Research ReportAbnormal neocortical development in mice lacking cGMP-dependent protein kinase I
Introduction
Cyclic GMP (cGMP)-dependent protein kinases are key signaling molecules that regulate diverse physiological mechanisms ranging from modulation of smooth muscle tone to synaptic potentiation. cGMP is produced by soluble guanylyl cyclase (sGC) and activates two different types of cGMP-dependent protein kinases (cGKI and cGKII) [3], [17], [22]. In adult rodent brain, cGKI has a restricted pattern of expression in the cerebellum and hippocampus with little detectable in the cortex [9], [10], [18], [26]. cGKI is also expressed in the peripheral nervous system, where it influences sensory neuron precursor proliferation [14] and guidance of sensory axons [36]. Analysis of cGKI knockout mice for neurological functions has shown that cGKI is required for the late phase of long-term potentiation (LTP) in the hippocampus [1], [24], [38] and for cerebellar long-term depression [11], but developmental expression and potential functions for cGKI in the cortex have not been studied. In contrast, cGKII, a membrane-bound kinase, is expressed widely in the adult brain, including the outer layers of the cerebral cortex [9], [18], [40].
To investigate a potential role for cGKI in brain development, we analyzed the developmental expression of cGKI in the mouse neocortex and the final positioning and dendritic projection of cortical pyramidal neurons in mutant mice deficient in cGKI. cGKI mutant mice are defective in regulation of smooth muscle tone and die early due to cardiovascular and gastrointestinal deficits [23], but neocortical development has not been studied. We found that cGKI in embryonic brain was expressed at highest levels in newly generated pyramidal neurons during migration to form cortical layers, where they extend neuronal processes and establish connectivity. Neuroanatomical analysis of adult cGKI null mutant mice indicated that cGKI contributes to migration/positioning, as well as dendritic orientation of cortical pyramidal neurons.
Section snippets
Immunofluorescence staining for cGKI in developing cortex
Wild type adult mice (Sv129 background) were analyzed for cGKI expression during the following embryonic and postnatal stages of development: embryonic day (E) 13.5, E14.5, E17.5, postnatal day (P) 0, and P28. Brains were fixed in 4% paraformaldehyde (PFA) in phosphate-buffered saline (PBS) for embryos or perfused with 4% PFA in (PBS) for postnatal mice then sectioned (14 μm) on a cryostat. Sections were washed with PBS, permeabilized in 0.1% Triton X-100 for 10 min at room temperature, and
cGKI is expressed in the developing mouse neocortex
To analyze a potential role of cGKI in cortical development, the expression of cGKI was analyzed in the mouse neocortex by immunofluorescence staining during embryonic and postnatal development. Immunoreactivity for cGKI became apparent at E13.5 in radially oriented processes in the ventricular zone (VZ; Fig. 1A), prior to the major period of radial locomotion of deep layer (V/VI) pyramidal neurons. Immunostaining for cGKI was most prominent at E14.5, when radial migration of pyramidal cell
Discussion
Analysis of the cortical phenotype of cGKI-minus mice shows that there are defects in neuronal positioning and dendrite orientation of cortical pyramidal neurons. Localized heterotopic collections of cortical neurons and invaginations of layer I seen in cGKI null mutant brain suggest that migration of cortical pyramidal neurons or their final placement depends on the function of cGKI during development of the neocortex. Aberrant orientation of apical dendrites of layer V pyramidal neurons was
Acknowledgments
This work was supported by NIH grants NS26620 and NSO49109, as well as P30NS045892, a grant to the University of North Carolina Neuroscience Center for support of the Confocal Microscopy Facility. We thank Chris Ingersoll for technical assistance.
References (40)
- et al.
FAK deficiency in cells contributing to the basal lamina results in cortical abnormalities resembling congenital muscular dystrophies
Neuron
(2003) - et al.
Cadherin-mediated differential cell adhesion controls slow muscle cell migration in the developing zebrafish myotome
Dev. Cell
(2003) - et al.
Altered distribution of dopaminergic neurons in the brain of L1 null mice
Brain Res. Dev. Brain Res.
(2001) - et al.
Close homolog of L1 modulates area-specific neuronal positioning and dendrite orientation in the cerebral cortex
Neuron
(2004) - et al.
Localization of the cGMP-dependent protein kinases in relation to nitric oxide synthase in the brain
J. Chem. Neuroanat.
(1999) - et al.
Imaging neuronal subsets in transgenic mice expressing multiple spectral variants of GFP
Neuron
(2000) - et al.
Distribution of IRAG and cGKI-isoforms in murine tissues
FEBS Lett.
(2004) - et al.
Ena/VASP proteins regulate cortical neuronal positioning
Curr. Biol.
(2002) - et al.
How do Abl family kinases regulate cell shape and movement?
Trends Cell Biol.
(2004) - et al.
Structure and physiological role of cGMP-dependent protein kinase
Biochim. Biophys. Acta
(1992)