Dicer Is Required for Embryonic Angiogenesis during Mou Development*
Received for publication,November 29,2004
Published,JBC Papers in Press,December 21,2004,DOI 10.1074/jbc.M413394200
Wei J.Yang,Derek D.Yang,Songqing Na,George E.Sandusky,Qing Zhang,and Genshi Zhao‡
From the Lilly Rearch Laboratories,Eli Lilly and Company,Indianapolis,Indiana 46285
Dicer is a multi-domain protein responsible for the generation of short interfering RNAs (siRNAs)from long double-stranded RNAs during RNA interference.It is also involved in the maturation of microRNAs,some of which are transcriptional regulators of developmental timing in nematodes.To asss the role of Dicer in mam-mals,we generated Dicer ex1/2mice with a deletion of the amino acid quences corresponding to the first and cond exons of the dicer gene via homologous recombi-nation.We found that Dicer ex1/2homozygous embryos displayed a retarded phenotype and died between days 12.5and 14.5of gestation.Thus,the results show that dicer ex1/2is verely hypomorphic and that Dicer is es-ntial for normal mou development.Interestingly,we also found that blood vesl formation/maintenance in dicer ex1/2embryos and yolk sacs were verely compro-
mid,suggesting a possible role for Dicer in angiogen-esis.This finding is consistent with the altered expres-sion of vegf ,flt1,kdr ,and tie1in the mutant embryos.Taken together,the results of this study indicate that Dicer exerts its function on mou embryonic angiogen-esis probably through its role in the processing of mi-croRNAs that regulate the expression levels of some critical angiogenic regulators in the cell.
RNA interference (RNAi)1is a post-transcriptional gene reg-ulation process that is conrved in organisms ranging from fungi to humans (1–5).When cells encounter long double-stranded RNA molecules,Dicer,a ribonuclea III type en-zyme,cleaves them into small interfering RNAs (siRNAs)of 21–23nucleotides.The siRNAs are incorporated into a mul-ticomponent protein complex known as RISC (RNA-induced silencing complex).The antin strand of siRNAs is believed to guide the RISC to locate its cognate mRNA molecule.As a result,the mRNAs are degraded (6–8).
As one of the critical enzymes of the RNAi pathway,Dicer was first identified from Drosophila embryo and S2cell ex-tracts as the initiation enzyme for RNAi (7).It is a large (ϳ220kDa),multi-domain protein that consists of an amino-terminal helica domain followed by a PAZ domain,two RNa III domains,and a C-terminal double-stranded RNA-binding do-main (dsRBD).The dsRBD and RNa II
I domains are involved
in the binding to and cleavage of long double-stranded RNAs,but the functions of other domains remain unclear (9).Genetic and biochemical studies have established that Dicer is required for the production of siRNAs from double-stranded RNAs in Caenorhabditis elegans ,Drosophila ,and humans.
In addition to its important role in the RNAi pathway,Dicer also plays pivotal roles in development.In C.elegans ,inacti-vation of the dcr-1(homolog of Drosophila Dicer)gene results in defects in the RNAi pathway as well as developmental ab-normalities similar to tho caud by the loss of function of let-7,a gene encoding a microRNA (miRNA)esntial for the control of developmental timing in this organism (10,11).This finding suggests that Dicer is also responsible for processing miRNA precursors,which often form a stem-loop structure creating a double-stranded RNA molecule (12).In addition,human Dicer was shown to be responsible for the generation of mature let-7in HeLa cells (13).Therefore,the function of Dicer in development may be well conrved during evolution.乳臭未干
Currently,there is an emerging interest in miRNAs becau,under normal conditions,miRNAs rather than siRNAs appear to have a major role in small RNA-mediated gene regulation.Therefore,the princ
iple role of Dicer in vivo may be involved in the biogenesis of mRNAs (14).Recently,ϳ90miRNAs have been identified from C.elegans ,Drosophila ,mou tissues,and human cell cultures.Among the,nine are found in more than one phylum (15–18).This finding suggests that miRNA-medi-ated post-transcriptional regulation may function in a wide variety of organisms.Furthermore,the large numbers of miR-NAs as well as the source from where they have been identified also suggest that miRNAs may be involved in the regulation of multiple physiological pathways (14).
To asss the function of Dicer in a mammalian system,we sought to generate Dicer-deficient mice via homologous recom-bination in embryonic stem cells.We show that dicer ex1/2em-bryos are growth and developmentally retarded and die be-tween embryonic day (E)12.5and 14.5.Therefore,Dicer is esntial for normal development.In addition,the mutant em-bryos and their yolk sacs are found to display defects in the blood vesl formation/maintenance,suggesting a role for Dicer in the regulation of embryonic angiogenesis via its function in the processing of miRNAs.
EXPERIMENTAL PROCEDURES
Construction of a dicer Targeting Vector—A DNA primer correspond-ing to the 5Ј-region of the mou dicer genomic quence was ud to screen a mou CITB BAC library by Rearch Geneti
cs (Carlsbad,CA).One positive clone (474E 20)was identified and confirmed by colony PCR using two primers,wjy1and wjy3,corresponding to mou dicer cDNA quences (wjy1,5Ј-AGCATGGCAGGCCTGCAG-3Ј;wjy3,5Ј-TGATGGGCCAGCTCTTTGG-3Ј).An ϳ10-kb BamHI-XhoI fragment from this BAC clone was ligated into a pBluescript plasmid (Strat-agene).The 1.2-kb HindIII-PmlI fragment containing exons 1and 2of dicer was then replaced by a neo expression castte.
Generation of dicer ex1/2Mice—The targeting construct was linearized at the XhoI site and introduced into mou embryonic stem cell E14via
*The costs of publication of this article were defrayed in part by the payment of page charges.This article must therefore be hereby marked “advertiment ”in accordance with 18U.S.C.Section 1734solely to indicate this fact.
‡To whom correspondence should be addresd:Cancer Rearch,Lilly Corporate Center,DC 0424,Indianapolis,IN 46285.Tel.:317-276-2040;Fax:317-276-6510;E-mail:Zhao_1
The abbreviations ud are:RNAi,RNA interference;siRNA,small interfering RNA;miRNA,microRNA;E,embryonic day;PECAM,plate-let endothelial cell adhesion molecule;RT,rev
er transcription;VEGF,vascular endothelial growth factor.
T HE J OURNAL OF B IOLOGICAL C HEMISTRY
Vol.280,No.10,Issue of March 11,pp.9330–9335,2005
©2005by The American Society for Biochemistry and Molecular Biology,Inc.Printed in U.S.A.
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electroporation.G418-resistant colonies lected were screened for the homologous recombination by Southern blotting analysis using a3Ј-flanking probe,a5Ј-flanking probe,and a neo probe.Both the3Ј-and 5Ј-probes were generated by PCR reaction off the BAC clone.The primers ud for the3Ј-probe were5Ј-TCTTCGTCGAAACGTACAAG-3Јand5Ј-TGGAAAGACCCTCATTCCAAG-3Ј.T
he primers for amplifying the5Ј-probe were5Ј-GTTATTACCACTAAATATCACG-3Јand5Ј-CT-GCCAAGGCTTTGTTTCAC-3Ј.The neo probe was a259-bp PstI frag-ment from a plasmid containing the neo castte.Three of six positive clones identified were microinjected into blastocysts and implanted into pudopregnant C57BL/6(B6)females.Chimeric males were mated with B6females(Harland)to yield heterozygous F1offspring,which were then intercrosd to produceϩ/ϩ,ϩ/Ϫ,Ϫ/Ϫoffspring.Tail DNA from mice was isolated using genomic isolation kit from Lamda Bio-technology,and genotyping was carried out by PCR reaction using the primers KOF(5Ј-AGCCATCTCCCCAGAAGTCC-3Ј),which was the for-ward primer common for both the wild-type and the targeted allele, KOR2(5Ј-CCAAAGAACGGAGCCGGTTG-3Ј),and KOR1(5Ј-CGTG-TAGGGTTCAGTCATTCGT-3Ј),which were designed for the amplifica-tion of the wild type and the targeted alleles,respectively.
Analysis of Embryos—Detection of vaginal plugs in the morning was taken as gestation day0.5(E0.5).Embryos were staged and discted from the pregnant females.Yolk sacs from different stage embryos were
dissolved in a PCR lysis buffer(50m M KCl,1.5m M MgCl
2,10m M
Tris-HCl,pH8.5,0.01%gelatin,0.45%Nonidet P-40,0.45%Tween,and 100g/ml proteina K)for4–12h.The proteina K was inactivated by incubation at95°C for5min prior to PCR analysis.PCR was carried out in20-l reaction mixtures using Taq Platinum(Invitrogen)accord-ing to the manufacturer’s suggestions.For histology,embryos were fixed overnight in3%paraformaldehyde in phosphate-buffered saline, dehydrated,and embedded in paraffin.Ten-micron ctions were stained with hematoxylin and eosin.
Northern Blotting Analysis—Total RNA was isolated from wild type, heterozygous,and mutant mou embryonic fibroblasts.Mesnger
RNA was purified using an Oligotex mRNA isolation kit(Qiagen).Each mRNA sample(5g)was parated in a glyoxal-agaro gel,trans-ferred to nylon membranes,and hybridized with a32P-labeled cDNA probe generated by PCR reaction using wjy1and wjy3under the con-ditions described earlier(19).The same probe was also ud to hybrid-ize to a membrane containing RNAs derived from different mice tissues obtained at different developmental stages(Clontech).
Antibody Generation and Western Blotting Analysis—A Dicer-spe-cific antibody was generated against a peptide corresponding to the C terminus of the mou Dicer protein with an amino acid
quence of H-Cys-Leu-Arg-Ser-Leu-Lys-Ala-Asn-Gln-Pro-Gln-Val-Pro-Asn-Ser-OH.Proteins(100g)from whole embryos of wild type or dicer ex1/2 embryos at E14.5were fractionated on an SDS-polyacrylamide gel (4–20%)and transferred onto polyvinylidene difluoride membranes. The membranes were probed with a rabbit anti-Tie1antibody(Re-arch Diagnostics,Inc.)at2g/ml,a rabbit anti-Glut1antibody(Alpha Diagnostics)at2g/ml,a mou anti-actin antibody(Sigma)at 1:10,000,a rabbit anti-Dicer antirum(Anaspec)at1:500,or a rabbit anti-PTEN antibody(Upstate)at1:1000and,subquently,with ap-propriate peroxida-conjugated condary antibodies.The signals were detected with a Supersignal West Femto maximum nsitivity substrate(Pierce).
TaqMan RT-PCR Analysis—Total RNA was isolated from wild type and mutant embryos using TRIzol(Invitrogen)according to the manu-facturer’s protocol.Mesnger RNA was purified using an Oligotex mRNA isolation kit(Qiagen).The mRNA(10ng)was subjected to quantitative RT-PCR analysis using300n M primers and200n M probe in25-l reaction mixtures.TaqMan probes and primers ud for the re-actions were as follows:ang-1probe,5Ј-CAGAAAACGGAGGGAGAAG-ATATAACCGGA-3Ј;ang-1forward primer,5Ј-GCAACCAGCGCCGAA-AT-3Ј;ang-1rever primer,5Ј-GGCACATTGCCCATGTTGA-3Ј;ang-2 probe,5Ј-ATACAAAGAGGGCTTCGGGAGCCCTC-3Ј;ang-2f
orward primer,5Ј-GACTTCCAGAGGACGTGGAAAG-3Ј;ang-2rever primer, 5Ј-CTCATTGCCCAGCCAGTACTC-3Ј;flt1probe,5Ј-ACACCTGTCGC-GTGAAGAGTGGGTC-3Ј;flt-1forward primer,5Ј-CAATGTGGAGAG-CCGAGACA-3Ј;flt-1rever primer,5Ј-GAGGTGTTGAAAGACTGGA-ACGA-3Ј;kdr probe,5Ј-CTGGCTCCTTCTTGTCATTGTCCTACGG-3Ј; kdr forward primer,5Ј-ACTGCAGTGATTGCCATGTTCT-3Ј;kdr re-ver primer,5Ј-TCATTGGCCCGCTTAACG-3Ј;tie-1probe,5Ј-ACCC-CGTGCTGGAGTGGGAGG-3Ј;tie-1forward primer,5Ј-CACAGCCTG-AGCCCTTGAGT-3Ј;tie-1rever primer,5Ј-CCTATGAGGTCCTCAA-AGGTGATG-3Ј;vegf probe,5Ј-CCGCTGATGCGCTGTGCAGG-3Ј;vegf forward primer,5Ј-CATCTTCAAGCCGTCCTGTGT-3Ј;vegf rever primer,5Ј-CTCCAGGGCTTCATCGTTACA-3Ј.The pten probe and primer ts were obtained as a20ϫmixture from Applied Biosystems.
In Situ Hybridization and Immunohistochemistry—Anϳ400-bp di-cer3Ј-cDNA fragment was generated by PCR reactions from an E11.5 limb cDNA library(Stratagene)using the primers5Ј-GACTTGCA-CACGGAGCAGTG-3Јand5Ј-GCATCTCCCAGGAATTCTAAG-3Ј.This fragment was cloned into a pGEM-T easy vector(Promega).Sen and antin digoxigenin-labeled riboprobes were synthesized using T7 and SP6RNA polymeras,respectively.E10.5embryos from white Swiss mice(Harland)were collected and procesd for in situ hybrid-ization as described(20)and photographed.PECAM immunohisto-chemistry of yolk sacs were performed as described(21).
猴和羊合不合RESULTS
The dicer Gene Is Expresd in Mou Embryos and Adult Tissues—To determine the relative levels of dicer expression during development,a dicer cDNA probe(400bp)was hybrid-ized to mRNAs isolated from mou embryos at various devel-opmental stages(Fig.1A).A relatively low level of the dicer transcript(ϳ6.5kb)was detected in7-day old embryos.Levels of this transcript were significantly higher in11-day embryos and remained constant through17-day embryos,suggesting that Dicer may function during this period of development. To further determine the spatial expression patterns of dicer in developing embryos,in situ hybridization analysis of RNA was performed.At E10.5,the dicer mRNA was detected at similar levels throughout the embryos(Fig.1D).
The expression of dicer in veral adult tissues was also examined.Interestingly,dicer was strongly expresd in the heart,liver,and kidney.This suggests that Dicer may have a more specialized function in the organs.Recently,Lagos-Quintana et al.(18)identified34novel miRNAs by tissue-specific cloning ofϳ21-nucleotide RNAs from adult mice,many of which were derived from the heart,kidney,and liver.Thus, this finding is consistent with expression profiles of dicer in the organs.Low levels of dicer expression were also obrved in the brain,spleen,lung,skeletal muscle,and testis(Fig.1B). Generation of dicer ex1/2via Gene Targeting—To determine the in vivo fu
nction of Dicer during development,a targeting vector was designed to disrupt the dicer by replacing the
first
F IG.1.Expression of dicer mRNA during development and in adult tissues.A and B,poly(A)mRNA from embryonic days7,11,15, and17(A)and adult mou tissues(MTN blots;Clontech)(B)were hybridized with a dicer3ЈcDNA probe as described under“Experimen-tal Procedures.”Anϳ6.5-kb dicer transcript is indicated by the arrow. C,E10.5embryos were hybridized with a dicer n probe.D,E10.5 embryos were hybridized with a dicer antin probe.The dicer mRNA was detected at similar levels throughout the embryos.
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two exons of the dicer gene with a neo expression castte through homologous recombination in embryonic stem cells (Fig.2A ).The construct was introduced into E14embryonic stem cells,which were cultured under standard lective con-ditions.G418resistant clones were analyzed via Southern blot-ting analysis using both 3Ј-and 5Ј-flanking probes (Fig.2A ).The 3Ј-probe detected an
8-kb DNA fragment from the targeted allele and a Ͼ14-kb fragment from the wild type allele (Fig.2B ).Among the 132clones screened,six contained the targeted allele.Southern blotting analysis with a neo probe further confirmed that all six were single integrants (data not shown).Cells of three targeted clones were microinjected into blasto-cysts of C57BL/6(B6)animals to generate germline chimeras.A PCR-bad assay was developed for identification of the possi-ble genotypes (Fig.2C ).A Northern blotting analysis with a cDNA probe from exons 1and 2revealed that the quences were abnt in the cells from homozygous mutant embryos (Fig.2D ).However,a smaller sized transcript was detectable in homozygous mutant cells with a 3Ј-cDNA probe (data not shown).A 5Ј-rapid amplification of cDNA end (RACE)analysis indicated that this transcript originated from exon 3(data not shown).A Western blotting analysis with a Dicer antibody detected a band from lysates of E14.5mutants (Fig.3A ).The translation of this protein is probably initiated from the AUG codon in exon 4,which is in frame with the start codon of dicer .To determine whether this truncated protein still retains func-tion,a Western blotting analysis was carried out to examine the expression level of veral miRNA targets.The pten and ang -1genes have been identified as targets that are subjected to miRNA regulation (22).Our Western blotting analysis showed that the expressions of both proteins were much higher in dicer ex1/2embryos than tho in wild type embryos (Fig.3A ).However,RT-PCR analysis showed that the transcript levels both genes in dicer ex1/2embryos were similar to tho in
wild type embryos (Fig.3B ).Thus,the results have confirmed that both the pten and the ang -1genes are subjected to post-tran-scriptional regulation by miRNAs.Together,the results show that the amino acid quence corresponding to the first two exons of dicer is esntial for the function of the protein and that the dicer ex1/2is a vere hypomorphic allele.
Developmental Defects in dicer ex1/2Embryos—Germline chi-meras were mated to B6females to generate heterozygous carriers of the mutant allele.Heterozygous animals were via-ble,healthy,and did not exhibit any overt abnormalities.To obtain homozygous mutants,heterozygous carriers were inter-crosd,and the resulting offspring were genotyped.Of 59mice born,none was a homozygous m
utant,indicating that dicer ex1/2animals were not viable.To further determine the timing of the lethality,embryos from heterozygous intercross were ana-lyzed at various developmental stages.At E10.5,dicer ex1/2em-bryos appeared morphologically normal (data not shown).How-ever,starting from E11.5,virtually all dicer ex1/2embryos were growth and developmentally retarded as compared with their wild type or heterozygous litter mates (Fig.4,A–D and F–I ).Histological analys of E12.5mutant embryos revealed that they had overtly normal structures,except that they were smaller and slightly underdeveloped (data not shown).The number of dicer ex1/2embryos obtained was within normal Mendelian distribution up to this stage,although resorbed embryos were found at the stages as well (Table I).Most of the dicer ex1/2embryos recovered at E13.5and E14.5were mor-phologically abnormal and nonviable.The few viable ones all had various degrees of edema (Fig.4,H and I ).Noticeably,the mutant embryos appeared to be very pale with smaller livers.The blood vesls in the mutants were thin and sub-optimally developed (arrowheads in Fig.4,D and I ).No viable embryos were retrieved beyond E14.5,indicating that dicer ex1/2embryos died between E12.5and 14.5.
Defects in the Yolk Sacs of dicer ex1/2Embryos—In addition to the embryonic phenotypes,yolk sacs from mutant embryos were abnormal as well.As early as E10.5a subt of dicer ex1/2yolk sacs start
ed to display phenotypes different from tho of wild type or heterozygous controls.In general,they appeared pale (Fig.5,A and D ),and this phenomenon became more pronounced in yolk sacs from older embryos.Microscopic ex-amination revealed that there were fewer blood vesls in the dicer ex1/2yolk sacs and that the vesls were thin,small,and less organized than tho of control yolk sacs (Fig.5,B ,C ,E ,and F ).In some extreme cas,there appeared to be no yolk sac blood vesls at all (data not shown).Together,the obrva-tions indicate that Dicer may be required for blood vesl development during embryogenesis.
团队合作的句子Impaired Angiogenesis in dicer ex1/2Embryos and Their Yolk Sacs—To analyze vascular network formation,yolk sacs from E11.5embryos were stained with anti-PECAM antibodies spe-cific to the endothelial cell.The blood vesls in yolk sacs from wild type embryos at this stage were well into vascular re-modeling and formed an orderly arranged network of blood vesls with large vitelline arteries and veins branching in a fractal pattern to smaller vesls (Fig.5G ).In contrast,the blood vesls in dicer ex1/2yolk sacs were thin and disorganized (Fig.5H ).However,the prence of a honeycomb-like primary vascular structure indicates that vasculogenesis and the initial steps of angiogenesis proceeded normally in the mutants.Hence,the obrvations suggested that in the abnce of a fully functional Dicer,either angiogenesis failed to go to com-pletion and/or that the for
med vesls could not be maintained or stabilized.Whole mount PECAM staining of E14.5embryos also revealed defects in the angiogenesis of mutant embryos proper.The tree-like blood vesl structures obrved in the wild type embryos (Fig.4E )were abnt in mutant embryos (Fig.4J ).Instead,there were many disorganized vascular structures.Again,the prence of vascular bedding suggests that Dicer is not required for vasculogenesis and early differ-entiation of the endothelial cells.The immunohistochemical data suggest that Dicer is required for the proper angiogenesis
F I
G .2.Gene targeting strategy.A ,the dicer wild type locus,the targeting vector,and the targeted allele.The targeting vector was designed to replace the first two exons of the dicer gene with a neo castte.Black boxes indicate exons.Hatched boxes reprent the two flanking probes ud in Southern blotting analysis.Arrow indicates the direction of transcription of the neo castte.Xh ,XhoI;B ,BamHI;
H ,HindIII;P ,PmlI;Sp ,SpeI.B ,Southern blot analysis of DNA extracted from embryonic stem clones.DNA was digested with Spe
I and hybrid-ized with the 3Ј-flanking probe.Arrows indicate the positions of the wild type (wt )and th
e mutant fragments,and the genotypes are indicated above each lane .C ,PCR analysis of DNA extracted from yolk sacs.The three primers generated specific products for the wild type (656bp)and the mutant allele (359bp).D ,Northern blotting analysis of mRNA extracted from murine embryonic fibroblasts of each genotype hybrid-ized with a dicer cDNA probe derived from exons 1and 2.The arrow indicates the 6.5-kb dicer transcript.
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of both the embryos and their yolk sacs.
Altered Expression of vegf,flt1,kdr,and tie1in dicer ex1/2Embryos—The vascular defects obrved in the dicer ex1/2em-bryos prompted us to examine the expression of veral crucial genes involved in embryonic angiogenesis.Mesnger RNA was isolated from pooled E14.5wild type and dicer ex1/2embryos and subjected to TaqMan RT-PCR analysis.Interestingly,the mRNA levels of vegf and the genes of its receptors,flt1and kdr ,in dicer ex1/2embryos were significantly higher than tho in w
ild type embryos.On the contrary,the mRNA level of tie-1,a receptor tyrosine kina gene,was lower in the mutant em-bryos (Fig.6A ).The results were confirmed by quantitative RT-PCR analysis of individual embryos at the same stage (data not shown).The reduction of the Tie-1protein level in dicer ex1/2embryos was further confirmed by Western blotting analysis (Fig.6B ).Thus,the results indicate that the impaired angio-genesis in dicer ex1/2embryos is probably due to the altered expression levels of angiogenesis regulators in the cell.The results also indicate that Dicer probably exerts its regulatory function on embryonic angiogenesis through the processing of miRNA molecules that control the expression levels of angio-genesis regulators.
DISCUSSION
We have attempted to disrupt the dicer gene via homologous recombination in embryonic stem cells.Becau exon 1con-
tains the putative translation initiation codon AUG,we rea-soned that an elimination of the first two exons of the mou dicer gene with a neo expression castte should abolish the expression of a functional Dicer protein.However,to our sur-pri we detected a protein with a size of ϳ220kDa in dicer ex1/2embryos.This protein is probably synthesized by using the in-frame AUG in exon 4,althou
gh this codon is not preceded with a connsus Kozak quence.As one of the primary roles for Dicer is to generate miRNAs,and becau hundreds of miRNA targets have recently been identified (22),we sought to investigate whether the truncated Dicer protein still retains function by examining the RNA and protein levels of the puta-tive miRNA target genes,pten and ang -1,in the mutant and wild type embryos.Our findings that the levels of both gene products were significantly higher in the mutant embryos than in the wild type embryos but that their transcript levels were similar clearly indicate that Dicer function is verely compro-mid in mutant embryos.Recently,Bernstein et al.also re-ported the embryonic lethality for their dicer knock-out ani-mals (23).In their study,however,the dicer Ϫ/Ϫembryos died around E7.5,which is much earlier than the time for dicer ex1/2embryos obtained in this study.Therefore,the results show that dicer ex1/2is a vere hypomorphic allele.
The abnce of live offspring of dicer ex1/2demonstrates that Dicer is esntial for normal development.This finding is con-sistent with the hypothesis that Dicer has a developmental role,probably through its ability to process miRNAs.Further analys of embryos between E10.5to 16.5revealed that the dicer ex1/2embryos die at E12.5–E14.5,with notable defects in vascular structures.The formation of blood vesls during em-bryonic development consists of two distinct pro
cess:1)vas-culogenesis,in which angioblasts differentiate and proliferate in situ to form the primary vascular network (24,25);and 2)angiogenesis,a process that modifies the primitive vesl structures through sprouting and vesl enlargement to form the branching patterns of the mature vasculature (26).In the abnce of Dicer,vasculogenesis in both the yolk sacs and the embryos ems to proceed normally.Therefore,Dicer may not be required for the differentiation and proliferation of endothe-lial cells.The initiation of angiogenesis is probably not
affected
F I
G .3.Compromid miRNA proc-essing in dicer ex1/2mutants.A ,West-ern blotting analysis of protein
s (100ug)extracted from whole embryos of wild type (ϩ/ϩ)and dicer ex1/2at E14.5.Blots were probed with anti-Dicer (top ),anti-PTEN (middle ),and anti-actin (bottom )antibodies.B ,TaqMan RT-PCR analysis of RNAs isolated from wild type (ϩ/ϩ)and dicer ex1/2embryos at E14.5.The white bars reprent wild type (wt ),and the black bars reprent
mutants.
F I
G .4.Retarded development and defective angiogenesis in dicer ex1/2embryos.Dicer deficient embryos (F–I )and wild type litter mates (A–D )at the same stage.Asterisk indicates the liver,and the black arrowhead indicates the blood vesls.Panel E shows PECAM staining of wild type embryos at E14.5.Panel J shows PECAM staining of dicer ex1/2embryos at E14.5.The stage and genotype of each embryo at the top and bottom of each panel.Scale bar ,1mm.
T ABLE I
Genotyping analysis of embryos obtained from
heterozygous intercross
Age/stage
Genotype
Resorbed
Total
ϩ/ϩ
ϩ/Ϫ
Ϫ/Ϫ
Weaning 20390NA a 59E16.5381b 012E15.56140525E14.5193412c 368E13.58234c 237E12.582910047E11.58145229E10.5
12247245
a Not applicable.
b
Embryos not viable.c
Some embryos not viable.
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by the abnce of Dicer either,becau the first sign of yolk sac defects aris at E10.5,and only a subt of dicer ex1/2yolk sacs appeared to be abnormal.Branching vesls can also be found in yolk sacs of some E14.5dicer ex1/2embryos.Also,in the mutant embryos proper the dorsal aortae,the intersomitic arteries,and the limb bud vasculature all appeared to be pres-ent.The obrvations indicate that Dicer is specifically re-quired for the maintenance of blood vesl integrity during late embryogenesis.
Analys of the expression of veral important regulators for embryonic vasculogenesis and angiogenesis revealed an alteration in their expression levels.Interestingly,the pheno-types of our dicer ex1/2embryos are similar to tho of the Tie1-deficient embryos.Both mutant embryos exhibit vascular de-fects,moderate edema,and die around days 13.5and 14.5of gestation.The receptor tyrosine kina Tie1has been shown to be required cell autonomously for the integrity and survival of vascular endothelial cells during late embryogenesis (27).Chi-meric analys indicate that Tie1is not required for the early differentiation of endothelial cells,nor is it required for the early pha of angiogenic vesl growth.Rather,Tie-1func-tions to support capillary endothelial cell proliferation (28).Recently,Tie-1was also shown to activate phosphatidylinositol 3-kina and Akt to inhibit apopto
sis (29).In E14.5dicer ex1/2embryos,the mRNA level of tie1was decread to ϳ28%of that in the wild type embryos.As a result,the TIE-1protein also decread dramatically in the mutant embryos.Therefore,this decread expression of Tie-1could lead to the apoptosis of endothelial cells,thereby causing the collap of blood vesls in the mutant embryos.
VEGF is one of the most important growth factors for blood vesl formation,as it is required for both vasculogenesis and angiogenic sprouting.Developing mou embryos are very n-sitive to the level of VEGF.A slight variation in the VEGF level during vesl formation can cau vere vascular defects,eventually leading to embryonic lethality.For example,disrup-tion of even a single allele of vegf in mice results in vere vascular abnormalities and embryonic lethality (30,31),whereas an inappropriate induction of VEGF results in the formation of immature and leaky vesls (32,33).
VEGF
F I
G .5.Morphology of dicer ex1/2yolk sacs.Wild type (A–C )and dicer ex1/2yolk sacs (D –F )at various stages.Panel G shows PECAM staining of E11.5yolk sac from wild type embryos.Panel
H depicts PECAM staining of E11.5yolk sac from dicer ex1/2embryos.Arrows indicates the blood vesls.The stage and genotypes are indicated at the bottom and top of each panel .Scale bar ,1
mm.
F I
G .6.Altered expression of vegf,flt1,kdr,and tie-1in dicer ؊/؊embryos.A ,TaqMan RT-PCR analysis of mRNA from pooled E14.5wild type (wt )(white bar )and dicer ex1/2embryos (mut ,mutant)(black bar )was performed as described under “Experimental Procedures.”Data were normalized according to the levels of -actin.B ,Western blotting analysis of proteins (100g)extracted from whole embryos of wild type (ϩ/ϩ)and dicer ex1/2at E14.5.Blots were probed with anti-Tie1(top ),anti-Glut1(middle ),and anti-actin (bottom ).
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mainly functions through its binding to the receptors Flt-1and Kdr.It has been shown that oxygen homeostasis plays an important role in the regulation of angiogenesis.The transcrip-tion of the VEGF gene is up-regulated via hypoxia-inducible factor-1under hypoxic conditions.Although incread VEGF expression in a hypoxic environment can promote vesl out-growth,the formed vesls tend
to be leaky,exhibiting disor-ganized patterns(34).Hypoxia also up-regulates Flt-1and Kdr in cultured cells(35,36).In E14.5dicer ex1/2embryos,vegf,flt-1, and kdr levels are significantly higher than tho in wild type embryos.This incread expression most likely results from the disrupted vesl structures,which subquently cau com-promid circulation and hypoxic condition in the mutant embryos.Consistent with this hypothesis,a dramatic increa in the level of Glut1,a known hypoxia-inducible factor-1target gene and a hypoxia marker,was obrved in the dicer ex1/2 embryos(Fig.6B).
Becau the primary function of Dicer in vivo is to process miRNAs,our results suggest that embryonic angiogenesis might also be regulated by miRNAs.Becau all miRNAs stud-ied so far em to act as post-transcriptional repressors of their targets,a defective Dicer is expected to cau a defect in the processing of miRNAs,which leads to overexpression of their target genes in the mutant embryos.This prediction has been confirmed,becau the expression levels of Ang-1and PTEN were significantly higher in the mutant embryos than tho in the wild type embryos.Thus,the dicer ex1/2embryos can be a very valuable system for the validation of miRNA targets in nature.Finally,miRNAs from the mir-80family have been implicated in the control of developmental cell death and/or cell proliferation(37).It is possible that miRNAs related to the mir-80family also regulate endothelial cell proliferation/death in mice during development.
Acknowledgments—We gratefully acknowledge Patricia Solenberg for providing the neo expression castte and Jingyong Zhao for help with PCR analysis.We thank Dr.Niles Fox for his advice and for sharing equipment.
REFERENCES
1.Fire,A.,Xu,S.,Montgomery,M.K.,Kostas,S.A.,Driver,S.E.,and Mello,
制作糖葫芦
C.C.(1998)Nature391,806–811
2.Bass,B.L.(2000)Cell101,235–238
3.Plasterk,R.H.,and Ketting,R.F.(2000)Curr.Opin.Genet.Dev.5,562–567
4.Hammond,S.M.,Caudy,A.A.,and Hannon,G.J.(2001)Nat.Rev.Genet.2,
110–119
5.Sharp,P.A.(2001)Genes Dev.15,485–490
6.Zamore,P.D.,Tuschl,T.,Sharp,P.A.,and Bartel,D.P.(2000)Cell101,25–33
7.Bernstein,E.,Caudy,A.A.,Hammond,S.M.,and Hannon,G.J.(2001)Nature
409,363–366
8.Nyka¨nen,A.,Haley,B.,and Zamore,P.D.(2001)Cell107,309–321
9.Zhang,H.,Kolb,F.A.,Brondani,V.,Billy,E.,and Filipowicz,W.(2002)EMBO
J.21,5875–5885
10.Ketting,R.F.,Fischer,S.E.J.,Bernstein,E.,Sijen,T.,Hannon,G.J.,and
Plasterk,R.H.A.(2001)Gen.Dev.15,2654–2659
11.Grishok,A.,Pasquinelli,A.E.,Conte,D.,Li,N.,Parrish,S.,Ha,I.,Baillie,
D.L.,Fire,A.,Ruvkun,G.,and Mello,C.C.(2001)Cell106,23–34
12.Pasquinelli,A.E.,Reinhart,B.J.,Slack,F.,Martindale,M.Q.,Kuroda,M.I.,
Maller,B.,Hayward,D.C.,Ball,E.E.,Degnan,B.,Muller,P.,Spring,J., Srinivasan,A.,Fishman,M.,Finnerty,J.
,Corbo,J.,Levine,M.,Leahy,P., Davidson,E.,and Ruvkun,G.(2000)Nature408,86–89
13.Hutva´gner,G.,McLachlan,J.,Pasquinelli,A.E.,Balint,E´.,Tuschl,T.,and
Zamore,P.D.(2001)Science293,834–838
14.Grosshans,H.,and Slack,F.J.(2002)J.Cell Biol.156,17–21
15.Lagos-Quintana,M.,Rauhurt,R.,Lendeckel,W.,and Tuschl,T.(2001)Science苏子的功效与作用
294,853–858
16.Lau,N.C.,Lim,L.P.,Weinstein,E.G.,and Bartel,D.P.(2001)Science294,
858–862
17.Lee,R.C.,and Ambros,V.(2001)Science294,862–864
18.Lagos-Quintana,M.,Rauhut,R.,Yalcin,A.,Meyer,J.,Lendeckel,W.,and
好作文大全Tuschl,T.(2002)Curr.Biol.12,735–739
19.Yang,W.,and Mansour,S.L.(1999)Dev.Dyn.215,108–116
20.Henrique,D.,Adam,J.,Myat,A.,Chitnis,A.,Lewis,J.,and Ish-Horowicz,D.
(1995)Nature375,787–790
21.Suri,C.,Jones,P.F.,Patan,S.,Maisonpierre,P.C.,Davis,S.,Sato,T.N.,and
Yancopoulos,G.D.(1996)Cell87,1171–1180
22.Lewis,B.P.,Shih,I.,Jones-Rhoades,M.W.,Bartel,D.P.,and Burge,C.B.
(2004)Cell115,787–798
23.Bersnstein,E.,Kim,S.Y.,Carmell,M.A.,Murchison,E.P.,Alcorn,H.,Li,
M.Z.,Mills,A.A.,Elledge,S.J.,Anderson,K.V.,and Hannon,G.J.(2003) Nat.Genet.35,213–215
24.Risau,W.,and Flamme,I.(1995)Annu.Rev.Cell Dev.Biol.11,73–91
25.Risau,W.(1997)Nature386,671–674
26.Yancopoulos,G.D.,Davis,S.,Gale,N.W.,Rudge,J.S.,Wiegand,S.J.,and
Holash,J.(2000)Nature407,242–248
27.Puri,M.C.,Rossant,J.,Alitalo,K.,Bernstein,A.,and Partanen,J.(1995)
EMBO J.14,5884–5891
28.Partanen,J.,Puri,M.C.,Schwartz,L.,Fischer,K.-D.,Bernstein,A.,and
Rossant,J.(1996)Development122,3013–3021
29.Kontos,C.D.,Cha,E.H.,York,J.D.,and Peters,K.G.(2002)Mol.Cell.Biol.
22,1704–1713
30.Carmeliet,P.,Ferreira,V.,Breier,G.,Pollefeyt,S.,Kieckens,L.,Gertnstein,
M.,Fahrig,M.,Vandenhoeck,A.,Harpal,K.,Eberhardt,C.,Declercq,C., Pawling,J.,Moons,L.,Collen,D.,Risau,W.,and Nagy,A.(1996)Nature 380,435–439
31.Ferrara,N.,Carver-Moore,K.,Chen,H.,Dowd,M.,Lu,L.,O’Shea,K.S.,
Powell-Braxton,L.,Hillan,K.J.,and Moore,M.W.(1996)Nature380, 439–442
32.Adamis,A.P.,Miller,J.W.,Bernal,M.T.,D’Amico,D.J.,Folkman,J.,Yeo,
T.K.,and Yeo,K.T.(1994)Am.J.Ophthalmol.118,445–450
33.Aiello,L.P.,Avery,R.L.,Arrigg,P.G.,Keyt,B.A.,Jampel,H.D.,Shah,S.T.,
Pasquale,L.R.,Thieme,H.,Iwamoto,M.A.,Park,J.E.,Nguyen,H.V., Aiello,L.M.,Ferrara,N.,and King,G.L.(1994)N.Engl.J.Med.331, 1480–1487
wrong反义词
34.Sohn,S.J.,Sarvis,B.K.,Cado,D.,and Winoto,A.(2002)J.Biol.Chem.277,
43344–43351
35.Gerber,H.P.,Condorelli,F.,Park,J.,and Ferrara,N.(1997)J.Biol.Chem.
272,23659–23667
36.Waltenberger,J.,Mayr,U.,Pentz,S.,and Hombach,V.(1996)Circulation94,
1647–1654
37.Carrington,J.C.,and Ambros,V.(2003)Science301,336–338
Targeted Disruption of Dicer in Mice9335
by guest, on November 7, 2011www.jbc
Downloaded from
