Abstract
One of the many physiological functions of cyclic guanosine 3′,5′ monophosphate (cGMP) signalling is the regulation of a specific mode of axonal branching. The bifurcation of axons from dorsal root ganglion (DRG) neurons at the dorsal root entry zone of the embryonic spinal cord is triggered by a cGMP signalling pathway comprising the ligand C-type natriuretic peptide (CNP), the cGMP-producing natriuretic peptide receptor 2 (Npr2), and the cGMP-dependent protein kinase Iα (cGKIα). Absence of any of these components causes a loss of bifurcation and sensory axons instead only turn in either a rostral or a caudal direction. In this chapter we describe a genetic strategy to study the impact of cGMP signalling on the arborization of individual DRG neurons in mice. Expression of an alkaline phosphatase (AP) reporter is selectively induced in Npr2-positive DRG neurons by tamoxifen-dependent activation of a Cre recombinase under the control of the Npr2 promoter. This approach might also be employed for the analysis of axonal branching in neuronal subsets expressing Npr2 elsewhere in the nervous system.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Acebes A, Ferrus A (2000) Cellular and molecular features of axon collaterals and dendrites. Trends Neurosci 23:557–565
Schmidt H, Rathjen FG (2010) Signalling mechanisms regulating axonal branching in vivo. Bioessays 32:977–985
Gibson DA, Ma L (2011) Developmental regulation of axon branching in the vertebrate nervous system. Development 138:183–195
Gallo G (2011) The cytoskeletal and signaling mechanisms of axon collateral branching. Dev Neurobiol 71:201–220
Ozaki S, Snider WD (1997) Initial trajectories of sensory axons toward laminar targets in the developing mouse spinal cord. J Comp Neurol 380:215–229
Schmidt H, Werner M, Heppenstall PA et al (2002) cGMP-mediated signaling via cGKIalpha is required for the guidance and connectivity of sensory axons. J Cell Biol 159:489–498
Schmidt H, Stonkute A, Juttner R et al (2007) The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord. J Cell Biol 179:331–340
Schmidt H, Stonkute A, Juttner R et al (2009) C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons. Proc Natl Acad Sci USA 106:16847–16852
Zhao Z, Wang Z, Gu Y et al (2009) Regulate axon branching by the cyclic GMP pathway via inhibition of glycogen synthase kinase 3 in dorsal root ganglion sensory neurons. J Neurosci 29:1350–1360
Zhao Z, Ma L (2009) Regulation of axonal development by natriuretic peptide hormones. Proc Natl Acad Sci USA 106:18016–18021
Golgi C (1873) Sulla struttura della sostanza grigia del cervello. Gazzetta Medica Italiana-Lombardia 33:244–246
Kristensson K, Olsson Y (1971) Retrograde axonal transport of protein. Brain Res 29:363–365
Lapper SR, Bolam JP (1991) The anterograde and retrograde transport of neurobiotin in the central nervous system of the rat: comparison with biocytin. J Neurosci Methods 39:163–174
Honig MG, Hume RI (1989) Dil and diO: versatile fluorescent dyes for neuronal labelling and pathway tracing. Trends Neurosci 12:333–1
Feng G, Mellor RH, Bernstein M et al (2000) Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP. Neuron 28:41–51
Birling MC, Gofflot F, Warot X (2009) Site-specific recombinases for manipulation of the mouse genome. Methods Mol Biol 561:245–263
Badea TC, Wang Y, Nathans J (2003) A noninvasive genetic/pharmacologic strategy for visualizing cell morphology and clonal relationships in the mouse. J Neurosci 23:2314–2322
Joyner AL, Zervas M (2006) Genetic inducible fate mapping in mouse: establishing genetic lineages and defining genetic neuroanatomy in the nervous system. Dev Dyn 235:2376–2385
Feil R, Wagner J, Metzger D et al (1997) Regulation of Cre recombinase activity by mutated estrogen receptor ligand-binding domains. Biochem Biophys Res Commun 237:752–757
Lobe CG, Koop KE, Kreppner W et al (1999) Z/AP, a double reporter for cre-mediated recombination. Dev Biol 208:281–292
Badea TC, Nathans J (2004) Quantitative analysis of neuronal morphologies in the mouse retina visualized by using a genetically directed reporter. J Comp Neurol 480:331–351
Badea TC, Hua ZL, Smallwood PM et al (2009) New mouse lines for the analysis of neuronal morphology using CreER(T)/loxP-directed sparse labeling. PLoS One 4:e7859
Tsuji T, Kunieda T (2005) A loss-of-function mutation in natriuretic peptide receptor 2 (Npr2) gene is responsible for disproportionate dwarfism in cn/cn mouse. J Biol Chem 280:14288–14292
Schmidt H, Rathjen FG (2011) DiI-labeling of DRG neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord. J Vis Exp 58:e3667
Acknowledgements
We thank Dr. Alistair Garratt (Max Delbrück Center, Berlin) for a critical reading of the manuscript. Work in the authors’ laboratory was supported by a grant from the Deutsche Forschungsgemeinschaft (SFB665).
Author information
Authors and Affiliations
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2013 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Schmidt, H., Ter-Avetisyan, G., Rathjen, F.G. (2013). A Genetic Strategy for the Analysis of Individual Axon Morphologies in cGMP Signalling Mutant Mice. In: Krieg, T., Lukowski, R. (eds) Guanylate Cyclase and Cyclic GMP. Methods in Molecular Biology, vol 1020. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-459-3_12
Download citation
DOI: https://doi.org/10.1007/978-1-62703-459-3_12
Published:
Publisher Name: Humana Press, Totowa, NJ
Print ISBN: 978-1-62703-458-6
Online ISBN: 978-1-62703-459-3
eBook Packages: Springer Protocols