Fouquet B et al. (1988), Expression of intermediate filament proteins du...

XB-ART-27170

Development 1988 Dec 01;1044:533-48.

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Expression of intermediate filament proteins during development of Xenopus laevis. III. Identification of mRNAs encoding cytokeratins typical of complex epithelia.

Fouquet B , Herrmann H , Franz JK , Franke WW .

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A Xenopus laevis mRNA encoding a cytokeratin of the basic (type II) subfamily that is expressed in postgastrulation embryos was cDNA-cloned and sequenced. Comparison of the deduced amino acid sequence of this polypeptide (513 residues, calculated mol. wt 55,454; Mr approximately 58,000 on SDS-PAGE) with those of other cytokeratins revealed its relationship to certain type II cytokeratins of the same and other species, but also remarkable differences. Using a subclone representing the 3'-untranslated portion of the 2.4 kb mRNA encoding this cytokeratin, designated XenCK55(5/6), in Northern blot experiments, we found that it differs from the only other Xenopus type II cytokeratin known, i.e. the simple epithelium-type component XenCK1(8), in that it is absent in unfertilized eggs and pregastrulation embryos. XenCK55(5/6) mRNA was first detected at gastrulation (stage 11) and found to rapidly increase during neurulation and further development. It was also identified in Xenopus laevis cultured kidney epithelial cells of the line A6 and in the adult animal where it is a major polypeptide in the oesophageal mucosa but absent in most other tissues examined. The pattern of XenCK55(5/6) expression during embryonic development was similar to that reported for the type I polypeptides of the 'XK81 subfamily' previously reported to be embryo-specific and absent in adult tissues. Therefore, we used a XK81 mRNA probe representing the 3'-untranslated region in Northern blots, S1 nuclease and hybrid-selection-translation assays and found the approximately 1.6 kb XK81 mRNA and the resulting protein of Mr approximately 48,000 not only in postgastrula embryos and tadpoles but also in the oesophagus of adult animals. Our results show that both these type II and type I cytokeratins are synthesized only on gastrulation and are very actively produced in early developmental stages but is continued in at least one epithelium of the adult organism. These observations raise doubts on the occurrence of Xenopus cytokeratins that are strictly specific for certain embryonic or larval stages and absent in the adult. They rather suggest that embryonically expressed cytokeratins are also produced in some adult tissues, although in a restricted pattern of tissue and cell type distribution.

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Species referenced: Xenopus laevis
Genes referenced: actl6a alb krt12.4 krt70 mt-tr ncoa5 tbx2 trna
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	Fig. 1. Nucleotide sequence and deduced amino acid sequence of Xenopus laevis cDNA clone pXenCK55(5/6) encoding a type II cytokeratin. Asterisk denotes stop codon. The poly(A) region and the portion of the 5'-untranslated region of the mRNA are not represented in the clones.
	Fig. 2. Amino acid comparison of Xenopus cytokeratin Xen55(5/6), Xenopus cytokeratin XenCKl(8) (taken from Franz & Franke, 1986), the partial sequence of human cytokeratin K5 (from Lersch & Fuchs, 1988) and human cytokeratin K6 (from Tyner et al. 1985). The asterisk in the XenCK55(5/6) sequence indicates the problem of designation in relation to the human cytokeratin catalogue of Moll et al. (1982a). This protein migrates with an apparent Mr of 58000 on SDS-PAGE and shares sequence features in the head and tail region with human cytokeratins K5 and K6. Sequences are aligned to obtain maximal homology, deletions introduced for this purpose are denoted by horizontal bars. Bold-faced letters denote amino acids identical in Xenopus cytokeratin XenCK55(5/6) and at least one of the other basic cytokeratins. The downward arrow indicates the start and the upward arrow the end of the cr-helical rod. The lines and dots above the sequence blocks indicate the extent of the coiled-coil subdomains CIA, Clb and C2 of the rod domain. The dots represent positions a and d of the heptade convention to maximize coiled-coil configuration. Note the absence of cysteines in both Xenopus cytokeratins, whereas 6 are present in human cytokeratin 6 and 3 in the partial sequence of human cytokeratin 5 (indicated by arrowheads).
	Fig. 3. Amino acid sequence comparison of the carboxyterminal region of Xenopus cytokeratin XenCK55(5/6) with that of various other cytokeratins of the same (type II) subfamily. Bovlll and BovIV are the respective bovine analogues to human cytokeratins 5 and 6. Human cytokeratins 1 (Johnson et al. 1985) and 4 (Leube et al. 1988) as well as the Xenopus type II cytokeratins 1(8) (Franz & Franke, 1986) and the type I cytokeratin XK81, here named HXenCK81Al' (Jonas et al. 1985) are shown for comparison. Sequences are aligned to achieve maximal homology for cytokeratins XenCK55(5/6) with Bovlll and BovIV. Boxes denote extensive sequence homology. Conservative exchanges have been included into the boxed area. The consensus sequence DGRKV found in certain type I and type II cytokeratins as well as in several nonkeratinous IF proteins has been underlined.
	Fig. 4. Identification of the polypeptide encoded by the XenCK55(5/6) mRNA, using in vitro translation of mRNA selected by hybridization to the cDNA clone pXenCK55(5/6), followed by two-dimensional coelectrophoresis of the radioactively labelled translation product with cytoskeletal proteins of oesophageal mucosa from adult Xenopus (first dimension: isoelectric focusing, direction denoted by horizontal arrow; second dimension: SDS-PAGE, vertical arrow). (A) Coomassie-blue-stained gel, major cytoskeletal proteins of oesophageal mucosa are denoted by brackets. Cytokeratins are numbered, the bracket without a number denotes an as yet unidentified cytoskeletal protein, a, actin; b, bovine serum albumin. (B) Autoradiograph of the gel shown in A, showing that the 35S-methionine-labelled products obtained after translation in vitro of the hybrid-selected embryonal stage-18 mRNA comigrate with the unlabelled oesophageal cytokeratin polypeptides 1 and 2. (C) Autoradiograph of a gel in parallel, showing the 35Smethionine- labelled product obtained after translation in vitro of the hybrid-selected mRNA from total RNA of oesophageal mucosa of the adult animal which comigrates with the oesophageal polypeptides 1 and 2.

	Fig. 6. Detection of mRNA encoding cytokeratin XenCK55(5/6) in various stages of development of Xenopus laevis by Northern blot hybridization. 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). 20^g of total RNA were loaded on a formaldehyde/agarose gel. RNAs were hybridized with a random-primed, 32P-labelled, 3'-specific probe synthesized from clone pXenCK55(5/6). Note reaction with a -2-4 kb RNA (lanes 5 to 8). The mRNA is first detected in gastrulae (stage 11, lane 4), as revealed after prolonged exposure and drastically increases in neurulae. There is no specific mRNA detectable in unfertilized eggs and pregastrulation stage (lanes 1-3 RNA loading and blot was controlled by photography).
	Fig. 7. Detection of mRNA encoding cytokeratin XenCK55(5/6) in different tissues of adult Xenopus laevis. Lane 1, ovary; lane 2, liver; lane 3, cardiac muscle; lane 4, skeletal muscle; lane 5, skin; lane 6, XLKE-A6 cell cultures; lane 7, oesophagus. 20 ng total RNA (lanes 3, 4 and 7) or l^g poly(A)+RNA (lanes 1, 2, 5 and 6) were loaded on a formaldehyde/agarose gel. RNAs were hybridized with a random-primed, 32Plabelled fragment of clone pXenCK55(5/6) representing the 3'-untranslated region of the mRNA. Bars indicate positions of 28S and 18S rRNA of Xenopus laevis. RNA of negative samples (lanes 1-4) has been controlled by reaction with other cDNA probes such as pXenViml, pXenDesl, pXLl/8 (Franz & Franke, 1986; Herrmann et al. 1988a,b).
	developmental stages and adult tissues of Xenopus laevis. (A) RNA blot analysis (Northern blot) of gel electrophoretically separated total RNA (50 JJ% each) from different tissues and embryonic stages: Lane 1, oocytes; lane 2, unfertilized eggs; lane 3, stage 18 (neural groove stage); lane 4, stage 42 (swimming tadpole); lane 5, adult oesophageal mucosa; lane 6, adult skeletal muscle; lane 7, cultured kidney epithelial cells of line A6. RNAs were hybridized with radioactively labelled, XenCK81-specific 60-mer polynucleotide (see Materials and Methods). There is a reaction with an approximately 1-6 kb RNA in stages 18 and 42 (lanes 3 and 4) and in the oesophageal epithelium (lane 5). Horizontal bars indicate positions of rRNAs co-electrophoresed for reference (from top to bottom: bovine 28S, E. coli 23S, bovine 18S and E. coli 16S rRNA). (B) SI nuclease protection analysis using poly(A)+RNA (5 fig each) from different embryonic stages and adult tissues and the radioactively labelled polynucleotide probe for XenCK81A. Lane 1, 60-mer polynucleotide alone; lane 2, not loaded; lane 3, unfertilized eggs; lane 4, stage 6-5 (morula); lane 5, stage 9 (fine cell blastula); lane 6, stage 18 (neurula groove stage); lane 7, stage 42 (swimming tadpole); lane 8, adult oesophageal epithelium; lane 9, adult epidermis; lane 10, cultured kidney epithelial cells of line XLKE-A6; lane 11, adult skeletal muscle; lane 12, ovary; lane 13, yeast tRNA; lane 14, assay without RNA added; lane 15, not loaded; lane 16, assay without SI nuclease, 300cpm loaded. Note the extremely strong reaction in lane 6 (stage 18) but also positive, although weaker signals in lanes 7 (tadpole) and 8 (adult oesophagus).
	Fig. 9. Immunofluorescence microscopy (A, phasecontrast optics; B, epifluorescence) of section through frozen oesophagus of adult Xenopus laevis, showing positive reaction with monoclonal cytokeratin antibody (KL1) in the epithelium (brackets) whereas the lamina proparia (Ip) is negative. Asterisks denote the oesophageal lumen. Bar, 50um
	Fig. 10. Two-dimensional gel electrophoresis of cytoskeletal proteins from oesophageal mucosa of adult Xenopus laevis as seen after staining with Coomassie blue (A), after immunoblot reaction with monoclonal antibody K,panl-8.136 reactive with type II cytokeratins (B, autoradiograph), or after identification of type I cytokeratins by specific binding of purified, [125I]-labelled cytokeratin 8 (A) from rat liver (C, autoradiograph). Separation of proteins was as described in Fig. 4. a, actin; b, bovine serum albumin. Brackets with numbers designate the polypeptides identified as cytokeratins, the bracket without a number denotes a yet unidentified component. Type II cytokeratin polypeptides are collectively identified by the reaction shown in B, type I cytokeratins by the cytokeratin binding assay (C).
	Fig. 11. Identification of the polypeptide encoded by XenCK81A mRNA, using in vitro translation of mRNA selected by hybridization to the cloned 60-mer polynucleotide, followed by two-dimensional coelectrophoresis of the translation product with cytoskeletal proteins of oesophageal mucosa from adult Xenopus. Separation of polypeptides was as described in Fig. 4. (A) Coomassie blue-staining. (B) Autoradiograph of the gel shown in A. Note that the [35S]methioninelabelled product of the in vitro translation from hybridselected embryonal stage-18 mRNA comigrates with the unlabelled oesophageal cytokeratin polypeptide 6 as seen in A.