Fig. 6. GST–Cdc42 or GST–Rac induced actin polymerization in WAVE2-deficient cell extracts. Kinetics of actin polymerization in the absence (blue) or presence of GTPγS-charged GST-Rac (green) or GTPγS-charged GST–Cdc42 (red). (A) Het (WAVE2^+/neo); (B) KO (WAVE2^neo/neo); (C) Rescue (WAVE2^neo/neo+RVWAVE2).

Fig. 5. (A) Western blot detection of WAVE1, WAVE2 and N-WASP in wild-type (WT) and knockout (KO) mouse embryonic fibroblasts (MEFs). The genotypes of the MEFs examined are depicted. The 84 kDa standard protein marker is shown. (B) RT–PCR analyses of WAVE1, WAVE2 and WAVE3 expression levels in WT and KO MEFs and in brain. β-actin is used as a control. PCRs were performed with 5-fold dilution of cDNAs from left to right.

Fig. 2. WAVE2 deficiency results in mild to severe growth retardation and profound morphological defects in embryos. (A–C) Micrographs of E11–E12 wild-type (wt), WAVE2^+/neo (Het) and WAVE2^neo/neo (mut) embryos derived from F1 WAVE^+/neo matings. Embryo littermates were photographed together at 20× (B and C) or 40× (A) magnification. The micrograph in (A) shows embryos fixed in Bouin’s Solution (Sigma); (B) and (C) show micrographs of unfixed embryos. Distinct growth retardations of mut versus wt embryos are apparent in (A) and (B). Branchial arches, limb buds, somites (A, B and C) and blood (B and C) are apparent. In (C), the mut embryo is comparable in size to wt and Het littermates but displays distinct conformational abnormalities: decreased size and malformation of cephalic (head) and caudal extremities and increased hemorrhaging. (D–I) High-powered hematoxylin-eosin staining of the transverse sections of WAVE2^+/neo (Het) and WAVE2^neo/neo (mut) embryo littermates. (D, E, F and G) Transverse section of 6–7 µm thickness taken from the cephalic region of WAVE2^+/neo (D and F) and WAVE2^neo/neo  (E and G). The forebrain (fb) is oriented at the top and the hindbrain (hb) at the bottom. More anterior section (D and E) displaying the three ventricles of the forebrain (fb) and later sections (F and G) displaying lens and cheek formation are shown. Significant malformations of ventricles in both the forebrain and hindbrain are apparent. (H and I) Transverse section of WAVE2^+/neo (H) and WAVE2^neo/neo (I) caudal somites. Arrows point to two of several regions that display unusual detachment of cells.

Fig. 4. WAVE2 is essential for lamellipodium and ruffle formation in mouse embryonic fibroblasts (MEFs). (A) Sub-confluent serum-starved wild-type (WT) and WAVE2 knockout (KO) MEFs were left unstimulated or stimulated with 20 ng/ml PDGF for 10 min. The leading edge and other areas of lamellipodia and ruffles are more obvious by cortactin staining (green panels) and detection of F-actin cytoskeleton by TRITC-phalloidin (red panels). KO cells show more distinct cytoskeletal defects. Note the absence of leading edge and aberrant lamellipodia formation (yellow arrows point to leading edge of lamellipodia) in unstimulated KO cells compared with WT cells. PDGF induces lamellipodia (yellow arrows), and circular ruffles (blue arrows) in KO MEFs are structurally malformed. Scale bar, 10 µm. (B) WAVE2 retroviral expression rescues the defect in lamellipodium formation. WAVE2 KO MEFs transduced with WAVE2 retrovirus (RVWAVE2), left unstimulated or stimulated with 20 ng/ml PDGF for 10 min. Cortactin staining is shown in red and F-actin in blue. Rescued KO MEFs exhibit a normal morphology with well-defined lamellipodium areas. (C) Membrane ruffle formation of WT, KO and Rescue (KO MEFs rescued with the WAVE2 retrovirus). One hundred cells of each condition were counted. Membrane ruffles are broken down into two distinct categories: lamellipodia and circular ruffles. Numbers represent an average of more than eight independent experiments performed for WT and KO and more than three experiments for the Rescue conditions. The average of these conditions with standard error bars is shown. (D) The chemotactic response of WT and KO primary MEFs to PDGF was examined under conditions containing (+PDGF) or lacking (-PDGF) PDGF at 10 ng/ml. The graph depicts the average of three experiments (42 fields of cells per condition) counted at two magnifications.

Fig. 1. Murine WAVE2 RNA expression and targeted disruption of WAVE2 in mice and cells. (A) Diagram of murine WAVE2 genomic locus and targeting strategy. The physical map of the entire WAVE2 genomic locus containing all eight exons is shown. Exons are indicated by small rectangular boxes. Genomic DNA fragments used to generate the targeting construct are hatched. The neomycin-resistance (Neo^r) and thymidine kinase (TK) genes used for positive and negative selections, the 5′ and 3′ probes, and the Xba1 restriction sites used for screening embryonic stem (ES) cell transfectants by Southern blot analyses are labeled. Deletion of the loxP-flanked neo^r cassette leaves a single loxP site and the GFP downstream of an intra-ribosomal entry sequence (IRES). (B) Generation of WAVE2^+/neo, WAVE2^neo/neo and WAVE2^–/neo ES cells and germline mice. Using the 5′ probe noted in the schematic in (A), homologous recombination results in the loss of a single 7.6 kb band and the appearance of a 8.6 kb targeted band or a 7 kb Cre deleted band. Genotypes of germline WAVE2^+/neo mice born from mating a WAVE2 chimeric male and a 129sv/ev wild-type female are shown. (C) Loss of WAVE2 protein and RNA expression in WAVE2-deficient ES cells. Western blot analyses of protein extracts from wild-type (+/+), WAVE2^+/neo (+/neo) and WAVE2^neo/neo (neo/neo) ES cells. WAVE2 runs at the 84 kDa molecular weight marker and is absent in WAVE2-deficient ES cells. Equal protein loading was determined using an antibody to Ku86 (Santa Cruz). Northern blot analysis was determined using a WAVE2 cDNA probe. Equal RNA loading was determined with a β-actin control probe.

Fig. 3. Growth rate of WAVE2-deficient cell lines. (A) Comparison of the growth rate of primary mouse embryonic fibroblasts (MEFs) derived from E11 embryo littermates, one wild-type (376-3+/+), two WAVE2^+/neo (376-5+/– and 376-9+/–) and three WAVE2^neo/neo MEFs (376-1–/–, 376-6–/– and 376-8–/–), and the growth rates of primary MEFs, untransduced (44–/– and 61-12–/–) or transduced with retrovirus expressing GFP (WTRVGFP and 44–/–RVGFP) or the WAVE2–GFP (61-12–/–RVW2 and 44–/–RVW2). Early passage cells were plated at 2 × 10⁴ per well in a 6-well dish in triplicate fashion. Cells were counted every 2 days up to 9 days in two independent experiments. The average of each experimental point with standard error bars is shown (hatched bars). Additional growth curves of primary MEFs derived from E9.5 embryo littermates gave similar results (data not shown). (B) The growth rate of WAVE2-deficient (KO) embryonic stem (ES) cells containing or lacking the loxP-flanked neo^r cassette are compared with wild-type (WT) and two Het ES cell lines. The genotypes of the ES cell lines are depicted. ES cells, depleted of MEFs, were plated at 2 × 10⁴ cells per well in a 6-well dish and counted in triplicate as described above. The average of each experimental point with standard error bars is shown (hatched bars). (C) Flow cytometric analyses of FITC-Annexin V binding versus Topro 3 uptake in WT versus KO MEFs. Upper left, damaged cells (Annexin V^–, Topro 3⁺); lower left, live cells (Annexin V^–, Topro 3^–); upper right, late apoptotic and dead cells (Annexin V⁺, Topro 3⁺); lower right, early apoptotic cells (Annexin V⁺, Topro 3^–). Numbers represent the average of three experiments. (D) WAVE2 KO MEFs contain fewer replicating cells. WT and KO MEFs were pulsed for 1.5 h with 20 µM bromodeoxyuridine (BrdU) and analyzed by flow cytometry. The average percentage of labeled cells among total live cells from three experiments is indicated by the box. (E) Kinetics of BrdU incorporation of WT versus KO MEFs. MEFs were continuously labeled with 100 µM BrdU for 48 h and analyzed by flow cytometry. The average percentage of BrdU-labeled cells among total live cells from three experiments with standard error bars is plotted at each time point.