Disparate progenitor cell populations contribute to maintenance and repair neurogenesis in the zebrafish olfactory epithelium.

Olfactory sensory neurons (OSNs) undergo constant turnover under physiological conditions but also regenerate efficiently following tissue injury. Maintenance and repair neurogenesis in the olfactory epithelium (OE) have been attributed to the selective activity of globose (GBCs) and horizontal basal cells (HBCs), respectively. In zebrafish, cells with GBC-like properties are localized to the peripheral margins of the sensory OE and contribute to OSN neurogenesis in the intact OE, while cells that resemble HBCs at the morphological and molecular level are more uniformly distributed. However, the contribution of these cells to the restoration of the injured OE has not been demonstrated. Here, we provide a detailed cellular and molecular analysis of the tissue response to injury and show that a dual progenitor cell system also exists in zebrafish. Zebrafish HBCs respond to the structural damage of the OE and generate a transient population of proliferative neurogenic progenitors that restores OSNs. In contrast, selective ablation of OSNs by axotomy triggers neurogenic GBC proliferation, suggesting that distinct signaling events activate GBC and HBC responses. Molecular analysis of differentially expressed genes in lesioned and regenerating OEs points toward an involvement of the canonical Wnt/ß-catenin pathway. Activation of Wnt signaling appears to be sufficient to stimulate mitotic activity, while inhibition significantly reduces, but does not fully eliminate, HBC responses. Zebrafish HBCs are surprisingly active even under physiological conditions with a strong bias toward the zones of constitutive OSN neurogenesis, suggestive of a direct lineage relationship between progenitor cell subtypes.

Diving into the streams and waves of constitutive and regenerative olfactory neurogenesis: insights from zebrafish.

The olfactory system is renowned for its functional and structural plasticity, with both peripheral and central structures displaying persistent neurogenesis throughout life and exhibiting remarkable capacity for regenerative neurogenesis after damage. In general, fish are known for their extensive neurogenic ability, and the zebrafish in particular presents an attractive model to study plasticity and adult neurogenesis in the olfactory system because of its conserved structure, relative simplicity, rapid cell turnover, and preponderance of neurogenic niches. In this review, we present an overview of the anatomy of zebrafish olfactory structures, with a focus on the neurogenic niches in the olfactory epithelium, olfactory bulb, and ventral telencephalon. Constitutive and regenerative neurogenesis in both the peripheral olfactory organ and central olfactory bulb of zebrafish is reviewed in detail, and a summary of current knowledge about the cellular origin and molecular signals involved in regulating these processes is presented. While some features of physiologic and injury-induced neurogenic responses are similar, there are differences that indicate that regeneration is not simply a reiteration of the constitutive proliferation process. We provide comparisons to mammalian neurogenesis that reveal similarities and differences between species. Finally, we present a number of open questions that remain to be answered.

TGFß Is Specifically Upregulated on Circulating CD14++ CD16+ and CD14+ CD16++ Monocytes in Patients with Atrial Fibrillation and Severe Atrial Fibrosis.

BACKGROUND/AIMS: Fibrotic remodeling of the atria plays a key role in the pathogenesis of atrial fibrillation (AF). As little is known about the contribution of circulating monocytes in atrial remodeling and the pathophysiology of AF, we investigated profibrotic factors in different subsets of circulating monocytes obtained from patients with atrial fibrillation undergoing catheter ablation. METHODS: A 3D high density voltage mapping was performed in sinus rhythm to evaluate the extent of low-voltage areas (LVAs) in the atria of 71 patients with persistent AF. Low-voltage was defined as signals of < 0.5mV during sinus rhythm. Prior to ablation, blood was drawn and monocytes were analyzed by FACS. Based on the expression of CD14 and CD16, three subgroups including CD14++ CD16- ('classical'), CD14++ CD16+ ('intermediate'), and CD14+ CD16++ ('non-classical') were analyzed for the expression of TGFb, CD147, and MMP-9, representing pivotal profibrotic pathways in myocardial remodeling. RESULTS: Expression of TGFb was increased in CD14+ monocytes of patients with extensive LVAs compared to patients with a low extend of LVAs. While CD14++ CD16- monocytes showed no difference, CD14++ CD16+ and CD14+ CD16++ monocytes showed a strong increase of TGFb abundance. Although CD147 and MMP-9 are strongly associated with myocardial fibrosis, we found no difference in expression between the two groups in any monocyte subsets. CONCLUSION: TGFb is specifically upregulated on CD14++ CD16+ and CD14+ CD16++ monocytes in patients with extensive LVAs undergoing catheter ablation.

Odorant receptor gene choice and axonal wiring in mice with deletion mutations in the odorant receptor gene SR1.

In the mouse, a mature olfactory sensory neuron (OSN) of the main olfactory epithelium (MOE) expresses one allele of one of the 1200 odorant receptor (OR) genes in the genome. The mechanisms that underlie the one receptor-one neuron rule remain poorly understood. A popular experimental paradigm for OR gene choice is to delete an OR coding region by gene targeting or in a transgene. Here we have applied this ∆OR paradigm to SR1, also known as MOR256-3 or Olfr124. This gene is expressed in OSNs of the MOE, and in ~50% of the OSNs of the septal organ. In heterozygous ∆SR1 mice, we observe an unprecedented biallelic expression rate of 30% at the SR1 locus. In homozygous ∆SR1 mice, we find a significant increase in the number of septal organ OSNs that undergo apoptosis. As a population, ∆SR1 OSNs project their axons to 81-85 glomeruli in each half of the OB, and coexpress at least 77 OR genes as evaluated by single-cell molecular analysis. There are no obvious or simple rules for the set of OR genes that are coexpressed with the ∆SR1 allele. The frequencies of coexpression are different for ∆SR1 OSNs in the septal organ compared to those in the MOE. We propose that there are as many as five scenarios for the fate of individual ∆SR1 OSNs.

HB-EGF promotes progenitor cell proliferation and sensory neuron regeneration in the zebrafish olfactory epithelium.

Maintenance and regeneration of the zebrafish olfactory epithelium (OE) are supported by two distinct progenitor cell populations that occupy spatially discrete stem cell niches and respond to different tissue conditions. Globose basal cells (GBCs) reside at the inner and peripheral margins of the sensory OE and are constitutively active to replace sporadically dying olfactory sensory neurons (OSNs). In contrast, horizontal basal cells (HBCs) are uniformly distributed across the sensory tissue and are selectively activated by acute injury conditions. Here we show that expression of the heparin-binding epidermal growth factor-like growth factor (HB-EGF) is strongly and transiently upregulated in response to OE injury and signals through the EGF receptor (EGFR), which is expressed by HBCs. Exogenous stimulation of the OE with recombinant HB-EGF promotes HBC expansion and OSN neurogenesis in a pattern that resembles the tissue response to injury. In contrast, pharmacological inhibition of HB-EGF membrane shedding, HB-EGF availability, and EGFR signaling strongly attenuate or delay injury-induced HBC activity and OSN restoration without affecting maintenance neurogenesis by GBCs. Thus, HB-EGF/EGFR signaling appears to be a critical component of the signaling network that controls HBC activity and, consequently, repair neurogenesis in the zebrafish OE.

SR1, a mouse odorant receptor with an unusually broad response profile.

The current consensus model in mammalian olfaction is that the detection of millions of odorants requires a large number of odorant receptors (ORs) and that each OR interacts selectively with a small subset of odorants, which are typically related in structure. Here, we report the odorant response properties of an OR that deviates from this model: SR1, a mouse OR that is abundantly expressed in sensory neurons of the septal organ and also of the main olfactory epithelium. Patch-clamp recordings reveal that olfactory sensory neurons (OSNs) that express SR1 respond to many, structurally unrelated odorants, and over a wide concentration range. Most OSNs expressing a gene-targeted SR1 locus that lacks the SR1 coding sequence do not show this broad responsiveness. Gene transfer in the heterologous expression system Hana3A confirms the broad response profile of SR1. There may be other mouse ORs with such broad response profiles.

Mechanisms of odorant receptor gene choice in Drosophila and vertebrates.

Odorant receptors are encoded by extremely large and divergent families of genes. Each receptor is expressed in a small proportion of neurons in the olfactory organs, and each neuron in turn expresses just one odorant receptor gene. This fundamental property of the peripheral olfactory system is widely conserved across evolution, and observed in vertebrates, like mice, and invertebrates, like Drosophila, despite their olfactory receptor gene families being evolutionarily unrelated. Here we review the progress that has been made in these two systems to understand the intriguing and elusive question: how does a single neuron choose to express just one of many possible odorant receptors and exclude expression of all others?

Purinergic signalling selectively modulates maintenance but not repair neurogenesis in the zebrafish olfactory epithelium.

Olfactory sensory neurons (OSNs) of the vertebrate olfactory epithelium (OE) undergo continuous turnover but also regenerate efficiently when the OE is acutely damaged by traumatic injury. Two distinct pools of neuronal stem/progenitor cells, the globose (GBCs), and horizontal basal cells (HBCs) have been shown to selectively contribute to intrinsic OSN turnover and damage-induced OE regeneration, respectively. For both types of progenitors, their rate of cell divisions and OSN production must match the actual loss of cells to maintain or to re-establish sensory function. However, signals that communicate between neurons or glia cells of the OE and resident neurogenic progenitors remain largely elusive. Here, we investigate the effect of purinergic signaling on cell proliferation and OSN neurogenesis in the zebrafish OE. Purine stimulation elicits transient Ca2+ signals in OSNs and distinct non-neuronal cell populations, which are located exclusively in the basal OE and stain positive for the neuronal stem cell marker Sox2. The more apical population of Sox2-positive cells comprises evenly distributed glia-like sustentacular cells (SCs) and spatially restricted GBC-like cells, whereas the more basal population expresses the HBC markers keratin 5 and tumor protein 63 and lines the entire sensory OE. Importantly, exogenous purine stimulation promotes P2 receptor-dependent mitotic activity and OSN generation from sites where GBCs are located but not from HBCs. We hypothesize that purine compounds released from dying OSNs modulate GBC progenitor cell cycling in a dose-dependent manner that is proportional to the number of dying OSNs and, thereby, ensures a constant pool of sensory neurons over time.

Patterned Arrangements of Olfactory Receptor Gene Expression in Zebrafish are Established by Radial Movement of Specified Olfactory Sensory Neurons.

Spatial restriction of olfactory receptor (OR) gene expression in peripheral sense organs is a common phenomenon across species, suggesting that zonal OR expression somehow contributes to olfactory function. In zebrafish OR expression patterns reminiscent of zones occur as concentric domains with preferred diameters for different ORs. However, the function and the developmental origin of the pattern are unknown. Here we investigate olfactory sensory neuron (OSN) neurogenesis in the adult zebrafish olfactory epithelium (OE) to understand how the zonally organized OR pattern is established and maintained during the lifetime of the animal. We find that OSNs are generated from two discontinuous proliferation zones located at the central and peripheral edge of the sensory OE. OSNs turn on OR expression soon after they exit mitosis and invade the sensory tissue, approaching each other from both ends of the OE. Biased generation of OSN subpopulations at both neurogenic sites and elimination of OSNs along their route across the OE generates the impression of OR-specific expression domains. We formulated a simple mathematical model based on exact parameters derived from our analysis of OSN neurogenesis, which accurately generates OR-like distributions without the need to invoke molecular signals to pattern the OE.

SIK2 is involved in the negative modulation of insulin-dependent muller cell survival and implicated in hyperglycemia-induced cell death.

PURPOSE: To investigate the role of the serine/threonine kinase SIK2, a member of the salt-inducible kinase (SIK) family, in insulin-dependent cell survival and hyperglycemia-induced cell death in Müller glia. METHODS: Expression studies were performed by RT-PCR, immunostaining, Northern blotting, and immunoblotting. Insulin-dependent changes in SIK2 activity were investigated by in vitro kinase assays in MIO-M1 Müller cell line. Akt activation was studied by immunoblotting and cell death by TUNEL assay. The potential role of SIK2 in insulin signaling was explored by overexpression and sh-RNA knock-down approaches. Effects of hyperglycemia were studied in vitro and in vivo in streptozotocin-injected rats. RESULTS: SIK2 expression was detected throughout adult retina, except for the outer nuclear layer. Insulin stimulation of MIO-M1 cells resulted in a rapid 2-fold increase of SIK2 activity, increased insulin receptor substrate 1 (IRS1)-SIK2 interaction, and reduced cell death. pAkt levels following insulin treatment were modulated by SIK2 activity. Under hyperglycemia, increased SIK2 activity/expression was concomitant to decreased Akt activation and enhanced apoptosis; whereas knockdown of SIK2 under normo- and hyperglycemic conditions resulted in a rapid increase in pAkt levels and blunted cell death. SIK2 overexpression under normoglycemia had an opposite effect. SIK2 activity increased significantly within 2 weeks of induction of hyperglycemia in the rat retina. CONCLUSIONS: Results indicate that SIK2 functions as a negative modulator of the insulin-dependent survival pathway and contributes to hyperglycemia-induced cell death of Müller glia in vitro. Although still hypothetical at this point, our study suggests that SIK2 could serve a similar role during the development of diabetic retinopathy in vivo and that it represents a potential target to control disease progression.

Born to run: patterning the Drosophila olfactory system.

The distributed, apparently random pattern of odorant receptor expression is thought to reflect a stochastic choice process. In this issue of Developmental Cell, Song et al. (2012) suggest that the Drosophila pattern is established secondarily from regionally specified cell populations through changes in transcription factor expression and cell migration.

The adverse effects of air pollution on the nervous system.

Exposure to ambient air pollution is a serious and common public health concern associated with growing morbidity and mortality worldwide. In the last decades, the adverse effects of air pollution on the pulmonary and cardiovascular systems have been well established in a series of major epidemiological and observational studies. In the recent past, air pollution has also been associated with diseases of the central nervous system (CNS), including stroke, Alzheimer's disease, Parkinson's disease, and neurodevelopmental disorders. It has been demonstrated that various components of air pollution, such as nanosized particles, can easily translocate to the CNS where they can activate innate immune responses. Furthermore, systemic inflammation arising from the pulmonary or cardiovascular system can affect CNS health. Despite intense studies on the health effects of ambient air pollution, the underlying molecular mechanisms of susceptibility and disease remain largely elusive. However, emerging evidence suggests that air pollution-induced neuroinflammation, oxidative stress, microglial activation, cerebrovascular dysfunction, and alterations in the blood-brain barrier contribute to CNS pathology. A better understanding of the mediators and mechanisms will enable the development of new strategies to protect individuals at risk and to reduce detrimental effects of air pollution on the nervous system and mental health.

Mapping of class I and class II odorant receptors to glomerular domains by two distinct types of olfactory sensory neurons in the mouse.

The repertoire of approximately 1200 odorant receptors (ORs) is mapped onto the array of approximately 1800 glomeruli in the mouse olfactory bulb (OB). The spatial organization of this array is influenced by the ORs. Here we show that glomerular mapping to broad domains in the dorsal OB is determined by two types of olfactory sensory neurons (OSNs), which reside in the dorsal olfactory epithelium. The OSN types express either class I or class II OR genes. Axons from the two OSN types segregate already within the olfactory nerve and form distinct domains of glomeruli in the OB. These class-specific anatomical domains correlate with known functional odorant response domains. However, axonal segregation and domain formation are not determined by the class of the expressed OR protein. Thus, the two OSN types are determinants of axonal wiring, operate at a higher level than ORs, and contribute to the functional organization of the glomerular array.

Local and cis effects of the H element on expression of odorant receptor genes in mouse.

From the approximately 1,200 odorant receptor (OR) genes in the mouse genome, an olfactory sensory neuron is thought to express only one gene. The mechanisms of OR gene choice are not understood. A 2.1 kilobase region (the H element) adjacent to a cluster of seven OR genes has been proposed as a trans- and pan-enhancer for OR gene expression. Here, we deleted the H element by gene targeting in mice. The deletion abolishes expression of a family of three OR genes proximal to H, and H operates in cis on these genes. Deletion of H has a graded effect on expression of a distal group of four OR genes, commensurate with genomic distance. There is no demonstrable effect on expression of OR genes located outside the cluster. Our findings are not consistent with the hypothesis of H as an essential trans-acting enhancer for genome-wide regulation of OR gene expression.

The Grueneberg ganglion of the mouse projects axons to glomeruli in the olfactory bulb.

First described in 1973, the Grueneberg ganglion (GG) is an arrow-shaped neuronal structure at the anterior end of the nasal cavity. It lines both sides of the nasal septum, within the nasal vestibule, close to the opening of the naris. The functions of the GG and the pattern of projections to the brain are not known. Here, we report that neurons of the mouse GG express olfactory marker protein, which is normally expressed in mature olfactory or vomeronasal sensory neurons. The approx. 500 cells in each GG are arranged in several densely packed cell clusters. Individual cells give rise to single axons, which fasciculate to form a nerve bundle that projects caudally. The axons terminate in glomeruli of the olfactory bulb, one or two large glomeruli associated with a semicircle of up to 10 smaller, somewhat diffusely organized glomeruli that surround the most anterior part of the accessory olfactory bulb. Development of the GG starts around embryonic day 16 and appears to be completed at birth; cell numbers then undergo a minor decrease during postnatal development. The strategic location of the GG, expression of olfactory marker protein, axonal projections to glomeruli at particular locations in the olfactory bulb and early development suggest that this neuronal structure performs specific chemosensory functions at neonatal stages.

Selective targeting of zebrafish olfactory receptor neurons by the endogenous OMP promoter.

The olfactory nervous system of fish, in particular zebrafish, has become a valid model for that of higher vertebrates. However, no genetic markers for olfactory specific cell types, e.g. the olfactory receptor neurons, have been established in this species. Olfactory marker protein (OMP) is a reliable marker for olfactory receptor neurons in several other vertebrates. We have cloned zOMP, the zebrafish homologue of olfactory marker protein. During development, zOMP is expressed exclusively in the olfactory placode, presumably in olfactory receptor neurons, as shown by in situ hybridization. In the adult nasal epithelium zOMP is found restricted to the sensory region. zOMP appears to be a single gene, without close family members. The 5'-flanking region lacks most of the expected regulatory sequence motifs, both general and cell type-specific ones. Nevertheless, it drives reporter gene expression strongly and specifically in olfactory receptor neurons during the whole developmental period examined. Thus the zOMP promoter constitutes a powerful tool which should be useful to selectively introduce a wide variety of genetic modifications into olfactory receptor neurons.

Selective imaging of presynaptic activity in the mouse olfactory bulb shows concentration and structure dependence of odor responses in identified glomeruli.

More chemicals can be smelled than there are olfactory receptors for them, necessitating a combinatorial representation by somewhat broadly tuned receptors. To understand the perception of odor quality and concentration, it is essential to establish the nature of the receptor repertoires that are activated by particular odorants at particular concentrations. We have taken advantage of the one-to-one correspondence of glomeruli and olfactory receptor molecules in the mouse olfactory bulb to analyze the tuning properties of a major receptor population by high resolution calcium imaging of odor responses selectively in the presynaptic compartment of glomeruli. We show that eighty different olfactory receptors projecting to the dorsal olfactory bulb respond to high concentrations of aldehydes with limited specificity. Varying ensembles of about 10 to 20 receptors encode any particular aldehyde at low stimulus concentrations with high specificity. Even normalized odor response patterns are markedly concentration dependent, caused by pronounced differences in affinity within the aldehyde receptor repertoire.