Comparison of Calcium Balancing Strategies During Hypothermic Acclimation of Tilapia (Oreochromis mossambicus) and Goldfish (Carassius auratus)

The body temperatures of teleost species fluctuate following changes in the aquatic environment. As such, decreased water temperature lowers the rates of biochemical reactions and affects many physiological processes, including active transport-dependent ion absorption. Previous studies have focused on the impacts of low temperature on the plasma ion concentrations or membrane transporters in fishes. However, very few in vivo or organism-level studies have been performed to more thoroughly elucidate the process of acclimation to low temperatures. In the present study, we compared the strategies for cold acclimation between stenothermic tilapia and eurythermic goldfish. Whole-body calcium content was more prominently diminished in tilapia than in goldfish after long-term cold exposure. This difference can be attributed to alterations in the transportation parameters for Ca2+ influx, i.e., maximum velocity (Vmax) and binding affinity (1/Km). There was also a significant difference in the regulation of Ca2+ efflux between the two fishes. Transcript levels for Ca2+ related transporters, including the Na+/Ca2+ exchanger and epithelial Ca2+ channel, were similarly regulated in both fishes. However, upregulation of plasma membrane Ca2+ATPase expression was more pronounced in goldfish than in tilapia. In addition, enhanced Na+/K+-ATPase abundance, which provides the major driving force for ion absorption, was only detected in tilapia, while upregulated Na+/K+-ATPase activity was only detected in goldfish. Based on the results of the present study, we have found that goldfish and tilapia differentially regulate gill epithelial plasma membrane Ca2+-ATPase (PMCA) expression and Na+/K+-ATPase activity in response to cold environments. These regulatory differences are potentially linked to more effective regulation of Ca2+ influx kinetics and better maintenance of whole body calcium content in goldfish than in tilapia

Reducing TRPC1 Expression through Liposome-Mediated siRNA Delivery Markedly Attenuates Hypoxia-Induced Pulmonary Arterial Hypertension in a Murine Model

We tested the hypothesis that Lipofectamine siRNA delivery to deplete transient receptor potential cation channel (TRPC) 1 protein expression can suppress hypoxia-induced pulmonary arterial hypertension (PAH) in mice. Adult male C57BL/6 mice were equally divided into group 1 (normal controls), group 2 (hypoxia), and group 3 (hypoxia + siRNA TRPC1). By day 28, right ventricular systolic pressure (RVSP), number of muscularized arteries, right ventricle (RV), and lung weights were increased in group 2 than in group 1 and reduced in group 3 compared with group 2. Pulmonary crowded score showed similar pattern, whereas number of alveolar sacs exhibited an opposite pattern compared to that of RVSP in all groups. Protein expressions of TRPCs, HIF-1α, Ku-70, apoptosis, and fibrosis and pulmonary mRNA expressions of inflammatory markers were similar pattern, whereas protein expressions of antifibrosis and VEGF were opposite to the pattern of RVSP. Cellular markers of pulmonary DNA damage, repair, and smooth muscle proliferation exhibited a pattern similar to that of RVSP. The mRNA expressions of proapoptotic and hypertrophy biomarkers displayed a similar pattern, whereas sarcomere length showed an opposite pattern compared to that of RVSP in all groups. Lipofectamine siRNA delivery effectively reduced TRPC1 expression, thereby attenuating PAH-associated RV and pulmonary arteriolar remodeling

A Genome-Wide Association Study Identifies Susceptibility Variants for Type 2 Diabetes in Han Chinese

To investigate the underlying mechanisms of T2D pathogenesis, we looked for diabetes susceptibility genes that increase the risk of type 2 diabetes (T2D) in a Han Chinese population. A two-stage genome-wide association (GWA) study was conducted, in which 995 patients and 894 controls were genotyped using the Illumina HumanHap550-Duo BeadChip for the first genome scan stage. This was further replicated in 1,803 patients and 1,473 controls in stage 2. We found two loci not previously associated with diabetes susceptibility in and around the genes protein tyrosine phosphatase receptor type D (PTPRD) (P = 8.54×10−10; odds ratio [OR] = 1.57; 95% confidence interval [CI] = 1.36–1.82), and serine racemase (SRR) (P = 3.06×10−9; OR = 1.28; 95% CI = 1.18–1.39). We also confirmed that variants in KCNQ1 were associated with T2D risk, with the strongest signal at rs2237895 (P = 9.65×10−10; OR = 1.29, 95% CI = 1.19–1.40). By identifying two novel genetic susceptibility loci in a Han Chinese population and confirming the involvement of KCNQ1, which was previously reported to be associated with T2D in Japanese and European descent populations, our results may lead to a better understanding of differences in the molecular pathogenesis of T2D among various populations

FGF signaling in mesoderm development and evolution in deuterostomes: insights from the hemichordate Ptychodera flava

Mesoderm is a crucial germ layer that contributes to the complexity of bilaterian animals. In chordates, mesodermal cells are first specified into dorsal notochord to support the body and bilateral somites that later differentiate into muscles. These two mesodermal structures are defining features of chordates and are believed to contribute to a better motility for their tadpole-type larvae comparing to non-chordate larvae that propel by cilia. Studies in various chordate species have demonstrated that fibroblast growth factor (FGF) signaling is essential for notochord development through the activation of brachyury at notochord in vertebrates and ascidian. FGF signaling also regulates somite and muscle development in vertebrates but not the formation of trunk muscle in ascidian. In non-chordate deuterostome such as sea urchin embryos, FGF signaling is required for muscle development, but not the expression of brachyury. The functional differences of FGF signaling in controlling muscle development lead to ambiguity in the ancient role of FGF signaling in deuterostomes, and how FGF signaling had evolved to a new function in controlling notochord development through brachyury in chordates. In order to understand the role of FGF signaling during deuterostome evolution, we investigated functions of FGF signaling in mesoderm development during embryogenesis and metamorphosis in a non-chordate marine animal Ptychodera flava, an indirect-developing hemichordate that possess larval morphology similar to echinoderms and adult body features that resemble chordates. We have identified five FGF ligands and three FGF receptors in P. flava. Phylogenetic analyses revealed that hemichordates possess a conserved FGF8/17/18 in addition to several putative hemichordate-specific FGFs. Further functional studies showed that the mesodermal cell fate is specified at the early gastrula stage, and then theses cells are differentiated stepwise into the hydroporic canal, the pharyngeal muscle, and the muscle string; notably, formation of the last two muscular structures are regulated by FGF signaling. Moreover, the transcription levels of FGF ligands and receptors were significantly increased during metamorphosis, and augmentation of FGF signaling accelerated the process, suggesting its' role in facilitating the transformation from cilia-driven swimming larvae into muscle-driven worms. These results support the ancestral role of FGF signaling in muscle development in deuterostomes. Further studies are in progress for elucidating how the novel role of FGF signaling in notochord development had evolved from its ancestral role in the lineage leading to chordates.Acknowledgment………………………………………….....…………...……………………i Abstract…………………………………………………………….....…...……………………..ii List of Tables Figures……………………………………………....……………………….iv List of Supplementary tables and Figures ………………….....………………....v Part 1. Evolutionary developmental biology (Evo-Devo), chordate evolution, and hemichordates…………………….…………………….....……….…..1 Part 2. FGF signaling repertoire of the indirect developing hemichordate Ptychodera flava……………………………………………….......…12 Abstract………………………………………………………………........……………………13 Introduction…………………………………………………………………......……………..14 Materials and Methods………………………………………….……………………….…17 Results and Discussion…..……………………………………..………………………...19 Conclusion……………………………………………………………….......…………………31 Part 3. Reiterative use of FGF signaling in mesoderm development during embryogenesis and metamorphosis in the hemichordate Ptychodera flava………….......……....……....……....……....……....…….........…39 Abstract……………………………........………………………………………………………40 Introduction………………………………......……………………………..…………………41 Materials and Methods……………………………….……………………………………44 Results……………………………………………........….……………………………….……47 Discussion…………………………………………………………………………………68 Conclusion…………………………………………………………………………………70 Part 4. Discussion and conclusion……..……………..…..…………………………76 Discussion ……………...………………………....……………………………………77 Conclusion ……………...…………………………......………………………………79 Part 5. The regulation of Pfbra and future prospect………….….………….81 Preliminary results ……...……………………………….............………………82 Future prospects ……...…………………………...…………………............…85 Appendix……...………………………………………………………………………….........86 Results of trials for optimizing the microinjection technology……87 Microinjection workflow in the hemichordate P. flava……......…...88 References…………………………………………………………………………………....…91 Resume……………………………………………………………………………………........10

Reiterative use of FGF signaling in mesoderm development during embryogenesis and metamorphosis in the hemichordate Ptychodera flava

Abstract Background Mesoderm is generally considered to be a germ layer that is unique to Bilateria, and it develops into diverse tissues, including muscle, and in the case of vertebrates, the skeleton and notochord. Studies on various deuterostome animals have demonstrated that fibroblast growth factor (FGF) signaling is required for the formation of many mesodermal structures, such as vertebrate somites, from which muscles are differentiated, and muscles in sea urchin embryos, suggesting an ancient role of FGF signaling in muscle development. However, the formation of trunk muscles in invertebrate chordates is FGF-independent, leading to ambiguity about this ancient role in deuterostomes. To further understand the role of FGF signaling during deuterostome evolution, we investigated the development of mesodermal structures during embryogenesis and metamorphosis in Ptychodera flava, an indirect-developing hemichordate that has larval morphology similar to echinoderms and adult body features that are similar to chordates. Results Here we show that genes encoding FGF ligands, FGF receptors and transcription factors that are known to be involved in mesoderm formation and myogenesis are expressed dynamically during embryogenesis and metamorphosis. FGF signaling at the early gastrula stage is required for the specification of the mesodermal cell fate in P. flava. The mesoderm cells are then differentiated stepwise into the hydroporic canal, the pharyngeal muscle and the muscle string; formation of the last two muscular structures are controlled by FGF signaling. Moreover, augmentation of FGF signaling during metamorphosis accelerated the process, facilitating the transformation from cilia-driven swimming larvae into muscle-driven worm-like juveniles. Conclusions Our data show that FGF signaling is required for mesoderm induction and myogenesis in the P. flava embryo, and it is reiteratively used for the morphological transition during metamorphosis. The dependence of muscle development on FGF signaling in both planktonic larvae and sand-burrowing worms supports its ancestral role in deuterostomes

Additional file 7: of Reiterative use of FGF signaling in mesoderm development during embryogenesis and metamorphosis in the hemichordate Ptychodera flava

Supplementary methods. (DOCX 77 kb

Additional file 9: of Reiterative use of FGF signaling in mesoderm development during embryogenesis and metamorphosis in the hemichordate Ptychodera flava

Perturbations of FGF signaling during sand-induced metamorphosis. The morphology of a wild type Spengel larva (A1) and the Spengel larvae treated with 10 μM (A2) or 20 μM (A3) of SU5402. The images were taken 2 days after treatments. The morphology of individuals after cultured for 2 days with sand containing 0.1% BSA (B1–2), 100 ng/ml (B3–4) or 200 ng/ml bFGF protein (B5–6). The Spengel larvae transformed into either Agassiz (B1, B3, B5) or juveniles (B2, B4, B6). A1-A3 and B1-B6 are shown in two different scales, according to the scale bars in A1 and B1, respectively. (PNG 5404 kb

Additional file 1: of Reiterative use of FGF signaling in mesoderm development during embryogenesis and metamorphosis in the hemichordate Ptychodera flava

Table S1. Accession numbers for genes/proteins used in this study. Table S2. Primers used for cloning. (DOCX 25 kb

Additional file 3: of Reiterative use of FGF signaling in mesoderm development during embryogenesis and metamorphosis in the hemichordate Ptychodera flava

Perturbations of FGF signaling after fertilization. Phenotypes of embryos at 43 hpf (A1-F1) and 73 hpf (A2-F2) after treatment with FGF signaling inhibitors (B1-D2) or bFGF protein (F1-F2) upon fertilization. Control embryos were treated with DMSO or 0.1% BSA. The concentrations of each drug or protein are indicated in each panel. All embryos are shown from a lateral view with the mouth on the left. All panels are shown in the same scale, according to the scale bar in A1. Abbreviations: me, mesoderm; en, endoderm. (PNG 2340 kb

Additional file 2: of Reiterative use of FGF signaling in mesoderm development during embryogenesis and metamorphosis in the hemichordate Ptychodera flava

Figure S1. Development of the mesodermal structures in P. flava. (A) The presumptive endomesoderm emerges as a thickened vegetal plate (yellow and red stripes) at the late blastula stage. (B) At the mid gastrula stage, the mesodermal cells (red) are specified at the tip of the archenteron (yellow). (C) At the late gastrula stage, the mesoderm develops into the protocoel that extends dorsally and forms a duct-like structure, the hydroporic canal (black arrow), which opens in the dorsal ectoderm to form a hydropore (black asterisk). (D) After hatching, the mesoderm of the tornaria larva further differentiates into the pharyngeal muscle (green arrow) and the muscle string (black arrowhead) that reaches to the anterior ectoderm. (E) At the Spengel larval stage, the protocoel is considerably enlarged, and two paired coeloms, the mesocoels (light purple) and metacoels (dark purple), form as two pairs of rings surrounding the stomach. (F) During metamorphosis, the protocoel forms a proboscis coelom at the Agassiz stage. (G) The transforming Agassiz has a more elongated posterior region, starts losing its cilia, and is incapable of swimming. (H) The juvenile has a typical tripartite body with an anterior proboscis, followed by a collar region and a trunk. The two black dots on the anterior ectoderm indicate the eye spots. Abbreviations: prob., proboscis; col., collar. (PNG 353 kb