Goda T et al. (2009), Xenopus Rnd1 and Rnd3 GTP-binding proteins are ...

XB-ART-40320

Dev Dyn 2009 Nov 01;23811:2867-76. doi: 10.1002/dvdy.22099.

Show Gene links Show Anatomy links

Xenopus Rnd1 and Rnd3 GTP-binding proteins are expressed under the control of segmentation clock and required for somite formation.

Goda T , Takagi C , Ueno N .

???displayArticle.abstract???
The process of segmentation in vertebrates is described by a clock and wavefront model consisting of a Notch signal and an fibroblast growth factor-8 (FGF8) gradient, respectively. To further investigate the segmentation process, we screened gene expression profiles for downstream targets of the segmentation clock. The Rnd1 and Rnd3 GTP-binding proteins comprise a subgroup of the Rho GTPase family that show a specific expression pattern similar to the Notch signal component ESR5, suggesting an association between Rnd1/3 and the segmentation clock. Rnd1/3 expression patterns are disrupted by overexpression of dominant-negative or active forms of Notch signaling genes, and responds to the FGF inhibitor SU5402 by a posterior shift analogous to other segmentation-related genes, suggesting that Rnd1/3 expressions are regulated by the segmentation clock machinery. We also show that antisense morpholino oligonucleotides to Rnd1/3 inhibit somite segmentation and differentiation in Xenopus embryos. These results suggest that Rnd1/3 are required for Xenopus somitogenesis.

???displayArticle.pubmedLink??? 19795516
???displayArticle.link??? Dev Dyn

Species referenced: Xenopus laevis
Genes referenced: actl6a cdc42 clock elob esr-5 gal.2 msgn1 myod1 notch1 odc1 pcdh8 rac1 rho.2 rhoa rnd1 rnd3 thy1
???displayArticle.antibodies??? Somite Ab1
???displayArticle.morpholinos??? rnd1 MO1 rnd3 MO1

???attribute.lit??? ???displayArticles.show???

	Figure 1. The expression of Rho GTPase family in Xenopus embryos. A-L: Embryos at neurula (A,C,E,G,I,K) or tail bud stages (B,D,F,H,J,L) were stained in whole-mount for the expression of ESR5 (A,B), Rnd1 (C,D), Rnd3 (E,F), Cdc42 (G,H), Rac1 (I,J), and RhoA (K,L). Red brackets in A-F show specific expression patterns of genes indicated. A,C,E,G,I,K: Embryos are oriented with anterior to the top, and dorsal views are shown. B,D,F,H,J,L: Embryos are oriented with anterior to the left, dorsal to the top, and lateral views are shown. M: Rnd1 and Rnd3 mRNA expressions at the different stages were detected by reverse transcriptase-polymerase chain reaction (RT-PCR). ODC (ornithine decarboxylase) was used as loading control. The numbers indicated on the top show staging of Xenopus embryo according to Nieuwkoop and Faber (1967). -RT, without reverse transcriptase. N: Schematic diagram shows Xenopus somitogenesis in neurula (Stage 17) and tail bud (stage 23) embryos (refer to Hausen and Riebesell, [1991]).
	Figure 2. Comparison of expression domains between Rnd1/3 and Notch signal components at somitomeres. A-J: Embryos at neurula stage were double-stained in whole-mount with digoxigenin (DIG) -labeled probes of Rnd1 (A,C,E,G) or Rnd3 (B,D,F,H-J) and fluorescein-labeled probes of ESR5 (A-D), Thy1 (E-H), or Rnd1 (I,J). After staining, embryos were bisected sagittally (C,D,G,H,J). Red arrows show expression domains of DIG-labeled Rnd1 or Rnd3, and blue arrows show expression domains of fluorescein-labeled ESR5, Thy1, or Rnd1. Embryos are oriented with anterior to the top (A,B,E,F,I) or left (C,D,G,H,J). Dorsal views (A,B,E,F,I) and sectional lateral views (C,D,G,H,J) are shown. K: Schematic diagram shows expression domains of ESR5, Thy1 (both are stained in blue), Rnd1 (stained in orange), and Rnd3 (stained in red) at Xenopus neurula stage. S1, most posterior somite; S0-SIII, somitomeres from anterior to posterior. Each somitomeres are divided into anterior and posterior half by dot lines.
	Figure 3. Disruption of Rnd1 and Rnd3 expression patterns by overexpression of dominant-negative or active form of Notch signal genes. A-L prime : Water (A-D,Q-T), mRNA of (E-H,U-X,I prime ,K prime ) DN-XDelta2, (I-L,Y-B prime ) MT-ICD, or (M-P,C prime -F prime ,J prime ,L prime ) Su(H)DBM was injected on one side of the four-cell stage embryos with green fluorescent protein (GFP) mRNA for tracer. At the neurula stage (A-F prime ) or tail bud stage (I prime -L prime ), GFP signal was observed in the injected embryos as a lineage trace (G prime ,H prime ), and stained in whole-mount for the expression of ESR5 (A,E,I,M), Thy1 (B,F,J,N), Rnd1 (C,G,K,O,Q,R,U,V,Y,Z,C prime ,D prime ), and Rnd3 (D,H,L,P,S,T,W,X,A prime ,B prime ,E prime ,F prime ,I prime -L prime ). Red arrows show gene expression domains (A-E prime ), and red brackets or arrowheads show Rnd3 expression domains in somite or presomitic mesoderm (PSM), respectively (I prime -L prime ). A-P: Embryos are oriented with anterior to the top, and dorsal views are shown. Left: Noninjected; right: injected side of mRNAs. Q-F prime : Embryos are oriented with anterior to the top, dorsal to the right (Q,S,U,W,Y,A prime ,C prime ,E prime ) or left (R,T,V,X,Z,B prime ,D prime F prime ), and lateral views are shown. I prime -L prime : Embryos are oriented with anterior to the left, dorsal to the top (K prime ,L prime ) or bottom (I prime ,J prime ), and lateral views are shown. N: noninjected; I: injected side of mRNAs.
	Figure 4. Posteriorly shift of Rnd1 and Rnd3 expression domains by fibroblast growth factor (FGF) inhibitor SU5402. A-H: Stage 14 embryos were incubated with (B,D,F,H) or without (A,C,E,G) 50 mu M SU5402 for 3.5 hr, and stained in whole-mount for the expression of Mes1 (A,B), Thy1 (C,D), Rnd1 (E,F), and Rnd3 (G,H). Red arrows or brackets show expression of genes indicated. All embryos are oriented with anterior to the top, and dorsal views are shown.
	Figure 5. Gene depletion of Rnd1 and Rnd3 disrupted somite formation. A-L: Antisense morpholino oligonucleotide (MO) for control (A,D,G,J), Rnd1 (B,E,H,K), or Rnd3 (C,F,I,L) was injected on one side of the four-cell stage embryos with nlacZ mRNA. At tail bud stage, all embryos were stained in blue with X-gal. A-F: Embryos were stained in whole-mount with monoclonal 12/101 antibody. A-F: Lateral views of noninjected side (A-C) and MO plus nlacZ injected side (D-F) are shown. G-I: Embryos were stained in longitudinal tissue section with monoclonal 12/101 antibody. Red brackets show each somitic unit. J-L: Embryos were stained in longitudinal tissue section with phalloidin. White brackets show each somitic unit. All images are positioned with anterior to the left. G-L: MO plus nlacZ injected side to the top and noninjected side to the bottom (indicated by + and -, respectively), and dorsal views are shown. M: The effect of Rnd3 MO on Rnd3 mRNA transcription was tested with in vitro transcription/translation system. Rnd3 MO inhibited the Rnd3-Vns transcription constructed to generate a fusion protein with eYFP (Vns) specifically, but not inhibited a rescue construct Rnd3-Vns6M. Vns was used as loading control.
	Figure 6. Expression patterns and levels of segmentation clock- and somite differentiation-related genes in Rnd1 or Rnd3 morpholino oligonucleotide (MO) injected embryos. A-O: Control (A-E), Rnd1 (F-J), or Rnd3 (K-O) MO was injected on one side of the four-cell stage embryos with green fluorescent protein (GFP) mRNA. Neurula stage embryos were stained in whole-mount for the expression of ESR5 (A,F,K), Thy1 (B,G,L), PAPC (C,H,M), MyoD (D,I,N), and actin (E,J,O). Red arrowheads or brackets show striped or nonstriped domains of each gene expression, respectively. All images are positioned with anterior to the top, MO and GFP mRNA injected side to the right, and dorsal views are shown. P: Embryos injected with control, Rnd1, or Rnd3 MO were analyzed by reverse transcriptase-polymerase chain reaction (RT-PCR) for gene expression levels indicated at the left column. OCD was used as loading control. Rhodamine was injected in embryos with MOs as a lineage trace. -RT, without reverse transcriptase.