Baldwin TJ et al. (1988), Regulation of acetylcholine receptor transcript...

XB-ART-27672

J Cell Biol 1988 Feb 01;1062:469-78.

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Regulation of acetylcholine receptor transcript expression during development in Xenopus laevis.

Baldwin TJ , Yoshihara CM , Blackmer K , Kintner CR , Burden SJ .

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The level of transcripts encoding the skeletal muscle acetylcholine receptor (AChR) was determined during embryonic development in Xenopus laevis. cDNAs encoding the alpha, gamma, and delta subunits of the Xenopus AChR were isolated from Xenopus embryo cDNA libraries using Torpedo AChR cDNAs as probes. The Xenopus AChR cDNAs have greater than 60% amino acid sequence homology to their Torpedo homologues and hybridize to transcripts that are restricted to the somites of developing embryos. Northern blot analysis demonstrates that a 2.3-kb transcript hybridizes to the alpha subunit cDNA, a 2.4-kb transcript hybridizes to the gamma subunit cDNA, and that two transcripts, of 1.9 and 2.5 kb, hybridize to the delta subunit cDNA. RNase protection assays demonstrate that transcripts encoding alpha, gamma, and delta subunits are coordinately expressed at late gastrula and that the amount of each transcript increases in parallel with muscle-specific actin mRNA during the ensuing 12 h. After the onset of muscle activity the level of actin mRNA per somite remains relatively constant, whereas the level of alpha subunit and delta subunit transcripts decrease fourfold per somite and the level of gamma subunit transcript decreases greater than 50-fold per somite. The decrease in amount of AChR transcripts per somite, however, occurs when embryos are paralyzed with local anaesthetic during their development. These results demonstrate that AChR transcripts in Xenopus are initially expressed coordinately, but that gamma subunit transcript levels are regulated differently than alpha and delta at later stages. Moreover, these results demonstrate that AChR transcript levels in Xenopus myotomal muscle cells are not responsive to electrical activity and suggest that AChR transcript levels are influenced by other regulatory controls.

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Species referenced: Xenopus laevis
Genes referenced: actc1 chrna1 chrnd chrng mt-tr trna
GO keywords: larval somatic muscle development

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	Figure 1. Restriction map, sequencing strategy, nucleotide sequence, and deduced amino acid sequence of the alpha, gamma, and delta subunits of the Xenopus laevis skeletal muscle AChR. (Le~) Restriction map and sequencing strategy of the alpha, gamma, and delta subunits. Numbers indicate the 5' terminal nucleotide generated by cleavage. (Below and right) Nucleotide and deduced amino acid sequence of the alpha, gamma, and delta subunits. Nucleotide 1 indicates the first nucleotide of the codon encoding the amino terminal residue in the mature protein and nucleotides to the 5' side of this amino terminal residue are indicated with negative numbers. The number of the nucleotide residue at the end of each line is provided. The predicted amino acid sequence is shown above the nucleotide sequence. Amino acid residues are numbered beginning with the amino terminal residue of the mature protein and the preceeding amino acids of the signal sequence are indicated with negative numbers. Since mRNA encoding each subunit is longer than the corresponding cDNA, these sequences are incomplete.
	Figure 2. Alpha subunit transcript is confined to the somites of Xenopus embryos. (a) Bright-field optics of Giemsa-stained transverse section of stage 26 Xenopus embryo illustrates the neural tube (nt), notochord (n), somites (s), and epidermis (ep). (b) Dark-field optics illustrates the position of the autoradiographic grains, which are highly concentrated over the somites and not present over other tissue at a level higher than background. Bar, 50 ~tm.
	Figure 3. Northern blot analysis of poly(A) + RNA from Xenopus embryos. 1 gg of poly(A) + RNA isolated from stage 41 Xenopus embryos was fractionated by eleetrophoresis in a formaldehyde agarose (1%) gel, transferred to Zetabind, and hybridized to 32p-labeled AChR alpha (a), gamma ('D or delta subunit (6) cDNA probes (Materials and Methods). This filter was washed at moderate stringency (Materials and Methods). The RNAs that hybridize to the alpha, gamma, and delta probes migrate at 2.3 (tt), 2.4 (~/), and 1.9/ 2.5 kb (6), respectively. Washing at higher stringency did not reduce hybridization to AChR transcripts. The positions of RNA standards (4.4, 2.4, and 1.4 kb) are indicated at right. The pattern of Northern blots from earlier stages (stages 20 and 25) were identical to those from stage 41 embryo RNA except that gamma subunit transcript levels were comparable to those of alpha and delta at earlier stages. The filter was exposed to x-ray film with an intensifying screen at -70~ for 4 d.
	Figure 4. AChR transcripts are expressed at late gastrula. RNA from 25 embryo equivalents was included in hybridizations with 32p-labeled AChR subunit probes, whereas RNA from 2.5 embryo equivalents was included in hybridizations with the 32p-labeled cardiac actin probe (AC). A single batch of RNA isolated from each stage was used in protection assays with each probe, except that a different batch of stage 18 RNA was used in the protection as~y with the delta subunit probe. This second batch of stage 18 RNA contained less total RNA per embryo and correspondingly less actin mRNA per embryo. Quantitation of transcript levels is presented in Table I. The positions of protected fragments are indicated with arrowheads; the positions of the minor protected fragments in hybridizations with the delta subunit probe are indicated with arrows. The standard curve for delta subunit RNA is illustrated (std) and includes 0.5, 2.0, 5.0, and 10 pg of synthetic delta RNA. Developmental stages are indicated at the top of each lane; neither AChR nor actin mRNA was detected in eggs (lane E). Gels were exposed to x-ray films with an intensifying screen at -70~ for 70 h (actin) or 7 d (AChR).
	Figure 5. Gamma subunit transcript levels are regulated differently than alpha and delta subunit transcript levels. RNA from 10 embryo equivalents was included in hybridizations with 32p-labeled AChR subunit probes, whereas RNA from 1 embryo equivalent was included in hybridizations with the 32p-labeled cardiac actin probe (AC). A single batch of RNA isolated from each stage was used in protection assays with each probe, except that a different batch of stage 25 RNA was used in the protection assay with the delta subunit probe. This second batch of stage 25 RNA contained less total RNA per embryo and correspondingly less actin mRNA per embryo. Quantitation of transcript levels is presented in Table I. Developmental stages are indicated at the top of each lane; neither AChR nor actin mRNA was detected in eggs (lane E). The positions of protected fragments are indicated with arrowheads. Gels were exposed to x-ray films with an intensifying screen at -70~ for 6.5 (actin) or 16 h (AChR).
	Figure 6. Embryos that remain nonmotile throughout development express normal levels of AChR transcripts. 50 gg of embryo RNA (10 embryo equivalents for all samples) was included in hybridizations with 32p-labeled AChR probes, whereas 5 gg of embryo RNA was included in hybridizations with 32p-labeled cardiac actin probe (AC). Developmental stages are indicated at the top of each lane; t indicates RNA from embryos that were paralyzed with tricaine. The positions of protected fragments are indicated with arrowheads. Gels were exposed to x-ray films with an intensifying screen at -70~ for 6.5 (actin) or 16 h (AChR).
	Figure 7. Alpha subunit transcript levels increase 100-fold in denervated adult muscle. The triceps femoris muscle of adult Xenopus was denervated and alpha subunit transcript levels in normal and denervated muscle were measured by RNase protection (Materials and Methods). The amount of alpha transcript in normal, innervated muscle (INN) is low and increases 100-fold in denervated muscle (DEN), whereas the amount of total mRNA as determined for Ef-1 alpha transcript in normal, innerva~xl (INN), and denervated muscle (DEN) is indistinguishable. There is no protection of either probe in hybridizations with tRNA. The positions of protected fragments are indicated with arrowheads. The gels were exposed to x-ray films with an intensifying screen at -70~ for 1 d.

References [+] :

Anderson, Nerve-induced and spontaneous redistribution of acetylcholine receptors on cultured muscle cells. 1977, Pubmed, Xenbase