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Redundancy of myostatin and growth/differentiation factor 11 function.

McPherron AC, Huynh TV, Lee SJ - BMC Dev. Biol. (2009)

Bottom Line: Despite their high degree of sequence identity, targeted mutations in these genes result in non-overlapping phenotypes affecting distinct biological processes.In order to investigate the possible functional redundancy of myostatin and Gdf11, we analyzed the effect of eliminating the functions of both of these signaling molecules.We also show that deletion of Gdf11 specifically in skeletal muscle in either Mstn+/+ or Mstn-/- mice does not affect muscle size, fiber number, or fiber type.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA. mcpherrona@niddk.nih.gov

ABSTRACT

Background: Myostatin (Mstn) and growth/differentiation factor 11 (Gdf11) are highly related transforming growth factor beta (TGFbeta) family members that play important roles in regulating embryonic development and adult tissue homeostasis. Despite their high degree of sequence identity, targeted mutations in these genes result in non-overlapping phenotypes affecting distinct biological processes. Loss of Mstn in mice causes a doubling of skeletal muscle mass while loss of Gdf11 in mice causes dramatic anterior homeotic transformations of the axial skeleton, kidney agenesis, and an increase in progenitor cell number in several tissues. In order to investigate the possible functional redundancy of myostatin and Gdf11, we analyzed the effect of eliminating the functions of both of these signaling molecules.

Results: We show that Mstn-/- Gdf11-/- mice have more extensive homeotic transformations of the axial skeleton than Gdf11-/- mice in addition to skeletal defects not seen in single mutants such as extra forelimbs. We also show that deletion of Gdf11 specifically in skeletal muscle in either Mstn+/+ or Mstn-/- mice does not affect muscle size, fiber number, or fiber type.

Conclusion: These results provide evidence that myostatin and Gdf11 have redundant functions in regulating skeletal patterning in mice but most likely not in regulating muscle size.

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Related in: MedlinePlus

Muscle-specific targeting of Gdf11 gene. (A) Representation of targeting strategy. The 3 exons are shown as boxes with coding sequences shaded black and the 3'UTR open. The targeting construct is represented by a thick line which contains loxP sequences with EcoRI restriction sites inserted into the second XbaI site in exon 1 and flanking a neo gene inserted into the EcoRI site downstream of the 3'UTR. Cre-mediated recombination of the loxP sites flanking the neo gene results in a Gdf11flox allele. Recombination of the Gdf11flox allele generates the Gdf11Δ2–3 allele. Oligonucleotide primers used for distinguishing alleles are labeled a, b, and c. A skeletal muscle-specific Cre expressing transgene was constructed using the MLC1 promoter/1/3 enhancer and an SV40 t antigen intron and poly adenylation signal. (B) Southern blot showing detection of Gdf11+, Gdf11flox, and Gdf11-(, from the original knockout line) alleles. (C) Northern blot analysis of MLC-Cre transgene expression in pectoralis and quadriceps muscles but not in other tissues. (D) Detection of Gdf11 alleles in genomic DNA in Gdf11flox/floxand Gdf11flox/floxMLC-Cre mice by PCR (right panel). Recombination was detected in quadriceps and pectoralis muscles of Gdf11flox/floxMLC-Cre mice but not in heart, brain, or kidney. No recombination was seen in Gdf11flox/floxmice. Left panel shows control reactions. (E) Northern blot analysis of skeletal muscle Gdf11 expression showing a strong decrease in expression in Gdf11flox/-MLC-Cre muscle compared to Gdf11flox/-muscle.
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Figure 2: Muscle-specific targeting of Gdf11 gene. (A) Representation of targeting strategy. The 3 exons are shown as boxes with coding sequences shaded black and the 3'UTR open. The targeting construct is represented by a thick line which contains loxP sequences with EcoRI restriction sites inserted into the second XbaI site in exon 1 and flanking a neo gene inserted into the EcoRI site downstream of the 3'UTR. Cre-mediated recombination of the loxP sites flanking the neo gene results in a Gdf11flox allele. Recombination of the Gdf11flox allele generates the Gdf11Δ2–3 allele. Oligonucleotide primers used for distinguishing alleles are labeled a, b, and c. A skeletal muscle-specific Cre expressing transgene was constructed using the MLC1 promoter/1/3 enhancer and an SV40 t antigen intron and poly adenylation signal. (B) Southern blot showing detection of Gdf11+, Gdf11flox, and Gdf11-(, from the original knockout line) alleles. (C) Northern blot analysis of MLC-Cre transgene expression in pectoralis and quadriceps muscles but not in other tissues. (D) Detection of Gdf11 alleles in genomic DNA in Gdf11flox/floxand Gdf11flox/floxMLC-Cre mice by PCR (right panel). Recombination was detected in quadriceps and pectoralis muscles of Gdf11flox/floxMLC-Cre mice but not in heart, brain, or kidney. No recombination was seen in Gdf11flox/floxmice. Left panel shows control reactions. (E) Northern blot analysis of skeletal muscle Gdf11 expression showing a strong decrease in expression in Gdf11flox/-MLC-Cre muscle compared to Gdf11flox/-muscle.

Mentions: We also sought to determine whether myostatin and Gdf11 are functionally redundant with respect to control of muscle mass. The increase in muscle mass in Mstn-/- mice is not present at birth (A.C.M. and S-J.L., unpublished observations) so the neonatal death of Gdf11-/- and Mstn-/- Gdf11-/- mice precludes a comparison of the skeletal muscle phenotype. We therefore generated a targeting construct containing a conditional deletion allele of the Gdf11 gene by inserting loxP recombination sites into intron 1 and flanking a neo gene downstream of the Gdf11 3' UTR (Gdf11flox-neo) (Figure 2A and 2B). Following homologous recombination in embryonic stem (ES) cells and injection of the targeted cells into blastocysts, we obtained chimeric mice that transmitted the Gdf11flox-neo allele through the germline. Mice carrying the Gdf11flox-neo allele were then crossed to EIIa-Cre transgenic mice to generate Gdf11flox/+ mice carrying one upstream and one downstream loxP site after removal of the neo gene. Recombination at the remaining loxP sites would be predicted to delete exons 2 and 3 (Gdf11Δ2–3), which would remove the biologically-active carboxy-terminal domain. To demonstrate that recombination of loxP sites produced a allele, we generated germline recombination of loxP sites in Gdf11flox/+ mice (Gdf11Δ2–3/+). Skeletal analysis performed on offspring from Gdf11Δ2–3/+ matings showed that Gdf11Δ2–3/+ and Gdf11Δ2–3/Δ2–3 mice had 1 and 5 extra thoracic vertebrae, respectively (data not shown). These thoracic vertebral numbers are identical to that of Gdf11+/- and Gdf11-/- mice [18] confirming that recombination of loxP sites in the Gdf11flox allele results in a allele.


Redundancy of myostatin and growth/differentiation factor 11 function.

McPherron AC, Huynh TV, Lee SJ - BMC Dev. Biol. (2009)

Muscle-specific targeting of Gdf11 gene. (A) Representation of targeting strategy. The 3 exons are shown as boxes with coding sequences shaded black and the 3'UTR open. The targeting construct is represented by a thick line which contains loxP sequences with EcoRI restriction sites inserted into the second XbaI site in exon 1 and flanking a neo gene inserted into the EcoRI site downstream of the 3'UTR. Cre-mediated recombination of the loxP sites flanking the neo gene results in a Gdf11flox allele. Recombination of the Gdf11flox allele generates the Gdf11Δ2–3 allele. Oligonucleotide primers used for distinguishing alleles are labeled a, b, and c. A skeletal muscle-specific Cre expressing transgene was constructed using the MLC1 promoter/1/3 enhancer and an SV40 t antigen intron and poly adenylation signal. (B) Southern blot showing detection of Gdf11+, Gdf11flox, and Gdf11-(, from the original knockout line) alleles. (C) Northern blot analysis of MLC-Cre transgene expression in pectoralis and quadriceps muscles but not in other tissues. (D) Detection of Gdf11 alleles in genomic DNA in Gdf11flox/floxand Gdf11flox/floxMLC-Cre mice by PCR (right panel). Recombination was detected in quadriceps and pectoralis muscles of Gdf11flox/floxMLC-Cre mice but not in heart, brain, or kidney. No recombination was seen in Gdf11flox/floxmice. Left panel shows control reactions. (E) Northern blot analysis of skeletal muscle Gdf11 expression showing a strong decrease in expression in Gdf11flox/-MLC-Cre muscle compared to Gdf11flox/-muscle.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2666675&req=5

Figure 2: Muscle-specific targeting of Gdf11 gene. (A) Representation of targeting strategy. The 3 exons are shown as boxes with coding sequences shaded black and the 3'UTR open. The targeting construct is represented by a thick line which contains loxP sequences with EcoRI restriction sites inserted into the second XbaI site in exon 1 and flanking a neo gene inserted into the EcoRI site downstream of the 3'UTR. Cre-mediated recombination of the loxP sites flanking the neo gene results in a Gdf11flox allele. Recombination of the Gdf11flox allele generates the Gdf11Δ2–3 allele. Oligonucleotide primers used for distinguishing alleles are labeled a, b, and c. A skeletal muscle-specific Cre expressing transgene was constructed using the MLC1 promoter/1/3 enhancer and an SV40 t antigen intron and poly adenylation signal. (B) Southern blot showing detection of Gdf11+, Gdf11flox, and Gdf11-(, from the original knockout line) alleles. (C) Northern blot analysis of MLC-Cre transgene expression in pectoralis and quadriceps muscles but not in other tissues. (D) Detection of Gdf11 alleles in genomic DNA in Gdf11flox/floxand Gdf11flox/floxMLC-Cre mice by PCR (right panel). Recombination was detected in quadriceps and pectoralis muscles of Gdf11flox/floxMLC-Cre mice but not in heart, brain, or kidney. No recombination was seen in Gdf11flox/floxmice. Left panel shows control reactions. (E) Northern blot analysis of skeletal muscle Gdf11 expression showing a strong decrease in expression in Gdf11flox/-MLC-Cre muscle compared to Gdf11flox/-muscle.
Mentions: We also sought to determine whether myostatin and Gdf11 are functionally redundant with respect to control of muscle mass. The increase in muscle mass in Mstn-/- mice is not present at birth (A.C.M. and S-J.L., unpublished observations) so the neonatal death of Gdf11-/- and Mstn-/- Gdf11-/- mice precludes a comparison of the skeletal muscle phenotype. We therefore generated a targeting construct containing a conditional deletion allele of the Gdf11 gene by inserting loxP recombination sites into intron 1 and flanking a neo gene downstream of the Gdf11 3' UTR (Gdf11flox-neo) (Figure 2A and 2B). Following homologous recombination in embryonic stem (ES) cells and injection of the targeted cells into blastocysts, we obtained chimeric mice that transmitted the Gdf11flox-neo allele through the germline. Mice carrying the Gdf11flox-neo allele were then crossed to EIIa-Cre transgenic mice to generate Gdf11flox/+ mice carrying one upstream and one downstream loxP site after removal of the neo gene. Recombination at the remaining loxP sites would be predicted to delete exons 2 and 3 (Gdf11Δ2–3), which would remove the biologically-active carboxy-terminal domain. To demonstrate that recombination of loxP sites produced a allele, we generated germline recombination of loxP sites in Gdf11flox/+ mice (Gdf11Δ2–3/+). Skeletal analysis performed on offspring from Gdf11Δ2–3/+ matings showed that Gdf11Δ2–3/+ and Gdf11Δ2–3/Δ2–3 mice had 1 and 5 extra thoracic vertebrae, respectively (data not shown). These thoracic vertebral numbers are identical to that of Gdf11+/- and Gdf11-/- mice [18] confirming that recombination of loxP sites in the Gdf11flox allele results in a allele.

Bottom Line: Despite their high degree of sequence identity, targeted mutations in these genes result in non-overlapping phenotypes affecting distinct biological processes.In order to investigate the possible functional redundancy of myostatin and Gdf11, we analyzed the effect of eliminating the functions of both of these signaling molecules.We also show that deletion of Gdf11 specifically in skeletal muscle in either Mstn+/+ or Mstn-/- mice does not affect muscle size, fiber number, or fiber type.

View Article: PubMed Central - HTML - PubMed

Affiliation: Genetics of Development and Disease Branch, National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, 9000 Rockville Pike, Bethesda, MD 20892, USA. mcpherrona@niddk.nih.gov

ABSTRACT

Background: Myostatin (Mstn) and growth/differentiation factor 11 (Gdf11) are highly related transforming growth factor beta (TGFbeta) family members that play important roles in regulating embryonic development and adult tissue homeostasis. Despite their high degree of sequence identity, targeted mutations in these genes result in non-overlapping phenotypes affecting distinct biological processes. Loss of Mstn in mice causes a doubling of skeletal muscle mass while loss of Gdf11 in mice causes dramatic anterior homeotic transformations of the axial skeleton, kidney agenesis, and an increase in progenitor cell number in several tissues. In order to investigate the possible functional redundancy of myostatin and Gdf11, we analyzed the effect of eliminating the functions of both of these signaling molecules.

Results: We show that Mstn-/- Gdf11-/- mice have more extensive homeotic transformations of the axial skeleton than Gdf11-/- mice in addition to skeletal defects not seen in single mutants such as extra forelimbs. We also show that deletion of Gdf11 specifically in skeletal muscle in either Mstn+/+ or Mstn-/- mice does not affect muscle size, fiber number, or fiber type.

Conclusion: These results provide evidence that myostatin and Gdf11 have redundant functions in regulating skeletal patterning in mice but most likely not in regulating muscle size.

Show MeSH
Related in: MedlinePlus