斑马鱼胚胎发育过程中FGF3基因的表达

为了解斑马鱼胚胎发育过程中FGF3基因的时空性表达情况,并探讨其对胚胎发育的调控作用,该研究分别提取2,4,8,12,24,36,48,72hpf斑马鱼胚胎的总RNA,经逆转录成cDNA,实时荧光定量PcR检测FGF3基因mRNA表达量;扩增FGF3基因特异片段,构建pGEM-T／FGF3基因片段重组质粒,经克隆及测序验证后,合成地高辛标记的反义RNA探针,以整体原位杂交法检测斑马鱼胚胎FGF3基因的空间性表达。结果显示：FGF3P基因在2hp胚胎就有表达,并持续至胚胎孵化,12hpf胚胎FGF3表达量达到高峰（P〈0．01）;胚胎发育过程中心表达部位以头、尾、咽弓为主。由此得出结论,FGF3主要在胚胎发育早期表达,其表达可能与胚胎脑、眼、耳、咽弓及尾部器官的发育调控有关。     

Opposing Nodal/Vg1 and BMP signals mediate axial patterning in embryos of the basal chordate amphioxus

The basal chordate amphioxus resembles vertebrates in having a dorsal, hollow nerve cord, a notochord and somites. However, it lacks extensive gene duplications, and its embryos are small and gastrulate by simple invagination. Here we demonstrate that Nodal/Vg1 signaling acts from early cleavage through the gastrula stage to specify and maintain dorsal/anterior development while, starting at the early gastrula stage, BMP signaling promotes ventral/posterior identity. Knockdown and gain-of-function experiments show that these pathways act in opposition to one another. Signaling by these pathways is modulated by dorsally and/or anteriorly expressed genes including Chordin, Cerberus, and Blimp1. Overexpression and/or reporter assays in Xenopus demonstrate that the functions of these proteins are conserved between amphioxus and vertebrates. Thus, a fundamental genetic mechanism for axial patterning involving opposing Nodal and BMP signaling is present in amphioxus and probably also in the common ancestor of amphioxus and vertebrates or even earlier in deuterostome evolution.     

Nodal Signaling Regulates the Bone Morphogenic Protein Pluripotency Pathway in Mouse Embryonic Stem Cells

Members of the transforming growth factor-β superfamily play essential roles in both the pluripotency and differentiation of embryonic stem (ES) cells. Although bone morphogenic proteins (BMPs) maintain pluripotency of undifferentiated mouse ES cells, the role of autocrine Nodal signaling is less clear. Pharmacological, molecular, and genetic methods were used to further understand the roles and potential interactions of these pathways. Treatment of undifferentiated ES cells with SB431542, a pharmacological inhibitor of Smad2 signaling, resulted in a rapid reduction of phosphorylated Smad2 and altered the expression of several putative downstream targets. Unexpectedly, inhibition of the Nodal signaling pathway resulted in enhanced BMP signaling, as assessed by Smad1/5 phosphorylation. SB431542-treated cells also demonstrated significant induction of the Id genes, which are known direct targets of BMP signaling and important factors in ES cell pluripotency. Inhibition of BMP signaling decreased the SB431542-mediated phosphorylation of Smad1/5 and induction of Id genes, suggesting that BMP signaling is necessary for some Smad2-mediated activity. Because Smad7, a known inhibitory factor to both Nodal and BMP signaling, was down-regulated following inhibition of Nodal-Smad2 signaling, the contribution of Smad7 to the cross-talk between the transforming growth factor-β pathways in ES cells was examined. Biochemical manipulation of Smad7 expression, through shRNA knockdown or inducible gene expression, significantly reduced the SB431542-mediated phosphorylation of Smad1/5 and induction of the Id genes. We conclude that autocrine Nodal signaling in undifferentiated mouse ES cells modulates the vital pluripotency pathway of BMP signaling.     

Alk3/Alk3b and Smad5 Mediate BMP Signaling during Lymphatic Development in Zebrafish

Lymphatic vessels are essential to regulate interstitial fluid homeostasis and diverse immune responses. A number of crucial factors, such as VEGFC, SOX18, PROX1, FOX2C, and GJC2, have been implicated in differentiation and/or maintenance of lymphatic endothelial cells (LECs). In humans, dysregulation of these genes is known to cause lymphedema, a debilitating condition which adversely impacts the quality of life of affected individuals. However, there are no currently available pharmacological treatments for lymphedema, necessitating identification of additional factors modulating lymphatic development and function which can be targeted for therapy. In this report, we investigate the function of genes associated with Bone Morphogenetic Protein (BMP) signaling in lymphatic development using zebrafish embryos. The knock-down of BMP type II receptors, Bmpr2a and Bmpr2b, and type I receptors, Alk3 and Alk3b, as well as SMAD5, an essential cellular mediator of BMP signaling, led to distinct lymphatic defects in developing zebrafish. Therefore, it appears that each constituent of the BMP signaling pathway may have a unique function during lymphatic development. Taken together, our data demonstrate that BMP signaling is essential for normal lymphatic vessel development in zebrafish.     

Transgenic Zebrafish for Studying Nervous System Development and Regeneration

1 tubulin gene expression is induced in the developing and regenerating CNS of vertebrates. Therefore, 1 tubulin gene expression may serve as a good probe for mechanisms underlying CNS development and regeneration. One approach to identify these mechanisms is to work backwards from the genome. This requires identification of 1 tubulin DNA sequences that mediate its developmental and regeneration-dependent expression pattern. Therefore, we generated transgenic zebrafish harboring a fragment of the 1 tubulin gene driving green fluorescent protein expression (GFP). In these fish, and similar to the endogenous gene, transgene expression was dramatically induced in the developing and regenerating nervous system. Although transgene expression generally declined during maturation of the nervous system, robust GFP expression was maintained in progenitor cells in the retinal periphery, lining brain ventricles and surrounding the central canal of the spinal cord. When these cells were cultured in vitro they divided and gave rise to new neurons. We also show that optic nerve crush in adult fish re-induced transgene expression in retinal ganglion cells. These studies identified a relatively small region of the 1 tubulin promoter that mediates its regulated expression pattern in developing and adult fish. This promoter will be extremely useful to investigators interested in targeting gene expression to the developing or regenerating nervous system. As adult transgenic fish maintain transgene expression in neural progenitors, these fish also provide a valuable resource of labeled adult neural progenitor cells that can be studied in vivo or in vitro. Finally, these fish should provide a unique in vivo system for investigating mechanisms mediating CNS development and regeneration.     

LncRNA-NEAT1对妊娠期高血压大鼠JAK2/STAT3信号通路、炎症反应和妊娠结局的影响

摘要 目的：探讨LncRNA-NEAT1对妊娠期高血压大鼠JAK2/STAT3信号通路、炎症反应和妊娠结局的影响。方法：采用注射L-精氨酸甲酯的方法构建妊娠期高血压大鼠模型。采用Western blot检测JAK2/STAT3信号通路蛋白表达;采用ELISA法检测炎症因子和血管内皮损伤因子。观察并记录大鼠24 h蛋白尿、尾静脉压和死胎率。结果：与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组JAK2、STAT3的蛋白表达水平明显更高（P＜0.05）;与模型组相比,LncRNA-NEAT1抑制组JAK2、STAT3的蛋白表达水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组JAK2、STAT3的蛋白表达水平明显更高（P＜0.05）;与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组ET-1和sICAM-1水平明显更高,而NO水平明显更低（P＜0.05）;与模型组相比,LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组ET-1和sICAM-1水平明显更高（P＜0.05）,而NO水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组ET-1和sICAM-1表达水平明显更高,而NO明显更低（P＜0.05）;与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组TNF-α、IL-1β、IL-18表达水平明显更高（P＜0.05）;与模型组相比,LncRNA-NEAT1抑制组TNF-α、IL-1β、IL-18表达水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组TNF-α、IL-1β、IL-18表达水平明显更高（P＜0.05）;与空白组相比,模型组、LncRNA-NEAT1过表达组、LncRNA-NEAT1抑制组尾静脉压和24h蛋白尿水平明显更高（P＜0.05）;与模型组相比,LncRNA-NEAT1抑制组尾静脉压和24 h蛋白尿表达水平明显更低（P＜0.05）,而LncRNA-NEAT1过表达组尾静脉压和24 h蛋白尿表达水平明显更高（P＜0.05）;LncRNA-NEAT1过表达组（21.56%）死胎率显著高于模型组（16.72%）和LncRNA-NEAT1抑制组（5.65%）。结论：妊娠期糖尿病大鼠LncRNA-NEAT1的表达下调可抑制JAK2/STAT3信号通路的表达并下调下游促炎因子的表达,进而缓解血管内皮损伤降低死胎率。     

Nucleolar GTPase NOG-1 Regulates Development,Fat Storage,and Longevity through Insulin/IGF Signaling in C. elegans

NOG1 is a nucleolar GTPase that is critical for 60S ribosome biogenesis. Recently, NOG1 was identified as one of the downstream regulators of target of rapamycin (TOR) in yeast. It is reported that TOR is involved in regulating lifespan and fat storage in Caenorhabditis elegans. Here, we show that the nog1 ortholog (T07A9.9: nog-1) in C. elegans regulates growth, development, lifespan, and fat metabolism. A green fluorescence protein (GFP) promoter assay revealed ubiquitous expression of C. elegans nog-1 from the early embryonic to the adult stage. Furthermore, the GFP-tagged NOG-1 protein is localized to the nucleus, whereas the aberrant NOG-1 protein is concentrated in the nucleolus. Functional studies of NOG-1 in C. elegans further revealed that nog-1 knockdown resulted in smaller broodsize, slower growth, increased life span, and more fat storage. Moreover, nog-1 over-expression resulted in decreased life span. Taken together, our data suggest that nog-1 in C. elegans may be an important player in regulating life span and fat storage via the insulin/IGF pathway.