Herrmann H et al. (1989), Expression of intermediate filament proteins du...

XB-ART-26932

Development 1989 Feb 01;1052:299-307.

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Expression of intermediate filament proteins during development of Xenopus laevis. II. Identification and molecular characterization of desmin.

Herrmann H , Fouquet B , Franke WW .

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During embryogenesis of avian and mammalian species the formation of intermediate filaments (IFs) containing desmin is characteristic for myogenesis. In view of important differences of patterns of IF protein expression in embryogenic pathways of amphibia on the one hand and birds and mammals on the other, we have decided to study the expression of desmin during early embryogenesis of Xenopus laevis by cDNA hybridization and antibody reactions. Here we describe the isolation of a cDNA clone encoding Xenopus desmin and the deduced amino acid sequence (458 residues; Mr 52,800) which displays a very high degree of conservation during vertebrate evolution from Xenopus to chicken and hamster, with a similar degree of sequence divergence between all three species compared. In addition, we have noted, by both cDNA-hybrid-selection-translation and immunoblotting of cytoskeletal proteins a second desmin-related polypeptide of Mr approximately 49,000. RNA (Northern) blot analyses show the occurrence of three different desmin mRNAs (1.9, 2.6 and 3.0 kb) which seem to represent different polyadenylation sites, displaying quantitative differences in different kinds of muscle tissues. During embryogenesis, desmin mRNA has first been detected in stage-14 embryos and then increases drastically to high levels at stage 18 and thereafter. Immunofluorescence microscopy using desmin-specific antibodies shows that this synthesis of desmin is restricted to somite tissue. The embryonic time course of synthesis of desmin and desmin mRNA is discussed in relation to those of other muscle proteins.

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Species referenced: Xenopus laevis
Genes referenced: actl6a alb ncoa5 tbx2 vim
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	Fig. 1. Identification of the polypeptide encoded by the cDNA clone pXenDesl, using in vitro translation of mRNA selected from stage-18 embryo RNA and twodimensional coelectrophoresis of the radioactively labelled translation products with cytoskeletal proteins from the muscular layer of Xenopus oesophagus (horizontal arrow, IEF; vertical arrow; SDS-PAGE). (A) Coomassie-brilliant-blue-stained gel loaded with cytoskeletal proteins from the muscular layer of oesophagus. (B) Autoradiograph of the gel shown in A, revealing [35S]methionine-labelled products of in vitro translation of mRNA selected by hybridization with clone pXenDesl. (C) Immunoblot detection of desmin in a parallel gel electrophoresis, using monoclonal antibody to desmin. (D) Ponceau-S-stained nitrocellulose replica from gels loaded with cytoskeletal proteins from stage-18 embryos. (E) Immunoblot detection of desmin using monoclonal antibody to desmin. Arrows indicate the reactive polypeptides. Bars in A and B designate positions of immunoreactive desmin polypeptides as identified from the immunoblot in C. The second series of spots of lower MT in B are either the corresponding proteolytic breakdown products or represent a genuine desmin-related polypeptide; in A and C additional, more acidic desmin variants are evident, compared to the components seen in the translational assay (B) and in the immunoblot of the cytoskeletal fraction of stage-18 embryos (E). a, Actin; b, bovine serum albumin.
	Fig. 2. RNA blot analysis of gel electrophoretically separated desmin mRNAs from various Xenopus tissues. Lane 1, cardiac muscle; lane 2, skeletal muscle; lane 3, ovarian tissue; lane 4, XLKE-A6 cells. 10ng of total RNA were loaded and hybridized with a random-primed 32P-labelled probe of pXenDesl. Bars indicate positions of 28S and 18S Xenopus rDNAs. Note the presence of two (in lane 1) and three (in lane 2) major mRNAs of different sizes.
	Fig. 3. Nucleotide sequence of clone pXenDesl and deduced amino acid sequence of Xenopus laevis desmin. Asterisk denotes stop codon. Three putative polyadenylation signals are marked by bars.
	Fig. 4. Amino acid sequence comparison of Xenopus desmin (Xen), chicken desmin (Chk; taken from Geisler & Weber, 1984) and hamster desmin (Ham; taken from Quax et al. 1985). Bold-faced letters denote amino acids identical in Xenopus desmin and in at least one of the other two species. Amino acid sequences have been aligned for maximal homology; insertions introduced for this purpose are denoted by horizontal bars. The downward arrow demarcates the start and the upward arrow the end of the <r-helical rod domain. The dots represent positions a and d of the heptade convention to maximize coiled-coil configuration. The rod-domain contains two non-a--helical interruptions of 11 and 43 amino acids, respectively, giving rise to coiled-coil subdomains 1A (CIA), IB (C1B) and 2 (C2). The arrowhead indicates an alteration in the heptade pattern that probaby results in a 'stutter' in coil 2.
	Fig. 5. Highly conserved sequence feature found near the JV-terminus of the head domains of vimentin, desmin and neurone-specific IF protein c73IF. Vimentin and desmin from Xenopus (Xen), chicken (Chk) and hamster (Ham) and rat c73IF protein (taken from Leonard et al. 1988) are aligned with respect to the conserved nonapeptide consensus sequence (SSYRRXFGG). Bold-faced letters designate identical amino acids in all three proteins, including a conservative serine to threonine exchange in c73IF. In hamster vimentin and rat protein c73IF, a similar sequence precedes the nonapeptide (underlined). The motif PSFS present in Xenopus and hamster desmin appears in a modified form in protein c73IF as well as in chicken desmin (overlined). Note that in the desmins the sequence preceding the nonapeptide is also very similar.
	Fig. 6. RNA blot analysis of gel electrophoretically separated RNAs from various stages of Xenopus laevis development. Total RNA was prepared from embryos of the specific embryonal stage, and 20/xg each were loaded per slot for formaldehyde/agarose gel electrophoresis. Blotted RNA was hybridized with a random-primed, 32Plabelled probe of pXenDesl. Lane 1, unfertilized eggs; lane 2, stage 6-5 (morula); lane 3, stage 9 (fine cell blastula); lane 4, stage 11 (gastrula); lane 5, stage 14 (neural plate stage); lane 6, stage 18 (neural groove stage); lane 7, stage 28; lane 8, stage 39; lane 9, stage 42 (swimming tadpole). Bars indicate positions of Xenopus 28S and 18S rRNA. Note that first reaction is seen in stage 14 (lane 5).
	Fig. 7. Immunofluorescence microscopy performed on cryostat sections of snap-frozen stage-18 embryos of Xenopus laevis using a monoclonal desmin antibody. (A,B) The same field is shown in epifiuorescence (A) and phase-contrast (B) optics. The somites are brightly stained, all other cell layers are negative, ec, ectoderm; en, endoderm; n, neural groove; no, notochord; s, somite. Bar, 50um.