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Dev Biol
2013 Jun 15;3782:74-82. doi: 10.1016/j.ydbio.2013.04.005.
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Ric-8A, a guanine nucleotide exchange factor for heterotrimeric G proteins, is critical for cranial neural crest cell migration.
Fuentealba J
,
Toro-Tapia G
,
Arriagada C
,
Riquelme L
,
Beyer A
,
Henriquez JP
,
Caprile T
,
Mayor R
,
Marcellini S
,
Hinrichs MV
,
Olate J
,
Torrejón M
.
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The neural crest (NC) is a transient embryonic structure induced at the border of the neural plate. NC cells extensively migrate towards diverse regions of the embryo, where they differentiate into various derivatives, including most of the craniofacial skeleton and the peripheral nervous system. The Ric-8A protein acts as a guanine nucleotide exchange factor for several Gα subunits, and thus behaves as an activator of signaling pathways mediated by heterotrimeric G proteins. Using in vivo transplantation assays, we demonstrate that Ric-8A levels are critical for the migration of cranial NC cells and their subsequent differentiation into craniofacial cartilage during Xenopus development. NC cells explanted from Ric-8A morphant embryos are unable to migrate directionally towards a source of the Sdf1 peptide, a potent chemoattractant for NC cells. Consistently, Ric-8A knock-down showed anomalous radial migratory behavior, displaying a strong reduction in cell spreading and focal adhesion formation. We further show that during in vivo and in vitro neural crest migration, Ric-8A localizes to the cell membrane, in agreement with its role as a G protein activator. We propose that Ric-8A plays essential roles during the migration of cranial NC cells, possibly by regulating cell adhesion and spreading.
Fig. 1. Ric-8A is required for migration of cranial NC cells in Xenopus tropicalis and Xenopus laevis. (A) Ric-8A is expressed in the NC migration routes at stage 24 (lateral view). (B) Western blot analysis showing that the Ric-8AMO efficiently blocks the translation of a Ric-8A synthetic mRNA. (C-K) Molecular characterization of Ric-8AMO injected embryos. Snail-2 was used as neural crets marker and chordin and MyoD were used as dorsal mesoderm markers. Blue signal in G and H shows β-galactosidase activity used as a lineage tracer. (C) Schematic drawing showing the injection procedure performed in X.t. (panels D, E and I-K). (D) Ric-8A down-regulation shows no effect over neural crest cells induction at stage 16 (dorsal view). (E) Ric-8A knock down inhibits NC cells migration into the three migratory routes at stage 22 (lateral views). (F) Schematic drawing showing the injection procedure performed in X.l. (panels G and H). (G) Ric-8A down-regulation shows no effect over neural crest cells induction at stage 16 (dorsal view). (H) Ric-8A knock down inhibits the migration of NC cells into the three migratory routes at stage 22 (lateral views). (I-I′) Ric-8A down-regulation does not affect chordin and MyoD expression at stage 10.25 (vegetal view). (J) Dorsal view showing the staining from the Tunel. (J′): Dorsal view from a magnification of the region boxed in J. (K) Co-injection of Ric-8MO with a Ric-8A synthetic mRNA is able to rescue the cranial NC cells migration defects at stage 22 (lateral view). Arrowheads in E, H and K show the three cranial NC migratory routes. Arrowheads in J′ indicate no difference in the Tunel staining between nis and is. The embryo images are representative of three independent in situ experiments analyzing 20-50 embryos injected for each condition analyzed. Abbreviations: nis, non-injected side; is, injected side.
Fig. 2. Ric-8A function displays a cell-autonomous behavior and is required for proper craniofacial development in Xenopus embryos. (A) Ventral view showing cartilage of a stage 42 embryo injected with the control morpholino (CtMO). (B) Ventral view showing cartilage of a stage 42 embryo injected with the Ric-8AMO. (C) Schematic drawing of the ventral cartilaginous elements of a normal tadpole. The embryo images are representative of three independent experiments analyzing 30 embryos injected for each condition analyzed. Abbreviations: nis, non-injected side; is, injected side; Ir, infrarostralcartilage; M, Meckel cartilage; CH, ceratohyalcartilage; CB, ceratobranchial cartilage and BH, basihyalcartilage. (D) Schematic drawing of the transplantation procedure used to assess cranial NC migration in vivo. (E) Cranial NC cells transplanted from embryos co-injected with the CtMO and rhodamine dextran (RD) display a normal migratory pattern. (F) Cranial NC cells transplanted from embryos co-injected with the Ric-8AMO and RD remain in the dorsal region of the embryo. The embryo images are representative of two independent experiments analyzing 15 embryos injected for each condition analyzed.
Fig. 3. Ric-8A is required for the migration of cranial NC cells toward Sdf1. In vitro attraction assays performed with cranial NC explanted from embryos co-injected with a mGFP mRNA and a CtMO (A), Ric-8AMO (B) or a mixture of the Ric-8AMO and Ric-8A mRNA (C). Explants from cranial NC cells were exposed to Sdf1 soaked beads (blue asterisk) and the panels show the migratory pattern after 0, 150 and 300 min. (D) Quantification of the results from three independent experiments indicating the average chemotaxis index for each category analyzed. At least 50 cells from 7 different explants were analyzed for each condition. Statistical significance is indicated by *** (P<0.001).
Fig. 4. Ric-8A is required for cranial NC cells spreading and migration by regulating cell adhesion in vivo and in vitro. (A) Cranial NC cells explanted from embryos co-injected with a CtMO and synthetic mRNAs coding for mGFP and nRFP display a normal migratory behavior. (B) Cranial NC cells transplanted from embryos co-injected with the Ric-8AMO and synthetic mRNAs coding mGFP and nRFP fail to spread onto fibronectin-coated dishes. (C) Quantification of the results shown in A and B calculated from three independent experiments indicating the average spreading index from 7 different explants for each category analyzed. Migratory behavior pictures were taken at times 0, 150 and 300 min. Statistical significance is indicated by *** (P<0.001). (D) Quantification of three independent cellular adhesion experiments from at least 20 to 30 explants for dish with two different MO concentrations, indicating the percentage of adherent explants. Statistical significance is indicated by *** (P<0.001). (E) Phospho-paxillin antibody staining (red) of cranial NC cells explanted from embryos injected with CtMO and counterstained with DAPI (blue). (F) Phospho-paxillin antibody staining (red) of cranial NC cells explanted from embryos injected with Ric-8AMO and counterstained with DAPI (blue). The images are representative of three independent experiments analyzing 10 explant for each condition analyzed. The same exposure time was applied to the pictures shown in E and F.
Fig. 5. Ric-8A localizes at the cell membrane of migrating cranial NC cells in vitro and in vivo. (A-C) Cranial NC cells explants from embryos co-injected with the mRNAs coding for 6Myc-Ric-8A and mGFP. The localization of 6Myc-Ric-8A is shown in A (red), the mGFP signal is shown in B (green) and the merged signal is shown in C. Nuclei were counterstained with DAPI (blue). The images are representative of three independent experiments analyzing 10 explants. (D-F) Cranial NC precursor cells from embryos co-injected with the mRNAs coding for 6Myc-Ric-8A and mGFP were transplanted into a WT host embryo. At migratory stages embryos were sectioned and analyzed. The localization of 6Myc-Ric-8A is shown in D (red), the mGFP signal is shown in E (green) and the merged signal is shown in F. Nuclei were counterstained with DAPI (blue). Arrows show the colocalization of 6Myc-Ric-8A and mGFP. The images are representative of two independent experiments analyzing 4 transplant sections. (G) Control explant from an embryo co-injected with mRNA coding for 6Myc-Ric-8A and mGFP processed as in A-C except that the primary antibody was omitted, the inset shows a higher magnification. (H) Control explants from an embryo injected only with mRNA coding for mGFP and processed as in A, the inset shows a higher magnification. (I) Model depicting the possible functions of Ric-8A during the directional migration of cranial NC cells attracted towards an Sdf1 source (green gradient). Protrusions (filopodia and lamellipodia) can be seen at the border of the group, in the cell toward Sdf1 gradient. Possible Ric-8A function are indicates in gray, and its roles investigated in this study are indicated in black. Arrows show the directionality of regulatory information. Double arrows refer to an unknown signaling direction. The blue gradient represents the cell polarization during migration.
