Meng F et al. (1997), Cloning and characterization of cDNAs encoding ...

XB-ART-15763

Mech Dev 1997 Oct 01;672:141-55.

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Cloning and characterization of cDNAs encoding the integrin alpha2 and alpha3 subunits from Xenopus laevis.

Meng F , Whittaker CA , Ransom DG , DeSimone DW .

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Integrins containing the alpha2 and alpha3 subunits associate with the beta1 subunit to form distinct receptors with partially overlapping adhesive specificities. We report the cloning and sequence of cDNAs that encode the Xenopus orthologues of integrins alpha2 and alpha3 and the expression of these subunits during embryogenesis. Integrin alpha2 and alpha3 mRNAs are first expressed in the dorsal mesoderm and developing notochord at gastrulation. We also show that alpha3 mRNAs are expressed in the entire marginal zone of gastrulae dorsalized with LiCl but that this localization is lost in embryos ventralized by ultraviolet light. Immunoblots reveal that the alpha3 protein is expressed throughout early development, however, the alpha2 protein is not detected until late tailbud stages. Injection of full-length alpha3 transcripts into the animal poles of fertilized eggs results in embryonic defects in paraxial mesoderm attributed to the failure of somites to form segments. Injection of the alpha3 transcripts into the vegetal pole and overexpression of a 5'-truncated alpha3 control construct have no apparent affect on development or somite formation. These data suggest that normal position-specific expression of integrins is important in maintaining the proper organization of tissues during early amphibian morphogenesis.

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Species referenced: Xenopus laevis
Genes referenced: itga2 itga3 sall1
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	Fig. 1. Schematic of integrin a3 and a2 proteins and the corresponding Xenopus cDNAs. The hatched boxes indicate the signal sequence, solid boxes indicate the transmembrane domains and the three shaded boxes indicate the locations of the metal-binding domains. The stippled box in a2 represents the putative I-domain. The a3 PCR3 cDNA was used to obtain G, D and J from an XTC cell cDNA library. Clones FF2 and MM were obtained from the stage 45 and 17 cDNA libraries, respectively. The a2 cDNAs A3 and B9 were both obtained from the stage 17 library. B, BamH1; H, Hind3; P, Pst1; R, EcoR1; S, Sal1; X, Xho1.
	Fig. 2. Comparison of the deduced amino acid sequences of the Xenopus (xa3, GenBank accession number L43057) and human (ha3, GenBank accession number M59911; Takada et al., 1991) integrin a3 subunits. Also aligned is the deduced sequence of the PCR fragment amplified from XTC cells (PCR3). Regions of identity with the complete xa3 sequence are indicated by hyphens. Gaps in the alignments are indicated by dots. Amino acid number 1 corresponds to the first amino acid in the mature a3 protein based on direct sequencing of the human a3 N-terminus (Takada et al., 1991). Position minus32 of ha3 corresponds to the initiating methionine and the beginning of the signal sequence. The single underline at the N-terminus of ha3 corresponds to the region of the human cDNA used to prepare the hxa3 construct. Conserved cysteines (C) in the extracellular domain of xa3 are indicated in bold; an additional cysteine in xa3 is underlined (C). Potential N-linked carbohydrate addition sites in the extracellular domain of xa3 are indicated by bold dots. The putative metal-binding domains are shaded and the predicted transmembrane domain boxed. The putative dibasic cleavage site in the extracellular domain (KRRRR) is marked by crosses (†). The C-terminal peptide used for antibody production is indicated by the double underline.
	Fig. 3. Alignment of the partial deduced amino acid sequence of the Xenopus integrin a2 subunit (xa2, GenBank accession number L43058) and the corresponding segment of the human integrin a2 subunit (ha2, GenBank accession number X17033; Takada and Hemler, 1989). Also aligned is the sequence deduced from a PCR amplified segment of the a2 subunit prepared from Xenopus embryonic stage 17 cDNA template (PCR2, GenBank accession number L10186; Whittaker and DeSimone, 1993). Regions of identity with the xa2 sequence are indicated by hyphens. Gaps in the alignments are indicated by dots. Amino acid numbering corresponds to the complete human a2 sequence (Takada and Hemler, 1989). Conserved cysteines (C) in the extracellular domain of xa2 are indicated in bold; an additional cysteine in xa2 is underlined (C). Potential N-linked carbohydrate addition sites in the extracellular domain of xa2 are indicated by bold dots. The putative metal-binding domains are shaded and the predicted transmembrane domain boxed. The C-terminal peptide used for antibody production is indicated by the double underline.
	Fig. 4. Northern blot analysis. Total RNAs (20 mg/each lane) from the embryonic stages indicated were separated on 1.2% denaturing agarose gels and transferred to nylon for hybridization. Following hybridization with a 32P-labeled a3 cDNA (A), the membrane was stripped and reprobed with an a2 cDNA (B). (C) Photograph of the ethidium bromide stained gel prior to transfer to nylon, demonstrating equal loading of RNA in each lane.
	Fig. 5. Spatial expression of integrin a2 and a3 mRNAs. Whole-mount in situ hybridization was performed on albino Xenopus embryos using antisense digoxygenin-labeled a2 (A–C) or a3 (D–F) transcripts. (A) Stage 11.5 gastrula, anterior to the right, dorsal to the top with staining of a2 in posterior notochord (arrowhead). (B) Stage 19 neurula with a2 staining localized to the posterior end of the notochord (arrowhead). (C) Slightly oblique dorsal view of a stage 32 tailbud embryo. a2 expression persists in the posterior tip of the notochord (arrowhead). (D) Dorsal lip view of a normal stage 10.5 control gastrula. The cells expressing a3 are the dorsal involuting mesoderm (arrowhead). (E) Side view of a gastrula stage embryo that has been dorsalized by exposure to LiCl. Arrowheads indicate the circumblastoporal expression of a3 encircling the yolk plug. (F) Side view of an embryo that has been ventralized by UV irradiation of the vegetal hemisphere. Embryos treated with UV light lack detectable a3 staining. Labeled sense transcripts for both a2 and a3 were included as specificity controls in parallel hybridizations for each experiment (data not shown). y, yolk plug; bc, blastocoel; ar, archenteron.
	Fig. 6. Characterization of antibodies. (A) Immunoblots of total protein extracts from stage 35 embryos were incubated with either the complete anti-a3 immune sera (D3F) or affinity purified antibody (D3FAP). XTC cells were first immunoprecipitated with the anti-b1 McAb 8C8, subjected to SDS-PAGE under reducing (+) and non-reducing (-) conditions and then immunoblotted. D3FAP recognizes 135–140 kDa doublet a3 bands from the total stage 35 embryo extract and a single 140 kDa a3 band in the b1 immunoprecipitate from XTC cells (non-reduced). The D3FAP immunoreactive C-terminal fragment of a3 runs off the 7% gel under reducing conditions (XTC + DTT lane). (B) Immunoblots of stage 45 embryo total protein extracts incubated with complete anti-Xenopus a2 immune sera (D5F) or affinity purified antibodies (D5FAP). The D5FAP antibody recognizes a single a2 protein of 160 kDa, which is not cleaved upon reduction (-). (C) Immunoblots of XTC cell extracts immunoprecipitated with D3FAP and incubated with either McAb 2F10 or 8C8. (D) XTC cell immunoblot of D3FAP immunoprecipitated material (1) or D3FAP immunodepleted extract (2). 2F10 recognizes the 140 kDa a3 band from the immunoprecipitate but not the immunodepleted supernatant. (E) Western blots of Xenopus a3 transfected (T) and non-transfected (N) mouse NIH 3T3 cells. 2F10 and D3FAP recognize the 140 kDa a3 protein in only the transfected cells. The anti-b1 antibody 363 blot is included as a loading control. All gel samples were prepared either in the presence (+) or absence (-) of dithiothreitol (DTT).
	Fig. 7. Developmental time course of a3 (A) and a2 (B) protein expression by Western blot of whole embryo extracts at the indicated stages. One embryo equivalent was loaded onto each lane of the gel. D3FAP (A) recognizes the putative 135 kDa immature form of a3 in the egg and in cleavage stage embryos. The 140 kDa form of the protein is first detected at the neurula stage. The levels of both forms increase as development proceeds. In contrast, the 160 kDa a2 protein is not detected by D5FAP until much later in development (B). 7% SDS-PAGE gels were run under non-reducing conditions.
	Fig. 8. (A) Schematic of transcripts used for a3 ectopic overexpression experiments. hxa3 encodes a full length chimeric transcript containing the human a3 signal sequence. txa3 represents a truncated, non-translatable form of the a3 transcript. (B) Western blots of total protein extracts derived from stage 9 embryos injected with the hxa3 or txa3 transcripts. D3FAP recognizes low levels of endogenous a3 in uninjected (U) and txa3 injected embryos. In contrast, high levels of a3 are detected in hxa3 injected embryos. The 8C8 Western blot is included as a control to demonstrate equal loading of samples from injected and non-injected embryos.
	Fig. 9. Overexpression of a3 results in shortening along the antero-posterior axis and disruptions in somite segmentation. (A,B,E) Embryos derived from control txa3 injected eggs. (C,D,F) Embryos derived from hxa3 injected eggs. (A,C) Whole-mount immunostaining with the somite-specific 12-101 antibody (Kintner and Brockes, 1984). (B,D,E,F) Hematoxylin and eosin stained paraffin sections of injected embryos. Control injected embryos display a normal pattern of somites (A,B) including well defined segments (E, arrowheads) and aligned myocyte nuclei (E, arrow). In contrast, hxa3 injected embryos are shortened along the antero-posterior axis and have poorly defined somitic segments (C,D). Although myocyte nuclei are often observed in alignment (F, arrow), segmental boundaries are not resolved in affected regions. The embryo in (D) shows a severe example of disruption of segmentation of the somitic fields on both sides of the embryo. Note the appearance of a compact but relatively normal notochord in this embryo. e, ectoderm; s, somite; n, notochord. The scale bar is 400 mm in (D) and 40 mm in (F).
	itga2 (integrin subunit alpha 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 19, lateral view, anterior right, dorsal up.
	itga2 (integrin subunit alpha 2) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior right, dorsal up.
	itga3 (integrin subunit alpha 3) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 10.5, vegetal view, dorsal up.