Thompson AC, Capellini TD, Guenther CA, Chan YF, Infante CR, Menke DB, Kingsley DM. A novel enhancer near the gene influences development and evolution of pelvic appendages in vertebrates. Elife. 2018;7. Publisher's VersionAbstract
Vertebrate pelvic reduction is a classic example of repeated evolution. Recurrent loss of pelvic appendages in sticklebacks has previously been linked to natural mutations in a pelvic enhancer that maps upstream of . The sequence of this upstream enhancer is not conserved to mammals, so we have surveyed a large region surrounding the mouse gene for other possible hind limb control sequences. Here we identify a new pelvic enhancer, , that maps downstream rather than upstream of drives expression in the posterior portion of the developing hind limb, and deleting the sequence from mice alters the size of several hind limb structures. sequences are broadly conserved from fish to mammals. A wild stickleback population lacking the pelvis has an insertion/deletion mutation that disrupts the structure and function of , suggesting that changes in this ancient enhancer contribute to evolutionary modification of pelvic appendages in nature.
Grinstein M, Dingwall HL, Shah RR, Capellini TD, Galloway JL. A robust method for RNA extraction and purification from a single adult mouse tendon. PeerJ. 2018;6 :e4664. Publisher's VersionAbstract
Background: Mechanistic understanding of tendon molecular and cellular biology is crucial toward furthering our abilities to design new therapies for tendon and ligament injuries and disease. Recent transcriptomic and epigenomic studies in the field have harnessed the power of mouse genetics to reveal new insights into tendon biology. However, many mouse studies pool tendon tissues or use amplification methods to perform RNA analysis, which can significantly increase the experimental costs and limit the ability to detect changes in expression of low copy transcripts. Methods: Single Achilles tendons were harvested from uninjured, contralateral injured, and wild type mice between three and five months of age, and RNA was extracted. RNA Integrity Number (RIN) and concentration were determined, and RT-qPCR gene expression analysis was performed. Results: After testing several RNA extraction approaches on single adult mouse Achilles tendons, we developed a protocol that was successful at obtaining high RIN and sufficient concentrations suitable for RNA analysis. We found that the RNA quality was sensitive to the time between tendon harvest and homogenization, and the RNA quality and concentration was dependent on the duration of homogenization. Using this method, we demonstrate that analysis of gene expression in single mouse tendons reduces the biological variation caused by pooling tendons from multiple mice. We also show successful use of this approach to analyze and gene expression changes in injured compared with uninjured control tendons. Discussion: Our work presents a robust, cost-effective, and straightforward method to extract high quality RNA from a single adult mouse Achilles tendon at sufficient amounts for RT-qPCR as well as RNA-seq. We show this can reduce variation and decrease the overall costs associated with experiments. This approach can also be applied to other skeletal tissues, as well as precious human samples.
Kiapour AM, Cao J, Young M, Capellini TD. The role of Gdf5 regulatory regions in development of hip morphology. PLoS One. 2018;13 (11) :e0202785. Publisher's VersionAbstract
Given GDF5 involvement in hip development, and osteoarthritis (OA) and developmental hip dysplasia (DDH) risk, here we sought to assess the role(s) of GDF5 and its regulatory sequence on the development of hip morphology linked to injury risk. The brachypodism (bp) mouse, which harbors a Gdf5 inactivating mutation, was used to survey how Gdf5 loss of function impacts the development of hip morphology. Two transgenic Gdf5 reporter BAC lines were used to assess the spatiotemporal expression of Gdf5 regulatory sequences. Each BAC line was also used to assess the functional roles of upstream and downstream sequence on hip morphology. bp/bp mice had shorter femora with smaller femoral heads and necks as well as larger alpha angles, smaller anterior offsets, and smaller acetabula, compared to bp/+ mice (p<0.04). Regulatory sequences downstream of Gdf5 drove strong prenatal (E17) expression and low postnatal (6 months) expression across regions of femoral head and acetabulum. Conversely, upstream regulatory sequences drove very low expression at E17 and no detectable expression at 6 months. Importantly, downstream, but not upstream Gdf5 regulatory sequences fully restored all the key morphologic features disrupted in bp/bp mice. Hip morphology is profoundly affected by Gdf5 absence, and downstream regulatory sequences mediate its effects by controlling Gdf5 expression during development. This downstream region contains numerous enhancers harboring risk variants related to hip OA, DDH, and dislocation. We posit that subtle alterations to morphology driven by changes in downstream regulatory sequence underlie this locus' role in hip injury risk.
Jagoda E, Lawson DJ, Wall JD, Lambert D, Muller C, Westaway M, Leavesley M, Capellini TD, Lahr MM, Gerbault P, et al. Disentangling Immediate Adaptive Introgression from Selection on Standing Introgressed Variation in Humans. Molecular Biology and Evolution. 2017;Dec 6. doi: 10.1093/molbev/msx314. Publisher's Version
Guo M, Liu Z, Willen J, Shaw CP, Richard D, Jagoda E, Doxey AC, Hirschhorn JN, Capellini TD. Epigenetic profiling of growth plate chondrocytes sheds insight into regulatory genetic variation influencing height. eLife. 2017;Dec 5;6. pii: e29329. doi: 10.7554/eLife.29329. Publisher's Version
Capellini TD, Chen H, Cao J, Doxey AC, Kiapour A, Schoor M, Kingsley DM. Ancient selection for derived alleles at a GDF5 enhancer influencing human growth and osteoarthritis risk. Nature Genetics. 2017;49 (8) :1202-1210. Publisher's Version
Capellini TD, Dingwall H. Combining genetic and developmental methods to study musculoskeletal evolution in primates. In: Building Bones. Cambridge University Press ; 2017.
Cao J, Wei C, Zhang S, Capellini TD, al et. Screening of reproduction-related single-nucleotide variations from MeDIP-seq data in sheep. Mol Reprod Dev. 2016;Nov;83 (11) :958-967. Publisher's Version
Reiter T, Jagoda E, Capellini TD. Dietary Variation and Evolution of Gene Copy Number among Dog Breeds. Plos One. 2016;Feb 10;11 (2) :e0148899. Publisher's Version
Young NM, Capellini TD, Roach NT, Alemseged Z. Reply to Almécija: A new direction for reconstructing our last common ancestor with chimpanzees. Proc Natl Acad Sci U S A. 2016;Feb 23;113 (8) :E945. Publisher's Version
Young NM, Capellini TD, Roach NT, Alemseged Z. Reply to Almécija: A new direction for reconstructing our last common ancestor with chimpanzees. Proc Natl Acad Sci U S A. 2016;Feb 23;113 (8) :E945. Publisher's Version
Chen H, Capellini TD, Schoor M, Mortlock D, Reddi H, Kingsley DM. Heads, Shoulders, Elbows, Knees, and Toes: Modular Gdf5 Enhancers Control Different Joints in the Vertebrate Skeleton. Plos Genetics. 2016;Nov 30;12 (11) ::e1006454. Publisher's Version
Young N, Capellini TD. Out on a limb: development and the evolution of the human appendages. In: Evolutionary Developmental Anthropology. Wiley Press ; 2016.
Young NM, Capellini TD, Roach NT, Alemseged Z. Reply to Melillo: Woranso-Mille is consistent with an australopithecine shoulder intermediate between African apes and Homo. Proc Natl Acad Sci U S A. 2015;Dec 29;112 (52) :E7160. Publisher's Version
Cao J, al et. DNA methylation Landscape of body size variation in sheep. Scientific Reports. 2015;Oct 16; 5 :13950. Publisher's Version
Young N, Capellini TD, Roach N, Alemseged Z. Evidence from Fossil Hominin Shoulders Supports an African Ape Morphotype of the Last Common Ancestor. Proc Natl Acad Sci U S A. 2015;Sep 22;112 (38) :11829-34. Publisher's Version
Sears KE, Capellini TD, Diogo R. On the serial homology of the pectoral and pelvic girdles of tetrapods. Evolution. 2015;Oct;69 (10) :2543-55. Publisher's Version
Koss M, Bolze A, Brendolan A, Saggese M, Capellini TD, Bojilova E, Boisson B, Prall OW, Elliott DA, Solloway M, et al. (2012) Congenital Asplenia in Mice and Humans with Mutations in a Pbx/Nkx2-5/p15 Module. Developmental Cell. 2012;22 :913-26.
Capellini TD, Zappavigna V, Selleri L. (2011) Pbx homeodomain proteins: TALEnted regulators of limb patterning and outgrowth. . Developmental Dynamics . 2011;240 :1063-86.