Localization of the expression of the α1(V) collagen gene during the embryonnary mouse development
1
98 Biology of the Cell CHARACTERIZATION OF A cDNA FOR A PROTEIN WITH MULTIPLE DOMAINS OF G-X-Y REPEATS IDENTIFIES A BOMBYX COLLAGENOUS PROTEIN ’ sea I CHAREYRE, Marie-Thtr&se BESSON and Georges BOSQIJET lJnivcrsit6 LYON I, C.G.M.C. U.M.R. 106 43, Bd du I1 Novembre 191%. 69622 VILLEURBANNE Cede.7 Using a differential screening strategy, a 2 kb cDNA was isolated from a differentiating Bombyx mori imaginai wing discs library (ClIA~YKI’ P., GUILLET C.. BESSON M.-T., FOURCHE J.. and BOSQUET C. (1993). Insecl Mel Biol.. 2. 239-246). The deduced primary structure of this cDNA resembles a collagen sequence, the first one isolated in Bombyx. The available reading frame of 1710 bp covers 395 amino acids of the collagenous domain (COL domain) and 175 amino acids of the non-collagenous C-terminal domain (NC1 domain). The domain organization of this Bombyx mori Collagen @mCol[) is different from most other known collagen tyccs. The partial COL homain hefines ten triple-helical domains sepa%ed.$y non-t$lc-helical re&ions. In the Gly-Xaa-Yaa triple-helical forming units, proline occurs almost exclusively-in the Yaa po$tion and glutamine is comtionly round in the Xaa position. The NC1 domain contains five cystein residues and two potential sites for N-linked glycosylation. Northern blots reveal a unique 7.4 kb mRNA in imagina! wing discs, epidermis and brain. BmColl mRNA accumulation parallels the elevation 01 the 20-hvdroxvecdvsone t20-OHE) titer in larvae. In virro studies revcal that this ac&mul&ion-is doie dependent and detectable within one hour in response to 20-OHE and/or cycloheximide. We propose that intrinsically unstable BmColl mRNAs are stabilized by 20-OHE which rcprcss the synthesis of a short-lived destabilizer protein. This hypothesis is in agrcemcnt with the presence in the 3’ untranslated region of the messengers o!’ 4 AUUUA motifs associated with unstability of mRNA in mammalian cells (SHAW Ci. and KAMEN R. (1986). Cell. 46. 659-667). We speculate that thcsc short sequences are recognized by an unidentified RNA-binding protein which would direct specific degradation of BmColl messengers. Thus, cycloheximide would promote BmColl mRNAs accumulation as a result o!’ the inhibition of synthesis of this protein. Similar 20-OHE regulatory pathway has never been taked into account in models for control of gcnc’ activity by 20-OHE. EXPRESSION OF A NEW FIBRILLAR COLLAGEN CLASS IN THE SEA URCHIN EMBRYO DURING SPICULOGENESIS LETHIAS Claire, EXPOSITO Jean-Yves, DESCOLLONGES Yvette, PENIN Francois and GARRONE Robert /nstifut de Sioiogie et Chimie des Protkines, 7 passage du Vercors, 69367 Lyon cedex 07, France In vertebrates, the best known subfamily of collagens is the so- called fibrillar collagen% All the fibrillar collagen chains contain a perfect triple helical region flanked by two non-collagenous propeptides. Contrary to the carboxypropeptides, the aminopropeptides greatly differ in size and composition. In sea urchin, we have previously characterized a fibrillar collagen chain which harbors a new N-propeptide configuration (Exposito J.Y., D’Alessio M. and Ramirez F., 1992, J. Biol. Chem., 267, 17404- 17408). This N-propeptide contains a region composed of 12 repeats of a new 4-cysteine motif. cDNA, Northern-blot analysis and RT-PCR experiments showed that this region undergoes alternative splicing. To understand the specific function of this region of the N-propeptide, we have produced in a bacterial system recombinant proteins corresponding to different subdomains of this region : repeat 2 and repeat 8 were chosen in relation with the different alternatively spliced isoforms. Polyclonal and monoclonal antibodies against these motifs were produced and used for immunolabeling. In the sea urchin embryo immunofluorescence experiments clearly indicate that the two motifs of the N-propeptide are associated with the formation of skeletal spicules. These results are confirmed in cultures of primary mesenchylme cells, which are able to elaborate spicules in vitro. The labeling is found at the surface of the spicules. These results suggest that this new motif present in N-propeptide is involved in the formation of sea urchin spicules. The biochemical composition of spicules is still unclear, but collagenous components are probably relatively rare in their organic matrix. Considering these data, we speculate that the role of this new motif is related to the indution or the maintenance of differentiation of mesenchymal cells involved in spiculogenesis. LOCALIZATIOR OF TlIE EXPRESSION Or: TllE al(V) COLLAGEN GENE DURING TI[E ERII~RYONNi\RY I\lOUSE DEVELOPMENT 1,E GUELLEC Dominiuur. *PEYSSON P~~scalr, PROST Chrisrophe, GARROTE Robert and **KARSENTY Girard To determine the spatial and rempoml pattern of the ex!“rssion of the moubr (II(V) collanen Eerie, we have Derforn~ed i/l situ hvbridization exoeriments examining mouse embryos at hifferent developmental stages, from 7.5 to the birth, using al(V) antisense riboprohe. The examination of the tvoe V collagen gene expression was compared with the disrrihution of type Ii&l I! collagen mRNAs. Image analysis was performed ro visualize the signal of hybridization on whole embryo sogirtal sections. Col5al expression started with a low intensity at 10.5 in the majority of mesitnchyma! cells. At this stage the highest level of transcripts was found in crph;llir mrsenchyme tissue, around dorsal aorta and in the n~antlihul:lr com!>onenl of the br;u~chia! arch. At 12.5 the expression of ColSn I \YIIS incrr;lsed in perlchondrium, between the undifferentiated vertrhr;d hodies, perironeal membrane and intestinal mesenchynie. The hilrhest level of I;lbelin!! was observed at lb.5 and it decreased from 18.5. The expression of the al(V) collagyn gent: was similar to rhe expression of Ihe a! (I) collagen gene, but very dltlerent from [hat of the nl(IJ) coll:rgcn gene which was csclusivslv exoressed 11, cartilaginous structures SuchYe n;lsn! septum. E\%&el’c;lr;il;igz, c;lrtil;\ce primordium of scapula and phalenge:tl oc,ci!)ir;ll. A hish expression ;f ColSal and Coil a! was observed in relation with the :\ppr:lrance of Ihe ossification center. A high labeling wa:, detected on pr‘riostrum and on fihrohlasts and osteoblasts of hone suggesting an import;tnt role of rype V collagen in rhe building of [his tissue. During Ihe skin dif!‘erenri;nion typeV collagen mRNAs were derected in srr;num grrmin:nivum constilured by flattened cells. As soon as these cells rook .I cube-like asncct. Co!.5:11 was no more expressed in these cells. Only lhe cells located underneath were labeled. This result indickltes Ihat migratring epithrli:~! cells are able to synthesize type V collacen. su ggcsting Ihat this coll:~gen would he necess;lrv for cell movement, wl;icli is in agrremenr wirh the wor!i of Slenn et ;ii. (!979).Nature, 271, 219-732). NORMAL HUMAN SKIN FIBROBLAST ADHESION ONTO COLLAGENE I MONOMERS OR FIBERS : CYTOSKELETON REORGANISATION AND PHOSPHORYLATION MERCIES Isabetle. GAILL FmnFoise and AUMAILLEY Monique Institut de Biologie et Chimie des ProtPines, CNl?S VPR 412. Lyorl In order to determine wether integrin-ligand interactions lead to different signals. we have studied the reorganization of the cytoskeleton of normal human skin fibroblasts attached to collagen 1 monomers or collagen I fibers. Scanning electron-microscopy observation shows well spread fibroblast on collagen I monomers. In contrast on collagen I fibers, fibroblast present a compact morphology and seem to align and connect together. Actin forms many stress fibers, as shown by immunofluorescence, in cells spread on collagen I monomers, vinculin and phosphotyrosyl-containing proteins colocalize with the actin stress fibers termini at sites or adhesion. A similar cytoskeleton organization is observed in fibroblasc attached to lihronectin. On collagen I fibers, cells exhibit a dense cellular body with fibrous cxtcnsions. Actin does not polymerize in stress fibers hut forms filaments and vinculin appears less condensed. Moreover phosphotyrosyl-containing proteins seem to bc concentrated along the axis of the collagen 1 fibers to which the cells attach. These observations suggest that signals transmitted by integrin-ligand !ead to different reorganizations of the cytoskeleton according to the molecular organization of the matricial subslrat.
Transcript of Localization of the expression of the α1(V) collagen gene during the embryonnary mouse development
98 Biology of the Cell
CHARACTERIZATION OF A cDNA FOR A PROTEIN WITH MULTIPLE DOMAINS OF G-X-Y REPEATS IDENTIFIES A
BOMBYX COLLAGENOUS PROTEIN
’ sea I CHAREYRE, Marie-Thtr&se BESSON and Georges BOSQIJET
lJnivcrsit6 LYON I, C.G.M.C. U.M.R. 106 43, Bd du I1 Novembre 191%. 69622 VILLEURBANNE Cede.7
Using a differential screening strategy, a 2 kb cDNA was isolated from a differentiating Bombyx mori imaginai wing discs library (ClIA~YKI’ P., GUILLET C.. BESSON M.-T., FOURCHE J.. and BOSQUET C. (1993). Insecl Mel Biol.. 2. 239-246). The deduced primary structure of this cDNA resembles a collagen sequence, the first one isolated in Bombyx. The available reading frame of 1710 bp covers 395 amino acids of the collagenous domain (COL domain) and 175 amino acids of the non-collagenous C-terminal domain (NC1 domain). The domain organization of this Bombyx mori Collagen @mCol[) is different from most other known collagen tyccs. The partial COL homain hefines ten triple-helical domains sepa%ed.$y non-t$lc-helical re&ions. In the Gly-Xaa-Yaa triple-helical forming units, proline occurs almost exclusively-in the Yaa po$tion and glutamine is comtionly round in the Xaa position. The NC1 domain contains five cystein residues and two potential sites for N-linked glycosylation.
Northern blots reveal a unique 7.4 kb mRNA in imagina! wing discs, epidermis and brain. BmColl mRNA accumulation parallels the elevation 01 the 20-hvdroxvecdvsone t20-OHE) titer in larvae. In virro studies revcal that this ac&mul&ion-is doie dependent and detectable within one hour in response to 20-OHE and/or cycloheximide. We propose that intrinsically unstable BmColl mRNAs are stabilized by 20-OHE which rcprcss the synthesis of a short-lived destabilizer protein. This hypothesis is in agrcemcnt with the presence in the 3’ untranslated region of the messengers o!’ 4 AUUUA motifs associated with unstability of mRNA in mammalian cells (SHAW Ci. and KAMEN R. (1986). Cell. 46. 659-667). We speculate that thcsc short sequences are recognized by an unidentified RNA-binding protein which would direct specific degradation of BmColl messengers. Thus, cycloheximide would promote BmColl mRNAs accumulation as a result o!’ the inhibition of synthesis of this protein. Similar 20-OHE regulatory pathway has never been taked into account in models for control of gcnc’ activity by 20-OHE.
EXPRESSION OF A NEW FIBRILLAR COLLAGEN CLASS IN THE SEA URCHIN EMBRYO DURING SPICULOGENESIS
LETHIAS Claire, EXPOSITO Jean-Yves, DESCOLLONGES Yvette, PENIN Francois and GARRONE Robert /nstifut de Sioiogie et Chimie des Protkines, 7 passage du Vercors,
69367 Lyon cedex 07, France
In vertebrates, the best known subfamily of collagens is the so- called fibrillar collagen% All the fibrillar collagen chains contain a perfect triple helical region flanked by two non-collagenous propeptides. Contrary to the carboxypropeptides, the aminopropeptides greatly differ in size and composition. In sea urchin, we have previously characterized a fibrillar collagen chain which harbors a new N-propeptide configuration (Exposito J.Y., D’Alessio M. and Ramirez F., 1992, J. Biol. Chem., 267, 17404- 17408). This N-propeptide contains a region composed of 12 repeats of a new 4-cysteine motif. cDNA, Northern-blot analysis and RT-PCR experiments showed that this region undergoes alternative splicing. To understand the specific function of this region of the N-propeptide, we have produced in a bacterial system recombinant proteins corresponding to different subdomains of this region : repeat 2 and repeat 8 were chosen in relation with the different alternatively spliced isoforms. Polyclonal and monoclonal antibodies against these motifs were produced and used for immunolabeling. In the sea urchin embryo immunofluorescence experiments clearly indicate that the two motifs of the N-propeptide are associated with the formation of skeletal spicules. These results are confirmed in cultures of primary mesenchylme cells, which are able to elaborate spicules in vitro. The labeling is found at the surface of the spicules. These results suggest that this new motif present in N-propeptide is involved in the formation of sea urchin spicules. The biochemical composition of spicules is still unclear, but collagenous components are probably relatively rare in their organic matrix. Considering these data, we speculate that the role of this new motif is related to the indution or the maintenance of differentiation of mesenchymal cells involved in spiculogenesis.
LOCALIZATIOR OF TlIE EXPRESSION Or: TllE al(V) COLLAGEN GENE DURING TI[E ERII~RYONNi\RY I\lOUSE DEVELOPMENT
1,E GUELLEC Dominiuur. *PEYSSON P~~scalr, PROST Chrisrophe, GARROTE Robert and **KARSENTY Girard
To determine the spatial and rempoml pattern of the ex!“rssion of the moubr (II(V) collanen Eerie, we have Derforn~ed i/l situ hvbridization exoeriments examining mouse embryos at hifferent developmental stages, from 7.5 to the birth, using al(V) antisense riboprohe. The examination of the tvoe V collagen gene expression was compared with the disrrihution of type Ii&l I! collagen mRNAs. Image analysis was performed ro visualize the signal of hybridization on whole embryo sogirtal sections. Col5al expression started with a low intensity at 10.5 in the majority of mesitnchyma! cells. At this stage the highest level of transcripts was found in crph;llir mrsenchyme tissue, around dorsal aorta and in the n~antlihul:lr com!>onenl of the br;u~chia! arch. At 12.5 the expression of ColSn I \YIIS incrr;lsed in perlchondrium, between the undifferentiated vertrhr;d hodies, perironeal membrane and intestinal mesenchynie. The hilrhest level of I;lbelin!! was observed at lb.5 and it decreased from 18.5. The expression of the al(V) collagyn gent: was similar to rhe expression of Ihe a! (I) collagen gene, but very dltlerent from [hat of the nl(IJ) coll:rgcn gene which was csclusivslv exoressed 11, cartilaginous structures SuchYe n;lsn! septum. E\%&el’c;lr;il;igz, c;lrtil;\ce primordium of scapula and phalenge:tl oc,ci!)ir;ll. A hish expression ;f ColSal and Coil a! was observed in relation with the :\ppr:lrance of Ihe ossification center. A high labeling wa:, detected on pr‘riostrum and on fihrohlasts and osteoblasts of hone suggesting an import;tnt role of rype V collagen in rhe building of [his tissue. During Ihe skin dif!‘erenri;nion typeV collagen mRNAs were derected in srr;num grrmin:nivum constilured by flattened cells. As soon as these cells rook .I cube-like asncct. Co!.5:11 was no more expressed in these cells. Only lhe cells located underneath were labeled. This result indickltes Ihat migratring epithrli:~! cells are able to synthesize type V collacen. su ggcsting Ihat this coll:~gen would he necess;lrv for cell movement, wl;icli is in agrremenr wirh the wor!i of Slenn et ;ii. (!979).Nature, 271, 219-732).
NORMAL HUMAN SKIN FIBROBLAST ADHESION
ONTO COLLAGENE I MONOMERS OR FIBERS :
CYTOSKELETON REORGANISATION AND PHOSPHORYLATION
MERCIES Isabetle. GAILL FmnFoise and AUMAILLEY Monique
Institut de Biologie et Chimie des ProtPines, CNl?S VPR 412. Lyorl In order to determine wether integrin-ligand interactions lead to different
signals. we have studied the reorganization of the cytoskeleton of
normal human skin fibroblasts attached to collagen 1 monomers or
collagen I fibers. Scanning electron-microscopy observation shows
well spread fibroblast on collagen I monomers. In contrast on
collagen I fibers, fibroblast present a compact morphology and seem to
align and connect together. Actin forms many stress fibers, as shown
by immunofluorescence, in cells spread on collagen I monomers,
vinculin and phosphotyrosyl-containing proteins colocalize with the
actin stress fibers termini at sites or adhesion. A similar cytoskeleton
organization is observed in fibroblasc attached to lihronectin. On
collagen I fibers, cells exhibit a dense cellular body with fibrous
cxtcnsions. Actin does not polymerize in stress fibers hut forms
filaments and vinculin appears less condensed. Moreover
phosphotyrosyl-containing proteins seem to bc concentrated along the
axis of the collagen 1 fibers to which the cells attach. These
observations suggest that signals transmitted by integrin-ligand !ead to
different reorganizations of the cytoskeleton according to the molecular
organization of the matricial subslrat.
