Onuma Y et al. (2009), Bestrophin genes are expressed in Xenopus devel...

XB-ART-39662

Biochem Biophys Res Commun 2009 Jul 03;3843:290-5. doi: 10.1016/j.bbrc.2009.04.117.

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Bestrophin genes are expressed in Xenopus development.

Onuma Y , Haramoto Y , Nejigane S , Takahashi S , Asashima M .

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The Bestrophin-1/VMD2 gene has been implicated in Best disease, a juvenile-onset vitelliform macular dystrophy. The Bestrophin proteins have anion channel activity, and the four mammalian members share sequence homologies in multiple transmembrane domains and an RFP-tripeptide motif. The expression patterns and functions of the Bestrophin genes in retinal pigment epithelium have been studied intensively, whereas little is known about their roles in vertebrate embryogenesis. This study examined the roles of four Xenopus tropicalis homologs of BEST genes. The xtBest genes showed spatially and temporally distinct expression. xtBest-2 was the only maternally expressed Best gene, and both xtBest-2 and the Xenopus laevis Best-2 gene were expressed at the edge of the blastopore lip including the organizer. Ectopic expression of xBest-2 caused defects in dorsal axis formation and in mesodermal gene expression during gastrulation. These results suggest a new role of the Bestrophin family genes in early vertebrate embryogenesis.

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Species referenced: Xenopus tropicalis Xenopus laevis
Genes referenced: best2 gsc nodal nodal5.2 not odc1 tbxt vegt
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	Fig. 1. Comparison of X. tropicalis Best gene expression patterns. (A) Temporal expressions of X. tropicalis Best genes. RNA was extracted from X. tropicalis embryos at the stages indicated above each lane. egg, Unfertilized egg; RT-, RT-PCR without reverse transcriptase in ODC reaction. (B) Expression profile of Best genes in X. tropicalis adult tissues. RNA was extracted from X. tropicalis embryos at the stages indicated above each lane. egg, Unfertilized egg; RT-, RT-PCR without reverse transcriptase in EF-1 a reaction. (Câ€“W) Spatial expression pattern of X. tropicalis Best genes analyzed by in situ hybridization. (C, J, Q) Stage 9 embryos in lateral view. (D, K, R) Stage 10.5 embryos in vegetal view. Arrowheads indicate the blastopore lip. (E, L, S) Stage 12 embryos in vegetal view. (F, M, T) Stage 20 embryos in dorsal view. (G, N, U) Stage 25 embryos. (H, O, V) Stage 30 embryos. (I, P, W) Stage 35 embryos. nc, neural crest; ae, anterior endoderm; pe, posterior endoderm; tb, tailbud; pd, pronephric duct; is, intersomatic tissue.
	Fig. 2. Conserved expression of Xenopus Best-2 gene in the organizer and blastopore lip. (A) Temporal expression of X. laevis Best-2 gene. RNA was extracted from X. laevis embryos at the stages indicated above each lane. egg, unfertilized egg; RT-, RT-PCR without reverse transcriptase. (Bâ€“J) Spatial expression of X. laevis Best-2 analyzed by in situ hybridization. (B) Stage 10.5 embryo in vegetal view. Arrowhead indicates the blastopore. The line represents the position of the section shown in (I). (C) Stage 12 embryo in vegetal view. The line represents the position of the section shown in (J). (D) Stage 15 embryo in dorsal view. The line represents the position of the section shown in (K). (E) Stage 20 embryo in dorsal view. (F) Stage 25 embryo. (G) Stage 30 embryo. The line represents the position of the section shown in (L). (H) Stage 35 embryo. (I) Sagittal section of a stage 10.5 embryo. The arrowhead indicates the blastopore. (J) Sagittal section of a stage 12 embryo. Arrowheads indicate the edge of the yolk plug. (K) Transverse section of a stage 15 embryo. (L) Transverse section of a stage 30 embryo. nc, neural crest; ae, anterior endoderm; pe, posterior endoderm; tb, tailbud; ar, archenteron roof; ca, cloaca. (M) xBest-2 is induced by nodal signaling. Animal caps were dissected from embryos injected with 20 pg of Xnr5 RNA or 500 pg of VegT RNA, and cultured until stage 20 for RT-PCR. Both Xnr5 and VegT induced expression of xBest-2. un, uninjected animal caps as a negative control; WE, whole embryo as a positive control; RT-, reverse transcriptase minus as a negative control.
	Fig. 3. Overexpression of xBest-2 causes gastrulation defect and interferes with the expression of mesendoderm genes. (AâC) Embryos were injected with 1 ng of xBest-2 RNA into the dorsal side (A, B) or the ventral side (C) at the 4-cell stage, and photographed at stage 35. (D) Uninjected control embryo at stage 35. (EâP) Embryos were injected with 1 ng of xBest-2 RNA into two dorsal (E, F, I, J, M, N) or two ventral (G, H, K, L, O, P) blastomeres at the 4-cell stage, and fixed at stage 10.5 for in situ hybridization analysis. Embryos are in dorsal view (E, F, M, N), ventral view (G, H, O, P), and vegetal view (I, J, K, L). (EâH) In situ hybridization of Xbra. (I, J) In situ hybridization of goosecoid (gsc). (KâP) In situ hybridization of Xnot. Black arrowheads indicate the blastopore lip. Red arrowheads indicate ectopic expression of Xnot.
	Fig. 4. Loss-of-function study of xBest-2. (A) Genomic structures including exon 1 (1) and exon 2 (2) of xBest-2a and xBest-2b. The translation initiation codon (ATG) and primer regions are indicated (Primer). The splice-site-targeted antisense morpholino oligonucleotides, xBest-2a MO and xBest-2b MO, were designed against the sequence at the boundary of exon 1/intron 1 of each gene. xBest-2a and xBest-2b intron 1 have different lengths. The pre-mRNAs of xBest-2a and xBest-2b generated by splice blocking have a termination codon immediately following exon 1. The intron 1 sequences are in lower case. Amino acid sequences are also indicated. (B) RT-PCR analysis of xBest-2 mRNA in MO-injected embryos. Embryos were injected with 20 ng of xBest-2a MO plus 20 ng of xBest-2b MO (B2), or 40 ng of standard control MO (SC), in the marginal region of both blastomeres at the 2-cell stage, and cultured for RT-PCR until various stages as indicated above each lane. The primer set was designed to span the first intron to detect splicing of the endogenous pre-mRNA for both xBest-2a and xBest-2b (corresponding to Primer in (A)). In xBest-2 MO-injected embryos, the mature mRNA (m) was decreased and nonspliced pre-mRNA (p1 and p2) was detected from stage 10.5. The mature xBest-2 mRNA was eliminated at stage 30 in xBest-2 MO-injected embryos. Splicing of xBest-2 pre-mRNA was not affected by control MO injection. (C) A control uninjected embryo at stage 35 (uninjected). (D) An embryo injected with 20 ng of xBest-2a MO plus 20 ng of xBest-2b MO (xBest-2 MOs). Disruption of xBest-2 did not affect the patterning of embryos at stage 35.
	Supplemental Fig. 1. Phylogenetic tree of Bestrophin proteins. Multiple protein sequence alignments were performed using MacVector software (MacVector Inc). The following sequences were used: hBEST1 (NM_004183), hBEST2 (NM_017682), hBEST3 (NM_032735), hBEST4 (NM_153274), mBest1 (NM_011913), mBest2 (NM_145388), mBest3 (NM_001007583), rBest1 (NM_001011940), rBest2 (NM_001108895), rBest3 (XM_235161), rBest4 (XM_001067201), xtBest-1 (AB495092), xtBest-2 (NM_203643), xtBest-3 (AB495093), xtBest-4 (NM_001123442), and dBest-1 (AAR99659). hBEST, human BEST. mBest, mouse Best. rBest, rat Best. dBest-1, Drosophila Bestrophin-1.
	best2 (bestrophin 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 20, dorsal view, anterior up.
	best2 (bestrophin 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.