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Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult.

Schweizer U, Gunnersen J, Karch C, Wiese S, Holtmann B, Takeda K, Akira S, Sendtner M - J. Cell Biol. (2002)

Bottom Line: In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult.Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons.Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical Neurobiology, University of Würzburg, 97080 Würzburg, Germany.

ABSTRACT
Members of the ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/cardiotrophin gene family are potent survival factors for embryonic and lesioned motoneurons. These factors act via receptor complexes involving gp130 and LIFR-beta and ligand binding leads to activation of various signaling pathways, including phosphorylation of Stat3. The role of Stat3 in neuronal survival was investigated in mice by Cre-mediated gene ablation in motoneurons. Cre is expressed under the neurofilament light chain (NF-L) promoter, starting around E12 when these neurons become dependent on neurotrophic support. Loss of motoneurons during the embryonic period of naturally occurring cell death is not enhanced in NF-L-Cre; Stat3(flox/KO) mice although motoneurons isolated from these mice need higher concentrations of CNTF for maximal survival in culture. In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult. These neurons, however, can be rescued by the addition of neurotrophic factors, including CNTF. Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons. Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.

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Spinal motoneurons do not depend on Stat3 signaling during the phase of naturally occurring cell death. (A) Embryonic motoneurons express Cre under control of the human NF-L promoter. E13 embryos from NF-L–Cre transgenic lacZ reporter mice were processed for β-galactosidase activity. A section through the lumbar spinal cord shows β-gal positive neurons (blue, arrows) only in the ventrolateral motor columns. (B) RT-PCR shows the presence of the recombined shortened Stat3 transcript lacking exon 22 as early as E12. Motoneurons from NF-L–Cre; Stat3flox/wt and NF-L–Cre; Stat3flox/KO mice were isolated, kept overnight in the presence of GDNF to recover and reexpress genes before total RNA was isolated. Sensory neurons from NF-L–Cre; Stat3flox/KO and NF-L–Cre; Stat3flox/wt mice were isolated at E14 and total RNA was prepared after 4 h in culture. (C) Embryonic motoneurons from NF-L–Cre; Stat3flox/KO mice require higher doses of CNTF for maximal survival in vitro. Motoneurons from E14 spinal cord of NF-L–Cre; Stat3flox/wt and NF-L–Cre; Stat3flox/KO mice were cultivated in the presence of BDNF (1 ng/ml), GDNF (0.1 ng/ml), and various concentrations of CNTF for 5 d. After 5 d in vitro, surviving motoneurons were counted and numbers expressed in relation to originally plated cells. Motoneurons from NF-L–Cre; Stat3flox/KO mice exhibited the same maximal survival response toward neurotrophic factors including CNTF as those from NF-L–Cre; Stat3flox/wt animals, indicating that Stat3 is not required for motoneuron survival during this critical period of development (asterisk indicates P < 0.05 ANOVA). Bar, 200 μm.
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fig2: Spinal motoneurons do not depend on Stat3 signaling during the phase of naturally occurring cell death. (A) Embryonic motoneurons express Cre under control of the human NF-L promoter. E13 embryos from NF-L–Cre transgenic lacZ reporter mice were processed for β-galactosidase activity. A section through the lumbar spinal cord shows β-gal positive neurons (blue, arrows) only in the ventrolateral motor columns. (B) RT-PCR shows the presence of the recombined shortened Stat3 transcript lacking exon 22 as early as E12. Motoneurons from NF-L–Cre; Stat3flox/wt and NF-L–Cre; Stat3flox/KO mice were isolated, kept overnight in the presence of GDNF to recover and reexpress genes before total RNA was isolated. Sensory neurons from NF-L–Cre; Stat3flox/KO and NF-L–Cre; Stat3flox/wt mice were isolated at E14 and total RNA was prepared after 4 h in culture. (C) Embryonic motoneurons from NF-L–Cre; Stat3flox/KO mice require higher doses of CNTF for maximal survival in vitro. Motoneurons from E14 spinal cord of NF-L–Cre; Stat3flox/wt and NF-L–Cre; Stat3flox/KO mice were cultivated in the presence of BDNF (1 ng/ml), GDNF (0.1 ng/ml), and various concentrations of CNTF for 5 d. After 5 d in vitro, surviving motoneurons were counted and numbers expressed in relation to originally plated cells. Motoneurons from NF-L–Cre; Stat3flox/KO mice exhibited the same maximal survival response toward neurotrophic factors including CNTF as those from NF-L–Cre; Stat3flox/wt animals, indicating that Stat3 is not required for motoneuron survival during this critical period of development (asterisk indicates P < 0.05 ANOVA). Bar, 200 μm.

Mentions: To test whether Cre is active in embryonic motoneurons, we analyzed NF-L–Cre transgenic lacZ reporter mice at embryonic day 13 (Fig. 2 A). At this stage Cre-mediated activation of the lacZ reporter gene was detectable in many spinal motoneurons (Fig. 2 A). We then isolated motoneurons from 12-d-old NF-L–Cre; Stat3flox/wt and NF-L–Cre; Stat3flox/KO embryos. After 1 d in culture, a prominent signal for Cre expression and a clear band for the recombined Stat3 gene was detectable, whereas a reduction of the wild-type transcript by at least 50% was apparent (Fig. 2 B). We have also isolated sensory neurons from E14 NF-L–Cre; Stat3flox/KO mice to test for Cre-mediated recombination of the floxed Stat3 allele. Although the NF-L promoter is only active in a subpopulation of DRG neurons (unpublished data), a prominent band corresponding to the recombined Stat3 (Fig. 2 B) was detectable. These data demonstrate that Stat3 gene deletion has already started around E12 in motoneurons, 2 d before these cells were isolated for testing survival responses to various neurotrophic factors.


Conditional gene ablation of Stat3 reveals differential signaling requirements for survival of motoneurons during development and after nerve injury in the adult.

Schweizer U, Gunnersen J, Karch C, Wiese S, Holtmann B, Takeda K, Akira S, Sendtner M - J. Cell Biol. (2002)

Spinal motoneurons do not depend on Stat3 signaling during the phase of naturally occurring cell death. (A) Embryonic motoneurons express Cre under control of the human NF-L promoter. E13 embryos from NF-L–Cre transgenic lacZ reporter mice were processed for β-galactosidase activity. A section through the lumbar spinal cord shows β-gal positive neurons (blue, arrows) only in the ventrolateral motor columns. (B) RT-PCR shows the presence of the recombined shortened Stat3 transcript lacking exon 22 as early as E12. Motoneurons from NF-L–Cre; Stat3flox/wt and NF-L–Cre; Stat3flox/KO mice were isolated, kept overnight in the presence of GDNF to recover and reexpress genes before total RNA was isolated. Sensory neurons from NF-L–Cre; Stat3flox/KO and NF-L–Cre; Stat3flox/wt mice were isolated at E14 and total RNA was prepared after 4 h in culture. (C) Embryonic motoneurons from NF-L–Cre; Stat3flox/KO mice require higher doses of CNTF for maximal survival in vitro. Motoneurons from E14 spinal cord of NF-L–Cre; Stat3flox/wt and NF-L–Cre; Stat3flox/KO mice were cultivated in the presence of BDNF (1 ng/ml), GDNF (0.1 ng/ml), and various concentrations of CNTF for 5 d. After 5 d in vitro, surviving motoneurons were counted and numbers expressed in relation to originally plated cells. Motoneurons from NF-L–Cre; Stat3flox/KO mice exhibited the same maximal survival response toward neurotrophic factors including CNTF as those from NF-L–Cre; Stat3flox/wt animals, indicating that Stat3 is not required for motoneuron survival during this critical period of development (asterisk indicates P < 0.05 ANOVA). Bar, 200 μm.
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fig2: Spinal motoneurons do not depend on Stat3 signaling during the phase of naturally occurring cell death. (A) Embryonic motoneurons express Cre under control of the human NF-L promoter. E13 embryos from NF-L–Cre transgenic lacZ reporter mice were processed for β-galactosidase activity. A section through the lumbar spinal cord shows β-gal positive neurons (blue, arrows) only in the ventrolateral motor columns. (B) RT-PCR shows the presence of the recombined shortened Stat3 transcript lacking exon 22 as early as E12. Motoneurons from NF-L–Cre; Stat3flox/wt and NF-L–Cre; Stat3flox/KO mice were isolated, kept overnight in the presence of GDNF to recover and reexpress genes before total RNA was isolated. Sensory neurons from NF-L–Cre; Stat3flox/KO and NF-L–Cre; Stat3flox/wt mice were isolated at E14 and total RNA was prepared after 4 h in culture. (C) Embryonic motoneurons from NF-L–Cre; Stat3flox/KO mice require higher doses of CNTF for maximal survival in vitro. Motoneurons from E14 spinal cord of NF-L–Cre; Stat3flox/wt and NF-L–Cre; Stat3flox/KO mice were cultivated in the presence of BDNF (1 ng/ml), GDNF (0.1 ng/ml), and various concentrations of CNTF for 5 d. After 5 d in vitro, surviving motoneurons were counted and numbers expressed in relation to originally plated cells. Motoneurons from NF-L–Cre; Stat3flox/KO mice exhibited the same maximal survival response toward neurotrophic factors including CNTF as those from NF-L–Cre; Stat3flox/wt animals, indicating that Stat3 is not required for motoneuron survival during this critical period of development (asterisk indicates P < 0.05 ANOVA). Bar, 200 μm.
Mentions: To test whether Cre is active in embryonic motoneurons, we analyzed NF-L–Cre transgenic lacZ reporter mice at embryonic day 13 (Fig. 2 A). At this stage Cre-mediated activation of the lacZ reporter gene was detectable in many spinal motoneurons (Fig. 2 A). We then isolated motoneurons from 12-d-old NF-L–Cre; Stat3flox/wt and NF-L–Cre; Stat3flox/KO embryos. After 1 d in culture, a prominent signal for Cre expression and a clear band for the recombined Stat3 gene was detectable, whereas a reduction of the wild-type transcript by at least 50% was apparent (Fig. 2 B). We have also isolated sensory neurons from E14 NF-L–Cre; Stat3flox/KO mice to test for Cre-mediated recombination of the floxed Stat3 allele. Although the NF-L promoter is only active in a subpopulation of DRG neurons (unpublished data), a prominent band corresponding to the recombined Stat3 (Fig. 2 B) was detectable. These data demonstrate that Stat3 gene deletion has already started around E12 in motoneurons, 2 d before these cells were isolated for testing survival responses to various neurotrophic factors.

Bottom Line: In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult.Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons.Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.

View Article: PubMed Central - PubMed

Affiliation: Institute for Clinical Neurobiology, University of Würzburg, 97080 Würzburg, Germany.

ABSTRACT
Members of the ciliary neurotrophic factor (CNTF)/leukemia inhibitory factor (LIF)/cardiotrophin gene family are potent survival factors for embryonic and lesioned motoneurons. These factors act via receptor complexes involving gp130 and LIFR-beta and ligand binding leads to activation of various signaling pathways, including phosphorylation of Stat3. The role of Stat3 in neuronal survival was investigated in mice by Cre-mediated gene ablation in motoneurons. Cre is expressed under the neurofilament light chain (NF-L) promoter, starting around E12 when these neurons become dependent on neurotrophic support. Loss of motoneurons during the embryonic period of naturally occurring cell death is not enhanced in NF-L-Cre; Stat3(flox/KO) mice although motoneurons isolated from these mice need higher concentrations of CNTF for maximal survival in culture. In contrast, motoneuron survival is significantly reduced after facial nerve lesion in the adult. These neurons, however, can be rescued by the addition of neurotrophic factors, including CNTF. Stat3 is essential for upregulation of Reg-2 and Bcl-xl expression in lesioned motoneurons. Our data show that Stat3 activation plays an essential role for motoneuron survival after nerve lesion in postnatal life but not during embryonic development, indicating that signaling requirements for motoneuron survival change during maturation.

Show MeSH
Related in: MedlinePlus