ANR5, an FGF target gene product, regulates gastrulation in Xenopus

Gastrulation is a morphogenetic process in which tightly coordinated cell and tissue movements establish the three germ layers (ectoderm, mesoderm, and endoderm) to define the anterior-to-posterior embryonic organization [1]. To elicit this movement, cells modulate membrane protrusions and undergo dynamic cell interactions. Here we report that ankyrin repeats domain protein 5 (xANR5), a novel FGF target gene product, regulates cell-protrusion formation and tissue separation, a process that develops the boundary between the ectoderm and mesoderm [2, 3], during Xenopus gastrulation. Loss of xANR5 function by antisense morpholino oligonucleotide (MO) caused a short trunk and spina bifida without affecting mesodermal gene expressions. xANR5-MO also blocked elongation of activin-treated animal caps (ACs) and tissue separation. The dorsal cells of xANR5-MO-injected embryos exhibited markedly reduced membrane protrusions, which could be restored by coinjecting active Rho. Active Rho also rescued the xANR5-MO-inhibited tissue separation. We further demonstrated that xANR5 interacted physically and functionally with paraxial protocadherin (PAPC), which has known functions in cell-sorting behavior, tissue separation, and gastrulation cell movements [4-6], to regulate early morphogenesis. Our findings reveal for the first time that xANR5 acts through Rho to regulate gastrulation and is an important cytoplasmic partner of PAPC, whose cytoplasmic partner was previously unknown.     

Phosphorylation-Dependent Ubiquitination of Paraxial Protocadherin (PAPC) Controls Gastrulation Cell Movements

Paraxial protocadherin (PAPC) has been shown to be involved in gastrulation cell movements during early embryogenesis. It is first expressed in the dorsal marginal zone at the early gastrula stage and subsequently restricted to the paraxial mesoderm in Xenopus and zebrafish. Using Xenopus embryos, we found that PAPC is also regulated at the protein level and is degraded and excluded from the plasma membrane in the axial mesoderm by the late gastrula stage. Regulation of PAPC requires poly-ubiquitination that is dependent on phosphorylation. PAPC is phosphorylated by GKS3 in the evolutionarily conserved cytoplasmic domain, and this in turn is necessary for poly-ubiquitination by an E3 ubiquitin ligase β-TrCP. We also show that precise control of PAPC by phosphorylation/ubiquitination is essential for normal Xenopus gastrulation cell movements. Taken together, our findings unveil a novel mechanism of regulation of a cell adhesion protein and show that this system plays a crucial role in vertebrate embryogenesis.     

The protocadherin PAPC establishes segmental boundaries during somitogenesis in xenopus embryos

BACKGROUND: One prominent example of segmentation in vertebrate embryos is the subdivision of the paraxial mesoderm into repeating, metameric structures called somites. During this process, cells in the presomitic mesoderm (PSM) are first patterned into segments leading secondarily to differences required for somite morphogenesis such as the formation of segmental boundaries. Recent studies have shown that a segmental pattern is generated in the PSM of Xenopus embryos by genes encoding a Mesp-like bHLH protein called Thylacine 1 and components of the Notch signaling pathway. These genes establish a repeating pattern of gene expression that subdivides cells in the PSM into anterior and posterior half segments, but how this pattern of gene expression leads to segmental boundaries is unknown. Recently, a member of the protocadherin family of cell adhesion molecules, called PAPC, has been shown to be expressed in the PSM of Xenopus embryos in a half segment pattern, suggesting that it could play a role in restricting cell mixing at the anterior segmental boundary. RESULTS: Here, we examine the expression and function of PAPC during segmentation of the paraxial mesoderm in Xenopus embryos. We show that Thylacine 1 and the Notch pathway establish segment identity one segment prior to the segmental expression of PAPC. Altering segmental identity in embryos by perturbing the activity of Thylacine 1 and the Notch pathway, or by treatment with a protein synthesis inhibitor, cycloheximide, leads to the predicted changes in the segmental expression of PAPC. By disrupting PAPC function in embryos using a putative dominant-negative or an activated form of PAPC, we show that segmental PAPC activity is required for proper somite formation as well as for maintaining segmental gene expression within the PSM. CONCLUSIONS: Segmental expression of PAPC is established in the PSM as a downstream consequence of segmental patterning by Thylacine 1 and the Notch pathway. We propose that PAPC is part of the mechanism that establishes the segmental boundaries between posterior and anterior cells in adjacent segments.     

Paraxial protocadherin mediates cell sorting and tissue morphogenesis by regulating C-cadherin adhesion activity

Little is known about how protocadherins function in cell adhesion and tissue development. Paraxial protocadherin (PAPC) controls cell sorting and morphogenetic movements in the Xenopus laevis embryo. We find that PAPC mediates these functions by down-regulating the adhesion activity of C-cadherin. Expression of exogenous C-cadherin reverses PAPC-induced cell sorting and gastrulation defects. Moreover, loss of endogenous PAPC results in elevated C-cadherin adhesion activity in the dorsal mesoderm and interferes with the normal blastopore closure, a defect that can be rescued by a dominant-negative C-cadherin mutant. Importantly, activin induces PAPC expression, and PAPC is required for activin-induced regulation of C-cadherin adhesion activity and explant morphogenesis. Signaling through Frizzled-7 is not required for PAPC regulation of C-cadherin, suggesting that C-cadherin regulation and Frizzled-7 signaling are two distinct branches of the PAPC pathway that induce morphogenetic movements. Thus, spatial regulation of classical cadherin adhesive function by local expression of a protocadherin is a novel mechanism for controlling cell sorting and tissue morphogenesis.     

The type II activin receptors are essential for egg cylinder growth, gastrulation, and rostral head development in mice.

The type II activin receptors, ActRIIA and ActRIIB, have been shown to play critical roles in axial patterning and organ development in mice. To investigate whether their function is required for mesoderm formation and gastrulation as implicated in Xenopus studies, we generated mice carrying both receptor mutations by interbreeding the ActRIIA and ActRIIB knockout mutants. We found that embryos homozygous for both receptor mutations were growth arrested at the egg cylinder stage and did not form mesoderm. Further analyses revealed that ActRIIA(-/-)ActRIIB(+/-) and about 15% of the ActRIIA(-/-) embryos failed to form an elongated primitive streak, resulting in severe disruption of mesoderm formation in the embryo proper. Interestingly, we observed similar gastrulation defects in ActRIIA(-/-)nodal(+/-) double mutants, which, if they developed beyond the gastrulation stage, displayed rostral head defects and cyclopia. These results provide genetic evidence that type II activin receptors are required for egg cylinder growth, primitive streak formation, and rostral head development in mice.     

Mouse paraxial protocadherin is expressed in trunk mesoderm and is not essential for mouse development

Paraxial protocadherin (PAPC) is a cell adhesion molecule that marks cells undergoing convergence-extension cell movements in Xenopus and zebrafish gastrulating embryos. Here a mouse homologue (mpapc) was identified and characterized. During early- to mid-gastrulation, mpapc is expressed in the primitive streak as the trunk mesoderm undergoes morphogenetic cell movements. At head-fold stage mpapc expression becomes localized to paraxial regions in which somites are formed in the segmental plate. At later stages, mpapc displays a complex expression pattern in cerebral cortex, olfactory bulb, inferior colliculus, and in longitudinal stripes in hindbrain. To analyze the effect of the loss of PAPC function during mouse development, a null allele of the mouse papc gene was generated. Homozygous animals show no defects in their skeleton and are viable and fertile.     

PI3K and Erk MAPK mediate ErbB signaling in Xenopus gastrulation

ErbB signaling regulates cell adhesion and movements during Xenopus gastrulation, but the downstream pathways involved have not been elucidated. In this study, we show that phosphatidylinositol-3 kinase (PI3K) and Erk mitogen-activated protein kinase (MAPK) mediate ErbB signaling to regulate gastrulation. Both PI3K and MAPK function sequentially in mesoderm specification and movements, and ErbB signaling is important only for the late phase activation of these pathways to control cell behaviors. Activation of either PI3K or Erk MAPK rescues gastrulation defects in ErbB4 morphant embryos, and restores convergent extension in the trunk mesoderm as well as coherent cell migration in the head mesoderm. The two signals preferentially regulate different aspects of cell behaviors, with PI3K more efficient in rescuing cell adhesion and spreading and MAPK more effective in stimulating the formation of filopodia. PI3K and MAPK also weakly activate each other, and together they modulate gastrulation movements. Our results reveal that PI3K and Erk MAPK, which have previously been considered as mesodermal inducing signals, also act downstream of ErbB signaling to participate in regulation of gastrulation morphogenesis.     

Xenopus paraxial protocadherin inhibits Wnt/β-catenin signalling via casein kinase 2β

Xenopus paraxial protocadherin (PAPC) regulates cadherin-mediated cell adhesion and promotes the planar cell polarity (PCP) pathway. Here we report that PAPC functions in the Xenopus gastrula as an inhibitor of the Wnt/β-catenin pathway. The intracellular domain of PAPC interacts with casein kinase 2 beta (CK2β), which is part of the CK2 holoenzyme. The CK2α/β complex stimulates Wnt/β-catenin signalling, and the physical interaction of CK2β with PAPC antagonizes this activity. By this mechanism, PAPC restricts the expression of Wnt target genes during gastrulation. These experiments identify a novel function of protocadherins as regulators of the Wnt pathway.     

PCNS: a novel protocadherin required for cranial neural crest migration and somite morphogenesis in Xenopus

Protocadherins (Pcdhs), a major subfamily of cadherins, play an important role in specific intercellular interactions in development. These molecules are characterized by their unique extracellular domain (EC) with more than 5 cadherin-like repeats, a transmembrane domain (TM) and a variable cytoplasmic domain. PCNS (Protocadherin in Neural crest and Somites), a novel Pcdh in Xenopus, is initially expressed in the mesoderm during gastrulation, followed by expression in the cranial neural crest (CNC) and somites. PCNS has 65% amino acid identity to Xenopus paraxial protocadherin (PAPC) and 42-49% amino acid identity to Pcdh 8 in human, mouse, and zebrafish genomes. Overexpression of PCNS resulted in gastrulation failure but conferred little if any specific adhesion on ectodermal cells. Loss of function accomplished independently with two non-overlapping antisense morpholino oligonucleotides resulted in failure of CNC migration, leading to severe defects in the craniofacial skeleton. Somites and axial muscles also failed to undergo normal morphogenesis in these embryos. Thus, PCNS has essential functions in these two important developmental processes in Xenopus.     

Role of glypican 4 in the regulation of convergent extension movements during gastrulation in Xenopus laevis

Coordinated morphogenetic cell movements during gastrulation are crucial for establishing embryonic axes in animals. Most recently, the non-canonical Wnt signaling cascade (PCP pathway) has been shown to regulate convergent extension movements in Xenopus and zebrafish. Heparan sulfate proteoglycans (HSPGs) are known as modulators of intercellular signaling, and are required for gastrulation movements in vertebrates. However, the function of HSPGs is poorly understood. We analyze the function of Xenopus glypican 4 (Xgly4), which is a member of membrane-associated HSPG family. In situ hybridization revealed that Xgly4 is expressed in the dorsal mesoderm and ectoderm during gastrulation. Reducing the levels of Xgly4 inhibits cell-membrane accumulation of Dishevelled (Dsh), which is a transducer of the Wnt signaling cascade, and thereby disturbs cell movements during gastrulation. Rescue analysis with different Dsh mutants and Wnt11 demonstrated that Xgly4 functions in the non-canonical Wnt/PCP pathway, but not in the canonical Wnt/beta-catenin pathway, to regulate gastrulation movements. We also provide evidence that the Xgly4 protein physically binds Wnt ligands. Therefore, our results suggest that Xgly4 functions as positive regulator in non-canonical Wnt/PCP signaling during gastrulation.     

Mesoderm-independent regulation of gastrulation movements by the Src tyrosine kinase in Xenopus embryo

In vitro studies have demonstrated the involvement of Src kinases in several aspects of cell scattering, including cell dissociation and motility. We have therefore sought to explore their functions in the context of the whole organism. Loss-of-function microinjection studies indicate that the ubiquitous Src, Fyn, and Yes tyrosine kinases are specifically implicated in Xenopus gastrulation movements. Injection of mRNAs coding for dominant negative forms of the ubiquitous members of the Src family, namely Fyn, Src, and Yes, perturbs gastrulation movements, resulting in the inability to close the blastopore. Injection of mRNA coding for Csk, a natural inhibitor of Src kinase activity, produces the same phenotypic alterations. The ubiquitous Src kinases have redundant functions in gastrulation movements since overexpression of one member of the family can compensate for the inhibition of another. Interfering mutants of the Src family also inhibit activin-induced morphogenetic movements of animal cap explants isolated from injected embryos. In contrast, these mutants do not interfere with mesoderm induction, as inferred from the presence of mesoderm derivatives and from the expression of early mesodermal markers in injected embryos. In addition, Src kinase activity measured by an in vitro kinase assay is elevated in gastrulating embryos and in FGF- and activin-treated animal caps, confirming the implication of Src enzymatic activity during gastrulation. Altogether, our results demonstrate that Src kinases are essential components of the machinery that drives gastrulation movements independent of mesoderm induction and suggest that Src activity is primarily implicated in cellular movements that take place during the process of cell intercalation.     

非洲爪蟾PAPC多克隆抗体的制备及其特异性鉴定

非洲爪蟾ParaxialProtocadherin(PAPC)是一个在爪蟾Spemann组织者特异表达的膜蛋白.它在爪蟾原肠运动阶段的汇聚延伸运动和体节发生阶段的体节边界形成,以及早期听泡的形态发生和细胞特化过程中都有重要的作用.为了研究PAPC基因在早期胚胎发育过程中的表达及其生物学功能,需要制备PAPC抗体.应用谷胱甘肽S-转移酶(glutathioneStransferase,GST)表达系统表达GST-PAPC融合蛋白,亲和纯化后用以免疫新西兰大白兔,获得PAPC多克隆抗体.免疫印迹分析发现,以1∶3000稀释的该多克隆抗体为一抗时,能够在转染了全长PAPC质粒的HEK293T细胞的蛋白质抽提物中,特异地识别出150ku的印迹条带.同时,GST-PAPC融合蛋白可以竞争性抑制该抗体对全长PAPC质粒转染细胞的蛋白质抽提物的特异性条带.用1∶500稀释的该抗体为一抗进行免疫荧光分析时,发现,PAPC多克隆抗体能够识别在HEK293T细胞中过表达以及爪蟾动物极细胞中过表达的PAPC蛋白,荧光信号定位在细胞膜上.免疫印迹分析证明,PAPC抗体能够识别爪蟾胚胎中内源表达的PAPC蛋白.     

The cerberus-related gene, Cerr1, is not essential for mouse head formation

The Xenopus cerberus gene encodes a secreted factor expressed in the Spemann organizer that can cause ectopic head formation when its mRNA is injected into Xenopus embryos. In mouse, the cerberus-related gene, Cerr1, is expressed in the anterior mesendoderm that underlies the presumptive anterior neural plate and its expression is downregulated in Lim1 headless embryos. To determine whether Cerr1 is required for head formation we generated a null mutation in Cerr1 by gene targeting in mouse embryonic stem cells. We found that head formation is normal in Cerr1(-/-) embryos and we detected no obvious phenotypic defects in adult Cerr1(-/-) mice. However, in embryonic tissue layer recombination assays, Cerr1(-/-) presomitic/somitic mesoderm, unlike Cerr1-expressing wild-type presomitic/somitic mesoderm, was unable to maintain expression of the anterior neural marker gene Otx2 in ectoderm explants. These findings suggest that establishment of anterior identity in the mouse may involve the action of multiple functionally redundant factors.     

FGF signal interpretation is directed by Sprouty and Spred proteins during mesoderm formation

Vertebrate gastrulation requires coordination of mesoderm specification with morphogenetic movements. While both of these processes require FGF signaling, it is not known how mesoderm specification and cell movements are coordinated during gastrulation. The related Sprouty and Spred protein families are recently discovered regulators of receptor tyrosine kinase signaling. We identified two genes for each family in Xenopus tropicalis: Xtsprouty1, Xtsprouty2, Xtspred1, and Xtspred2. In gain- and loss-of-function experiments we show that XtSprouty and XtSpred proteins modulate different signaling pathways downstream of the FGF receptor (FGFR), and consequently different developmental processes. Notably, XtSproutys inhibit morphogenesis and Ca(2+) and PKCdelta signaling, leaving MAPK activation and mesoderm specification intact. In contrast, XtSpreds inhibit MAPK activation and mesoderm specification, with little effect on Ca(2+) or PKCdelta signaling. These differences, combined with the timing of their developmental expression, suggest a mechanism to switch FGFR signal interpretation to coordinate mesoderm formation and cell movements during gastrulation.     

The cytoplasmic tyrosine kinase Arg regulates gastrulation via control of actin organization

Coordinated cell movements are crucial for vertebrate gastrulation and are controlled by multiple signals. Although many factors are shown to mediate non-canonical Wnt pathways to regulate cell polarity and intercalation during gastrulation, signaling molecules acting in other pathways are less investigated and the connections between various signals and cytoskeleton are not well understood. In this study, we show that the cytoplasmic tyrosine kinase Arg modulates gastrulation movements through control of actin remodeling. Arg is expressed in the dorsal mesoderm at the onset of gastrulation, and both gain- and loss-of-function of Arg disrupted axial development in Xenopus embryos. Arg controlled migration of anterior mesendoderm, influenced cell decision on individual versus collective migration, and modulated spreading and protrusive activities of anterior mesendodermal cells. Arg also regulated convergent extension of the trunk mesoderm by influencing cell intercalation behaviors. Arg modulated actin organization to control dynamic F-actin distribution at the cell-cell contact or in membrane protrusions. The functions of Arg required an intact tyrosine kinase domain but not the actin-binding motifs in its carboxyl terminus. Arg acted downstream of receptor tyrosine kinases to regulate phosphorylation of endogenous CrkII and paxillin, adaptor proteins involved in activation of Rho family GTPases and actin reorganization. Our data demonstrate that Arg is a crucial cytoplasmic signaling molecule that controls dynamic actin remodeling and mesodermal cell behaviors during Xenopus gastrulation.     

Inhibition of the cell cycle is required for convergent extension of the paraxial mesoderm during Xenopus neurulation

Coordination of morphogenesis and cell proliferation is essential during development. In Xenopus, cell divisions are rapid and synchronous early in development but then slow and become spatially restricted during gastrulation and neurulation. One tissue that transiently stops dividing is the paraxial mesoderm, a dynamically mobile tissue that forms the somites and body musculature of the embryo. We have found that cessation of cell proliferation is required for the proper positioning and segmentation of the paraxial mesoderm as well as the complete elongation of the Xenopus embryo. Instrumental in this cell cycle arrest is Wee2, a Cdk inhibitory kinase that is expressed in the paraxial mesoderm from mid-gastrula stages onwards. Morpholino-mediated depletion of Wee2 increases the mitotic index of the paraxial mesoderm and this results in the failure of convergent extension and somitogenesis in this tissue. Similar defects are observed if the cell cycle is inappropriately advanced by other mechanisms. Thus, the low mitotic index of the paraxial mesoderm plays an essential function in the integrated cell movements and patterning of this tissue.     

Xenopus paraxial protocadherin has signaling functions and is involved in tissue separation

Protocadherins have homophilic adhesion properties and mediate selective cell-cell adhesion and cell sorting. Knockdown of paraxial protocadherin (PAPC) function in the Xenopus embryo impairs tissue separation, a process that regulates separation of cells of ectodermal and mesodermal origin during gastrulation. We show that PAPC can modulate the activity of the Rho GTPase and c-jun N-terminal kinase, two regulators of the cytoskeletal architecture and effectors of the planar cell polarity pathway. This novel signaling function of PAPC is essential for the regulation of tissue separation. In addition, PAPC can interact with the Xenopus Frizzled 7 receptor, and both proteins contribute to the development of separation behavior by activating Rho and protein kinase Calpha.     

Characterization of sFRP2‐like in amphioxus: insights into the evolutionary conservation of Wnt antagonizing function

Wnt signaling plays a key role in embryonic patterning and morphogenetic movements. The secreted Frizzled‐related proteins (sFRPs) antagonize Wnt signaling, but their roles in development are poorly understood. To determine whether function of sFRPs is conserved between amphioxus and vertebrates, we characterized sFRP2‐like function in the amphioxus, Branchiostoma belcheri tsingtauense (B. belcheri). As in other species of Branchiostome, in B. belcheri, expression of sFRP2‐like is restricted to the mesendoderm during gastrulation and to the anterior mesoderm and endoderm during neurulation. Functional analyses in frog (Xenopus laevis) indicate that amphioxus sFRP2‐like potently inhibits both canonical and non‐canonical Wnts. Thus, sFRP‐2 probably functions in amphioxus embryos to inhibit Wnt signaling anteriorly. Moreover, dorsal overexpression of amphioxus sFRP2‐like in Xenopus embryos, like inhibition of Wnt11, blocks gastrulation movements. This implies that sFRP2‐like may also modulate Wnt signaling during gastrulation movements in amphioxus.