Tomomi Haremaki

Integration of multiple signal transducing pathways on Fgf response elements of the Xenopus caudal homologue Xcad3.

Early neural patterning along the anteroposterior (AP) axis appears to involve a number of signal transducing pathways, but the precise role of each of these pathways for AP patterning and how they are integrated with signals that govern neural induction step is not well understood. We investigate the nature of Fgf response element (FRE) in a posterior neural gene, Xcad3 (Xenopus caudal homologue) that plays a crucial role of posterior neural development. We provide evidence that FREs of Xcad3 are widely dispersed in its intronic sequence and that these multiple FREs comprise Ets-binding and Tcf/Lef-binding motifs that lie in juxtaposition. Functional and physical analyses indicate that signaling pathways of Fgf, Bmp and Wnt are integrated on these FREs to regulate the expression of Xcad3 in the posterior neural tube through positively acting Ets and Sox family transcription factors and negatively acting Tcf family transcription factor(s).

Xema, a foxi-class gene expressed in the gastrula stage Xenopus ectoderm, is required for the suppression of mesendoderm.

The molecular basis of vertebrate germ layer formation has been the focus of intense scrutiny for decades, and the inductive interactions underlying this process are well defined. Only recently, however, have studies demonstrated that the regulated inhibition of ectopic germ layer formation is also crucial for patterning the early vertebrate embryo. We report here the characterization of Xema (Xenopus Ectodermally-expressed Mesendoderm Antagonist), a novel member of the Foxi-subclass of winged-helix transcription factors that is involved in the suppression of ectopic germ layer formation in the frog, Xenopus laevis. Xema transcripts are restricted to the animal pole ectoderm during early Xenopus development. Ectopic expression of Xema RNA inhibits mesoderm induction, both by growth factors and in the marginal zone, in vivo. Conversely, introduction of antisense morpholino oligonucleotides directed against the Xema transcript stimulates the expression of a broad range of mesodermal and endodermal marker genes in the animal pole. Our studies demonstrate that Xema is both necessary and sufficient for the inhibition of ectopic mesendoderm in the cells of the presumptive ectoderm, and support a model in which Fox proteins function in part to restrict inappropriate germ layer development throughout the vertebrate embryo.

Vertebrate Ctr1 coordinates morphogenesis and progenitor cell fate and regulates embryonic stem cell differentiation

Embryogenesis involves two distinct processes. On the one hand, cells must specialize, acquiring fates appropriate to their positions (differentiation); on the other hand, they must physically construct the embryo through coordinated mechanical activity (morphogenesis). In early vertebrate development, fibroblast growth factor (FGF) regulates multiple embryonic events, including germ layer differentiation and morphogenesis; the cellular components that direct FGF signaling to evoke these different responses remain largely unknown. We show here that the copper transporter 1 (Ctr1) protein is a critical router of FGF signals during early embryogenesis. Ctr1 both promotes the differentiation and inhibits the morphogenesis of mesoderm and neurectoderm in embryos of the frog Xenopus laevis, thereby coordinating normal development. Signal sorting by Ctr1 involves the activation of the Ras–MAP kinase cascade and appears to be independent of its role in copper transport. Mouse embryonic stem (ES) cells deficient for Ctr1 (Ctr1−/−) retain characteristics of pluripotency under conditions that favor differentiation in wild-type ES cells, indicating a conserved role for Ctr1 during amphibian and mammalian cell fate determination. Our studies support a model in which vertebrate Ctr1 functions as a key regulator of the differentiation capacity of both stem and progenitor cell populations.

β-Catenin-Independent Activation of TCF1/LEF1 in Human Hematopoietic Tumor Cells through Interaction with ATF2 Transcription Factors

The role of Wnt signaling in embryonic development and stem cell maintenance is well established and aberrations leading to the constitutive up-regulation of this pathway are frequent in several types of human cancers. Upon ligand-mediated activation, Wnt receptors promote the stabilization of β-catenin, which translocates to the nucleus and binds to the T-cell factor/lymphoid enhancer factor (TCF/LEF) family of transcription factors to regulate the expression of Wnt target genes. When not bound to β-catenin, the TCF/LEF proteins are believed to act as transcriptional repressors. Using a specific lentiviral reporter, we identified hematopoietic tumor cells displaying constitutive TCF/LEF transcriptional activation in the absence of β-catenin stabilization. Suppression of TCF/LEF activity in these cells mediated by an inducible dominant-negative TCF4 (DN-TCF4) inhibited both cell growth and the expression of Wnt target genes. Further, expression of TCF1 and LEF1, but not TCF4, stimulated TCF/LEF reporter activity in certain human cell lines independently of β-catenin. By a complementary approach in vivo, TCF1 mutants, which lacked the ability to bind to β-catenin, induced Xenopus embryo axis duplication, a hallmark of Wnt activation, and the expression of the Wnt target gene Xnr3. Through generation of different TCF1-TCF4 fusion proteins, we identified three distinct TCF1 domains that participate in the β-catenin-independent activity of this transcription factor. TCF1 and LEF1 physically interacted and functionally synergized with members of the activating transcription factor 2 (ATF2) family of transcription factors. Moreover, knockdown of ATF2 expression in lymphoma cells phenocopied the inhibitory effects of DN-TCF4 on the expression of target genes associated with the Wnt pathway and on cell growth. Together, our findings indicate that, through interaction with ATF2 factors, TCF1/LEF1 promote the growth of hematopoietic malignancies in the absence of β-catenin stabilization, thus establishing a new mechanism for TCF1/LEF1 transcriptional activity distinct from that associated with canonical Wnt signaling.

Regulation of vertebrate embryogenesis by the exon junction complex core component Eif4a3

The establishment and maintenance of cellular identity are ultimately dependent upon the accurate regulation of gene expression, the process by which genetic information is used to synthesize functional gene products. The post‐transcriptional, pre‐translational regulation of RNA constitutes RNA processing, which plays a prominent role in the modulation of gene expression in differentiated animal cells. The multi‐protein Exon Junction Complex (EJC) serves as a critical signaling hub within the network that underlies many RNA processing events. Here, we identify a requirement for the EJC during early vertebrate embryogenesis. Knockdown of the EJC component Eukaryotic initiation factor 4a3 (Eif4a3) in embryos of the frog Xenopus laevis results in full‐body paralysis, with defects in sensory neuron, pigment cell, and cardiac development; similar phenotypes are seen following knockdown of other “core” EJC protein constituents. Our studies point to an essential role for the EJC in the development of neural plate border derivatives. Developmental Dynamics 239:1977–1987, 2010.

Eif4a3 is required for accurate splicing of the Xenopus laevis ryanodine receptor pre-mRNA.

The Exon Junction Complex (EJC) plays a critical role in multiple posttranscriptional events, including RNA subcellular localization, nonsense-mediated decay (NMD), and translation. We previously reported that knockdown of the EJC core component Eukaryotic initiation factor 4a3 (Eif4a3) results in full-body paralysis of embryos of the frog, Xenopus laevis. Here, we explore the cellular and molecular mechanisms underlying this phenotype. We find that cultured muscle cells derived from Eif4a3 morphants do not contract, and fail to undergo calcium-dependent calcium release in response to electrical stimulation or treatment with caffeine. We show that ryr (ryanodine receptor) transcripts are incorrectly spliced in Eif4a3 morphants, and demonstrate that inhibition of Xenopus Ryr function similarly results in embryonic paralysis. These results suggest that the EJC mediates muscle cell function via regulation of pre-mRNA splicing during early vertebrate embryogenesis.

Xmab21l3 mediates dorsoventral patterning in Xenopus laevis.

Specification of the dorsoventral (DV) axis is critical for the subsequent differentiation of regional fate in the primary germ layers of the vertebrate embryo. We have identified a novel factor that is essential for dorsal development in embryos of the frog Xenopus laevis. Misexpression of Xenopus mab21-like 3 (Xmab21l3) dorsalizes gastrula-stage mesoderm and neurula-stage ectoderm, while morpholino-mediated knockdown of Xmab21l3 inhibits dorsal differentiation of these embryonic germ layers. Xmab21l3 is a member of a chordate-specific subclass of a recently characterized gene family, all members of which contain a conserved, but as yet ill-defined, Mab21 domain. Our studies suggest that Xmab21l3 functions to repress ventralizing activity in the early vertebrate embryo, via regulation of BMP/Smad and Ras/ERK signaling.

Xmc mediates Xctr1-independent morphogenesis in Xenopus laevis†

In the frog, Xenopus laevis, fibroblast growth factor (FGF) signaling is required for both mesoderm formation and the morphogenetic movements that drive the elongation of the notochord, a dorsal mesodermal derivative; the coordination of these distinct roles is mediated by the Xenopus Ctr1 (Xctr1) protein: maternal Xctr1 is required for mesodermal differentiation, while the subsequent loss of Xctr1 promotes morphogenesis. The signaling cascade activated by FGF in the presence of Ctr1 has been well characterized; however, the Xctr1‐independent, FGF‐responsive network remains poorly defined. We have identified Xenopus Marginal Coil (Xmc) as a gene whose expression is highly enriched following Xctr1 knockdown. Zygotic initiation of Xmc expression in vivo coincides with a decrease in maternal Xctr1 transcripts; moreover, Xmc loss‐of‐function inhibits Xctr1 knockdown‐mediated elongation of FGF‐treated animal cap explants, implicating Xmc as a key effector of Xctr1‐independent gastrular morphogenesis. Developmental Dynamics 238:2382–2400, 2009.

Cloning and spatiotemporal expression of Xenopus laevis Apolipoprotein CI

Apolipoprotein CI (ApoCI) belongs to the Apolipoprotein superfamily, members of which are involved in lipid transport, uptake and homeostasis. Excessive ApoCI has been implicated in atherosclerosis and Alzheimer’s disease in humans. In this study we report the isolation of Xenopus laevis apoCI and describe the expression pattern of this gene during early development, using reverse transcription polymerase chain reaction and whole mount in situ hybridization. Xenopus apoCI is enriched in the dorsal ectoderm during gastrulation, and is subsequently expressed in sensory placodes, neural tube and cranial neural crest. These data suggest as yet uncharacterized roles for ApoCI during early vertebrate embryogenesis.