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Adhesion of cells to extracellular matrix proteins is mediated, in large part, by transmembrane receptors of the integrin family. The identification of specific integrins expressed in early embryos is an important first step to understanding the roles of these receptors in developmental processes. We have used polymerase chain reaction methods and degenerate oligodeoxynucleotide primers to identify and clone Xenopus integrin alpha subunits from neurula-stage (stage 17) cDNA. Partial cDNAs encoding integrin subunits alpha 2, alpha 3, alpha 4, alpha 5, alpha 6 and an alpha IIb-related subunit were cloned and used to investigate integrin mRNA expression in early embryos by RNase protection assay and whole-mount in situ hybridization methods. Considerable integrin diversity is apparent early in development with integrins alpha 2, alpha 3, alpha 4, alpha 5 and alpha 6 each expressed by the end of gastrulation. Both alpha 3 and alpha 5 are expressed as maternal mRNAs. Zygotic expression of alpha 2, alpha 3, alpha 4 and alpha 6 transcripts begins during gastrulation. Integrin alpha 5 is expressed at relatively high levels during cleavage, blastula and gastrula stages suggesting that it may represent the major integrin expressed in the early embryo. We demonstrated previously that integrin beta 1 protein synthesis remains constant following induction of stage 8animal cap cells with activin (Smith, J. C., Symes, K., Hynes, R. O. and DeSimone, D. W. (1990) Development 108, 289-298.). Here we report that integrin alpha 3, alpha 4 and alpha 6 mRNA levels increase following induction with 10 U/ml activin-A whereas alpha 5, beta 1 and beta 3 mRNA levels remain unchanged. Whole-mount in situ hybridization reveals that alpha 3 mRNAs are expressed by cells of the involuting mesoderm in the dorsal lip region of early gastrulae. As gastrulation proceeds, alpha 3 expression is localized to a stripe of presumptive notochordal cells along the dorsal midline. In neurulae, alpha 3 mRNA is highly expressed in the notochord but becomes progressively more restricted to the caudalmost portion of this tissue as development proceeds from tailbud to tadpole stages. In addition, alpha 3 is expressed in the forebrain region of later stage embryos. These data suggest that integrin-mediated adhesion may be involved in the process of mesoderm involution at gastrulation and the organization of tissues during embryogenesis.
Fig. 1. Deduced amino acid sequences of Xenopus integrin a subunit partial cDNAs. Schematic illustrates the general structural features
of a typical integrin a subunit. I domain box indicates the location of a 180 amino acid homologous segment present in the extracellular
domains of some a subunits. Transmembrane domain (filled box), location of disulfide-bonded postranslational cleavage site present in
some a subunits and the N-terminal signal sequence (hatched box) are also indicated. The degenerate oligodeoxynucleotide primers (see
Materials and methods and Erle et al., 1991) used in the PCR amplification (AF and AR) are located in the highly conserved region of the
a subunit cation-binding domains (shaded boxes). Small arrows delineate the amplified regions obtained by PCR of Xenopus neurulastage
cDNA. Deduced amino acid sequences of amplified regions (minus 5¢ and 3¢ oligo sequences) were aligned using the PILEUP
program of the GCG sequence analysis software package (Devereux et al., 1984). Assignments of a subunit identities are based on
pairwise comparisons with all known, published integrin a subunits sequences (see Fig. 2 and Table 1). Only 5- and 6-fold amino acid
identities are highlighted. , conserved Asp residues of cation binding motifs; , location of Gly-Ala-Pro sequence conserved in all
integrin a subunits.
Fig. 2. Dendrogram of pairwise comparisons of Xenopus a
subunit cDNAs with published integrin a subunit sequences.
Deduced amino acid sequences of PCR-amplified Xenopus partial
cDNAs were compared to the corresponding regions of published
integrin a subunits using the PILEUP program of the GCG
sequence analysis software package (Devereux et al., 1984).
Progressive pairwise alignments result in the clustering of the
most highly related pairs of sequences. Distance along horizontal
axis is proportional to differences between clustered sequences
and provides an approximate measure of amino acid similarity.
Distance along the vertical axis has no significance (i.e.,
placement of clusters from top to bottom is in no particular order).
The assignment of Xenopus a subunit identity is based on best %
similarity score with known sequences (Table 1). The putative
Xenopus a2, a3, a4, a5, a6 and aIIb subunits are indicated by the
arrowheads. Sequence data for other a subunits: rat a1 (Ignatius et
al., 1990); human a2 (Takada and Hemler, 1989); human a3
(Tsuji et al., 1991; Takada et al., 1991); hamster a3 (Tsuji et al.,
1990); human a4 (Takada et al., 1989); mouse a4 (Neuhaus et al.,
1991); human a5 (Argraves et al., 1987); human a6 (Tamura et
al., 1990); chicken a6, (deCurtis et al., 1991); rat a7 (Song et al.,
1992); chicken a8 (Bossy et al., 1991); human mac-1 (Arnaout et
al., 1988); mouse mac-1 (Pytela, 1988); human p150 (Corbi et al.,
1990); human lfa-1 (Larson et al., 1989); human aV (Suzuki et al.,
1987); chicken aV (Bossy and Reichardt, 1990); human aIIb
(Poncz et al., 1987); Drosophila PS-2 (Bogaert et al., 1987).
Fig. 3. Developmental time course of a subunit mRNA
expression determined by RNase protection analysis.10 embryo
equivalents of total RNA per lane were hybridized with 32Plabelled
antisense transcripts followed by digestion with RNase.
Protected fragments are indicated by the arrowheads. The larger
sized undigested probes run with each set of reactions are not
shown. RNase protections of integrin b1 and EF1-a (not shown)
were included as controls for RNA loading at each stage. The a3,
a5 and b1 subunits are present in egg and cleavage-stage embryos
as maternal mRNAs. Accumulation of a2, a4 and a6 subunit
mRNAs is first noted during gastrulation with aIIb appearing
during neurulation. The a2, a3, a4, a5, a6 and b1 autoradiograms
were exposed for 2 days and the aIIb for 11 days. Embryos staged
according to Nieuwkoop and Faber, 1967. Approximate size of
protected fragments indicated by arrowheads for a2, a4, a5, a6,
and aIIb is 245 nt. The a3 protected fragment is about 460 nt and
b1 is 531 nt. t, tRNA control lane.
Fig. 4. Integrin a subunit mRNA expression during gastrulation,
mesoderm induction and in XTC cells. RNase protection analyses
of total RNAs obtained from embryos, activin-A induced and
uninduced animal caps, and the Xenopus XTC cell line. (A)
Embryonic stages 8-13. 10 embryo equivalents of mRNA
hybridized with 32P-labelled antisense transcripts for Xenopus
integrins a2, a3, a4, a5, a6, b1, b3 and for EF1-a. Equal quantities
(mass) of animal cap RNA (20 μg/lane, 35 μg in a4 sample) were
used in the activin-A-induced (I; 10 units/ml activin-A) and
uninduced (U) samples. X, 10 μg total RNA was used for each
XTC sample except for a4 which contained 40 μg. EF1-a probe
was included in each protection assay as a control to normalize for
RNA loading (only 1 representative example of EF1-a, from the
a5 experiment, is shown). (B) Control for induction experiments
shown in A. Brachyury is expressed in induced (I) but not in
uninduced (U) animal caps. EF1-a mRNA levels are the same in
both the induced and uninduced samples. No Xbra mRNA is
detected in XTC cells (X). P, undigested Xbra probe. Sizes of
protected fragments for a subunits and the b1 subunit are same as
in Fig. 3. Approximate sizes of other protected fragments are
indicated by arrowheads: b3, 450 nt; Xbra, 410 nt; and EF1-a, 75
nt. Each assay was exposed to film for 48 hours with the exception
of a2 and a4, which were exposed for 8 days.
Fig. 5. Spatial expression of integrin a 3 mRNA. Whole-mount in situ hybridiztion was performed on albino Xenopus embryos using digoxigenin-labelled a3 antisense (A-K) and sense (L) transcripts. Hybridization signal visualized as alkaline phosphatase chromogenic
reaction product. Anterior end of embryos is oriented to the right in D-K. (A) Gastrula stage 10.5, vegetal pole view; staining of individual cells in dorsal lip region (arrow). (B) Stage 10.5, side view of dorsal involuting mesoderm (arrow). Arrowheads in B and D indicate faint background staining of outer ectodermal layer also observed with gastrula sense controls (L). Blastocoel roof in B is partially collapsed (arrowhead). (C) Dorsal and (D) side view of stage 12 embryo. Note intense staining of the presumptive notochord (arrows). (E) Dorsal and (F) side view of stage 20 embryo. Expression of a 3 observed along entire length of notochord. Anterior progression of staining extends to the prechordal plate (F). (G) Stage 24, side view. Expression of a3 mRNA in the notochord (arrow) is decreased in the anterior portion. (H) Stage 28 and (I) stage 31 embryos, side views. Staining limited to caudal portion of notochord in the developing tail bud (I). Increased staining detected in the head with pronounced expression at the level of the forebrain (arrowheads). (J) Detail of a stage 31 tail bud. Note intense staining at the caudal tip of the notochord (arrow). (K) Stage 39, optical section of anterior end of embryo visualized by differential interference contrast microscopy. Arrowheads indicate borders of a3 mRNA expression in the prosencephalon. (L) Representative tail bud, neurula and gastrula hybridized with a3 sense transcript. Note faint background staining in ectoderm of gastrula and head of tail bud-stage embryo. Scale bar in A, same as in C,D,J,K, equals 100 fliD. Scale bar in B, same as in E-H, equals 200 fliD. Scale bars in I and L equal400 fliD. Y, yolk plug in A-D
