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Biochem Biophys Res Commun
2008 Jan 11;3652:214-20. doi: 10.1016/j.bbrc.2007.10.146.
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Characterization of a novel Xenopus SH3 domain binding protein 5 like (xSH3BP5L) gene.
Hu ZG
,
Chen Y
,
Zhou Q
,
Lv XY
,
Zhang Z
,
Wang YD
,
Xiao Y
,
Guo H
,
Liu YH
,
Tan RZ
,
Li QW
,
Bian GH
,
Wei YQ
.
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SH3 domain binding protein 5 like (xSH3BP5L) gene encodes a protein that is a new found member of SH3 domain binding protein family which has been implicated at multiple levels of biological functions. Here, we have characterized Xenopus SH3 domain binding protein 5 like (xSH3BP5L) gene in the development of Xenopus laevis. Transcripts of xSH3BP5L were detected at all stages of development and in numerous adult tissues. Whole-mount in situ hybridization demonstrated that xSH3BP5L is expressed at the animal pole from stage-2 onward. Interestingly, translational inhibition of xSH3BP5L using antisense morpholino oligonucleotides (MOs) and overexpression of xSH3BP5L in Xenopus embryos resulted in failed or delayed blastopore closure. Taken together, these data suggested that xSH3BP5L is required for normal embryogenesis of blastopore closure in X. laevis.
Fig. 1. Sequence analysis of xSH3BP5L. (A) Comparison of SH3BP5L proteins from Xenopus laevis (BC084800), mouse (NM_024480), and human (NM_030645). Dark and gray backgrounds represent identical amino acids. White letters on gray background indicate amino acid residues that are the sh3bp5 conserved domain. (B) Phylogenetic analysis of SH3BP5L family members using DNAstar MegAlign. Zebrafish (NM_213160); Rattus norvegicus (XM_001075049); Bos taurus (BC119925).
Fig. 2. Expression patterns of xSH3BP5L. (A) Temporal expression profile of xSH3BP5L analyzed by RT-PCR. Transcripts of xSH3BP5L were detected from unfertilized egg to tadpole. RT-PCR using an ornithine decarboxylase (ODC)-specific primer was carried out in parallel to control the amount of input RNA (ODC (RT+)]. RT-PCR without reverse transcriptase showed no contamination of genomic DNA (ODC (RT−)]. (B) Tissue expression pattern of xSH3BP5L in adult frog analyzed by RT-PCR. (C–G) Whole-mount in situ hybridization reveals spatial distribution patterns of xSH3BP5L mRNA. Bars, 250 μm. (C,D) xSH3BP5L expression is detectable in animal pole cells of blastula embryos. (E) xSH3BP5L mRNA is expressed in the ventral–lateral mesoderm during early gastrulation. (F) Expression of xSH3BP5L in the head. (G) Lateral view of a stage-29 embryo, showing that xSH3BP5L mRNA is detected predominantly in eye, but faintly in bronchial arch, otic vesicle, and pronephros (arrowheads in G). (H) Embryo hybridized with Xmyod antisense probe showing muscle signals (black arrowheads). (I) Control hybridization at stage-30 with sense xSH3BP5L probe. Ba, bronchial arches; ev, eye vesicle; m, muscle; ov, otic vesicle; pr, pronephros.
Fig. 3. Effect of xSH3BP5L MOs microinjection. Uninjected embryos (A) and embryos microinjected with 20 ng control MO (B) showed normal at stage-15. (C) Embryos microinjected with 20 ng xSH3BP5L MO exhibited delayed blastopore closure (black arrowheads). (D) The delayed blastopore closure was rescued in the embryos co-injected with 400 pg xSH3BP5L mRNA (black arrowheads). (E) Blastopore closure formation was inhibited differently in the embryos co-injected with 800 pg xSH3BP5L mRNA. (F) The xSH3BP5L MO targeting sequence localized to 5′ UTR of xSH3BP5L mRNA (sequence in red box) and the in vitro transcription start codon of ATG. (G) The frequency of phenotypes seen in experiment. (For interpretation of the references to color in this figure legend, the reader is referred to the web version of this article.)
Fig. 4. Overexpression of xSH3BP5L in X. laevis. (A) Uninjected embryos showed normal at stage-15. (B) Microinjection of 400 pg xSH3BP5L mRNA caused failed blastopore closure (black arrowheads).
sh3bp5l (SH3-binding domain protein 5 like) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, blastoporal view, dorsal up.
sh3bp5l (SH3-binding domain protein 5 like) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 28, lateral view, anteriorleft, dorsal up.
