Background
Xenopus embryonic skin is composed of the superficial layer with defined apicobasal polarity and the inner layer lacking the apical domain. Multiciliated cells (MCCs) originate in the inner layer of the epidermal ectoderm and subsequently migrate to the surface. How MCCs acquire the apicobasal polarity and intercalate into the superficial layer during neurulation is largely unknown. As Rab11-dependent vesicle trafficking has been implicated in ciliary membrane assembly and in apical domain formation in epithelial cells, we assessed the involvement of Rab11 in MCC development.
Results
Here we report that Rab11 is specifically enriched and becomes apically polarized in skin MCCs. Interference with Rab11 function by overexpression of a dominant negative mutant or injection of a specific morpholino oligonucleotide inhibited MCC intercalation into the superficial layer. Dominant negative Rab11-expressing MCC precursors revealed intrinsic apicobasal polarity, characterized by the apical domain that is not normally observed in inner layer cells. Despite the presence of the apical domain, the cells with inhibited Rab11 function were randomly oriented relative to the plane of the tissue, thereby demonstrating a defect in planar polarity.
Conclusion
These results establish a requirement for Rab11 in MCC development and support a two-step model, in which the initial polarization of MCC precursors is critical for their integration into the superficial cell layer.
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).
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.
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