![[Feedback]](/icons/banner/feedbackACT.gif){kind=icon}
![[For Subscribers]](/icons/banner/subscriberACT.gif){kind=icon}
![[Archive]](/icons/banner/archiveACT.gif){kind=icon}
![[Search]](/icons/banner/searchACT.gif){kind=icon}
![[Contents]](/icons/banner/tocACT.gif){kind=icon}
|
|
|
|
|
||
Vol. 19, Issue 5, 1883-1892, May 2008
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
Department of Experimental Medical Science, Lund Strategic Research Center for Stem Cell Biology and Cell Therapy, Lund University, 22184 Lund, Sweden
Submitted December 11, 2007;
Revised February 5, 2008;
Accepted February 8, 2008
Monitoring Editor: Marianne Bronner-Fraser
 |
ABSTRACT |
|---|
 TOP ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
|
INTRODUCTION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Lawrence and Struhl, 1996). Cells within each parasegment are sorted out from neighboring parasegments by the establishment of parasegment boundaries that are visible as transient grooves at the anterior of each stripe of cells expressing the segment polarity gene engrailed (en) (Vincent and O'Farrell, 1992). Another boundary, the segment boundary, forms posterior to each stripe of en expression. Formation of segment boundaries begins shortly after the initiation of germ band retraction, and is associated with distinct morphologic changes in segment border cells. These changes include apical constriction of the most posterior en-expressing cells that result in a local folding termed segmental groove. Formation of segmental grooves requires activity of the segment polarity genes en, hedgehog (hh), and wingless (wg). Embryos mutant for either en or hh fail to form segment boundaries, and continuous expression of these genes is essential for maintenance of the boundaries. Anterior to en-expressing cells, a wg-mediated inhibitory signal prevents cells from forming a groove (Larsen et al., 2003). However, how these patterning genes direct morphogenesis, and which cytoskeletal regulators control cell shape during this process is currently unknown.
Rho-guanosine triphosphatases (GTPases) play an important role in the control of cell shape changes that are driven by rearrangement of the actin cytoskeleton. In Drosophila, Rho1 has been shown to regulate cell shape and adhesion in many processes, such as cellularization (Padash Barmchi et al., 2005), gastrulation (Barrett et al., 1997; Häcker and Perrimon, 1998), epidermal development (Bloor and Kiehart, 2002), dorsal closure, segmentation, and head involution (Magie et al., 1999) as well as during epithelial folding in wing imaginal discs (Nikolaidou and Barrett, 2004). Activation of Rho1 requires the exchange of guanosine diphosphate for guanosine triphosphate, which is catalyzed by guanine nucleotide exchange factors (GEFs) such as DRhoGEF2.
The role of DRhoGEF2 in epithelial morphogenesis has been studied in most detail in early Drosophila embryos. During gastrulation, DRhoGEF2 regulates apical cell constriction that drives the invagination of mesodermal and endodermal primordia (Barrett et al., 1997; Häcker and Perrimon, 1998). Evidence identifying factors that connect DRhoGEF2 to the actin cytoskeleton has emerged from genetic studies. In the ventral mesoderm DRhoGEF2 and the Rho-effector Rho-kinase (DRok) are required for apical localization of myosin II, and mutants in both genes result in similar phenotypes (Dawes-Hoang et al., 2005). In Drosophila Schneider (S2) cells, DRhoGEF2-mediated cell contraction can be inhibited by DRok inactivation (Rogers et al., 2004). Thus, a Rho-effector pathway including DRok, the regulatory subunit of myosin light chain phosphatase (myosin binding subunit) (Mizuno et al., 2002; Tan et al., 2003) and myosin II that has been described in other systems (Amano et al., 1996) is thought to control acto-myosin contraction in response to DRhoGEF2 activation.
DRhoGEF2 is also required for stabilization and contraction of actomyosin filaments during blastoderm cellularization (Grosshans et al., 2005; Padash Barmchi et al., 2005), and mutants in DRhoGEF2 and DRok have similar defects during this process (Dawes-Hoang et al., 2005). DRok may therefore act as a DRhoGEF2-effector in a broad cellular context.
Another Rho1-effector, that plays an important role in early embryos and regulates F-actin nucleation and polymerization together with the actin binding protein Profilin (Wallar and Alberts, 2003) in a pathway parallel to DRok (Watanabe et al., 1997) is the Formin homology protein Diaphanous (Dia) (Afshar et al., 2000). In mammalian systems, mDia1 and the DRok homologue Rho-kinase (ROCK) have been shown to work concurrently during stress fiber formation. Interestingly, mDia1 and ROCK can induce stress fibers of different thicknesses and densities depending on the balance between their individual activities, indicating that the two Rho-effector pathways may be independently regulated (Watanabe et al., 1999).
It has been hypothesized that Rho-effector pathway specificity may be conferred by individual GEFs such as DRhoGEF2. However, whether DRhoGEF2 can activate Dia or signals specifically through DRok is currently unclear.
Here, we show that DRhoGEF2 and Dia regulate cell shape changes during segmental groove morphogenesis. Overexpression of DRhoGEF2 in epidermal cells is sufficient to induce cell contraction and premature formation of segmental grooves. Activation of the Formin Dia results in strengthening of cell junctions, and controls F-actin levels and cell shape in a manner distinct from DRhoGEF2. Our data suggest that DRhoGEF2 may select the outcome of Rho1-activation to regulate cortical myosin II recruitment and constriction of acto-myosin fibers but not cell adhesion or F-actin polymerization.
|
MATERIALS AND METHODS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
), dia5 (Castrillon and Wasserman, 1994), and sqh-GFP (Royou et al., 2004). The following Gal4 drivers and their responders were used: prd-Gal4, en-Gal4-GFP, pnr-gal4 (Bloomington Drosophila Stock Center, Department of Biology, Indiana University, Bloomington, IN), twi-Gal4 (gift from A. Michelson, Department of Medicine, Brigham and Women's Hospital, Harvard Medical School, Boston, MA), wg-Gal4 (gift from N. Perrimon, Department of Genetics, HHMI, Harvard Medical School, Boston, MA), UAS-DRhoGEF2-RE (Padash Barmchi et al., 2005), and UAS-diaCA-HA (Somogyi and Rørth, 2004). Germline clones (GLCs) of DRhoGEF2L(2)04291 and dia5 were generated by using the autosomal flippase-mediated recombination dominant-female-sterile technique (Chou and Perrimon, 1996). Females carrying DRhoGEF2L(2)04291 GLCs were crossed to DRhoGEF2L(2)04291/Cyo-GFP males. Embryos maternally and zygotically mutant for DRhoGEF2L(2)0429 were identified by the absence of green fluorescent protein (GFP). Females carrying dia5 germline clones were crossed to dia5/CyO males and kept at 18°C.
Immunolocalization
For phalloidin stainings, embryos were dechorionated in 50% bleach, fixed in 4 ml of HEPES, 1 mM EGTA, 20 mM Mgso4, 4 or 8% formaldehyde, and 5 ml of heptane for 20 min, and then they were devitellinized by hand, washed in phosphate-buffered saline plus 0.1% Tween 20 (PBT), and incubated in rhodamine-conjugated phalloidin (Invitrogen, Carlsbad, CA) for 1 h, washed three times in PBT, and mounted on a microscope slide. Otherwise embryos were fixed as described above and methanol devitellinized. Primary antibodies used were as follows: rabbit anti-DRhoGEF2 (gift from S. Rogers, Department of Biology, The University of North Carolina at Chapel Hill, Chapel Hill, NC; 1/1000), rabbit anti-Dia (gift from S. Wasserman, Section of Cell and Developmental Biology, University of California at San Diego, La Jolla, CA; 1/2000), rat anti-hemagglutinin (HA) high-affinity (Roche Diagnostics, Indianapolis, IN; 1/100), rabbit anti-Sqh (gift from Tien Hsu, Medical University of South Carolina, Hollings Cancer Center, Charleston, SC; 1/50), mouse anti-Arm (Developmental Studies Hybridoma Bank, University of Iowa, Iowa City, IA; 1/200), rat anti-E-Cad (gift from H. Oda, JT Biohistory Research Hall, Osaka, Japan; 1/250), guinea pig anti-odd (gift from C. Rauskolb, Waksman Institute, Rutgers, the State University of New Jersey, Piscataway, NJ; 1/1000), or mouse anti-En (Developmental Studies Hybridoma Bank; 1/50). The secondary antibodies used were cyanine (Cy)2-goat anti-rabbit immunoglobulin (Ig)G, Rhodamine Red-X goat anti-mouse IgG, Cy2-goat anti-mouse IgG, Cy2-goat anti-rat IgG, Rhodamine Red-X goat anti-rat IgG, Rhodamine Red-X goat anti-rabbit IgG (Jackson ImmunoResearch Laboratories, West Gove, PA), and Alexa 647-goat anti-rabbit IgG (Invitrogen). Images were obtained from a laser scanning confocal microscope (model TCS SP2; Leica, Wetzlar, Germany), and they were processed with Adobe Photoshop software (Mountain View, CA).
Scanning Electron Microscopy
Embryos were collected at timed intervals, dechorionated, fixed in 2.5% glutaraldehyde for 2 h, and devitellinized with methanol. For visualizing filopodia, embryos were devitellinized by hand, using a needle. Embryos were then washed twice in 0.1 M sodium cacodylate, pH 7.4, postfixed in 1% osmiumtetroxide and 0.1 M sodium cacodylate, pH 7.4, for 1 h, and dehydrated in an ethanol series. After exchange of ethanol against carbon dioxide in a critical point dryer, embryos were mounted on stubs, gold-palladium coated in a sputter coater, and photographed in a JEOL LV5600 scanning electron microscope (SEM; JEOL, Tokyo, Japan).
|
RESULTS |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
; Rogers et al., 2004; Dawes-Hoang et al., 2005). We investigated the localization of DRhoGEF2 with respect to F-actin and myosin II at the segmental grooves that first become apparent in the embryonic epidermis during stage 12, shortly after the onset of germband retraction. Groove formation occurs first in thoracic segments by dorsal-ventral elongation of the tracheal pits that are in register with segmental grooves, and it gradually proceeds toward more posterior segments in the course of germband retraction during stage 12. Formation of segmental grooves is initiated by apical constriction of a single cell row in each segment corresponding to the posterior-most en-expressing cells (Larsen et al., 2003). At the onset of groove formation, DRhoGEF2, F-actin, and myosin II are distributed along the apicolateral cortex in all epidermal cells (Figure 1, E–E''). Levels of F-actin and to a much lesser degree DRhoGEF2 were increased in groove founder cells (Figure 1, A–D', arrowheads) that constrict apically and change shape from cuboidal to wedge shaped. Interestingly, F-actin accumulation was polarized in the plane of the epithelium because F-actin levels were consistently highest at the posterior boundary of groove founder cells (Figure 1, C, C', D, and D'). A significant enrichment or polarization of myosin II in groove founder cells could not consistently be detected. Apical constriction of groove founder cells during early stage 12 results in the formation of shallow grooves. As the groove deepens, DRhoGEF2, F-actin, and myosin II begin to accumulate basally at the interface between epidermal cells and the underlying mesoderm (Figure 1, F–F'', arrows). Concomitantly, the epidermis is organized into a stratified columnar epithelium with a defined baseline. Grooves have a symmetrical appearance with the apically constricted groove founder cell at the center flanked by three to four cells on either side. No additional cells enter the groove after this stage; however, during the second half of stage 12 the groove continues to deepen as the basal end of cells moves further inside. Simultaneously, groove founder cells and their immediate neighbors elongate approximately twofold along their apical-basal axis (Figure 1, G, H, and H', arrow).
|
Groove regression temporally overlaps with the differentiation of denticles, which are F-actin–rich protrusions on the apical surface of cells in the ventral epidermis that together form the denticle belts of the mature larva. The cells that constrict during segmental groove regression coincide largely with cells that form denticles. It has been reported that denticle-forming cells undergo shape changes and accumulate F-actin and myosin II at their apical ends. After initial uniform distribution (Figure 1, I and J, arrows), F-actin and myosin II condense into polarized aggregates (Figure 1, K–N) at the posterior cell boundary that subsequently differentiate into denticles (Price et al., 2006; Walters et al., 2006). In contrast, DRhoGEF2 remains cortically enriched in more apicolateral regions (Figure 1, L' and N'), and it is not found in F-actin condensations (Figure 1, O–O'' and P–P'', brackets). In addition, DRhoGEF2 accumulation also occurs in cells of the lateral epidermis that undergo the same cell shape changes as ventral cells, but do not form denticles (Figure 1Q, arrows). These observations are consistent with the view that DRhoGEF2 accumulation may play a role in the regulation of cell shape rather than denticle formation.
Embryos lacking DRhoGEF2 fail to recruit myosin II to the apical membrane of ventral furrow cells during gastrulation (Nikolaidou and Barrett, 2004; Dawes-Hoang et al., 2005). To investigate whether DRhoGEF2 is sufficient to recruit myosin II to the cell membrane, we expressed a UAS-DRhoGEF2-RE transgene (hereafter referred to as DRhoGEF2) (Padash Barmchi et al., 2005) in the epidermis by using prd-Gal4. This resulted in cortical accumulation of myosin II in cells of the prd domain (Figure 1, R–R''), suggesting that DRhoGEF2 may regulate apical myosin II localization also during segmental groove morphogenesis. An effect of DRhoGEF2 on F-actin accumulation could not be detected (Figure 3).
DRhoGEF2 Is Required for Formation and Regression of Segmental Grooves
DRhoGEF2 is maternally supplied to the embryo, and ubiquitously expressed in the zygote. In embryos that are zygotic mutant for the null allele DRhoGEF2l(2)04291, segmental grooves form but occasionally individual grooves fail to regress (Figure 4B), suggesting that DRhoGEF2 may play a role in groove regression. Among embryos derived from DRhoGEF2l(2)04291-mutant germline clones, two phenotypically distinct groups can be distinguished that correspond to maternally (DRhoGEF2M) or maternally and zygotically (DRhoGEF2MZ) mutant embryos. Embryos from either group develop with severe defects during early embryogenesis that make analysis of segmental groove formation very difficult. We therefore stained these embryos for En, which highlights the position at which segmental grooves form in the wild type. Our analysis revealed that despite the severe distortion of the epidermis that occurs in the absence of maternal DRhoGEF2, most segmental grooves form (Figure 2A). In contrast, fully developed grooves that are several cell diameters in depth were not observed in DRhoGEF2MZ mutants (Figure 2B). Analysis at the cellular levels showed that DRhoGEF2MZ embryos form shallow indentations in the epidermis that occasionally, but not always, coincide with the posterior-most en-expressing cell. However, cell shapes are randomized and cell size is irregular with many cells abnormally enlarged. Apical constriction of groove founder cells is not observed, and subsequent morphologic changes in epithelial cells such as apical-basal cell elongation do not occur (Figure 2C, arrowheads). F-actin is often distributed irregularly at the cortex of cells throughout the epithelium (Figure 2D, arrowheads). These results show that DRhoGEF2 is required for F-actin organization and the regulation of cell shape changes at the segmental grooves in the embryonic epidermis.
|
|
).
|
The premature onset of groove invagination and the preservation of normal groove morphology suggest that increased DRhoGEF2-induced contractility at least in part enhances normal aspects of cell behavior and that it can be used to modulate the forces contributing to groove formation. A question that has remained unanswered in the context of groove formation is whether grooves are formed autonomously by shape changes of epidermal cells or whether pulling forces in underlying tissues contribute to furrowing. To address this issue, we expressed DRhoGEF2 exclusively in the mesoderm by using twi-Gal4 as a driver. Interestingly, no effect on groove formation was observed. Grooves did not form prematurely (Figure 3F), groove depth remained unchanged (Figure 3G), and groove regression occurred normally as in wild type (Figure 3H). In contrast, when DRhoGEF2 was overexpressed exclusively in the dorsal ectoderm by using pnr-Gal4, the DRhoGEF2-expressing areas of the epidermis formed deep grooves that persisted throughout dorsal closure of the embryo (Figures 3I and 4, E, F, and F'). These observations suggest that modulation of contractile force in the ectoderm, but not in the underlying mesodermal tissue, can contribute to groove invagination in a cell-autonomous manner.
Role of Diaphanous in Segmental Groove Morphogenesis
To identify other cytoskeletal regulators involved in segmental groove morphogenesis, we investigated Dia, which plays a role in other DRhoGEF2-dependent processes such as pole cell formation and cellularization (Afshar et al., 2000). Dia is distributed throughout the epidermis and enriched at the vertices of epithelial cells, were elevated levels of the adherens junction (AJ) protein Armadillo (Arm) and F-actin are also found (Figure 5, A and A', arrowhead; and C and C'). We found that Dia accumulates apicolaterally in groove founder cells at the onset of groove invagination (Figure 5, B and B', arrowhead). Similar to F-actin, Dia accumulation is polarized to the posterior boundary of groove founder cells consistent with the view that Dia may control F-actin nucleation in these cells (Figure 5, C and C').
|
). We therefore analyzed the strong hypomorphic allele dia5 in which dia protein levels are severely reduced (Figure 5, D and D'). Embryos derived from females homozygous for dia5 in the germline develop with severe morphologic defects and die at the end of embryogenesis. Staining of these embryos for En and F-actin suggested that segmentation occurs normally, however, cells throughout the epidermis were enlarged, irregular in shape and many cells were multinucleated (Figure 5G, arrowheads). F-actin was localized at the cortex but often distributed in irregular aggregates (Figure 5, D' and E'). In addition, the homotypic adherens junction protein E-Cadherin (E-Cad) was lost from the cell cortex in some areas (Figure 5, E–E''), suggesting a role for dia in the maintenance of adherens junctions. In the majority of embryos, no segmental grooves were observed and a coordinated sequence of specific cell shape changes at the segment boundaries could not be identified (Figure 5, H and H'). In a small subset of embryos (
25%) grooves formed despite the abnormal shape of cells, which is likely due to partial rescue by paternally contributed dia (Figure 5, I and I'). We conclude from these observations that dia is required for segmental groove formation.
To investigate the function of Dia in groove founder cells and to compare the specific role of Dia to DRhoGEF2, we expressed constitutive active diaCA (Somogyi and Rørth, 2004) in the embryonic epidermis. Expression of diaCA by using prd-Gal4 resulted in premature formation of segmental grooves that were significantly deeper than in the wild type (Figure 6A), and they persisted at stages when wild-type grooves had regressed (Figure 4D). Expression of diaCA by using wg-Gal4 was able to induce apical constriction in cells that do not constrict in the wild type (Figure 6B, arrowheads, and C–F'), showing that similar to DRhoGEF2, the ability of diaCA to induce cell shape changes is not restricted to cells in the groove.
|
Dia has previously been implicated in maintenance of myosin II at contractile acto-myosin filaments (Dean et al., 2005). To investigate whether Dia affects myosin II in the embryonic epidermis, we expressed diaCA by using wg-Gal4 and we found that myosin II levels were increased at the membrane of diaCA-expressing cells (Figure 6, G and G', arrowheads).
Similar to previous reports diaCA-expression induced extensive filopodia formation at the surface of cells (Figures 4, I and J, arrowhead; and 6, J and J', arrowhead). In the large amnioserosa cells, Dia was localized at the apical tip of filopodia (Figure 6I, arrowhead) where it has also been described in other systems (Schirenbeck et al., 2005; Williams et al., 2007). Filopodia can also be induced by inactivation of DRok downstream of RhoV14 (Williams et al., 2007) consistent with the possibility that a balance of activity between the Dia and Rho-kinase pathways may regulate filopodia formation. We found that DRhoGEF2 mutant cells in the invaginating ventral furrow, and to a lesser degree also cells in the epidermis (data not shown), form large numbers of filopodia-like protrusions (Figure 4M) consistent with the view that DRhoGEF2 may inhibit filopodia, whereas Dia may promote their formation. To test this hypothesis, we coexpressed DRhoGEF2 and diaCA using prd-Gal4. Interestingly, this resulted in the formation of extremely deep segmental grooves (Figure 6, K and K'), indicating an additive effect of Dia and DRhoGEF2. Cells took on a rounded shape characteristic for DRhoGEF2 overexpression (Figure 4K) and at the same time they accumulated high levels of F-actin that was uniformly distributed at the cell cortex (compare Figure 6, J and J' with L and L'). Formation of filopodia was not observed. Together, our data suggest that activation of DRhoGEF2 or Dia results in distinctly different reorganizations of the actin-based cytoskeleton.
|
DISCUSSION |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Subsequent to apical constriction, groove founder cells and their immediate neighbors elongate their apical-basal axis and extend their basal sides inward. Apical-basal elongation does not occur when DRhoGEF2 or diaCA are overexpressed, suggesting that activation of these factors may counteract this specific change in cell shape. Inward movement of the epithelial baseline is not affected, and it occurs to a similar depth as in wild type, suggesting that it is not resulting from a pushing force generated by cell elongation. Expression of DRhoGEF2 in underlying mesodermal cells does not affect groove formation. Thus, cell autonomous bending of the epithelium rather than a pulling force originating in underlying tissues may be the main contributor to groove invagination.
It has been suggested that germband retraction that occurs in parallel to segmental groove formation may contribute to the forces that cause groove founder cells to invaginate (Larsen et al., 2003). In DRhoGEF2 mutants, the posterior midgut primordium remains at the posterior pole due to failure of the endoderm to invaginate (Barrett et al., 1997; Häcker and Perrimon, 1998). Consequently, the germband is thrown into deep folds that illustrate the pressure exerted on the epithelial sheet. Yet, segmental grooves do not form in these embryos. Furthermore, local expression of DRhoGEF2 causes the formation of deep grooves in expressing cells but not in neighboring tissues. These observations do not exclude that global tissue rearrangements support segmental groove invagination, but they suggest that the major contributing force to groove formation is due to autonomous action of epidermal cells.
At the onset of stage 13 when germband retraction has completed, DRhoGEF2, F-actin, and myosin II accumulate apically in four to five cells posterior to the grooves. These cells subsequently constrict apically, which may bend the epithelium in an outward direction. Thus, groove regression may be actively driven by actomyosin-based constriction of epidermal cells. This is supported by previous findings showing that in zipper mutants, which partially lack nonmuscle myosin, grooves occasionally do not regress (Larsen et al., 2003).
Groove regression coincides with the formation of denticles in ventral epidermal cells posterior to the groove (Price et al., 2006; Walters et al., 2006). Denticle forming cells undergo cell shape changes that are followed by polarized accumulation of F-actin, myosin II, and Dia into condensations at the posterior cell boundary. Our analysis showed that DRhoGEF2 in contrast to Dia and myosin II, does not polarize to the F-Actin condensations and DRhoGEF2 mutants are able to form denticles (Padash Barmchi et al., 2005). DRhoGEF2 is therefore unlikely to play a role in denticle formation. We propose that the narrowing of cells along the anterior-posterior axis that occurs throughout the ventral and lateral epidermis before hair formation may be due to DRhoGEF2-controlled cell contraction and may contribute to the regression of segmental grooves.
In conclusion, our data suggest that groove invagination and regression are active processes driven by acto-myosin-based contractile filaments in epidermal cells that are regulated by DRhoGEF2 and Dia in a concentration-dependent manner.
DRhoGEF2 Effector Pathways in Segmental Groove Morphogenesis
The small GTPase Rho1 regulates acto-myosin constriction through a pathway including Rho-kinase (Winter et al., 2001), the regulatory subunit of myosin light chain phosphatase (Mizuno et al., 2002; Tan et al., 2003) and myosin II. DRhoGEF2 regulates cell contraction in different developmental contexts and DRhoGEF2-mediated recruitment of myosin II to the apical cortex and cell contraction require Rho-kinase (Rogers et al., 2004; Dawes-Hoang et al., 2005), suggesting that DRhoGEF2 controls acto-myosin contraction through the Rho-kinase pathway.
Rho1 also regulates F-actin nucleation and polymerization through a pathway including Dia and the actin-binding protein Profilin (Wallar and Alberts, 2003). In cell culture systems, mammalian Dia1 has been shown to act as a positive feedback regulator of Rho-activity through direct interaction with the C terminus of the mammalian DRhoGEF2 homologue LARG (Kitzing et al., 2007). However, it is not clear whether Dia can act as an effector of DRhoGEF2 during morphogenesis in vivo. Drosophila Dia is required in processes such as metaphase furrow invagination, pole cell formation and cellularization (Afshar et al., 2000) that are also defective in DRhoGEF2 mutants consistent with the idea that both proteins may function in concert.
Our data suggest that DRhoGEF2 and Dia are required for segmental groove morphogenesis. Interestingly, morphologic changes elicited by DiaCA at the epithelial and cellular level were distinct from those observed with DRhoGEF2. Whereas DRhoGEF2-overexpression caused cells to contract and take on a rounded shape, thereby reducing contact with each other, diaCA-expressing cells remained tightly packed and columnar. In addition, cells showed increased levels of the junctional proteins β-catenin and E-cadherin, suggesting a strengthening of cell–cell contacts. Consistent with this, mammalian Dia1 has been implicated in maintenance and stabilization of adherens junctions. In contrast, Rho-kinase-mediated generation of contractile force leads to the physical disruption of cell–cell contacts and cell rounding reminiscent of DRhoGEF2 overexpression (Sahai and Marshall, 2002). Another distinguishing feature between DRhoGEF2 and dia is the accumulation of F-actin in response to diaCA expression that is consistent with the role of Dia in nucleation and elongation of F-actin filaments. F-actin accumulation was not observed in response to DRhoGEF2-overexpression consistent with the view that DRhoGEF2 regulates F-actin contraction but not polymerization. We find that DRhoGEF2 and Dia have qualitatively different effects on F-actin remodeling, supporting the view that they may be connected to the actin cytoskeleton by distinct Rho-effector pathways. Despite the differences, overexpression of either DRhoGEF2 or diaCA in some instances seems to result in increased contractility. In DRhoGEF2, this is likely due to direct regulation of acto-myosin contractility through the Rho-kinase pathway. In DiaCA, the increased levels of actin elicited by Dia activation might indirectly promote the formation of acto-myosin filaments. Alternatively, DiaCA could affect contractility through its effect on myosin.
Expression of either diaCA or DRhoGEF2 led to increased myosin II levels. In Schneider cells, DRok activity and myosin phosphorylation were found to be required for myosin II recruitment to contractile acto-myosin fibers, whereas Dia was required for maintenance of myosin at contractile rings (Dean et al., 2005). Similar to Rho-kinase, DRhoGEF2 is required for apical recruitment of myosin II in the ventral furrow during gastrulation (Dawes-Hoang et al., 2005). A role of Dia in this process has not been reported but it is possible that DRhoGEF2 and Dia may regulate different aspects of myosin function also in the Drosophila embryo.
In summary, we find that DRhoGEF2 and Dia are required for segmental groove formation and that they may act in concert to regulate a series of specific cell shape changes that lead to groove invagination. However, clear differences in the response of cells to activation of DRhoGEF2 or Dia at the morphologic and molecular level suggest that both genes may regulate the actin cytoskeleton through distinct effector pathways.
Note added in proof. We state that a role for Dia in gastrulation has not been reported. Since submission of this manuscript Homem and Peifer (2008) have reported such a role and also describe a function for Dia in the regulation of myosin and adherens junctions similar to our data.
|
ACKNOWLEDGMENTS |
|---|
|
Footnotes |
|---|
* Present address: Department of Zoology, Life Sciences Institute, 2350 Health Sciences Mall, Vancouver, BC, Canada V6T 1Z3. 
Address correspondence to: Udo Häcker (udo.hacker{at}med.lu.se)
|
REFERENCES |
|---|
|
TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
|---|
Amano, M., Ito, M., Kimura, K., Fukata, Y., Chihara, K., Nakano, T., Matsuura, Y., and Kaibuchi, K. (1996). Phosphorylation and activation of myosin by Rho-associated kinase (Rho-kinase). J. Biol. Chem 271, 20246–20249.
Barrett, K., Leptin, M., and Settleman, J. (1997). The Rho GTPase and a putative RhoGEF mediate a signaling pathway for the cell shape changes in Drosophila gastrulation. Cell 91, 905–915.[CrossRef][Medline]
Bloor, J. W., and Kiehart, D. P. (2002). Drosophila RhoA regulates the cytoskeleton and cell-cell adhesion in the developing epidermis. Development 129, 3173–3183.
Castrillon, D. H., and Wasserman, S. A. (1994). Diaphanous is required for cytokinesis in Drosophila and shares domains of similarity with the products of the limb deformity gene. Development 120, 3367–3377.[Abstract]
Chou, T. B., and Perrimon, N. (1996). The autosomal FLP-DFS technique for generating germline mosaics in Drosophila melanogaster. Genetics 144, 1673–1679.[Abstract]
Dawes-Hoang, R. E., Parmar, K. M., Christiansen, A. E., Phelps, C. B., Brand, A. H., and Wieschaus, E. F. (2005). Folded gastrulation, cell shape change and the control of myosin localization. Development 132, 4165–4178.
Dean, S. O., Rogers, S. L., Stuurman, N., Vale, R. D., and Spudich, J. A. (2005). Distinct pathways control recruitment and maintenance of myosin II at the cleavage furrow during cytokinesis. Proc. Natl. Acad. Sci. USA 102, 13473–13478.
Grosshans, J., Wenzl, C., Herz, H. M., Bartoszewski, S., Schnorrer, F., Vogt, N., Schwarz, H., and Muller, H. A. (2005). RhoGEF2 and the formin Dia control the formation of the furrow canal by directed actin assembly during Drosophila cellularisation. Development 132, 1009–1020.
Häcker, U., and Perrimon, N. (1998). DRhoGEF2 encodes a member of the Dbl family of oncogenes and controls cell shape changes during gastrulation in Drosophila. Genes Dev 12, 274–284.
Homem, C. C., and Peifer, M. (2008). Diaphanous regulates myosin and adherens junctions to control cell contractility and protrusive behavior during morphogenesis. Development 135, (6), 1005–1018.
Kitzing, T. M., Sahadevan, A. S., Brandt, D. T., Knieling, H., Hannemann, S., Fackler, O. T., Grosshans, J., and Grosse, R. (2007). Positive feedback between Dia1, LARG, and RhoA regulates cell morphology and invasion. Genes Dev 21, 1478–1483.
Larsen, C. W., Hirst, E., Alexandre, C., and Vincent, J. P. (2003). Segment boundary formation in Drosophila embryos. Development 130, 5625–5635.
Lawrence, P. A., and Struhl, G. (1996). Morphogens, compartments, and pattern: lessons from Drosophila? Cell 85, 951–961.[CrossRef][Medline]
Magie, C. R., Meyer, M. R., Gorsuch, M. S., and Parkhurst, S. M. (1999). Mutations in the Rho1 small GTPase disrupt morphogenesis and segmentation during early Drosophila development. Development 126, 5353–5364.[Abstract]
Martinez-Arias, A., and Lawrence, P. A. (1985). Parasegments and compartments in the Drosophila embryo. Nature 313, 639–642.[CrossRef][Medline]
Mizuno, T., Tsutsui, K., and Nishida, Y. (2002). Drosophila myosin phosphatase and its role in dorsal closure. Development 129, 1215–1223.
Nikolaidou, K. K., and Barrett, K. (2004). A Rho GTPase signaling pathway is used reiteratively in epithelial folding and potentially selects the outcome of Rho Activation. Curr. Biol 14, 1822–1826.[CrossRef][Medline]
Padash Barmchi, M., Rogers, S., and Häcker, U. (2005). DRhoGEF2 regulates actin organization and contractility in the Drosophila blastoderm embryo. J. Cell Biol 168, 575–585.
Price, M. H., Roberts, D. M., McCartney, B. M., Jezuit, E., and Peifer, M. (2006). Cytoskeletal dynamics and cell signaling during planar polarity establishment in the Drosophila embryonic denticle. J. Cell Sci 119, 403–415.
Rogers, S. L., Wiedemann, U., Häcker, U., Turck, C., and Vale, R. D. (2004). Drosophila RhoGEF2 associates with microtubule plus ends in an EB1-dependent manner. Curr. Biol 14, 1827–1833.[CrossRef][Medline]
Royou, A., Field, C., Sisson, J. C., Sullivan, W., and Karess, R. (2004). Reassessing the role and dynamics of nonmuscle myosin II during furrow formation in early Drosophila embryos. Mol. Biol. Cell 15, 838–850.
Sahai, E., and Marshall, C. J. (2002). ROCK and Dia have opposing effects on adherens junctions downstream of Rho. Nat. Cell Biol 4, 408–415.[CrossRef][Medline]
Schirenbeck, A., Bretschneider, T., Arasada, R., Schleicher, M., and Faix, J. (2005). The Diaphanous-related formin dDia2 is required for the formation and maintenance of filopodia. Nat. Cell Biol 7, 619–625.[CrossRef][Medline]
Somogyi, K., and Rorth, P. (2004). Evidence for tension-based regulation of Drosophila MAL and SRF during invasive cell migration. Dev. Cell 7, 85–93.[CrossRef][Medline]
Tan, C., Stronach, B., and Perrimon, N. (2003). Roles of myosin phosphatase during Drosophila development. Development 130, 671–681.
Vincent, J. P., and O'Farrell, P. H. (1992). The state of engrailed expression is not clonally transmitted during early Drosophila development. Cell 68, 923–931.[CrossRef][Medline]
Wallar, B. J., and Alberts, A. S. (2003). The formins: active scaffolds that remodel the cytoskeleton. Trends Cell Biol 13, 435–446.[CrossRef][Medline]
Walters, J. W., Dilks, S. A., and DiNardo, S. (2006). Planar polarization of the denticle field in the Drosophila embryo: roles for Myosin II (zipper) and fringe. Dev. Biol 297, 323–339.[CrossRef][Medline]
Watanabe, N., Kato, T., Fujita, A., Ishizaki, T., and Narumiya, S. (1999). Cooperation between mDia1 and ROCK in Rho-induced actin reorganization. Nat. Cell Biol 1, 136–143.[CrossRef][Medline]
Watanabe, N., Madaule, P., Reid, T., Ishizaki, T., Watanabe, G., Kakizuka, A., Saito, Y., Nakao, K., Jockusch, B. M., and Narumiya, S. (1997). p140mDia, a mammalian homolog of Drosophila diaphanous, is a target protein for Rho small GTPase and is a ligand for profilin. EMBO J 16, 3044–3056.[CrossRef][Medline]
Williams, M. J., Habayeb, M. S., and Hultmark, D. (2007). Reciprocal regulation of Rac1 and Rho1 in Drosophila circulating immune surveillance cells. J. Cell Sci 120, 502–511.
Winter, C. G., Wang, B., Ballew, A., Royou, A., Karess, R., Axelrod, J. D., and Luo, L. (2001). Drosophila Rho-associated kinase (Drok) links Frizzled-mediated planar cell polarity signaling to the actin cytoskeleton. Cell 105, 81–91.[CrossRef][Medline]
This article has been cited by other articles:
|
|
 |
|
  S. J. Warner and G. D. Longmore Distinct functions for Rho1 in maintaining adherens junctions and apical tension in remodeling epithelia J. Cell Biol., June 15, 2009; 185(6): 1111 - 1125. [Abstract] [Full Text] [PDF]
|
|
|
|
 |
|
 R. L. Webb, M.-N. Zhou, and B. M. McCartney A novel role for an APC2-Diaphanous complex in regulating actin organization in Drosophila Development, April 15, 2009; 136(8): 1283 - 1293. [Abstract] [Full Text] [PDF]
|
|
| ||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||||
