Gfi1.1 regulates hematopoietic lineage differentiation during zebrafish embryogenesis

(GFI1 ) 生长因素独立 1 为成年造血的干细胞(HSC ) 的哺乳动物的淋巴细胞和 neutrophils 和维护的成熟是重要的。在胚胎的造血作用的 GFI1 的角色是很好描绘的更少。通过一幅 enhancer 陷井屏幕和生物信息学分析，我们识别了 Gfi1 (命名 gfi1.1 ) 的一个斑马鱼相当或相同的事物并且在胚胎发育期间分析了它的功能。内长的 gfi1.1 基因和在它的 genomic 地点附近插入的 GFP 记者基因的表示在斑马鱼胚胎的造血的房间被检测。Morpholino (瞬间) gfi1.1 击倒 scl， lmo2， c-myb， mpo， rag1， gata1 和血红素高山的减少的表示哈 embryonic-1 (hbae1 ) ，以及胚胎的血红素的全部的数量，而是 pu.1 和 l-plastin 的增加的表示。在一样的条件下面，瞬间注射没影响涉及的标记脉管并且专业版肾的开发。相反地，在经由信使 rna 的 gfi1.1 的表示上，注射提高了 gata1 的表示，但是禁止了 pu.1 的表示。这些调查结果建议 Gfi1.1 在调整胚胎的 erythroid 和 myeloid 系决心的平衡起一个关键作用，并且也在斑马鱼期间为淋巴细胞和 granulocytes 的区别被要求胚胎开始。

从“毛顺灯楼”到万寿宫灯——历代元宵花灯述略

每逢元宵佳节,火树银花不夜天,分外热闹。然而,许多人存在一个误区:认为正月十五的声声爆竹、灯影婆娑是为了庆祝春节的圆满结束,实则不然。“元宵节”的正式称谓是“上元节”,和七月十五的“中元节”、十月十五的“下元节”合称“三元”。只是由于上元之期与春节相隔仅半月,兼有赏花灯、吃元宵、打灯谜等丰富多彩的庆祝活动,故而在百姓心中拥有特殊地位,人气也直线飙升,逐渐将中元、下元两节远远地甩在了后面。由是可知,元宵节并非春节的附属,而是与新春、端午、中秋并列的四大传统节日之一,其起源可以上溯至汉代。

暖阁春初入 长遣四时寒——古代宫廷的取暖生活

"孟冬十月,北风徘徊",每个人心中都渴望一处温暖的所在,躲避刺骨的严寒。寻常百姓会在家中点起炉火,挂上门帘,以提高室温。然而,对于生活在深宫禁苑的皇室而言,取暖就成了大问题。以明清两代帝王居住的紫禁城为例,占地面积多达七十二万平方米,建筑面积在十五万平方米左右,七十余座琼楼玉宇分布其间。置身于偌大的皇家建筑群中,帝后、妃嫔们究竟会采用何种手段御寒,渡过寒冷漫长的冬日呢?

SNX17 regulates Notch pathway and pancreas development through the retromer-dependent recycling of Jag1

Background:Notch is one of the most important signaling pathways involved in cell fate determination.Activation of the Notch pathway requires the binding of a membrane-bound ligand to the Notch receptor in the adjacent cell which induces proteolytic cleavages and the activation of the receptor.A unique feature of the Notch signaling is that processes such as modification,endocytosis or recycling of the ligand have been reported to play critical roles during Notch signaling,however,the underlying molecular mechanism appears context-dependent and often controversial.Results:Here we identified SNX17 as a novel regulator of the Notch pathway.SNX17 is a sorting nexin family protein implicated in vesicular trafficking and we find it is specifically required in the ligand-expressing cells for Notch signaling.Mechanistically,SNX17 regulates the protein level of Jag1a on plasma membrane by binding to Jag1a and facilitating the retromer-dependent recycling of the ligand.In zebrafish,inhibition of this SNX17-mediated Notch signaling pathway results in defects in neurogenesis as well as pancreas development.Conclusions:Our results reveal that SNX17,by acting as a cargo-specific adaptor,promotes the retromer dependent recycling of Jag1a and Notch signaling and this pathway is involved in cell fate determination during zebrafish neurogenesis and pancreas development.

Glucokinase activator improves glucose tolerance and induces hepatic lipid accumulation in mice with diet-induced obesity

Background and aims:Type 2 diabetes mellitus remains a substantial medical problem with increasing global prevalence.Pharmacological research is becoming increasingly focused on personalized treatment strategies.Drug development based on glucokinase(GK)activation is an important strategy for lowering blood glucose.This study aimed to investigate the effect of GK activation on glucose and lipid metabolism in diet-induced obese mice.Materials and methods:Mice were fed with a high-fat diet(HFD)for 16 weeks to induce obesity,followed by a GK activator(GKA,AZD1656)or vehicle treatment by gavage for 4 weeks.The effect of GKA treatment on glucose metabolism was evaluated using glucose and insulin tolerance tests.Hepatic lipid accumulation was assessed by hematoxylin and eosin staining,Oil Red O staining,and transmission electron microscopy.The underlying mechanism of GK activation in glucose and lipid metabolism in the liver was studied using transcriptomic analysis,with a mechanistic study in mouse livers in vivo and AML12 cells in vitro.Results:GK activation by GKA treatment improved glucose tolerance in HFD-fed mice while increasing hepatic lipid accumulation.Transcriptomic analysis of liver tissues indicated the lipogenesis and protein kinase RNA-like endoplasmic reticulum kinase(PERK)-unfolded protein response(UPR)pathway activations in GKA-treated HFD-fed mice.Inhibition of the ACC activity,which is an important protein in lipogenesis,attenuated GKA treatment-induced lipid accumulation and PERK-UPR activation in vitro.Conclusions:GK activation improved glucose tolerance and insulin sensitivity while inducing hepatic lipid accumulation by increasing the lipogenic gene expression,which subsequently activated the hepatic PERK-UPR signaling pathway.

Efficient Gene Targeting in Zebrafish Mediated by a Zebrafish-Codon-Optimized Cas9 and Evaluation of Off-Targeting Effect

Targeted genome modifications with the Cas9/gRNA system derived from the clustered regularly interspaced short palin- dromic repeat/CRISPR-associated （CRISPR/Cas） system have been successfully used in cultured human cells as well as in most model organisms, including zebrafish （Danio rerio）, mouse, and fruit fly （Chang et al., 2013; Cong et al., 2013; Gratz et al., 2013; Hwang et al., 2013; Jao et al., 2013; Shen et al., 2013; Wei et al., 2013）. Its application in zebrafish is particu- larly attractive due to the ease of handling this organism and the simple application of this method by direct injection of Cas9/ gRNA. However, the information about its specificity in this organism is very limited and needs further evaluation. In addition, it is conceivable that a Cas9 mRNA optimized for zebrafish codon preference could enhance its activity.

Engineering vascularized skeletal muscle tissue with transcriptional factor ETV2-induced autologous endothelial cells

Dear Editor,Newly engineered tissues often fail to function due to insufficient blood vessels formation.Autologous vascular endothelial cells are ideal sources for tissue engineering but are often of limited availability.We first identified that transcriptional factor Etv2(ets variant 2,initially named as Etsrp)was specifically expressed in the zebrafish vascular endothelial cells and was required for vascular development in zebrafish(Sumanas and Lin,2006).

Reverse Genetic Approaches in Zebrafish

Zebrafish（Danio rerio） is a well-established vertebrate animal model.A comprehensive collection of reverse genetics tools has been developed for studying gene function in this useful organism.Morpholino is the most widely used reagent to knock down target gene expression post-transcriptionally.For a long time,targeted genome modification has been heavily relied on large-scale traditional forward genetic screens,such as ENU（N-ethyl-N-nitrosourea） mutagenesis derived TILLING（Targeting Induced Local Lesions IN Genomes） strategy and pseudo-typed retrovirus mediated insertional mutagenesis.Recently,engineered endonucleases,including ZFNs（zinc finger nucleases） and TALENs（transcription activator-like effector nucleases）,provide new and efficient strategies to directly generate site-specific indel mutations by inducing double strand breaks in target genes.Here we summarize the major reverse genetic approaches for loss-of-function studies used and emerging in zebrafish,including strategies based on genome-wide mutagenesis and methods for site-specific gene targeting.Future directions and expectations will also be discussed.

A Zebrafish Model of 5q-Syndrome Using CRISPR/Cas9 Targeting RPS14 Reveals a p53-Independent and p53-Dependent Mechanism of Erythroid Failure

5q-syndrome is a distinct form of myelodysplastic syndrome（MDS） where a deletion on chromosome 5 is the underlying cause.MDS is characterized by bone marrow failures,including macrocytic anemia.Genetic mapping and studies using various models support the notion that ribosomal protein S14（RPS14） is the candidate gene for the erythroid failure.Targeted disruption of RPS14 causes an increase in p53 activity and p53-mediated apoptosis,similar to what is observed with other ribosomal proteins.However,due to the higher risk for cancer development in patients with ribosome deficiency,targeting the p53 pathway is not a viable treatment option.To better understand the pathology of RPS14 deficiency in 5q-deletion,we generated a zebrafish model harboring a mutation in the RPS14 gene.This model mirrors the anemic phenotype seen in 5q-syndrome.Moreover,the anemia is due to a late-stage erythropoietic defect,where the erythropoietic defect is initially p53-independent and then becomes p53-dependent.Finally,we demonstrate the versatility of this model to test various pharmacological agents,such as RAP-011,L-leucine,and dexamethasone in order to identify molecules that can reverse the anemic phenotype.

Identification and expression analysis of mical family genes in zebrafish

Mical （molecule interacting with CasL） represent a conserved family of cytosolic multidomain proteins that has been shown to be as- sociated with a variety of cellular processes, including axon guidance, cell movement, cell-cell junction formation, vesicle trafficking and cancer cell metastasis. However, the expression and function of these genes during embryonic development have not been comprehen- sively characterized, especially in vertebrate species, although some limited in vivo studies have been carried out in neural and muscula- ture systems of Drosophila and in neural systems of vertebrates. So far, no mical family homologs have been reported in zebrafish, an ideal vertebrate model for the study of developmental processes. Here we report eight homologs of mical family genes in zebrafish and their expression profiles during embryonic development. Consistent with the findings in Drosophila and mammals, most zebra_fish mical family genes display expression in neural and musculature systems. In addition, five mical homologs are detected in heart, and one, mi- call2a, in blood vessels. Our data established an important basis for further functional studies of mical family genes in zebrafish, and suggest a possible role for mical genes in cardiovascular development.

RNA methylation regulates hematopoietic stem and progenitor cell development

Methylation of adenosine base on the nitrogen-6 position （N6-methyladenosine, m^6A） is the most common and abundant modification on mRNA transcripts. This post-transcriptional modification was first described in the 1970s in hepatoma cells （Desrosiers et al., 1974）.

ETV2 mediates endothelial transdifferentiation of glioblastoma

Glioblastoma multiforme(GBM)is characterized by extensive endothelial hyperplasia.Recent studies suggest that a subpopulation of endothelial cells originates via vasculogenesis by the transdifferentiation of GBM tumor cells into endothelial cells(endotransdifferentiation).The molecular mechanism underlying this process remains poorly defined.Here,we show that the expression of ETS variant 2(ETV2),a master regulator of endothelial cell development,is highly correlated with malignancy.Functional studies demonstrate that ETV2 is sufficient and necessary for the transdifferentiation of a subpopulation of CD133+/Nestin+GBM/neural stem cells to an endothelial lineage.Combinational studies of ChIP-Seq with gain-of-function RNA-Seq data sets suggest that ETV2,in addition to activating vascular genes,represses proneural genes to direct endo-transdifferentiation.Since endotransdifferentiation by ETV2 is VEGF-A independent,it likely accounts for the observed resistance of GBM tumor cells to antiangiogenesis therapy.Further characterization of the regulatory networks mediated by ETV2 in endo-transdifferentiation of GBM tumor cells should lead to the identification of more effective therapeutic targets for GBM.

Questions about NgAgo

Dear Editor： Gao et al. published data in Nature Biotechnology （Nat Biotechnol. 2016 May 2） showing that DNA-guided genome editing using the Natronobacterium gregoryi Argonaute （NgAgo） protein targeted 47 mammalian genomic loci with a 100% success rate and an efficiency of 21.3%-41.3% at various targets. This report led us to test NgAgo＇s utility in various cells and organisms such as mouse and zebrafish for gene editing.

Erratum to： Questions about NgAgo

In the original publication of this article the Figure 1E legend has been incorrectly published.

Optimized Target-AID system efficiently induces single base changes in zebrafish

CRISPR-Cas system has been widely adapted as a platform for genome editing in various eukaryotic organisms, including zebrafish（Cong et al., 2013; Hwang et al., 2013）. One of the important applications of CRISPR-Cas9 system is to produce double-strand DNA breaks（DSBs） at targeted sites with guide RNA（gRNA）.

ETV2 expression increases the efficiency of primitive endothelial cell derivation from human embryonic stem cells

Background:Endothelial cells line the luminal surface of blood vessels and form a barrier between the blood and other tissues of the body.Ets variant 2(ETV2)is transiently expressed in both zebrafish and mice and is necessary and sufficient for vascular endothelial cell specification.Overexpression of this gene in early zebrafish and mouse embryos results in ectopic appearance of endothelial cells.Ectopic expression of ETV2 in later development results in only a subset of cells responding to the signal.Findings:We have examined the expression pattern of ETV2 in differentiating human embryonic stem cells(ESCs)to determine when the peak of ETV2 expression occurs.We show that overexpression of ETV2 in differentiating human ESC is able to increase the number of endothelial cells generated when administered during or after the endogenous peak of gene expression.Conclusions:Addition of exogenous ETV2 to human ESCs significantly increased the number of cells expressing angioblast genes without arterial or venous specification.This may be a viable solution to generate in vitro endothelial cells for use in research and in the clinic.