XB-ART-19917Mech Dev April 1, 1995; 50 (2-3): 187-99.
Integrin alpha 5 during early development of Xenopus laevis.
The full length sequence of the Xenopus integrin alpha 5 subunit is reported. Analysis of cloned cDNA fragments reveals that alternative polyadenylation of alpha 5 mRNA occurs in the embryo. Furthermore, a variant form of the alpha 5 mRNA is expressed which encodes an integrin alpha 5 subunit with a truncated cytoplasmic domain. Integrin alpha 5 mRNA and protein are expressed in oocytes, eggs and throughout development. Spatial expression of alpha 5 mRNAs is first detected by whole mount in situ hybridization in presumptive neural crest cells and in the somitic mesoderm from the midgastrula stage onwards. In contrast, the alpha 5 protein is present on newly formed plasma membranes beginning at first cleavage. During neurulation, the integrin alpha 5 subunit disappears from the outer layer of the ectoderm, the notochord and the neural tube and accumulates in the sensorial layer of the ectoderm, the somites and the neural crest cells. These results provide evidence for the position specific regulation of alpha subunit expression in early vertebrate embryos.
PubMed ID: 7619730
Species referenced: Xenopus laevis
Genes referenced: fn1 itga5 itgb1 mcf2 tbx2 xa-1
Antibodies: Itga5 Ab1 Itgb1 Ab1
Article Images: [+] show captions
|Fig. 3. mRNA expression of the Xenopus integrin Q~ subunit. RNase protection analysis was performed with �P-labeled antisense RNA probes for Xas (A) and XaStr (C) corresponding to the regions indicated in Fig. I. These probes were hybridized to total RNA isolated from five embryos of the indicated stages, or to IO pg of total RNA from the A6 cell lines (A). Control tRNA samples were included into each experiment without RNaseA (-) treatment to show the intact, undigested probe and with RNase treatment (+) to identify unprotected but incompletely digested probe fragments. RNA samples from the same preparations were also hybridized to a & probe. As it is known that this RNA is present in similar amounts in the embryos of the different stages (Gawantka et al.. 1992), this experiment serves as an internal control for the RNA preparations (B). The autoradiograms shown in (A) and (B) were exposed for 3 days and the autoradiogram shown in (C) for 30 days. Embryos were staged according to Nieuwkoop and Faber (1967).|
|Fig. 4. RT-PCR analysis of the expression of the Xa,tr mRNA in different embryonic stages. With the gene specific primers indicated in Fig. 1, cDNA fragments corresponding either to Xa, (A, u) or Xczstr (A, tr) were amplified from reverse transcribed mRNA ofthe indicated stages. For the amplification of the Xcu, cDNA fragment (position: 23&:3183 BP; length: 837), the PCR was performed with 30 cycles, whereas for the Xastr cDNA fragment (position: 1081-1985; length: 904), 45 cycles were required. PCR products isolated from stage I and 23 were digested with the restriciton enzyme HaeIll and yielded the predicted pattern (B, XolS-HaeIII fragments: 52, 141, 167, 180 and 297 bp; X+r-Ha&I fragments: 141, 280, 234 and 349 bp).|
|Fig. 5. Localization of integrin aS mRNA during embryogenesis. Whole mount in situ hybridization with digoxygenin-rUTP-labeled antisense riboprobes was used to determine the localization of integrin q mRNAs throughout development. In each panel, arrows indicate expression in neural crest cells and arrowheads point to staining within the somitic mesoderm. (A) Dorsal view, stage 1 I .5-12 gastrula, oriented with anterior end at the top of the panel. (B) Optical section of (A). The approximate plane of section is indicated by the line in (A). (C) Frontal view, stage 15 neurula, dorsal to the top. Integrin q transcripts are detected in the neural crest (arrow) and paraxial mesoderm (arrowhead). (D) Side view, stage 14 neuruia, anterior to the right. (E) Optical section, stage 17 neurula, dorsal is to the top. Arrow indicates trunk neural crest cells, arrowhead marks the somitic mesoderm. (F) Dorsal view, stage 19 neurula, anterior to the left. Cranial neural crest cells (black arrow) are intensely stained and located lateral to the unstained neural tube. Trunk neural crest forms a single line of cells along the dorsal midline of the embryo (white arrow). (G) Side view, stage 21 neurula, anterior to the right and dorsal at the top. The somatic staining of the integrin os is concentrated around the nuclei of the post-rotation somites (arrowhead). Intensely stained cranial neural crest cells are visible in the anterior part of the embryo (arrow). (H) Optical section, stage 22 neurula, dorsal to the top. Trunk neural crest a single line of cells dorsal to the neural tube (arrow). Staining within the somites is also visible (arrowhead). (I) Dorsal view, stage 23 neurula, anterior to the top. Staining is localized in discrete foci in the post-rotation somitic mesoderm (black arrowhead) and is diffuse in the pre-rotation mesoderm (white arrow). (J) Side view, stage 32 tailbud embryo, anterior to the right. Note weaker staining in anterior somites. (K) High magnification view of the tailbud of the embryo in (J). Integrin (r5 expression in the tail remains restricted to the neural crest (arrow) and somites (arrowhead).|
|Fig. 6. Characterization of the q antiserum. Inununoprecipitations were performed, either with the anti q antiserum (lanes B and C) or with a 8, specific mAB 8C8 (lanes A, D and E). The precipitates were analyzed by immunoblotting with a polyclonal antiserum directed against the 8, subunit (lanes A and B), or with the anti q antiserum (lanes C-E). Electrophoresis was carried out under reducing (lanes A-D) or under non-reducing conditions (lane E). The signal in the lower part of (B) and (C) indicate IgGs which leaked from the precipitation beads. The positions (bars) and size (M, x 10m3) of relative molecular weight markers are indicated.|
|Fig. 7. Temporal expression of the Xenopus integrin a5 protein during development. Extracts from embryos of different stages (eight embryos per lane) were electrophoresed under non-reducing conditions and immunoblotted with the antiserum anti-q (A) or with mAB 8C8 (B). Developmental stages and the positions (bars) and size (Mr x 10-3) of relative molecular weight markers are indicated.|
|Fig. 8. Distribution of the 0~s integrin subunit in the embryo during cleavage, blastula and gastrula stages. Embryos were whole mount stained with the anti os antiserum, embedded in glycolmethacrylate and sectioned. (A) Detail of a stage 6 embryo stained with the anti cq antiserum preabsorbed with the peptide used for the immunization. The speckled signal is also seen in all following pictures and is regarded as background. (B) Phase contrast image of the region of contact between two blastomeres of a stage 5 embryo, animal view. (C) Same embryo as in (B) immunostained. (D) Transverse section of a stage 7 embryo. (E) Dorsal blastopore lip region of a stage 10.5 embryo, with apically constricted bottle cells. (F) Blastopore region of a stage I3 embryo. ae, archenteron; an, animal pole region; bl, blastocoel; dbl, dorsal blastopore lip region; me, mesoderm; vg, vegatal pole region; yp, yolk plug. Bars: 50 um.|
|Fig. 9. Distribution of (Ye integrin in the embryo during neurula and tadpole stages. (A) Transverse section of a stage 20 embryo. (B) Transverse section through a stage 30 embryo. Arrows indicate the ectoderm/mesoderm and mesodermlendoderm boundaries. ae, archenteron; en, endoderm; no, notochord; nt, neural tube; pm, paraxial mesoderm; p, pronephros. Histological procedure as in Fig. 8. Bars: 50 um.|
|Itga5 Ab1 staining of a NF stage 30 Xenopus laevis embryo. Coronal section, Dorsal up.|
|Itga5 (integrin, alpha 5 (fibronectin receptor, alpha polypeptide)) gene expression in a Xenopus laevis embryo as assayed by in situ hybridization, NF stage 19, dorsal view, anterior left.|
|Itga5 (integrin, alpha 5 (fibronectin receptor, alpha polypeptide)) gene expression in a Xenopus laevis embryo as assayed by in situ hybridization, NF stage 32. Lateral view: Dorsal up, anterior left.|