Ransom DG et al. (1993), Integrin expression in early amphibian embryos:...

XB-ART-22069

Dev Biol 1993 Nov 01;1601:265-75. doi: 10.1006/dbio.1993.1304.

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Integrin expression in early amphibian embryos: cDNA cloning and characterization of Xenopus beta 1, beta 2, beta 3, and beta 6 subunits.

Ransom DG , Hens MD , DeSimone DW .

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During embryogenesis cells modulate their adhesion to other cells and the surrounding extracellular matrix, in part, through the combination of integrins they express. In order to identify integrins that may mediate morphogenetic cell movements in the early Xenopus embryo, we have used polymerase chain reaction methods to isolate cDNAs encoding Xenopus integrin beta subunits. Based on deduced amino acid sequence, they are identified as homologs of human integrins beta 1, beta 2, beta 3 and beta 6. We also report the cloning and sequencing of cDNAs covering the complete coding region of Xenopus beta 3. Embryonic patterns of expression for these integrin beta subunit mRNAs have been examined both by RNase protection analysis and by whole mount in situ hybridization. In the early embryo the beta 1 subunit is encoded by a maternally transcribed mRNA expressed in all cells, but is most abundant in ectoderm and mesoderm. In contrast, Xenopus beta 3 mRNA is detected in the epidermis, bottle cells of the neural groove, and a subset of cells arising from the ventral blood islands. The beta 2 and beta 6 mRNAs are expressed at high levels in late tailbud stages, although very low levels of beta 6 are also detected in eggs and early embryos. These data provide evidence that multiple integrins are expressed at the earliest stages of vertebrate development coincident with the onset of morphogenesis.

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Species referenced: Xenopus laevis
Genes referenced: gcg itgb1 itgb3 mt-tr tbx2 trna uqcc6

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	FIG. 1. Cloning of Xenopu.~ integrin {j subunit cDNAs by PCR amplification. The deduced atnino acid sequences of amplified Xeno-pus integrin cDNAs ar<) aligned with their human homologs below a schematic representation of a typical {j snbunit. Arrowheads indicate regions amplified (stippled box) u~ing degenerate oligodeoxynucleotide primers (B" and BR). The locations of the signal sequence (hatched box), cy3teine·rich regions (vertical lined boxes), and the transmembrane domain (black box) are indicated. Gaps in the {3, sequence correspond to regions not represented in the amplified eDNA. GcnBank Accession Nnmbers for nneleotidc sequences are L13.S92 (fJ2), LJ.3591 (fJ8), and L13468 (116).
	FIG. 2. A dendrogram of amino acid similarities between the Xeno· pits and human integrin {:J subunits along the region amplified by PCR. The results of BESTFIT and PILEUP comparisons (GCG version 7; Devereux et al... 1984) were used to generate a dendrogram illustrating the <lpproximale percentage s imilarities b€tween the predicted amino acid sequences of the amplified Xe1W}rus integrin cDNAs and previously published human {3 subunits. The distance along the X axis is proportional to the degree of similarity between sequences. Sequence data for human integrin fJ subunits: {J1 (Argraves et aL, 1987), {32 (Ki· shimoto et at., 1987), [j, (Fitzgerald et al., 1987), (.J, (Hogervorst et al., 1990), #5 (.McLean eta!, 1990), tJ. (Sheppard et aL,1990), {J7 (Erie et al, 1991), and [38 (Moyle et. aL, 1991 ).
	FIG. 3. Map of Xenopu.s integrin {J3 subunit: cDNAs. A s-chematic diagram of the predicted. protein (s-ee Fig. 1) is aligned with cDNAs obtained from the XTC cell line eDNA library. The positio11s of the PCR ))roduet used for cloning and RNase protections (Xtl8) and the probe used for N ortherns and in situ hybridizations (X,83- E.i.P) are indicated. Arrows \'epreser.t. sequenced ngions. Rt!presentative Testriction sites are also marked. Tbe sites are (H), Him:IIII, (P) Psti, (R) EcoRI, and (S) Sal!.
	Flo. 4. Comparison of Xemrpus and human integrin {38 subunit protein sequences. The amino acid sequences of Xenorms and human integrin {33 (Fitzgerald et ctL, 1987) are aligned for comparison. Identities are indicated by dashes in the human sequence and gaps are marked by dots. Cysteines are highlighted by shaded blocks. Arrows mark the predicted (von Heijne, 1986) $ignal sequence cleavage sites. Arrowheads denote potential sites of N·linked glycosylation. The transmembrane domains are underlined. Aspartic acid 143 (Dl45 in Xenopus (33) is marked by a star and lysine 151 in human {38 (K149 in Xenopus) is marked by a solid box. The nuei()Otide sequence for Xenqms Ps has been deposited with GenBank under Accession No. Ll3591.
	FlO. 5. Northern blot analysis reveals multiple integrin {33 transcripts. Northern blot analysis was performed on 10 pg of poly(A) selected RNA isolated from gastrula (stage 11.5), neurula (stage 17), t ail bud (stage 33), and tadpole (stag•J 45) stage embryos. In (A), the blot was hybridized at high stringency to a eDNA probe derived from t he coding region of Xenorms 11 •• (B) The same blot as in (A) was stripped and reprobcd with an EFl·a eDNA (Krieg et aJ, , 198!1).
	FIG. 6. Developmental expression of Xenopus integrin f3 subunit mRNAs. RNase protection analyses of intcgrins {31 , ,B2 , ,83 , and P~ were performed using 10 embryo equivalents of total RNA per assay. Arrows indicate the positions of the protected prol>cs. Control tRNA samples containing no embryonic RNA are also included with each ex11criment to identify unprotected but incompletely digested probe fragments. The EFI ·<T probe was included in all protection assays as an internal control. The EFl·<r panel shows a representative time course. Undigested probes (P) arc larger than the protected fragment.s due to the preSI!nce of vector sequence included in the transcripts. Arrowheads mark the positions of fully protected probes. The lanes are:(P) full-lenl.'th transcript prior to digestion; (t) lO !Lg tRNA control; (1) egg; (8, \1) blastula; (10, 11, 12) gastrula; (13, 17, 25) neurula; (35) tailbud: and (45} tadpole.
	FIG. 7. Spatial patterns of integrin {J1 mRNA expression analyzed by whole mount i-n situ hybridization. Whole mount in situ hybrid izations demonstrate that #1 n1RNA is expressed in a wide variety of embryonic tissues. (A) Vegetal view of a stage 11 gastrula; note increased levels of expression along the blastopore (arrow). (B) Dorsal view of a stage 20 neurula, staining in the notochord, somites. and brain. (C) Side view of the same embryo, staining in dorsal mesodermal and ect~ermal derivatives. (D) Side view of a stage 25 neurula with reduced levels of expre~:~sion in the notochord. Arrows show areas of pronounced expression in the brain. (E) Detail of the head of the same embryo in (D); arrows indicate staining in the branchial arches. (F) Stage 28 neurula with high levels of expression in the posterior notochord (arrows). (G) Stage 33/34 tailbud embryo with decreased levels of expression in the notochord and myotomes but continued staining at the tipo( the tail (arrow). (H) Stage 37/38 tail bud embryo with abundant expression of {J1 mRNA in the region of the heart (arrow) and decreased signal in dorsal structures.
	Fie. 8. Spatial pattet·ns of int.lgrin {33 mRNA expression analyzed by whole mount in situ hybridization. Whole n10unt in situ hybridizations reveal a more restricted pattern of integrin 13, mRNA expression in comparison to that of /31 • (A) Side view of a stage 17 neurula; note staining in a line of cells in the neural groove at the anterior end (arrow). (B) Dorsal view of the same embryo in (A), staining in the neural groove (arrow) and neural folds (arrowheads). (C) Dorsal view of a stage 18 neurula; {J3 mRNA expression in the neural groove (arrow) decreases beginning at the posterior end of the embryo and proceeding anteriorly. Arrowheads indicate staining along the neural folds. (D) Side view of a stage 30 embryo that has begun to express /33 mRNA in a subset ofventral blood island cells (arrow). (E) Side view of a stage :32/33 tailbud embryo before the onset of blood circulation; note the broadly distributed mass of stained ventral cells (arrows). (F} Stage 33/34 embryo after circulation has begun. The /13-positive cells are now found scattered throughout the embryo. (G, H) Stage 35/36 and 37/38 embryos have an increased number of cells expressing Ps associated with major blood vessels. (H) Note the line of stained cells along dorsal blood vessels (arrowheads) and in the regi()n o£ the heart (arrow}.
	itgb3 ( integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 17, lateral view, anterior left, dorsal up.
	itgb3 ( integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 30, lateral view, anterior left, dorsal up.
	itgb3 ( integrin, beta 3 (platelet glycoprotein IIIa, antigen CD61)) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 38, lateral view, anterior left, dorsal up.