Roles of Xenopus chemokine ligand CXCLh (XCXCLh) in early embryogenesis

Several chemokine molecules control cell movements during early morphogenesis. However, it is unclear whether chemokine molecules affect cell fate. Here, we identified and characterized the CXC‐type chemokine ligand in Xenopus laevis, Xenopus CXCLh (XCXCLh), during early embryogenesis. XCXCLh is expressed in the dorsal vegetal region at the gastrula stage. Both overexpression and knockdown of XCXCLh in the dorsal region inhibited gastrulation. XCXCLh contributed to the attraction of mesendodermal cells and accelerated the reassembly of scratched culture cells. Also, XCXCLh contributed to early endodermal induction. Overexpression of VegTmRNA or high concentrations of calcium ions induced XCXCLh expression. XCXCLh may play roles in both cell movements and differentiation during early Xenopus embryogenesis.     

The Xenopus platelet-derived growth factor α receptor: cDNA Cloning and demonstration that mesoderm induction establishes the lineage-specific pattern of ligand and receptor gene expression

We have cloned the Xenopus PDGF α receptor cDNA and have used this clone, along with cDNA encoding PDGF A, to examine their expression pattern in Xenopus embryos and to determine the factors responsible for lineage specificity. Recombinant Xenopus α receptor expressed in COS cells exhibits PDGF-A-dependent tyrosine kinase activity. We find that receptor mRNA is present in cultured marginal zone tissue explants and in animal cap tissue induced to form mesoderm either by grafting to vegetal tissue or by treatment with recombinant activin A. In contrast, PDGF A mRNA is expressed in cultured, untreated animal cap tissue and is suppressed by mesoderm induction. These results suggest that ectodermally produced PDGF A may act on the mesoderm during gastrulation and that mesoderm induction establishes the tissue pattern of ligand and receptor expression. © 1993Wiley-Liss, Inc.     

The involvement of Eph-Ephrin signaling in tissue separation and convergence during Xenopus gastrulation movements

In Xenopus gastrulation, the involuting mesodermal and non-involuting ectodermal cells remain separated from each other, undergoing convergent extension. Here, we show that Eph–ephrin signaling is crucial for the tissue separation and convergence during gastrulation. The loss of EphA4 function results in aberrant gastrulation movements, which are due to selective inhibition of tissue constriction and separation. At the cellular levels, knockdown of EphA4 impairs polarization and migratory activity of gastrulating cells but not specification of their fates. Importantly, rescue experiments demonstrate that EphA4 controls tissue separation via RhoA GTPase in parallel to Fz7 and PAPC signaling. In addition, we show that EphA4 and its putative ligand, ephrin-A1 are expressed in a complementary manner in the involuting mesodermal and non-involuting ectodermal layers of early gastrulae, respectively. Depletion of ephrin-A1 also abrogates tissue separation behaviors. Therefore, these results suggest that Eph receptor and its ephrin ligand might mediate repulsive interaction for tissue separation and convergence during early Xenopus gastrulation movements.     

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.     

Evolutionary conservation, developmental expression, and genomic mapping of mammalian Twisted gastrulation

The twisted gastrulation gene (tsg) encodes a secreted protein required for the correct specification of dorsal midline cell fate during gastrulation in Drosophila. We report that tsg homologs from human, mouse, zebrafish, and Xenopus share 72–98% identity at the amino acid level and retain all 24 cysteine residues from Drosophila. In contrast to Drosophila where tsg expression is limited to early embryos, expression is found throughout mouse and human development. In Drosophila, tsg acts in synergy with decapentaplegic (dpp), a member of the TGF-β family of secreted proteins. The vertebrate orthologs of dpp, BMP-2 and -4, are crucial for gastrulation and neural induction, and aberrant signaling by BMPs and other TGF-β family members results in developmental defects including holoprosencephaly (HPE). Interestingly, human TSG maps to the HPE4 locus on Chromosome 18p11.3, and our analysis places the gene within 5 Mbp of TG-interacting factor (TGIF). Received: 21 August 2000 / Accepted: 9 March 2001     

Planar and vertical induction of anteroposterior pattern during the development of the amphibian central nervous system

In amphibians and other vertebrates, neural development is induced in the ectoderm by signals coming from the dorsal mesoderm during gastrulation. Classical embryological results indicated that these signals follow a “vertical” path, from the involuted dorsal mesoderm to the overlying ectoderm. Recent work with the frog Xenopus laevis, however, has revealed the existence of “planar” neural-inducing signals, which pass within the continuous sheet or plane of tissue formed by the dorsal mesoderm and presumptive neurectoderm. Much of this work has made use of Keller explants, in which dorsal mesoderm and ectoderm are cultured in a planar configuration with contact along only a single edge, and vertical contact is prevented. Planar signals can induce the full anteroposterior (A-P) extent of neural pattern, as evidenced in Keller explants by the expression of genes that mark specific positions along the A-P axis. In this review, classical and modern molecular work on vertical and planar inductionwill be discussed. This will be followed by a discussion of various models for vertical induction and planar induction. It has been proposed that the A-P pattern in the nervous system is derived from a parallel pattern of inducers in the dorsal mesoderm which is “imprinted” vertically onto the overlying ectoderm. Since it is now known that planar signals can also induce A-P neural pattern, this kind of model must be reassessed. The study of planar induction of A-P pattern in Xenopus embryos provides a simple, manipulable, two-dimensional system in which to investigate pattern formation. © 1993 John Wiley & Sons, Inc.     

Dorsoventral Differences in Cell-Cell Interactions Modulate the Motile Behaviour of Cells from the Xenopus Gastrula

When groups of cells from the inner marginal zone (mesendoderm) of the early Xenopus gastrula are placed on a fibronectin-coated substratum, the explants of the dorsal region spread into monolayers whereas those from the ventral region, though they adhere to the substratum, do not show this spreading reaction. This different behaviour is not reflected in the in vitro behaviour of the respective cells kept in isolation. No difference between dorsal and ventral cells was observed, when they were tested for lamellipodia-driven spreading, movement over the substratum or properties of integrin- and cadherin-mediated adhesion. However, cell contacts between individual dorsal cells are significantly less stable than those between ventral cells. The higher flexibility of the cell-cell contacts seems to determine the spreading behaviour of the dorsal explants, which includes lamellipodia-driven outward movement of the peripheral cells, rearrangements of the cells, building up a horizontal tension within the aggregate and intercalation of cells from above into the bottom layer. Ventral explants lack these properties. Staining for F-actin revealed a decisive difference of the supracellular organisation of the cytoskeleton that underlies the morphology of the different types of explants. Evidence for a higher flexibility of cell-cell contacts in the dorsal mesendoderm was also obtained in SEM studies on gastrulating embryos. Dorsal mesendodermal cells show stronger protrusive activity as compared to ventral mesendodermal cells. The meaning of these observations for the mechanisms of morphogenetic movements during gastrulation is central to the discussion.     

Cdc2-like kinase 2 (Clk2) promotes early neural development in Xenopus embryos

Neural induction and patterning in vertebrates are regulated during early development by several morphogens, such as bone morphogenetic proteins (BMPs) and fibroblast growth factors (FGFs). Ventral ectoderm differentiates into epidermis in response to BMPs, whereas BMP signaling is tightly inhibited in the dorsal ectoderm which develops into neural tissues. Here, we show that Cdc2-like kinase 2 (Clk2) promotes early neural development and inhibits epidermis differentiation in Xenopus embryos. clk2 is specifically expressed in neural tissues along the anterior-posterior axis during early Xenopus embryogenesis. When overexpressed in ectodermal explants, Clk2 induces the expression of both anterior and posterior neural marker genes. In agreement with this observation, overexpression of Clk2 in whole embryos expands the neural plate at the expense of epidermal ectoderm. Interestingly, the neural-inducing activity of Clk2 is increased following BMP inhibition and activation of the FGF signaling pathway in ectodermal explants. Clk2 also downregulates the level of p-Smad1/5/8 in cooperation with BMP inhibition, in addition to increasing the level of activated MAPK together with FGF. These results suggest that Clk2 plays a role in early neural development of Xenopus possibly via modulation of morphogen signals such as the BMP and FGF pathways.     

Reprogramming of HeLa cells upon DNMT3L overexpression mimics carcinogenesis

Previously we had discovered loss of DNA methylation at the DNMT3L promoter, an enzymatically-inactive DNA methyltransferase, in squamous cell carcinoma of cervix indicating association between cancer and DNMT3L. This study extends this correlation further by identifying the role of DNMT3L in nuclear reprogramming, an event central to the process of carcinogenesis. We show that in cervical cancer cell lines, overexpression of DNMT3L, which functions by regulating the activity of DNMT3A and DNMT3B, increased cellular proliferation and anchorage-independent growth. Importantly, increased DNMT3L expression resulted in changed morphology of cells but this change was gradual and observed only after several passages. Interestingly, confluent cultures of DNMT3L-overexpressing HeLa cell colonies had characteristics of iPS cells. Concomitant with the morphological changes, expression pattern of genes important in nuclear reprogramming, development and cell cycle were observed to have significantly changed. Many imprinted genes, the known targets of DNMT3L, were downregulated. The slow nature of morphological changes and genome-wide nuclear reprogramming observed upon DNMT3L overexpression reinforces its role in carcinogenesis.     

NEDD4L regulates convergent extension movements in Xenopus embryos via Disheveled-mediated non-canonical Wnt signaling

During the early vertebrate body plan formation, convergent extension (CE) of dorsal mesoderm and neurectoderm is coordinated by the evolutionarily conserved non-canonical Wnt/PCP signaling. Disheveled (Dvl), a key mediator of Wnt/PCP signaling, is essential for the medial–lateral polarity formation in the cells undergoing convergent extension movements. NEDD4L, a highly conserved HECT type E3 ligase, has been reported to regulate the stability of multiple substrates including Dvl2. Here we demonstrate that NEDD4L is required for the cellular polarity formation and convergent extension in the early Xenopus embryos. Depletion of NEDD4L in early Xenopus embryos results in the loss of mediolateral polarity of the convergent-extending mesoderm cells and the shortened body axis, resembling those defects caused by the disruption of non-canonical Wnt signaling. Depletion of xNEDD4L also blocks the elongation of the animal explants in response to endogenous mesoderm inducing signals and partially compromises the expression of Brachyury. Importantly, reducing Dvl2 expression can largely rescue the cellular polarity and convergent extension defects in NEDD4L-depleted embryos and explants. Together with the data that NEDD4L reduces Dvl2 protein expression in the frog embryos, our findings suggest that regulation of Dvl protein levels by NEDD4L is essential for convergent extension during early Xenopus embryogenesis.     

Role of fibroblast growth factors as inducing agents in early embryonic development

To assess the potential role of a molecule in development we need to know three things: 1) what are the biological activities of the molecule, 2) what is its expression pattern, and 3) what are the consequences of removing it from the embryo? In the case of the FGF family in Xenopus embryos we have quite a lot of information about all three questions. Most members of the family can induce mesoderm from isolated animal caps, thus mimicking the natural “ventral vegetal” inducing signal operative in the blastula. This activity can be exerted on isolated, disaggregated cells and does not involve a change in division rate. When overexpressed from injected mRNA, the activity of FGFs depends largely on whether or not they possess a signal sequence, showing the importance of secretion in the inductive process. In addition to the mesoderm-inducing activity, there are effects of overexpression on whole embryos which lead to a suppression of anterior structures. Three types of FGF have so far been cloned from Xenopus: direct homologs of each of the mammalian types FGF-2 and FGF-3, and eFGF (“embryonic FGF”), which is equidistant in sequence from mammalian FGF-4 and FGF-6. Attempts to find homologs of mammalian FGF-5 and FGF-7 in Xenopus have proved unsuccessful. All three types of Xenopus FGF are expressed in early development. FGF-2 and eFGF are present in the oocyte and fertilized egg, and are thus both available at the time of mesoderm induction. FGF-3 and eFGF are both expressed from the embryonic genome during gastrulation and concentrated in the forming mesoderm. FGF-2 is expressed from the embryonic genome during neurulation in the brain, and a little later in the branchial arch mesenchyme and in the forming myotomes. These expression patterns suggest that there are several functions for the FGFs. The most successful strategy for inhibition of the FGF system has been the use of a dominant negative receptor construct introduced by Kirschner and colleagues. Overexpression of this construct can abolish the FGF responsiveness of animal caps. In whole embryos, the absence of FGF signaling causes a reduction, although not a total ablation, of mesoderm formation. There is also a severe effect on axis formation in which formation of the posterior parts is reduced consequent on an inhibition of invagination and elongation of the dorsal mesoderm. Thus, the present evidence suggests that the FGF system contributes to, although is not solely responsible for, mesoderm induction in vivo. It is also necessary for normal gastrulation movements, particularly in the dorsal mesoderm, and is likely to have several later functions, particularly in development of the central nervous system and the myotomes. © 1994 Wiley-Liss, Inc.     

PV.1 induced by FGF-Xbra functions as a repressor of neurogenesis in Xenopus embryos

During Xenopus early development, FGF signaling is involved in mesoderm formation and neurogenesis by modulating various signaling cascades. FGF-MAPK signaling induces Xbra expression, which maintains mesodermal fate through an autocatalytic-loop. Interestingly, previous reports have demonstrated that basic FGF (bFGF) treatment alone does not induce neurogenesis in ectodermal explants, even though FGF signaling inhibits BMP signaling via phosphorylation in Smad1 linker region. In addition, the overexpression of dominantnegative Xbra induces neurogenesis in ectodermal explants. However, the detailed mechanism underlying these phenomena has not yet been clarified. In this work, we showed that bFGF-Xbra signaling increased the PV.1 expression. DN-Xbra was found to decrease PV.1 expression, and the co-injection of PV.1 with DN-Xbra reduced neurogenesis in ectodermal explants. Furthermore, the knockdown of PV.1 induced neurogenesis in bFGF-treated ectodermal explants. Taken together, our results demonstrate that FGF-Xbra signaling induces PV.1 expression and that PV.1 functions as a neural repressor in the FGF-treated ectoderm. [BMB Reports 2014; 47(12): 673-678]