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。

Single-cell analysis reveals an Angpt4-initiated EPDC-EC-CM cellular coordination cascade during heart regeneration

Mammals exhibit limited heart regeneration ability,which can lead to heart failure after myocardial infarction.In contrast,zebrafish exhibit remarkable cardiac regeneration capacity.Several cell types and signaling pathways have been reported to participate in this process.However,a comprehensive analysis of how different cells and signals interact and coordinate to regulate cardiac regeneration is unavailable.We collected major cardiac cell types from zebrafish and performed high-precision single-cell transcriptome analyses during both development and post-injury regeneration.We revealed the cellular heterogeneity as well as the molecular progress of cardiomyocytes during these processes,and identified a subtype of atrial cardiomyocyte exhibiting a stem-like state which may transdifferentiate into ventricular cardiomyocytes during regeneration.Furthermore,we identified a regeneration-induced cell(RIC)population in the epicardium-derived cells(EPDC),and demonstrated Angiopoietin 4(Angpt4)as a specific regulator of heart regeneration.angpt4 expression is specifically and transiently activated in RIC,which initiates a signaling cascade from EPDC to endocardium through the Tie2-MAPK pathway,and further induces activation of cathepsin K in cardiomyocytes through RA signaling.Loss of angpt4 leads to defects in scar tissue resolution and cardiomyocyte proliferation,while overexpression of angpt4 accelerates regeneration.Furthermore,we found that ANGPT4 could enhance proliferation of neonatal rat cardiomyocytes,and promote cardiac repair in mice after myocardial infarction,indicating that the function of Angpt4 is conserved in mammals.Our study provides a mechanistic understanding of heart regeneration at single-cell precision,identifies Angpt4 as a key regulator of cardiomyocyte proliferation and regeneration,and offers a novel therapeutic target for improved recovery after human heart injuries.

新时代中国科技期刊出版的机遇与挑战

基于中国科学院学部主办的以"新时代的科技出版"为主题的科学与技术前沿论坛的主要研讨成果,结合文献调研,对我国科技期刊的现状、面临的机遇和挑战等进行综述,并提出对策建议.本文从期刊和论文的开放获取出版、开放同行评审、开放数据、预印本平台、期刊的经营模式等方面论述了开放科学背景下的科技期刊出版,并从数字出版与传播、大数据、区块链、人工智能、基于各种新型技术和传播媒介的互动与融合等方面讨论了新技术如何推动科技出版创新发展.与国际一流科技期刊相比较,我国科技期刊在吸引高水平研究成果的能力、学术不端行为防范与处理、品牌声誉与影响力等方面都还有很大差距.在当前我国科学研究快速发展和建设世界一流期刊的良好出版环境和政策背景下,我国科技期刊面临着前所未有的发展机遇.本文提出新时代中国科技期刊的发展对策建议主要如下:(1)关注开放科学的发展趋势,融入开放科学的发展环境;(2)加强国际合作,在合作中不断发展;(3)创新科技出版服务理念,扩展出版新技术和新方式;(4)加强出版伦理规范建设,增强处理学术不端问题的能力;(5)加强科技出版人才队伍建设,提升科学传播者的科学素养;(6)寻求社会效益和市场效益的共赢,积极探索可持续发展模式.

High efficient gene targeting in rice field eel Monopterus albus by transcription activator-like effector nucleases

Genome editing plays an important role in the research of gene functions,gene correction and cell replacement therapy.Traditionally,genome editing is mainly dependent on the technology of homologous recombination(HR)and embryonic stem(ES)cell lines in mammals[1].However,genome editing is severely limited by the low efficiency of HR and lack of ES cell lines in most organisms.Recently,the development of zinc finger nucleases(ZFNs),

Time course of foreign gene integration and expression in transgenic fish embryos

Using a nuclear transplantation approach, the integration and expression of the green fluorescent protein (GFP) gene in the embryogenesis of transgenic loach (Misgurnus anguillicaudatus Cantor) have been studied. The GFP gene expression is first observed at the gastrula stage, which is consistent with the initiation of cell differentiation of fish embryos. The time course of the foreign gene expression is correlated with the regulatory sequences. The expression efficiency also depends on the gene configuration: the expression of pre-integrating circular plasmid at early embryos is higher than that of the linear plasmid. The integration of the GFP gene is first detected at the blastula stage and lasts for quite a long period. When two types of different plasmids are co-injected into fertilized eggs, the behavior of their integration and expression is not identical.

Surrogate production of genome-edited sperm from a different subfamily by spermatogonial stem cell transplantation

The surrogate reproduction technique,such as inter-specific spermatogonial stem cells(SSCs)transplantation(SSCT),provides a powerful tool for production of gametes derived from endangered species or those with desirable traits.However,generation of genome-edited gametes from a different species or production of gametes from a phylogenetically distant species such as from a different subfamily,by SSCT,has not succeeded.Here,using two small cyprinid fishes from different subfamilies,Chinese rare minnow(gobiocypris rarus,for brief:Gr)and zebrafish(danio rerio),we successfully obtained Gr-derived genome-edited sperm in zebrafish by an optimized SSCT procedure.The transplanted Gr SSCs supported the host gonadal development and underwent normal spermatogenesis,resulting in a reconstructed fertile testis containing Gr spermatids and zebrafish testicular somatic cells.Interestingly,the surrogate spermatozoa resembled those of host zebrafish but not donor Gr in morphology and swimming behavior.When pou5f3 and chd knockout Gr SSCs were transplanted,Gr-derived genome-edited sperm was successfully produced in zebrafish.This is the first report demonstrating surrogate production of gametes from a different subfamily by SSCT,and surrogate production of genome-edited gametes from another species as well.This method is feasible to be applied to future breeding of commercial fish and livestock.

Designing future farmed fishes using genome editing

Aquaculture,the most rapidly growing food production sector,has exhibited tremendous expansion over the past several decades and it currently accounts for more than half of the global fish production for human consumption.To ensure sustainable aquaculture,it is necessary to breed farmed fish species(strains)with valuable economic traits,such as high production,high quality,disease resistance,and stress tolerance.Modem fish genetic breeding techniques largely rely on key processes including understanding the genetic basis of economically important traits to the generation and selection of favorable genotypes.

Nuclear transplantation in different strains of zebrafish

Single later blastula nuclei from AB strain of zebrafish (Danio rerio) were transplanted into enucleated unfertilized eggs of Long fin strain. Of 1119 cloning embryos, 14 reconstructed embryos developed into fry. DNA fingerprinting systems of the cloned fish were similar to those of the nuclear donor fish, but were distinctly different from those of the nuclear recipient fish. It confirmed that the genetic material originated from nuclear donor cell other than from nuclear recipient egg. The research suggested that the basic technique for nuclear transplantation performed with different strains of zebrafish has made a breakthrough. It should be helpful for the study of some important developmental problems such as gene function, the regulation of gene expression during animal development, the developmental potential of a nucleus and the interactions between the donor nucleus and the recipient cytoplasm, etc.

The integration characteristics of the exogenous growth hormone gene in a transgenic common carp(Cyprinus carpio L.) with fast-growth performance

The genetic stability and expression efficiency of exogenous genes in transgenic animals are closely related to integration site and copy number. In our laboratory, by transgenic manipulation and subsequent test crosses, we established an ‘‘all-fish'' growth hormone(GH)transgenic common carp family that exhibits fast growth.In this present study, genome walking, real-time quantitative polymerase chain reaction, and fluorescence in situ hybridization techniques were applied to identify the integration characteristics of the exogenous grass carp GH gene in the transgenic common carp. The exogenous GH genes, in the form of two complete and one incomplete tandem repeats, were found to have integrated into an ATrich region near the end of a chromosome pair. We hypothesize that the high efficiency of exogenous GH gene expression might be due to the low copy number in the genome and the AT-rich integration site.

Chromosomal in situ hybridization of Cyprinus carpio genomic DNA repetitive sequence CR1

Common carp Cyprinus carpio genomic DNA repetitive sequence CR1 has been DIG-labeled and hybridized in situ against chromosomes of red common carp ( Cyprinus carpio L. Xingguo red var.). It is found that the repetitive sequence CR1 is mainly localized at the cen-tromeric regions of chromosomes of the red common carp. The application of the

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.

Efficient generation of zebrafish maternal-zygotic mutants through transplantation of ectopically induced and Cas9/gRNA targeted primordial germ cells

The clustered regularly interspaced short palindromic repeats(CRISPR)/Cas9 technology has been widely utilized for knocking out genes involved in various biological processes in zebrafish. Despite this technology is efficient for generating different mutations, one of the main drawbacks is low survival rate during embryogenesis when knocking out some embryonic lethal genes. To overcome this problem, we developed a novel strategy using a combination of CRISPR/Cas9 mediated gene knockout with primordial germ cell(PGC) transplantation(PGCT) to facilitate and speed up the process of zebrafish mutant generation, particularly for embryonic lethal genes. Firstly, we optimized the procedure for CRISPR/Cas9 targeted PGCT by increasing the efficiencies of genome mutation in PGCs and induction of PGC fates in donor embryos for PGCT. Secondly, the optimized CRISPR/Cas9 targeted PGCT was utilized for generation of maternal-zygotic(MZ) mutants of tcf7l1a(gene essential for head development), pou5f3(gene essential for zygotic genome activation) and chd(gene essential for dorsal development) at F1 generation with relatively high efficiency. Finally, we revealed some novel phenotypes in MZ mutants of tcf7l1 a and chd, as MZtcf7l1 a showed elevated neural crest development while MZchd had much severer ventralization than its zygotic counterparts. Therefore, this study presents an efficient and powerful method for generating MZ mutants of embryonic lethal genes in zebrafish. It is also feasible to speed up the genome editing in commercial fishes by utilizing a similar approach by surrogate production of CRISPR/Cas9 targeted germ cells.

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.

ASER:Animal Sex Reversal Database

Sex reversal,representing extraordinary sexual plasticity during the life cycle,not only triggers reproduction in animals but also affects reproductive and endocrine system-related diseases and cancers in humans.Sex reversal has been broadly reported in animals;however,an integrated resource hub of sex reversal information is still lacking.Here,we constructed a comprehensive database named ASER(Animal Sex Reversal)by integrating sex reversal-related data of 18 species from teleostei to mammalia.We systematically collected 40,018 published papers and mined the sex reversal-associated genes(SRGs),including their regulatory networks,from 1611 core papers.We annotated homologous genes and computed conservation scores for whole genomes across the 18 species.Furthermore,we collected available RNA-seq datasets and investigated the expression dynamics of SRGs during sex reversal or sex determination processes.In addition,we manually annotated 550 in situ hybridization(ISH),fluorescence in situ hybridization(FISH),and im-munohistochemistry(IHC)images of SRGs from the literature and described their spatial expression in the gonads.Collectively,ASER provides a unique and integrated resource for researchers to query and reuse organized data to explore the mechanisms and applications of SRGs in animal breeding and human health.The ASER database is publicly available at http://aser.ihb.ac.cn/.

Bi-FoRe:an efficient bidirectional knockin strategy to generate pairwise conditional alleles with fluorescent indicators

Gene expression labeling and conditional manipulation of gene function are important for elaborate dissection of gene function.However,contemporary generation of pairwise dual-function knockin alleles to achieve both conditional and geno-tagging effects with a single donor has not been reported.Here we first developed a strategy based on a flipping donor named FoRe to generate conditional knockout alleles coupled with fluorescent allele-labeling through NHEJ-mediated unidirectional targeted insertion in zebrafish facilitated by the CRISPR/Cas system.We demonstrated the feasibility of this strategy at sox10 and isl1 loci,and successfully achieved Cre-induced conditional knockout of target gene function and simultaneous switch of the fluorescent reporter,allowing generation of genetic mosaics for lineage tracing.We then improved the donor design enabling efficient one-step bidirectional knockin to generate paired positive and negative conditional alleles,both tagged with two different fluorescent reporters.By introducing Cre recombinase,these alleles could be used to achieve both conditional knockout and conditional gene restoration in parallel;furthermore,differential fluorescent labeling of the positive and negative alleles enables simple,early and efficient realtime discrimination of individual live embryos bearing different genotypes prior to the emergence of morphologically visible phenotypes.We named our improved donor as Bi-FoRe and demonstrated its feasibility at the sox10 locus.Furthermore,we eliminated the undesirable bacterial backbone in the donor using minicircle DNA technology.Our system could easily be expanded for other applications or to other organisms,and coupling fluorescent labeling of gene expression and conditional manipulation of gene function will provide unique opportunities to fully reveal the power of emerging single-cell sequencing technologies.

Father of biological cloning in China

Dizhou Tong(T.C.Tung)is a well-familiar name in China,as there is a story about him in one of the elementary school textbooks.The story describes how he came from a poor family and could not go to middle school until the age of 17,and how his resolution and diligence made him a successful biologist.But in the academic community,he is remembered and respected for another reason—the outstanding achievement of cross-species cloning in fish.

Progress in research on fish sex determining genes

Research on fish sex determining genes and their mode of action is not only important for elucidating the mechanisms of vertebrate sex determination,but also for fish monosex control breeding.This paper briefly summarizes our current knowledge about vertebrate sex determination,and reviews the progress of research on fish sex determining genes,including their function and their mechanism of action.In recent years,the number of newly uncovered fish sex determining genes has grown rapidly.In contrast,no sex determining genes have been identified in Cyprinid fishes,which constitute most of the aquaculture fish species in China.Technologies such as high-throughput sequencing and gene editing will provide important technical support for the study of sex determining genes in fish,and play an important role in establishing accurate sex-controlling breeding techniques and the creation of new fish germplasm.