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Flamingo, a cadherin-type receptor involved in the Drosophila planar polarity pathway, can block signaling via the canonical wnt pathway in Xenopus laevis.
Morgan R
,
El-Kadi AM
,
Theokli C
.
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The Flamingo gene encodes a seven-pass transmembrane receptor of the cadherin super family and is one of a growing number of components identified as being necessary for the establishment of planar polarity in the Drosophila wing. Although vertebrate homologues of Flamingo have been identified in both man and mice, no function has as yet been ascribed to them. Here, we report the cloning of the Xenopus homologue of Flamingo (XFmi). XFmi is expressed in the dorsal ectoderm during gastrulation and in the forebrain and midbrain subsequently. We show that ectopic expression of the murine Flamingo gene can prevent the wnt mediated posteriorisation of the neural plate by interfering with the canonical wnt signalling pathway.
Fig. 1. XFmi expression during early Xenopus development. RT-PCR analysis of XFmi expression in embryos of different developmental stages, as shown. Oo, oocyte. Stage 2 is a 4 cell embryo; 10, early gastrula; 11, mid gastrula; 12.5, late gastrula / early neurula; 17, late neurula; 20, early tail bud. ODC, ornithine decarboxylase (loading control).
Fig. 2. In situ hybridisation analysis of XFmi expression in whole embryos. (A) Sagittal section through an early gastrula (stage 10+) embryo. Faint expression of XFmi can be seen in the zone of internal involution just above the dorsal blastopore groove, and also in the ectodermal cells closer to the animal pole (the vegetal pole of the embryo is at the bottom, the animal pole at the top). (B) Dorsal view of the same stage embryo (10+). XFmi expression begins at a defined border above the dorsal blastopore lip (marked by the two lines), corresponding to the involuted cells. (C) Dorsal view of two early neurula (stage 13) embryos, the posterior end (marked by yolk plug; arrow heads) is on the right. No expression is detectable at this stage. (D) Lateral view of a mid neurula (stage 17) embryo, dorsal side uppermost. A faint staining is apparent at the anterior end of the embryo. (E) Lateral view of an early tail bud (stage 22) embryo, anterior to left. (F) Lateral view of a tail bud (stage 26) embryo, anterior to left. (G) Enlargement of the head of the embryo shown in (E). (H) Dorsal - anterior view of the embryo shown in (F), with the anterior-most end upwards. The anterior limit of XFmi expression is indicated by the two lines. A group of cells scattered around the midline, and anterior to this line, also express XFmi and are indicated by *. (I) Section through the embryo shown in (F) across indicated plane (�I�). The dorsal side is at the top. (J) Section through the embryo shown in (F) across the indicated plane (�J�). The dorsal side is at the top. Abbreviations: cg, cement gland; dbg, dorsal blastopore grove; dbl, dorsal blastopore lip; e, ectoderm; en, endoderm; ey, eye; fg, foregut; fp, floor plate; h, headmesenchyme; ms, mesencephalon; nc, notochord; ps, prosencephalon; rc, rhombencephalon; sc, spinal chord; zii, zone of internal involution.
Fig. 3. Ectopic expression of MFmi results in the anteriorisation of the early embryo. Fertilised Xenopus eggs were injected with MFmi RNA (Fmi+), or with a control transcript lacking the translation start site (Fmi-). Embryos were allowed to develop to the late gastrula stage (11.5) and analyzed for the expression of Otx2 by in situ hybridization (blue / purple staining). The embryos are viewed with the blastopore (BP) uppermost in the top two panels, and with the blastopore to the left in the bottom two. The arrow head marks the position of the dorsal blastopore lip.
Fig. 4. Fmi can block the posteriorisation of neural tissue by Wnt3A. (A) Schematic overview of the experimental procedure. Fertilised Xenopus eggs were injected with various combinations of RNAs (1) and cultured until the early gastrula stage (2). At this point the animal cap was removed (3) and cultured until sibling embryos had reached the early tail bud stage. Total RNA was extracted and analysed for marker gene expression by RT-PCR. (B) RTPCR analysis of caps expressing a combination of different injected RNAs, as shown (�TREATMENT�). The expression of various marker genes was analysed by RT-PCR (�MARKER�). a/s MFmi, morpholino oligo with complementary sequence to the translation start site of the MFmi RNA. XOtx2, XHoxb1, XHoxb4 and XHoxb9 are arranged in anterior (Ant) to posterior (Post) order, as indicated by the double headed arrow.
Fig. 5. Flamingo does not block the FGF mediated posteriorisation of the neural plate. RT-PCR analysis of caps expressing a combination of different injected RNAs, as shown ('TREATMENT'). The expression of various marker genes was analysed by RT-PCR ('MARKER'). XOtx2, XHoxb1, XHoxb4 and XHoxb9 are arranged in anterior (Ant) to posterior (Post) order, as indicated by the double headed arrow.
Fig. 6. MFmi blocks the up-regulation of Xnr3 and Xsia by Xwnt8. RTPCR analysis of caps expressing a combination of different injected RNAs, as shown. a/s MFmi, morpholino oligo with complementary sequence to the translation start site of the MFmi RNA.
Fig. 7. Hoxb4 and Fmi repress each others expression. RT-PCR analysis of gene expression in tail bud stage embryos that have developed from eggs injected with different RNAs, as shown above each column. NIC, noninjected control. a/s, co-injection of a morpholino oligo complementary to the translation start site of the injected RNA. -RT, control without reverse transcription. XEf1α is included as a loading control.
