BioMed Research International: Developmental Biology The latest articles from Hindawi © 2017 , Hindawi Limited . All rights reserved. An Integrative Developmental Genomics and Systems Biology Approach to Identify an In Vivo Sox Trio-Mediated Gene Regulatory Network in Murine Embryos Sun, 28 May 2017 08:33:05 +0000 Embryogenesis is an intricate process involving multiple genes and pathways. Some of the key transcription factors controlling specific cell types are the Sox trio, namely, Sox5, Sox6, and Sox9, which play crucial roles in organogenesis working in a concerted manner. Much however still needs to be learned about their combinatorial roles during this process. A developmental genomics and systems biology approach offers to complement the reductionist methodology of current developmental biology and provide a more comprehensive and integrated view of the interrelationships of complex regulatory networks that occur during organogenesis. By combining cell type-specific transcriptome analysis and in vivo ChIP-Seq of the Sox trio using mouse embryos, we provide evidence for the direct control of Sox5 and Sox6 by the transcriptional trio in the murine model and by Morpholino knockdown in zebrafish and demonstrate the novel role of Tgfb2, Fbxl18, and Tle3 in formation of Sox5, Sox6, and Sox9 dependent tissues. Concurrently, a complete embryonic gene regulatory network has been generated, identifying a wide repertoire of genes involved and controlled by the Sox trio in the intricate process of normal embryogenesis. Wenqing Jean Lee, Sumantra Chatterjee, Sook Peng Yap, Siew Lan Lim, Xing Xing, Petra Kraus, Wenjie Sun, Xiaoming Hu, V. Sivakamasundari, Hsiao Yun Chan, Prasanna R. Kolatkar, Shyam Prabhakar, and Thomas Lufkin Copyright © 2017 Wenqing Jean Lee et al. All rights reserved. Txndc9 Is Required for Meiotic Maturation of Mouse Oocytes Wed, 24 May 2017 00:00:00 +0000 Txndc9 (thioredoxin domain containing protein 9) has been shown to be involved in mammalian mitosis; however, its function in mammalian oocyte meiosis remains unclear. In this study, we initially found that Txndc9 is expressed during meiotic maturation of mouse oocytes and higher expression of Txndc9 mRNA and protein occurred in germinal vesicle (GV) stage. By using confocal scanning, we observed that Txndc9 localized at both nucleus and cytoplasm, especially at spindle microtubules. Specific depletion of Txndc9 by siRNA in mouse oocyte resulted in decreasing the rate of first polar body extrusion and increasing abnormal spindle assemble. Moreover, knockdown of Txndc9 in germinal vesicle (GV) stage oocytes led to higher level of reactive oxygen species (ROS) and lower level of antioxidant glutathione (GSH) as compared with control oocytes, which indicated that Txndc9 may be involved in mediating the redox balance. In summary, our results demonstrated that Txndc9 is crucial for mouse oocyte maturation by regulating spindle assembly, polar body extrusion, and redox status. Fanhua Ma, Liming Hou, and Liguo Yang Copyright © 2017 Fanhua Ma et al. All rights reserved. Feeder Cell Type Affects the Growth of In Vitro Cultured Bovine Trophoblast Cells Wed, 24 May 2017 00:00:00 +0000 Trophectoderm cells are the foremost embryonic cells to differentiate with prospective stem-cell properties. In the current study, we aimed at improving the current approach for trophoblast culture by using granulosa cells as feeders. Porcine granulosa cells (PGCs) compared to the conventional mouse embryonic fibroblasts (MEFs) were used to grow trophectoderm cells from hatched bovine blastocysts. Isolated trophectoderm cells were monitored and displayed characteristic epithelial/cuboidal morphology. The isolated trophectoderm cells expressed mRNA of homeobox protein (CDX2), cytokeratin-8 (KRT8), and interferon tau (IFNT). The expression level was higher on PGCs compared to MEFs throughout the study. In addition, primary trophectoderm cell colonies grew faster on PGCs, with a doubling time of approximately 48 hrs, compared to MEFs. PGCs feeders produced a fair amount of 17β-estradiol and progesterone. We speculated that the supplementation of sex steroids and still-unknown factors during the trophoblasts coculture on PGCs have helped to have better trophectoderm cell’s growth than on MEFs. This is the first time to use PGCs as feeders to culture trophectoderm cells and it proved superior to MEFs. We propose PGCs as alternative feeders for long-term culture of bovine trophectoderm cells. This model will potentially benefit studies on the early trophoblast and embryonic development in bovines. Islam M. Saadeldin, Ahmed Abdelfattah-Hassan, and Ayman Abdel-Aziz Swelum Copyright © 2017 Islam M. Saadeldin et al. All rights reserved. Postneonatal Mortality and Liver Changes in Cloned Pigs Associated with Human Tumor Necrosis Factor Receptor I-Fc and Human Heme Oxygenase-1 Overexpression Wed, 19 Apr 2017 09:11:22 +0000 Soluble human tumor necrosis factor (shTNFRI-Fc) and human heme oxygenase 1 (hHO-1) are key regulators for protection against oxidative and inflammatory injury for xenotransplantation. Somatic cells with more than 10 copy numbers of shTNFRI-Fc and hHO-1 were employed in somatic cell nuclear transfer to generate cloned pigs, thereby resulting in seven cloned piglets. However, produced piglets were all dead within 24 hours after birth. Obviously, postnatal death with liver apoptosis was reported in the higher copy number of shTNFRI-Fc and hHO-1 piglets. In liver, the transcript levels of ferritin heavy chain, light chain, transferrin, and inducible nitric oxide synthase were significantly highly expressed compared to those of lower copy number of shTNFRI-Fc and hHO-1 piglets (). Also, H2O2 contents were increased, and superoxide dismutase was significantly lower in the higher copy number of shTNFRI-Fc and hHO-1 piglets (). These results indicate that TNFRI-Fc and hHO-1 overexpression may apparently induce free iron in the liver and exert oxidative stress by enhancing reactive oxygen species production and block normal postneonatal liver metabolism. Geon A. Kim, Jun-Xue Jin, Sanghoon Lee, Anukul Taweechaipaisankul, Hyun Ju Oh, Joing-Ik Hwang, Curie Ahn, Islam M. Saadeldin, and Byeong Chun Lee Copyright © 2017 Geon A. Kim et al. All rights reserved. Proteomic Analysis of Fetal Ovaries Reveals That Primordial Follicle Formation and Transition Are Differentially Regulated Tue, 07 Feb 2017 11:37:36 +0000 Primordial follicle formation represents a critical phase of the initiation of embryonic reproductive organ development, while the primordial follicle transition into primary follicle determines whether oestrus or ovulation will occur in female animals. To identify molecular mechanism of new proteins which are involved in ovarian development, we employed 2D-DIGE to compare the protein expression profiles of primordial follicles and primary follicles of fetal ovaries in pigs. Fetal ovaries were collected at distinct time-points of the gestation cycle (g55 and g90). The identified proteins at the g55 time-point are mainly involved in the development of anatomical structures [reticulocalbin-1 (RCN1), reticulocalbin-3 (RCN3)], cell differentiation (actin), and stress response [heterogeneous nuclear ribonucleoprotein K (HNRNPK)]. Meanwhile, at the g90 stage, the isolated proteins with altered expression levels were mainly associated with cell proliferation [major vault protein (MVP)] and stress response [heat shock-related 70 kDa protein 2 (HSPA2)]. In conclusion, our work revealed that primordial follicle formation is regulated by RCN1, RCN3, actin, and HNRNPK, while the primordial follicle transformation to primary follicle is regulated by MVP and HSPA2. Therefore, our results provide further information for the prospective understanding of the molecular mechanism(s) involved in the regulation of the ovarian follicle development. Mengmeng Xu, Long Che, Zhenguo Yang, Pan Zhang, Jiankai Shi, Jian Li, Yan Lin, Zhengfeng Fang, Lianqiang Che, Bin Feng, De Wu, and Shengyu Xu Copyright © 2017 Mengmeng Xu et al. All rights reserved. Morphokinetic Characteristics and Developmental Potential of In Vitro Cultured Embryos from Natural Cycles in Patients with Poor Ovarian Response Tue, 20 Dec 2016 11:21:32 +0000 Background. Patients with poor ovarian response to ovarian hyperstimulation represent an interesting group for studying the impact of embryo cleavage irregularities on clinical outcome since all embryos, regardless of their quality, are usually transferred to the uterus. The aim of our study was to follow the morphokinetics of fertilized oocytes from natural cycles in poor responders. Methods. Zygotes from 53 cycles were cultured in vitro for 3 days. The morphokinetics of their development and transfer outcomes were retrospectively analyzed for the normally and irregularly cleaved embryos. Results. Of all embryos, 30.2% had single and 20.8% multiple cleavage irregularities with the following prevalence: developmental arrest 30.2%, direct cleavage to more than two cells 24.5%, chaotic cleavage 13.2%, and reverse cleavage 11.3%. These embryos had longer pronuclear phases, first cytokinesis, second embryo cell cycles, and less synchronized divisions. The transfer of normally developing embryos resulted in an implantation rate of 30.8% and a delivery rate of 23.1%, but irregularly cleaved embryos did not implant. Conclusions. The use of time-lapse microscopy in poor responder patients identified embryos with cleavage abnormalities that are related with no or extremely low implantation potential. Gained information about embryo quality is important for counselling patients about their expectations. N. Hojnik, V. Vlaisavljević, and B. Kovačič Copyright © 2016 N. Hojnik et al. All rights reserved. The Effect of Advanced Motherhood on Newborn Offspring’s Hippocampal Neural Stem Cell Proliferation Mon, 05 Sep 2016 08:58:07 +0000 Objective. To investigate the effect of advanced motherhood on rat hippocampal neural stem cell proliferation. Methods. Female parents were subdivided into control and old mother group by age, and neural stem cells were cultured from hippocampal tissues for 24 h newborn offspring. The diameter and numbers of neurospheres were examined by microscopy, and differences in proliferation were examined by EdU immunofluorescence, CCK-8 assay, and cell cycle analysis. Results. The number of neurospheres in the old mother group after culture was lower than the control group. Additionally, neurospheres’ diameter was smaller than that of the control group (). The EdU positive rate of the old mother group was lower than that of the control group (). CCK-8 assay results showed that the absorbance values for the old mother group were lower than that of the control group at 48 h and 72 h (). The proportions of cells in the S and G2/M phases of the cell cycle for the older mother group were less than that found for the control group (). Conclusion. The proliferation rates of hippocampal NSCs seen in the older mother group were lower than that seen in the control group. Bo Li, Ping Duan, Xuefei Han, Wenhai Yan, and Ying Xing Copyright © 2016 Bo Li et al. All rights reserved. The Regulatory Roles of MicroRNAs in Bone Remodeling and Perspectives as Biomarkers in Osteoporosis Thu, 17 Mar 2016 16:29:54 +0000 MicroRNAs are involved in many cellular and molecular activities and played important roles in many biological and pathological processes, such as tissue formation, cancer development, diabetes, neurodegenerative diseases, and cardiovascular diseases. Recently, it has been reported that microRNAs can modulate the differentiation and activities of osteoblasts and osteoclasts, the key cells that are involved in bone remodeling process. Meanwhile, the results from our and other research groups showed that the expression profiles of microRNAs in the serum and bone tissues are significantly different in postmenopausal women with or without fractures compared to the control. Therefore, it can be postulated that microRNAs might play important roles in bone remodeling and that they are very likely to be involved in the pathological process of postmenopausal osteoporosis. In this review, we will present the updated research on the regulatory roles of microRNAs in osteoblasts and osteoclasts and the expression profiles of microRNAs in osteoporosis and osteoporotic fracture patients. The perspective of serum microRNAs as novel biomarkers in bone loss disorders such as osteoporosis has also been discussed. Mengge Sun, Xiaoya Zhou, Lili Chen, Shishu Huang, Victor Leung, Nan Wu, Haobo Pan, Wanxin Zhen, William Lu, and Songlin Peng Copyright © 2016 Mengge Sun et al. All rights reserved. Dhcr7 Regulates Palatal Shelf Fusion through Regulation of Shh and Bmp2 Expression Tue, 15 Mar 2016 12:36:22 +0000 The aim of this study was to investigate the effect of the 7-dehydrocholesterol reductase (Dhcr7) gene and identify signaling pathways involved in regulation of embryonic palatogenesis. The expression of Dhcr7 and its protein product were examined during murine normal embryonic palatogenesis via a reverse transcription polymerase chain reaction (RT-PCR) and Western blot (WB). RNA interference (RNAi) technology was used to inhibit Dhcr7 expression in a palatal shelf culture in vitro. The effects of Dhcr7 on palatogenesis and palatal fusion were examined by scanning electron microscopy (SEM). The expression changes of Dhcr7, Sonic Hedgehog (Shh), and bone morphogenetic protein-2 (Bmp2) were measured by RT-PCR and WB after Dhcr7 gene silencing and the addition of exogenous cholesterol. The results showed that the palatal shelf failed to complete normal development and fusion when Dhcr7 expression was inhibited. The inhibitory effect study of RNAi on the development of the palatal shelf supported that cholesterol supplementation did not alter the silencing of Dhcr7. Shh and Bmp2 expressions were reduced after Dhcr7 gene silencing, and administration of exogenous cholesterol did not affect Dhcr7 expression; however Shh and Bmp2 expressions increased. We conclude that Dhcr7 plays a role in growth of the palatal shelf and can regulate palatogenesis through alterations in the levels of Shh and Bmp2. Wen-lin Xiao, Dai-zun Zhang, Hong Xu, and Cui-zhu Zhuang Copyright © 2016 Wen-lin Xiao et al. All rights reserved. The Alternative Faces of Macrophage Generate Osteoclasts Mon, 08 Feb 2016 11:35:51 +0000 The understanding of how osteoclasts are generated and whether they can be altered by inflammatory stimuli is a topic of particular interest for osteoclastogenesis. It is known that the monocyte/macrophage lineage gives rise to osteoclasts (OCs) by the action of macrophage colony stimulating factor (M-CSF) and receptor activator of nuclear factor-kB ligand (RANKL), which induce cell differentiation through their receptors, c-fms and RANK, respectively. The multinucleated giant cells (MGCs) generated by the engagement of RANK/RANKL are typical OCs. Nevertheless, very few studies have addressed the question of which subset of macrophages generates OCs. Indeed, two main subsets of macrophages are postulated, the inflammatory or classically activated type (M1) and the anti-inflammatory or alternatively activated type (M2). It has been proposed that macrophages can be polarized in vitro towards a predominantly M1 or M2 phenotype with the addition of granulocyte macrophage- (GM-) CSF or M-CSF, respectively. Various inflammatory stimuli known to induce macrophage polarization, such as LPS or TNF-, can alter the type of MGC obtained from RANKL-induced differentiation. This review aims to highlight the role of immune-related stimuli and factors in inducing macrophages towards the osteoclastogenesis choice. N. Lampiasi, R. Russo, and F. Zito Copyright © 2016 N. Lampiasi et al. All rights reserved. CMP-Neu5Ac Hydroxylase Null Mice as a Model for Studying Metabolic Disorders Caused by the Evolutionary Loss of Neu5Gc in Humans Mon, 19 Oct 2015 08:01:53 +0000 The purpose of this study was to identify the modification/turnover of gene products that are altered in humans due to evolutionary loss of Neu5Gc. CMP-Neu5Ac hydroxylase- (Cmah-) deficient mice show the infiltration of Kupffer cells within liver sinusoids, whereas body and liver weight develop normally. Pathway analysis by use of Illumina MouseRef-8 v2 Expression BeadChip provided evidence that a number of biological pathways, including the glycolysis, gluconeogenesis, TCA cycle, and pentose phosphate pathways, as well as glycogen metabolism-related gene expression, were significantly upregulated in Cmah-null mice. The intracellular glucose supply in Cmah-null mice resulted in mitochondrial dysfunction, oxidative stress, and the advanced glycation end products accumulation that could further induce oxidative stress. Finally, low sirtuin-1 and sirtuin-3 gene expressions due to higher NADH/NAD in Cmah-null mice decreased Foxo-1 and MnSOD gene expression, suggesting that oxidative stress may result in mitochondrial dysfunction in Cmah-null mouse. The present study suggests that mice with CMAH deficiency can be taken as an important model for studying metabolic disorders in humans. Deug-Nam Kwon, Yun-Jung Choi, Ssang-Goo Cho, Chankyu Park, Han Geuk Seo, Hyuk Song, and Jin-Hoi Kim Copyright © 2015 Deug-Nam Kwon et al. All rights reserved. Age-Specific Gene Expression Profiles of Rhesus Monkey Ovaries Detected by Microarray Analysis Wed, 02 Sep 2015 13:31:58 +0000 The biological function of human ovaries declines with age. To identify the potential molecular changes in ovarian aging, we performed genome-wide gene expression analysis by microarray of ovaries from young, middle-aged, and old rhesus monkeys. Microarray data was validated by quantitative real-time PCR. Results showed that a total of 503 (60 upregulated, 443 downregulated) and 84 (downregulated) genes were differentially expressed in old ovaries compared to young and middle-aged groups, respectively. No difference in gene expression was found between middle-aged and young groups. Differentially expressed genes were mainly enriched in cell and organelle, cellular and physiological process, binding, and catalytic activity. These genes were primarily associated with KEGG pathways of cell cycle, DNA replication and repair, oocyte meiosis and maturation, MAPK, TGF-beta, and p53 signaling pathway. Genes upregulated were involved in aging, defense response, oxidation reduction, and negative regulation of cellular process; genes downregulated have functions in reproduction, cell cycle, DNA and RNA process, macromolecular complex assembly, and positive regulation of macromolecule metabolic process. These findings show that monkey ovary undergoes substantial change in global transcription with age. Gene expression profiles are useful in understanding the mechanisms underlying ovarian aging and age-associated infertility in primates. Hengxi Wei, Xiangjie Liu, Jihong Yuan, Li Li, Dongdong Zhang, Xinzheng Guo, Lin Liu, and Shouquan Zhang Copyright © 2015 Hengxi Wei et al. All rights reserved. Isolation, Culture, and Characterization of Chicken Cartilage Stem/Progenitor Cells Sun, 16 Aug 2015 06:43:41 +0000 A chondrocyte progenitor population isolated from the surface zone of articular cartilage has become a promising cell source for cell-based cartilage repair. The cartilage-derived stem/progenitor cells are multipotent stem cells, which can differentiate into three cell types in vitro including adipocytes, osteoblasts, and chondrocytes. Much work has been done on cartilage stem/progenitor cells (CSPCs) from people, horses, and cattle, but the relatively little literature has been published about these cells in chickens. In our work, CSPCs were isolated from chicken embryos in incubated eggs for 20 days. In order to inquire into the biological characteristics of chicken CSPCs, immunofluorescence, reverse transcription-polymerase chain reaction (RT-PCR), and flow cytometry were adopted to detect the characteristic surface markers of CSPCs. Primary CSPCs were subcultured to passage 22 and, for purpose of knowing the change of cell numbers, we drew the growth curves. Isolated CSPCs were induced to adipocytes, osteoblasts, and chondrocytes. Our results suggest that we have identified and characterised a novel cartilage progenitor population resident in chicken articular cartilage and CSPCs isolated from chickens possess similar biological characteristics to those from other species, which will greatly benefit future cell-based cartilage repair therapies. Lu Li, Yuehui Ma, Xianglong Li, Xiangchen Li, Chunyu Bai, Meng Ji, Shuang Zhang, Weijun Guan, and Junjie Li Copyright © 2015 Lu Li et al. All rights reserved. The dnd RNA Identifies Germ Cell Origin and Migration in Olive Flounder (Paralichthys olivaceus) Thu, 28 May 2015 13:27:45 +0000 The present study obtained a germ cell-specific marker dead end (dnd) in olive flounder (Paralichthys olivaceus) named Podnd. The tissue-specific expressions of Podnd transcripts were present in testis and ovary but were not detectable in other somatic tissues detected. SISH showed that Podnd expressed only in germ cells at different developmental stages but not in surrounding somatic cells. The expression of Podnd during embryonic development at 16 different stages revealed that the relative expression of Podnd transcript fluctuated at a high level in the cleavage stages, gradually decreased through subsequent development, and reached the lowest at late gastrula stage till it was nearly undetectable. The Podnd transcripts localization and migration were similar to zebrafish. Further research on the specification migration mechanism of PGCs and the role of germ cell during gonadal development in olive flounder would improve our understanding of germline development. Xueying Wang, Qinghua Liu, Yongshuang Xiao, Yang Yang, Yanfeng Wang, Zongcheng Song, Feng You, Hao An, Zhizhong Xiao, Shihong Xu, Daoyuan Ma, and Jun Li Copyright © 2015 Xueying Wang et al. All rights reserved. Transcriptional Activity of Human Endogenous Retroviruses in Human Peripheral Blood Mononuclear Cells Thu, 05 Feb 2015 09:37:45 +0000 Human endogenous retroviruses (HERVs) have been implicated in human physiology and in human pathology. A better knowledge of the retroviral transcriptional activity in the general population and during the life span would greatly help the debate on its pathologic potential. The transcriptional activity of four HERV families (H, K, W, and E) was assessed, by qualitative and quantitative PCR, in PBMCs from 261 individuals aged from 1 to 80 years. Our results show that HERV-H, HERV-K, and HERV-W, but not HERV-E, are transcriptionally active in the test population already in the early childhood. In addition, the transcriptional levels of HERV-H, HERV-K, and HERV-W change significantly during the life span, albeit with distinct patterns. Our results, reinforce the hypothesis of a physiological correlation between HERVs activity and the different stages of life in humans. Studies aiming at identifying the factors, which are responsible for these changes during the individual’s life, are still needed. Although the observed phenomena are presumably subjected to great variability, the basal transcriptional activity of each individual, also depending on the different ages of life, must be carefully considered in all the studies involving HERVs as causative agents of disease. Emanuela Balestrieri, Francesca Pica, Claudia Matteucci, Rossella Zenobi, Roberta Sorrentino, Ayele Argaw-Denboba, Chiara Cipriani, Ilaria Bucci, and Paola Sinibaldi-Vallebona Copyright © 2015 Emanuela Balestrieri et al. All rights reserved. Different Short-Term Mild Exercise Modalities Lead to Differential Effects on Body Composition in Healthy Prepubertal Male Rats Wed, 28 Jan 2015 09:39:03 +0000 Physical activity has a vital role in regulating and improving bone strength. Responsiveness of bone mass to exercise is age dependent with the prepubertal period suggested to be the most effective stage for interventions. There is a paucity of data on the effects of exercise on bone architecture and body composition when studied within the prepubertal period. We examined the effect of two forms of low-impact exercise on prepubertal changes in body composition and bone architecture. Weanling male rats were assigned to control (CON), bipedal stance (BPS), or wheel exercise (WEX) groups for 15 days until the onset of puberty. Distance travelled via WEX was recorded, food intake measured, and body composition quantified. Trabecular and cortical microarchitecture of the femur were determined by microcomputed tomography. WEX led to a higher lean mass and reduced fat mass compared to CON. WEX animals had greater femoral cortical cross-sectional thickness and closed porosity compared to CON. The different exercise modalities had no effect on body weight or food intake, but WEX significantly altered body composition and femoral microarchitecture. These data suggest that short-term mild voluntary exercise in normal prepubertal rats can alter body composition dependent upon the exercise modality. D. M. Sontam, M. H. Vickers, J. M. O’Sullivan, M. Watson, and E. C. Firth Copyright © 2015 D. M. Sontam et al. All rights reserved. ECR-MAPK Regulation in Liver Early Development Thu, 18 Dec 2014 00:11:25 +0000 Early growth is connected to a key link between embryonic development and aging. In this paper, liver gene expression profiles were assayed at postnatal day 22 and week 16 of age. Meanwhile another independent animal experiment and cell culture were carried out for validation. Significance analysis of microarrays, qPCR verification, drug induction/inhibition assays, and metabonomics indicated that alpha-2u globulin (extracellular region)-socs2 (-SH2-containing signals/receptor tyrosine kinases)-ppp2r2a/pik3c3 (MAPK signaling)-hsd3b5/cav2 (metabolism/organization) plays a vital role in early development. Taken together, early development of male rats is ECR and MAPK-mediated coordination of cancer-like growth and negative regulations. Our data represent the first comprehensive description of early individual development, which could be a valuable basis for understanding the functioning of the gene interaction network of infant development. Xiu-Ju Zhao and Hexian Zhuo Copyright © 2014 Xiu-Ju Zhao and Hexian Zhuo. All rights reserved. MicroRNA Dysregulation in Liver and Pancreas of CMP-Neu5Ac Hydroxylase Null Mice Disrupts Insulin/PI3K-AKT Signaling Thu, 28 Aug 2014 00:00:00 +0000 CMP-Neu5Ac hydroxylase (Cmah)-null mice fed with a high-fat diet develop fasting hyperglycemia, glucose intolerance, and pancreatic β-cell dysfunction and ultimately develop characteristics of type 2 diabetes. The precise metabolic role of the Cmah gene remains poorly understood. This study was designed to investigate the molecular mechanisms through which microRNAs (miRNAs) regulate type 2 diabetes. Expression profiles of miRNAs in Cmah-null mouse livers were compared to those of control mouse livers. Liver miFinder miRNA PCR arrays () showed that eight miRNA genes were differentially expressed between the two groups. Compared with controls, seven miRNAs were upregulated and one miRNA was downregulated in Cmah-null mice. Specifically, miR-155-5p, miR-425-5p, miR-15a-5p, miR-503-5p, miR-16-5p, miR-29a-3p, and miR-29b-3p were significantly upregulated in the liver and pancreas of Cmah-null mice. These target miRNAs are closely associated with dysregulation of insulin/PI3K-AKT signaling, suggesting that the Cmah-null mice could be a useful model for studying diabetes. Deug-Nam Kwon, Byung-Soo Chang, and Jin-Hoi Kim Copyright © 2014 Deug-Nam Kwon et al. All rights reserved. Characterization of Zebrafish Pax1b and Pax9 in Fin Bud Development Wed, 13 Aug 2014 07:18:54 +0000 Both Pax1 and Pax9 belong to the important paired box gene family (PAX), which mainly participates in animal development and sclerotome differentiation. To date, the precise molecular mechanism and related signaling pathway of Pax1 remain unclear. In our study, microinjection of morpholino- (MO-) modified antisense oligonucleotides against pax1b induced pectoral fin bud defects. Furthermore, we demonstrate that the phenotypes caused by the knockdown of Pax1b in zebrafish could not be phenocopied by pax9 MO and could not be rescued by either Pax1a or Pax9 overexpression. We further find that Pax1b affects the expression of col2a1, Uncx4.1, Noggin3, and aggrecan, confirming the role of Pax1b in chondrocyte differentiation and bone maturation. Moreover, we identify an interaction between PAX1 and FOXO1 and find that the interaction was enhanced under hypoxia stress. Together, this evidence for cell death caused by pax1b knockdown provides new insight into the role of the Pax protein family in cell fate determination and tissue specification. Xuemei Chen, Huizhe Huang, Hua Wang, Fengjin Guo, Xiaogang Du, Linqiang Ma, Liang Zhao, Zhuma Pan, Haibo Gui, Taixian Yuan, Xin Liu, Lin Song, Yiquan Wang, Junling He, Han Lei, and Rui Gao Copyright © 2014 Xuemei Chen et al. All rights reserved. Hox Transcription Factors: Modulators of Cell-Cell and Cell-Extracellular Matrix Adhesion Mon, 21 Jul 2014 00:00:00 +0000 Hox genes encode homeodomain-containing transcription factors that determine cell and tissue identities in the embryo during development. Hox genes are also expressed in various adult tissues and cancer cells. In Drosophila, expression of cell adhesion molecules, cadherins and integrins, is regulated by Hox proteins operating in hierarchical molecular pathways and plays a crucial role in segment-specific organogenesis. A number of studies using mammalian cultured cells have revealed that cell adhesion molecules responsible for cell-cell and cell-extracellular matrix interactions are downstream targets of Hox proteins. However, whether Hox transcription factors regulate expression of cell adhesion molecules during vertebrate development is still not fully understood. In this review, the potential roles Hox proteins play in cell adhesion and migration during vertebrate body patterning are discussed. Yasushi Taniguchi Copyright © 2014 Yasushi Taniguchi. All rights reserved. Morphometric Approach to Pulp Fibroblast Development in Tooth Germ Wed, 25 Jun 2014 11:00:10 +0000 This paper builds a morphometric framework for the analysis of dental pulp fibroblast evolution during tooth development. We investigated 15 tooth germs (cases) organized, by histological criteria, in three groups corresponding to cap, early bell, and late bell stages, respectively. Each group comprised five cases. The morphometric description used the following parameters: area , perimeter —automatically extracted by a color segmentation technique, and form factor (FF)—calculated as . The designed framework operated at inter- and intragroup levels. The intergroup analysis quantified the differences between groups, in the sense of a relative distance (RD) adequately defined by mean-value scaling. We showed that the stage of early bell is approximately 5 times closer to late bell than to cap. The quantification procedure required concomitant information about , parameters (as P versus A dependences, or FF values), whereas the procedure failed for A or P separately used. The intragroup analysis quantified the similarity of the cases belonging to the same stage. We proved that, unlike the intergroup tests, the individual exploitation of all three descriptors A, P, and FF is effective, yielding highly compatible results. Within any group, most cases presented RDs less than 10% from the group mean value, regardless of the descriptor type. Irina-Draga Căruntu, Sergiu Daniel Săvinescu, and Cornelia Amălinei Copyright © 2014 Irina-Draga Căruntu et al. All rights reserved. Circadian System Development and Plasticity Mon, 16 Jun 2014 07:22:58 +0000 Martin Fredensborg Rath, Yoav Gothilf, Mario Guido, and Estela Muñoz Copyright © 2014 Martin Fredensborg Rath et al. All rights reserved. Early Stages of we/we wal/wal Mouse Hair Morphogenesis: Light and Fluorescent Microscopy of the Whole-Mount Epidermis Tue, 03 Jun 2014 12:03:39 +0000 In adult skin, hair follicles cyclically self-renew in a manner that recapitulates embryonic hair follicle morphogenesis. The most common pathology of hair in adults is alopecia, which is hair loss to different extent. There are a number of murine models of alopecia including spontaneous mutations. In the present study, we worked with double homozygous we/we wal/wal mice which demonstrate symptoms closely resembling human alopecia. Using whole-mount preparations of epidermis of E18.5 embryos we show that hair follicle defects can be revealed as early as during embryonic morphogenesis in these mutants. The number of hair follicles was reduced almost 1.5-fold in mutant skin. The shape of the early stage small follicles was altered in mutant animals as compared to control ones. Additionally, follicles of mutant embryos were wider at the point of conjunction with interfollicular epidermis. We believe that the mutant mice studied represent a fascinating model to address the problem of hair loss. We demonstrated alterations in the morphogenesis of embryonic hair follicle in we/we wal/wal double homozygous mice developing alopecia postnatally. We suppose that incorrect morphogenesis of hair follicles during embryogenesis is closely related to alopecia in the adult life. Unveiling the mechanisms involved in altered embryogenesis may elucidate the pathogenesis of alopecia. Alexandra Rippa, Olga Leonova, Vladimir Popenko, Andrey Vasiliev, Vasily Terskikh, and Ekaterina Vorotelyak Copyright © 2014 Alexandra Rippa et al. All rights reserved. Morphological Changes in the Suprachiasmatic Nucleus of Aging Female Marmosets (Callithrix jacchus) Mon, 02 Jun 2014 06:40:11 +0000 The suprachiasmatic nuclei (SCN) are pointed to as the mammals central circadian pacemaker. Aged animals show internal time disruption possibly caused by morphological and neurochemical changes in SCN components. Some studies reported changes of neuronal cells and neuroglia in the SCN of rats and nonhuman primates during aging. The effects of senescence on morphological aspects in SCN are important for understanding some alterations in biological rhythms expression. Therefore, our aim was to perform a comparative study of the morphological aspects of SCN in adult and aged female marmoset. Morphometric analysis of SCN was performed using Nissl staining, NeuN-IR, GFAP-IR, and CB-IR. A significant decrease in the SCN cells staining with Nissl, NeuN, and CB were observed in aged female marmosets compared to adults, while a significant increase in glial cells was found in aged marmosets, thus suggesting compensatory process due to neuronal loss evoked by aging. Rovena Clara G. J. Engelberth, Kayo Diogenes de A. Silva, Carolina V. de M. Azevedo, Elaine Cristina Gavioli, Jose Ronaldo dos Santos, Joacil Germano Soares, Expedito S. Nascimento Junior, Judney C. Cavalcante, Miriam Stela M. O. Costa, and Jeferson S. Cavalcante Copyright © 2014 Rovena Clara G. J. Engelberth et al. All rights reserved. Early Appearance of Nonvisual and Circadian Markers in the Developing Inner Retinal Cells of Chicken Tue, 20 May 2014 07:14:09 +0000 The retina is a key component of the vertebrate circadian system; it is responsible for detecting and transmitting the environmental illumination conditions (day/night cycles) to the brain that synchronize the circadian clock located in the suprachiasmatic nucleus (SCN). For this, retinal ganglion cells (RGCs) project to the SCN and other nonvisual areas. In the chicken, intrinsically photosensitive RGCs (ipRGCs) expressing the photopigment melanopsin (Opn4) transmit photic information and regulate diverse nonvisual tasks. In nonmammalian vertebrates, two genes encode Opn4: the Xenopus (Opn4x) and the mammalian (Opn4m) orthologs. RGCs express both Opn4 genes but are not the only inner retinal cells expressing Opn4x: horizontal cells (HCs) also do so. Here, we further characterize primary cultures of both populations of inner retinal cells (RGCs and HCs) expressing Opn4x. The expression of this nonvisual photopigment, as well as that for different circadian markers such as the clock genes Bmal1, Clock, Per2, and Cry1, and the key melatonin synthesizing enzyme, arylalkylamine N-acetyltransferase (AA-NAT), appears very early in development in both cell populations. The results clearly suggest that nonvisual Opn4 photoreceptors and endogenous clocks converge all together in these inner retinal cells at early developmental stages. Nicolás M. Díaz, Luis P. Morera, Daniela M. Verra, María A. Contin, and Mario E. Guido Copyright © 2014 Nicolás M. Díaz et al. All rights reserved. Circadian Modulation of the Cl− Equilibrium Potential in the Rat Suprachiasmatic Nuclei Sun, 18 May 2014 08:45:30 +0000 The suprachiasmatic nuclei (SCN) constitute a circadian clock in mammals, where -amino-butyric acid (GABA) neurotransmission prevails and participates in different aspects of circadian regulation. Evidence suggests that GABA has an excitatory function in the SCN in addition to its typical inhibitory role. To examine this possibility further, we determined the equilibrium potential of GABAergic postsynaptic currents (EGABA) at different times of the day and in different regions of the SCN, using either perforated or whole cell patch clamp. Our results indicate that during the day most neurons in the dorsal SCN have an EGABA close to −30 mV while in the ventral SCN they have an EGABA close to −60 mV; this difference reverses during the night, in the dorsal SCN neurons have an EGABA of −60 mV and in the ventral SCN they have an EGABA of −30 mV. The depolarized equilibrium potential can be attributed to the activity of the Na(+)-K(+)-2Cl(−) (NKCC) cotransporter since the equilibrium potential becomes more negative following addition of the NKCC blocker bumetanide. Our results suggest an excitatory role for GABA in the SCN and further indicate both time (day versus night) and regional (dorsal versus ventral) modulation of EGABA in the SCN. Javier Alamilla, Azucena Perez-Burgos, Daniel Quinto, and Raúl Aguilar-Roblero Copyright © 2014 Javier Alamilla et al. All rights reserved. Regulation of Melanopsins and Per1 by α-MSH and Melatonin in Photosensitive Xenopus laevis Melanophores Tue, 13 May 2014 11:20:06 +0000 α-MSH and light exert a dispersing effect on pigment granules of Xenopus laevis melanophores; however, the intracellular signaling pathways are different. Melatonin, a hormone that functions as an internal signal of darkness for the organism, has opposite effects, aggregating the melanin granules. Because light functions as an important synchronizing signal for circadian rhythms, we further investigated the effects of both hormones on genes related to the circadian system, namely, Per1 (one of the clock genes) and the melanopsins, Opn4x and Opn4m (photopigments). Per1 showed temporal oscillations, regardless of the presence of melatonin or α-MSH, which slightly inhibited its expression. Melatonin effects on melanopsins depend on the time of application: if applied in the photophase it dramatically decreased Opn4x and Opn4m expressions, and abolished their temporal oscillations, opposite to α-MSH, which increased the melanopsins’ expressions. Our results demonstrate that unlike what has been reported for other peripheral clocks and cultured cells, medium changes or hormones do not play a major role in synchronizing the Xenopus melanophore population. This difference is probably due to the fact that X. laevis melanophores possess functional photopigments (melanopsins) that enable these cells to primarily respond to light, which triggers melanin dispersion and modulates gene expression. Maria Nathália de Carvalho Magalhães Moraes, Luciane Rogéria dos Santos, Nathana Mezzalira, Maristela Oliveira Poletini, and Ana Maria de Lauro Castrucci Copyright © 2014 Maria Nathália de Carvalho Magalhães Moraes et al. All rights reserved. The Effect of Different Photoperiods in Circadian Rhythms of Per3 Knockout Mice Thu, 08 May 2014 13:45:18 +0000 The aim of this study was to analyse the circadian behavioural responses of mice carrying a functional knockout of the Per3 gene () to different light : dark (L : D) cycles. Male adult wild-type (WT) and mice were kept under 12-hour light : 12-hour dark conditions (12L : 12D) and then transferred to either a short or long photoperiod and subsequently released into total darkness. All mice were exposed to both conditions, and behavioural activity data were acquired through running wheel activity and analysed for circadian characteristics during these conditions. We observed that, during the transition from 12L : 12D to 16L : 8D, mice take approximately one additional day to synchronise to the new L : D cycle compared to WT mice. Under these long photoperiod conditions, mice were more active in the light phase. Our results suggest that mice are less sensitive to light. The data presented here provides further evidence that Per3 is involved in the suppression of behavioural activity in direct response to light. D. S. Pereira, D. R. van der Veen, B. S. B. Gonçalves, S. Tufik, M. von Schantz, S. N. Archer, and M. Pedrazzoli Copyright © 2014 D. S. Pereira et al. All rights reserved. Timing Embryo Segmentation: Dynamics and Regulatory Mechanisms of the Vertebrate Segmentation Clock Wed, 07 May 2014 11:16:48 +0000 All vertebrate species present a segmented body, easily observed in the vertebrate column and its associated components, which provides a high degree of motility to the adult body and efficient protection of the internal organs. The sequential formation of the segmented precursors of the vertebral column during embryonic development, the somites, is governed by an oscillating genetic network, the somitogenesis molecular clock. Herein, we provide an overview of the molecular clock operating during somite formation and its underlying molecular regulatory mechanisms. Human congenital vertebral malformations have been associated with perturbations in these oscillatory mechanisms. Thus, a better comprehension of the molecular mechanisms regulating somite formation is required in order to fully understand the origin of human skeletal malformations. Tatiana P. Resende, Raquel P. Andrade, and Isabel Palmeirim Copyright © 2014 Tatiana P. Resende et al. All rights reserved. Homeobox Genes and Melatonin Synthesis: Regulatory Roles of the Cone-Rod Homeobox Transcription Factor in the Rodent Pineal Gland Wed, 30 Apr 2014 11:48:25 +0000 Nocturnal synthesis of melatonin in the pineal gland is controlled by a circadian rhythm in arylalkylamine N-acetyltransferase (AANAT) enzyme activity. In the rodent, Aanat gene expression displays a marked circadian rhythm; release of norepinephrine in the gland at night causes a cAMP-based induction of Aanat transcription. However, additional transcriptional control mechanisms exist. Homeobox genes, which are generally known to encode transcription factors controlling developmental processes, are also expressed in the mature rodent pineal gland. Among these, the cone-rod homeobox (CRX) transcription factor is believed to control pineal-specific Aanat expression. Based on recent advances in our understanding of Crx in the rodent pineal gland, we here suggest that homeobox genes play a role in adult pineal physiology both by ensuring pineal-specific Aanat expression and by facilitating cAMP response element-based circadian melatonin production. Kristian Rohde, Morten Møller, and Martin Fredensborg Rath Copyright © 2014 Kristian Rohde et al. All rights reserved. Nuclear Distribution of RNA Polymerase II and mRNA Processing Machinery in Early Mammalian Embryos Tue, 29 Apr 2014 13:21:37 +0000 Spatial distribution of components of nuclear metabolism provides a significant impact on regulation of the processes of gene expression. While distribution of the key nuclear antigens and their association with the defined nuclear domains were thoroughly traced in mammalian somatic cells, similar data for the preimplantation embryos are scanty and fragmental. However, the period of cleavage is characterized by the most drastic and dynamic nuclear reorganizations accompanying zygotic gene activation. In this minireview, we try to summarize the results of studies concerning distribution of major factors involved in RNA polymerase II-dependent transcription, pre-mRNA splicing mRNA export that have been carried out on early embryos of mammals. Irina O. Bogolyubova and Dmitry S. Bogolyubov Copyright © 2014 Irina O. Bogolyubova and Dmitry S. Bogolyubov. All rights reserved. Functional Development of the Circadian Clock in the Zebrafish Pineal Gland Wed, 16 Apr 2014 13:35:12 +0000 The zebrafish constitutes a powerful model organism with unique advantages for investigating the vertebrate circadian timing system and its regulation by light. In particular, the remarkably early and rapid development of the zebrafish circadian system has facilitated exploring the factors that control the onset of circadian clock function during embryogenesis. Here, we review our understanding of the molecular basis underlying functional development of the central clock in the zebrafish pineal gland. Furthermore, we examine how the directly light-entrainable clocks in zebrafish cell lines have facilitated unravelling the general mechanisms underlying light-induced clock gene expression. Finally, we summarize how analysis of the light-induced transcriptome and miRNome of the zebrafish pineal gland has provided insight into the regulation of the circadian system by light, including the involvement of microRNAs in shaping the kinetics of light- and clock-regulated mRNA expression. The relative contributions of the pineal gland central clock and the distributed peripheral oscillators to the synchronization of circadian rhythms at the whole animal level are a crucial question that still remains to be elucidated in the zebrafish model. Zohar Ben-Moshe, Nicholas S. Foulkes, and Yoav Gothilf Copyright © 2014 Zohar Ben-Moshe et al. All rights reserved. Synchronization by Food Access Modifies the Daily Variations in Expression and Activity of Liver GABA Transaminase Mon, 07 Apr 2014 11:46:16 +0000 Daytime restricted feeding (DRF) is an experimental protocol that influences the circadian timing system and underlies the expression of a biological clock known as the food entrained oscillator (FEO). Liver is the organ that reacts most rapidly to food restriction by adjusting the functional relationship between the molecular circadian clock and the metabolic networks. -Aminobutyric acid (GABA) is a signaling molecule in the liver, and able to modulate the cell cycle and apoptosis. This study was aimed at characterizing the expression and activity of the mostly mitochondrial enzyme GABA transaminase (GABA-T) during DRF/FEO expression. We found that DRF promotes a sustained increase of GABA-T in the liver homogenate and mitochondrial fraction throughout the entire day-night cycle. The higher amount of GABA-T promoted by DRF was not associated to changes in GABA-T mRNA or GABA-T activity. The GABA-T activity in the mitochondrial fraction even tended to decrease during the light period. We concluded that DRF influences the daily variations of GABA-T mRNA levels, stability, and catalytic activity of GABA-T. These data suggest that the liver GABAergic system responds to a metabolic challenge such as DRF and the concomitant appearance of the FEO. Dalia De Ita-Pérez, Isabel Méndez, Olivia Vázquez-Martínez, Mónica Villalobos-Leal, and Mauricio Díaz-Muñoz Copyright © 2014 Dalia De Ita-Pérez et al. All rights reserved. Developmental Stage-Specific Regulation of the Circadian Clock by Temperature in Zebrafish Thu, 27 Mar 2014 00:00:00 +0000 The circadian clock enables animals to adapt their physiology and behaviour in anticipation of the day-night cycle. Light and temperature represent two key environmental timing cues (zeitgebers) able to reset this mechanism and so maintain its synchronization with the environmental cycle. One key challenge is to unravel how the regulation of the clock by zeitgebers matures during early development. The zebrafish is an ideal model for studying circadian clock ontogeny since the process of development occurs ex utero in an optically transparent chorion and many tools are available for genetic analysis. However, the role played by temperature in regulating the clock during zebrafish development is poorly understood. Here, we have established a clock-regulated luciferase reporter transgenic zebrafish line (Tg (−3.1) per1b::luc) to study the effects of temperature on clock entrainment. We reveal that under complete darkness, from an early developmental stage onwards (48 to 72 hpf), exposure to temperature cycles is a prerequisite for the establishment of self-sustaining rhythms of zfper1b, zfaanat2, and zfirbp expression and also for circadian cell cycle rhythms. Furthermore, we show that following the 5–9 somite stage, the expression of zfper1b is regulated by acute temperature shifts. Kajori Lahiri, Nadine Froehlich, Andreas Heyd, Nicholas S. Foulkes, and Daniela Vallone Copyright © 2014 Kajori Lahiri et al. All rights reserved. Neuropeptide Y in the Adult and Fetal Human Pineal Gland Mon, 17 Mar 2014 10:06:59 +0000 Neuropeptide Y was isolated from the porcine brain in 1982 and shown to be colocalized with noradrenaline in sympathetic nerve terminals. The peptide has been demonstrated to be present in sympathetic nerve fibers innervating the pineal gland in many mammalian species. In this investigation, we show by use of immunohistochemistry that neuropeptide Y is present in nerve fibers of the adult human pineal gland. The fibers are classical neuropeptidergic fibers endowed with large boutons en passage and primarily located in a perifollicular position with some fibers entering the pineal parenchyma inside the follicle. The distance from the immunoreactive terminals to the pinealocytes indicates a modulatory function of neuropeptide Y for pineal physiology. Some of the immunoreactive fibers might originate from neurons located in the brain and be a part of the central innervation of the pineal gland. In a series of human fetuses, neuropeptide Y-containing nerve fibers was present and could be detected as early as in the pineal of four- to five-month-old fetuses. This early innervation of the human pineal is different from most rodents, where the innervation starts postnatally. Morten Møller, Pansiri Phansuwan-Pujito, and Corin Badiu Copyright © 2014 Morten Møller et al. All rights reserved. An Immunocytochemical Study of Interchromatin Granule Clusters in Early Mouse Embryos Wed, 11 Sep 2013 13:48:30 +0000 Interchromatin granule clusters (IGCs) are universal nuclear domains. Their molecular composition and functions were studied in detail in somatic cells. Here, we studied IGCs in the nuclei of early mouse embryos during zygotic gene activation (ZGA). We found that the size of IGCs gradually increases during realization of ZGA events. Using immunocytochemical approaches, we showed that the molecular composition of IGCs is also modified in mouse embryos. The hyperphosphorylated form of RNA polymerase II and the transcription factor TFIID have been revealed in IGCs before the end of ZGA. Association of these factors with IGCs became more noticeable during ZGA realization. Our data suggest that IGCs in early mouse embryos have some functional peculiarities connected most probably with IGC formation de novo. We believe that IGCs in early mouse embryos not only are storage sites of splicing factors but also may be involved in mRNA metabolism and represent the multifunctional nuclear domains. Irina Bogolyubova and Dmitry Bogolyubov Copyright © 2013 Irina Bogolyubova and Dmitry Bogolyubov. All rights reserved. Antioxidant, Antiproliferative, and Antiangiogenesis Effects of Polyphenol-Rich Seaweed (Sargassum muticum) Tue, 03 Sep 2013 16:21:23 +0000 In the present study, we evaluated the effect of brown seaweeds Sargassum muticum methanolic extract (SMME), against MCF-7 and MDA-MB-231 breast cancer cell lines proliferation. This algae extract was also evaluated for reducing activity and total polyphenol content. The MTT assay results indicated that the extracts were cytotoxic against breast cancer cell lines in a dose-dependent manner, with IC50 of 22 μg/ml for MCF-7 and 55 μg/ml for MDA-MB-231 cell lines. The percentages of apoptotic MCF-7-treated cells increased from 13% to 67% by increasing the concentration of the SMME. The antiproliferative efficacy of this algal extract was positively correlated with the total polyphenol contents, suggesting a causal link related to extract content of phenolic acids. Cell cycle analysis showed a significant increase in the accumulation of SMME-treated cells at sub-G1 phase, indicating the induction of apoptosis by SMME. Further apoptosis induction was confirmed by Hoechst 33342 and AO/PI staining. Also SMME implanted in vivo into fertilized chicken eggs induced dose-related antiangiogenic activity in the chorioallantoic membrane (CAM). Our results imply a new insight on the novel function of Sargassum muticum polyphenol-rich seaweed in cancer research by induction of apoptosis, antioxidant, and antiangiogenesis effects. Farideh Namvar, Rosfarizan Mohamad, Javad Baharara, Saeedeh Zafar-Balanejad, Fahimeh Fargahi, and Heshu Sulaiman Rahman Copyright © 2013 Farideh Namvar et al. All rights reserved. Regulatory Interactions between Androgens, Hoxb5, and TGFβ Signaling in Murine Lung Development Tue, 03 Sep 2013 11:09:37 +0000 Androgens enhance airway branching but delay alveolar maturation contributing to increased respiratory morbidity in prematurely born male infants. Hoxb5 protein positively regulates airway branching in developing lung. In other organs, androgen regulation intersects with Hox proteins and TGFβ-SMAD signaling, but these interactions have not been studied in the lung. We hypothesized that androgen alteration of airway branching early in lung development requires Hoxb5 expression and that these androgen-Hoxb5 interactions occur partially through regional changes in TGFβ signaling. To evaluate acute effects of androgen and TGFβ on Hoxb5, E11 whole fetal mouse lungs were cultured with dihydrotestosterone (DHT) with/without Hoxb5 siRNA or TGFβ inhibitory antibody. Chronic in utero DHT exposure was accomplished by exposing pregnant mice to DHT (subcutaneous pellet) from E11 to E18. DHT’s ability to enhance airway branching and alter phosphorylated SMAD2 cellular localization was partially dependent on Hoxb5. Hoxb5 inhibition also changed the cellular distribution of SMAD7 protein. Chronic in utero DHT increased Hoxb5 and altered SMAD7 mesenchymal localization. TGFβ inhibition enhanced airway branching, and Hoxb5 protein cellular localization was more diffuse. We conclude that DHT controls lung airway development partially through modulation of Hoxb5 protein expression and that this level of regulation involves interactions with TGFβ signaling. MaryAnn V. Volpe, Sujatha M. Ramadurai, Sana Mujahid, Thanhxuan Vong, Marcia Brandao, Karen T. Wang, Lucia D. Pham, and Heber C. Nielsen Copyright © 2013 MaryAnn V. Volpe et al. All rights reserved. Expression and Cellular Distribution of INHA and INHB before and after In Vitro Cultivation of Porcine Oocytes Isolated from Follicles of Different Size Tue, 20 Nov 2012 15:00:20 +0000 Cumulus-oocyte-complexes (COCs) were collected from small (<3 mm), medium (3–5 mm), and large (>5 mm) porcine follicles, and the INHA and INHB expression and cellular localization were studied. Developmentally competent (BCB+) COCs were cultured for 44 h. Samples of mRNA were isolated before and after in vitro maturation (IVM) from oocytes collected from follicles of different size for RQ-PCR assay. The INHA and INHB protein distribution within the oocytes was observed by confocal microscopy. INHA mRNA expression was increased in oocytes from large compared to medium and small follicles before IVM (), and to oocytes of small follicles after IVM (). The INHB expression was not different before IVM, but the IHNB mRNA level was gradually higher in oocytes from large follicles after IVM (). INHA was not differently expressed before IVM; however, in large follicle oocytes the protein was distributed in the peripheral area of the cytoplasm; in oocytes from small follicles it was in the entire cytoplasm. After IVM, INHA was strongly expressed in oocytes from small follicles and distributed particularly in the zona pellucida (ZP). Similarly and both before and after IVM, INHB protein was highly expressed in small follicle oocytes and within the cytoplasm. In summary, INHs can be recognized as a marker of porcine oocyte quality. Bartosz Kempisty, Marta Jackowska, Magdalena Woźna, Paweł Antosik, Hanna Piotrowska, Piotr Zawierucha, Dorota Bukowska, Jędrzej M. Jaśkowski, Michał Nowicki, and Klaus P. Brüssow Copyright © 2012 Bartosz Kempisty et al. All rights reserved. Selection of Ovine Oocytes by Brilliant Cresyl Blue Staining Wed, 23 May 2012 10:40:09 +0000 Sheep oocytes derived from the ovaries collected from the slaughterhouse are often used for research on in vitro embryo production, animal cloning, transgenesis, embryonic stem cells, and other embryo biotechnology aspects. Improving the in vitro culture efficiency of oocytes can provide more materials for similar studies. Generally, determination of oocyte quality is mostly based on the layers of cumulus cells and cytoplasm or cytoplasm uniformity and colors. This requires considerable experience to better identify oocyte quality because of the intense subjectivity involved (Gordon (2003), Madison et al. (1992) and De Loos et al. (1992)). BCB staining is a function of glucose-6-phosphate dehydrogenase (G6PD) activity, an enzyme synthesized in developing oocytes, which decreases in activity with maturation. Therefore, unstained oocytes (BCB−) are high in G6PD activity, while the less mature oocytes stains are deep blue (BCB+) due to insuffcient G6PD activity to decolorize the BCB dye. Liqin Wang, Jiapeng Lin, Juncheng Huang, Jing Wang, Yuncheng Zhao, and Tong Chen Copyright © 2012 Liqin Wang et al. All rights reserved. A Symphony of Regulations Centered on p63 to Control Development of Ectoderm-Derived Structures Sun, 22 May 2011 16:15:45 +0000 The p53-related transcription factor p63 is critically important for basic cellular functions during development of the ectoderm and derived structure and tissues, including skin, limb, palate, and hair. On the one side, p63 is required to sustain the proliferation of keratinocyte progenitors, while on the other side it is required for cell stratification, commitment to differentiate, cell adhesion, and epithelial-mesenchymal signaling. Molecules that are components or regulators of the p63 pathway(s) are rapidly being identified, and it comes with no surprise that alterations in the p63 pathway lead to congenital conditions in which the skin and other ectoderm-derived structures are affected. In this paper, we summarize the current knowledge of the molecular and cellular regulations centered on p63, derived from the comprehension of p63-linked human diseases and the corresponding animal models, as well as from cellular models and high-throughput molecular approaches. We point out common themes and features, that allow to speculate on the possible role of p63 downstream events and their potential exploitation in future attempts to correct the congenital defect in preclinical studies. Luisa Guerrini, Antonio Costanzo, and Giorgio R. Merlo Copyright © 2011 Luisa Guerrini et al. All rights reserved. Immunohistochemical Localisation of PDE5 in Rat Lung during Pre- and Postnatal Development Thu, 20 Aug 2009 16:13:26 +0000 In mammalian lung, at the transition to extrauterine life, NO/cGMP signal transduction system is known to play crucial roles in the regulation of vascular resistance and is supposed to act in angiogenesis. PDE5, which is the most abundant cGMP metabolizing enzyme within the lung, is highly expressed in the perinatal period, but its localisation in the different pulmonary cells is still poorly known. In our research, PDE5 immunohistochemical distribution was investigated in foetal and neonatal rat lung. The highest expression of PDE5 was found in cells randomly located in the stroma; in newborns, in particular, many cells in the intersaccular walls were heavily labelled, while much lower staining levels were shown by smooth myocytes belonging to vessels and airways. On the basis of their immunoreactivity for 𝛼-SM actin and/or desmin, most of the heavily PDE5-positive cells were identified as interstitial myofibroblasts and transitional pericytes, while only a few were interpreted as interstitial lipofibroblasts. Angela Scipioni, Mauro Giorgi, Valeria Nuccetelli, and Stefania Stefanini Copyright © 2009 Angela Scipioni et al. All rights reserved.