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Spatial and Quantitative Detection of BMP Activity in Mouse Embryonic Limb Buds

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Bone Morphogenetic Proteins

Part of the book series: Methods in Molecular Biology ((MIMB,volume 1891))

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Abstract

Modulation of bone morphogenetic protein (BMP) activity is essential to the progression of limb development in the mouse embryo. Genetic disruption of BMP signaling at various stages of limb development causes defects ranging from complete limb agenesis to oligodactyly, polydactyly, webbing, and chondrodysplasia. To probe the state of BMP signaling in early limb buds, we designed two sets of primers to measure both spatially and quantitatively the transcription of nine key genes indicative of canonical BMP activity. One set is used to generate digoxigenin (DIG)-labeled antisense RNA probes for whole-mount mRNA in situ hybridization, while the second set is used for SYBR® Green-based quantitative PCR on limb bud cDNA. Here we describe step-by-step protocols for both methods around this specific set of genes.

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Acknowledgments

The authors are grateful Dr. Rolf Zeller for forwarding the invitation to write this chapter and to Dr. Licia Selleri for providing the material and reagents necessary for generating and testing the mRNA in situ hybridization probes.

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Authors and Affiliations

  1. Department of Morphology, Area of Histology and Embryology, Piracicaba Dental School, University of Campinas, Campinas, SP, Brazil

    Marcelo Rocha Marques

  2. Department of Orofacial Sciences and Program in Craniofacial Biology, University of California, San Francisco, San Francisco, CA, USA

    Marcelo Rocha Marques & Jean-Denis Bénazet

Authors
  1. Marcelo Rocha Marques
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  2. Jean-Denis Bénazet
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Editor information

Editors and Affiliations

  1. New Jersey Medical School, Rutgers Biomedical and Health Sciences, Newark, NJ, USA

    Melissa B. Rogers

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Marques, M.R., Bénazet, JD. (2019). Spatial and Quantitative Detection of BMP Activity in Mouse Embryonic Limb Buds. In: Rogers, M. (eds) Bone Morphogenetic Proteins. Methods in Molecular Biology, vol 1891. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-8904-1_15

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  • DOI: https://doi.org/10.1007/978-1-4939-8904-1_15

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  • Publisher Name: Humana Press, New York, NY

  • Print ISBN: 978-1-4939-8903-4

  • Online ISBN: 978-1-4939-8904-1

  • eBook Packages: Springer Protocols

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