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Murine Limb Bud Organ Cultures for Studying Musculoskeletal Development

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Skeletal Development and Repair

Part of the book series: Methods in Molecular Biology ((MIMB,volume 2230))

Abstract

The biological signals that coordinate the three-dimensional outgrowth and patterning of the vertebrate limb bud have been well delineated. These include a number of vital embryonic signaling pathways, including the fibroblast growth factor, WNT, transforming growth factor, and hedgehog. Collectively these signals converge on multiple progenitor populations to drive the formation of a variety of tissues that make up the limb musculoskeletal system, such as muscle, tendon, cartilage, stroma, and bone. The basic mechanisms regulating the commitment and differentiation of diverse limb progenitor populations has been successfully modeled in vitro using high density primary limb mesenchymal or micromass cultures. However, this approach is limited in its ability to more faithfully recapitulate the assembly of progenitors into organized tissues that span the entire musculoskeletal system. Other biological systems have benefitted from the development and availability of three-dimensional organoid cultures which have transformed our understanding of tissue development, homeostasis and regeneration. Such a system does not exist that effectively models the complexity of limb development. However, limb bud organ cultures while still necessitating the use of collected embryonic tissue have proved to be a powerful model system to elucidate the molecular underpinning of musculoskeletal development. In this methods article, the derivation and use of limb bud organ cultures from murine limb buds will be described, along with strategies to manipulate signaling pathways, examine gene expression and for longitudinal lineage tracking.

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Acknowledgements

This work was supported by the following grants: Canadian Institutes of Health (CIHR) PJT-149026 (T.M.U.) and PJT-148816 (T.M.U.). M.A. was supported by a UBC graduate scholarship.

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Authors and Affiliations

  1. Biomedical Research Centre, University of British Columbia, Vancouver, BC, Canada

    Martin Arostegui & T. Michael Underhill

  2. Department of Cellular and Physiological Sciences, University of British Columbia, Vancouver, BC, Canada

    Martin Arostegui & T. Michael Underhill

  3. School of Biomedical Engineering, University of British Columbia, Vancouver, BC, Canada

    T. Michael Underhill

Authors
  1. Martin Arostegui
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  2. T. Michael Underhill
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Correspondence to T. Michael Underhill .

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Editors and Affiliations

  1. Department of Orthopaedic Surgery and Cell Biology, Duke Cellular, Developmental, and Genome Laboratories, Duke University School of Medicine, Durham, USA

    Matthew J. Hilton

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Arostegui, M., Underhill, T.M. (2021). Murine Limb Bud Organ Cultures for Studying Musculoskeletal Development. In: Hilton, M.J. (eds) Skeletal Development and Repair. Methods in Molecular Biology, vol 2230. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1028-2_8

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  • DOI: https://doi.org/10.1007/978-1-0716-1028-2_8

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  • Publisher Name: Humana, New York, NY

  • Print ISBN: 978-1-0716-1027-5

  • Online ISBN: 978-1-0716-1028-2

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