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Mech Dev
2002 Apr 01;1131:3-14. doi: 10.1016/s0925-4773(01)00664-5.
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Xenopus marginal coil (Xmc), a novel FGF inducible cytosolic coiled-coil protein regulating gastrulation movements.
Frazzetto G
,
Klingbeil P
,
Bouwmeester T
.
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Gastrulation in vertebrates is a highly dynamic process driven by convergent extension movements of internal mesodermal cells, under the regulatory activity of the Spemann-Mangold or gastrula organizer. In a large-scale screen for genes expressed in the organizer, we have isolated a novel gene, termed Xmc, an acronym for Xenopus marginal coil. Xmc encodes a protein containing two widely spaced evolutionarily non-conserved coiled coils. Xmc protein is found in vesicular aggregates in the cytoplasm and associated with the inner plasma membrane. We show that Xmc is expressed in a dynamic fashion around the blastoporal circumference, in mesodermal cells undergoing morphogenetic movements, in a pattern similar to FGF target genes. Likewise, Xmc expression can be induced by ectopic XeFGF signaling and the early mesodermal expression is dependent on FGF receptor-mediated signaling. Morpholino-mediated translational 'knock-down' of Xmc results in embryos that display a reduced elongation of the antero-posterior axis and in a pronounced inhibition of morphogenetic movements in embryos and dorsal marginal zone explants. Xmc loss-of-function does not interfere with mesoderm induction or maintenance per se. Our results suggest that Xmc is a novel FGF target gene that is required for morphogenetic movements during gastrulation in Xenopus.
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11900970
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Fig. 2. Dynamic spatialemporal expression of Xmc during embryonic development. (A) RT-PCR analysis of Xmc expression during embryonic development (stages according to Nieuwkoop and Faber, 1967). Histone H4 serves as internal loading control. (B) Whole-mount in situ hybridization analysis of Xmc expression. Spatial expression during early (B, C), mid (D, E) and late (F) gastrulation (vegetal views), neurulation (G, dorsal and lateral view, respectively; anterior to the left), and tailbud and tadpole stage (K). Asterisks indicate the elevated bilateral dorsal expression and stippled lines indicate the margin of organizer explants used for the subtraction screen. Abbreviations: me, mesenchyme; lp, lateral plate and so, somite.
Fig. 3. Comparative expression of Xmc, Xcad2 and Xbra. Spatial expression of Xmc (A), Xcad2 (B) and Xbra (C) in whole mount-stained mid-gastrula embryos
(vegetal view) and transverse sections thereof. White lines indicate the plane of sectioning. The red arrows mark the animal limit of marginal zone expression
and black arrowheads indicate the blastopore (dorsal is to the right).
Fig. 4. FGF signaling is necessary and sufficient to activate Xmc expression. (A) Whole-mount in situ hybridization analysis for Xmc and Xbra expres- sion in control and XeFGF-injected albino cap explants. Embryos were injected with ,160 pg of XeFGF mRNA in one animal blastomere at the two-cell stage. (B) RT-PCR analysis for Xmc and Xbra expression in control caps and animal caps injected with increasing doses of XeFGF (in two-fold increments from 10 to 40 pg). EF-1a serves as loading control. (C) Expression of Xmc in control and dnFGFR-injected mid-gastrula (stages 112) and early neurula embryos (stage 14). Embryos were injected with 400 pg of dnFGFR mRNA in four blastomeres at the four- cell stage.
Fig. 5. Subcellular localization of Xmc and deletion constructs in animal cap explants. Confocal images showing XmcA, Xmc.NH2A and Xmc.COOHHA distribution (green channel), cortical actin visualized by rhodamin phalloidin (red channel) and the overlay. Embryos were injected with 800 pg of mRNA in two animal blastomeres at the two-cell stage. (A) Full-length Xmc is localized in a vesicular pattern in the cytoplasm (red arrowheads) and associated with the inner cell membrane (white arrows). (D) Xmc.NH2 is quantitatively recruited to the plasma membrane. At high doses, cells tend to round up and dissociate (see inset). (G) Xmc.COOH is localized in a diffuse perinuclear manner.
Fig. 6. Xmc loss-of-function results in gastrulation defects. Fluorescent images of control (A), Xmc.GFP-injected (B) and Xmc.GFP 1 Xmc morpholino- injected (C) neurula embryos. Embryos were injected with 800 pg Xmc.GFP mRNA with or without 8 ng morpholino in two animal blastomeres at the two-cell stage. Co-injection efficiently abolishes Xmc.GFP translation. Lateral (D) and dorsal (D00) views of stage 25 control embryos (D, D0) or embryos injected with ,10 ng of Xmc morpholino in two marginal blastomeres at the four-cell stage (E, E0, G0). Injection of Xmc morpholinos results in three classes of phenotypic alterations. Class I embryos (E, E0) display a marked kink in the primary axis, class II embryos (F, F0) have a shortened axis and class III embryos (G, G0) display severe gastrulation defects. (H) Quantitation of the phenotypic alterations. Histogram showing the frequencies of the phenotypes and the reduction after co-injection of Xmc morpholino with the Xmc.rescue construct.
Fig. 7. Xmc morpholino directly inhibits convergent extension along the antero-posterior axis and in DMZ explants. (A, B) Lineage tracing experiments showing that when co-injecting Xmc morpholino and GFP in one dorsal blastomere, the direction of the kink along the antero-posterior axis follows the site injected with the morpholino, as indicated by GFP. Morphological analysis of control (C), Xmc morpholino-injected (D) and Xmc morpholino 1 Xmc.rescue mRNA-injected (E) DMZ explants. (F) RT-PCR analysis for Xbra and XmyoD expression in DMZ explants cultivated till stage 11 and a-actin expression in DMZ explants cultured till stage 25. (G) In situ hybridization for Xbra at stage 10.51 in whole embryos injected with Xmc morpholino . In all cases, embryos were injected with ,8 ng Xmc morpholino with or without ,1 ng Xmc.rescue mRNA.
