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

Methods Mol Biol. 2019;1891:201-219. doi: 10.1007/978-1-4939-8904-1_15.

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.

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

MeSH terms

  • Animals
  • Bone Morphogenetic Proteins / genetics
  • Bone Morphogenetic Proteins / metabolism*
  • Gene Expression Regulation, Developmental
  • In Situ Hybridization
  • Limb Buds / embryology*
  • Limb Buds / metabolism*
  • Mice
  • Real-Time Polymerase Chain Reaction
  • Signal Transduction

Substances

  • Bone Morphogenetic Proteins