Expression Patterns of Grainyhead-Like 2 and Ovo-Like 2 in Mouse Mammary Gland Development During Pregnancy, Lactation, and Weaning.

Grainyhead-like 2 (Grhl2) is a transcription factor that regulates cell adhesion genes in mammary ductal development and serves as a repressor of the epithelial-mesenchymal transition. Conversely, Ovo-like2 (Ovol2) is a target gene of Grhl2 but functions as a substitute in Grhl2-deficient mice, facilitating successful epithelial barrier formation and lumen expansion in kidney-collecting ductal epithelial cells. Our objective was to examine the expression patterns of Grhl2, Ovol2, and their associated genes during the intricate phases of mouse mammary gland development. The mRNA expression of Grhl2 and Ovol2 increased after pregnancy. We observed Grhl2 protein presence in the epithelial cell's region, coinciding with acini formation, and its signal significantly correlated with E-cadherin (Cdh1) expression. However, Ovol2 was present in the epithelial region without a correlation with Cdh1. Similarly, Zeb1, a mesenchymal transcription factor, showed Cdh1-independent expression. Subsequently, we explored the interaction between Rab25, a small G protein, and Grhl2/Ovol2. The expressions of Grhl2 and Ovol2 exhibited a strong correlation with Rab25 and claudin-4, a tight junction protein. These findings suggest that Grhl2 and Ovol2 may collaborate to regulate genes associated with cell adhesion and are crucial for maintaining epithelial integrity during the different phases of mammary gland development.

Adipocytes and Stromal Cells Regulate Brown Adipogenesis Through Secretory Factors During the Postnatal White-to-Brown Conversion of Adipose Tissue in Syrian Hamsters.

Brown adipose tissue (BAT) is a specialized tissue that regulates non-shivering thermogenesis. In Syrian hamsters, interscapular adipose tissue is composed primarily of white adipocytes at birth, which is converted to BAT through the proliferation and differentiation of brown adipocyte progenitors and the simultaneous disappearance of white adipocytes. In this study, we investigated the regulatory mechanism of brown adipogenesis during postnatal BAT formation in hamsters. Interscapular adipose tissue of a 10-day-old hamster, which primarily consists of brown adipocyte progenitors and white adipocytes, was digested with collagenase and fractioned into stromal-vascular (SV) cells and white adipocytes. SV cells spontaneously differentiated into brown adipocytes that contained multilocular lipid droplets and expressed uncoupling protein 1 (Ucp1), a marker of brown adipocytes, without treatment of adipogenic cocktail such as dexamethasone and insulin. The spontaneous differentiation of SV cells was suppressed by co-culture with adipocytes or by the addition of white adipocyte-conditioned medium. Conversely, the addition of SV cell-conditioned medium increased the expression of Ucp1. These results indicate that adipocytes secrete factors that suppress brown adipogenesis, whereas SV cells secrete factors that promote brown adipogenesis. Transcriptome analysis was conducted; however, no candidate suppressing factors secreted from adipocytes were identified. In contrast, 19 genes that encode secretory factors, including bone morphogenetic protein (BMP) family members, BMP3B, BMP5, and BMP7, were highly expressed in SV cells compared with adipocytes. Furthermore, the SMAD and MAPK signaling pathways, which represent the major BMP signaling pathways, were activated in SV cells, suggesting that BMPs secreted from SV cells induce brown adipogenesis in an autocrine manner through the SMAD/MAPK signaling pathways. Treatment of 5-day-old hamsters with type I BMP receptor inhibitor, LDN-193189, for 5 days reduced p38 MAPK phosphorylation and drastically suppressed BAT formation of interscapular adipose tissue. In conclusion, adipocytes and stromal cells regulate brown adipogenesis through secretory factors during the postnatal white-to-brown conversion of adipose tissue in Syrian hamsters.

Expression of Grainyhead-like 2 in the Process of Ductal Development of Mouse Mammary Gland.

Grainyhead-like 2 (Grhl2) is a transcription factor regulating cell adhesion genes. Grhl2 acts as an epithelial-mesenchymal transition suppressor, and it is a proto-oncogene involved in estrogen-stimulated breast cancer proliferation. However, its expression during ovarian hormone-dependent mammary ductal development remains obscure. We here examined Grhl2 expression in the mammary gland of normal and steroid-replaced ovariectomized mice. Grhl2 protein signals were detected in both the mammary luminal epithelial and myoepithelial nuclei. The ratio and density of Grhl2-positive nuclei increased after the onset of puberty and progressed with age, whereas Grhl2-negative epithelial cells were detected in mature ducts. Claudin 3, claudin 4, claudin 7, and E-cadherin gene expression in the mammary gland was upregulated, and their expression was highly correlated with Grhl2 gene expression. Furthermore, Grhl2 mRNA expression and ductal lumen width were significantly increased by the combined treatment of estrogen and progesterone compared with estrogen alone. These results suggest that Grhl2 expressed in the luminal epithelial and myoepithelial cells from the early phase of ductal development, controlling the expression of cell adhesion molecules to establish functional ducts.

RNAi-based screens uncover a potential new role for the orphan neuropeptide receptor Moody in Drosophila female germline stem cell maintenance.

Reproduction is highly sensitive to changes in physiology and the external environment. Neuropeptides are evolutionarily conserved signaling molecules that regulate multiple physiological processes. However, the potential reproductive roles of many neuropeptide signaling pathways remain underexplored. Here, we describe the results of RNAi-based screens in Drosophila melanogaster to identify neuropeptides/neuropeptide receptors with potential roles in oogenesis. The screen read-outs were either the number of eggs laid per female per day over time or fluorescence microscopy analysis of dissected ovaries. We found that the orphan neuropeptide receptor encoded by moody (homologous to mammalian melatonin receptors) is likely required in somatic cells for normal egg production and proper germline stem cell maintenance. However, the egg laying screens had low signal-to-noise ratio and did not lead to the identification of additional candidates. Thus, although egg count assays might be useful for large-scale screens to identify oogenesis regulators that result in dramatic changes in oogenesis, more labor-intensive microscopy-based screen are better applicable for identifying new physiological regulators of oogenesis with more subtle phenotypes.

Role of brown adipose tissue in body temperature control during the early postnatal period in Syrian hamsters and mice.

Brown adipose tissue (BAT) contributes to non-shivering thermogenesis and plays an important role in body temperature control. The contribution of BAT thermogenesis to body temperature control in a non-cold environment was evaluated using developing hamsters. Immunostaining for uncoupling protein 1 (UCP1), a mitochondrial protein responsible for BAT thermogenesis, indicated that interscapular fat tissue had matured as BAT at day 14. When pups were placed on a thermal plate kept at 23°C, the body surface temperature decreased in day 7- and 10-day-old pups but was maintained at least for 15 min in 14-day-old pups, indicating that hamsters are unable to maintain their body temperature until around day 14 even in a non-cold environment. Body temperature maintenance was also evaluated in UCP1-deficient mice. BAT analysis showed that the UCP1 protein level in Ucp1+/- Hetero mice was 61.3 ± 1.4% of that in wild-type (WT) mice and was undetected in Ucp1-/- knockout (KO) mice. When 12-day-old pups were place on a thermal plate at 23°C, body surface temperature was maintained for at least 15 min in WT and Hetero mice but gradually dropped by 2.4 ± 0.2°C in 15 min in KO mice. It is concluded that BAT thermogenesis is indispensable for body temperature maintenance in pups of hamsters and mice, even in the non-cold circumstances. The early life poikilothermy and the later acquirement of homeothermy in hamsters may be because of the postnatal development of BAT.

Impaired adrenergic agonist-dependent beige adipocyte induction in obese mice.

Brown adipocytes, which exist in brown adipose tissue (BAT), are activated by adrenergic stimulation, depending on the activity of uncoupling protein 1 (UCP1). Beige adipocytes emerge from white adipose tissue (WAT) in response to chronic adrenergic stimulation. We investigated obesity-related changes in responses of both types of adipocytes to adrenergic stimulation in mice. Feeding of mice with high-fat diets (HFD: 45%-kcal fat) for 14 weeks resulted in significantly higher body and WAT weight compared to feeding with normal diets (ND: 10%-kcal fat). Injection with ß3-adrenergic receptor agonist CL316,243 (CL; 0.1 mg/kg, once a day) for one week elevated the mRNA and protein expression levels of UCP1 in BAT, irrespective of diet. In WAT, CL-induced UCP1 expression in ND mice; however, the responses to CL treatment were attenuated in HFD mice, indicating that CL-induced browning of WAT was impaired in obese mice. Flow cytometric analysis revealed a significant decrease in platelet-derived growth factor receptor (PDGFR) α-expressing beige adipocyte progenitors in WAT of HFD mice compared with those of ND mice. Expression of PDGF-B, a PDGFRα ligand, increased in WAT following CL-injection in ND mice, but not in HFD mice. Treatment of mice with a PDGFR inhibitor significantly decreased CL-dependent UCP1 protein induction in WAT. Our study demonstrates that ß3-adrenergic stimulation-dependent beige adipocyte induction in WAT is impaired by obesity in mice, potentially due to obesity-dependent reduction in the number of PDGFRα-expressing progenitors and decreased PDGF-B expression.

Role of macrophages in depot-dependent browning of white adipose tissue.

Sympathetic stimulation induces beige adipocytes in white adipose tissue (WAT), known as browning of WAT. In this study, exposure of mice to cold ambient temperature (10 °C) for 24 h induced the mRNA expression of uncoupling protein 1 (UCP1), a marker for beige adipocytes, in inguinal WAT, but not in perigonadal WAT. Thus, we examined the role of macrophages in depot-dependent WAT browning in mice. Flowcytometric analysis showed that total number of macrophages was higher in perigonadal WAT than in inguinal WAT. Cold exposure failed to change the expression of macrophage marker genes in inguinal WAT; however, it increased the mRNA expression of CD11c and tumor necrosis factor-α in perigonadal WAT, indicating that proinflammatory M1 macrophage is activated. The removal of macrophages using clodronate significantly enhanced cold-induced UCP1 mRNA expression in perigonadal WAT. These results suggest that M1 macrophages are involved in the phenotype of perigonadal WAT that hardly undergo browning.

Retinoic acid modulates lipid accumulation glucose concentration dependently through inverse regulation of SREBP-1 expression in 3T3L1 adipocytes.

It is well known that retinoic acid (RA) suppresses adipogenesis, although there are some contradicting reports. In this study, we examined the effect of extracellular glucose on RA-induced suppression of adipogenesis in 3T3L1 cell culture. When the cells were cultured in normal glucose medium (NG), the addition of RA suppressed lipid accumulation. However, when cultured in high glucose medium (HG), addition of RA to the cells enhanced lipid accumulation. These changes were accompanied by parallel alterations in fatty acid synthase (FAS) and sterol regulatory element-binding protein (SREBP)-1 gene expression. Transfection of SREBP-1 siRNA suppressed RA-induced enhancement of lipid accumulation and FAS expression in the cells cultured with HG. Transfection of the nuclear form of SREBP-1a cDNA into the cells cultured with NG inhibited RA-induced suppression of lipid accumulation and FAS expression. Moreover, RA- and HG-induced SREBP-1a expression occurred at the early phase of adipogenesis and was dependent on glucocorticoid to induce liver X receptor (LXR) ß, peroxisomal proliferator-activated receptor (PPAR) γ and retinoid X receptor (RXR), the key nuclear factors influencing the SREBP-1a gene expression. These results suggest that RA suppresses and enhances lipid accumulation through extracellular glucose concentration-dependent modulation of SREBP-1 expression.

Adipocyte Metabolic Pathways Regulated by Diet Control the Female Germline Stem Cell Lineage in Drosophila melanogaster.

Nutrients affect adult stem cells through complex mechanisms involving multiple organs. Adipocytes are highly sensitive to diet and have key metabolic roles, and obesity increases the risk for many cancers. How diet-regulated adipocyte metabolic pathways influence normal stem cell lineages, however, remains unclear. Drosophila melanogaster has highly conserved adipocyte metabolism and a well-characterized female germline stem cell (GSC) lineage response to diet. Here, we conducted an isobaric tags for relative and absolute quantification (iTRAQ) proteomic analysis to identify diet-regulated adipocyte metabolic pathways that control the female GSC lineage. On a rich (relative to poor) diet, adipocyte Hexokinase-C and metabolic enzymes involved in pyruvate/acetyl-CoA production are upregulated, promoting a shift of glucose metabolism toward macromolecule biosynthesis. Adipocyte-specific knockdown shows that these enzymes support early GSC progeny survival. Further, enzymes catalyzing fatty acid oxidation and phosphatidylethanolamine synthesis in adipocytes promote GSC maintenance, whereas lipid and iron transport from adipocytes controls vitellogenesis and GSC number, respectively. These results show a functional relationship between specific metabolic pathways in adipocytes and distinct processes in the GSC lineage, suggesting the adipocyte metabolism-stem cell link as an important area of investigation in other stem cell systems.

Analysis of pain level in cases treated with Invisalign aligner: comparison with fixed edgewise appliance therapy.

BACKGROUND: The aim of this study was to evaluate and compare the difference in the level of pain using the visual analog scale (VAS) between cases treated with the edgewise appliance and Invisalign. In addition, the cause of pain and discomfort in the Invisalign cases was identified. METHODS: The sample consisted of 145 cases for the edgewise group (EG; n=55), Invisalign group (IG; n=38), and edgewise and Invisalign group (EIG; n=52). VAS scores were collected during the first three stages (first stage: 0 to 7 days, second stage: 14 to 21 days, and third stage: 28 to 35 days) and at the end of the treatment (overall VAS score). Evaluation of the cause of pain was categorized into three different types of problem (category 1: non-smoothed marginal ridge or missing materials, category 2: deformation of attachments, and Category 3: deformation of the tray). Statistical comparison of VAS scores between groups was performed by two-way analysis of variance. RESULTS: A significantly higher VAS score was observed at 3 and 4 days after, at 1, 2, and 3 days after, and at 2 and 3 days after in stages 1, 2, and 3, respectively, in EG compared to EIG and IG. A significant difference was observed in overall VAS scores between EG and IG in intensity of pain, number of days that pain lasted, and discomfort level. Only intensity of pain resulted in a significant difference between EG and EIG. Most of the causes of problem in the Invisalign cases were deformation of the tray. CONCLUSIONS: Invisalign may offer less pain compared to the edgewise appliance during the initial stages of treatment. In the use of Invisalign, deformation of tray must be carefully checked to avoid pain and discomfort for the patients.

gone early, a novel germline factor, ensures the proper size of the stem cell precursor pool in the Drosophila ovary.

In order to sustain lifelong production of gametes, many animals have evolved a stem cell-based gametogenic program. In the Drosophila ovary, germline stem cells (GSCs) arise from a pool of primordial germ cells (PGCs) that remain undifferentiated even after gametogenesis has initiated. The decision of PGCs to differentiate or remain undifferentiated is regulated by somatic stromal cells: specifically, epidermal growth factor receptor (EGFR) signaling activated in the stromal cells determines the fraction of germ cells that remain undifferentiated by shaping a Decapentaplegic (Dpp) gradient that represses PGC differentiation. However, little is known about the contribution of germ cells to this process. Here we show that a novel germline factor, Gone early (Goe), limits the fraction of PGCs that initiate gametogenesis. goe encodes a non-peptidase homologue of the Neprilysin family metalloendopeptidases. At the onset of gametogenesis, Goe was localized on the germ cell membrane in the ovary, suggesting that it functions in a peptidase-independent manner in cell-cell communication at the cell surface. Overexpression of Goe in the germline decreased the number of PGCs that enter the gametogenic pathway, thereby increasing the proportion of undifferentiated PGCs. Inversely, depletion of Goe increased the number of PGCs initiating differentiation. Excess PGC differentiation in the goe mutant was augmented by halving the dose of argos, a somatically expressed inhibitor of EGFR signaling. This increase in PGC differentiation resulted in a massive decrease in the number of undifferentiated PGCs, and ultimately led to insufficient formation of GSCs. Thus, acting cooperatively with a somatic regulator of EGFR signaling, the germline factor goe plays a critical role in securing the proper size of the GSC precursor pool. Because goe can suppress EGFR signaling activity and is expressed in EGF-producing cells in various tissues, goe may function by attenuating EGFR signaling, and thereby affecting the stromal environment.

Egfr signaling controls the size of the stem cell precursor pool in the Drosophila ovary.

In many animals, germline progenitors are kept undifferentiated to give rise to germline stem cells (GSCs), enabling continuous production of gametes throughout animal life. In the Drosophila ovary, GSCs arise from a subset of primordial germ cells (PGCs) that stay undifferentiated even after gametogenesis has started. How a certain population of PGCs is protected against differentiation, and the significance of its regulatory mechanisms on GSC establishment remain elusive. Here we show that epidermal growth factor receptor (Egfr) signaling in somatic stromal intermingled cells (ICs), activated by its ligand produced in germ cells, controls the size of the PGC pool at the onset of gametogenesis. Egfr signaling in ICs limits the number of cells that express the heparan sulfate proteoglycan Dally, which is required for the movement and stability of the locally-produced stromal morphogen, Decapentaplegic (Dpp, a BMP2/4 homologue). Dpp is received by PGCs and maintains them in an undifferentiated state. Altering Egfr signaling levels changes the size of the PGC pool and affects the number of GSCs established during development. While excess GSC formation is compensated by the adult stage, insufficient GSC formation can lead to adult ovarioles that completely lack GSCs, suggesting that ensuring an absolute size of the PGC pool is crucial for the GSC system.

Negative modulation of bone morphogenetic protein signaling by Dullard during wing vein formation in Drosophila.

Studies in Xenopus have shown that the C-terminal domain phosphatase-like domain (CPD) phosphatase Dullard is essential for proper neural development via inhibition of bone morphogenetic protein (BMP) signaling receptors. In contrast, the orthologous budding yeast Nem1 and human Dullard have been shown to dephosphorylate the phosphatidate phosphatases yeast Smp2/Pah1 and human Lipin, and the relationship between phospholipid metabolism and BMP signaling remain unsolved. Here we report evidence that the Dullard-Lipin phosphatase cascade in Drosophila can regulate BMP signaling, most likely by affecting the function of the nuclear envelope. Manipulating expression levels of either the Drosophila Dullard gene, d-dullard (ddd) or the Lipin gene, DmLpin affected wing vein formation in a manner suggesting a negative effect on BMP signaling. Furthermore, both genes exhibit genetic interaction with BMP signaling pathway components, and can affect the levels of phosphorylated-Mothers against dpp (p-Mad). Although changing ddd expression levels did not have an obvious effect on overall nuclear envelope morphology as has been shown for yeast nem1, the nuclear import machinery components Importin-ß and RanGAP were mislocalized and membrane lipid staining was altered in cells overexpressing ddd. Considering the known genetic interaction between Nup84 complex nucleoporins and nem1 in yeast, and the recently reported requirement for components from the orthologous nucleoporin complex in the nuclear translocation of Drosophila Mad (Chen & Xu 2010), it is likely that the role of Drosophila Dullard in regulating membrane lipid homeostasis is conserved and is critical for normal BMP signaling.

Supercooled micro flows and application for asymmetric synthesis.

Supercooled micro flow in microchannels was demonstrated. In order to clarify fundamental properties of the supercooling state in microchannels, freezing temperature was measured in the microchannels having widths ranging from 70 microm to 300 microm. The freezing temperature decreased with decreasing width of the microchannel when the microchannel wall was chemically modified with octadecylsilane group. The lowest freezing temperature was observed as -28 degrees C for water in the 70 microm wide microchannel. By contrast, the freezing temperature was -15 degrees C and did not depend on width when a microchannel with a bare glass surface was used. Next, the freezing point was measured with flow rates ranging from 0.1 to 2.0 microl min(-1) and no dependence on flow rate was observed. Then, the supercooled micro flow was applied to an asymmetric reaction in micro two-phase flow of aqueous and CH2Cl2 phases. As expected from thermodynamic prediction, enantiomeric selectivity increased in the supercooled state of water.

Surface modification method of microchannels for gas-liquid two-phase flow in microchips.

A capillarity restricted modification method for microchannel surfaces was developed for gas--liquid microchemical operations in microchips. In this method, a microstructure combining shallow and deep microchannels and the principle of capillarity were utilized for chemical modification of a restricted area of a microchannel. A hydrophobic--hydrophilic patterning in microchannels was prepared as an example for guiding gas and liquid flows along the respective microchannels. Validity of the patterning was confirmed by measuring aqueous flow leak pressure from the hydrophilic microchannel to the hydrophobic one. The leak pressure of 7.7-1.1 kPa agreed well with that predicted theoretically from the Young-Laplace equation for the microchannel depth of 8.6-39 microm. In an experiment to demonstrate usefulness and effectiveness of the method, an air bubble was first introduced into the hydrophilic microchannel and purged from the hydrophobic-hydrophilic patterned microchannels. Next, the patterning structure was applied to remove dissolved oxygen by contacting the aqueous flow with a nitrogen flow. The concentration of dissolved oxygen decreased with contact time, and its time course agreed well with numerical simulation. These demonstrations showed that the proposed patterning method can be used in general microfluidic gas-liquid operations.