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Characterization of F3 receptors involved in axonal growth
CHARACTERlZATION
OF F3 RECEPTORS AXONAL GROWTH
REVEST J.M~, BU’I-TIGLIONE
INVOLVED
IN
de Marseille.
LGPD,
CNRS,
Guidance of axons toward their cellular targets during embryonic development is controled by molecules expressed in their environment. Such molecules are exvressed either on neirrhbouring cells or in the extracellular matrix. We ark workin on a modermo!ecu!& named F3 and belonaing to the immunoglobulin 61~) superfamily. F3 exists both in soluble anld membrane ancho;ed form. 7-he glycan-phosphatidylinositol anchored form fullfils the operational criteria of an adhesion molecule and mediates Ca++ independent heterophlic interactions via its Fibronectin tvne III domains. The soluble form vromotes neurite outgrowth v&its Ig domains. Then F3 is a p!urifur&onal molecule likdv to interact with several lieands (DURBEC P., GENNARINI G. BU’I?IGLIONE M. GOMEZ S. &d ROUGON G. (1993) Eur.J. Narrosci.. 6,461-472).
Mv uhD nroiect consists in characterizine the F3 recevtors involved in’ ne&te &growth activities. The strategy choosen con&ted to construct a soluble chimeric formof F3 in which the FN III domains and GPI anchor have been replaced by the Fc fragment of the human IgGI: F3Fc. This protein had been transient1 expressed by eucaryotic Cos 7 cells. After metabolic labelline with 3r S Methionin. we verified that the protein is synthetized under drmeric form in nonreducing conditions and with a molecular weight of 120 kDa under reducing conditions. We showed that .F3Fc is lble to promote neurite outg;owt!i of sensory neurons similarly to wild-type F3. The same experiments are underway for cerebellum neurons. The chimera F3Fc is able to bind to five-daysold mouse cerebellum neurons maintained for six days in culture. To decide if these receotors. essentialv exoressed bv neurites. are alreadv identified moleculds, we perfomed @turbati& experiments using antibodies such as anti-L1 and anti-NCAM. The characterization of the receptors wi!! be carried on using biochemica! techniques such as crosslinking. We will also try to characterize the second messenger systems activated after binding of F3Fc to the neurons.
AC~IVI’I’IES
A RETROVIRAI.. v ftrslimf de hioh& MARSEILLE.
M., ROUGON G.
Institut de Biologie du Developpement 99.0, Luminy, MARSEILLE
h4OKPlIOGENE1’IC
OI: trrW2J MOLE~CULE USli\ri(; VECTOR TECHNIQUll
blOIWAU, II., CIWZAL., G., and I~OUGON, L;. clrr de mlo~~prnrr.~rl de ~Afcm d/r, LGPK), i’R’K.S. YY-I.!. LciMiA
3
The functional properties of the nervous system tfepend ~.rrt~callq 1.1:li: intricate network of naut-ona! sonneclions that is gent:~atcd ililt i:r~ development. The patterning of these connections ernergtis, in large lid’ L from precise and coordinated adhesive interactions between ~.lcv~f,i;?i:~~ neurons and their cellular environment. mCD23 is a membrane glycoprotein whose cxprcssion is rrgula~ell ire !:I~> mouse nervous system during development (NEDLILEC, J ,. t’ft?‘ii~~:?; :: _ MOREAU. -G. (IY92)
H., UAIII3bX J.. NAQUET. P.. ~A~VKE-SAI~KAII.II. Eur. J. Biwlrcm. 203; -t33-J-12) and is testficted
(KUCIltEK, ZANETrA
S., KOtIGON, G., hlAKS1IAL. P., I.EIII\lANN. J.P. (IY8Y) Nuuruscicnclr 13; 11 I-124)
c.
ANII
K(Iu~;::‘?,
to the pefiud whi:ii iteuroblasts are differentiating and migrating towards their final tlesiimt::J:t S,
l:WUEX,
,X ,>%I:
According to its structure (a small pcpt& h~dvrly gly~osyi;~u:J ;,I,~! anchored to the membrane by a glycosyl phosphatitiylinositol group) aurt-it\ i/z viva localization, we nosrulated that mCD24 could. nlav, B ~-+:~li-$3 modulating cell-cell interadions during neun~rial diffcrenti&n This hypothesis was first tested itr rifw. We showed. in collaboratiw wni! Dr. Cohen (Guy’s Hospital, London), that ncurire outgrowth was~inhiln!e~r when central or peripheral neurons were cultured on a monolayer oi Ci; glial cells transfected with a vector containing mCDZ-I, but not on :t::s control cells. Secondly, to study how rnorphogenetic events are influclr<~\.: by the correct spatio-temporal expression of mCD2J. we used reuovuu.~ mediated gene transfer to perturbate its expression in developing ni~~!~~ embryos. In collaboration with Dr. Price (SmithKline Ececham, U.K.), 1 have introduced the cDNA coding for mCD24 in a retrovirai vectorivec;:~: 1704 gift from Dr Major, SI Louis, USA). I prepareed high titres it infectious viruses by introducing this construct into a packaging celt trm: These viral particles were injected in the nervous system of rats and ~ILC embryos. The presence and the behavior of infected cells was examined 1:~ immunostaining at selected times following infection. In conclusion: a) the majority of infected cells are of neuronal l~hrnutyl~~: i/ is then possible that an ectopic expression of mCD2-l by asirocytes \voufil induce cell death. b) since infected neurons are found isolated, an ;ut:ic! expresston of mCD24 by neurol-riasts might induce them to kave 11rex:! cycle and prevent further division. c) a prolonged expresiion of rnCD:; might prevent normal neumnal funclion and induce cell tleirlh
SATELLITE CELL ACTIVATION AND EVOLUTION FOLLOWING.MUSCLE DENERVATION IN TWO STRAINS OF TURKEY. CHEREL Yan, BAKOU Serge, WYERS Monique. ZNRA
HYPERTROPHY AS A RESPONSE TO DENERVATION OF MULTI-INNERVATED FIBRRS INTWO STRXNSOF T&JR-&C-Y. CHEREf.Yan, BAYOU Serge, WYERS Monique. INRA URA, ~~~~~
URA. Ecole Nnrionale
Nationale
V&frinaire,
CP 3013, 44087 NANTES
Cedex
01
Six-week-old male turkeys from two strains were used in this study. The Heavy Weight (HW) strain was selected on criteria of weight and muscle mass. Light weight (LW) strain was not selected on body weight. Post natal hypetplasia was observed only in Heavy Weight turkey muscles. Denervation by sciatic nerve section was performed. -6astrocnemius lateralis, Tibialis craniulis and P&n&s muscles were removed at days 3, IO, 17, and 2 I. Immunostaining with PCNA/cyclin (Proliferating Cell Nuclear Antigen : nuclear antigen expressed during the S phase) monoclona! antibody (DAKO, PC 10) revealed satellite cell activation. In HW strain, satellite cell activation was observed at day 10. The activation increased until day 2 1 and reached 85 5~6 activated satellite RI j, 7 in Plutzraris, and 61 f 8 cells per 100 fibers in Tihialis crwicdis, per100 fibers in Castrocnenrius Irderctlix. In LW strain, activated satellite cells number remained less than 9 per 100 fibers (similar to control) in Tibia/is cmnifzlis. It was constant (almost 30 per 100) in Planfaris. In Gastrocnemius, at day 3, it was similar to control and reached 30 per 100 fibers after day 10. Activated satellite cells were distributed randomly on the sections, showing no correspondance to regeneration features of necrotic fibers In order to determine the evolution of these satellite cells, turkeys were treated from day IO to day 31 by intraperitoneal injections of BrdU (Bromo deoxyuridine). Labelled nuclei (revealed by BrdU antibodies (Amersham) were only observed in large size fibers at day 35, showing incorporation of satellite cells into myofibers. Turkey satellite cell activation following denervation was similar to those observed in mammals. In turkey, the rate was different from one muscle to another and was higher in HW strain than in LW-strain. In vitro satellite cells proliferation was similar in HW and LW strain (Magras, unpublished). The differences observed between HW and LW could be related to a greater ability of HW muscle fibers to release mitogens in response to denervation.
V&rinuire,
CP 3013.44087NANTES
Cedex 01
Six-week-old male turkeys from two strains, were used in this studv. lftc Heavy Weight (HW) strain was selected on criteria of weight and muscle mass. Light weight (LW) strain was not selected on body weight. Pus: natal hyperplasia -was observed only in Heavy Weight turkey musc1e.s (Cherei et al, 1994). Denervation by sciatic nerve section was performed. Gmirucnemr:~~ 1aterali.z Tibia&s cranialis and Planturis muscles were removed at days 3, IO, 17, and 2 !..histochemical techniques(ATPases, NADH-TR) were used, and fiber diameter was measured. Gastrocnemius latefalis and Tibialis uunialis muscles were compohrii by a mixture of multi-innervated, slow-twitch fibers (type 1). and focally-innervated,. fast-twitch fibers (type 11). Pkrntaris muscle was composed of type-II fibers intermixed with multi-innervated, slow-tonic fibers (type III). Similar atrophy was observed in the two strains III focally-innervated fibers (type II) after denervation. This atrophy was observed at day 5 and reached 40 % at day 2 I Hypertrophy was observed in multi-innervated ftbers (type 1 dnd type III). In LW strain, type I fiber hypertrophy was apparent at clay 10 and ahead. It reached 70 % at day 21. In HW strain, hypertsophy was observed later (day 17 for Tihialk and day 2 1 for Grtsrrocneuri& Type III fibers revealed transient atrophy at day 3. in L.W strain turkey+, followed by hypertrophy at day 10 and ahead. whereas in HW stratrr hypertrophy was only observed at day 2 1. Turkey fast-twitch fiber atrophy was similar to the one -observed ~1 mammals (Schmalbruch ef ~1 1991). In mammals, muIt!-innervated fibers only exist in extra-ocular muscles, and these fibers showed hypertrophy after denervation (Cluistianssen rr (II 1992). In our study, denervation hypertrophy was faster in LW strain I: revealed a greater ability of myofibri! synthesis in LW strain musclc~ following denervation. CHEREL
e-f al (lYY4)
Growth.
511. 157. ID5
CHRISTIANSSENernl(1992) Exp. Mol. Partml 56.87.‘)~ SCHMALBRUCHcr rrl (1991)1 Phvsiol.44t. 233.~3,
OF F3 RECEPTORS AXONAL GROWTH
REVEST J.M~, BU’I-TIGLIONE
INVOLVED
IN
de Marseille.
LGPD,
CNRS,
Guidance of axons toward their cellular targets during embryonic development is controled by molecules expressed in their environment. Such molecules are exvressed either on neirrhbouring cells or in the extracellular matrix. We ark workin on a modermo!ecu!& named F3 and belonaing to the immunoglobulin 61~) superfamily. F3 exists both in soluble anld membrane ancho;ed form. 7-he glycan-phosphatidylinositol anchored form fullfils the operational criteria of an adhesion molecule and mediates Ca++ independent heterophlic interactions via its Fibronectin tvne III domains. The soluble form vromotes neurite outgrowth v&its Ig domains. Then F3 is a p!urifur&onal molecule likdv to interact with several lieands (DURBEC P., GENNARINI G. BU’I?IGLIONE M. GOMEZ S. &d ROUGON G. (1993) Eur.J. Narrosci.. 6,461-472).
Mv uhD nroiect consists in characterizine the F3 recevtors involved in’ ne&te &growth activities. The strategy choosen con&ted to construct a soluble chimeric formof F3 in which the FN III domains and GPI anchor have been replaced by the Fc fragment of the human IgGI: F3Fc. This protein had been transient1 expressed by eucaryotic Cos 7 cells. After metabolic labelline with 3r S Methionin. we verified that the protein is synthetized under drmeric form in nonreducing conditions and with a molecular weight of 120 kDa under reducing conditions. We showed that .F3Fc is lble to promote neurite outg;owt!i of sensory neurons similarly to wild-type F3. The same experiments are underway for cerebellum neurons. The chimera F3Fc is able to bind to five-daysold mouse cerebellum neurons maintained for six days in culture. To decide if these receotors. essentialv exoressed bv neurites. are alreadv identified moleculds, we perfomed @turbati& experiments using antibodies such as anti-L1 and anti-NCAM. The characterization of the receptors wi!! be carried on using biochemica! techniques such as crosslinking. We will also try to characterize the second messenger systems activated after binding of F3Fc to the neurons.
AC~IVI’I’IES
A RETROVIRAI.. v ftrslimf de hioh& MARSEILLE.
M., ROUGON G.
Institut de Biologie du Developpement 99.0, Luminy, MARSEILLE
h4OKPlIOGENE1’IC
OI: trrW2J MOLE~CULE USli\ri(; VECTOR TECHNIQUll
blOIWAU, II., CIWZAL., G., and I~OUGON, L;. clrr de mlo~~prnrr.~rl de ~Afcm d/r, LGPK), i’R’K.S. YY-I.!. LciMiA
3
The functional properties of the nervous system tfepend ~.rrt~callq 1.1:li: intricate network of naut-ona! sonneclions that is gent:~atcd ililt i:r~ development. The patterning of these connections ernergtis, in large lid’ L from precise and coordinated adhesive interactions between ~.lcv~f,i;?i:~~ neurons and their cellular environment. mCD23 is a membrane glycoprotein whose cxprcssion is rrgula~ell ire !:I~> mouse nervous system during development (NEDLILEC, J ,. t’ft?‘ii~~:?; :: _ MOREAU. -G. (IY92)
H., UAIII3bX J.. NAQUET. P.. ~A~VKE-SAI~KAII.II. Eur. J. Biwlrcm. 203; -t33-J-12) and is testficted
(KUCIltEK, ZANETrA
S., KOtIGON, G., hlAKS1IAL. P., I.EIII\lANN. J.P. (IY8Y) Nuuruscicnclr 13; 11 I-124)
c.
ANII
K(Iu~;::‘?,
to the pefiud whi:ii iteuroblasts are differentiating and migrating towards their final tlesiimt::J:t S,
l:WUEX,
,X ,>%I:
According to its structure (a small pcpt& h~dvrly gly~osyi;~u:J ;,I,~! anchored to the membrane by a glycosyl phosphatitiylinositol group) aurt-it\ i/z viva localization, we nosrulated that mCD24 could. nlav, B ~-+:~li-$3 modulating cell-cell interadions during neun~rial diffcrenti&n This hypothesis was first tested itr rifw. We showed. in collaboratiw wni! Dr. Cohen (Guy’s Hospital, London), that ncurire outgrowth was~inhiln!e~r when central or peripheral neurons were cultured on a monolayer oi Ci; glial cells transfected with a vector containing mCDZ-I, but not on :t::s control cells. Secondly, to study how rnorphogenetic events are influclr<~\.: by the correct spatio-temporal expression of mCD2J. we used reuovuu.~ mediated gene transfer to perturbate its expression in developing ni~~!~~ embryos. In collaboration with Dr. Price (SmithKline Ececham, U.K.), 1 have introduced the cDNA coding for mCD24 in a retrovirai vectorivec;:~: 1704 gift from Dr Major, SI Louis, USA). I prepareed high titres it infectious viruses by introducing this construct into a packaging celt trm: These viral particles were injected in the nervous system of rats and ~ILC embryos. The presence and the behavior of infected cells was examined 1:~ immunostaining at selected times following infection. In conclusion: a) the majority of infected cells are of neuronal l~hrnutyl~~: i/ is then possible that an ectopic expression of mCD2-l by asirocytes \voufil induce cell death. b) since infected neurons are found isolated, an ;ut:ic! expresston of mCD24 by neurol-riasts might induce them to kave 11rex:! cycle and prevent further division. c) a prolonged expresiion of rnCD:; might prevent normal neumnal funclion and induce cell tleirlh
SATELLITE CELL ACTIVATION AND EVOLUTION FOLLOWING.MUSCLE DENERVATION IN TWO STRAINS OF TURKEY. CHEREL Yan, BAKOU Serge, WYERS Monique. ZNRA
HYPERTROPHY AS A RESPONSE TO DENERVATION OF MULTI-INNERVATED FIBRRS INTWO STRXNSOF T&JR-&C-Y. CHEREf.Yan, BAYOU Serge, WYERS Monique. INRA URA, ~~~~~
URA. Ecole Nnrionale
Nationale
V&frinaire,
CP 3013, 44087 NANTES
Cedex
01
Six-week-old male turkeys from two strains were used in this study. The Heavy Weight (HW) strain was selected on criteria of weight and muscle mass. Light weight (LW) strain was not selected on body weight. Post natal hypetplasia was observed only in Heavy Weight turkey muscles. Denervation by sciatic nerve section was performed. -6astrocnemius lateralis, Tibialis craniulis and P&n&s muscles were removed at days 3, IO, 17, and 2 I. Immunostaining with PCNA/cyclin (Proliferating Cell Nuclear Antigen : nuclear antigen expressed during the S phase) monoclona! antibody (DAKO, PC 10) revealed satellite cell activation. In HW strain, satellite cell activation was observed at day 10. The activation increased until day 2 1 and reached 85 5~6 activated satellite RI j, 7 in Plutzraris, and 61 f 8 cells per 100 fibers in Tihialis crwicdis, per100 fibers in Castrocnenrius Irderctlix. In LW strain, activated satellite cells number remained less than 9 per 100 fibers (similar to control) in Tibia/is cmnifzlis. It was constant (almost 30 per 100) in Planfaris. In Gastrocnemius, at day 3, it was similar to control and reached 30 per 100 fibers after day 10. Activated satellite cells were distributed randomly on the sections, showing no correspondance to regeneration features of necrotic fibers In order to determine the evolution of these satellite cells, turkeys were treated from day IO to day 31 by intraperitoneal injections of BrdU (Bromo deoxyuridine). Labelled nuclei (revealed by BrdU antibodies (Amersham) were only observed in large size fibers at day 35, showing incorporation of satellite cells into myofibers. Turkey satellite cell activation following denervation was similar to those observed in mammals. In turkey, the rate was different from one muscle to another and was higher in HW strain than in LW-strain. In vitro satellite cells proliferation was similar in HW and LW strain (Magras, unpublished). The differences observed between HW and LW could be related to a greater ability of HW muscle fibers to release mitogens in response to denervation.
V&rinuire,
CP 3013.44087NANTES
Cedex 01
Six-week-old male turkeys from two strains, were used in this studv. lftc Heavy Weight (HW) strain was selected on criteria of weight and muscle mass. Light weight (LW) strain was not selected on body weight. Pus: natal hyperplasia -was observed only in Heavy Weight turkey musc1e.s (Cherei et al, 1994). Denervation by sciatic nerve section was performed. Gmirucnemr:~~ 1aterali.z Tibia&s cranialis and Planturis muscles were removed at days 3, IO, 17, and 2 !..histochemical techniques(ATPases, NADH-TR) were used, and fiber diameter was measured. Gastrocnemius latefalis and Tibialis uunialis muscles were compohrii by a mixture of multi-innervated, slow-twitch fibers (type 1). and focally-innervated,. fast-twitch fibers (type 11). Pkrntaris muscle was composed of type-II fibers intermixed with multi-innervated, slow-tonic fibers (type III). Similar atrophy was observed in the two strains III focally-innervated fibers (type II) after denervation. This atrophy was observed at day 5 and reached 40 % at day 2 I Hypertrophy was observed in multi-innervated ftbers (type 1 dnd type III). In LW strain, type I fiber hypertrophy was apparent at clay 10 and ahead. It reached 70 % at day 21. In HW strain, hypertsophy was observed later (day 17 for Tihialk and day 2 1 for Grtsrrocneuri& Type III fibers revealed transient atrophy at day 3. in L.W strain turkey+, followed by hypertrophy at day 10 and ahead. whereas in HW stratrr hypertrophy was only observed at day 2 1. Turkey fast-twitch fiber atrophy was similar to the one -observed ~1 mammals (Schmalbruch ef ~1 1991). In mammals, muIt!-innervated fibers only exist in extra-ocular muscles, and these fibers showed hypertrophy after denervation (Cluistianssen rr (II 1992). In our study, denervation hypertrophy was faster in LW strain I: revealed a greater ability of myofibri! synthesis in LW strain musclc~ following denervation. CHEREL
e-f al (lYY4)
Growth.
511. 157. ID5
CHRISTIANSSENernl(1992) Exp. Mol. Partml 56.87.‘)~ SCHMALBRUCHcr rrl (1991)1 Phvsiol.44t. 233.~3,