Brian McNeill

Progenitor cell proliferation in the retina is dependent on Notch-independent Sonic hedgehog/Hes1 activity

Sonic hedgehog (Shh) is an indispensable, extrinsic cue that regulates progenitor and stem cell behavior in the developing and adult mammalian central nervous system. Here, we investigate the link between the Shh signaling pathway and Hes1, a classical Notch target. We show that Shh-driven stabilization of Hes1 is independent of Notch signaling and requires the Shh effector Gli2. We identify Gli2 as a primary mediator of this response by showing that Gli2 is required for Hh (Hedgehog)-dependent up-regulation of Hes1. We also show using chromatin immunoprecipitation that Gli2 binds to the Hes1 promoter, which suggests that Hes1 is a Hh-dependent direct target of Gli2 signaling. Finally, we show that Shh stimulation of progenitor proliferation and cell diversification requires Gli2 and Hes1 activity. This paper is the first demonstration of the mechanistic and functional link between Shh, Gli, and Hes1 in the regulation of progenitor cell behavior.

Chemoreceptor plasticity and respiratory acclimation in the zebrafish Danio rerio.

SUMMARY The goals of this study were to assess the respiratory consequences of exposing adult zebrafish Danio rerio to chronic changes in water gas composition (hypoxia, hyperoxia or hypercapnia) and to determine if any ensuing effects could be related to morphological changes in branchial chemoreceptors. To accomplish these goals, we first modified and validated an established non-invasive technique for continuous monitoring of breathing frequency and relative breathing amplitude in adult fish. Under normal conditions 20% of zebrafish exhibited an episodic breathing pattern that was composed of breathing and non-breathing (pausing/apneic) periods. The pausing frequency was reduced by acute hypoxia (PwO2<130 mmHg) and increased by acute hyperoxia (PwO2>300 mmHg), but was unaltered by acute hypercapnia. Fish were exposed for 28 days to hyperoxia (PwO2>350 mmHg), or hypoxia (PwO2=30 mmHg) or hypercapnia (PwCO2=9 mmHg). Their responses to acute hypoxia or hypercapnia were then compared to the response of control fish kept for 28 days in normoxic and normocapnic water. In control fish, the ventilatory response to acute hypoxia consisted of an increase in breathing frequency while the response to acute hypercapnia was an increase in relative breathing amplitude. The stimulus promoting the hyperventilation during hypercapnia was increased PwCO2 rather than decreased pH. Exposure to prolonged hyperoxia decreased the capacity of fish to increase breathing frequency during hypoxia and prevented the usual increase in breathing amplitude during acute hypercapnia. In fish previously exposed to hyperoxia, episodic breathing continued during acute hypoxia until PwO2 had fallen below 70 mmHg. In fish chronically exposed to hypoxia, resting breathing frequency was significantly reduced (from 191±12 to 165±16 min–1); however, the ventilatory responses to hypoxia and hypercapnia were unaffected. Long-term exposure of fish to hypercapnic water did not markedly modify the breathing response to acute hypoxia and modestly blunted the response to hypercapnia. To determine whether branchial chemoreceptors were being influenced by long-term acclimation, all four groups of fish were acutely exposed to increasing doses of the O2 chemoreceptor stimulant, sodium cyanide, dissolved in inspired water. Consistent with the blunting of the ventilatory response to hypoxia, the fish pre-exposed to hyperoxia also exhibited a blunted response to NaCN. Pre-exposure to hypoxia was without effect whereas prior exposure to hypercapnia increased the ventilatory responses to cyanide. To assess the impact of acclimation to varying gas levels on branchial O2 chemoreceptors, the numbers of neuroepithelial cells (NECs) of the gill filament were quantified using confocal immunofluorescence microscopy. Consistent with the blunting of reflex ventilatory responses, fish exposed to chronic hyperoxia exhibited a significant decrease in the density of NECs from 36.8±2.8 to 22.7±2.3 filament–1.

Characterization of a branchial epithelial calcium channel (ECaC) in freshwater rainbow trout (Oncorhynchus mykiss).

SUMMARY The entry of calcium (Ca2+) through an apical membrane epithelial calcium channel (ECaC) is thought to a key step in piscine branchial Ca2+ uptake. In mammals, ECaC is a member of the transient receptor potential (TRP) gene family of which two sub-families have been identified, TRPV5 and TPRV6. In the present study we have identified a single rainbow trout (Oncorhynchus mykiss) ECaC (rtECaC) that is similar to the mammalian TRPV5 and TRPV6. Phylogenetic analysis of the protein sequence suggests that an ancestral form of the mammalian genes diverged from those in the lower vertebrates prior to the gene duplication event that gave rise to TRPV5 and TRPV6. The putative model for Ca2+ uptake in fish proposes that the mitochondria-rich cell (also termed ionocyte or chloride cell) is the predominant or exclusive site of transcellular Ca2+ movements owing to preferential localisation of ECaC to the apical membrane of these cells. However, the results of real-time PCR performed on enriched gill cell populations as well as immunocytochemistry and in situ hybridisation analysis of enriched cells, cell cultures and whole gill sections strongly suggest that ECaC is not exclusive to mitochondria-rich cells but that it is also found in pavement cells. Not only was ECaC protein localized to areas of the gill normally having few mitochondria-rich cells, but there was also no consistent co-localization of ECaC- and Na+/K+-ATPase-positive (a marker of mitochondria rich cells) cells. Taken together, the results of the present study suggest that although ECaC (mRNA and protein) does exist in trout gill, its cellular distribution is more extensive than previously thought, thus suggesting that Ca2+ uptake may not be restricted to mitochondria-rich cells as was proposed in previous models.

Circulating catecholamines and cardiorespiratory responses in hypoxic lungfish (Protopterus dolloi): a comparison of aquatic and aerial hypoxia.

Circulating catecholamine levels and a variety of cardiorespiratory variables were monitored in cannulated bimodally breathing African lungfish (Protopterus dolloi) exposed to aquatic or aerial hypoxia. Owing to the purported absence of external branchial chemoreceptors in lungfish and the minor role played by the gill in O2 uptake, it was hypothesized that plasma catecholamine levels would increase only during exposure of fish to aerial hypoxia. The rapid induction of aquatic hypoxia (final PWo2 = 25.9 ± 1.6 mmHg) did not affect the levels of adrenaline (A) or noradrenaline (NA) within the plasma. Similarly, none of the measured cardiorespiratory variables—including heart rate (fH), blood pressure, air‐breathing frequency (fV), O2 consumption (Mo2), CO2 excretion (Mco2), or blood gases—were influenced by acute aquatic hypoxia. In contrast, however, the rapid induction of aerial hypoxia (inspired Po2 = 46.6 ± 3.3 mmHg) caused a marked increase in the circulating levels of A (from 7.9 ± 2.0 to 18.8 ± 6.1 nmol L−1) and NA (from 7.7 ± 2.2 to 19.7 ± 6.3 nmol L−1) that was accompanied by significant deceases in Mo2, arterial Po2 (Pao2), and arterial O2 concentration (Cao2). Air‐breathing frequency was increased (by approximately five breaths per hour) during aerial hypoxia and presumably contributed to the observed doubling of pulmonary Mco2 (from 0.25 ± 0.04 to 0.49 ± 0.07 mmol kg−1 h−1); fH and blood pressure were unaffected by aerial hypoxia. An in situ perfused heart preparation was used to test the possibility that catecholamine secretion from cardiac chromaffin cells was being activated by a direct localized effect of hypoxia. Catecholamine secretion from the chromaffin cells of the heart, while clearly responsive to a depolarizing concentration of KCl (60 mmol L−1), was unaffected by the O2 status of the perfusion fluid. The results of this study demonstrate that P. dolloi is able to mobilize stored catecholamines and increase fV during exposure to aerial hypoxia while remaining unresponsive to aquatic hypoxia. Thus, unlike in exclusively water‐breathing teleosts, P. dolloi would appear to rely solely on internal/airway O2 chemoreceptors for initiating catecholamine secretion and cardiorespiratory responses.

The interactive effects of hypoxia and nitric oxide on catecholamine secretion in rainbow trout (Oncorhynchus mykiss).

SUMMARY Experiments were performed to test the hypothesis that exposure of rainbow trout to repetitive hypoxia would result in a decreased capacity of chromaffin cells to secrete catecholamines owing to increased production of nitric oxide (NO), a potent inhibitor of catecholamine secretion. A partial sequence of trout neuronal nitric oxide synthase (nNOS) was cloned and its mRNA was found to be present in the posterior cardinal vein (PCV), the predominant site of chromaffin cells in trout. Using heterologous antibodies, nNOS and endothelial NOS (eNOS) were localized in close proximity to the chromaffin cells of the PCV. Exposure of trout to acute hypoxia (5.33 kPa for 30 min) in vivo resulted in significant increases in plasma catecholamine and NO levels. However, after 4 days of twice-daily exposures to hypoxia, the elevation of plasma catecholamine levels during hypoxia was markedly reduced. Associated with the reduction in plasma catecholamine levels during acute hypoxia was a marked increase in basal and hypoxia-evoked circulating levels of NO that became apparent after 2-4 days of repetitive hypoxia. The capacity of the chromaffin cells of the hypoxia-exposed fish to secrete catecholamine was assessed by electrical stimulation of an in situ saline-perfused PCV preparation. Compared with control (normoxic) fish, the PCV preparations derived from fish exposed to repeated hypoxia displayed a significant reduction in electrically evoked catecholamine secretion that was concomitant with a marked increased in NO production. This additional rise in NO secretion in preparations derived from hypoxic fish was prevented after adding NOS inhibitors to the perfusate; concomitantly, the reduction in catecholamine secretion was prevented. The increased production of NO during hypoxia in vivo and during electrical stimulation in situ was consistent with significant elevations of nNOS mRNA and protein; eNOS protein was unaffected. These results suggest that the reduced capacity of trout chromaffin cells to secrete catecholamines after repeated hypoxia reflects an increase in the expression of nNOS and a subsequent increase in NO production during chromaffin-cell activation.

Hydrogen sulfide stimulates catecholamine secretion in rainbow trout (Oncorhynchus mykiss)

We tested the hypothesis that endogenously produced hydrogen sulfide (H(2)S) can potentially contribute to the adrenergic stress response in rainbow trout by initiating catecholamine secretion from chromaffin cells. During acute hypoxia (water Po(2) = 35 mmHg), plasma H(2)S levels were significantly elevated concurrently with a rise in circulating catecholamine concentrations. Tissues enriched with chromaffin cells (posterior cardinal vein and anterior kidney) produced H(2)S in vitro when incubated with l-cysteine. In both tissues, the production of H(2)S was eliminated by adding the cystathionine beta-synthase inhibitor, aminooxyacetate. Cystathionine beta-synthase and cystathionine gamma-lyase were cloned and sequenced and the results of real-time PCR demonstrated that with the exception of white muscle, mRNA for both enzymes was broadly distributed within the tissues that were examined. Electrical field stimulation of an in situ saline-perfused posterior cardinal vein preparation caused the appearance of H(2)S and catecholamines in the outflowing perfusate. Perfusion with the cholinergic receptor agonist carbachol (1 x 10(-6) M) or depolarizing levels of KCl (1 x 10(-2) M) caused secretion of catecholamines without altering H(2)S output, suggesting that neuronal excitation is required for H(2)S release. Addition of H(2)S (at concentrations exceeding 5 x 10(-7) M) to the perfusion fluid resulted in a marked stimulation of catecholamine secretion that was not observed when Ca(2+)-free perfusate was used. These data, together with the finding that H(2)S-induced catecholamine secretion was unaltered by the nicotinic receptor blocker hexamethonium, suggest that H(2)S is able to directly elicit catecholamine secretion via membrane depolarization followed by Ca(2+)-mediated exocytosis.

Fooling a freshwater fish: how dietary salt transforms the rainbow trout gill into a seawater gill phenotype

SUMMARY Numerous fish species, including rainbow trout (Oncorhynchus mykiss), are able to inhabit both freshwater and seawater and routinely migrate between the two environments. One of the most critical adjustments allowing such successful migrations is a remodelling of the gill in which a suite of morphological and molecular changes ensure optimal function in the face of reversing requirements for salt and water balance. The remodelling leads to specific freshwater and seawater gill phenotypes that are readily identified by the orientation and/or quantities of specific ion transporters and the presence or absence of specific cell types. The proximate cues promoting gill phenotypic plasticity are unknown. Here, by assessing the consequences of a salt-enriched diet (in the absence of any changes in external salinity) in the freshwater rainbow trout, we demonstrate that internal salt loading alone, is able to induce various elements of the seawater gill phenotype. Specifically, we show upregulation of three ion transport genes, cystic fibrosis transmembrane conductance regulator (CFTR), Na+/K+/2Cl- co-transporter (NKCC1) and Na+/K+-ATPase, which are essential for ionic regulation in seawater, and the appearance of chloride cell-accessory cell complexes, which are normally restricted to fish inhabiting seawater. These data provide compelling evidence that gill remodelling during migration from freshwater to seawater may involve sensing of elevated levels of internal salt.

Methylglyoxal-Induced Endothelial Cell Loss and Inflammation Contribute to the Development of Diabetic Cardiomyopathy

The mechanisms for the development of diabetic cardiomyopathy remain largely unknown. Methylglyoxal (MG) can accumulate and promote inflammation and vascular damage in diabetes. We examined if overexpression of the MG-metabolizing enzyme glyoxalase 1 (GLO1) in macrophages and the vasculature could reduce MG-induced inflammation and prevent ventricular dysfunction in diabetes. Hyperglycemia increased circulating inflammatory markers in wild-type (WT) but not in GLO1-overexpressing mice. Endothelial cell number was reduced in WT-diabetic hearts compared with nondiabetic controls, whereas GLO1 overexpression preserved capillary density. Neuregulin production, endothelial nitric oxide synthase dimerization, and Bcl-2 expression in endothelial cells was maintained in the hearts of GLO1-diabetic mice and corresponded to less myocardial cell death compared with the WT-diabetic group. Lower receptor for advanced glycation end products and tumor necrosis factor-α (TNF-α) levels were also observed in GLO1-diabetic versus WT-diabetic mice. Over a period of 8 weeks of hyperglycemia, GLO1 overexpression delayed and limited the loss of cardiac function. In vitro, MG and TNF-α were shown to synergize in promoting endothelial cell death, which was associated with increased angiopoietin 2 expression and reduced Bcl-2 expression. These results suggest that MG in diabetes increases inflammation, leading to endothelial cell loss. This contributes to the development of diabetic cardiomyopathy and identifies MG-induced endothelial inflammation as a target for therapy.

Suppressor of Fused Is Required to Maintain the Multipotency of Neural Progenitor Cells in the Retina

The morphogen sonic hedgehog (Shh) plays a crucial role in development of the CNS, including the neural retina. Suppressor of fused (Sufu) has been recently identified as a critical regulator of Hh signaling in mammals. However, the precise roles that Sufu plays in the regulation of proliferation and cell-fate decisions in neural progenitors is unknown. Here, we have addressed these questions by conditionally deleting Sufu in mouse multipotent retinal progenitor cells (RPCs). Sufu deletion in RPCs results in transient increases in Hh activity and proliferation followed by developmentally premature cell-cycle exit. Importantly, we demonstrate a novel role for Sufu in the maintenance of multipotency in RPCs. Sufu-null RPCs downregulate transcription factors required to specify or maintain RPC identity (Rax, Vsx2) and multipotency (Pax6) but continue to express the neural progenitor marker Sox2. These cells fail to express retinal lineage-specific transcription factors, such as Math5, and adopt an amacrine or horizontal cell fate at the expense of all other classes of retinal neurons. Genetic elimination of Gli2 in Sufu-null RPCs attenuates Hh pathway activity and restores multipotency in neural progenitors. These data provide novel evidence that Sufu-mediated antagonism of Hh/Gli2 signaling is required to maintain RPC multipotency and identity.

Glyoxalase-1 overexpression in bone marrow cells reverses defective neovascularization in STZ-induced diabetic mice

AIMS Methylglyoxal (MG) accumulates in diabetes and impairs neovascularization. This study assessed whether overexpressing the MG-metabolizing enzyme glyoxalase-1 (GLO1) in only bone marrow cells (BMCs) could restore neovascularization in ischaemic tissue of streptozotocin-induced diabetic mice. METHODS AND RESULTS After 24 h of hyperglycaemic and hypoxic culture, BMCs from GLO1 overexpressing and wild-type (WT) diabetic mice were compared for migratory potential, viability, and mRNA expression of anti-apoptotic genes (Bcl-2 and Bcl-XL). In vivo, BMCs from enhanced green fluorescent protein (eGFP) mice that overexpress GLO1 were used to reconstitute the BM of diabetic mice (GLO1-diabetics). Diabetic and non-diabetic recipients of WT GFP(+) BM served as controls (WT-diabetics and non-diabetics, respectively). Following hindlimb ischaemia, the mobilization of BMCs was measured by flow cytometry. In hindlimbs, the presence of BM-derived angiogenic (GFP(+)CXCR4(+)) and endothelial (GFP(+)vWF(+)) cells and also arteriole density were determined by immunohistochemistry. Hindlimb perfusion was measured using laser Doppler. GLO1-BMCs had superior migratory potential, increased viability, and greater Bcl-2 and Bcl-XL expression, compared with WT BMCs. In vivo, the mobilization of pro-angiogenic BMCs (CXCR4(+), c-kit(+), and Flk(+)) was enhanced post-ischaemia in GLO1-diabetics compared to WT-diabetics. A greater number of GFP(+)CXCR4(+) and GFP(+)vWF(+) BMCs incorporated into the hindlimb tissue of GLO1-diabetics and non-diabetics than in WT-diabetics. Arteriole and capillary density and perfusion were also greater in GLO1-diabetics and non-diabetics. CONCLUSION This study demonstrates that protection from MG uniquely in BM is sufficient to restore BMC function and neovascularization of ischaemic tissue in diabetes and identifies GLO1 as a potential therapeutic target.