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

  • Marcelo Rocha Marques
  • Jean-Denis Bénazet
Part of the Methods in Molecular Biology book series (MIMB, volume 1891)


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.

Key words

Limb bud BMP signaling Grem1 SHH FGF Whole-mount mRNA in situ hybridization Quantitative PCR Mouse 



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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Copyright information

© Springer Science+Business Media, LLC, part of Springer Nature 2019

Authors and Affiliations

  • Marcelo Rocha Marques
    • 1
    • 2
  • Jean-Denis Bénazet
    • 2
  1. 1.Department of Morphology, Area of Histology and Embryology, Piracicaba Dental SchoolUniversity of CampinasCampinasBrazil
  2. 2.Department of Orofacial Sciences and Program in Craniofacial BiologyUniversity of California, San FranciscoSan FranciscoUSA

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