Research Article 2197 Rock2 controls TGFβ signaling and inhibits mesoderm induction in zebrafish embryos

Yu Zhang1, Xiang Li1, Jingjing Qi1, Jialu Wang1, Xingfeng Liu1, Haiwen Zhang1, Sheng-Cai Lin2 and Anming Meng1,3,* 1Protein Science Laboratory of Ministry of Education, Department of Biological Sciences and Biotechnology, Tsinghua University, Beijing 100084, China 2School of Life Sciences, Xiamen University, Xiamen, Fujian 361005, China 3State Key Laboratory of Biomembrane and Membrane Bioengineering, Institute of Zoology, Chinese Academy of Sciences, Beijing 100101, China *Author for correspondence (e-mail: [email protected])

Accepted 16 March 2009 Journal of Cell Science 122, 2197-2207 Published by The Company of Biologists 2009 doi:10.1242/jcs.040659

Summary The Rho-associated serine/threonine kinases Rock1 and Rock2 induction of mesoderm by ectopic Nodal signals. By contrast, play important roles in cell contraction, adhesion, migration, overexpression of a dominant negative form of zebrafish rock2a, proliferation and apoptosis. Here we report that Rock2 acts as dnrock2a, has an opposite effect on mesoderm induction, a negative regulator of the TGFβ signaling pathway. suggesting that Rock2 are endogenous inhibitors for Mechanistically, Rock2 binds to and accelerates the lysosomal mesoderm induction. Thus, our data have unraveled previously degradation of TGFβ type I receptors internalized from the cell unidentified functions of Rock2, in controlling TGFβ signaling surface in mammalian cells. The inhibitory effect of Rock2 on as well as in regulating embryonic patterning. TGFβ signaling requires its kinase activity. In zebrafish embryos, injection of rock2a mRNA attenuates the expression of mesodermal markers during late blastulation and blocks the Key words: Rho kinases, Rock2, TGFβ, Zebrafish, Embryo

Introduction microtubules (Salinas, 2007; Soldati and Schliwa, 2006). RhoA, a The transforming growth factor β (TGFβ) member of the Ras homology family of small GTPases, is a key pathway, which involves multiple ligands, including Activin, Nodal, regulator of the cytoskeleton during cell morphogenesis and Vg1 and GDFs, is a key player during early development of motility (Etienne-Manneville, 2004; Ridley, 2006). The Rho- vertebrate embryos. Among the TGFβ ligands, Nodal proteins are associated serine/threonine kinases Rock1 (also termed Rok1 and

Journal of Cell Science absolutely required for mesendoderm induction and the Rokβ) and Rock2 (also termed Rok2, Rokα and Rho kinase) are establishment of left-right asymmetry (Schier, 2003; Shen, 2007; immediate downstream targets of RhoA (Amano et al., 1997; Tian and Meng, 2006). These ligands initiate signal transduction Ishizaki et al., 1996; Leung et al., 1995; Matsui et al., 1996); they by binding to type I and type II serine/threonine kinase receptors phosphorylate a variety of substrates, including myosin light chain on the plasma membrane, which results in the activation of type I (Amano et al., 1996; Totsukawa et al., 2000), the myosin-binding receptors (Shi and Massague, 2003). The activated receptors subunit (MYPT1) of the myosin-associated form of phosphorylate the downstream effectors Smad2 and/or Smad3, and phosphatase 1 (Velasco et al., 2002), LIM kinases (Ohashi et al., the phosphorylated Smad2 or Smad3 form complexes with Smad4 2000; Sumi et al., 2001), adducin (Fukata et al., 1999) and Zipper- and translocate into the nucleus to control the expression of target interacting (Hagerty et al., 2007). Rho kinases may (Jia et al., 2008; Schmierer and Hill, 2007). The multiple intervene with intracellular transduction and propagation of various steps that are involved in the TGFβ signal transduction pathway extracellular signals by assisting intracellular targeting of the allow for precise regulation by many modulators through diverse signaling pathway components. Several reports have shown that mechanisms (Chen and Meng, 2004). RhoA and Rho kinases can be activated by TGFβ signaling and are Intracellular trafficking of internalized TGFβ receptors has been required for mediating the effects of TGFβ signals in epithelial-to- found to play a role in regulating TGFβ signaling. The membrane- mesenchymal transdifferentiaion, actin filament rearrangement and anchored receptors are internalized by clathrin-dependent and clathrin- growth inhibition (Bhowmick et al., 2001; Edlund et al., 2002; independent processes (Anders et al., 1997; Di Guglielmo et al., 2003; Kamaraju and Roberts, 2005). However, it is largely unknown Lu et al., 2002; Mitchell et al., 2004; Zwaagstra et al., 2001). The whether RhoA and Rho kinases function to down-regulate TGFβ internalized receptors are then recycled back to the membrane or signal transduction. degraded via proteasomal or lysosomal pathways (Di Guglielmo et Rho kinases have been found to be important regulators of cell al., 2003; Kavsak et al., 2000; Mitchell et al., 2004). Distinct targeting contraction, adhesion and migration as well as proliferation and of the internalized receptors, a process regulated by multiple factors, apoptosis (Riento and Ridley, 2003; Shi and Wei, 2007). Many studies determines the duration of the intracellular signaling, and thus has using Rho kinase inhibitors have indicated that Rho kinases are profound effects on vertebrate embryonic development (Jullien and involved in various physiological and pathological processes (Shi and Gurdon, 2005; Shim et al., 2006; Zhang et al., 2004). Wei, 2007; Wettschureck and Offermanns, 2002). Developmental Targeting and trafficking of intracellular proteins, including roles of Rho kinases have been examined in mice by knockout internalized receptors, rely on functions of actin-filaments and approaches. Rock1 null mice died soon after birth with failure of the 2198 Journal of Cell Science 122 (13)

closure of the eyelids and the ventral body wall (Shimizu et al., 2005). Rock2a on TGFβ signaling using the ARE-luciferase reporter that is Most Rock2-deficient mouse embryos died in uterus with placental driven by Activin responsive elements (Huang et al., 1995). The dysfunction and intrauterine growth retardation; the few survivors expression of ARE-luciferase in Hep3B cells was stimulated by the were born runts and developed without gross anomalies (Thumkeo addition of TGFβ1 (Fig. 1A) or by transfection of the constitutively et al., 2003). In Xenopus and zebrafish, Rock2 is required for cell active (ca) form of type I receptors ALK5 (Fig. 1B) or ALK4 (data convergence and extension movements during gastrulation (Marlow not shown), whereas the reporter activity was inhibited by co- et al., 2002). These reports indicate that Rock1 and Rock2 play transfection of zebrafish Rock2a in a dose-dependent manner. The important roles in regulating cell movements in vertebrate embryos. inhibitory effect of zebrafish Rock2a on ARE-luciferase reporter However, it remains unclear as to whether they are involved in expression was also observed in HepG2 cells (Fig. 1C). Furthermore, embryonic patterning during early development. overexpression of ROCK2 also inhibited ARE-luciferase We have previously demonstrated that the late-endosomal protein reporter expression (Fig. 1C). The inhibitory effect of human ROCK2 Dpr2 binds to and facilitates lysosomal degradation of TGFβ type overexpression on ARE-luciferase expression in response to TGFβ1 I receptors, and that zebrafish Dpr2 functions to inhibit mesoderm stimulation was blocked in the presence of the Rock kinase inhibitor induction by Nodal signals during early embryogenesis (Zhang et Y-27632 in Hep3B cells (data not shown), indicating that the Rock al., 2004). The function of Dpr2 is evolutionarily conserved in catalytic activity is required. These results imply that Rock2 functions vertebrates (Su et al., 2007). In this study, we identified Rock2 as to impede TGFβ signal transduction, and that this function is binding partner of Dpr2. We disclose that, like Dpr2, Rock2 evolutionarily conserved. attenuates TGFβ signaling by promoting lysosomal degradation of To investigate the effects of endogenous ROCK2 on TGFβ TGFβ type I receptors in mammalian cells. In zebrafish embryos, signaling, we made two siRNA constructs, pSilencer-ROCK2-siRNA1 Rock2 inhibits mesendoderm induction by Nodal signals. Thus, our and pSUPER-ROCK2-siRNA2 that were designed to target different findings have revealed novel functions of Rock2, in signaling regions of human ROCK2 mRNA. Transfection of either of the transduction and in embryonic development. constructs resulted in a decrease in HA-ROCK2 expression levels (Fig. 1D). Quantitative RT-PCR analysis revealed that transfection in Results HEK293T cells with these siRNAs caused significant reduction of Rock2 negatively regulates TGFβ signaling in mammalian cells endogenous ROCK2 mRNA levels but had little effect on ROCK1 Using a yeast two-hybrid screen to search for Dpr2-interacting factors mRNA levels (data not shown). These results indicate that both in zebrafish, we identified Rock2a as a Dpr2-binding partner. We ROCK2-siRNAs were able to effectively and specifically target then looked at whether Rock2a also plays a role in regulating TGFβ ROCK2. When endogenous ROCK2 in Hep3B cells was knocked signal transduction. We first investigated the effects of zebrafish down with pSilencer-ROCK2-siRNA1 or pSuper-ROCK2-siRNA2, Journal of Cell Science

Fig. 1. Rock2 inhibits TGFβ signaling in mammalian cells. (A-C) Co-expression of fish Rock2a (A-C) or human ROCK2 (C) attenuated TGFβ1-stimulated (A) or caALK5-induced (B,C) expression of the ARE-luciferase reporter in a dose-dependent manner in Hep3B (A,B) or HepG2 (C) cells. Doses: ARE-luciferase, 0.4 μg; caALK5, 0.1 μg; fish Rock2a, 0.2 μg (+) or 0.4 μg (++); human ROCK2, 1 μg (+) and 2 μg (++); and TGFβ1, 50 pM. (D) Efficiency of siRNAs targeting human ROCK2. HA-ROCK2 and GST plasmids were transiently transfected into HEK293T cells with ROCK2-specific siRNAs, control siRNA or empty vector as indicated. The expression of HA-ROCK2 and GST in cell lysates were detected by immunoblotting with anti-HA and anti-GST antibodies, respectively. (E) Knockdown of human ROCK2 significantly enhanced the response of the ARE-luciferase reporter to TGFβ1 stimulation (100 pM) in Hep3B cells. (F) Effect of ROCK2-siRNA1 on TGFβ 1-stimulated ARE-luciferase reporter expression was blocked by co-expression of the ROCK2 mutant ROCK2 (PM) in Hep3B cells. The relative luciferase activity from ctr siRNA transfection was used as the control for statistical comparisons. For all reporter assays, data represent the mean ± s.d. of the triplicates after being normalized to Renilla activity. *P<0.05; **P<0.01. Rock2 inhibits TGFβ signaling 2199

TGFβ1 induction of the ARE-luciferase reporter expression was in human HEK293T cells (data not shown). Overexpressed HA- further enhanced (Fig. 1E). Furthermore, the effect of ROCK2- ROCK2 bound to a small amount of endogenous ALK5 without the siRNA1 on TGFβ1-stimulated ARE-luciferase reporter expression stimulation of exogenous TGFβ1, but their association was intensified was blocked by co-expression of the ROCK2 mutant ROCK2 (PM) dramatically by the addition of TGFβ1 (Fig. 2A), suggesting that (Fig. 1F), which contains six mutations within the ROCK2-siRNA1 Rock2 preferentially associates with the activated receptor. recognition site and is thus resistant to ROCK2-siRNA1. These results Next, we tested whether overexpression of Rock2 promoted confirm that ROCK2 is able to inhibit TGFβ signaling in human cells. degradation of ALK4 or ALK5. When fish Rock2a was co- overexpressed, protein levels of caALK4 or caALK5 were markedly Rock2 promotes degradation of TGFβ type I receptors via reduced in a dose-dependent fashion (Fig. 2B). Even in the presence lysosomal pathway of cycloheximide (CHX), a protein synthesis inhibitor, transfection Dpr2 has been found to inhibit TGFβ signaling by accelerating the of fish Rock2a resulted in apparent reduction of the caALK5 level degradation of TGFβ type I receptors (Su et al., 2007; Zhang et al., (Fig. 2C) and led to a shortened half-life for caALK5 (Fig. 2C’). 2004). We speculated that Rock2 might regulate TGFβ signaling This implied that the Rock2a-induced caALK5 reduction was not through a similar mechanism. To test this, we first examined the due to impaired transcription or translation. Similarly, transfection physical interaction between Rock2 and TGFβ type I receptors by of human ROCK2 caused reduction of the caALK5 level in the co-immunoprecipitation. We found that overexpressed fish Rock2a presence of CHX (Fig. 2D), suggesting a conserved function of could associate with co-expressed wild-type (ALK5-FLAG) and Rock2 from fish to human. Consistent with these observations, constitutively active (caALK5- FLAG) forms of the receptor ALK5 knockdown of ROCK2 in human HEK293T cells by ROCK-siRNAs Journal of Cell Science

Fig. 2. Rock2 binds to and accelerates degradation of ALK5. (A) Co-immunoprecipitation detected association of endogenous ALK5 with overexpressed HA- ROCK2. Note that the addition of 100 pM TGFβ1 promoted the ALK5-ROCK2 association. (B) The amount of caALK4 or caALK5 decreased by co-transfection of fish Rock2a in a dose-dependent manner. HA-Rock2a dose: +, 1 μg; ++, 2 μg. Lysates of transfected cells were immunoblotted with anti-HA to detect caALK4 or caALK5 and Rock2a, and with anti-GFP to detect the internal standard GFP. (C) Rock2a transfection accelerated the degradation of caALK5 protein in the presence of 100 μg/ml cycloheximide (CHX). The duration of CHX treatment is indicated in hours. GFP levels were used as controls. Control assays using DMSO and the short-lived protein mouse Tob1 were performed, but the data are not shown here. (C’) Time-course of caALK5 turnover. Three independent experiments were performed as described in C. The intensity of caALK5 and GFP bands were scanned and quantified using ImageJ. After normalization with the internal control (GFP) the relative quantity of caALK5 (% of time zero) was indicated as the mean ± s.d. of three independent experiments at different time points. (D) Overexpression of human ROCK2 induced caALK5 degradation in a dose-dependent manner in the presence of CHX. HA-ROCK2 dose: +, 1 μg; ++, 2 μg. (E) Knockdown of endogenous ROCK2 resulted in higher levels of caALK5 protein. GST was used as a control. All the experiments were performed in HEK293T cells. TCL, total cell lysates; IP, immunoprecipitation. 2200 Journal of Cell Science 122 (13)

gave rise to an increase in the amount of overexpressed caALK5 Effect of Rock2 on TGFβ signaling depends on its kinase activity (Fig. 2E). Thus, Rock2 controls TGFβ signaling by accelerating As a serine/threonine kinase, Rock2 exerts its biological effects by the degradation of TGFβ type I receptors. phosphorylating substrates (Riento and Ridley, 2003). We then We then made efforts to identify the pathway(s) through which looked at whether the kinase activity of Rock2 was required for its Rock2 promotes degradation of ALK4 and ALK5. We found that ability to inhibit TGFβ signaling. We first used the ROCK kinase the presence of the lysosomal inhibitors NH4Cl, bafilomycin A1 or inhibitors Y27632 (Ishizaki et al., 2000) and HA1077 (Davies et chloroquine, but not of the proteasome inhibitor MG132, prevented al., 2000) to address this issue. Treatment with either of the inhibitors Rock2a-induced caALK5 degradation (Fig. 3A). We obtained in mammalian Hep3B cells resulted in elevated levels of TGFβ1- similar results for caALK4 (data not shown). Thus, Rock2 promotes induced ARE-luciferase reporter expression (Fig. 3C). The presence the receptor degradation through the lysosomal pathway but not the of Y27632 or HA1077 also caused an increase in caALK5-HA levels proteasomal pathway. We then adopted a biotinylation method (Lu and attenuated the HA-ROCK2-induced caALK5-HA degradation et al., 2002) to investigate the effect of Rock2 on the degradation (Fig. 3D; data not shown for HA1077 treatment). These results of TGFβ receptors anchored on the cell surface. In the precipitates suggest that the kinase activity of ROCKs is required for the pulled-down with streptavidin 3 hours after activation of inhibitory effect on TGFβ signal transduction. We further tested internalization, the amount of endogenous cell surface ALK5, the role of Rock2 kinase activity in the inhibition of TGFβ signaling detected with anti-ALK5 antibody, was markedly reduced by by using several mutant forms of human ROCK2 (Amano et al., overexpression of HA-ROCK2 (Fig. 3B). Taking these results 1997; Amano et al., 1999; Tan et al., 2002), including the together, we hypothesize that Rock2 binds to and promotes the constitutively active mutant ROCK2-CAT, which retains the N- degradation of endogenous ALK5 internalized from the cell surface. terminal region (6-553 aa) only, ROCK2-CAT KD (identical to Journal of Cell Science

Fig. 3. Rock2 promotes the lysosomal degradation of ALK5 depending on its own kinase activity. (A) Lysosomal inhibitors, but not proteasomal inhibitor, attenuated caALK5 degradation induced by fish Rock2a. HEK293T cells were co-transfected with 2 μg of HA-Rock2a or with empty vector as a control. At 24 hours post-transfection, cells were treated with the proteasomal inhibitor MG132 (25 μM), with the lysosomal inhibitors NH4Cl (NC, 25 mM), bafilomycin A1 (BF, 500 nM) or chloroquine (Chlq, 100 μM), or not treated for 6 hours and then harvested for anti-HA and anti-GFP immunoblotting to detect expression levels of caALK5-HA, HA-Rock2 or GFP, respectively. (B) ROCK2 promotes the turnover of biotinylated endogenous cell surface ALK5 in HeLa cells. The incubation times at 37°C in the presence of 100 pM TGFβ1 following biotin labeling at 4°C, which allowed internalization before harvest, are indicated. Note that the cell surface ALK5 amount was remarkably lower in the cells transfected with Rock2 than in the control cells following 3-hour internalization. TCL, total cell lysates; IP, immunoprecipitation. (C) TGFβ1 (50 pM)-stimulated ARE-luciferase reporter expression in Hep3B cells was intensified by treatment with Y27632 (10 μM) or HA1077 (20 μM). (D) Inhibition of Rock kinase activity with 10 μM Y27632 counteracted the effect of ROCK2 on the degradation of overexpressed ALK5 protein in HEK293T cells. Cells were stimulated by 100 pM TGFβ1 in the presence of 100 μg/ml CHX, and were collected after 6 hours for protein detection by western blotting. (E) Effect of different ROCK2 deletion mutants on caALK5-induced ARE-luciferase reporter expression in Hep3B cells. The mutants are described in the text. **P<0.01; ***P<0.001; asterisk with a line through, P>0.1. (F) Co-transfection of dnRock2a, which is a dominant negative form of fish Rock2a, elevated caALK5 levels in HEK293T cells in a dose-dependent manner. DnRock2a doses: +, 1 μg; ++, 2 μg. Protein levels were examined by western blotting. Rock2 inhibits TGFβ signaling 2201

ROCK2-CAT except for loss of kinase activity) and the dominant demonstrated in zebrafish that rock2a plays a role in cell negative mutant ROCK2-RB/PH(TT), which carries the C-terminal convergence and extension movements during gastrulation by region (1125-1388 aa) but lacks RhoA-binding activity. Like the mediating non-canonical Wnt11 signaling (Marlow et al., 2002). full-length ROCK2, ROCK2-CAT overexpression inhibited the To avoid interpretation ambiguity due to abnormal convergence and caALK5-stimulated ARE-luciferase reporter expression in Hep3B extension movements, we examined the expression of the Nodal cells (Fig. 3E). By contrast, overexpression of ROCK2-CAT KD targets goosecoid (gsc), chordin (chd) and no tail (ntl) at the sphere had no effect, but ROCK2-RB/PH(TT) overexpression enhanced stage, well before the onset of gastrulation, following injection of the reporter expression (Fig. 3E). Furthermore, overexpression of zebrafish rock2a mRNA into one-cell embryos. As shown in Fig. dnRock2a, a dominant negative mutant of zebrafish Rock2a that is 4A,A’, overexpression of rock2a led to reduction or elimination of similar to ROCK2-RB/PH(TT) (Marlow et al., 2002), elevated gsc, chd and ntl expression in the dorsal margin where dorsal caALK5-HA protein levels in HEK293T cells (Fig. 3F). Taken mesoderm precursors are located, whereas injection of lacZ mRNA together, these data support the idea that the kinase activity of Rock2 had little effect on gsc and ntl expression. Unexpectedly, injection is required for regulating TGFβ type I receptor degradation. of lacZ mRNA also caused reduction of chd expression, which might have occurred through unknown mechanisms. To confirm the in rock2a overexpression inhibits mesoderm induction in situ hybridization results, we performed quantitative RT-PCR zebrafish embryos analysis. As shown in Fig. 4B, rock2a overexpression caused As shown above, fish Rock2a is able to downregulate TGFβ significant reduction in gsc, ntl and chd transcript levels, which signaling by facilitating the degradation of ALK4 and ALK5 excludes the possibility that the change of the maker expression receptors in mammalian cells. We wondered whether Rock2a could patterns is owing to abnormal convergent extension movements. also regulate TGFβ signaling in zebrafish embryos. Because Nodal By contrast, neither quantitative RT-PCR nor whole-mount in situ signaling is essential for mesoderm induction (Tian and Meng, hybridization detected any decrease following rock2a 2006), we looked at whether rock2a had an effect on mesoderm overexpression, even if no increase, in the expression of bozozok induction in zebrafish embryos. A previous study by Marlow et al. (boz) (Fig. 4B,C), which is a direct target of canonical Wnt/β-catenin Journal of Cell Science

Fig. 4. Overexpression of rock2a inhibits mesodermal induction in zebrafish embryos. (A) Overexpression of rock2a downregulated expression of gsc, ntl and chd. Zebrafish embryos were injected at the one-cell stage with 400 pg of rock2a or lacZ mRNA. The expression of marker genes (named on the top of each panel) in injected or wild-type (WT) embryos was detected by whole-mount in situ hybridization at the sphere stage. For each marker, embryos were shown in animal-pole view (‘A’, left) and dorsal view (‘D’, right). Note that lacZ mRNA injection had little effect on gsc and ntl expression but interfered with chd expression through unknown mechanisms (three independent experiments produced similar results). (A’) Statistical data for A. Percentages of embryos with variable expression levels (in different colors) of indicated genes were calculated. The number of observed embryos (n) is indicated below each bar. (B) rock2a overexpression resulted in decrease of gsc, ntl and chd transcript amounts. Embryos were injected with 400 pg of rock2a or lacZ mRNA at the one-cell stage, and were analyzed at the sphere stage by real-time PCR for gsc, ntl, chd and boz expression. actin was also examined as an internal control. The fold change in transcript levels (y-axis) is given relative to control embryos and the data represent the mean ± s.d. from triplicates. Note that boz transcript level was not decreased but increased by rock2a overexpression. Statistical significance levels: *P<0.05; **P<0.01. (C) The expression pattern of boz, a canonical Wnt target, was unaltered in embryos injected with rock2a. 2202 Journal of Cell Science 122 (13)

signaling (Leung et al., 2003; Ryu et al., 2001). This implies that sphere stage (Fig. 5A). The inductive effect of sqt was obviously rock2a does not downregulate canonical Wnt/β-catenin signaling compromised by co-injection of rock2a mRNA. Zebrafish taram- in vivo. Thus, rock2a overexpression negatively regulates the a encodes a TGFβ-related type I receptor required for Nodal expression of the Nodal targets during mesoderm induction, signaling, and overexpression of its constitutively active form tar* irrespective of the canonical Wnt signaling. induces mesendodermal marker expression (Aoki et al., 2002; Renucci et al., 1996). We found that induction of gsc, chd and ntl rock2a overexpression impedes mesoderm induction activity of expression by injection of tar* mRNA was also inhibited by co- Nodal signals injection of rock2a mRNA (Fig. 5B). Lefty1 (also known as Antivin) The next issue to investigate was whether rock2a inhibited is a potent antagonist of Nodal ligands, and its overexpression in mesoderm induction by antagonizing Nodal signaling in zebrafish zebrafish embryos eliminates the formation of most mesendodermal embryos. As reported before (Rebagliati et al., 1998), tissues (Cheng et al., 2000; Thisse and Thisse, 1999). When 10 pg overexpression of squint (sqt), a zebrafish Nodal , caused of lefty1 mRNA was injected, the expression levels of gsc, chd and dramatic expansion of gsc, chd and ntl expression domains at the ntl were decreased at the sphere stage (Fig. 5C). When the same Journal of Cell Science

Fig. 5. Overexpression of rock2a impedes mesoderm induction of Nodal signals. Expression of marker genes in embryos injected with the indicated mRNAs or in wild-type (WT) embryos was detected by whole-mount in situ hybridization at the sphere stage. For each marker, embryos are shown in animal-pole view (‘A’, left) and dorsal view (‘D’, right). (A,B) rock2a overexpression inhibited sqt-induced (A) or tar*-induced (B) ectopic expression of the marker genes gsc, ntl and chd. Doses: sqt mRNA, 0.25 pg; tar* mRNA, 0.6 pg; rock2a mRNA, 200 pg. (C) rock2a overexpression intensified the effect of lefty1. Embryos were injected with 10 pg lefty1 mRNA alone or together with 200 pg rock2a mRNA. Note that lacZ mRNA injections at a comparable dose served as controls. (A’-C’) Statistical data for A-C, respectively. (D) Induction of endogenous phospho-Smad2 by sqt was suppressed by rock2a co-injection. Embryos were injected at the one-cell stage with 0.5 pg of sqt mRNA alone or in combination with 400 pg of rock2a mRNA and lysed at the sphere stage for immunoblotting. Phospho-Smad2 and total Smad2 were detected with anti-phospho-Smad2 and anti-Smad2 antibodies, respectively. Rock2 inhibits TGFβ signaling 2203

amount of lefty1 mRNA was co-injected with 200 pg of rock2a and Rock2a in regulating TGFβ signaling were interdependent. Like mRNA, most of the injected embryos showed further decrease in the full-length Rock2a, transfection of HA-Rock2aΔDpr2, a Rock2a the expression levels of these markers. Taken together, these data mutant that was depleted of 1068-1110 aa and failed to interact indicate that rock2a overexpression is able to block mesoderm with Dpr2 (data not shown), attenuated the caALK5-stimulated induction by ectopic Nodal signaling and to enhance the inhibitory expression of the ARE-luciferase reporter in Hep3B cells (Fig. 7C), effect of lefty1 on mesoderm induction in vivo. suggesting that the effect of Rock2a is independent of its Dpr2- To obtain direct evidence of the effects of rock2a on Nodal binding ability. When Dpr2 in HEK293T cells was knocked down signaling in fish embryos, we examined phosphorylated Smad2 by Dpr2-siRNA1 (Su et al., 2007), co-expression of HA-Rock2a levels after overexpression of sqt and rock2a. As shown in Fig. 5D, was still able to effectively accelerate caALK5-HA degradation (Fig. injection of sqt mRNA led to an increase in the phospho-Smad2 7D). Thus, it appears that Rock2a inhibits TGFβ signaling in a Dpr2- levels, but co-injection with rock2a mRNA compromised the independent manner. By contrast, transfection of Dpr2 had little stimulatory effect of sqt, while the total amount of Smad2 remained effect on the TGFβ1-stimulated ARE-luciferase reporter expression comparable. These results demonstrate that rock2a downregulates in Hep3B cells upon knockdown of endogenous ROCK2 by the Nodal signaling upstream of Smad2 phosphorylation in fish pSilencer-ROCK2-siRNA1 (Fig. 7E). Consistent with this embryos, which is consistent with the fact that Rock2 accelerates observation, overexpression of Dpr2 failed to promote caALK5- the turnover of TGFβ receptors in mammalian cells. HA degradation when ROCK2 was knocked down in HEK293T cells (Fig. 7F). These data imply that the inhibitory effect of Dpr2 Endogenous Rock2 is required for mesendoderm induction in on TGFβ signaling relies on ROCK2 function. zebrafish embryos We then investigated whether ROCK kinase activity is required A previous study carried out by Marlow and coworkers (Marlow for binding to and facilitating the function of Dpr2. We generated et al., 2002) indicated that knockdown of rock2a with antisense a ROCK2 mutant, ROCK2K121G, by mutating K121 to G121 at morpholino oligonucleotides in zebrafish embryos fails to cause the ATP-binding site. Like wild-type ROCK2, ROCK2K121G was any detectable phenotypes, probably due to the presence of the able to bind to mouse Dpr2 (mDpr2) (Fig. 7G), suggesting that the abundant maternal deposit of Rock2 proteins (Marlow et al., 2002). kinase activity of ROCK2 is not required for associating with Dpr2. Because the dominant negative mutant dnrock2a can attenuate the However, the presence of the ROCK kinase inhibitor Y-27632 degradation of TGFβ receptor by interfering with endogenous reduced the inhibitory effect of fish Dpr2 overexpression on the ROCK2 in mammalian cells (Fig. 3F), we evaluated the biological TGFβ1-stimulated ARE-luciferase expression (Fig. 7H). Thus, the function of endogenous Rock2 proteins by injecting dnrock2a kinase activity of ROCK2 is dispensable for binding to Dpr2 but mRNA into one-cell embryos and examining gsc, chd and ntl is necessary for assisting the function of Dpr2 in the control of TGFβ expression using whole-mount in situ hybridization and quantitative signaling. RT-PCR analysis at the sphere stage. In situ patterns of these markers were not altered obviously by dnrock2a overexpression (data not Discussion shown). However, quantitative RT-PCR analysis revealed that In this study, we have demonstrated that Rock2, which was overexpression of dnrock2a resulted in significant increases in ntl identified as a binding partner of Dpr2, acts to inhibit TGFβ signal

Journal of Cell Science and chd transcript levels, although the gsc transcript level was only transduction in mammalian cells. Mechanistically, Rock2 binds to marginally increased (Fig. 6A). These results suggest that and promotes the degradation of endocytosed TGFβ type I receptors endogenous Rock2 is required for mesoderm induction during the via the lysosomal pathway. In zebrafish embryos, rock2a early development of zebrafish embryos. overexpression inhibits mesoderm induction by Nodal signals. We also took another approach, dnrock2a and lefty1 co-injection, Interference with endogenous Rock2 proteins by injecting a to investigate the requirement of endogenous Rock2 for dominant negative mutant (dnrock2a) of rock2a compromises the mesendoderm induction. As shown in Fig. 6B,B’, lefty1-induced inhibitory effect of lefty1 on mesendoderm marker expression, reduction of gsc, ntl and chd expression at the sphere stage was suggesting an involvement of endogenous Rock2 proteins in compromised by co-injection of dnrock2a mRNA. Overexpression mesendoderm induction during early embryogenesis. Thus, our of lefty1 led to reduction of the expression of the endodermal marker study discovered a novel role of Rock2 in modulating TGFβ/Nodal sox32 (also a Nodal target) at the 75% epiboly stage and of ntl signaling and in controlling mesendoderm induction. expression in the axial mesoderm at the bud stage, but caused Rock1 and Rock2 have been found to play pivotal roles in cell expansion of the expression domains of the ventral ectodermal motility. They regulate actin-cytoskeleton assembly and cell marker gata2 at the 80% epiboly stage (Fig. 6C,C’). These effects contractility by phosphorylating specific substrates, which of lefty1 overexpression were also alleviated by co-expression of ultimately exert their effects on the cytoskeleton (Riento and dnrock2a. Our observations support the idea that endogenous Ridley, 2003). We found that Rock2 could promote lysosomal Rock2 proteins play an inhibitory role in mesendoderm induction degradation of endocytosed TGFβ type I receptors. Hypothetically, during zebrafish embryogenesis. this effect of Rock2 may be associated with cytoskeleton-dependent intracellular trafficking of the endosomes containing TGFβ receptor Rock2 inhibits TGFβ signaling independent of Dpr2 towards lysosomes. Several studies have shown that RhoA and Rho To confirm the functional relationship between Rock2 and Dpr2, kinases regulate endocytosis of transferrin and epidermal growth we first tested their physical interaction by co-immunoprecipitation factor receptors (Lamaze et al., 1996), low-density lipoprotein assay. As shown in Fig. 7A, overexpressed HA-Rock2a in receptors (Hrboticky et al., 2002), and equine herpesvirus 1 HEK293T cells was co-immunoprecipitated with Myc-Dpr2, and (Frampton et al., 2007). Kaneko et al. demonstrated that Rho kinases vice versa. Moreover, endogenous human ROCK2 was found to might inhibit endocytosis of epidermal growth factor receptors by associate with overexpressed Myc-tagged mouse Dpr2 in HEK293T phosphorylating endophilin A1, a key regulator of clathrin-mediated cells (Fig. 7B). We next investigated whether the functions of Dpr2 endocytosis (Kaneko et al., 2005). Accelerated degradation of the 2204 Journal of Cell Science 122 (13)

endocytosed TGFβ receptors by Rock2 might be ascribed to its receptor(s) located on endosomes and presents such receptors to ability to phosphorylate substrates that facilitate transportation of Rock2, which promotes movement of such ‘labeled’ endosomes the TGFβ-receptor-bound endosomes. Further studies are needed towards lysosomes along the cytoskeleton. Most probably, other to test this hypothesis. unidentified adaptor proteins may have a function similar to Dpr2, On the one hand, we found that Rock2 was physically associated which recognizes and presents TGFβ receptors to Rock2. with Dpr2 and TGFβ type I receptors; on the other hand, Dpr2 is In zebrafish, rock2a is maternally expressed and its transcripts primarily located in the late endosomes, and it also binds to are distributed in all blastomeres, implying that it may be involved endocytosed TGFβ type I receptors (Zhang et al., 2004). What then in specification of cell fate during early embryogenesis. A previous is the relationship between Rock2 and Dpr2 in controlling TGFβ study found that knockdown of rock2a with antisense morpholinos signaling? We note that Dpr2-induced degradation of TGFβ failed to cause any detectable defects in early zebrafish embryos, receptors is largely Rock2-dependent in mammalian cells, whereas even in convergence and extension movements during gastrulation the effects of Rock2 on TGFβ-receptor degradation are independent (Marlow et al., 2002). One reason for this could be the presence of of Dpr2 function. We hypothesize that Dpr2 binds to TGFβ the abundant maternally derived Rock2a protein that is not targetable Journal of Cell Science

Fig. 6. Interference with endogenous Rock2 proteins promotes mesodermal induction in zebrafish embryos. (A) Real-time RT-PCR analysis of mesodermal makers in embryos injected with dnrock2a. Embryos were injected at the one-cell stage with 400 pg of dnrock2a or lacZ mRNA, and total RNAs were extracted at the sphere stage for real-time RT-PCR analysis. (B,C) The expression changes of marker genes caused by ectopic expression of lefty1 (20 pg) were neutralized by 50 pg of dnrock2a mRNA co-injection. The expression of the marker genes (indicated on the top) was detected by whole-mount in situ hybridization at the indicated stages. Embryos are shown in animal-pole views with dorsal to the right (‘A’), in dorsal views with animal pole to the top (‘D’), or in lateral views with dorsal to the right (‘L’). Corresponding statistical data are presented in B’ and C’. Percentages of embryos with variable expression levels of indicated genes are shown in different colors. The number of observed embryos (n) is indicated below each bar. *P<0.05; **P<0.01. Rock2 inhibits TGFβ signaling 2205

by morpholinos. Another reason could be the presence of other rock other studies have found that RhoA and Rho kinases positively genes in the zebrafish genome, e.g. rock2b, rock2bl and probably regulate convergence and extension movements by mediating non- multiple genes, which play redundant functions, in addition canonical Wnt signaling in zebrafish and Xenopus gastrulas (Jopling to rock2a. Further genetic studies are needed to disclose the and den Hertog, 2005; Kim and Han, 2005; Marlow et al., 2002; requirement of endogenous Rho kinases in embryonic patterning. Zhu et al., 2006). Our unpublished data indicate that Rock2a may Rho kinases act on many different substrates, implying that they also be a positive regulator of the canonical Wnt/β-catenin signaling may regulate multiple signaling pathways. Although Rock2 proved pathway. Since these signaling pathways are all involved in to be a negative regulator of TGFβ signaling in this study, several embryonic patterning, weak phenotypes caused by genetic Journal of Cell Science

Fig. 7. Interdependence of Rock2 and Dpr2 functions on TGFβ signaling. (A) Physical interaction between zebrafish Rock2a and Dpr2 was verified by reciprocal co-immunoprecipitation in HEK293T cells. (B) Interaction between human endogenous ROCK2 and overexpressed mouse Dpr2 (Myc-mDpr2) was verified by co- immunoprecipitation assay in HEK293T cells. Cells lysates were subjected to immunoprecipitation with anti-Myc antibody, and then the immunoprecipitates together with total cell lysates were immunoblotted with anti-ROCK2 and anti-Myc antibodies, respectively. (C) Rock2aΔDpr2 defective in Dpr2 binding retained the ability to inhibit caALK5-induced ARE-luciferase expression in Hep3B cells. (D) Knockdown of endogenous Dpr2 by siRNA had no impact on Rock2a- induced caALK5 degradation. (E) Dpr2-induced suppression of the ARE-luciferase activity was compromised by ROCK2 knockdown. Hep3B cells were transfected with 0.3 μg of Dpr2 and 0.6 μg of ROCK2-siRNA1, and were stimulated 36 hours later with 50 pM TGFβ1 for an additional 12 hours before analysis. (F) Dpr2-induced caALK5 degradation was diminished by knockdown of endogenous ROCK2. Immunoblotting in D and F was performed essentially as described in the Materials and Methods. (G) Interaction between mouse Dpr2 (Myc-mDpr2) and ROCK2K121G (KG) or ROCK2 (WT) was verified by co- immunoprecipitation assay in HEK293T cells. ROCK2K121G was the kinase-dead full-length ROCK2 mutant. (H) Fish Dpr2-inhibited ARE-luciferase reporter expression was restored by Y-27632 treatment. At 30 hours post transfection, Hep3B cells were stimulated by 100 pM TGFβ1 with Y-27632 (10 μM) pretreatment or not, and then incubated for 16 more hours before harvest. TCL, total cell lysates; IP, immunoprecipitation. **P<0.01; asterisk with a line through, P>0.1 2206 Journal of Cell Science 122 (13)

manipulations of Rock activity in zebrafish, as shown in this study, required, cells were starved for 6 hours and then stimulated with 100 pM TGFβ1 for 2 hours before harvest. Cell lysate preparation, immunoprecipitation and western may be expected. It will be interesting to investigate how RhoA blotting were preformed as described previously (Zhang et al., 2004). and Rocks differentially regulate different signaling pathways. Biotinylation analysis of cell surface receptors Materials and Methods At 36 hours post-transfection, control cells or Rock2-transfected HeLa cells were labeled with 0.5 mg/ml NHS-SS-biotin (Pierce, Rockford, IL) for 40 min at 4°C, and Constructs β pCS2-Rock2a and pCS2+MT-dnRock2a were generous gifts from Lilianna Solnica- then were incubated at 37°C in the presence of 100 pM TGF 1 for 0 or 3 hours Krezel (Vanderbilt University Medical Center, Nashville, TN); ROCK2 (human cDNA before harvested. The biotinylated cell surface proteins were precipitated with clone KIAA0619) was a kind gift from KAZUSA DNA Research Institute (Chiba, streptavidin beads (Pierce) and then eluted. ALK5 in the eluted proteins was detected Japan). Zebrafish Rock2 and human ROCK2 were subcloned into pCMV5 with the by immunoblotting using anti-ALK5 antibody (Santa Cruz Biotechnology, Santa Cruz, addition of an HA tag at the N-terminus. N- or C-terminal deletion mutants of Rock2 CA). were generated according to standard molecular techniques. For human ROCK2 mutants, CAT encoding the catalytic domain of ROCK2 (6-553 aa) was inserted into Fish and embryonic analysis pCMV5 with an HA tag, and RB/PH (TT) (941-1388 aa, with the point mutations Wild-type Tuebingen strain was used. Embryos were incubated in Holtfreter’s solution N1036T and K1037T) inserted into pCMV5 with a FLAG tag (Amano et al., 1997; at 28.5°C and staged according to Kimmel and coworkers (Kimmel et al., 1995). Amano et al., 1999; Tan et al., 2002). ROCK2 (PM), a human ROCK2 mutant, was RNA synthesis, microinjection and whole-mount in situ hybridization were performed made by mutating the ROCK2-siRNA1-recognizing sequence to GTAAG - essentially as described previously (Zhao et al., 2003). TCGGTCAACACGCG (substituted bases are underlined). Deletion and point mutations were generated by a PCR-based QuickChange Site-Directed-Mutagenesis Quantitative real-time RT-PCR (Stratagene, La Jolla, CA) method with Pfu polymerase. All of the inserts were verified Total RNA was extracted from a pool of 120 injected embryos at the sphere stage by by sequencing. The details of clone strategy and primer sequences are available upon RNeasy Mini Kit (Qiagen, Valencia, CA). According to the manufacturer’s instructions request. (Promega), cDNAs were synthesized from 2 μg RNA using oligo-dT primer in a Two siRNAs targeting human ROCK2 were designed and constructed by vector- volume of 20 μl. The volume of each cDNA sample was brought up to 100 μl in dH2O based siRNA technology. Their targeting sequences are 5Ј-GCAAATCTGT - and then quantitative real-time PCR was carried out with SYBR green I (OPE, TAATACTCG-3Ј (for ROCK2-siRNA1) and 5Ј-ATCAGACAGCATCCTTTCT-3 Ј Shanghai, China) in the Mϫ3000P real-time PCR system (Stratagene). Expression (for ROCK2-siRNA2). Control (ctr) siRNA was designed to target the sequence levels of β-actin were analyzed in parallel as an internal control and each sample was 5Ј-AGCGGACTAAGTCCATTGC-3Ј, and was used as a negative control. run in triplicate. Primers for boz were 5Ј-TACTGCTGCTGCGTTCCA-3Ј (forward) Oligonucleotides of ROCK2-siRNA1 and ctr siRNA were inserted into ApaI/EcoRI and 5Ј-TGCTCCGTCTGGTTGTCG-3Ј (reverse); primers for other genes were as sites of the vector pSilencer 1.0-U6 (Ambion, Austin, TX), and those of ROCK2- described by others (Liu et al., 2005; Xiong et al., 2006). For testing ROCK2-siRNAs siRNA2, ROCK2 as well as ctr siRNA were cloned into BglII/HindIII sites of the specificity in HEK293T cells, primers for human ROCK1 were 5Ј-GACC - vector pSUPER.retro (OligoEngine, Seattle, WA). Dpr2-siRNA1 was reported TGTAACCCAAGGAGATG-3Ј (forward) and 5Ј-CAGGAAAGTG GTAGAGT - previously by Su et al. (Su et al., 2007). GTAGGA-3Ј (reverse), those for human ROCK2 were 5Ј-CAACTGT GAG - GCTTGTATGAAG-3Ј (forward) and 5Ј-TGCAAGGT GC TATAATCTCCTC-3Ј Yeast two-hybrid screen (reverse), and those for the control gene GAPDH were 5Ј-ATCACTGCCACCCA- Total RNAs were extracted from zebrafish embryos at the one-cell, 40% epiboly, 5- GAAGAC-3Ј (forward) and 5Ј-CAGTGAGCTTC CC GTTCAG-3 Ј (reverse). somite and 24-hour stages using Trizol reagent. The cDNA library was constructed using the BD Matchmaker Yeast Two-Hybrid System (Clontech, Palo Alto, CA) Detection of phospho-Smad2 in fish embryos according to the manufacturer’s instructions. Uninjected or injected embryos at the sphere stage were dechorionized, deyolked To screen Drp2-interacting proteins, the Drp2 N-terminal region of 479 amino and resuspended in the TNE solution (4 μl per embryo), which contained 100 mM acids was cloned into the bait vector pGBKT7. Yeast strain AH109 transformed with NaF, 1 mM orthovanadate (NaVO4), and a protease inhibitor cocktail (Roche the bait was mated with Y187 cells that were pre-transformed with the zebrafish Diagnostics GmbH, Mannheim, Germany). Total proteins were separated by 12% cDNA library, followed by screening with the BD Matchmaker Two-Hybrid System SDS-PAGE gels, immunoblotted with anti-phospho-Smad2 and anti-Smad2/3 (Clontech). antibodies (Cell Signaling Technology, Danvers, MA). Journal of Cell Science Cell culture and transient transfection We are grateful to Zhenguo Wu and Min Li for suggestions and HEK293T and HeLa cells were maintained in DMEM with 10% fetal bovine serum comments. We acknowledge members of Y. G. Chen’s laboratory (FBS, Hyclone, South Logan, UT) in a water-saturated atmosphere of 5% CO2. Hep3B and HepG2 cells were cultured in the same conditions, except that DMEM was (Tsinghua University, Beijing, China) for technical assistance and replaced with MEM. Transfection for HEK293T and HepG2 cells was performed reagents. We thank Lilianna Solnica-Krezel for providing zebrafish using the calcium-phosphate precipitation method, and transfection for Hep3B and rock2 cDNA and Frederic Rosa (Ecole Normale Supérieure, Paris, HeLa cells was carried out using lipofectamine (Gibco BRL, Gaithersburg, MD). To France) for providing taram constructs. This work was supported by keep a consistent total amount of transfected plasmids for each sample in one grants from the National Basic Research Program of China experiment, corresponding empty vector was added to the amount required. 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