analysis
(Monserrate and Baker Brachmann,
2007; Vidal et al.,
2006) to observe morphogenesis progression between 25 and 30 hours
APF (Fig. 3A-D; see Movies 1, 2
in the supplementary material). To facilitate these studies, we generated two
transgenic lines that reduced tkv activity through RNA interference:
tkv-IR1 targets within the 5' region and tkv-IR2
within the 3' region of the tkv mRNA. The phenotypes of the two
lines were identical, except that the phenotypes observed in flies with
tkv-IR1 were consistently stronger than in those with
tkv-IR2 (data not shown). The following observations further
validated the specificity of our tkv-IR constructs: (1) expressing
either tkv-IR line with the wing pouch driver scalloped-gal4
(sd>tkv-IR) or the eye driver (GMR>tkv-IR) phenocopied
dpp and tkv loss-of-function phenotypes
(Terracol and Lengyel, 1994)
(Fig. 2); (2) removing a
functional genomic copy of tkv significantly enhanced the
sd>tkv-IR2 phenotype; and (3) wing imaginal discs from
sd>tkv-IR larvae showed significant downregulation of the levels
of the phosphorylated form of Mad (p-Mad) in the wing pouch region (data not
shown).
To visualize tkv development in living tissues, multiple copies of the transgene were targeted specifically to the eye (GMR>tkv-IR). The most common and striking phenotype observed within developing GMR>tkv-IR eyes was a failure to maintain stable IPC:IPC contacts (see Movie 2 in the supplementary material). Neighbors established contact but then broke apart leading to direct contact between primary pigment cells from adjacent ommatidia (Fig. 3A-D; see Movie 2 in the supplementary material) in a manner that was not seen in control retinas (see Movie 1 in the supplementary material). This failure to maintain contact was briefly preceded by dissolution of the visible IPC:IPC adherens junction (Fig. 3A-D; see Movie 2 in the supplementary material). Further, these abnormal IPC:IPC interactions were accompanied by aberrant changes in cell shape that included abnormal expansions and/or reductions of their apical profiles. IPC:IPC contacts were often later reformed, reducing the severity of the final phenotype (Fig. 3D). These aberrant phenotypes repeated themselves across the retina over the time of visualization (see Movie 2 in the supplementary material). They were consistent with the abnormal IPC:IPC contacts observed in dpp, tkv and Mad mutants (Fig. 2), and in 25 hours APF tkv and Mad mutant clones (Fig. 3E-J), which frequently exhibited premature clearing of the IPC-IPC DE-cadherin junctions suggestive of junction dissolution. RT-PCR results indicated that shg expression levels were not detectably altered (data not shown). Together, these results indicate that Dpp signaling is required to maintain normal IPC:IPC contacts, junction stability and cell shape during morphogenesis of the pupal retina.
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;
Sekelsky et al., 1995). Clones
of the null allele Mad12 led to 2°/3° patterning
defects that mimicked those observed in dpp and tkv mutants
(Fig. 2P). These results
indicate that Dpp-dependent IPC patterning in the pupal retina requires
classical pathway signaling that includes Mad activity.
Nuclear levels of p-Mad serve as a readout of Dpp signal transduction
activity (Tanimoto et al.,
2000). Our data suggest that, in the context of the pupal retina,
primary pigment cells might act as a source of Dpp that is then provided to
surrounding cells to influence their patterning. To further test this
hypothesis, we utilized an antibody specific for p-Mad (see Fig. S1 in the
supplementary material) to identify the cells that exhibit active Dpp
signaling. Fig. 4 presents a
time course of Dpp pathway activity in the pupal retina. Consistent with our
ligand/receptor expression pattern and phenotypic results, p-Mad was detected
in cone cells, IPCs and sensory bristles but not in primary pigment cells.
IPCs contained high levels of p-Mad during the period of maximal IPC
patterning (20-26 hours APF; Fig.
4B,E; data not shown). Subsequently, IPC levels decreased at 28
hours APF and were undetectable by 31 hours APF
(Fig. 4H,K). Mad activity in
cone cells and bristles was evident after 20 hours APF and remained unchanged
through all stages examined (Fig.
4A,D,G,J); we did not observe consistent defects when dpp
activity was reduced in these cells and the functional relevance of Mad
activation in either cell type is unclear.
Consistent with our phenotypic analysis, therefore, IPCs exhibited a dynamic pattern of Dpp activity that was highest at the time of active cell rearrangement and was then rapidly downregulated.
Dpp signaling works in opposition to Rst during IPC patterning
No significant genetic modifier interactions were observed (data not shown)
between components of the Dpp pathway and Notchfa-g,
EgfrEl or winglesscx4 which were
previously implicated in IPC patterning
(Cagan and Ready, 1989a;
Cordero et al., 2004;
Freeman, 1996;
Miller and Cagan, 1998).
The results from our phenotypic analysis and in situ visualization indicated that mutations in the Dpp pathway affected cell shape, cell movements and cell-cell contacts, making Rst an attractive candidate to mediate Dpp function during IPC patterning. Consistent with this view, removing one genomic copy of tkv strongly suppressed the rough eye phenotype of rst3 mutants; removing one copy of Mad also produced a milder but significant suppression (Fig. 5). Independent tkv and Mad alleles gave similar results (data not shown). These results suggest that Rst and Dpp are functionally linked and that they act in opposition during patterning of the pupal retina.
Next, we assessed the epistatic relationship between Rst and Dpp signaling. We found no changes in Rst protein levels or localization when Dpp pathway activity was reduced (see Fig. S2A,B in the supplementary material). By contrast, we observed a striking failure of genotypically rst3 and rstCT pupal eyes to properly downregulate Dpp pathway activity: the normal reduction in p-Mad within IPCs at 28 hours APF did not occur (n=7 retinas for each genotype; Fig. 6, compare E,H with B). We could not unambiguously compare early-stage p-Mad in IPCs owing to its normally high levels. Levels of p-Mad in cone cells and bristle organs were not affected, providing a further, internal control. Together, these data indicate that Rst acts as a negative regulator of Dpp signaling activity as IPC patterning in the pupal retina progresses. In this scheme, loss of Rst activity leads to heightened Dpp pathway activity that in turn leads to defects in IPC patterning. Further supporting this model, expression of activated Tkv (GMR>tkvQ253D) in the developing eye led to a robust and fairly specific phenotype in the IPCs that at least partially phenocopied the defects observed in rst mutant retinas (Fig. 6J-L).
|
). Indeed, we observed that Tkv surface presence
correlated with p-Mad levels: the levels of IPC surface-associated Tkv were
highest during the period of maximal IPC patterning and decreased subsequently
(Fig. 7A-F). In rst
mutant retinas, surface Tkv protein failed to decrease as patterning
progressed (Fig. 7G-L). Mutant
retinas for the null allele rstCT showed uniform IPC
patterning defects accompanied by general upregulation of cell surface Tkv
(Fig. 7G-I). Mutants for the
hypomorphic allele rst3, which is subject to position
effect variegation (PEV) (Tanenbaum et
al., 2000), showed highest levels of cell surface Tkv in the areas
with strongest IPC patterning defects (Fig.
7J-L). Co-immunoprecipitation experiments from tissue failed to
detect physical interaction between Rst and Tkv (not shown), suggesting that
the regulation of Tkv protein by Rst might require additional intermediaries.
Furthermore, we saw no change in the levels of tkv transcript in
control versus rst mutant retinas (see Fig. S3 in the supplementary
material). Together, these data suggest that Rst opposes Dpp signaling by
regulating the levels of cell surface-associated Tkv protein.
Dpp signaling works in conjunction with DE-cadherin and Rho1 during IPC patterning
DE-cadherin, along with -catenin and Armadillo (also known as
ß-catenin), constitute the core components of the adherens junctions and
can help mediate cell-cell adhesion and cell rearrangement
(Peifer and Wieschaus, 1990;
Tepass et al., 1996;
Uemura et al., 1996). Notably,
we identified defects in adherens junction coherence and function when Dpp
pathway activity was reduced (Fig.
3; see Movie 2 in the supplementary material). To further explore
the relationship between Dpp and DE-cadherin, we tested null alleles of
components of the Dpp pathway in trans to a null allele of shotgun
(shgR69). Control retinas heterozygous for
tkv8, Mad12 or shgR69 were
essentially wild type except for infrequent 2°/3° defects
(Fig. 8C). The trans
heterozygous combinations tkv8/shgR69
and Mad12/shgR69 led to a significant
increase in the percentage of 2°/3° defects
(Fig. 8A-C). This genetic
enhancement is consistent with our observation that DE-cadherin and Rst act in
opposition (Fig. 1I-P) and,
together with the junction phenotype observed in tkv and Mad
mutant retinas (Fig. 3; see
Movie 2 in the supplementary material), further supports a model in which Dpp
signaling regulates IPC patterning at least in part by regulating
DE-cadherin-mediated cell adhesion in the retina.
Members of the Rho family of small GTPases - Rac, Rho and Cdc42 - have been
linked to regulation of the actin cytoskeleton in diverse organisms
(Van Aelst and D'Souza-Schorey,
1997). Rho1 interacts with DE-cadherin-associated proteins and
regulates cadherin-based cell junctions
(Magie et al., 2002). Using a
lower copy number of tkv-IR (GMR>tkv-IR) to direct a mild
IPC patterning phenotype (Fig.
8D), we found that removing one copy of Rho1
(Rho172F) led to significant enhancement of IPC patterning
defects (Fig. 8E; 60% of at
least 15 retinas scored). This functional relationship is not due to
regulation of Rho1 expression by Tkv activity, as Rho1 protein or
transcript levels were not altered in a tkv mutant background (see
Fig. S2C,D in the supplementary material; data not shown). Conversely, loss of
Rho1 (or DE-cadherin) activity did not alter p-Mad levels (data not shown).
This effect was specific for Rho1 because removing one copy of
Cdc42 or a Rac1, Rac2, Mtl triple heterozygote did not
detectably modify the tkv-IR phenotype (not shown). Together, these
results indicate that Dpp signaling cooperates with DE-cadherin and Rho1 to
regulate dynamic IPC morphogenesis, movement and cell-cell contacts during
morphogenesis of the pupal retina.
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). Driving tkv-IR using the wing pouch driver
scalloped-gal4 (sd>tkv-IR) yielded a classical
tkv loss-of-function wing vein phenotype
(Terracol and Lengyel, 1994)
that included thickening of wing veins L2 and L5 and of the intersection
between L5 and the posterior cross vein
(Fig. 9E-H; data not shown).
Removing one genomic copy of either shg
(Fig. 9I-L) or Rho1
(Fig. 9M-P) significantly
enhanced these wing vein phenotypes to levels comparable to those observed
when removing one genomic copy of tkv (data not shown). These
phenotypes were a result of defects in cell fate determination as assessed
with molecular markers specific for vein and intervein cells
(Fig. 9). As in the eye,
removing one copy of Cdc42 or a Rac1, Rac2, Mtl triple
mutant did not detectably modify the tkv-IR phenotype in the wing
(not shown). These data suggest that the functional connection between Dpp
signaling and DE-cadherin and Rho1 is not exclusive to the eye, but is also
relevant to the role of this pathway during cell fate decisions in the
wing. |
DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Shen and Dahmann, 2005), but
the function of Dpp signaling during maturation of developing epithelia is not
fully understood. Here, we show that reducing the activity of components of
the Dpp pathway leads to abnormal IPC shape and organization within the
ommatidial hexagonal pattern (Fig.
2). This activity is linked to fine regulation of apical junction
components and is required to maintain stable cell-cell contacts during cell
movements within the epithelium. The expression of Dpp in primary pigment
cells (Fig. 2C,D) and the
segregation of its receptors to the neighboring IPCs suggest a model in which
Dpp acts in the primaries to organize local IPCs through the dynamic control
of apical junctions. This view is supported by the dynamic changes in p-Mad
activity in the neighboring IPCs, which is highest during the stage (20-26
hours APF) when IPCs rearrangements are maximal
(Fig. 4).
The role of Dpp in cellular morphogenesis during epithelial development is
poorly understood. Therefore, we took advantage of the unique stereotyped
pattern of the pupal retina to study cell behavior as morphogenesis
progresses, focusing on events at the single-cell level. In situ visualization
experiments suggest that IPCs with reduced Tkv activity are incapable of
maintaining their cell-cell contacts and are subject to aberrant changes in
their cell shape (Fig. 3; see
Movie 2 in the supplementary material). Further emphasizing the link with
cellular adhesion, this function of Dpp signaling involves DE-cadherin and
Rho1 (Fig. 8), which are
essential regulators of cell adhesion and cell shape
(Magie et al., 2002).
IPCs require a balance between Rst and Dpp signaling
We provide several lines of evidence indicating that Rst is a negative
regulator of Dpp signaling (Figs
5,
6 and
7). Previous work has
demonstrated that Rst directs IPC movements through selective cell adhesion:
IPCs seek to maximize their Rst-mediated contacts with primaries while
decreasing contacts with their neighbors
(Bao and Cagan, 2005)
(Fig. 1A-F). Additionally,
reducing Rst activity leads to a failure of initial cell movement (D.E.L., S.
Bao and R.C., unpublished). Consistent with these results, Rst activity
opposes DE-cadherin-mediated cell adhesion
(Fig. 1I-P). One model to
account for these observations is that cells require a balance between cell
movement provided by Hbs-Rst and the stability of cell-cell contacts provided
by Dpp signaling. Our live visualization supports the view that reducing Dpp
activity leaves cells with an imbalance, as IPCs move toward their proper
positions but fail to stabilize cell-cell contacts or lock stably into their
final positions (Fig. 3; see
Movie 2 in the supplementary material). Furthermore, downregulation of Dpp
signaling leads to unstable DE-cadherin IPC-IPC junctions
(Fig. 3). Conversely, loss of
rst results in loss of cell movements, which can be compensated by
either reducing cell adhesion (Fig.
1M-P) or Dpp signaling activity
(Fig. 5), again supporting the
importance of maintaining a balance between the Hbs-Rst and the
Dpp-DE-cadherin systems. Perhaps Dpp (and, by extension, BMP) activity is
utilized in the adult for similar functions - for example, as a
`proof-reading' mechanism to remove aberrant cells from an epithelium.
Is Dpp signaling a general regulator of cell adhesion and cell shape?
Our results in the wing raise the interesting possibility that regulation
of DE-cadherin and Rho1-dependent cell shape and cell adhesion might be a
characteristic of Dpp pathway activity common to other biological systems.
Similar to the pupal retina, epithelial cells in the wing disc with reduced
Dpp signaling displayed abnormal morphologies and were unable to maintain
their positions. In the case of the wing, these defects were manifested as
viable cysts of mutant cells that were basally excluded from the epithelium
(Gibson and Perrimon, 2005;
Shen and Dahmann, 2005). The
mechanisms involved in such cell behaviors remain unknown. Our results suggest
that the role of Dpp signaling during wing patterning also involves
DE-cadherin and Rho1 (Fig. 9).
Our experiments do not distinguish whether the defects in wing cell fates are
a direct or a secondary effect of altered cell adhesion, although altering
DE-cadherin activity by itself was not sufficient to cause such defects (data
not shown). Cell adhesion and cell fate have been related previously: for
example, Rho-dependent cell shape changes can influence fate decisions in stem
cells (McBeath et al., 2004).
Despite the commonalities observed, tissue-specific factors are likely to
regulate Dpp-dependent epithelial patterning: for example, Rst does not appear
to have a role in wing development, and we did not observe changes in retinal
Tubulin distribution reported for the wing
(Gibson and Perrimon, 2005)
(see Fig. S2E-H in the supplementary material).
Dpp is the closest ortholog of vertebrate BMP2/4, and it appears to be
active during cellular morphogenesis in a number of contexts including the
developing vertebrate eye (Belecky-Adams et
al., 2002; Furuta and Hogan,
1998). Interestingly, and similar to our observations for IPCs
(Fig. 4), fiber cells in the
developing vertebrate lens show high levels of p-SMAD activity during the
period of cell elongation. Loss of the Type I receptor ALK3 (also known as
BMPR1A) or expression of the inhibitor noggin led to abnormal morphogenesis of
these fiber cells including mispositioning and failure to elongate
(Beebe et al., 2004);
requirements for E-cadherin (also known as cadherin 1) and RHOA function have
not been explored.
Finally, Rst does regulate developmental processes other than IPC
patterning. For example, Rst is expressed in retinal axons and is required for
correct targeting of those axons into the larval brain lobes
(Schneider et al., 1995).
Interestingly, Dpp signaling also has a role in this process
(Yoshida et al., 2005). We
have observed genetic interactions between rst3 and
members of the Dpp pathway in the arrangement of these descending axons (L.
Wickline and R.C., unpublished), raising the intriguing possibility that the
two systems act together in axon targeting as well.
Summary and future directions
Our results provide evidence to support a model in which the Dpp pathway
acts as an intermediary between the Rst and DE-cadherin adhesion systems. A
balanced interplay between these three systems is essential to regulate
epithelial cell movements, cell shape and cell-cell contacts during
morphogenesis of the pupal retina (Fig.
10).
Several questions emerge from our study. For example, our data suggest that Rst acts on Dpp signaling by regulating surface-associated Tkv. Immunoprecipitation experiments failed to identify a physical interaction between Rst and Tkv (not shown), suggesting intermediate steps remain to be identified. Also, the transcription factor Mad is required to regulate IPC patterning (Fig. 2P; Fig. 4; Fig. 5; Fig. 8B), but the transcriptional targets that link Dpp signaling to DE-cadherin and Rho1 are unknown. A better understanding of the links between these three pathways should help shed light on the mechanisms that regulate the fine cellular events required during patterning of developing epithelia.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/134/10/1861/DC1
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ACKNOWLEDGMENTS |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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