Pharmacological and Genetic Disruption of C-Type Natriuretic Peptide (nppcl) Expression in Zebrafish (Danio rerio) Causes Stunted Growth during Development.

Human patients with mutations within NPPC or NPR2 genes (encoding C-type natriuretic peptide (CNP) and guanylyl cyclase-B (GC-B), respectively) display clinical signs associated with skeletal abnormalities, such as overgrowth or short stature. Mice with induced models of Nppc or Npr2 deletion display profound achondroplasia, dwarfism and early death. Recent pharmacological therapies to treat short stature are utilizing long-acting CNP analogues, but the effects of manipulating CNP expression during development remain unknown. Here, we use Danio rerio (zebrafish) as a model for vertebrate development, employing both pharmacological and reverse genetics approaches to alter expression of genes encoding CNP in zebrafish. Four orthologues of CNP were identified in zebrafish, and spatiotemporal expression profiling confirmed their presence during development. Bioinformatic analyses suggested that nppcl is the most likely the orthologue of mammalian CNP. Exogenous CNP treatment of developing zebrafish embryos resulted in impaired growth characteristics, such as body length, head width and eye diameter. This reduced growth was potentially caused by increased apoptosis following CNP treatment. Expression of endogenous nppcl was downregulated in these CNP-treated embryos, suggesting that negative feedback of the CNP system might influence growth during development. CRISPR knock-down of endogenous nppcl in developing zebrafish embryos also resulted in impaired growth characteristics. Collectively, these data suggest that CNP in zebrafish is crucial for normal embryonic development, specifically with regard to growth.

Embryonic and postnatal neurogenesis produce functionally distinct subclasses of dopaminergic neuron.

Most neurogenesis in the mammalian brain is completed embryonically, but in certain areas the production of neurons continues throughout postnatal life. The functional properties of mature postnatally generated neurons often match those of their embryonically produced counterparts. However, we show here that in the olfactory bulb (OB), embryonic and postnatal neurogenesis produce functionally distinct subpopulations of dopaminergic (DA) neurons. We define two subclasses of OB DA neuron by the presence or absence of a key subcellular specialisation: the axon initial segment (AIS). Large AIS-positive axon-bearing DA neurons are exclusively produced during early embryonic stages, leaving small anaxonic AIS-negative cells as the only DA subtype generated via adult neurogenesis. These populations are functionally distinct: large DA cells are more excitable, yet display weaker and - for certain long-latency or inhibitory events - more broadly tuned responses to odorant stimuli. Embryonic and postnatal neurogenesis can therefore generate distinct neuronal subclasses, placing important constraints on the functional roles of adult-born neurons in sensory processing.

A role for CaV1 and calcineurin signaling in depolarization-induced changes in neuronal DNA methylation.

Direct manipulations of neuronal activity have been shown to induce changes in DNA methylation (DNAm), although little is known about the cellular signaling pathways involved. Using reduced representation bisulfite sequencing, we identify DNAm changes associated with moderate chronic depolarization in dissociated rat hippocampal cultures. Consistent with previous findings, these changes occurred primarily in the vicinity of loci implicated in neuronal function, being enriched in intergenic regions and underrepresented in CpG-rich promoter regulatory regions. We subsequently used 2 pharmacological interventions (nifedipine and FK-506) to test whether the identified changes depended on 2 interrelated signaling pathways known to mediate multiple forms of neuronal plasticity. Both pharmacological manipulations had notable effects on the extent and magnitude of depolarization-induced DNAm changes indicating that a high proportion of activity-induced changes are likely to be mediated by calcium entry through L-type CaV1 channels and/or downstream signaling via the calcium-dependent phosphatase calcineurin.

A distinct subtype of dopaminergic interneuron displays inverted structural plasticity at the axon initial segment.

The axon initial segment (AIS) is a specialized structure near the start of the axon that is a site of neuronal plasticity. Changes in activity levels in vitro and in vivo can produce structural AIS changes in excitatory cells that have been linked to alterations in excitability, but these effects have never been described in inhibitory interneurons. In the mammalian olfactory bulb (OB), dopaminergic interneurons are particularly plastic, undergoing constitutive turnover throughout life and regulating tyrosine hydroxylase expression in an activity-dependent manner. Here we used dissociated cultures of rat and mouse OB to show that a subset of bulbar dopaminergic neurons possess an AIS and that these AIS-positive cells are morphologically and functionally distinct from their AIS-negative counterparts. Under baseline conditions, OB dopaminergic AISs were short and located distally along the axon but, in response to chronic 24 h depolarization, lengthened and relocated proximally toward the soma. These activity-dependent changes were in the opposite direction to both those we saw in non-GABAergic OB neurons and those reported previously for excitatory cell types. Inverted AIS plasticity in OB dopaminergic cells was bidirectional, involved all major components of the structure, was dependent on the activity of L-type CaV1 calcium channels but not on the activity of the calcium-activated phosphatase calcineurin, and was opposed by the actions of cyclin-dependent kinase 5. Such distinct forms of AIS plasticity in inhibitory interneurons and excitatory projection neurons may allow considerable flexibility when neuronal networks must adapt to perturbations in their ongoing activity.

Expression of guanylyl cyclase-B (GC-B/NPR2) receptors in normal human fetal pituitaries and human pituitary adenomas implicates a role for C-type natriuretic peptide.

C-type natriuretic peptide (CNP/Nppc) is expressed at high levels in the anterior pituitary of rats and mice and activates guanylyl cyclase B receptors (GC-B/Npr2) to regulate hormone secretion. Mutations in NPR2/Npr2 can cause achondroplasia, GH deficiency, and female infertility, yet the normal expression profile within the anterior pituitary remains to be established in humans. The current study examined the expression profile and transcriptional regulation of NPR2 and GC-B protein in normal human fetal pituitaries, normal adult pituitaries, and human pituitary adenomas using RT-PCR and immunohistochemistry. Transcriptional regulation of human NPR2 promoter constructs was characterized in anterior pituitary cell lines of gonadotroph, somatolactotroph, and corticotroph origin. NPR2 was detected in all human fetal and adult pituitary samples regardless of age or sex, as well as in all adenoma samples examined regardless of tumor origin. GC-B immunoreactivity was variable in normal pituitary, gonadotrophinomas, and somatotrophinomas. Maximal transcriptional regulation of the NPR2 promoter mapped to a region within -214 bp upstream of the start site in all anterior pituitary cell lines examined. Electrophoretic mobility shift assays revealed that this region contains Sp1/Sp3 response elements. These data are the first to show NPR2 expression in normal human fetal and adult pituitaries and adenomatous pituitary tissue and suggest a role for these receptors in both pituitary development and oncogenesis, introducing a new target to manipulate these processes in pituitary adenomas.

Molecular characterisation and functional interrogation of a local natriuretic peptide system in rodent pituitaries, alphaT3-1 and LbetaT2 gonadotroph cells.

In the pituitary, C-type natriuretic peptide (CNP) has been implicated as a gonadotroph-specific factor, yet expression of the CNP gene (Nppc) and CNP activity in gonadotrophs is poorly defined. Here, we examine the molecular expression and putative function of a local gonadotroph natriuretic peptide system. Nppc, along with all three natriuretic peptide receptors (Npr1, Npr2 and Npr3), was expressed in both alphaT3-1 and LbetaT2 cells and primary mouse pituitary tissue, yet the genes for atrial-(ANP) and B-type natriuretic peptides (Nppa and Nppb) were much less abundant. Putative processing enzymes of CNP were also expressed in alphaT3-1 cells and primary mouse pituitaries. Transcriptional analyses revealed that the proximal 50 bp of the murine Nppc promoter were sufficient for GNRH responsiveness, in an apparent protein kinase C and calcium-dependent manner. Electrophoretic mobility shift assays showed Sp1/Sp3 proteins form major complexes within this region of the Nppc promoter. CNP protein was detectable in rat anterior pituitaries, and electron microscopy detected CNP immunoreactivity in secretory granules of gonadotroph cells. Pharmacological analyses of natriuretic peptide receptor activity clearly showed ANP and CNP are potent activators of cGMP production. However, functional studies failed to reveal a role for CNP in regulating cell proliferation or LH secretion. Surprisingly, CNP potently stimulated the human glycoprotein hormone alpha-subunit promoter in LbetaT2 cells but not in alphaT3-1 cells. Collectively, these findings support a role for CNP as the major natriuretic peptide of the anterior pituitary, and for gonadotroph cells as the major source of CNP expression and site of action.