👤 Masaaki Kume

Aniridia, driven by PAX6 mutations, causes aniridia-associated keratopathy (AAK), a progressive condition linked to limbal stem cell deficiency. A major hurdle to developing targeted therapies for AAK is the incomplete understanding of the molecular abnormalities in affected corneas. To address this, we leveraged Pax6± (Pax6 het) mice, a model of AAK, and applied single-cell RNA sequencing (scRNA-seq) to profile the transcriptomic changes at a single-cell resolution. ScRNA-seq of corneal/limbal tissues of wild type (WT) and Pax6 het mice were conducted. Immunostaining was performed to examine the expression of specific markers for stem cells. ScRNA-seq identified a quiescent limbal epithelial stem cell (LESC)-like cell cluster and an early transient amplifying cell (eTAC)-like cluster. An increase in the cell numbers in these two clusters in the Pax6 het mouse corneas was observed. Immunostaining detected a marked increase in markers for these two clusters including Tmem176b, Apoe, and Krt15 in the corneal epithelium of Pax6 het mice, suggesting an increase of these LESC/eTA-like cells into the corneal epithelium. The Pax6 deficiency inhibited the expression of genes involved in cell proliferation in the eTAC-like cluster as well as the expression of genes related to corneal epithelial cell fate and differentiation compared with WT mice. Our single cell transcriptome of the limbus and cornea of Pax6 het mice indicates that AAK may be due to the increase of dysfunctional stem/eTACs with defects in committing to a corneal epithelial cell fate and differentiation. Show less

Hog1 of Saccharomyces cerevisiae is activated by hyperosmotic stress, and this leads to cell-cycle delay in G1, but the mechanism by which cells restart from G1 delay remains elusive. We found that Whi3, a negative regulator of G1 cyclin, counteracted Hog1 in the restart from G1 delay caused by osmotic stress. We have found that phosphorylation of Ser-568 in Whi3 by RAS/cAMP-dependent protein kinase (PKA) plays an inhibitory role in Whi3 function. In this study we found that the phosphomimetic Whi3 S568D mutant, like the Δwhi3 strain, slightly suppressed G1 delay of Δhog1 cells under osmotic stress conditions, whereas the non-phosphorylatable S568A mutation of Whi3 caused prolonged G1 arrest of Δhog1 cells. These results indicate that Hog1 activity is required for restart from G1 arrest under osmotic stress conditions, whereas Whi3 acts as a negative regulator for this restart mechanism. Show less

The Start/G1 phase in the cell cycle is an important period during which cells determine their developmental fate, onset of mitotic progression, or the switch to developmental stages in response to both external and internal signals. In the budding yeast Saccharomyces cerevisiae, Whi3, a negative regulator of the G1 cyclins, has been identified as a positive regulator of cell size control and is involved in the regulation of Start. However, the regulatory pathway of Whi3 governing the response to multiple signals remains largely unknown. Here, we show that Whi3 is phosphorylated by the Ras/cAMP-dependent protein kinase (PKA) and that phosphorylation of Ser-568 in Whi3 by PKA plays an inhibitory role in Whi3 function. Phosphorylation of Whi3 by PKA led to its decreased interaction with CLN3 G1 cyclin mRNA and was required for the promotion of G1/S progression. Furthermore, we demonstrate that the phosphomimetic S568D mutation of Whi3 prevented the developmental fate switch to sporulation or invasive growth. Thus, PKA modulated the function of Whi3 by phosphorylation, thus implicating PKA-mediated modulation of Whi3 in multiple cellular events. Show less

Acquired heart diseases, such as valve disease, are major causes of human morbidity and mortality. However, the pathological mechanisms underlying these diseases are largely unknown. Our aim is to identify the role of the hairy and enhancer of split-related (Hesr)-2 gene in the adult heart. Echocardiography detected heart dysfunctions indicative of aortic valve anomalies, stenosis, and regurgitation, in ≈59% of >12-month-old Hesr2 knockout survivor mice. Morphological and histological analyses revealed thickened semilunar valves with increased fibrotic areas, indicating that sclerotic degeneration of valves is the main cause of aortic valve disease. The expression of osteogenic genes, such as osteopontin and sclerostin, were upregulated in the mutants, and the overexpression of sclerostin in endothelial cells resulted in thickened semilunar valves with increased fibrotic areas, similar to that seen in the Hesr2 knockout mice, suggesting that Hesr2 can regulate osteogenic gene expression in valves. Reduced left ventricular function, which may be caused by increased ventricular interstitial fibrosis, and enlarged myocardial cell size without ventricular wall thickening were found in both aortic valve stenosis/regurgitation-positive (33%) and aortic valve stenosis/regurgitation-negative (38%) subpopulations in 12-month-old survivor mice. Dilated left ventricular internal dimensions were specifically detected in the aortic valve stenosis/regurgitation-positive subpopulation, thus suggesting that the degeneration of cardiomyocytes is influenced by irregular hemodynamics. These data revealed that survivor mice lacking the Hesr2 gene exhibit fibrosis in the aortic valve and ventricle in adulthood, thus suggesting that Hesr2 plays an important role in maintaining the homeostasis of the aortic valve and ventricle. Show less

Recent studies have shown that in the developing embryo, arterial and venous identity is established by genetic mechanisms before circulation begins. Vascular endothelial growth factor (VEGF) signaling and its downstream Notch pathway play critical roles in arterial cell fate determination. We have recently shown that Foxc1 and Foxc2, two closely related Fox transcription factors, are essential for arterial cell specification during development by directly inducing the transcription of Delta-like 4 (Dll4), a ligand for Notch receptors. However, the basic mechanisms whereby the VEGF and Notch signaling pathways control transcriptional regulation of arterial-specific genes have yet to be elucidated. In the current study, we examined whether and how Foxc transcription factors are involved in VEGF and Notch signaling in induction of Dll4 as well as the Notch target gene Hey2 in endothelial cells. We found that Foxc1 and Foxc2 directly activate the Hey2 promoter via Foxc binding elements. Significantly, Foxc2 physically and functionally interacts with a Notch transcriptional activation complex containing Su(H) and Notch intracellular domain to induce Hey2 promoter activity. Moreover, activation of the Dll4 and Hey2 promoters is induced by VEGF in conjunction with either Foxc1 or Foxc2 more than by either component alone. VEGF-activated PI3K and ERK intracellular pathways modulate the transcriptional activity of Foxc proteins in Dll4 and Hey2 induction. Our new findings demonstrate that Foxc transcriptional factors interact with VEGF and Notch signaling to regulate arterial gene expression in multiple steps of the VEGF-Dll4-Notch-Hey2 signaling pathway. Show less

The mRNA expression of GPRC5B, an orphan G protein-coupled receptor, is induced by retinoic acid (RA). Because RA plays critical roles in embryonic development, reproductive functions, metabolism and homeostasis, GPRC5B is also considered crucial in these physiological events. We investigated the changes in expression of GPRC5B and RA receptor (RAR) alpha mRNAs and immunohistochemical localization of their proteins in the murine placenta and yolk sac at 13.5, 15.5 and 17.5 days post coitus. Stable levels of GPRC5B and RARalpha mRNAs were detected in the placenta and yolk sac. In the placenta, GPRC5B was present in maternal and fetal vascular endothelial cells, stromal cells, fibroblast-like cells and glycogen cells. A strong reaction to RARalpha was detected in maternal and fetal vascular endothelial cells and stromal cells. The levels of GPRC5B and RARalpha proteins in maternal and fetal vascular endothelial cells decreased with gestation. In the yolk sac, GPRC5B and RARalpha proteins were detected in vascular endothelial cells, but their levels did not change during the gestation period. These findings indicate that GPRC5B is involved in RA-dependent morphogenesis/angiogenesis and regulation of extracellular matrix synthesis in the murine placenta and yolk sac. Show less

We examined the expression profiles of doxorubicin-resistant K562 cells by serial analysis of gene expression (SAGE) to identify novel and/or partially characterized genes that might be related to drug resistance in human leukemia. SAGE complementary DNA (cDNA) libraries were constructed from K562 and doxorubicin-resistant K562 (K562/ADM) cells, and concatamer sequences were analyzed with SAGE 2000 software. We used 9792 tags in the identification of 1076 different transcripts, 296 of which were similarly expressed in K562 and K562/ADM cells. There were 343 genes more actively expressed in K562/ADM than in parental K562 cells and 437 genes expressed less often in K562/ADM cells. K562/ADM cells showed increased expression of well-known genes, including the genes for spectrin beta, eukaryotic translation initiation factor 1A (EIF1A), RAD23 homolog B, laminin receptor 1, and polyA-, RAN-, and PAI-1 messenger RNA-binding proteins. K562/ADM cells showed decreased expression of the genes for fatty acid desaturase 1 (FADS1), hemoglobin epsilon 1, N-myristoyltransferase 1, hemoglobin alpha 2, NADH dehydrogenase Fe-S protein 6, heat shock 90-kDa protein, and karyopherin beta 1. Quantitative reverse transcription-polymerase chain reaction analysis confirmed the increased expression of EIF1A and the decreased expression of FADS1 in K562/ADM cells. Prior to this investigation, such differences in the expression of these genes in doxorubicin-resistant leukemia cells were unknown. Although we do not provide any evidence in the present report for the potential roles of these genes in drug resistance, SAGE may provide a perspective into our understanding of drug resistance in human leukemia that is different from that provided by cDNA microarray analysis. Show less