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

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

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

AbstractThe 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

Cloning and spatiotemporal expression of <i>Xenopus laevis Apolipoprotein CI</i>

<div><p>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 <i>Xenopus laevis apoCI</i> and describe the expression pattern of this gene during early development, using reverse transcription polymerase chain reaction and whole mount <i>in situ</i> hybridization. <i>Xenopus apoCI</i> 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.</p></div

Spatial expression of <i>apoCI</i> mRNA.

<p>Vegetal (A, B) and lateral (A’, B’) views of stage 10 (A, A’) and stage 11 (B, B’) embryos, respectively; <u><i>a</i></u><i>poCI</i> transcript is localized to the dorsal ectoderm. Dotted lines in A, A’ mark the dorsal blastopore lip and dotted lines in B, B’ indicate the blastopore. Dorsal is to left in A, A’, B; dorsal is at 10 o’clock in B’. (C, D) Anterior views of <i>apoCI</i> expression in stage 13 and stage 15 embryos, respectively (dorsal to the top). Dotted line in D marks the pan-placodal primordium. (C’, D’) Dorsal views of embryos in C and D, respectively; anterior is down. (E) Anterior view of a stage 16 embryo; the anterior crescent of <i>apoCI</i> expression has expanded laterally. (F-L) Dorsoanterior views of stage 15 embryos; anterior is down and dorsal is to the top. (F) <i>apoCI</i> expression. (G) <i>slug</i> expression. (H) <i>apoCI</i> (purple) and <i>slug</i> (light blue) coexpression; arrows indicate region of overlap (cranial neural crest). (I) <i>pax6</i> expression. (J) <i>apoCI</i> (purple) and <i>pax6</i> (light blue) coexpression. (K) <i>otxA</i> expression. (L) <i>apoCI</i> (purple) and <i>otxA</i> (light blue) coexpression. (M, N) Anterior (M, dorsal is to the top) and dorsal (N, anterior is down) views showing <i>apoCI</i> expression in a stage 21 embryo. (O) Transverse section revealing expression of <i>apoCI</i> in the dorsal neural tube at stage 22; star indicates the neural tube. (P, Q) Lateral views of <i>apoCI</i> expression in stage 27 and stage 33 embryos, respectively; anterior is to the left. Arrow in Q marks liver primordium. No signal was detected with an <i>apoCI</i> sense strand probe (data not shown).</p

Temporal expression of a<i>poCI</i> mRNA.

<p>RT-PCR of RNA isolated from whole embryos between stage 4 and stage 32. <i>Chordin</i> serves as a staging control and <i>Ornithine decarboxylase</i> (ODC) as a control for input RNA levels.</p