Rho-kinase regulates tissue morphogenesis via non-muscle myosin and LIM-kinase during Drosophiladevelopment
© Verdier et al; licensee BioMed Central Ltd. 2006
Received: 14 June 2006
Accepted: 01 August 2006
Published: 01 August 2006
The Rho-kinases (ROCKs) are major effector targets of the activated Rho GTPase that have been implicated in many of the Rho-mediated effects on cell shape and movement via their ability to affect acto-myosin contractility. The role of ROCKs in cell shape change and motility suggests a potentially important role for Rho-ROCK signaling in tissue morphogenesis during development. Indeed, in Drosophila, a single ROCK ortholog, DRok, has been identified and has been found to be required for establishing planar cell polarity.
We have examined a potential role for DRok in additional aspects of tissue morphogenesis using an activated form of the protein in transgenic flies. Our findings demonstrate that DRok activity can influence multiple morphogenetic processes, including eye and wing development. Furthermore, genetic studies reveal that Drok interacts with multiple downstream effectors of the Rho GTPase signaling pathway, including non-muscle myosin heavy chain, adducin, and Diaphanous in those developmental processes. Finally, in overexpression studies, we determined that Drok and Drosophila Lim-kinase interact in the developing nervous system.
These findings indicate widespread diverse roles for DRok in tissue morphogenesis during Drosophila development, in which multiple DRok substrates appear to be required.
Rho-kinases (also known as ROKs or ROCKs) were the first Rho GTPase-binding effectors to be identified and were initially characterized as mediators of the formation of RhoA-induced stress fibers and focal adhesions [1, 2]. ROCKs are serine-threonine kinases that are most homologous to myotonic dystrophy kinase (DMPK) and citron kinase. They are comprised of a kinase domain at the N-terminus, followed by a coiled-coil domain containing a Rho-binding domain and a Pleckstrin-homology domain (PH) .
In non-muscle cells, ROCKs control a variety of cellular processes downstream of Rho, many of which depend upon actin cytoskeleton organization and cell contractility. These include cell-matrix and cell-cell adhesion, cell migration, neurite retraction and outgrowth, and cytokinesis.
Expression of a dominant-negative form of ROCK or treatment of cells with the selective pharmacologic ROCK inhibitor Y-27632 inhibits LPA-induced and Rho-induced formation of actin stress fibers and focal adhesions, implicating ROCKs in Rho-dependent signaling pathways to the cytoskeleton .
Several downstream substrates of ROCK that mediate such biological activity have been identified. The regulatory myosin-light-chain (MLC) of the protein myosin II is one substrate that is essential in regulating actomyosin contractility [4, 5]. MBS, the myosin-binding subunit of the myosin-light-chain phosphatase (MLCP) has also been established as a mediator of ROCK function . MLCP dephosphorylates MLC, implicating it as a negative regulator of acto-myosin contractility. ROCK phosphorylates MBS, consequently inhibiting its phosphatase activity and resulting in higher MLC phosphorylation . Thus, there is a dual regulation of myosin II phosphorylation by ROCK; i.e., directly through MLC and through MBS, to exert its biological effects on actomyosin contractility.
Another ROCK substrate implicated in actin dynamics is LIMK (Lim-kinase). LIMKs are serine/threonine kinases that can regulate actin filament assembly. They are directly phosphorylated by ROCK, consequently increasing LIMK's kinase activity towards cofilin, an actin-binding and -depolymerizing protein in its unphosphorylated state, and which regulates the turnover of actin filaments [8, 9].
In light of its prominent role in Rho-dependent cytoskeletal dynamics, ROCK function has also been studied in the context of tissue morphogenesis in several multicellular model organisms where it has been implicated in various developmental processes, including organogenesis in higher vertebrates such as chicken and mouse , embryo elongation and cytokinesis in C. elegans [11–13], and gastrulation in zebrafish . ROCK has also been shown to function downstream of the Wnt/planar cell polarity pathway to ensure convergent extension cell movements during vertebrate gastrulation in the Xenopus embryo .
Here, we describe studies to address DRok-mediated signaling pathways in various aspects of tissue morphogenesis in developing Drosophila. By expressing a constitutively activated form of DRok, we observed disruption of normal development in various tissues, and these phenotypes can be suppressed by reducing the activity of known Rho pathway components through genetic interactions, suggesting that the observed phenotypes reflect functions of endogenous DRok. In an unbiased genetic screen to identify suppressors of a DRok-induced developmental defect, we isolated several alleles of a non-muscle myosin heavy chain ortholog (zipper). We also found that the Rho-kinase substrate, Lim-kinase, is likely to mediate functions of DRok in the developing nervous system. These studies collectively reveal that DRok mediates multiple aspects of tissue morphogenesis during development through multiple downstream effectors.
Results and discussion
Expression of a constitutively active form of DRok in various Drosophilatissues results in morphogenesis defects in developing tissues
The full-length ROCK protein is folded in such a way that the C-terminus binds to and negatively regulates the kinase activity of the N-terminus . Therefore, to investigate downstream functions of DRok activity, we generated a mutant form of DRok predicted to be constitutively active, which consists of the isolated kinase region (DRok-cat for DRok-catalytic), sharing 65% identity with the corresponding isolated domain of mammalian ROCK1 (Fig. 1B). The latter has been previously reported to function as a constitutively active protein . The activated form of DRok was expressed in various tissues of transgenic flies, under the control of a UAS promoter, using the UAS-GAL4 transactivation system, or directly in the eye under the control of the eye-specific GMR-promoter.
Phenotypes generated by different expression levels of DRok-cat in many Drosophila tissues
3rd instar larval discs
Viable. Rough eyes
Viable. Rough eyes
Viable no phenotype
Viable no phenotype
Viable no phenotype
Viable no phenotype
Viable no phenotype
Viable no phenotype
Wing discs-posterior half
Viable no phenotype
Viable no phenotype
Slightly rough eyes
Slightly rough eyes
In addition, it is worth mentioning that although it is formally possible that phenotypes generated by expression of GMR-Drok-cat could result from non-specific secondary effects due to the engineered expression of the mutant DRok-cat protein, the biological relevance of these phenotypes has been demonstrated and will be described below with the analysis of genetic interactions between Drok and other components of the Rho GTPase pathway in this DRok-cat-expression model.
Ubiquitous expression of DRok-cat using actin-GAL4 or tubulin-GAL4 drivers results in lethality at embryonic or early larval stages, independently of the level of DRok-cat expression. Similar results are observed when DRok-cat is specifically expressed in early embryogenesis: daughterless-GAL4 (da-GAL4) and prd-GAL4 are two embryonic drivers that produce a larval or embryonic lethal phenotype when driving expression of DRok-cat. Targeted expression of DRok-cat to some tissues, such as third instar larval discs (69B-GAL4), epidermal leading edge cells in embryogenesis (LE-GAL4), or the central nervous system (elav-GAL4, 1407-GAL4), results in lethality depending on the dosage of expressed DRok-cat. Together, these findings suggest that DRok activity must be tightly regulated during embryogenesis. Notably, genetic disruption of Drok (a null allele of the gene) has previously been reported, and zygotic mutant animals die at the larval stage, indicating that Drok is an essential Drosophila gene , and suggesting that excessive or insufficient DRok activity is incompatible with normal development.
Excessive DRok activity disrupts both ommatidial structure and crossvein formation in the developing eye and wing, respectively
Expression of DRok-cat in the posterior half of the wing, using en-GAL4 as a driver, results in disappearance of the crossveins, suggesting that DRok may play a role in crossvein formation (Fig. 2H, arrows). Unlike the eye phenotype, this is not rescued by overexpression of p35 in the wing, indicating that the absence of crossveins is probably not due to apoptosis of crossvein cells (data not shown). In addition, expression of DRok-cat does not produce a wing hair polarity phenotype, unlike Drok2 mutant clones . As described above, it is possible that, upon expression of activated DRok in the posterior half of the wing, multiple integrated signaling pathways are activated to produce a visible crossvein phenotype rather than hair polarity defects. The presence or absence of crossveins in wing development has been reported to depend upon an inductive signal from the dorsal wing epithelium that is required for the formation of vein tissue in the ventral wing epithelium. That process has been shown to involve the products of the crossvein (cv), cv-2, decapentaplegic (dpp), glass bottom boat genes, and other components of the Bone Morphogenetic Protein (BMP)-like signaling pathway . Therefore, to further characterize the DRok-cat crossveinless wing phenotype, we tested genetic interactions between Drok and the above genes but did not detect any interaction in the en-GAL4-induced DRok-cat expression background. This might reflect the fact that modifications of the DRok-cat phenotype, which is due to expression of a constitutively activated form, would only be expected using mutations that affect pathway components downstream of DRok.
Drokgenetically interacts with members of the Rho GTPase signaling pathways in the eye and in the wing
DRok interacts with zipper, the Drosophilanon-muscle myosin heavy chain, in the eye and in the wing
sqh, unlike zipper, does not interact with constitutively activated Drok
As previously mentioned, Zipper is orthologous to members of the family of non-muscle myosin heavy chain proteins. Myosin heavy chains are chemo-mechanical motors which drive contraction of the actin cytoskeleton. These proteins bind to non-muscle myosin light chains (MLC) and their assembly forms myosin II molecules . Rho-kinase has been reported to regulate the phosphorylation of the non-muscle myosin regulatory light chain (MLC), primarily at Ser-19 and secondarily at Thr-18 both in vitro and in vivo. Phosphorylation of MLC at these sites induces a conformational change that allows myosin II to form filaments and increases its actin-activated ATPase activity [3, 4]. In Drosophila, the regulation by DRok of the phophorylation of Sqh, the Drosophila ortholog of MLC has also been demonstrated and Sqh and Zipper have been shown to both participate in establishing wing hair number in planar epithelial polarity . In addition, Drok, sqh and zipper have been reported to interact genetically during another Drosophila developmental process, namely dorsal closure . We therefore tested whether sqh also interacts with Drok in our overexpression system. Loss of one copy of the sqh gene (using the loss-of-function sqh2 allele) does not suppress the two-copy GMR-Drok-cat-induced rough eye phenotype, and has no effect on the single-copy GMR-Drok-cat-induced ommatidial disruption (Fig. 4G). In addition, heterozygosity for sqh2 does not even partially rescue the wing crossvein phenotype (data not shown). One possible explanation is that the inability of sqh2 to rescue the wing phenotype or the GMR-Drok-cat eye phenotype is due to perdurance of maternal Sqh, which lasts longer than that of maternal Zipper in development: homozygous sqh2 (null allele) mutant animals die at the 3rd instar larval stage, whereas homozygous zip1 (null allele) animals die at the embryonic stage. As a result, the absence of rescue of the wing or the GMR-Drok-cat eye phenotype by sqh2, not by zip1, may reflect a differential perdurance of Sqh and Zipper maternal products. Another possibility is that Drok might interact genetically with zipper independently of the Drok-sqh known interaction in a particular biological context. Thus, it is possible that DRok directly regulates Zipper in addition of phosphorylating Sqh. Unpublished studies from Acanthamoeba castellanii have shown that incorporation of 32Pi into crude extracts of A. castellanii myosin II heavy chain is significantly decreased in the presence of the Rho-kinase inhibitor Y-27632 or antibodies against ROCK, suggesting that Rho-kinase might be regulating the phosphorylation of myosin II heavy chain in vitro, in A. castellanii. However, in trying to address the biochemical nature of the genetic interaction between Drok and zipper in mammalian cells, we were unable to detect phosphorylation of nmMHC by Rho-kinase in vitro in a kinase assay or in vivo in a 32Pi incorporation assay, although several putative consensus sites for Rho-kinase phosphorylation have been found in the nmMHC amino acid sequence (data not shown). Thus, in addition to the Drok-sqh-zip interaction reported so far, there may be an interaction between Zipper and DRok via a Sqh-independent pathway, but this interaction may not be regulated by phosphorylation, rather by other signaling events. Interestingly, in the eye disc, DRok has been suggested previously as not being the major kinase responsible for the phosphorylation of Sqh, as phospho-Sqh staining was not altered in Drok2 mutant clones . Another kinase might phosphorylate Sqh specifically in the eye disc, which could explain the lack of detected genetic interaction between Drok and sqh in our system.
Overall, we have shown genetic evidence that levels of Zipper, the Drosophila non-muscle myosin heavy chain, limit the actions of DRok during Drosophila development, and that Zipper, in addition to Sqh, is an important downstream player in mediating DRok's biological effects during various developmental processes, such as in the eye and in the wing.
DRok interacts with Lim-kinase in the developing nervous system
Among the Rho-kinase substrates that have been strongly implicated in neural development are the Lim-kinases. The single Drosophila Lim-kinase (DLimk) is required for proper synapse formation and proper regulation of its activity is necessary for normal axon growth [32, 33]. To determine whether DRok-mediated activation of DLimk plays a role in proper neural development, we crossed transgenic flies expressing activated DRok with flies over-expressing DLimk to examine phenotypes in the developing nervous system. First, using GMR-driven transgenes to identify a potential interaction in the developing eye, we observed that while overexpression of DLimk causes no detectable effects on eye development (Fig. 5J), co-expression of DLimk with activated DRok results in a dramatic disruption of eye development associated with a severe morphology defect of the external eye and a reduced overall eye size (Fig. 5L). Since the effects of a single-copy DRok-cat transgene on exterior eye structures in this setting are relatively mild (Fig. 5K), this finding is consistent with a synergistic interaction between these two proteins, and suggests that a DRok-DLimk signal may be influencing normal development. Second, a similar synergistic interaction between DRok and DLimk was observed in the developing central nervous system. Using an elav-GAL4 driver to express UAS-linked Drok-cat and Dlimk in developing neurons, it was observed that neither protein alone causes any detectable effect on the appearance of the embryonic nervous system (Fig. 5N, 5O), whereas co-expression of the proteins results in the appearance of breaks along the ventral nerve cord (Fig. 5P). These findings suggest that DLimk is likely to mediate at least some of the DRok-dependent functions in the developing nervous system.
In conclusion, our genetic analysis of DRok in development, using ovexpression studies in the eye, in the wing and in the CNS indicates that stringent regulation of DRok activity is required for various developmental processes, such as photoreceptor maintenance and wing vein formation. In addition, our overexpression system has revealed zipper, the Drosophila nonmuscle myosin heavy chain, as a strong genetic interactor of DRok, as seen in other reported developmental events such as dorsal closure and wing planar cell polarity, confirming that myosin II is a key downstream mediator of Rho-kinase biological effects in several morphogenetic processes. Moreover, we have shown that DRok interacts with another target protein, DLimk, to influence some other aspects of tissue morphogenesis, including CNS development.
Drosophilastrains and transgenes
Drosophila stocks were maintained at 25°C. Generated stocks include GMR-Drok-cat, UAS-Drok-cat, GMR-Dlimk and UAS-Dlimk. The Drok cDNA was isolated in a yeast two-hybrid screen of a Drosophila embryo Matchmaker™ cDNA library in pACT2 vector (Clontech) with constitutively active RhoL63 as bait. A GMR-Drok-cat transgene was generated by subcloning the catalytic domain (amino acids 1–506) of DRok (DRok-cat) from the Drok-pBSK plasmid (Hinc II site) into the pGMR vector (Stu I site). A UAS-Drok-cat was then constructed by subcloning from the Drok-cat-pGMR plasmid (EcoR I site) into the pUAST vector (EcoR I site). The Drosophila limk coding sequence was subcloned into the pGMR and pUAST vectors and transformed into Drosophila as described previously . GAL4 drivers include engrailed-GAL4 (en-GAL4), actin5c-GAL4, tubulin-GAL4, daughterless-GAL4 (da-GAL4), prd-GAL4, 69B-GAL4, LE-GAL4, elav-GAL4, 1407-GAL4, Cy6-GAL4, and eyeless-GAL4. Other stocks utilized include sqh2 and zip1 (kindly provided by Daniel Kiehart), GMR-p35, hts1103 (from the Bloomington Stock Center), dia k07135 (Bloomington), and Rho1720 (Bloomington).
Microscopy and immunochemistry
For adult eye images, sections, and scanning electron micrographs (SEMs), genotypes were as follows: Ore R ; GMR-Drok-cat/+; GMR-Drok-cat/GMR-Drok-cat; GMR-Drok-cat1-GMR-Drok-cat2/+; zip1/+, GMR-Drok-cat1-GMR-Drok-cat2/+; sqh2/+, GMR-Drok-cat1-GMR-Drok-cat2/+; GMR-p35/+, GMR-Drok-cat/+; hts1103/+, GMR-Drok-cat/+; dia k07135 GMR-Drok-cat/+; GMR-Dlimk/+; GMR-Dlimk/GMR-Drok-cat. Adult eye sections were performed according to standard protocols . For adult wings, the genotypes were as follows: engr-GAL4/+, UAS-Drok-cat/+; zip1/en-GAL4, UAS-Drok-cat/+; hts1103/en-GAL4, UAS-Drok-cat/+; rho1720/en-GAL4, UAS-Drok-cat/+; dia k07135 /en-GAL4, UAS-Drok-cat/+. Staining of dissected third instar larval imaginal eye discs was performed as described previously . Staining of embryonic CNS (central nervous system) was carried out as described previously . The following antibodies were used: mouse anti-Elav (1:50, Developmental Studies Hybridoma Bank – DSHB), mouse 24B10 (anti-Chaoptin) (1:50, DHSB), mouse monoclonal BP102 (1:200, DHSB). Rhodamine-phalloidin (Sigma) was used at 1:200. Immunofluorescence images were collected on a Carl Zeiss Axiovert 100 M confocal microscope.
Mutagenesis was performed using ethyl methane sulfonate (EMS) as described previously . Females carrying two copies of the GMR-Drok-cat transgene on each chromosome III were mated to mutagenized males and F1 male progeny was screened for suppression of the two copy-GMR-Drok-cat-induced rough eye phenotype. Stocks were made from each male carrying a putative suppressor mutation.
We are grateful to members of the Settleman laboratory for helpful discussions. We thank Alexey Veraksa for assistance with confocal microscopy. Reagents were generously provided by Daniel Kiehart. Some Drosophila stocks and antibodies were obtained from the Bloomington Stock Center and Developmental Studies Hybridoma Bank, respectively. This work was supported by NIH grant RO1 GM60466 to J.S.
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