Lallier TE et al. (1996), Integrin alpha 6 expression is required for ear...

XB-ART-17919

Development 1996 Aug 01;1228:2539-54. doi: 10.1242/dev.122.8.2539.

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Integrin alpha 6 expression is required for early nervous system development in Xenopus laevis.

Lallier TE , Whittaker CA , DeSimone DW .

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The integrin alpha 6 subunit pairs with both the beta 1 and beta 4 subunits to form a subfamily of laminin receptors. Here we report the cDNA cloning and primary sequence for the Xenopus homologue of the mammalian integrin alpha 6 subunit. We present data demonstrating the spatial and temporal expression of alpha 6 mRNA and protein during early development. Initially, alpha 6 transcripts are expressed in the dorsal ectoderm and future neural plate at the end of gastrulation. Later in development, alpha 6 mRNAs are expressed in a variety of neural derivatives, including the developing sensory placodes (otic and olfactory) and commissural neurons within the neural tube. Integrin alpha 6 is also expressed in the elongating pronephric duct as well as a subset of the rhombencephalic neural crest, which will form the Schwann cells lining several cranial nerves (VII, VIII and X). In vivo expression of an alpha 6 antisense transcript in the animal hemisphere leads to a reduction in alpha 6 protein expression, a loss of adhesion to laminin, and severe defects in normal development. In 35% of cases, reduced levels of alpha 6 expression result in embryos that complete gastrulation normally but arrest at neurulation prior to the formation of the neural plate. In an additional 22% of cases, embryos develop with severe axial defects, including complete loss of head or tail structures. In contrast, overexpression of the alpha 6 subunit by injection of full-length mRNA has no apparent effect on embryonic development. Co-injection of antisense and sense plasmid constructs results in a partial rescue of the antisense-generated phenotypes. These data indicate that the integrin alpha 6 subunit is critical for the early development of the nervous system in amphibians.

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Species referenced: Xenopus laevis
Genes referenced: fn1 itga6 itgb1 mt-tr tbx2 trna
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	Fig. 1. Cloning of Xenopus a6 cDNAs. Five cDNA clones were isolated from a Xenopus XTC-cell cDNA library, encompassing the entire coding region of the Xenopus homolog of the integrin a6 subunit. Two of these cDNAs (Xa6-A1 and Xa6-H1) were used to construct a clone containing the full-length coding region (Xa6-A1H1). Above this construct is a schematic representation of the a6 protein. The putative signal sequence (diagonal lines), divalent cation-binding domains (dark boxes), transmembrane domain (vertical lines) and B cytoplasmic domain variant (cross hatched) are also represented. The original PCR product (Whittaker and DeSimone, 1993) used to isolate these cDNAs and the probe used for RNAse protection assays are indicated above the peptide schematic. Arrowheads represent restriction endonuclease cleavage sites (H) HindIII, (E) EcoRI, (P) PstI, (X) XbaI.
	Fig. 2. Comparison of the deduced amino acid sequence of the Xenopus integrin a6 subunit and its human (Tamura et al., 1990), mouse (Hierick et al., 1993) and chick (de Curtis et al., 1991) homologues. Amino acid identities are indicated by dashes and gaps are marked by dots. The three (3) conserved metal-binding domains are indicated by gray shading. Amino acid numbers correspond to the Xenopus a6 sequence beginning with the putative first amino acid (phenylalanine) of the mature protein. Conserved cysteines (*) and potential sites of N-glycosylation (.) are indicated above the Xenopus sequence. A single non-paired cysteine within the Xenopus sequence is indicated (+), along with lone cysteines within the other vertebrate homologues (C in bold). The transmembrane domain is indicated by double overline, the conserved A cytoplasmic domain by underline, and the B cytoplasmic domain by double underline. The putative cleavage site is indicated by (Â¯), note the histidine (H in bold) in the Xenopus sequence. The nucleotide sequence for Xenopus integrin a6 has been deposited in GenBank under Accession No. L35051.
	Fig. 3. (A) Northern blot analysis of Xenopus integrin a6 mRNA. Equivalent amounts of poly(A)- selected mRNA from various stage Xenopus embryos were probed with the Xa6-A1 clone (corresponding to the 5Â¢ end of the coding region). Two a6 mRNAs (6.7 and 6.1 kb respectively) are detected throughout development, although the relative levels of expression vary between early (stage 11 and 17) and late stages (45). (B) RNAse protection analysis of the two Xenopus integrin a6 subunit cytoplasmic variants. Levels of expression for each Xenopus a6 mRNA were detected using a 495 nt probe (387 nt complementary to the Xenopus a6 mRNA and 108 nt of flanking plasmid RNA) that overlaps both the A and B cytoplasmic variants (cyto-A and cyto-B) of Xenopus a6. As a control for equivalent RNA loading, a 167 nt antisense integrin b1 probe was added to each sample. 10 embryo equivalents of total RNA isolated from various stage embryos (stages 10-40), RNA from a Xenopus cell line (XTC), in vitro transcribed Xenopus cyto-A integrin a6 sense transcript (Sa6), and tRNA control (t) were assayed using both probes. Protected RNA fragments of 387 nt (cyto-A) and 198 nt (cyto-B) were detected in all embryo and XTC cell RNA samples. The protected fragment in the Sa6 sample is slightly larger than the endogenous a6 mRNA due to the presence of vector sequences in the sense transcript that are also in the antisense probe. (P) lanes of undigested probes.
	Fig. 4. Localization of integrin a6 mRNA in early Xenopus embryos by whole-mount in situ hybridization. Stage 11.5 (A, dorsal side at top) and 12.5 (B, anterior at right) Xenopus embryos express a6 mRNA in their dorsal ectoderm (y, yolk plug). (C) During neurulation (stage 15, dorsal view, anterior at right), a6 mRNA is localized to the central region of the neural plate and the preplacodal ectoderm outlining the neural ridge, with elevated expression in the future hindbrain. Transverse sections of equivalently staged embryos reveal a6 mRNA in the thickening neural epithelium (arrowheads in G-I) as well as in the caudal ectoderm (I). (D) Lateral view of stage 15 embryo, reveals prominent a6 mRNA expression throughout the neural tube, as well as in the caudal ectoderm surrounding the closed blastopore (arrowheads). (E) Lateral view of a late neurula (stage 24), a6 mRNA is expressed throughout the neural tube, with prominent expression in the hindbrain, otic vesicle (arrowhead) and optic lobe (arrow), as well as the notochord (n). (F) Longitudinal view of similar stage embryo reveals that expression in the neural tube is restricted to the ventricular region. In addition, elevated levels of signal in the hindbrain represent expression in the otic vesicle and possibly rhombencephalic neural crest lateral to the neural tube (arrowheads).
	NIHFig. 5. Localization of integrin a6 subunit mRNA in stage 32 tailbud Xenopus embryos by whole-mount in situ hybridization. Views of stage 32 Xenopus embryos probed with either antisense (A,C-K) or sense (B) digoxigenin-labeled transcripts. The rostral end of each embryo is to the right in A-H). (C) Integrin a6 mRNA is expressed in discreet regions throughout the developing nervous system including the Schwann cells of the seventh, eighth and tenth (VII, VIII and X) cranial nerves (arrows), the olfactory placode (op), the otic vesicle (ov), (D) the trigeminal ganglia (tg) and lens (l), and (F) inter-neurons in the spinal cord (in). In the developing brain (H), a6 mRNA is expressed in the cells of the ventricular layer (v). In the developing kidney (E), integrin a6 mRNA is expressed in the pronephros (pn) and (G) the elongating pronephric duct (pnd). Transverse sections of whole-mount stained embryos are presented at the levels of the (I) hindbrain, (J) rostral spinal cord and (K) mid-thoracic region. a6 mRNA in the neural tube is detected in two rows of laterally symmetrical interneurons whose cell bodies reside midway along the dorsal-ventral axis of the neural tube (arrowheads). (I) In rostral regions of the embryo, a6 mRNA is present in the otic vesicle (ov) and within the pronephros (pn), and (J) more caudally in the pronephric duct (pnd).
	Fig. 6. (A) The expression of Xenopus integrin a6 mRNA in transfected NIH-3T3 cells was assayed by northern blot analysis. RNA isolated from NIH-3T3 cells, either transfected with the Xenopus integrin a6 subunit (B7, A8) or mock-transfected (3T3) was separated by agarose gel electrophoresis and probed with the 2100 bp cDNA, Xa6-A1. Transfected cells express an mRNA of 3.9 kb (arrow), which is recognized by the Xenopus integrin a6 probe. Mock transfected cells do not express this mRNA. (B) The expression of Xenopus integrin a6 protein by transfected NIH-3T3 cells was assayed by immunoblot analysis. Protein extracts from Xenopus a6 transfected (B7), Xenopus a3 transfected (Xa3: Meng, Whittaker and DeSimone, unpublished data) and mocktransfected (3T3) cells, and the Xenopus cell line (XTC) were separated by SDS-PAGE and immunoblotted with an antisera to the conserved cytoplasmic region of the integrin a6 A variant. NIH-3T3 cells express very low levels of endogenous integrin a6 protein, while transfected cells express high levels of the Xenopus a6 subunit (arrow). (C) Possible cleavage of Xenopus a6 protein into heavy and light chains was investigated by immunblot analysis. Samples from mock transfected (3T3), Xenopus a6 transfected (B7) and XTC cells (XTC) were either solubilized in the presence (+DTT) or absence (-DTT) of 1 mM dithiothreitol. Proteins were separated by 10% SDS-PAGE, and immunoblotted with the a6cytA antisera (arrow). Only intact Mr 128,000 a6 protein is detected under both reducing and non-reducing conditions.
	Fig. 7. The ligand-binding specificity of the Xenopus integrin a6 subunit was assayed by cell attachment assay. (A) NIH-3T3 cells transfected with the Xenopus integrin a6 subunit and mock transfectants, were allowed to adhere to substrata coated with fibronectin (FN), laminin (LN), collagen types I (Col I) and IV (Col IV), and ovalbumin (ov). Only a6 transfected cells bind to laminin. Xenopus a6 transfected and mock-transfected NIH-3T3 cells were assayed for their ability to adhere to fibronectin (FN) and laminin (LN) substrata presented in alternating 50 mm stripes on the tissue culture plate. (B) Mock transfected cells adhere only to the FNcoated stripes, while (C) Xenopus a6 transfected cells adhere to both FN- and LN-coated stripes.
	Fig. 8. Expression of the a6 subunit during development. Extracts from embryos at various stages of development were analyzed by immunoblot using an antisera directed against a human integrin a6 cytoplasmic domain peptide. The a6-specific protein of Mr 128,000 (arrow) is first detected in stage 13 (neural plate) embryos. Increased expression is noted through early tailbud-stage (stage 30) embryos. No protein was detected prior to or during mid-gastrulation (stage 11). (C) Control extract from NIH-3T3 cells transfected with Xenopus a6 integrin.
	Fig. 9. Expression of a6 antisense RNA disrupts neurulation and normal axial development. Single cell embryos were injected with control CSKA (A,C) and Xa6-CSKA-A antisense plasmids (B,D-H). Control embryos develop normally to early neurula (A, stage 16) and tailbud (C, stage 32) stages, while antisense-injected embryos exhibit a range of defects at these stages (B,D-F). (B) At early stages, approximately 50% of embryos lack neural plates. (D) Later in development (stage 32), 35% of embryos have failed to neurulate, while (F) approximately 10% of embryos fail to gastrulate properly. (E) Another 20% have severe, but regionally localized, axial defects such as the lack of tail structures, the lack of head structures and/or curved neural axis. Antisense-injected embryos can be rescued to normal or near-normal phenotypes by injection of either Xenopus a6 mRNA (G, Xa6-Sp64T) or sense plasmid constructs (H, Xa6- CSKA-S). The variability of rescued phenotypes is evident in representative embryos (H).
	Fig. 10. The distribution of plasmid driven antisense and sense transcripts in early embryos was determined by in situ hybridization. None of these embryos have been cleared so that transcripts in the superficial cells could be delineated more clearly. Both sense (A,D) and antisense (B,E) transcripts are detected in late blastula/early gastrula-stage embryos derived from plasmid-injected single cell embryos. The transcripts are routinely detected in cells representing 15-20% of the total surface area of the embryo. In embryos that exhibit severe neurulation defects (i.e., neurulation arrest, Fig. 9D) following antisense Xa6-CSKA-A injection (C, F), transcripts are localized in the dorsal region of the embryo, often restricted to the midline region of the presumptive neural plate and its underlying mesoderm.
	Fig. 11. Endogenous a6 protein levels are reduced in embryos expressing antisense a6 RNAs. Protein levels for the integrin (A) a6, (B) a3 and (C) b1 subunits were assayed by immunoblot analyses (AÂ¢ represents a longer exposure of A). Embryos were assayed at two developmental stages corresponding to early neurula (stage 16) and early tailbud (stage 32). In order to increase detection of a6 protein levels from stage 16 embryos, 4 embryo equivalents were assayed per lane. Stage 32 samples represent 1 embryo equivalent per lane. Embryos were injected at the single cell stage with control CSKA plasmid containing no insert (P), full-length Xenopus a6 integrin in the sense orientation (S), or partial length Xenopus a6 integrin in the antisense orientation (AS). Antisense-injected stage 32 embryos were further subdivided into groups representing the three major phenotypes observed, as listed in Table 2 and shown in Fig. 9; gastrulation defects (AG), no neural plate (AN) and defects of axial structures (AA). (D) Relative protein expression was quantified by densitometric scans of fluorograms from four separate experiments. Relative expression of a6 integrin protein is presented as mean (Â±s.e.m.) values from the four sets of densitometric data. Densitometric data was normalized within each experiment, with the value for control (P)-injected embryos being 100%. Data from stage 16 and stage 32 embryos were calculated separately.
	Fig. 12. Endogenous a6 protein levels were reduced consistently relative to b1 protein levels in separate groups of embryos expressing a6 antisense transcripts. (A) Extracts of Xenopus a6 integrin transfected cells (C), uninjected control (control) and sibling embryos injected with Xa6-CSKA-A (antisense) were analyzed by western blot using the a6 antisera (a6cytoA) and a monoclonal antibody to b1 integrin (8C8). The levels of a6 protein are reproducibly reduced relative to the b1 subunit in each of three sets of pooled embryos (2 embryos per lane). (B) Approximately 80% of a6 the protein is eliminated in antisense-treated embryos, while less than 10% of the b1 protein was reduced. Data in (B) represents pooled densitometric scans from three separate experiments (3 samples per experiment as shown in A) involving simultaneous detection with both the a6cytoA and 8C8 antibodies.
	Fig. 13. Expression of antisense a6 transcripts reduces embryonic cell attachment to laminin, but not fibronectin. Cells were isolated from Xenopus embryos injected with Xa6-CSKA-A (antisense) or uninjected siblings (control) by manually dissecting the presumptive neural plate and associated mesoderm from stage 13 embryos in Ca2+/Mg2+-free media. Cells were allowed to adhere to fibronectinor laminin-coated substrata (A). Relative cell attachment was determined by counting the number of adherent cells on each substratum for twelve non-overlapping microscopic fields in three experiments. These data were normalized so that attachment to fibronectin equals 100% (B).
	itga6 (integrin, alpha 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 11.5, lateral view, anterior left, dorsal up.
	itga6 (integrin, alpha 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 15, dorsal view, anterior left.
	itga6 (integrin, alpha 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 24, lateral view, anterior left, dorsal up.
	itga6 (integrin, alpha 6) gene expression in Xenopus laevis embryo, assayed via in situ hybridization, NF stage 32, lateral view, anterior left, dorsal up.