Transgenic over-expression of growth differentiation factor 11 propeptide in skeleton results in transformation of the seventh cervical vertebra into a thoracic vertebra.
ABSTRACT: Growth differentiation factor 11 (GDF11) is one of the significant genes that control skeletal formation. Knockout of GDF11 function causes abnormal patterning of the anterior/posterior axial skeleton. The mRNA of GDF11 is initially translated to a precursor protein that undergoes a proteolytic cleavage to generate the C-terminal peptide or mature GDF11, and the N-terminal peptide named GDF11 propeptide. The propeptide can antagonize GDF11 activity in vitro. To investigate the effects of GDF11 propeptide on GDF11 function in vivo, we generated transgenic mice that over-express the propeptide cDNA in skeletal tissue. The transgenic mice showed formation of extra ribs on the seventh cervical vertebra (C7) as a result of transformation of the C7 vertebra into a thoracic vertebra. The GDF11 propeptide transgene mRNA was detected in tail tissue in embryos and was highly expressed in tail and calvaria bones after birth. A high frequency of C7 rib formation was noticed in the transgenic mouse line with a high level of transgene expression. The anterior boundaries of Hoxa-4 and Hoxa-5 mRNA in situ expressions showed cranial shifts from their normal prevertebra locations in transgenic embryos. These results demonstrated significant effects of GDF11 propeptide transgene on vertebral formation, which are likely occurring through depressing GDF11 function and altered locations of Hoxa-4 and Hoxa-5 expression.
Project description:BACKGROUND:Growth differentiation factor 11 (GDF11) is a member of the transforming growth factor β superfamily. The GDF11 propeptide, which is derived from the GDF11 precursor protein, blocks the activity of GDF11 and its homolog, myostatin, which are both potent inhibitors of muscle growth. Thus, treatment with GDF11 propeptide may be a potential therapeutic strategy for diseases associated with muscle atrophy like sarcopenia and the muscular dystrophies. Here, we evaluate the impact of GDF11 propeptide-Fc (GDF11PRO-Fc) gene delivery on skeletal muscle in normal and dystrophic adult mice. METHODS:A pull-down assay was used to obtain physical confirmation of a protein-protein interaction between GDF11PRO-Fc and GDF11 or myostatin. Next, differentiated C2C12 myotubes were treated with AAV6-GDF11PRO-Fc and challenged with GDF11 or myostatin to determine if GDF11PRO-Fc could block GDF11/myostatin-induced myotube atrophy. Localized expression of GDF11PRO-Fc was evaluated via a unilateral intramuscular injection of AAV9-GDF11PRO-Fc into the hindlimb of C57BL/6J mice. In mdx mice, intravenous injection of AAV9-GDF11PRO-Fc was used to achieve systemic expression. The impact of GDF11PRO-Fc on muscle mass, function, and pathological features were assessed. RESULTS:GDF11PRO-Fc was observed to bind both GDF11 and myostatin. In C2C12 myotubes, expression of GDF11PRO-Fc was able to mitigate GDF11/myostatin-induced atrophy. Following intramuscular injection in C57BL/6J mice, increased grip strength and localized muscle hypertrophy were observed in the injected hindlimb after 10 weeks. In mdx mice, systemic expression of GDF11PRO-Fc resulted in skeletal muscle hypertrophy without a significant change in cardiac mass after 12 weeks. In addition, grip strength and rotarod latency time were improved. Intramuscular fibrosis was also reduced in treated mdx mice; however, there was no change seen in central nucleation, membrane permeability to serum IgG or serum creatine kinase levels. CONCLUSIONS:GDF11PRO-Fc induces skeletal muscle hypertrophy and improvements in muscle strength via inhibition of GDF11/myostatin signaling. However, GDF11PRO-Fc does not significantly improve the dystrophic pathology in mdx mice.
Project description:Circulating polypeptides and proteins have been implicated in reversing or accelerating aging phenotypes, including growth/differentiation factor 8 (GDF8), GDF11, eotaxin, and oxytocin. These proteoforms, which are defined as the protein products arising from a single gene due to alternative splicing and PTMs, have been challenging to study. Both GDF8 and GDF11 have known antagonists such as follistatin (FST), and WAP, Kazal, immunoglobulin, Kunitz, and NTR domain-containing proteins 1 and 2 (WFIKKN1, WFIKKN2). We developed a novel multiplexed SRM assay using LC-MS/MS to measure five proteins related to GDF8 and GDF11 signaling, and in addition, eotaxin, and oxytocin. Eighteen peptides consisting of 54 transitions were monitored and validated in pooled human plasma. In 24 adults, the mean (SD) concentrations (ng/mL) were as follows: GDF8 propeptide, 11.0 (2.4); GDF8 mature protein, 25.7 (8.0); GDF11 propeptide, 21.3 (10.9); GDF11 mature protein, 16.5 (12.4); FST, 29.8 (7.1); FST cleavage form FST303, 96.4 (69.2); WFIKKN1, 38.3 (8.3); WFIKKN2, 32.2 (10.5); oxytocin, 1.9 (0.9); and eotaxin, 2.3 (0.5). This novel multiplexed SRM assay should facilitate the study of the relationships of these proteoforms with major aging phenotypes.
Project description:WFIKKN1 and WFIKKN2 are large extracellular multidomain proteins consisting of a WAP, a follistatin, an immunoglobulin, two Kunitz-type protease inhibitor domains, and an NTR domain. Recent experiments have shown that WFIKKN2 protein binds mature GDF8/myostatin and myostatin propeptide and inhibits the biological activity of myostatin (Hill, J. J., Qiu, Y., Hewick, R. M., and Wolfman, N. M. (2003) Mol. Endocrinol. 17, 1144-1154). Here we show that the paralogue of this protein, WFIKKN1, also binds to both myostatin and myostatin propeptide and that both WFIKKN1 and WFIKKN2 bind GDF11, the growth and differentiation factor most closely related to myostatin, with high affinity. Structure-function studies on WFIKKN1 have revealed that the follistatin domain is primarily responsible for the binding of mature growth factor, whereas the NTR domain contributes most significantly to the interaction with myostatin propeptide. Analysis of the evolutionary histories of WFIKKN1/WFIKKN2 and GDF8/GDF11 proteins indicates that the functional association of an ancestral WFIKKN protein with an ancestor of GDF8/11 may date back to cephalochordates/urochordates. Although duplication of the corresponding genes gave rise to WFIKKN1/WFIKKN2 and GDF8/GDF11 in early vertebrates, the data presented here suggest that there is significant functional overlap of the paralogous proteins.
Project description:Murine Hoxd-3 (Hox 4.1) genomic DNA and cDNA and Hoxa-3 (Hox 1.5) cDNA were cloned and sequenced. The homeodomains of Hoxd-3 and Hoxa-3 and regions before and after the homeodomain are highly conserved. Both Hoxa-3 and Hoxa-3 proteins have a proline-rich region that contains consensus amino acid sequences for binding to Src homology 3 domains of some signal transduction proteins. Northern blot analysis of RNA from 8- to 11-day-old mouse embryos revealed a 4.3-kb species of Hoxd-3 RNA, whereas a less abundant 3.0-kb species of Hoxd-3 RNA was found in RNA from 9- to 11-day-old embryos. Two species of Hoxd-3 poly(A)+ RNA, 4.3 and 6.0 kb in length, were found in poly(A)+ RNA from adult mouse kidney, but not in RNA from other adult tissues tested. Hoxd-3 mRNA was detected by in situ hybridization in 12-, 14-, and 17-day-old mouse embryos in the posterior half of the myelencephalon, spinal cord, dorsal root ganglia, first cervical vertebra, thyroid gland, kidney tubules, esophagus, stomach, and intestines.
Project description:Growth and differentiation factor 11 (GDF11; BMP11) is a circulating cytokine in the transforming growth factor beta (TGF-?) superfamily. Treatment with recombinant GDF11 (rGDF11) protein has previously been shown to reverse skeletal muscle dysfunction in aged mice. However, the actions of GDF11 in skeletal muscle are still not fully understood. Because GDF11 activates the TGF-?-SMAD2/3 pathway, we hypothesized that GDF11 overexpression would inhibit skeletal muscle growth. To test this hypothesis, we generated recombinant adeno-associated virus serotype 9 (AAV9) vectors harboring the gene for either human GDF11 (AAV9-GDF11) or human IgG1 Fc-fused GDF11 propeptide (AAV9-GDF11Pro-Fc-1) to study the effects of GDF11 overexpression or blockade on skeletal muscle growth and function in vivo. After intravenous administration of AAV9-GDF11 into neonatal C57BL/6J mice, we observed sustained limb muscle growth inhibition along with reductions in forelimb grip strength and treadmill running endurance at 16 weeks. Conversely, treatment with AAV9-GDF11Pro-Fc-1 led to increased limb muscle mass and forelimb grip strength after 28 weeks, although a difference in the total body mass/muscle mass ratio was not observed between treatment and control groups. In sum, our results suggest GDF11 overexpression has an inhibitory effect on skeletal muscle growth.
Project description:We compared the effectiveness of postural reduction and instrumental reduction in the treatment of thoracolumbar and lumbar vertebra fracture in 40 patients. Under general anaesthesia, postural reduction in a stretching prone position was first performed. Instrumental reduction and fixation were then conducted. Radiographs were made after each reduction. Comparisons between the two films and the fracture films were made based on the changes in prevertebral height of both the fractured vertebra and the adjacent superior and inferior intervertebral spaces. It was found that the recovery of the prevertebral height in postural and instrumental reductions was basically identical. The recovery of the prevertebral height in the intervertebral spaces was more significant in instrumental reduction. Both reductions were ineffective in patients whose compression of the diseased vertebra was more than two-thirds of the normal. In cases of lower lumbar vertebra fractures, the effect of both reductions was unsatisfactory. Our findings indicated that the effectiveness of the reduction of vertebra fracture depends on the quantitative change of the spongy bone of the injured vertebra. Instrumental reduction only exerts an indirect tension. Postural reduction is effective in reducing thoracolumbar vertebral fracture, while instrumental reduction exerts only a relatively weak effect but it is particularly useful to maintain the result of postural reduction.
Project description:Hoxa9 is expressed in hematopoietic stem and progenitor cells, although this expression is usually diminished as these cells undergo differentiation. In addition, aberrant expression of Hoxa9 is strongly associated with both T cell and myeloid leukemia in mice and humans. Despite this strong association, enforced expression of Hoxa9 in murine bone marrow or thymus has only shown a modest ability to transform cells. To investigate this question, we used Vav regulatory elements to generate a transgenic mouse that targets Hoxa9 overexpression to all hematopoietic tissues. High-level expression of the Hoxa9 transgene in the hematopoietic compartment was associated with embryonic lethality, as no pups from founders that expressed high levels of the transgene were born live. However, offspring of an additional founder line, which expressed lower levels of Hoxa9, developed a precursor T cell lymphoblastic leukemia/lymphoma, accompanied by spontaneous Notch1 mutations. In contrast to most murine models of leukemia associated with Hoxa9 overexpression, the Vav-Hoxa9 mice did not overexpress other Hoxa cluster genes, mir196b (a microRNA that is embedded in the Hoxa locus), Meis1, or Pbx3. The Hoxa9 transgenic mouse reported in this study provides a suitable system for the study of Hoxa9 collaborators that drive myeloid and lymphoid malignant transformation.
Project description:The hagfish, a group of extant jawless fish, are known to lack true vertebrae and, for this reason, have often been excluded from the group Vertebrata. However, it has yet to be conclusively shown whether hagfish lack all vertebra-like structures, and whether their somites follow developmental processes and patterning distinct from those in lampreys and gnathostomes. Here we report the presence of vertebra-like cartilages in the in-shore hagfish, Eptatretus burgeri. These elements arise as small nodules occupying anatomical positions comparable to those of gnathostome vertebrae. Examination of hagfish embryos suggests that the ventromedial portion of a somite transforms into mesenchymal cells that express cognates of Pax1/9 and Twist, strikingly similar to the pattern of sclerotome development in gnathostomes. We conclude that the vertebra-like elements in the hagfish are homologous to gnathostome vertebrae, implying that this animal underwent secondary reduction of vertebrae in most of the trunk.
Project description:Introduction:A prior study showed the touched vertebra (TV), defined as the most cephalad thoracolumbar/lumbar vertebra “touched” by the center sacral vertical line (CSVL), as a potential landmark vertebra & recommended lowest instrumented vertebra (LIV) as well. We evaluated a large cohort of Lenke type 1 & 2 cases to determine if selecting the TV as the LIV will produce optimal positioning at a min. 5 yrs postoperative. Our hypothesis was that it would and that fusing short of the TV would lead to a suboptimal result.Material and Methods:299 pts with Lenke 1 (n = 207) or Lenke 2 (n = 92) AIS curves at a min. 5 yr f/u were evaluated. The TV was selected on the preoperative x-ray by 2 independent examiners & confirmed for agreement. The LIV selected was compared to the preoperative TV as well as the LIV-CSVL distance at min. 5 yr f/u. Comparison was made on the LIV-CSVL distance in pts fused short of the TV, to the TV or distal to the TV using standard statistical software.Results:When comparing the entire cohort, differences in 5 yr LIV-CSVL absolute values between the 3 groups approached, but did not reach, significance ( = .055). In a subanalysis of the lumbar A modifiers (n = 161), main effect ANOVA indicated a significant difference among the 3 groups ( = .002). Post hoc comparison revealed that pts fused short of the TV (TV-1) had significantly greater LIV-CSVL distance values than those fused to the TV ( = .006) & those fused distal to the TV (TV+1, = .002). There was no significant difference among the 3 groups when looking at lumbar B (n = 76, p = 0.424) & lumbar C (n = 62, = .326) modifiers.Conclusion:Selecting the touched vertebra (TV) as the LIV for Lenke type 1A & 2A curves produced optimal LIV positioning at a minimum 5 yrs postoperative, while fusing short of the TV showed statistically increased LIV- CSVL translation. Understanding & utilizing the TV rule assists the surgeon in proper LIV selection in Lenke type 1A & 2A curve patterns for AIS.
Project description:We have identified an ethylnitrosourea (ENU)-induced recessive mouse mutation (Vcc) with a pleiotropic phenotype that includes cardiac, tracheoesophageal, anorectal, anteroposterior patterning defects, exomphalos, hindlimb hypoplasia, a presacral mass, renal and palatal agenesis, and pulmonary hypoplasia. It results from a C470R mutation in the proprotein convertase PCSK5 (PC5/6). Compound mutants (Pcsk5(Vcc/null)) completely recapitulate the Pcsk5(Vcc/Vcc) phenotype, as does an epiblast-specific conditional deletion of Pcsk5. The C470R mutation ablates a disulfide bond in the P domain, and blocks export from the endoplasmic reticulum and proprotein convertase activity. We show that GDF11 is cleaved and activated by PCSK5A, but not by PCSK5A-C470R, and that Gdf11-deficient embryos, in addition to having anteroposterior patterning defects and renal and palatal agenesis, also have a presacral mass, anorectal malformation, and exomphalos. Pcsk5 mutation results in abnormal expression of several paralogous Hox genes (Hoxa, Hoxc, and Hoxd), and of Mnx1 (Hlxb9). These include known Gdf11 targets, and are necessary for caudal embryo development. We identified nonsynonymous mutations in PCSK5 in patients with VACTERL (vertebral, anorectal, cardiac, tracheoesophageal, renal, limb malformation OMIM 192350) and caudal regression syndrome, the phenotypic features of which resemble the mouse mutation. We propose that Pcsk5, at least in part via GDF11, coordinately regulates caudal Hox paralogs, to control anteroposterior patterning, nephrogenesis, skeletal, and anorectal development.