clc is co-expressed with clf or cntfr in developing mouse muscles
© de Bovis et al; licensee BioMed Central Ltd. 2005
Received: 21 July 2004
Accepted: 31 January 2005
Published: 31 January 2005
The ciliary neurotrophic factor (CNTF) receptor is composed of two signalling receptor chains, gp130 and the leukaemia inhibitory factor receptor, associated with a non-signalling CNTF binding receptor α component (CNTFR). This tripartite receptor has been shown to play important roles in the development of motor neurons, but the identity of the relevant ligand(s) is still not clearly established. Recently, we have identified two new ligands for the CNTF receptor complex. These are heterodimeric cytokines composed of cardiotrophin-like cytokine (CLC) associated either with the soluble receptor subunit cytokine-like factor-1 (CLF) or the soluble form of the binding receptor itself (sCNTFR).
Here we show that, during development, clc is expressed in lung, kidney, vibrissae, tooth, epithelia and muscles during the period of development corresponding to when motoneuron loss is observed in mice lacking a functional CNTF receptor. In addition, we demonstrate that it is co-expressed at the single cell level with clf and cntfr, supporting the idea that CLC might be co-secreted with either CLF or sCNTFR.
This expression pattern is in favor of CLC, associated either with CLF or sCNTFR, being an important player in the signal triggered by the CNTF receptor being required for motoneuron development.
CLC (cardiotrophin-like cytokine) shares homology with CNTF (ciliary neurotrophic factor) and CT-1 (cardiotrophin-1) and requires co-expression with either CLF (cytokine-like factor-1) or the soluble form of the CNTFR to be secreted [1, 2]. The CLC-CLF heterodimer displays activities only on cells expressing a functional CNTF receptor  and therefore CLC is likely to be part of the developmentally important second ligand for CNTFR. The existence of such a second ligand has been suggested by the phenotype of mice lacking any of the three receptor subunits comprising the functional CNTF receptor complex (LIFRβ, gp130 and CNTFR) which exhibit significant reductions in motoneuron number [3–5] whereas CNTF-deficient mice have no motoneuron loss during development . There are however two prerequisites for CLC to play a major role in motoneuron development: 1) CLC must be expressed in the environment of motoneurons during development. 2) As it cannot be secreted alone, it must be co-expressed with either CLF or sCNTFR, in the same cell.
Results and Discussion
Developmental expression of clc
RT-PCR analysis of clc, clf and cntfr expressiona
0.113 ± 0.02
0.936 ± 0.12
0.232 ± 0.003
24.84 ± 2.13
90.25 ± 2.71
6.72 ± 0.76
3.12 ± 0.12
1.2 ± 0.09
495 ± 17.1
77.1 ± 4.64
28.3 ± 10.9
2.6 ± 0.83
11.4 ± 0.75
9.25 ± 0.79
1050 ± 65.3
524 ± 85.8
425 ± 47.5
57.9 ± 4.02
4.08 ± 0.65
16.4 ± 0.48
2290 ± 490
2240 ± 184
43.8 ± 15.6
5.55 ± 0.12
6.75 ± 1.15
61.4 ± 4.59
51.8 ± 9.49
19.8 ± 3.12
0.191 ± 0.02
0.129 ± 0.03
0.071 ± 0.001
0.034 ± 0.09
0.047 ± 0.002
0.038 ± 0.03
1.04 ± 0.12
0.317 ± 0.09
150 ± 3.39
74.3 ± 16.4
216 ± 2.01
236 ± 16.1
0.183 ± 0.01
0.123 ± 0.01
61.2 ± 6.25
6.13 ± 0.03
109 ± 27.4
Co-expression of clc, clf and cntfr in the developing muscle
Clc is expressed in developing muscles during the period of motoneuron loss in mice lacking a functional CNTF receptor and it is co-expressed with both CLF and CNTFR. This expression pattern is in favor of the hypothesis that CLC is an important player in the signal triggered by the CNTF receptor and that is required for motoneuron development. In addition, our results show that in the kidney, clc is expressed in cells neighboring those expressing clf or cntfr but it is not co-expressed with these genes suggesting either the possible existence of an additional protein capable of inducing secretion of CLC or that CLC is not secreted in these cells and therefore not functional. Because genetic deletion of cntf fails to perturb neuronal development before birth, we can hypothesize some functional redundancies in vivo that will require the analysis of double or triple knockout mice for CNTFR ligands to clarify their respective involvement in mouse neural development.
RT and real time PCR
Total RNA was extracted using Trizol reagent (Invitrogen) from E16.5 or E18.5 mouse tissues according to the manufacturer's instructions. Complementary cDNA was synthesised from 2 μg of RNA by random hexamer priming using MMLV reverse transcriptase (Promega). Quantitative PCR was performed using a capillary real-time LightCycler (Roche Diagnostics), and the data analysed using "Fit Point Method" (Roche Diagnostics). For comparison of gene expression levels, all quantifications were normalized to endogenous gapdh to account for variability in the initial concentration of RNA and for differences in the efficiency of the reverse transcription reactions. The following primers were designed to amplify mouse clc: 5'-GCTACCTGGAGCATCAACT-3', 5'-GGTGACTGTACGCCTCATAG-3'; clf: 5'-CAGTCAGGAGACAATCTGGT-3', 5'-ACGTGAGATCCTTCATGTTC-3'; cntfr: 5'-CTACATCCCCAATACCTACA-3', 5'-GTGAATTCGTCAAAGGTGAT-3'; gapdh: 5'-TGCGACTTCAACAGCAACTC-3', 5'-CTTGCTCAGTGTCCTTGCTG-3'. Results are expressed in fmole of cDNA/μgRNA.
Plasmid cDNA clones were linearized and transcribed with T7 or T3 polymerase using digoxigenin (Dig) or fluorescein (Fluo)labeling reagents (Roche Diagnostics). Probes were used at a concentration of 500 ng/ml. The cntfr clone was as previously described  and the mouse clf  and clc probes corresponded to the isolated cDNAs.
In situ hybridization
In situ hybridization was performed as described previously  on 20 μm-thick frozen transverse cryostat sections prepared from mouse embryos fixed with 4% paraformaldehyde in PBS, and cryopreserved in 15% sucrose in PBS before embedding in OCT compound (Miles). Alternatively, 100 μm-thick vibratome sections were prepared from fixed embryos embedded in glutaraldehyde/gelatin. After hybridization overnight at 70°C with Dig-labeled riboprobes, the slides were washed twice in 1X SSC, 50% formamide at 70°C for 30 min and blocked in the presence of 4% blocking reagent (Roche Diagnostics) and 20% inactivated sheep serum. The slides were then incubated with anti-Dig-alkaline-phosphatase (AP)-conjugated antibody (1/5000, Roche Diagnostics), washed and revealed by NBT/BCIP staining.
In order to confirm that muscle fibers, per se, express clc and clf, double in situ hybridization / immunohistochemistry was carried out as described  on sections from E16.5 MLCnlacZ mice, which express the nlacZ reporter gene under the control of a muscle-specific myosin light chain promoter. After in situ hybridization, slides were rinsed in PBT (PBS, 0.1% Triton), and sections were successively incubated for 1 h with blocking solution containing 2% BSA, 2% heat-inactivated donkey serum in PBT and then overnight at 4°C with rabbit anti-β-galactosidasel (1/1000, Cappel). After three washes in PBT, slides were incubated 1 h at RT with a biotin donkey anti-mouse secondary antibody. Slides were then washed in PBS, and TBS (50 mM Tris-HCl, 0.15 M NaCl, pH 7.6), and incubated for 30 min at RT in ABC streptavidin/HRP in TBS. Staining was revealed with DAB (D4293, Sigma) in the presence of H2O2.
Double in situ hybridization was performed as described previously . Briefly, Dig- and Fluo-labeled probes were mixed in hybridization buffer and applied to sections. After hybridization at 70°C overnight and washing at 65°C, the first probe was revealed using a 1:2000 dilution of anti-Fluo-alkaline phosphatase (AP)- conjugate (Roche Diagnostics) and Fast Red (Sigma) as a substrate. Sections were photographed at this stage. After AP inactivation with 0.1 M glycine, pH 2.2, the second probe was revealed using a 1:5000 dilution of anti-Dig-AP and NBT/BCIP staining. Fast Red precipitates were then removed by incubating the slides in increasing concentrations of ethanol culminating in two final incubations in 100% ethanol for 10 min before cleaning with Histoclear and mounting with Eukitt (VWR, Strasbourg, France). Photomicrographs of the NBT/BCIP results were then taken for comparison with those showing the Fast Red results on the same sections.
We thank members of INSERM U.623 and U.564 for many helpful discussions and encouraging support. This work was funded by INSERM, CNRS, the Association Française contre les Myopathies (AFM), the post-genome program from Région Pays-de-la-Loire, the Canadian Institutes of Health Research (IRSC) and the Multiple Sclerosis Scientific Research Foundation (SP). Damien Derouet was supported by INSERM and the Région Pays-de-la-Loire.
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