Advertisement

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

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

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.

Key words

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

Notes

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.

References

  1. 1.
    Zeller R, López-Ríos J, Zuniga A (2009) Vertebrate limb bud development: moving towards integrative analysis of organogenesis. Nat Rev Genet 10:845–858CrossRefGoogle Scholar
  2. 2.
    Bénazet JD, Zeller R (2009) Vertebrate limb development: moving from classical morphogen gradients to an integrated 4-dimensional patterning system. Cold Spring Harb Perspect Biol 1:a001339CrossRefGoogle Scholar
  3. 3.
    Bénazet JD, Bischofberger M, Tiecke E et al (2009) A self-regulatory system of interlinked signaling feedback loops controls mouse limb patterning. Science 323:1050–1053CrossRefGoogle Scholar
  4. 4.
    Niswander L, Jeffrey S, Martin GR et al (1994) A positive feedback loop coordinates growth and patterning in the vertebrate limb. Nature 371:609–612CrossRefGoogle Scholar
  5. 5.
    Laufer E, Nelson CE, Johnson RL et al (1994) Sonic hedgehog and Fgf-4 act through a signaling cascade and feedback loop to integrate growth and patterning of the developing limb bud. Cell 79:993–1003CrossRefGoogle Scholar
  6. 6.
    Benazet JD, Zeller R (2013) Dual requirement of ectodermal Smad4 during AER formation and termination of feedback signaling in mouse limb buds. Genesis 51:660–666PubMedGoogle Scholar
  7. 7.
    Ahn K, Mishina Y, Hanks MC et al (2001) BMPR-IA signaling is required for the formation of the apical ectodermal ridge and dorsal-ventral patterning of the limb. Development 128:4449–4461PubMedGoogle Scholar
  8. 8.
    Soshnikova N, Zechner D, Huelsken J et al (2003) Genetic interaction between Wnt/beta-catenin and BMP receptor signaling during formation of the AER and the dorsal-ventral axis in the limb. Genes Dev 17:1963–1968CrossRefGoogle Scholar
  9. 9.
    Pajni-Underwood S, Wilson CP, Elder C et al (2007) BMP signals control limb bud interdigital programmed cell death by regulating FGF signaling. Development 134:2359–2368CrossRefGoogle Scholar
  10. 10.
    Lewandoski M, Sun X, Martin GR (2000) Fgf8 signalling from the AER is essential for normal limb development. Nat Genet 26:460–463CrossRefGoogle Scholar
  11. 11.
    Moon AM, Capecchi MR (2000) Fgf8 is required for outgrowth and patterning of the limbs. Nat Genet 26:455–459CrossRefGoogle Scholar
  12. 12.
    Bénazet JD, Pignatti E, Nugent A et al (2012) Smad4 is required to induce digit ray primordia and to initiate the aggregation and differentiation of chondrogenic progenitors in mouse limb buds. Development 139:4250–4260CrossRefGoogle Scholar
  13. 13.
    Ovchinnikov DA, Selever J, Wang Y et al (2006) BMP receptor type IA in limb bud mesenchyme regulates distal outgrowth and patterning. Dev Biol 295:103–115CrossRefGoogle Scholar
  14. 14.
    Zúñiga A, Haramis AP, McMahon AP et al (1999) Signal relay by BMP antagonism controls the SHH/FGF4 feedback loop in vertebrate limb buds. Nature 401:598–602CrossRefGoogle Scholar
  15. 15.
    Khokha MK, Hsu D, Brunet LJ et al (2003) Gremlin is the BMP antagonist required for maintenance of Shh and Fgf signals during limb patterning. Nat Genet 34:303–307CrossRefGoogle Scholar
  16. 16.
    Michos O, Panman L, Vintersten K et al (2004) Gremlin-mediated BMP antagonism induces the epithelial-mesenchymal feedback signaling controlling metanephric kidney and limb organogenesis. Development 131:3401–3410CrossRefGoogle Scholar
  17. 17.
    te Welscher P, Zuniga A, Kuijper S et al (2002) Progression of vertebrate limb development through SHH-mediated counteraction of GLI3. Science 298:827–830CrossRefGoogle Scholar
  18. 18.
    Mariani FV, Ahn CP, Martin GR (2008) Genetic evidence that FGFs have an instructive role in limb proximal-distal patterning. Nature 453:401–405CrossRefGoogle Scholar
  19. 19.
    Selever J, Liu W, Lu MF et al (2004) Bmp4 in limb bud mesoderm regulates digit pattern by controlling AER development. Dev Biol 276:268–279CrossRefGoogle Scholar
  20. 20.
    Verheyden JM, Sun X (2008) An Fgf/Gremlin inhibitory feedback loop triggers termination of limb bud outgrowth. Nature 454:638–641CrossRefGoogle Scholar
  21. 21.
    Scherz PJ, Harfe BD, McMahon AP et al (2004) The limb bud Shh-Fgf feedback loop is terminated by expansion of former ZPA cells. Science 305:396–399CrossRefGoogle Scholar
  22. 22.
    Lopez-Rios J, Speziale D, Robay D et al (2012) GLI3 constrains digit number by controlling both progenitor proliferation and BMP-dependent exit to chondrogenesis. Dev Cell 22:837–848CrossRefGoogle Scholar
  23. 23.
    Harfe BD, Scherz PJ, Nissim S et al (2004) Evidence for an expansion-based temporal Shh gradient in specifying vertebrate digit identities. Cell 118:517–528CrossRefGoogle Scholar
  24. 24.
    Ahn S, Joyner AL (2004) Dynamic changes in the response of cells to positive hedgehog signaling during mouse limb patterning. Cell 118:505–516CrossRefGoogle Scholar
  25. 25.
    Bandyopadhyay A, Tsuji K, Cox K et al (2006) Genetic analysis of the roles of BMP2, BMP4, and BMP7 in limb patterning and skeletogenesis. PLoS Genet 2:e216CrossRefGoogle Scholar
  26. 26.
    Raspopovic J, Marcon L, Russo L et al (2014) Modeling digits. Digit patterning is controlled by a Bmp-Sox9-Wnt Turing network modulated by morphogen gradients. Science 345:566–570CrossRefGoogle Scholar
  27. 27.
    Suzuki T, Hasso SM, Fallon JF (2008) Unique SMAD1/5/8 activity at the phalanx-forming region determines digit identity. Proc Natl Acad Sci U S A 105:4185–4190CrossRefGoogle Scholar
  28. 28.
    Retting KN, Song B, Yoon BS et al (2009) BMP canonical Smad signaling through Smad1 and Smad5 is required for endochondral bone formation. Development 136:1093–1104CrossRefGoogle Scholar
  29. 29.
    Yoon BS, Ovchinnikov DA, Yoshii I et al (2005) Bmpr1a and Bmpr1b have overlapping functions and are essential for chondrogenesis in vivo. Proc Natl Acad Sci U S A 102:5062–5067CrossRefGoogle Scholar
  30. 30.
    Wong YL, Behringer RR, Kwan KM (2012) Smad1/Smad5 signaling in limb ectoderm functions redundantly and is required for interdigital programmed cell death. Dev Biol 363:247–257CrossRefGoogle Scholar
  31. 31.
    Choi KS, Lee C, Maatouk DM et al (2012) Bmp2, Bmp4 and Bmp7 are co-required in the mouse AER for normal digit patterning but not limb outgrowth. PLoS One 7:e37826CrossRefGoogle Scholar
  32. 32.
    Galli A, Robay D, Osterwalder M et al (2010) Distinct roles of Hand2 in initiating polarity and posterior Shh expression during the onset of mouse limb bud development. PLoS Genet 6:e1000901CrossRefGoogle Scholar

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

Personalised recommendations