CNP-induced cGMP signalling in neural differentiation and functional integration

Hannes Schmidt, Tübingen

A cGMP signalling pathway triggered by binding of C-type natriuretic peptide (CNP) to its receptor guanylyl cyclase B (GC-B; also named Npr2) has been linked by genetic evidence to a remarkable variety of physiological functions including skeletal bone growth, oocyte maturation, cardiac growth, fat metabolism, and gastrointestinal function. Furthermore, our own investigations demonstrated that the CNP/GC-B/cyclic guanosine monophosphate (cGMP)/cGMP-dependent protein kinase type I (cGKI) signalling pathway is essential for sensory axon branching at the dorsal root entry zone of the spinal cord during embryonic development. In the course of these studies we generated genetic reporter mouse lines for CNP and GC-B, in which the wild-type alleles were replaced by a NLS-lacZ-cassette enabling a detailed analysis of the expression patterns of the ligand and its receptor. We observed an interesting distribution of both proteins in the subventricular zone (SVZ) of the lateral ventricles and in the dentate gyrus (DG) of the hippocampus, the two regions in the mammalian brain where active neurogenesis proceeds during postnatal stages. In this project, we will investigate the function of CNP-induced cGMP signalling in the context of neural differentiation and functional integration into neural circuitry. We will first determine the identity of the cell populations expressing CNP and GC-B in the SVZ and the DG. Immunological studies using 5-bromo-2'€™-deoxyuridine (BrdU)-injected GC-B-NLS-LacZ reporter mice should help to answer the question, whether or not GC-B expressing cells in the adult brain derive from dividing neuronal stem cells (NSCs). A genetic strategy allowing the fluorescent labelling of GC-B positive cells by the induction of Cre recombinase under the control of the GC-B promoter will facilitate the analysis of these cells, for example, their electrophysiological properties or the identification of specific cGMP effector proteins. In a second set of experiments we will investigate the effects of impaired cGMP signalling on proliferation, survival, maturation and synaptic integration of new-born cells in mutant mouse models for both CNP and GC-B. Furthermore, comparative expression profiling of heterozygous and homozygous GC-B knockout dentate granule cells (DGCs) will be performed to uncover cGMP-dependent transcriptional programmes involved in neural differentiation and function of GC-B expressing DGCs. These studies should provide new insights about the role of CNP/GC-B/cGMP signalling during the process of functional integration of GC-B expressing cells into neuronal networks. 

Fig. 1. AcGMP signalling pathway controls sensory axon bifurcation. (A) Binding of the ligand C-type natriuretic peptide (CNP) to its receptor , the guanylate cyclase GC-B, stimulates the generation of cyclic GMP from GTP and the subsequent activation of the serine/threonine kinase cGKIα in embryonic dorsal root ganglion (DRG) neurons.(B) Schematic representation of the complementary distribution of CNP in the dorsal part of the embryonic spinal cord and GC-B and cGKIα in DRG neurons. (C) Tamoxifin-induced activation of an alkaline phosphatase-reporter in GC-B-expressing DRG neurons reveals a loss of axon bifurcation in GC-B deficient mice. Scale bar 200 μm. (C) Hannes Schmidt

Project-related publications

PI of this project; PIs of other FOR 2060 projects are in bold.

1.    Schmidt H, Peters S, Frank K, Wen L, Feil R, Rathjen FG. Dorsal root ganglion axon bifurcation tolerates increased cyclic GMP levels: the role of phosphodiesterase 2A and scavenger receptor Npr3. Eur J Neurosci. 2016; doi: 10.1111/ejn.13434. [Pubmed] [Project 1] [Project 6]

2.    Ter-Avetisyan G, Rathjen FG, Schmidt H. Bifurcation of axons from cranial sensory neurons is disabled in the absence of Npr2-induced cGMP signaling. J Neurosci. 2014;34:737–47. [Project 6] [pubmed]

3.    Schmidt H, Ter-Avetisyan G, Rathjen FG. A genetic strategy for the analysis of individual axon morphologies in cGMP signalling mutant mice. Methods Mol Biol. 2013;1020:193-204. [Project 6] [pubmed]

4.    Ter-Avetisyan G, Tröster P, Schmidt H, Rathjen FG. cGMP signalling and axonal branching in the spinal cord. Future Neurology. 2012;7:639-51. [Project 6] [Abstract]

5.    Schmidt H, Rathjen FG. DiI-labeling of DRG neurons to study axonal branching in a whole mount preparation of mouse embryonic spinal cord. J Vis Exp. 2011; 58:e3667. [Project 6] [pubmed]

6.    Schmidt H, Rathjen FG. Signalling mechanisms regulating axonal branching in vivo. BioEssays. 2010;32:977-85. [Project 6] [pubmed]

7.    Schmidt H, Stonkute A, Jüttner R, Schäffer S, Koesling D, Friebe A, Rathjen FG. C-type natriuretic peptide (CNP) is a bifurcation factor for sensory neurons. Proc Natl Acad Sci U S A. 2009;106:16847-52. [Project 2Project 3 and Project 6] [pubmed]

8.    Schmidt H, Stonkute A, Jüttner R, Schäffer S, Buttgereit J, Feil R, Hofmann F, Rathjen FG. The receptor guanylyl cyclase Npr2 is essential for sensory axon bifurcation within the spinal cord. J Cell Biol. 2007;179:331-40. [Project 1 and Project 6] [pubmed]