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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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Generation and characterization of NF-L–Cre. (A) The coding region of exon 1 within a 6-kb human NF-L gene fragment was replaced by the open reading frame of bacteriophage P1 Cre recombinase leaving regulatory sequences within NF-L introns intact. (B) Cre recombinase is specifically expressed in brain and spinal cord. RT-PCR on total RNA derived from adult cerebral cortex, brainstem, spinal cord, and sciatic nerve detects Cre message only brain and spinal cord, but not in sciatic nerve, liver, spleen, and kidney (latter three not shown). (C) Transgenic Cre recombinase is enzymatically active and recombines a reporter allele in vivo. NF-L–Cre mice were cross-bred with a lac Z reporter strain. Cre-mediated recombination of the reporter gene was detected via PCR on genomic DNA by a 580-bp product as opposed to the 1,700-bp product of the unrecombined reporter allele. Cre- mediated recombination is therefore specific for brain and spinal cord. (D) Cre recombinase expression is neuron- specific. In situ hybridization detects Cre expression in the facial nucleus and other brain regions (unpublished data). The transgenic Cre allele is solely expressed in neurons but not in surrounding glia. (E) Cre-mediated site-specific recombination in facial motoneurons. Brain sections from mice transgenic for NF-L–Cre and for the lacZ reporter allele were stained with X-Gal for β-galactosidase activity reflecting Cre-mediated activation of the lacZ reporter gene. 53.7 ± 12.9% (mean ± SD; n = 3) of motoneurons in the adult facial nucleus appeared X-Gal positive. (F) Breeding scheme to produce mice with a neuron-specific Stat3 gene ablation. Mice with the indicated genotypes were crossed to produce mice lacking functional Stat3 in motoneurons (NF-L–Cre; Stat3flox/KO) or mice carrying at least one wild-type Stat3 allele (NF-L–Cre; Stat3flox/wt) at a 50% ratio. Bar, 100 μm.
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fig1: Generation and characterization of NF-L–Cre. (A) The coding region of exon 1 within a 6-kb human NF-L gene fragment was replaced by the open reading frame of bacteriophage P1 Cre recombinase leaving regulatory sequences within NF-L introns intact. (B) Cre recombinase is specifically expressed in brain and spinal cord. RT-PCR on total RNA derived from adult cerebral cortex, brainstem, spinal cord, and sciatic nerve detects Cre message only brain and spinal cord, but not in sciatic nerve, liver, spleen, and kidney (latter three not shown). (C) Transgenic Cre recombinase is enzymatically active and recombines a reporter allele in vivo. NF-L–Cre mice were cross-bred with a lac Z reporter strain. Cre-mediated recombination of the reporter gene was detected via PCR on genomic DNA by a 580-bp product as opposed to the 1,700-bp product of the unrecombined reporter allele. Cre- mediated recombination is therefore specific for brain and spinal cord. (D) Cre recombinase expression is neuron- specific. In situ hybridization detects Cre expression in the facial nucleus and other brain regions (unpublished data). The transgenic Cre allele is solely expressed in neurons but not in surrounding glia. (E) Cre-mediated site-specific recombination in facial motoneurons. Brain sections from mice transgenic for NF-L–Cre and for the lacZ reporter allele were stained with X-Gal for β-galactosidase activity reflecting Cre-mediated activation of the lacZ reporter gene. 53.7 ± 12.9% (mean ± SD; n = 3) of motoneurons in the adult facial nucleus appeared X-Gal positive. (F) Breeding scheme to produce mice with a neuron-specific Stat3 gene ablation. Mice with the indicated genotypes were crossed to produce mice lacking functional Stat3 in motoneurons (NF-L–Cre; Stat3flox/KO) or mice carrying at least one wild-type Stat3 allele (NF-L–Cre; Stat3flox/wt) at a 50% ratio. Bar, 100 μm.

Mentions: To study Stat3 function in postnatal motoneurons, we have generated a transgenic mouse line expressing the bacteriophage P1 Cre recombinase under control of the human NF-L promoter and intragenic sequences. Previous studies (Leconte et al., 1994; Akassoglou et al., 1997) have shown that the human NF-L promoter and its intragenic sequences can drive heterologous expression of transgenes in mice. Therefore, we have replaced the coding region of the first exon of the human NF-L gene by a CrepA cassette, leaving 2 kb of the 5′ promoter and 3 kb of downstream sequence intact (Fig. 1 A). Two lines of transgenic mice were established and various tissues were tested for Cre mRNA expression by reverse transcription (RT)-PCR. One line of transgenic mice exhibited Cre expression in brain and spinal cord but not in sciatic nerve or organs such as kidney, liver, and spleen (Fig. 1 B; unpublished data). To control whether Cre expressed under the NF-L promoter is functional, we cross-bred NF-L–Cre with a lacZ reporter strain (Akagi et al., 1997) and analyzed double transgenic mice at various embryonic and postnatal stages. Using a PCR-based assay (Akagi et al., 1997), we show that Cre expressed from the NF-L–Cre transgene is functional and recombines a loxP-flanked reporter gene specifically in neural tissues (Fig. 1 C). To test whether Cre expression is specific for neurons, we performed in situ hybridization with a probe specific for Cre mRNA. As shown in Fig. 1 D, the Cre transgene is expressed in motoneurons of the facial nucleus, but not in the surrounding glia. Cre expression was also detected in hippocampal and cortical neurons (unpublished data). We then tested the cell type specificity of Cre-mediated recombination by staining of brain slices and whole organs for β-galactosidase. Again, X-gal staining was confined to neurons, in particular spinal and brain stem motoneurons (Fig. 1 E and unpublished data). Neuron-specific inactivation of the Stat3 gene was achieved by cross-breeding NF-L–Cre transgenic mice with mice carrying a floxed Stat3 gene (Takeda et al., 1998) according to the scheme depicted in Fig. 1 F. In these mice, Cre-mediated recombination leads to removal of exon 22 of Stat3, which contains the functionally relevant tyrosine residue. Due to usage of a cryptic splice site, part of the following exon containing the regulatory serine phosphorylation site is also removed (Takeda et al., 1998). Inclusion of a Stat3 allele resulted in 50% offspring devoid of functional Stat3 in a significant subpopulation of motoneurons (NF-L–Cre; Stat3flox/KO) and 50% offspring carrying one functional allele of Stat3 in motoneurons (NF-L–Cre; Stat3flox/wt).


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)

Generation and characterization of NF-L–Cre. (A) The coding region of exon 1 within a 6-kb human NF-L gene fragment was replaced by the open reading frame of bacteriophage P1 Cre recombinase leaving regulatory sequences within NF-L introns intact. (B) Cre recombinase is specifically expressed in brain and spinal cord. RT-PCR on total RNA derived from adult cerebral cortex, brainstem, spinal cord, and sciatic nerve detects Cre message only brain and spinal cord, but not in sciatic nerve, liver, spleen, and kidney (latter three not shown). (C) Transgenic Cre recombinase is enzymatically active and recombines a reporter allele in vivo. NF-L–Cre mice were cross-bred with a lac Z reporter strain. Cre-mediated recombination of the reporter gene was detected via PCR on genomic DNA by a 580-bp product as opposed to the 1,700-bp product of the unrecombined reporter allele. Cre- mediated recombination is therefore specific for brain and spinal cord. (D) Cre recombinase expression is neuron- specific. In situ hybridization detects Cre expression in the facial nucleus and other brain regions (unpublished data). The transgenic Cre allele is solely expressed in neurons but not in surrounding glia. (E) Cre-mediated site-specific recombination in facial motoneurons. Brain sections from mice transgenic for NF-L–Cre and for the lacZ reporter allele were stained with X-Gal for β-galactosidase activity reflecting Cre-mediated activation of the lacZ reporter gene. 53.7 ± 12.9% (mean ± SD; n = 3) of motoneurons in the adult facial nucleus appeared X-Gal positive. (F) Breeding scheme to produce mice with a neuron-specific Stat3 gene ablation. Mice with the indicated genotypes were crossed to produce mice lacking functional Stat3 in motoneurons (NF-L–Cre; Stat3flox/KO) or mice carrying at least one wild-type Stat3 allele (NF-L–Cre; Stat3flox/wt) at a 50% ratio. Bar, 100 μm.
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fig1: Generation and characterization of NF-L–Cre. (A) The coding region of exon 1 within a 6-kb human NF-L gene fragment was replaced by the open reading frame of bacteriophage P1 Cre recombinase leaving regulatory sequences within NF-L introns intact. (B) Cre recombinase is specifically expressed in brain and spinal cord. RT-PCR on total RNA derived from adult cerebral cortex, brainstem, spinal cord, and sciatic nerve detects Cre message only brain and spinal cord, but not in sciatic nerve, liver, spleen, and kidney (latter three not shown). (C) Transgenic Cre recombinase is enzymatically active and recombines a reporter allele in vivo. NF-L–Cre mice were cross-bred with a lac Z reporter strain. Cre-mediated recombination of the reporter gene was detected via PCR on genomic DNA by a 580-bp product as opposed to the 1,700-bp product of the unrecombined reporter allele. Cre- mediated recombination is therefore specific for brain and spinal cord. (D) Cre recombinase expression is neuron- specific. In situ hybridization detects Cre expression in the facial nucleus and other brain regions (unpublished data). The transgenic Cre allele is solely expressed in neurons but not in surrounding glia. (E) Cre-mediated site-specific recombination in facial motoneurons. Brain sections from mice transgenic for NF-L–Cre and for the lacZ reporter allele were stained with X-Gal for β-galactosidase activity reflecting Cre-mediated activation of the lacZ reporter gene. 53.7 ± 12.9% (mean ± SD; n = 3) of motoneurons in the adult facial nucleus appeared X-Gal positive. (F) Breeding scheme to produce mice with a neuron-specific Stat3 gene ablation. Mice with the indicated genotypes were crossed to produce mice lacking functional Stat3 in motoneurons (NF-L–Cre; Stat3flox/KO) or mice carrying at least one wild-type Stat3 allele (NF-L–Cre; Stat3flox/wt) at a 50% ratio. Bar, 100 μm.
Mentions: To study Stat3 function in postnatal motoneurons, we have generated a transgenic mouse line expressing the bacteriophage P1 Cre recombinase under control of the human NF-L promoter and intragenic sequences. Previous studies (Leconte et al., 1994; Akassoglou et al., 1997) have shown that the human NF-L promoter and its intragenic sequences can drive heterologous expression of transgenes in mice. Therefore, we have replaced the coding region of the first exon of the human NF-L gene by a CrepA cassette, leaving 2 kb of the 5′ promoter and 3 kb of downstream sequence intact (Fig. 1 A). Two lines of transgenic mice were established and various tissues were tested for Cre mRNA expression by reverse transcription (RT)-PCR. One line of transgenic mice exhibited Cre expression in brain and spinal cord but not in sciatic nerve or organs such as kidney, liver, and spleen (Fig. 1 B; unpublished data). To control whether Cre expressed under the NF-L promoter is functional, we cross-bred NF-L–Cre with a lacZ reporter strain (Akagi et al., 1997) and analyzed double transgenic mice at various embryonic and postnatal stages. Using a PCR-based assay (Akagi et al., 1997), we show that Cre expressed from the NF-L–Cre transgene is functional and recombines a loxP-flanked reporter gene specifically in neural tissues (Fig. 1 C). To test whether Cre expression is specific for neurons, we performed in situ hybridization with a probe specific for Cre mRNA. As shown in Fig. 1 D, the Cre transgene is expressed in motoneurons of the facial nucleus, but not in the surrounding glia. Cre expression was also detected in hippocampal and cortical neurons (unpublished data). We then tested the cell type specificity of Cre-mediated recombination by staining of brain slices and whole organs for β-galactosidase. Again, X-gal staining was confined to neurons, in particular spinal and brain stem motoneurons (Fig. 1 E and unpublished data). Neuron-specific inactivation of the Stat3 gene was achieved by cross-breeding NF-L–Cre transgenic mice with mice carrying a floxed Stat3 gene (Takeda et al., 1998) according to the scheme depicted in Fig. 1 F. In these mice, Cre-mediated recombination leads to removal of exon 22 of Stat3, which contains the functionally relevant tyrosine residue. Due to usage of a cryptic splice site, part of the following exon containing the regulatory serine phosphorylation site is also removed (Takeda et al., 1998). Inclusion of a Stat3 allele resulted in 50% offspring devoid of functional Stat3 in a significant subpopulation of motoneurons (NF-L–Cre; Stat3flox/KO) and 50% offspring carrying one functional allele of Stat3 in motoneurons (NF-L–Cre; Stat3flox/wt).

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