Activation of the planar cell polarity formin DAAM1 leads to inhibition of endothelial cell proliferation, migration, and angiogenesis

Rong Jua,1,2, Pasquale Cironea,1, Shengda Linb, Hilary Griesbachb, Diane C. Slusarskib, and Craig M. Crewsa,c,d,3

Departments of aMolecular, Cellular, and Developmental Biology, cPharmacology, and dChemistry, Yale University, New Haven, CT 06511; and bDepartment of Biology, University of Iowa, Iowa City, IA 52242

Edited by Robert Langer, Massachusetts Institute of Technology, Cambridge, MA, and approved March 4, 2010 (received for review February 3, 2010) The Wnt/planar cell polarity (PCP) pathway regulates directed cell followed by the diaphanous inhibitory domain (DID), two formin movement during development and was recently found to play a homology (FH1 and FH2) domains, and a diaphanous autor- critical role in endothelial cell proliferation and angiogenesis egulatory domain (DAD) at the C terminus (9) (Fig. 1A). [Zhang Y, et al. (2006) Chem Biol 13:1001–1009; Masckauchan TN, FH1 and FH2 domains coordinate to induce nucleation et al. (2006) Mol Biol Cell 17:5163–5172]. However, the mechanisms and polymerization (10, 11). In the inactive state, the autor- by which PCP signaling components regulate angiogenesis remain egulatory domain of a DRF is bound to the DID domain (12). unknown. We report that expression of a constitutively active C- Association of GTP-bound Rho or Cdc42 with the GBD results in terminal domain of Dishevelled-associated activator of morpho- the release of this inactive conformation, thus permitting the FH genesis 1 (DAAM1) selectively inhibited endothelial cell prolifera- domains to catalyze actin nucleation and filament elongation (13, tion. Moreover, this activated construct suppressed endothelial 14). In addition to actin polymerization, the most widely studied cell migration and the ability to form coordinated networks in vivo mammalian formin, mDia1, stabilizes microtubules (MTs) through and in vitro. Although constitutively active DAAM1 (CDAAM1) its C-terminal FH2 domain (15). induced both actin polymerization and microtubule (MT) stabiliza- Given that loss of PCP signaling suppressed endothelial cell tion, the stabilization of MTs alone was sufficient to inhibit endo- proliferation and migration, thus inhibiting angiogenesis (4), we thelial cell growth selectively. Inhibition of actin polymerization next examined the role of DAAM1 in endothelial cells and alone by jasplakinolide treatment failed to reproduce the inhibi- angiogenesis. We report here that constitutively active DAAM1 tory effects of CDAAM1. These results indicate that DAAM1 regu- inhibited endothelial cell growth in a cell type-selective manner. lates endothelial cell growth through MT stabilization in a cell Moreover, we found that CLASP1α, a plus end-tracking pro- type-selective manner and suggest that PCP signaling plays a piv- tein (+TIP) that induces MT stabilization independent of actin otal role in angiogenesis by regulating MT stabilization. polymerization, also inhibited proliferation of endothelial cells selectively. Jasplakinolide, a chemical inducer of actin polymer- fi Wnt signaling | microtubule stabilization | zebra sh | dishevelled- ization, did not suppress endothelial cell proliferation. In addition, associated activator of morphogenesis 1 | HUVEC activated DAAM1 blocked endothelial cell migration and network formation in vitro. These findings were further validated in vivo, ngiogenesis is the process by which blood vessels grow from because activated DAAM1 inhibited angiogenesis in zebrafish Aexisting vessels and is involved in many pathological con- embryos. We therefore conclude that PCP signaling through ditions such as wound healing, inflammation, rheumatoid arthri- DAAM1 coordinates cell growth and migration in endothelial tis, endometriosis, diabetic retinopathy, macular degeneration, cells predominantly through MT assembly and stabilization. and tumor growth (1). TNP-470, a potent small-molecule angio- genesis inhibitor, has shown promising efficacy in vivo (2). Re- Results cently, our laboratory reported that TNP-470 disrupts planar cell CDAAM1 Selectively Inhibits Endothelial Cell Proliferation. Our find- polarity (PCP) signaling (3) and that inhibition of PCP signaling by ings pertaining to the role of PCP signaling in angiogenesis (4) either chemical or genetic means disrupts angiogenesis (4). This prompted us to investigate further downstream PCP signaling signaling pathway is one of several Wnt-driven signaling cascades. mediators to gain more detailed mechanistic insights into Wnt, as a ligand, interacts with a seven-transmembrane-spanning angiogenesis. In this study, we focused on DAAM1, which is receptor Frizzled (Fz) to transduce signaling through a scaffolding activated in the PCP pathway independent of other Wnt sig- Dishevelled (Dvl). Depending on the downstream effec- naling cascades (8). Because the N-terminal (NDAAM1) and C- tors of Dvl, Wnt signaling can be classified as either β-catenin- terminal (CDAAM1) domains of DAAM1 (Fig. 1A) exert dependent (canonical) or β-catenin-independent (noncanonical). dominant-negative and dominant-positive (constitutively active) This latter class encompasses a number of signaling cascades, functions on PCP signaling, respectively (8), we compared the 2+ including the Wnt/Ca and PCP pathways (5). The PCP pathway effects of PCP signaling on endothelial cell proliferation by was first studied in the cellular patterning of Drosophila wing epithelia (6) but has been shown to be involved in polarized cell movements such as convergent extension and gastrulation in Author contributions: R.J., P.C., D.C.S., and C.M.C. designed research; R.J., P.C., S.L., and vertebrates (7). Downstream of Dvl, effectors in the PCP pathway H.G. performed research; R.J. and P.C. contributed new reagents/analytic tools; R.J., P.C., signal through the small GTP-binding protein Rho to coordinate D.C.S., and C.M.C. analyzed data; and R.J., P.C., D.C.S., and C.M.C. wrote the paper. actin polymerization and organization, giving rise to polarized cell The authors declare no conflict of interest. movements (5). This article is a PNAS Direct Submission. Recently, binding of the formin Dishevelled-associated activator 1R.J. and P.C. contributed equally to this work. of morphogenesis 1 (DAAM1) to Dvl and the small GTPase Rho 2Present address: Department of Internal Medicine, Yale University, New Haven, has been shown to coordinate Wnt signaling cues (8). DAAM1 is CT 06511. a member of a subgroup of , the diaphanous-related for- 3To whom correspondence should be addressed. E-mail: [email protected]. mins (DRFs), and contains multiple regulatory domains. These This article contains supporting information online at www.pnas.org/cgi/content/full/ domains include an N-terminal GTPase-binding domain (GBD), 1001075107/DCSupplemental.

6906–6911 | PNAS | April 13, 2010 | vol. 107 | no. 15 www.pnas.org/cgi/doi/10.1073/pnas.1001075107 Downloaded by guest on October 2, 2021 GBD DID DD FH1 FH2 true for other cell types that form continuous anisotropic tissues A 1 550 1077 Full-length DAAM1 FLAG in vivo including smooth muscle cells (SMC) and Madin-Darby

490 1077 canine kidney (MDCK) cells. These cell types were not sensitive CDAAM1 (DP) HA to the expression of CDAAM1 (Fig. S1). Therefore, the growth 1 233 NDAAM1 (DN) HA inhibition resulting from the expression of constitutively active

945 1077 CDAAM1 was indeed selective for endothelial cells. GFP DAAMDAD (DP) Because we were interested in the selectivity of endothelial cells to perturbations in PCP signaling and NDAAM1 appeared 1 2 3 4 DAAM1 shRNA Scramble B C CDAAM1 HUVEC MPE to inhibit cell proliferation in a cell-unselective manner, we Actin 40 40 HUVEC U2OS focused on the cellular effects mediated by DAAM1 activation by 40 30 30 CDAAM1 expression. To rule out the possibility that CDAAM1- 20 20 * 30 mediated endothelial cell proliferation inhibition was an artifact ** 10 10 ** ** 20 ** ** ** ** of CDAAM1 overexpression, we subsequently tested whether % BrdU-positive nuclei % BrdU-positive nuclei 0 0 10 endogenous DAAM1 activation resulted in similar endothelial GFP GFP DAAM1 DAAM1 NDAAM1 CDAAM1 NDAAM1 CDAAM1 % BrdU-positive nuclei 0 cell growth inhibition as seen for CDAAM1 expression. Pre- viously, expression of the C-terminally localized DAD domain of DAAM1 Scramble DAAM1 Scramble shRNA shRNA mDia1 has been shown to relieve autoinhibition of endogenous U2OS Dermal Fibroblasts HUVEC U2OS 40 40 D40 mDia (10). Taking advantage of the fact that, like mDia1,

30 30 30 DAAM1 also possesses a DAD domain (17, 18), we expressed the 20 20 DAAM1 DAD domain fused with GFP in HUVECs and control ** 20 * 10 10 U2OS cells. DAAM1 DAD expression produced similar growth 0 10

0 % BrdU-positive nuclei % BrdU-positive nuclei ** inhibition to that observed on CDAAM1 expression in HUVECs GFP GFP % BrdU-positive nuclei 0 DAAM1 DAAM1 NDAAM1 CDAAM1 NDAAM1 CDAAM1 but not in U2OS cells (Fig. 1D). These results suggest that acti- GFP GFP vation of endogenous DAAM1 by DAD can also inhibit the DAAMDAD DAAMDAD growth of endothelial cells in a similar manner as through over- Fig. 1. Activation of DAAM1 selectively inhibits endothelial cell growth. (A) expression of CDAAM1. Graphical representation of various DAAM1 constructs. (B) Growth effects of various DAAM1 domains in nonendothelial and endothelial cells. Cells were CDAAM1 Induces Actin Polymerization and MT Stabilization. Because transfected with NDAAM1, CDAAM1, and full-length DAAM1 for 48 h; pulse- formin are known to affect both actin polymerization labeled with BrdU for 1 h; and then probed for the epitope (either FLAG- or and MT stabilization (8, 15, 19), we next determined which of HA-tag as appropriate) and for BrdU incorporation as described in Materials these two activities (or both) could affect proliferation in an and Methods. Vector-expressing GFP served as a negative control for all endothelial cell-selective manner. We first investigated whether experiments. Error bars indicate SEM. (C) shRNA-mediated knockdown of DAAM1 indiscriminately inhibits proliferation of both endothelial and U2OS CDAAM1-induced actin polymerization in endothelial cells was cells. The immunoblot illustrates an approximately >70% knockdown of HA- distinguishable from that in nonendothelial cells. CDAAM1 CDAAM1 by shRNA3 and shRNA4. BrdU incorporation experiments were induced actin polymerization in both endothelial (HUVEC) cells performed using shRNA3 against DAAM1. (D) Effect of DAAM1 DAD domain (Fig. 2A) and nonendothelial (U2OS) cells (Fig. 2B) without any on HUVEC and U2OS cell proliferation. Each graph represents an average of discernible differences. three independent experiments (duplicate experiments for C with over 100 Based on the high degree of homology between mDia1 and cells each). Statistical significance was determined by one-way ANOVA and DAAM1 (20), we next speculated that CDAAM1 expression < < denoted for *P 0.05 and **P 0.001. DD, dimerization domain; DP, dom- could induce MT stabilization in a manner similar to that observed inant positive; DN, dominant negative. on expression of the constitutively active mDia1 (15). To test this hypothesis, we first examined if CDAAM1 could differentially expressing NDAAM1 and CDAAM1. BrdU incorporation into change overall MT structures in HUVECs and U2OS cells. No both human umbilical vein endothelial cells (HUVECs) and dramatic MT organizational changes were observed in CDAAM1- murine pulmonary endothelial (MPE) cells was diminished on expressing HUVECs or in U2OS cells compared with their respective controls (Fig. 2 C and D). DAAM1 activation, there- overexpression of DAAM1 or its truncated mutants relative to fore, did not affect overall MT organization, nor were there any GFP controls (Fig. 1B). To test if other cell types would respond outstanding cell type distinguishing features observed. in a similar manner, we introduced these constructs into human Although the overall MT organization was not affected, it was primary dermal fibroblasts and human U2OS osteosarcoma cells. still possible that activated DAAM1 could stabilize MTs in a man- Interestingly, neither CDAAM1 nor full-length DAAM1 signif- ner similar to mDia. In most cells, the subset of MTs possessing icantly inhibited proliferation in these cells, although NDAAM1 carboxyl-terminal detyrosinated tubulin (Glu tubulin) is more stable blocked cell growth (Fig. 1B). Also, the loss of function of than other MTs (21). Because the FH1/FH2 domains of mDia1 are DAAM1 by shRNA-mediated knockdown indiscriminately able to increase stable MTs (15), we examined whether there was an inhibited both HUVEC and U2OS proliferation in a similar increase in MT stabilization in CDAAM1-expressing HUVECs and manner as the dominant negative NDAAM1 construct (Fig. 1C). U2OS cells as measured by anti-Glu-tubulin immunofluorescence. fl fi This is re ected in vivo, where zebra sh embryos injected with As shown in Fig. 2 E and F, we found that CDAAM1 stabilized MTs an antisense morpholino to DAAM1 display notochord defects in both HUVECs and U2OS cells proportional to the expression (16). Similar, although more pronounced, defects were observed levels of CDAAM1 (Fig. 2E). with zebrafish NDAAM1 or in fish injected with morpholinos to However, there is an apparent difference in stable MT organ- both DAAM1 and DAAM2 (16). Therefore, whereas the anti- ization between these two cell types. Stabilized MTs in endo- proliferative effects of DAAM1 activation appear to be endo- thelial cells were predominantly localized in the perinuclear thelial cell-specific, the loss-of-function data in vitro and in vivo region, whereas stable MTs were dispersed throughout the cyto- support a broader role for DAAM1 in a variety of cell types, plasm within U2OS cells (Fig. 2E, Glu-tubulin panel). Quantifi- including endothelial cells. cation of cells displaying a typical perinuclear-to-lamellipodia However, because the proliferation inhibition resulting from orientation over total cells illustrated that CDAAM disrupted

up-regulation via expression of CDAAM1 appeared to be se- this orientation selectively and significantly in endothelial cells CELL BIOLOGY lective for endothelial cells (Fig. 1B), we tested whether this was (Fig. 2F). Interestingly, the stable MTs were strongly colocalized

Ju et al. PNAS | April 13, 2010 | vol. 107 | no. 15 | 6907 Downloaded by guest on October 2, 2021 HUVEC U2OS A GFP/HA Actin Merge B GFP/HA Actin Merge GFP GFP

CDAAM1(HA) Fig. 2. DAAM1 activation affects both actin and MT CDAAM1(HA) networks. (A) Expression of CDAAM1 on actin structure in HUVECs. (B) Expression of CDAAM1 on actin structure HUVEC U2OS C HA alpha-tubulin Merge D HA alpha-tubulin Merge in U2OS cells. (C) Expression of CDAAM1 on overall MTs in HUVECs. (D) Expression of CDAAM1 on overall MTs in U2OS cells. (E) Expression of CDAAM1 on stable MTs in HUVECs and U2OS cells. Arrows indicate trans- Control Control fected cells. Note that in E, in HUVECs (Upper), the formation of stable MTs was proportional to expression of CDAAM1. In addition, both cell types displayed spontaneous formation of stable Glu-tubules. In each experiment, cells were transfected with CDAAM1 con- CDAAM1(HA) CDAAM1(HA) structs for 24–48 h and then double-stained with anti- HA and with phalloidin, anti-α-tubulin, or anti-Glu- Glu-tubulin HA Glu-tubulin Merge E F HUVEC U2OS tubulin antibodies. (F) Quantification of HUVECs and 100 U2OS cells displaying perinuclear-to-lamellipodia ori-

80 entation of stabilized MTs. Commonly, spontaneous or

** (by adenoviral vector) CDAAM1-induced stabilized MTs 60 were observed to orient from the perinuclear region

40 outward. In HUVECs particularly, Glu-tubulin was pre- dominantly perinuclear in most cells without projecting U2OS HUVEC 20 distally from the nucleus. The graph represents pooled < 0 data from two independent experiments (**P 0.001). Control CDAAM1 (HA) % Cells with Oriented Glu-Tubulin GFP CDAAM1 GFP CDAAM1 (Scale bar = 50 μm.)

with CDAAM1 in HUVECs and only moderately so in U2OS growth (Fig. 3D, Left). Although GFP-CLASP1α also induced cells, suggesting a transport relation in endothelial cells. This MT stabilization in U2OS cells (Fig. 3C, Lower), it did not inhibit relation is supported by data, whereby DAAM1 has been shown cell growth (Fig. 3D, Right). Despite the recent report that to mediate the endocytosis of EphB (16). Therefore, although CLASPs bind the actin network (25), our results indicate that DAAM1 can activate both actin polymerization and MT stabili- CLASP1α did not induce actin polymerization in MPE cells zation, the coordination of stabilized MTs appears to vary ac- (Fig. S2). Taken together, these results suggest that MT stabili- cording to cell type, which is likely responsible for the varying zation, rather than actin polymerization alone or a combination of effects of DAAM activation on proliferation. the two, selectively inhibits endothelial cell growth. Therefore, endothelial cell growth arrest by CDAAM1 is most likely medi- MT Stabilization Rather Than Actin Polymerization Inhibits Endo- ated through MT stabilization. thelial Cell Proliferation. Because CDAAM1 expression had anti- proliferative effects in endothelial cells but not U2OS cells, CDAAM1 Inhibits Endothelial Cell Migration and Tube Formation. although inducing actin polymerization and MT stabilization in Because angiogenesis is a multicellular process that requires not both, we hypothesized that endothelial cells may respond to only cell proliferation but migration and intercellular coordina- cytoskeletal changes (either actin polymerization, MT stabiliza- tion (26), it was important to assess if DAAM1 activation could tion, or a combination of both) differently than other cell types. affect these complex processes. To test this, MPE cells express- To test this hypothesis, we used agents that induced either actin ing CDAAM1 were examined in migration and network for- polymerization or MT stabilization independent of each other. As mation assays. Expression of CDAAM1 drastically suppressed with previous observations (22), treatment of HUVECs with the endothelial cell migration compared with the control (Fig. S3A). actin polymerization reagent jasplakinolide drastically induced Not surprisingly, CDAAM1 also drastically reduced the number actin polymerization at 10 nM (Fig. 3A). Nevertheless, the drug- of cell extensions formed within cell networks on Matrigel (BD induced actin polymerization, even at higher doses, failed to BioSciences) membranes (Fig. S3B). Because in vitro endothelial inhibit cell proliferation as measured by BrdU staining (Fig. 3B). cell network formation correlates with angiogenic potential (27), Clearly, actin polymerization alone was insufficient to reproduce these data suggest that proper coordination of DAAM1 activities CDAAM1-mediated endothelial cell growth inhibition. is required for angiogenesis. To test if MT stabilization could inhibit endothelial cell pro- liferation independent of actin polymerization, we introduced CDAAM1 Affects Angiogenesis in Vivo. To address the role of GFP-fused CLASP1α into our cell lines. CLASP1α, a +TIP, has DAAM1 in angiogenesis in vivo, we used fli:GFP transgenic been shown to induce MT stabilization (23, 24) but has not been zebrafish expressing GFP specifically within vascular endothelial reported to inhibit cell proliferation. Expression of GFP- cells (28). First, we investigated whether manipulation of DAAM1 CLASP1α induced MT stabilization in MPE cells as indicated by activity by CDAAM1 RNA microinjection induced gastrulation the appearance of Glu-tubulin (Fig. 3C, Upper). Furthermore, defects as readout for reduced PCP signaling in zebrafish. Com- BrdU incorporation assays revealed that expression of GFP- pared with WT (Fig. 4A), CDAAM1 RNA injection at a low dose CLASP1α in MPE cells significantly decreased their proliferation (0.2 ng of RNA) generated moderate defects with obvious tail compared with controls (Fig. 3D, Left). Adding jasplakinolide in kinks and shorter anterior-posterior (A-P) length (Fig. 4C), al- addition to GFP-CLASP1α expression did not further inhibit though at a higher injection dose (0.4 ng), more severe con-

6908 | www.pnas.org/cgi/doi/10.1073/pnas.1001075107 Ju et al. Downloaded by guest on October 2, 2021 Jasplakinolide DA SE A Control 10 nM 50 nM A B Wildtype PCV C D SE CDAAM1

B Low dose

E F DA CDAAM1 High dose

% BrdU-positive nuclei PCV 0 nM 10 nM 50 nM G H Jasplakinolide

C Glu-Tubulin CLASP1α Glu-Tubulin Merge MPE fi Control Fig. 4. CDAAM1 inhibits angiogenesis in zebra sh. (A and B)WTembryos demonstrating normal Fli-1 expression. (C and D) Embryos injected with 2ngofCDAAM1RNA.(E and F) Embryos injected with 4 ng of CDAAM1

U2OS RNA. A, C,andE (arrows) reveal the A-P length as readout for the PCP pathway. B, D,andF display vascular endothelial cell staining in the somite Control region. (G and H) Confocal images with Fli1-EGFP expression overlaid on lineage tracer identifying transplant location. Zebrafish embryo manipu- MPE U2OS D 30 40 lation, RNA preparation, and imaging are described in Materials and +50 nM Jasplakinolide Methods. (Scale bars = 100 μm.)

30 20

20 SEs were thinner and more diffuse, and some even disappeared * (Fig. 4D). Moreover, CDAAM1-injected embryos with more 10 ** 10 severe defects lacked SE outgrowth and had gaps in the DA and % BrdU-positive nuclei % BrdU-positive nuclei PCV (Fig. 4 E and F). 0 0 To rule out the possibility that the C-E defects contributed to the GFP CLASP1α GFP CLASP1α GFP CLASP1α vascular disruptions, we performed transplantation of CDAAM-1- Fig. 3. MTs rather than actin network changes are responsible for endo- expressing cells into WT hosts. A majority of the host embryos (12 thelial cell growth inhibition. (A) Jasplakinolide (10 or 50 nM, 16 h) induced of 13) had transplanted cells outside of the somite/intersegmental actin polymerization. (B) Jasplakinolide did not affect endothelial cell region and showed normal vascularization (Fig. 4G). Of the host growth. HUVECs were treated with either 10 or 50 nM of jasplakinolide for embryos with CDAAM-1 expression in the somite/intersegmental 16 h and then stained with either phalloidin or anti-BrdU antibody. S-phase region, all (5 of 5) showed dramatic alterations to vascularization cells were counted as in Fig. 1B. Error bars indicate SEM, representing an average of three independent experiments. (C) CLASP1α induces stable MTs (Fig. 4H). Because the vascular disruptions observed in the trans- in MPE cells. MPE cells were transfected with GFP-CLASP1α.After24h plant model were limited to the region of transplanted cells, the posttransfection, stable MTs were visualized with anti-Glu-tubulin anti- predominant effect of CDAAM on angiogenesis is within endo- body. Transfected cells were visualized with an anti-GFP antibody because thelial cells in an autonomous manner. CDAAM1-induced phe- GFP was quenched by methanol fixation. Arrows indicate transfected cells. notypes are also highly penetrant compared with control injections (D)CLASP1α inhibited MPE but not U2OS cell growth. Cells were transfected (Table 1). Taken together, the data indicate that overactivation with GFP-CLASP1α for 24 h, with or without 50 nM jasplakinolide for of DAAM1 signaling disrupts A-P extension of the embryo and fi another 24 h, and cells were then xed and visualized with anti-BrdU angiogenesis in vivo. antibody. The percentage of S-phase cells among GFP-positive cells was counted as in Fig.1B. Error bars indicate SEM; averages of three inde- Discussion pendent experiments are displayed. Statistical analyses by one-way ANOVA were performed to yield respective P values (*P < 0.05; **P < 0.001). (Scale In this study, we found that CDAAM1 expression selectively bar = 50 μm.) inhibited cell growth in endothelial cells. In addition, we show that, like the constitutively active mDia1, constitutively active CDAAM1 induces MT stabilization, indicating that MT stabili- vergence extension (C-E) defects were observed (Fig. 4E). These zation may be a central function for formin proteins. More results indicated that overactivation of DAAM1 affects the vas- importantly, aberrant MT stabilization, rather than actin poly- cularization of these embryos in a manner consistent with non- merization mediated by expression of CDAAM1, is responsible canonical Wnt signaling inhibition such as in pipetail (Wnt5 for the growth inhibition of endothelial cells. This report shows knockout) zebrafish (4). Analysis of an fli-1-driven vascular that up-regulation of PCP signaling mediated by activation of endothelial cell marker in the injected embryos revealed clear DAAM1 can lead to growth inhibition in a cell-selective manner. vascular defects to the trunk and tail in CDAAM1-injected This study provides a mechanism by which precise Wnt/PCP zebrafish (Fig. 4 C and D). In a lateral view of a control 28-h signaling regulates endothelial cell proliferation, because both postfertilization (hpf) embryo, normal vasculature in the trunk overactivation (i.e., CDAAM1 expression) and underactivation displayed a dorsal aorta (DA) and posterior cardinal vein (PCV) (i.e., NDAAM1 expression) resulted in proliferation sup-

with intersegmental vessels (SEs) extending dorsally (Fig. 4B). In pression. This may explain the conflicting results from two CELL BIOLOGY CDAAM1-injected embryos with moderate tail kink defects, the groups studying the roles of Wnt signaling in endothelial cells.

Ju et al. PNAS | April 13, 2010 | vol. 107 | no. 15 | 6909 Downloaded by guest on October 2, 2021 Table 1. Statistical representation of defects caused by CDAAM1 RNA Normal Mild-moderate defects Severe defects % Defects

GFP, 0.5 ng 32 ——0 CDAAM1, 0.2 ng 8 32 5 82 CDAAM1, 0.4 ng 9 17 6 72

Goodwin et al. (29) found that Wnt5a decreased cell growth and an essential role in neuronal guidance. Answers to these ques- capillary length of bovine aortic endothelial cells, whereas others tions will determine whether the role of DAAM1 in MT stabi- (30) reported that Wnt5a signaling promoted endothelial cell lization is confined only to endothelial cells and angiogenesis. proliferation and survival. Under our experimental conditions, we failed to detect endothelial cell growth inhibition or stim- Materials and Methods ulation with Wnt5a-conditioned medium (4). One possibility is Cell Culture and Transfection. HUVECs and human dermal fibroblasts were that endothelial cells may have developed certain mechanisms to obtained from the Yale Cell Culture Core facility. MPE cells were isolated as restrain Wnt5a signaling. For instance, we observed that almost described previously (39). HUVECs were cultured in EGM-2 (Lonza). MPE cells, all Fz4, the presumed Wnt receptor for the PCP pathway, when human dermal fibroblasts, SMCs, MDCKs, and U2OS cells were cultured in overexpressed in endothelial cells, is localized within punctuate DMEM with 10% (vol/vol) FBS. Cells were transfected with Lipofectamine aggregates or in the perinuclear region rather than on the plasma 2000 (Invitrogen) or Fugene HD (BD BioScience) according to the manu- ’ membrane (Fig. S4). Conversely, Fz4 was found to localize on facturer s instructions. MISSION shRNA lentiviral vectors (Sigma) were used ’ the plasma membrane when expressed in HEK293 cells (31). to deliver scrambled or anti-DAAM1 shRNA according to the manufacturer s This may suggest that endothelial cells maintain PCP signaling at instructions. Finally, adenoviral vectors (pAd/MV/V5-DEST; Invitrogen) were used in certain experiments (according to the manufacturer’s protocol) in a proper level by regulating the availability of Wnt receptors. which chemical transfections were insufficiently effective (as described in Several clinical studies from MT-targeted agents (MTAs) have the text). PCR and plasmid construction are further detailed in SI Text and indicated that both MT stabilization and depolymerization Table S1. increase MT dynamics specifically in endothelial cells at low doses and inhibit endothelial cell proliferation, migration, and Immunofluorescence and BrdU Double-Staining. The reader is referred to SI network formation on Matrigel (32–34). These results suggest Text for details. that endothelial cells may likely differ from other cell types with respect to MT composition, such as by tubulin isotypes or pro- Migration Assays and Tube/Network Formation Assay on Matrigel. Migration teins that regulate MT dynamics (33). In this study, we demon- assays were performed in a 24-well plate (BD Biocoat Control Inserts, catalog strated that MT stabilization mediated by CDAAM1 or CLASPα no. 354578; BD Biosciences) according to the manufacturer’s instructions. can selectively inhibit similar functions as MTAs, further sup- Matrigel assays were performed as described in the product literature (27). porting the hypothesis that endothelial cells may regulate MT Details are provided in SI Text. networks differently than other cell types. Our present study shows that overactivation of DAAM1 Microinjection of Zebrafish. Zebrafish (Danio rerio) embryos collected from inhibits angiogenesis. This inhibition occurs at multiple levels: natural matings were processed using standard techniques (40). RNA was Not only is endothelial cell proliferation inhibited but migration introduced into one to eight cell-stage embryos with a pressure injector. and network formation are suppressed. Moreover, zebrafish data CDAAM RNA was synthesized from linearized templates using the mMes- sage mMachine kit (Ambion). Injected embryos were analyzed for mor- show that injection of constitutively active CDAAM1 results in phological defects and fixed (4% formaldehyde) at 28 hpf. Whole-mount in diminished and miscoordinated vasculature, a phenotype similar situ hybridization was performed using antisense Fli1 riboprobe. The Fli1 to the Wnt5 loss-of-function mutant (pipetail, ppt). Clearly in riboprobe was generated using primers Fli-1F1 (5′-CTGCTGCTCCTTTACC- vivo, overactive PCP signaling can be as detrimental as dimin- CAAG-3′) and Fli-1R1 (5′-AGCTCCAGTATGGGGTTGTG-3′), which were ampli- ished Wnt signaling, indicating that the PCP signaling pathway fied by PCR from embryonic cDNA and cloned into pCRII TOPO vector. must be properly coordinated in cellular and developmental Antisense Fli1 riboprobe was then made through a digestion with BamH1 processes. Studies investigating other PCP mediators such as and in vitro transcription with T7 RNA polymerase. Embryos were photo- vang/stbm support this hypothesis (35, 36), because either over- graphed using a Zeiss Axiocam. expression or inhibition of vang/stbm in Xenopus led to a fore- For the transplantation experiment, donor Fli1:EGFP embryos were shortened embryo with disrupted convergent extension. microinjected with cDAAM RNA and 0.5 pmol lissamine-conjugated standard Overall, we show that perturbations in PCP signaling via control morpholino (MO) as a lineage tracer ( Tools, LLC). Donor and DAAM1 overactivation inhibit angiogenesis through MT dis- host Fli1:EGFP embryos were manually dechorionated before trans- organization. These results provide a possible mechanism by plantation. At the sphere stage, 20–30 cells were removed from donor which PCP signaling governs angiogenesis and offer unique embryos and transplanted into the blastodermal margin of each host opportunities to inhibit pathological angiogenesis without neg- embryo. At 30 hpf, host embryos were anesthetized with tricaine (4 mg/mL) ative collateral effects. Nevertheless, further questions remain. and imaged on a Leica SP2 confocal microscope. Datasets were collected fi × For example, the mechanistic steps leading from ectopic MT from the trunk region focusing on the SEs at a magni cation of 20. stabilization to endothelial cell cycle arrest have yet to be Statistical Analysis. GraphPad Prism v4.0b software was used to perform all determined. Moreover, it remains uncertain how DAAM1 can ’ fl statistical analyses. Student s t tests and one- or two-way ANOVA analyses affect the polarity of cells and, in turn, in uence their migration were followed by Tukey’s multiple comparison tests or Bonferroni’s posttests and ability to coordinate with other cells. Also, the selective as appropriate (*P < 0.05; **P < 0.01). inhibitory effects on endothelial cells by DAAM1 activation suggest a unique role for DAAM1 in endothelial cells not shared ACKNOWLEDGMENTS. We thank Drs. Taavi Neklesa, Tim Corson, and with other similar cell types. Finally, given the fact that DAAM1 Stephanie Leuenroth-Quinn for critical comments and discussion. We also is strongly expressed in the differentiating nervous system (37) thank Drs. Greg Gundersen and Niels Galjart for the kind gifts of the Glu- tubulin antibody and the GFP-CLASP1α plasmid, respectively. This work was and the PCP/Wnt cascade is implicated in the growth and supported by National Institutes of Health Grants CA083049 (to C.M.C.) and guidance of axons and dendrites (38), it will be interesting to CA112369 (to D.C.S.). P.C. is the recipient of a Leukemia and Lymphoma examine whether MT stabilization or actin polymerization plays Society Fellowship.

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