Shp2 knockdown and Noonan/LEOPARD mutant Shp2-induced gastrulation defects

Shp2 is a cytoplasmic protein-tyrosine phosphatase that is essential for normal development. Activating and inactivating mutations have been identified in humans to cause the related Noonan and LEOPARD syndromes, respectively. The cell biological cause of these syndromes remains to be determined. We have used the zebrafish to assess the role of Shp2 in early development. Here, we report that morpholino-mediated knockdown of Shp2 in zebrafish resulted in defects during gastrulation. Cell tracing experiments demonstrated that Shp2 knockdown induced defects in convergence and extension cell movements. In situ hybridization using a panel of markers indicated that cell fate was not affected by Shp2 knock down. The Shp2 knockdown-induced defects were rescued by active Fyn and Yes and by active RhoA. We generated mutants of Shp2 with mutations that were identified in human patients with Noonan or LEOPARD Syndrome and established that Noonan Shp2 was activated and LEOPARD Shp2 lacked catalytic protein-tyrosine phosphatase activity. Expression of Noonan or LEOPARD mutant Shp2 in zebrafish embryos induced convergence and extension cell movement defects without affecting cell fate. Moreover, these embryos displayed craniofacial and cardiac defects, reminiscent of human symptoms. Noonan and LEOPARD mutant Shp2s were not additive nor synergistic, consistent with the mutant Shp2s having activating and inactivating roles in the same signaling pathway. Our results demonstrate that Shp2 is required for normal convergence and extension cell movements during gastrulation and that Src family kinases and RhoA were downstream of Shp2. Expression of Noonan or LEOPARD Shp2 phenocopied the craniofacial and cardiac defects of human patients. The finding that defective Shp2 signaling induced cell movement defects as early as gastrulation may have implications for the monitoring and diagnosis of Noonan and LEOPARD syndrome.     

Phosphoproteomics-Mediated Identification of Fer Kinase as a Target of Mutant Shp2 in Noonan and LEOPARD Syndrome

Noonan syndrome (NS) and LEOPARD syndrome (LS) cause congenital afflictions such as short stature, hypertelorism and heart defects. More than 50% of NS and almost all of LS cases are caused by activating and inactivating mutations of the phosphatase Shp2, respectively. How these biochemically opposing mutations lead to similar clinical outcomes is not clear. Using zebrafish models of NS and LS and mass spectrometry-based phosphotyrosine proteomics, we identified a down-regulated peptide of Fer kinase in both NS and LS. Further investigation showed a role for Fer during development, where morpholino-based knockdown caused craniofacial defects, heart edema and short stature. During gastrulation, loss of Fer caused convergence and extension defects without affecting cell fate. Moreover, Fer knockdown cooperated with NS and LS, but not wild type Shp2 to induce developmental defects, suggesting a role for Fer in the pathogenesis of both NS and LS.     

PZR Coordinates Shp2 Noonan and LEOPARD Syndrome Signaling in Zebrafish and Mice

Noonan syndrome (NS) is an autosomal dominant disorder caused by activating mutations in the PTPN11 gene encoding Shp2, which manifests in congenital heart disease, short stature, and facial dysmorphia. The complexity of Shp2 signaling is exemplified by the observation that LEOPARD syndrome (LS) patients possess inactivating PTPN11 mutations yet exhibit similar symptoms to NS. Here, we identify “protein zero-related” (PZR), a transmembrane glycoprotein that interfaces with the extracellular matrix to promote cell migration, as a major hyper-tyrosyl-phosphorylated protein in mouse and zebrafish models of NS and LS. PZR hyper-tyrosyl phosphorylation is facilitated in a phosphatase-independent manner by enhanced Src recruitment to NS and LS Shp2. In zebrafish, PZR overexpression recapitulated NS and LS phenotypes. PZR was required for zebrafish gastrulation in a manner dependent upon PZR tyrosyl phosphorylation. Hence, we identify PZR as an NS and LS target. Enhanced PZR-mediated membrane recruitment of Shp2 serves as a common mechanism to direct overlapping pathophysiological characteristics of these PTPN11 mutations.     

Mouse model of Noonan syndrome reveals cell type- and gene dosage-dependent effects of Ptpn11 mutation

Noonan syndrome is a common human autosomal dominant birth defect, characterized by short stature, facial abnormalities, heart defects and possibly increased risk of leukemia. Mutations of Ptpn11 (also known as Shp2), which encodes the protein-tyrosine phosphatase Shp2, occur in approximately 50% of individuals with Noonan syndrome, but their molecular, cellular and developmental effects, and the relationship between Noonan syndrome and leukemia, are unclear. We generated mice expressing the Noonan syndrome-associated mutant D61G. When homozygous, the D61G mutant is embryonic lethal, whereas heterozygotes have decreased viability. Surviving Ptpn11(D61G/+) embryos ( approximately 50%) have short stature, craniofacial abnormalities similar to those in Noonan syndrome, and myeloproliferative disease. Severely affected Ptpn11(D61G/+) embryos ( approximately 50%) have multiple cardiac defects similar to those in mice lacking the Ras-GAP protein neurofibromin. Their endocardial cushions have increased Erk activation, but Erk hyperactivation is cell and pathway specific. Our results clarify the relationship between Noonan syndrome and leukemia and show that a single Ptpn11 gain-of-function mutation evokes all major features of Noonan syndrome by acting on multiple developmental lineages in a gene dosage-dependent and pathway-selective manner.     

Distinct and Overlapping Functions of ptpn11 Genes in Zebrafish Development

The PTPN11 (protein-tyrosine phosphatase, non-receptor type 11) gene encodes SHP2, a cytoplasmic PTP that is essential for vertebrate development. Mutations in PTPN11 are associated with Noonan and LEOPARD syndrome. Human patients with these autosomal dominant disorders display various symptoms, including short stature, craniofacial defects and heart abnormalities. We have used the zebrafish as a model to investigate the role of Shp2 in embryonic development. The zebrafish genome encodes two ptpn11 genes, ptpn11a and ptpn11b. Here, we report that ptpn11a is expressed constitutively and ptpn11b expression is strongly upregulated during development. In addition, the products of both ptpn11 genes, Shp2a and Shp2b, are functional. Target-selected inactivation of ptpn11a and ptpn11b revealed that double homozygous mutants are embryonic lethal at 5–6 days post fertilization (dpf). Ptpn11a-/-ptpn11b-/- embryos showed pleiotropic defects from 4 dpf onwards, including reduced body axis extension and craniofacial defects, which was accompanied by low levels of phosphorylated Erk at 5 dpf. Interestingly, defects in homozygous ptpn11a-/- mutants overlapped with defects in the double mutants albeit they were milder, whereas ptpn11b-/- single mutants did not show detectable developmental defects and were viable and fertile. Ptpn11a-/-ptpn11b-/- mutants were rescued by expression of exogenous ptpn11a and ptpn11b alike, indicating functional redundance of Shp2a and Shp2b. The ptpn11 mutants provide a good basis for further unravelling of the function of Shp2 in vertebrate development.     

RPTPα and PTPε signaling via Fyn/Yes and RhoA is essential for zebrafish convergence and extension cell movements during gastrulation

Convergence and extension (C&E) cell movements are essential to shape the body axis during vertebrate gastrulation. We have used the zebrafish to assess the role of the receptor protein-tyrosine phosphatases, RPTPα and PTPε, in gastrulation cell movements. Both RPTPα and PTPε knockdown and ptpra^−/− embryos show defects in C&E movements. A method was developed to track gastrulation cell movements using confocal microscopy in a quantitative manner and ptpra^−/− embryos displayed reduced convergence as well as extension speeds. RPTPα and PTPε knockdowns cooperated with knockdown of a well known factor in C&E cell movement, non-canonical Wnt11. RPTPα and PTPε dephosphorylate and activate Src family kinases in various cell types in vitro and in vivo. We found that Src family kinase phosphorylation was enhanced in ptpra^−/− embryos, consistent with reduced Src family kinase activity. Importantly, both ptpra^−/− and RPTPα and PTPε knockdown induced C&E defects were rescued by active Fyn and Yes. Moreover, active RhoA rescued the RPTPα and PTPε knockdown and ptpra^−/− induced gastrulation cell movement defects as well. Our results demonstrate that RPTPα and PTPε are essential for C&E movements in a signaling pathway parallel to non-canonical Wnts and upstream of Fyn, Yes and RhoA.     

Ror2 Receptor Mediates Wnt11 Ligand Signaling and Affects Convergence and Extension Movements in Zebrafish

The receptor-tyrosine kinase Ror2 acts as an alternative receptor or co-receptor for Wnt5a and mediates Wnt5a-induced convergent extension movements during embryogenesis in mice and Xenopus as well as the polarity and migration of several cell types during development. However, little is known about whether Ror2 function is conserved in other vertebrates or is involved in other non-canonical Wnt ligands in vivo. In this study we demonstrated that overexpression of dominant-negative ror2 (ror2-TM) mRNA in zebrafish embryos resulted in convergence and extension defects and incompletely separated eyes, which is consistent with observations from slb/wnt11 mutants or wnt11 knockdown morphants. Moreover, the co-injection of ror2-TM mRNA and a wnt11 morpholino or the coexpression of ror2 and wnt11 in zebrafish embryos synergetically induced more severe convergence and extension defects. Transplantation studies further demonstrated that the Ror2 receptor responded to the Wnt11 ligand and regulated cell migration and cell morphology during gastrulation. DnRor2 inhibited the action of Wnt11, which was revealed by a decreased percentage of Wnt11-induced convergence and extension defects. Ror2 physically interacts with Wnt11. The intracellular Tyr-647 and Ser-863 sites of Ror2 are essential for mediating the action of Wnt11. Dishevelled and RhoA act downstream of Wnt11-Ror2 to regulate convergence and extension movements. Overall, our data suggest an important role of Ror2 in mediating Wnt11 signaling and in regulating convergence and extension movements in zebrafish.     

Functions of Shp2 in cancer

Diagnostics and therapies have shown evident advances. Tumour surgery, chemotherapy and radiotherapy are the main techniques in treat cancers. Targeted therapy and drug resistance are the main focus in cancer research, but many molecular intracellular mechanisms remain unknown. Src homology region 2‐containing protein tyrosine phosphatase 2 (Shp2) is associated with breast cancer, leukaemia, lung cancer, liver cancer, gastric cancer, laryngeal cancer, oral cancer and other cancer types. Signalling pathways involving Shp2 have also been discovered. Shp2 is related to many diseases. Mutations in the ptpn11 gene cause Noonan syndrome, LEOPARD syndrome and childhood leukaemia. Shp2 is also involved in several cancer‐related processes, including cancer cell invasion and metastasis, apoptosis, DNA damage, cell proliferation, cell cycle and drug resistance. Based on the structure and function of Shp2, scientists have investigated specific mechanisms involved in cancer. Shp2 may be a potential therapeutic target because this phosphatase is implicated in many aspects. Furthermore, Shp2 inhibitors have been used in experiments to develop treatment strategies. However, conflicting results related to Shp2 functions have been presented in the literature, and such results should be resolved in future studies.     

Diverse biochemical properties of Shp2 mutants. Implications for disease phenotypes

Mutations in the Src homology 2 (SH2)-containing protein-tyrosine phosphatase Shp2 (PTPN11) underlie half of the cases of the autosomal dominant genetic disorder Noonan syndrome, and somatic Shp2 mutations are found in several hematologic and solid malignancies. Earlier studies of small numbers of mutants suggested that disease-associated mutations cause constitutive (SH2 binding-independent) activation and that cancer-associated mutants are more active than those associated with Noonan syndrome. We have characterized a larger panel of Shp2 mutants and find that this "activity-centric" model cannot explain the behaviors of all pathogenic Shp2 mutations. Instead, enzymatic, structural, and mathematical modeling analyses show that these mutants can affect basal activation, SH2 domain-phosphopeptide affinity, and/or substrate specificity to varying degrees. Furthermore, there is no absolute correlation between the mutants' extents of basal activation and the diseases they induce. We propose that activated mutants of Shp2 modulate signaling from specific stimuli to a subset of effectors and provide a theoretical framework for understanding the complex relationship between Shp2 activation, intracellular signaling, and pathology.     

PTPN11 (Shp2) mutations in LEOPARD syndrome have dominant negative, not activating, effects

Multiple lentigines/LEOPARD syndrome (LS) is a rare, autosomal dominant disorder characterized by Lentigines, Electrocardiogram abnormalities, Ocular hypertelorism, Pulmonic valvular stenosis, Abnormalities of genitalia, Retardation of growth, and Deafness. Like the more common Noonan syndrome (NS), LS is caused by germ line missense mutations in PTPN11, encoding the protein-tyrosine phosphatase Shp2. Enzymologic, structural, cell biological, and mouse genetic studies indicate that NS is caused by gain-of-function PTPN11 mutations. Because NS and LS share several features, LS has been viewed as an NS variant. We examined a panel of LS mutants, including the two most common alleles. Surprisingly, we found that in marked contrast to NS, LS mutants are catalytically defective and act as dominant negative mutations that interfere with growth factor/Erk-mitogen-activated protein kinase-mediated signaling. Molecular modeling and biochemical studies suggest that LS mutations contort the Shp2 catalytic domain and result in open, inactive forms of Shp2. Our results establish that the pathogenesis of LS and NS is distinct and suggest that these disorders should be distinguished by mutational analysis rather than clinical presentation.     

Distinct functions for ERK1 and ERK2 in cell migration processes during zebrafish gastrulation

The MAPKs are key regulatory signaling molecules in many cellular processes. Here we define differential functions for ERK1 and ERK2 MAPKs in zebrafish embryogenesis. Morpholino knockdown of ERK1 and ERK2 resulted in cell migration defects during gastrulation, which could be rescued by co-injection of the corresponding mRNA. Strikingly, Erk2 mRNA cross-rescued ERK1 knockdown, but erk1 mRNA was unable to compensate for ERK2 knockdown. Cell-tracing experiments revealed a convergence defect for ERK1 morphants without a severe posterior-extension defect, whereas ERK2 morphants showed a more severe reduction in anterior-posterior extension. These defects were primary changes in gastrulation cell movements and not caused by altered cell fate specification. Saturating knockdown conditions showed that the absence of FGF-mediated dual-phosphorylated ERK2 from the blastula margin blocked initiation of epiboly, actin and tubulin cytoskeleton reorganization processes and further arrested embryogenesis, whereas ERK1 knockdown had only a mild effect on epiboly progression. Together, our data define distinct roles for ERK1 and ERK2 in developmental cell migration processes during zebrafish embryogenesis.     

Fyn/Yes and non-canonical Wnt signalling converge on RhoA in vertebrate gastrulation cell movements

Convergent extension (CE) cell movements during gastrulation mediate extension of the anterior-posterior body axis of vertebrate embryos. Non-canonical Wnt5 and Wnt11 signalling is essential for normal CE movements in vertebrate gastrulation. Here, we show that morpholino (MO)-mediated double knock-down of the Fyn and Yes tyrosine kinases in zebrafish embryos impaired normal CE cell movements, resembling the silberblick and pipetail mutants, caused by mutations in wnt11 and wnt5, respectively. Co-injection of Fyn/Yes- and Wnt11- or Wnt5-MO was synergistic, but wnt11 or wnt5 RNA did not rescue the Fyn/Yes knockdown or vice versa. Remarkably, active RhoA rescued the Fyn/Yes knockdown as well as the Wnt11 knockdown, indicating that Fyn/Yes and Wnt11 signalling converged on RhoA. Our results show that Fyn and Yes act together with non-canonical Wnt signalling via RhoA in CE cell movements during gastrulation.     

Role of lbx2 in the noncanonical Wnt signaling pathway for convergence and extension movements and hypaxial myogenesis in zebrafish

It has been suggested that mouse lbx1 is essential for directing hypaxial myogenic precursor cell migration. In zebrafish, the expression of lbx1a, lbx1b, and lbx2 has been observed in pectoral fin buds. It has also been shown that knocking down endogenous lbx2 in zebrafish embryos diminishes myoD expression in the pectoral fin bud. However, downstream lbxs signals remain largely unexplored. Here, we describe a previously unknown function of zebrafish lbx2 (lbx2) during convergent extension (CE) movements. The abrogation of the lbx2 function by two non-overlapping morpholino oligonucleotides (MOs) resulted in the defective convergence and extension movements in morphants during gastrulation. Our transplantation studies further demonstrated that the overexpression of lbx2 autonomously promotes CE movements. Expression of wnt5b is significantly reduced in lbx2 morphants. We have demonstrated that application of the wnt5b MO, a dominant-negative form of disheveled (Dvl) and a chemical inhibitor of Rho-associated kinase Y27632 in zebrafish embryos have effects reminiscent that are of the CE and hypaxial myogenesis defects observed in lbx2 morphants. Moreover, the CE and hypaxial mesoderm defects seen in lbx2 morphants can be rescued by co-injection with wnt5b or RhoA mRNA. However, this reduced level of active RhoA and hypaxial myogenesis defects in the embryos injected with the dominant-negative form of Dvl mRNA cannot be effectively restored by co-injection with lbx2 mRNA. Our results suggest that the key noncanonical Wnt signaling components Wnt5, Dvl, and RhoA are downstream effectors involved in the regulative roles of lbx2 in CE movement and hypaxial myogenesis during zebrafish embryogenesis.     

Essential role for Csk upstream of Fyn and Yes in zebrafish gastrulation

Morphogenetic cell movements during gastrulation shape the vertebrate embryo bodyplan. Non-canonical Wnt signaling has been established to regulate convergence and extension cell movements that mediate anterior-posterior axis elongation. In recent years, many other factors have been implicated in the process by modulation of non-canonical Wnt signaling or by different, unknown mechanisms. We have found that the Src family kinases, Fyn and Yes, are required for normal convergence and extension cell movements in zebrafish embryonic development and they signal in parallel to non-canonical Wnts, eventually converging on a common downstream factor, RhoA. Here, we report that Csk, a negative regulator of Src family kinases has a role in gastrulation cell movements as well. Csk knock down induced a phenotype that was similar to the defects observed after knock down of Fyn and Yes, in that gastrulation cell movements were impaired, without affecting cell fate. The Csk knock down phenotype was rescued by simultaneous partial knock down of Fyn and Yes. We conclude that Csk acts upstream of Fyn and Yes to control vertebrate gastrulation cell movements.     

Pair-wise regulation of convergence and extension cell movements by four phosphatases via RhoA

Various signaling pathways regulate shaping of the main body axis during early vertebrate development. Here, we focused on the role of protein-tyrosine phosphatase signaling in convergence and extension cell movements. We identified Ptpn20 as a structural paralogue of PTP-BL and both phosphatases were required for normal gastrulation cell movements. Interestingly, knockdowns of PTP-BL and Ptpn20 evoked similar developmental defects as knockdown of RPTPα and PTPε. Co-knockdown of RPTPα and PTP-BL, but not Ptpn20, had synergistic effects and conversely, PTPε and Ptpn20, but not PTP-BL, cooperated, demonstrating the specificity of our approach. RPTPα and PTPε knockdowns were rescued by constitutively active RhoA, whereas PTP-BL and Ptpn20 knockdowns were rescued by dominant negative RhoA. Consistently, RPTPα and PTP-BL had opposite effects on RhoA activation, both in a PTP-dependent manner. Downstream of the PTPs, we identified NGEF and Arhgap29, regulating RhoA activation and inactivation, respectively, in convergence and extension cell movements. We propose a model in which two phosphatases activate RhoA and two phosphatases inhibit RhoA, resulting in proper cell polarization and normal convergence and extension cell movements.     

Has2 is required upstream of Rac1 to govern dorsal migration of lateral cells during zebrafish gastrulation

The large extracellular polysaccharide Hyaluronan (HA) and its synthesizing enzymes (Has) have been implicated in regulating the migratory potential of metastatic cancer cells. Here, we analyze the roles of zebrafish Has2 in normal development. Antisense morpholino oligonucleotide (MO)-mediated knockdown of zebrafish Has2 leads to the loss of HA, and severe migratory defects during gastrulation, somite morphogenesis and primordial germ cell migration. During gastrulation, ventrolateral cells of has2 morphant embryos fail to develop lamellipodia and to migrate dorsally, resulting in a blockage of dorsal convergence, whereas extension of the dorsal axis is normal. The effect is cell autonomous, suggesting that HA acts as an autocrine signal to stimulate the migration of HA-generating cells. Upon ectopic expression in axial cells, has2 causes the formation of supernumerary lamellipodia and a blockage of axis extension. Epistasis analyses with constitutively active and dominant-negative versions of the small GTPase Rac1 suggest that HA acts by Rac1 activation, rather than as an essential structural component of the extracellular matrix. Together, our data provide evidence that convergence and extension are separate morphogenetic movements of gastrulation. In addition, they suggest that the same HA pathways are active to auto-stimulate cell migration during tumor invasion and vertebrate embryogenesis.     

The role of Ppt/Wnt5 in regulating cell shape and movement during zebrafish gastrulation

Wnt genes play important roles in regulating patterning and morphogenesis during vertebrate gastrulation. In zebrafish, slb/wnt11 is required for convergence and extension movements, but not cell fate specification during gastrulation. To determine if other Wnt genes functionally interact with slb/wnt11, we analysed the role of ppt/wnt5 during zebrafish gastrulation. ppt/wnt5 is maternally provided and zygotically expressed at all stages during gastrulation. The analysis of ppt mutant embryos reveals that Ppt/Wnt5 regulates cell elongation and convergent extension movements in posterior regions of the gastrula, while its function in more anterior regions is largely redundant to that of Slb/Wnt11. Frizzled-2 functions downstream of ppt/wnt5, indicating that it might act as a receptor for Ppt/Wnt5 in this process. The characterisation of the role of Ppt/Wnt5 provides insight into the functional diversity of Wnt genes in regulating vertebrate gastrulation movements.     

New Approaches to Prevent LEOPARD Syndrome-associated Cardiac Hypertrophy by Specifically Targeting Shp2-dependent Signaling

In LEOPARD syndrome (LS) patients, mutations in the protein tyrosine phosphatase Shp2 cause hypertrophic cardiomyopathy. The prohypertrophic effects of mutant Shp2 are mediated downstream by hyperactivation of mammalian target of rapamycin. Our goal was to further define the signaling cascade that is essential for the underlying pathomechanism, thus expanding the list of potential future therapeutic targets.Using cultured neonatal rat cardiomyocytes with adenoviral gene delivery and pharmacological inhibitors, we found that hypertrophy induced by a particularly aggressive LS mutation in Shp2 depends on hyperactivation of Akt and focal adhesion kinase as well as mammalian target of rapamycin. Dissecting domain-specific functions of Shp2 using double and truncation mutants, we determined that the hypertrophic effects of mutant Shp2 depend on the two SH2 domains and on an intact catalytic center. The latter finding prompted us to test the efficacy of a Shp2 inhibitor targeted directly at the catalytic pocket. This compound, PHPS1, effectively prevented mutant Shp2-induced hypertrophy. In summary, we identified three novel targets for pharmacological therapy of LS-associated cardiac hypertrophy. Of particular importance is the finding that intervention directly at the mutant Shp2 protein is effective because this would facilitate custom-tailored therapeutic approaches for patients carrying LS mutations in Shp2.     

Comparative phosphoproteomics of zebrafish Fyn/Yes morpholino knockdown embryos

The coordinated movement of cells is indispensable for normal vertebrate gastrulation. Several important players and signaling pathways have been identified in convergence and extension (CE) cell movements during gastrulation, including non-canonical Wnt signaling. Fyn and Yes, members of the Src family of kinases, are key regulators of CE movements as well. Here we investigated signaling pathways in early development by comparison of the phosphoproteome of wild type zebrafish embryos with Fyn/Yes knockdown embryos that display specific CE cell movement defects. For quantitation we used differential stable isotope labeling by reductive amination of peptides. Equal amounts of labeled peptides from wild type and Fyn/Yes knockdown embryos were mixed and analyzed by on-line reversed phase TiO(2)-reversed phase LC-MS/MS. Phosphorylated and non-phosphorylated peptides were quantified, and significant changes in protein expression and/or phosphorylation were detected. We identified 348 phosphoproteins of which 69 showed a decrease in phosphorylation in Fyn/Yes knockdown embryos and 72 showed an increase in phosphorylation. Among these phosphoproteins were known regulators of cell movements, including Adducin and PDLIM5. Our results indicate that quantitative phosphoproteomics combined with morpholino-mediated knockdowns can be used to identify novel signaling pathways that act in zebrafish development in vivo.     

Ptenb mediates gastrulation cell movements via Cdc42/AKT1 in zebrafish

Phosphatidylinositol 3-kinase (PI3 kinase) mediates gastrulation cell migration in zebrafish via its regulation of PIP(2)/PIP(3) balance. Although PI3 kinase counter enzyme PTEN has also been reported to be essential for gastrulation, its role in zebrafish gastrulation has been controversial due to the lack of gastrulation defects in pten-null mutants. To clarify this issue, we knocked down a pten isoform, ptenb by using anti-sense morpholino oligos (MOs) in zebrafish embryos and found that ptenb MOs inhibit convergent extension by affecting cell motility and protrusion during gastrulation. The ptenb MO-induced convergence defect could be rescued by a PI3-kinase inhibitor, LY294002 and by overexpressing dominant negative Cdc42. Overexpression of human constitutively active akt1 showed similar convergent extension defects in zebrafish embryos. We also observed a clear enhancement of actin polymerization in ptenb morphants under cofocal microscopy and in actin polymerization assay. These results suggest that Ptenb by antagonizing PI3 kinase and its downstream Akt1 and Cdc42 to regulate actin polymerization that is critical for proper cell motility and migration control during gastrulation in zebrafish.