Abhijit Bugde

PAX7 expression defines germline stem cells in the adult testis

Spermatogenesis is a complex, multistep process that maintains male fertility and is sustained by rare germline stem cells. Spermatogenic progression begins with spermatogonia, populations of which express distinct markers. The identity of the spermatogonial stem cell population in the undisturbed testis is controversial due to a lack of reliable and specific markers. Here we identified the transcription factor PAX7 as a specific marker of a rare subpopulation of A(single) spermatogonia in mice. PAX7+ cells were present in the testis at birth. Compared with the adult testis, PAX7+ cells constituted a much higher percentage of neonatal germ cells. Lineage tracing in healthy adult mice revealed that PAX7+ spermatogonia self-maintained and produced expanding clones that gave rise to mature spermatozoa. Interestingly, in mice subjected to chemotherapy and radiotherapy, both of which damage the vast majority of germ cells and can result in sterility, PAX7+ spermatogonia selectively survived, and their subsequent expansion contributed to the recovery of spermatogenesis. Finally, PAX7+ spermatogonia were present in the testes of a diverse set of mammals. Our data indicate that the PAX7+ subset of A(single) spermatogonia functions as robust testis stem cells that maintain fertility in normal spermatogenesis in healthy mice and mediate recovery after severe germline injury, such as occurs after cancer therapy.

Evaluation of the corneal effects of topical ophthalmic fluoroquinolones using in vivo confocal microscopy

Purpose: To compare the effects of several fluoroquinolone antibiotics on the corneal epithelium and stroma using in vivo confocal microscopy. Methods: Five antibiotic solutions were evaluated: 1) 0.3% ofloxacin (Oflox) solution with 0.005% benzalkonium chloride (BAC); 2) 0.3% gatifloxacin (Gati) solution with 0.005% BAC; 3) 0.3% ciprofloxacin (Cipro) solution with 0.006% BAC; 4) 0.5% levofloxacin (Levo) with 0.005% BAC; and 5) 0.5% moxifloxacin (Moxi) solution with no BAC. Preservative-free artificial tears (Tears) were used as a control. New Zealand white rabbits were used for this study (six per solution group). Ten days prior to exposure to any solution, central corneal epithelial thickness and stromal thickness were measured using in vivo confocal microscopy through focusing. Images of the superficial epithelium were also acquired. Both eyes of each rabbit then received one drop of the assigned solution six times the first day and then four times per day for 6 days. On day 7, in vivo confocal microscopy was repeated. Results: A significant decrease in epithelial thickness was induced by 7 days of exposure to Levo, Gati, Oflox, and Cipro (P < 0.05, two-way repeated-measures ANOVA, Tukey test). Tears and Moxi, which do not contain BAC, did not induce significant changes in epithelial thickness. No significant changes in stromal thickness were detected (P = 0.266), and no keratocyte activation was observed for any of the solutions evaluated. Conclusion: We have previously used confocal microscopy to establish a correlation between epithelial thinning (due to superficial cell loss) and slight ocular irritation. The results of this study suggest that Moxi induces less damage to the corneal epithelium than other antibiotic solutions, perhaps because it does not contain BAC.

Identification and Functional Characterization of TMEM16A, a Ca2+-activated Cl− Channel Activated by Extracellular Nucleotides, in Biliary Epithelium

Cl− channels in the apical membrane of biliary epithelial cells (BECs) provide the driving force for ductular bile formation. Although a cystic fibrosis transmembrane conductance regulator has been identified in BECs and contributes to secretion via secretin binding basolateral receptors and increasing [cAMP]i, an alternate Cl− secretory pathway has been identified that is activated via nucleotides (ATP, UTP) binding apical P2 receptors and increasing [Ca2+]i. The molecular identity of this Ca2+-activated Cl− channel is unknown. The present studies in human, mouse, and rat BECs provide evidence that TMEM16A is the operative channel and contributes to Ca2+-activated Cl− secretion in response to extracellular nucleotides. Furthermore, Cl− currents measured from BECs isolated from distinct areas of intrahepatic bile ducts revealed important functional differences. Large BECs, but not small BECs, exhibit cAMP-stimulated Cl− currents. However, both large and small BECs express TMEM16A and exhibit Ca2+-activated Cl− efflux in response to extracellular nucleotides. Incubation of polarized BEC monolayers with IL-4 increased TMEM16A protein expression, membrane localization, and transepithelial secretion (Isc). These studies represent the first molecular identification of an alternate, noncystic fibrosis transmembrane conductance regulator, Cl− channel in BECs and suggest that TMEM16A may be a potential target to modulate bile formation in the treatment of cholestatic liver disorders.

Initiation of purinergic signaling by exocytosis of ATP-containing vesicles in liver epithelium

Extracellular ATP represents an important autocrine/paracrine signaling molecule within the liver. The mechanisms responsible for ATP release are unknown, and alternative pathways have been proposed, including either conductive ATP movement through channels or exocytosis of ATP-enriched vesicles, although direct evidence from liver cells has been lacking. Utilizing dynamic imaging modalities (confocal and total internal reflection fluorescence microscopy and luminescence detection utilizing a high sensitivity CCD camera) at different scales, including confluent cell populations, single cells, and the intracellular submembrane space, we have demonstrated in a model liver cell line that (i) ATP release is not uniform but reflects point source release by a defined subset of cells; (ii) ATP within cells is localized to discrete zones of high intensity that are ∼1 μm in diameter, suggesting a vesicular localization; (iii) these vesicles originate from a bafilomycin A1-sensitive pool, are depleted by hypotonic exposure, and are not rapidly replenished from recycling of endocytic vesicles; and (iv) exocytosis of vesicles in response to cell volume changes depends upon a complex series of signaling events that requires intact microtubules as well as phosphoinositide 3-kinase and protein kinase C. Collectively, these findings are most consistent with an essential role for exocytosis in regulated release of ATP and initiation of purinergic signaling in liver cells.

Regulation of Purinergic Signaling in Biliary Epithelial Cells by Exocytosis of SLC17A9-dependent ATP-enriched Vesicles

ATP in bile is a potent secretogogue, stimulating biliary epithelial cell (BEC) secretion through binding apical purinergic receptors. In response to mechanosensitive stimuli, BECs release ATP into bile, although the cellular basis of ATP release is unknown. The aims of this study in human and mouse BECs were to determine whether ATP release occurs via exocytosis of ATP-enriched vesicles and to elucidate the potential role of the vesicular nucleotide transporter SLC17A9 in purinergic signaling. Dynamic, multiscale, live cell imaging (confocal and total internal reflection fluorescence microscopy and a luminescence detection system with a high sensitivity charge-coupled device camera) was utilized to detect vesicular ATP release from cell populations, single cells, and the submembrane space of a single cell. In response to increases in cell volume, BECs release ATP, which was dependent on intact microtubules and vesicular trafficking pathways. ATP release occurred as stochastic point source bursts of luminescence consistent with exocytic events. Parallel studies identified ATP-enriched vesicles ranging in size from 0.4 to 1 μm that underwent fusion and release in response to increases in cell volume in a protein kinase C-dependent manner. Present in all models, SLC17A9 contributed to ATP vesicle formation and regulated ATP release. The findings are consistent with the existence of an SLC17A9-dependent ATP-enriched vesicular pool in biliary epithelium that undergoes regulated exocytosis to initiate purinergic signaling.

WNK1 is required for mitosis and abscission

WNK [with no lysine (K)] protein kinases are found in all sequenced multicellular and many unicellular organisms. WNKs influence ion balance. Two WNK family members are associated with a single gene form of hypertension. RNA interference screens have implicated WNKs in survival and growth, and WNK1 is essential for viability of mice. We found that the majority of WNK1 is localized on cytoplasmic puncta in resting cells. During cell division, WNK1 localizes to mitotic spindles. Therefore, we analyzed mitotic phenotypes in WNK1 knockdown cells. A large percentage of WNK1 knockdown cells fail to complete cell division, displaying defects in mitotic spindles and also in abscission and cell survival. One of the best-characterized WNK1 targets is the protein kinase OSR1 (oxidative stress responsive 1). OSR1 regulates ion cotransporters, is activated in response to osmotic stress by WNK family members, and is largely associated with WNK1. In resting cells, the majority of OSR1, like WNK1, is on cytoplasmic puncta. OSR1 is also in nuclei. In contrast to WNK1, however, OSR1 does not concentrate around spindles during mitosis and does not show a WNK1-like localization pattern in mitotic cells. Knockdown of OSR1 has only a modest effect on cell survival and does not lead to spindle defects. We conclude that decreased cell survival associated with loss of WNK1 is attributable to defects in chromosome segregation and abscission and is independent of the effector kinase OSR1.

Regulation of mechanosensitive biliary epithelial transport by the epithelial Na(+) channel.

Intrahepatic biliary epithelial cells (BECs), also known as cholangiocytes, modulate the volume and composition of bile through the regulation of secretion and absorption. While mechanosensitive Cl– efflux has been identified as an important secretory pathway, the counterabsorptive pathways have not been identified. In other epithelial cells, the epithelial Na+ channel (ENaC) has been identified as an important contributor to fluid absorption; however, its expression and function in BECs have not been previously studied. Our studies revealed the presence of α, β, and γ ENaC subunits in human BECs and α and γ subunits in mouse BECs. In studies of confluent mouse BEC monolayers, the ENaC contributes to the volume of surface fluid at the apical membrane during constitutive conditions. Further, functional studies using whole‐cell patch clamp of single BECs demonstrated small constitutive Na+ currents, which increased significantly in response to fluid‐flow or shear. The magnitude of Na+ currents was proportional to the shear force, displayed inward rectification and a reversal potential of +40 mV (ENa+ = +60 mV), and were abolished with removal of extracellular Na+ (N‐methyl‐d‐glucamine) or in the presence of amiloride. Transfection with ENaCα small interfering RNA significantly inhibited flow‐stimulated Na+ currents, while overexpression of the α subunit significantly increased currents. ENaC‐mediated currents were positively regulated by proteases and negatively regulated by extracellular adenosine triphosphate. Conclusion: These studies represent the initial characterization of mechanosensitive Na+ currents activated by flow in biliary epithelium; understanding the role of mechanosensitive transport pathways may provide strategies to modulate the volume and composition of bile during cholestatic conditions. (Hepatology 2016;63:538–549)

Bile acids stimulate cholangiocyte fluid secretion by activation of transmembrane member 16A Cl- channels

Bile acids stimulate a bicarbonate‐rich choleresis, in part, through effects on cholangiocytes. Because Cl− channels in the apical membrane of cholangiocytes provide the driving force for secretion and transmembrane member 16A (TMEM16A) has been identified as the Ca2+‐activated Cl− channel in the apical membrane of cholangiocytes, the aim of the present study was to determine whether TMEM16A is the target of bile‐acid–stimulated Cl− secretion and to identify the regulatory pathway involved. In these studies of mouse, rat, and human biliary epithelium exposure to ursodeoxycholic acid (UDCA) or tauroursodeoxycholic acid (TUDCA) rapidly increased the rate of exocytosis, ATP release, [Ca2+]i, membrane Cl− permeability, and transepithelial secretion. Bile‐acid–stimulated Cl− currents demonstrated biophysical properties consistent with TMEM16A and were inhibited by pharmacological or molecular (small‐interfering RNA; siRNA) inhibition of TMEM16A. Bile acid–stimulated Cl− currents were not observed in the presence of apyrase, suramin, or 2‐aminoethoxydiphenyl borate (2‐APB), demonstrating that current activation requires extracellular ATP, P2Y, and inositol 1,4,5‐trisphosphate (IP3) receptors. TUDCA did not activate Cl− currents during pharmacologic inhibition of the apical Na+‐dependent bile acid transporter (ASBT), but direct intracellular delivery of TUDCA rapidly activated Cl− currents. Conclusion: Bile acids stimulate Cl− secretion in mouse and human biliary cells through activation of membrane TMEM16A channels in a process regulated by extracellular ATP and [Ca2+]i. These studies suggest that TMEM16A channels may be targets to increase bile flow during cholestasis. (Hepatology 2018;68:187‐199).

Tissue clearing of both hard and soft tissue organs with the PEGASOS method

Tissue clearing technique enables visualization of opaque organs and tissues in 3-dimensions (3-D) by turning tissue transparent. Current tissue clearing methods are restricted by limited types of tissues that can be cleared with each individual protocol, which inevitably led to the presence of blind-spots within whole body or body parts imaging. Hard tissues including bones and teeth are still the most difficult organs to be cleared. In addition, loss of endogenous fluorescence remains a major concern for solvent-based clearing methods. Here, we developed a polyethylene glycol (PEG)-associated solvent system (PEGASOS), which rendered nearly all types of tissues transparent and preserved endogenous fluorescence. Bones and teeth could be turned nearly invisible after clearing. The PEGASOS method turned the whole adult mouse body transparent and we were able to image an adult mouse head composed of bones, teeth, brain, muscles, and other tissues with no blind areas. Hard tissue transparency enabled us to reconstruct intact mandible, teeth, femur, or knee joint in 3-D. In addition, we managed to image intact mouse brain at sub-cellular resolution and to trace individual neurons and axons over a long distance. We also visualized dorsal root ganglions directly through vertebrae. Finally, we revealed the distribution pattern of neural network in 3-D within the marrow space of long bone. These results suggest that the PEGASOS method is a useful tool for general biomedical research.