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Tetraspanins comprise a large family of integral membrane proteins involved in the regulation of cell adhesion, migration and fusion. In humans it consists of 33 members divided in four subfamilies. Here, we examined the spatial and temporal gene expression of four related tetraspanins during the embryonic development of Xenopus laevis by quantitative real-time PCR and in situ hybridization: Tspan3 (encoded by the gene Tm4sf8 gene) Tspan4 (encoded by the gene Tm4sf7), Tspan5 (encoded by the gene Tm4sf9) and Tspan7 (encoded by the gene Tm4sf2). These genes appeared first in the vertebrates during the evolution and are conserved across different species. In humans, they were associated with several diseases such as sclerosis, mental retardation and cancer; however their physiological role remained unclear. This work provides a comprehensive comparative analysis of the expression of these tetraspanins during the development of X. laevis. The more closely related tetraspanins Tspan3, Tspan4 and Tspan7 exhibited very similar spatial expression patterns, albeit differing in their temporal occurrence. The corresponding transcripts were found in the dorsal animal ectoderm at blastula stage. At early tailbud stages (stage 26) the genes were expressed in the migrating cranial neural crest located in the somites, developing eye, brain, and in otic vesicles. In contrast, Tspan5 appeared first at later stages of development and was detected prominently in the notochord. These data support close relatedness of Tspan3, Tspan4 and Tspan7. The expression of these tetraspanins in the cells with a high migratory potential, e.g. neural crest cells, suggests their role in the regulation of migration processes, characteristic for tetraspanin family members, during development. Similarity of the expression profiles might indicate at least partial functional redundancy, which is in concordance with earlier findings of tissue-limited or absent phenotypes in the knock-down studies of tetraspanins family members performed.
Fig. 2. Phylogenetic analysis of the tetraspanins Tspan3, Tspan4, Tspan5 and Tspan7. The phylogram was generated using ClustalW2 algorithm on the EBI public server http://www.ebi.ac.uk/Tools/phylogeny/clustalw2_phylogeny/ and includes Danio rerio, Xenopus laevis, Xenopus tropicalis, Mus musculus, Rattus norvegicus and Homo sapiens homologs of the tetraspanins analyzed.
Fig. 3. Real-time RT-PCR analysis of Tspan3 expression in different embryonic stages. The total RNA was isolated from the group of 10 embryos on the corresponding stage of development. Samples were normalized to levels of ornithine decarboxylase (ODC). Error bars indicate standard error.
Fig. 4. Expression of Tspan3 during Xenopus development. (A, B and E–H) Whole mount in situ hybridization of Tspan3 encoding gene Tm4sf8 on stage 8–32 Xenopus embryos as indicated. (I) Control in situ hybridization with sense probe for Tm4sf8 at stage 32. Vegetal views (A–C), anterior view (E), dorsal view, anterior to up (F), lateral views, anterior to left (G–I). (C) Schematic representation of a stage 11.5 embryo modified from Nieuwkoop and Faber (1967). (D) Semi-sections of embryos shown in C as indicated. (J and K) Vibratome sections of embryos shown in H as indicated. CNC = cranial neural crest, DMZ = dorsal marginal zone, NF = neural fold, OC = optic cup, OV = otic vesicle, PSM = pre-somitic mesoderm, SO = somites, TNC = trunk neural crest. Scale bar 450 μm (A–I) and 100 μm (J and K).
Fig. 5. Real-time RT-PCR analysis of Tspan4 expression in different embryonic stages. The total RNA was isolated from the group of 10 embryos on the corresponding stage of development. Samples were normalized to levels of ornithine decarboxylase (ODC). Error bars indicate standard error.
Fig. 6. Expression of Tspan4 during Xenopus development. (A, B and E–H) Whole mount in situ hybridization of Tspan4 encoding gene Tm4sf7 on stage 8–31 Xenopus embryos as indicated. (I) Control in situ hybridization with sense probe for Tm4sf7 at stage 31. Vegetal views (A–C), anterior view (E), dorsal view, anterior to up (F), lateral views, anterior to left (G–I). (C) Schematic representation of a stage 11.5 embryo modified from Nieuwkoop and Faber (1967). (D) Semi-sections of embryos shown in C as indicated. (J and K) Vibratome sections of embryos shown in H as indicated. CNC = cranial neural crest, DMZ = dorsal marginal zone, NF = neural fold, OC = optic cup, OV = otic vesicle, PSM = pre-somitic mesoderm, SO = somites, TNC = trunk neural crest. Scale bar 450 μm (A–I) and 100 μm (J and K).
Fig. 7. Real-time RT-PCR analysis of Tspan7 expression in different embryonic stages. The total RNA was isolated from the group of 10 embryos on the corresponding stage of development. Samples were normalized to levels of ornithine decarboxylase (ODC). Error bars indicate standard error.
Fig. 8. Expression of Tspan7 during Xenopus development. (A, B and E–H) Whole mount in situ hybridization of Tspan7 encoding gene Tm4sf2 on stage 8–31 Xenopus embryos as indicated. (I) Control in situ hybridization with sense probe for Tm4sf2 at stage 31. Vegetal views (A–C), anterior view (E), dorsal view, anterior to up (F), lateral views, anterior to left (G–I). (C) Schematic representation of a stage 11.5 embryo modified from Nieuwkoop and Faber (1967). (D) Semi-sections of embryos shown in C as indicated. (J) Vibratome section of embryo shown in H as indicated. CNC = cranial neural crest, NF = neural fold, OC = optic cup, OV = otic vesicle, PSM = pre-somitic mesoderm, SO = somites, NO = notochord. Scale bar 450 μm (A–I) and 100 μm (J).
Fig. 9. Real-time RT-PCR analysis of Tspan5 expression in different embryonic stages. The total RNA was isolated from the group of 10 embryos on the corresponding stage of development. Samples were normalized to levels of ornithine decarboxylase (ODC). Error bars indicate standard error.
Fig. 10. Expression of Tspan5 during Xenopus development. (A) Whole-mount in situ hybridization for Tspan5 encoding gene Tm4sf9 on Xenopus embryos at stage 32, 34 and 37, lateral view, anterior to left. (B) Transverse vibratome section corresponding to the line indicated in panel B. NT, neural tube; NC, notochord. (C) Crop area corresponding to the dashed square indicated in panel C. (D) Whole-mount in situ hybridization for sense Tm4sf9 at stage 32 as negative control, lateral view, anterior to left. NT = neural tube, N0 = notochord, BR = brain, OV = otic vesicle, BA = branchial arches. Scale bar 450 μm (A, D, E and F) and 100 μm (B and C).
Graphical abstract figure, no caption given.
See XB-IMG-79068/Fig 10 for gene expression curation of tspan5 at NF stage 32 (far left) and XB-IMG-79062/Fig. 4 for gene expression curation of tspan3 at NF stage 26, (far right).
