Abstract
Endochondral ossification is a process essential for the formation of the vertebrate skeleton. Indian Hedgehog (IHH) is a key regulator of this process. So far, monitoring IHH expression in whole-mount developing skeletal structures has been hampered by the permeability and the opacity of the tissue. Whole-mount preparations require advanced techniques of fixation, clearing, and staining. We describe a reliable method for fixing, immunostaining, and clearing whole-mount developing cartilages that allows for the detection of IHH in the developing skeleton of avian embryos. The fixation process ensures a proper preservation of cellular structures and, especially, the antigenicity of the tissue, allowing the antibody labelling of IHH. This protocol reveals specific cell staining in localized regions of the developing cartilage, facilitating the study of IHH function during key periods of skeletogenesis.
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References
Bisgard JD, Bisgard ME (1935) Longitudinal growth of long bones. Arch Surg 31(4):568–578
Fell HB (1925) The histogenesis of cartilage and bone in the long bones of the embryonic fowl. J Morphol 40(3):417–459
Crombrugghe B, Lefebvre V, Nakashima K (2001) Regulatory mechanisms in the pathways of cartilage and bone formation. Curr Opin Cell Biol 13(6):721–728
Maes C, Kobayashi T et al (2010) Osteoblast precursors, but not mature osteoblasts, move into developing and fractured bones along with invading blood vessels. Dev Cell 19(2):329–344
Vortkamp A, Lee K et al (1996) Regulation of rate of cartilage differentiation by Indian hedgehog and PTH-related protein. Science 273(5275):613–622
Mak K, Chen M et al (2006) Wnt/{beta}-catenin signaling interacts differentially with Ihh signaling in controlling endochondral bone and synovial joint formation. Development 133(18):3695
Chung U, Schipani E et al (2001) Indian hedgehog couples chondrogenesis to osteogenesis in endochondral bone development. J Clin Invest 107(3):295–304
Pathi S, Rutenberg J et al (1999) Interaction of Ihh and BMP/Noggin signaling during cartilage differentiation. Dev Biol 209(2):239–253
Naski MC, Colvin JS et al (1998) Repression of hedgehog signaling and BMP4 expression in growth plate cartilage by fibroblast growth factor receptor 3. Development 125(24):4977–4988
Kronenberg H, Lee K et al (1997) Parathyroid hormone-related protein and Indian hedgehog control the pace of cartilage differentiation. J Endocrinol 154(3 Suppl):S39–S45
Minina E, Wenzel H et al (2001) BMP and Ihh/PTHrP signaling interact to coordinate chondrocyte proliferation and differentiation. Development 128(22):4523–4534
Koziel L, Wuelling M et al (2005) Gli3 acts as a repressor downstream of Ihh in regulating two distinct steps of chondrocyte differentiation. Development 132(23):5249–5260
Karp SJ, Schipani E et al (2000) Indian hedgehog coordinates endochondral bone growth and morphogenesis via parathyroid hormone related-protein-dependent and-independent pathways. Development 127(3):543–548
Long F, Chung U-i et al (2004) Ihh signaling is directly required for the osteoblast lineage in the endochondral skeleton. Development 131(6):1309–1318
Hammond CL, Schulte-Merker S (2009) Two populations of endochondral osteoblasts with differential sensitivity to Hedgehog signalling. Development 136(23):3991–4000
Zhou J, Meng J et al (2007) IHH and FGF8 coregulate elongation of digit primordia. Biochem Biophys Res Commun 363(3):513–518
Nagashima H, Sugahara F et al (2009) Evolution of the turtle body plan by the folding and creation of new muscle connections. Science 325(5937):193–196
Klymkowsky MW, Hanken J (1991) Whole-mount staining of Xenopus and other vertebrates. Methods Cell Biol 36:419–441
Hama H, Kurokawa H et al (2011) Scale: a chemical approach for fluorescence imaging and reconstruction of transparent mouse brain. Nat Neurosci 14(11):1481–1488
Ke M-T, Fujimoto S, Imai T (2013) SeeDB: a simple and morphology-preserving optical clearing agent for neuronal circuit reconstruction. Nat Neurosci 16(8):1154–1161
Kuwajima T, Sitko AA et al (2013) ClearT: a detergent- and solvent-free clearing method for neuronal and non-neuronal tissue. Development 140(6):1364–1368
Johnson RL, Tabin CJ (1997) Molecular models for vertebrate limb development. Cell 90:979–990
Saunders JW (1948) The proximo‐distal sequence of origin of the parts of the chick wing and the role of the ectoderm. J Exp Zool 108(3):363–403
Sherman A, McGrew M et al (2010) The Roslin Institute Transgenic Chicken Facility: developments in chicken transgenesis. Transgenic Res 19(1):151
Dent JA, Klymkowsky MW (1989) Whole-mount analyses of cytoskeletal reorganization and function during oogenesis and early embryogenesis in Xenopus. In: Schatten H, Schatten G (eds) The cell biology of fertilization. Academic, New York, NY, pp 63–103
Hamburger V, Hamilton HL (1951) A series of normal stages in the development of the chick embryo. J Morphol 88(1):49–92
Acknowledgment
This work was supported by FONDAP 15090007 (V.P.), Fondecyt grant 1110237 (V.P.), Fondecyt doctoral grant 24100058 (J.B.)
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Botelho, J.F., Smith-Paredes, D., Verónica, P.A. (2015). Efficient Detection of Indian Hedgehog During Endochondral Ossification by Whole-Mount Immunofluorescence. In: Riobo, N. (eds) Hedgehog Signaling Protocols. Methods in Molecular Biology, vol 1322. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-2772-2_14
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DOI: https://doi.org/10.1007/978-1-4939-2772-2_14
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-2771-5
Online ISBN: 978-1-4939-2772-2
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