Qing Zhang

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 assess the role of Dicer in mammals, we generated Dicerex1/2 mice with a deletion of the amino acid sequences corresponding to the first and second exons of the dicer gene via homologous recombination. We found that Dicerex1/2 homozygous embryos displayed a retarded phenotype and died between days 12.5 and 14.5 of gestation. Thus, these results show that dicerex1/2 is severely hypomorphic and that Dicer is essential for normal mouse development. Interestingly, we also found that blood vessel formation/maintenance in dicerex1/2 embryos and yolk sacs were severely compromised, suggesting a possible role for Dicer in angiogenesis. This finding is consistent with the altered expression of vegf, flt1, kdr, and tie1 in the mutant embryos. Taken together, the results of this study indicate that Dicer exerts its function on mouse embryonic angiogenesis probably through its role in the processing of microRNAs that regulate the expression levels of some critical angiogenic regulators in the cell. 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 assess the role of Dicer in mammals, we generated Dicerex1/2 mice with a deletion of the amino acid sequences corresponding to the first and second exons of the dicer gene via homologous recombination. We found that Dicerex1/2 homozygous embryos displayed a retarded phenotype and died between days 12.5 and 14.5 of gestation. Thus, these results show that dicerex1/2 is severely hypomorphic and that Dicer is essential for normal mouse development. Interestingly, we also found that blood vessel formation/maintenance in dicerex1/2 embryos and yolk sacs were severely compromised, suggesting a possible role for Dicer in angiogenesis. This finding is consistent with the altered expression of vegf, flt1, kdr, and tie1 in the mutant embryos. Taken together, the results of this study indicate that Dicer exerts its function on mouse embryonic angiogenesis probably through its role in the processing of microRNAs that regulate the expression levels of some critical angiogenic regulators in the cell. RNA interference (RNAi) 1The abbreviations used are: RNAi, RNA interference; siRNA, small interfering RNA; miRNA, microRNA; E, embryonic day; PECAM, platelet endothelial cell adhesion molecule; RT, reverse transcription; VEGF, vascular endothelial growth factor. is a post-transcriptional gene regulation process that is conserved in organisms ranging from fungi to humans (1Fire A. Xu S. Montgomery M.K. Kostas S.A. Driver S.E. Mello C.C. Nature. 1998; 391: 806-811Crossref PubMed Scopus (11908) Google Scholar, 2Bass B.L. Cell. 2000; 101: 235-238Abstract Full Text Full Text PDF PubMed Scopus (340) Google Scholar, 3Plasterk R.H. Ketting R.F. Curr. Opin. Genet. Dev. 2000; 5: 562-567Crossref Scopus (86) Google Scholar, 4Hammond S.M. Caudy A.A. Hannon G.J. Nat. Rev. Genet. 2001; 2: 110-119Crossref PubMed Scopus (717) Google Scholar, 5Sharp P.A. Genes Dev. 2001; 15: 485-490Crossref PubMed Scopus (659) Google Scholar). When cells encounter long double-stranded RNA molecules, Dicer, a ribonuclease III type enzyme, cleaves them into small interfering RNAs (siRNAs) of 21–23 nucleotides. These siRNAs are incorporated into a multicomponent protein complex known as RISC (RNA-induced silencing complex). The antisense strand of siRNAs is believed to guide the RISC to locate its cognate mRNA molecule. As a result, the mRNAs are degraded (6Zamore P.D. Tuschl T. Sharp P.A. Bartel D.P. Cell. 2000; 101: 25-33Abstract Full Text Full Text PDF PubMed Google Scholar, 7Bernstein E. Caudy A.A. Hammond S.M. Hannon G.J. Nature. 2001; 409: 363-366Crossref PubMed Scopus (3819) Google Scholar, 8Nykänen A. Haley B. Zamore P.D. Cell. 2001; 107: 309-321Abstract Full Text Full Text PDF PubMed Scopus (835) Google Scholar). As one of the critical enzymes of the RNAi pathway, Dicer was first identified from Drosophila embryo and S2 cell extracts as the initiation enzyme for RNAi (7Bernstein E. Caudy A.A. Hammond S.M. Hannon G.J. Nature. 2001; 409: 363-366Crossref PubMed Scopus (3819) Google Scholar). It is a large (∼220 kDa), multi-domain protein that consists of an amino-terminal helicase domain followed by a PAZ domain, two RNase III domains, and a C-terminal double-stranded RNA-binding domain (dsRBD). The dsRBD and RNase III domains are involved in the binding to and cleavage of long double-stranded RNAs, but the functions of other domains remain unclear (9Zhang H. Kolb F.A. Brondani V. Billy E. Filipowicz W. EMBO J. 2002; 21: 5875-5885Crossref PubMed Scopus (481) Google Scholar). 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, inactivation of the dcr-1 (homolog of Drosophila Dicer) gene results in defects in the RNAi pathway as well as developmental abnormalities similar to those caused by the loss of function of let-7, a gene encoding a microRNA (miRNA) essential for the control of developmental timing in this organism (10Ketting R.F. Fischer S.E.J. Bernstein E. Sijen T. Hannon G.J. Plasterk R.H.A. Gen. Dev. 2001; 15: 2654-2659Crossref PubMed Scopus (1453) Google Scholar, 11Grishok A. Pasquinelli A.E. Conte D. Li N. Parrish S. Ha I. Baillie D.L. Fire A. Ruvkun G. Mello C.C. Cell. 2001; 106: 23-34Abstract Full Text Full Text PDF PubMed Scopus (1524) Google Scholar). 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 (12Pasquinelli 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. Ruvkun G. Nature. 2000; 408: 86-89Crossref PubMed Scopus (1879) Google Scholar). In addition, human Dicer was shown to be responsible for the generation of mature let-7 in HeLa cells (13Hutvágner G. McLachlan J. Pasquinelli A.E. Balint É. Tuschl T. Zamore P.D. Science. 2001; 293: 834-838Crossref PubMed Scopus (2190) Google Scholar). Therefore, the function of Dicer in development may be well conserved during evolution. Currently, there is an emerging interest in miRNAs because, under normal conditions, miRNAs rather than siRNAs appear to have a major role in small RNA-mediated gene regulation. Therefore, the principle role of Dicer in vivo may be involved in the biogenesis of mRNAs (14Grosshans H. Slack F.J. J. Cell Biol. 2002; 156: 17-21Crossref PubMed Scopus (120) Google Scholar). Recently, ∼90 miRNAs have been identified from C. elegans, Drosophila, mouse tissues, and human cell cultures. Among these, nine are found in more than one phylum (15Lagos-Quintana M. Rauhurt R. Lendeckel W. Tuschl T. Science. 2001; 294: 853-858Crossref PubMed Scopus (3975) Google Scholar, 16Lau N.C. Lim L.P. Weinstein E.G. Bartel D.P. Science. 2001; 294: 858-862Crossref PubMed Scopus (2697) Google Scholar, 17Lee R.C. Ambros V. Science. 2001; 294: 862-864Crossref PubMed Scopus (2339) Google Scholar, 18Lagos-Quintana M. Rauhut R. Yalcin A. Meyer J. Lendeckel W. Tuschl T. Curr. Biol. 2002; 12: 735-739Abstract Full Text Full Text PDF PubMed Scopus (2779) Google Scholar). This finding suggests that miRNA-mediated post-transcriptional regulation may function in a wide variety of organisms. Furthermore, the large numbers of miRNAs 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 (14Grosshans H. Slack F.J. J. Cell Biol. 2002; 156: 17-21Crossref PubMed Scopus (120) Google Scholar). To assess the function of Dicer in a mammalian system, we sought to generate Dicer-deficient mice via homologous recombination in embryonic stem cells. We show that dicerex1/2 embryos are growth and developmentally retarded and die between embryonic day (E) 12.5 and 14.5. Therefore, Dicer is essential for normal development. In addition, the mutant embryos and their yolk sacs are found to display defects in the blood vessel formation/maintenance, suggesting a role for Dicer in the regulation of embryonic angiogenesis via its function in the processing of miRNAs. Construction of a dicer Targeting Vector—A DNA primer corresponding to the 5′-region of the mouse dicer genomic sequence was used to screen a mouse CITB BAC library by Research Genetics (Carlsbad, CA). One positive clone (474 E 20) was identified and confirmed by colony PCR using two primers, wjy1 and wjy3, corresponding to mouse dicer cDNA sequences (wjy1, 5′-AGCATGGCAGGCCTGCAG-3′; wjy3, 5′-TGATGGGCCAGCTCTTTGG-3′). An ∼10-kb BamHI-XhoI fragment from this BAC clone was ligated into a pBluescript plasmid (Stratagene). The 1.2-kb HindIII-PmlI fragment containing exons 1 and 2 of dicer was then replaced by a neo expression cassette. Generation of dicerex1/2 Mice—The targeting construct was linearized at the XhoI site and introduced into mouse embryonic stem cell E14 via electroporation. G418-resistant colonies selected were screened for the homologous recombination by Southern blotting analysis using a 3′-flanking probe, a 5′-flanking probe, and a neo probe. Both the 3′- and 5′-probes were generated by PCR reaction off the BAC clone. The primers used for the 3′-probe were 5′-TCTTCGTCGAAACGTACAAG-3′ and 5′-TGGAAAGACCCTCATTCCAAG-3′. The primers for amplifying the 5′-probe were 5′-GTTATTACCACTAAATATCACG-3′ and 5′-CT-GCCAAGGCTTTGTTTCAC-3′. The neo probe was a 259-bp PstI fragment from a plasmid containing the neo cassette. Three of six positive clones identified were microinjected into blastocysts and implanted into pseudopregnant C57BL/6 (B6) females. Chimeric males were mated with B6 females (Harland) to yield heterozygous F1 offspring, which were then intercrossed to produce +/+, +/-, -/- offspring. Tail DNA from mice was isolated using genomic isolation kit from Lamda Biotechnology, and genotyping was carried out by PCR reaction using the primers KOF (5′-AGCCATCTCCCCAGAAGTCC-3′), which was the forward primer common for both the wild-type and the targeted allele, KOR2 (5′-CCAAAGAACGGAGCCGGTTG-3′), and KOR1 (5′-CGTGTAGGGTTCAGTCATTCGT-3′), which were designed for the amplification of the wild type and the targeted alleles, respectively. Analysis of Embryos—Detection of vaginal plugs in the morning was taken as gestation day 0.5 (E0.5). Embryos were staged and dissected from the pregnant females. Yolk sacs from different stage embryos were dissolved in a PCR lysis buffer (50 mm KCl, 1.5 mm MgCl2, 10 mm Tris-HCl, pH 8.5, 0.01% gelatin, 0.45% Nonidet P-40, 0.45% Tween, and 100 μg/ml proteinase K) for 4–12 h. The proteinase K was inactivated by incubation at 95 °C for 5 min prior to PCR analysis. PCR was carried out in 20-μl reaction mixtures using Taq Platinum (Invitrogen) according to the manufacturer's suggestions. For histology, embryos were fixed overnight in 3% paraformaldehyde in phosphate-buffered saline, dehydrated, and embedded in paraffin. Ten-micron sections were stained with hematoxylin and eosin. Northern Blotting Analysis—Total RNA was isolated from wild type, heterozygous, and mutant mouse embryonic fibroblasts. Messenger RNA was purified using an Oligotex mRNA isolation kit (Qiagen). Each mRNA sample (5 μg) was separated in a glyoxal-agarose gel, transferred to nylon membranes, and hybridized with a 32P-labeled cDNA probe generated by PCR reaction using wjy1 and wjy3 under the conditions described earlier (19Yang W. Mansour S.L. Dev. Dyn. 1999; 215: 108-116Crossref PubMed Scopus (24) Google Scholar). The same probe was also used to hybridize to a membrane containing RNAs derived from different mice tissues obtained at different developmental stages (Clontech). Antibody Generation and Western Blotting Analysis—A Dicer-specific antibody was generated against a peptide corresponding to the C of the mouse Dicer protein with an amino acid sequence of μg) from embryos of wild type dicerex1/2 embryos at were on an and transferred The were with a antibody at 2 a antibody at 2 a mouse antibody at a at a antibody at with The were with a Analysis—Total RNA was isolated from wild type and mutant embryos using (Invitrogen) according to the manufacturer's Messenger RNA was purified using an Oligotex mRNA isolation kit (Qiagen). The mRNA was to analysis using primers and probe in reaction and primers used for the were as probe, forward reverse probe, forward reverse probe, forward reverse probe, forward reverse probe, forward reverse probe, forward reverse The probe and primer were obtained as a from In and dicer fragment was generated by PCR from an cDNA library using the primers and This fragment was into a and antisense were using and RNA respectively. embryos from mice (Harland) were and for in as described D. J. A. A. J. D. Nature. PubMed Scopus Google and of yolk sacs were as described C. S. S. Cell. Full Text Full Text PDF PubMed Scopus Google Scholar). The dicer in Embryos and the levels of dicer expression during a dicer cDNA probe was hybridized to mRNAs isolated from mouse embryos at developmental stages of the dicer was in embryos. of this were in embryos and through suggesting that Dicer may function during this of development. To the expression of dicer in in analysis of RNA was the dicer mRNA was at similar levels the embryos The expression of dicer in tissues was also Interestingly, dicer was in the and This suggests that Dicer may have a more function in these Recently, M. Rauhut R. Yalcin A. Meyer J. Lendeckel W. Tuschl T. Curr. Biol. 2002; 12: 735-739Abstract Full Text Full Text PDF PubMed Scopus (2779) Google identified miRNAs by of RNAs from of which were derived from the and Thus, this finding is consistent with expression of dicer in these levels of dicer expression were also in the and Generation of dicerex1/2 via the in vivo function of Dicer during a targeting was designed to the dicer by the first two exons of the dicer gene with a neo expression through homologous recombination in embryonic stem cells The construct was introduced into E14 embryonic stem which were under clones were via Southern blotting analysis using both 3′- and 5′-flanking The 3′-probe an DNA fragment from the targeted and a fragment from the wild type Among the clones six the targeted Southern blotting analysis with a neo probe confirmed that six were of targeted clones were microinjected into blastocysts of to generate was for of the possible Northern blotting analysis with a cDNA probe from exons 1 and 2 that these sequences were in the cells from homozygous mutant embryos a was in homozygous mutant cells with a probe amplification of cDNA analysis that this from Western blotting analysis with a Dicer antibody a from of The of this protein is probably from the in which is in with the of To this protein a Western blotting analysis was carried out to the expression of miRNA The and have been identified as that are to miRNA regulation I. Bartel D.P. Cell. Full Text Full Text PDF Scopus Google Scholar). Western blotting analysis that the of both were in dicerex1/2 embryos than those in wild type embryos analysis that the levels both in dicerex1/2 embryos were similar to those in wild type embryos Thus, these results have confirmed that both the and the are to post-transcriptional regulation by miRNAs. these results show that the amino acid sequence corresponding to the first two exons of dicer is essential for the function of the protein and that the dicerex1/2 is a hypomorphic miRNA processing in dicerex1/2 Western blotting analysis of from embryos of wild type and dicerex1/2 at were with and analysis of RNAs isolated from wild type and dicerex1/2 embryos at The wild type and the in dicerex1/2 were mated to B6 females to generate heterozygous of the mutant were and To homozygous heterozygous were and the were mice was a homozygous that dicerex1/2 were To the timing of the embryos from heterozygous were at developmental dicerex1/2 embryos normal from dicerex1/2 embryos were growth and developmentally retarded as with their wild type heterozygous and of mutant embryos that they normal that they were and The of dicerex1/2 embryos obtained was normal to this embryos were found at these stages as well of the dicerex1/2 embryos at and were and The of and these mutant embryos to be with The blood in these were and in and embryos were that dicerex1/2 embryos died between and analysis of embryos obtained from heterozygous embryos embryos Embryos embryos in a in the Yolk of dicerex1/2 addition to the embryonic yolk sacs from mutant embryos were as As as a of dicerex1/2 yolk sacs to display different from those of wild type heterozygous In they and and this more in yolk sacs from embryos. that there were blood in the dicerex1/2 yolk sacs and that these were and than those of control yolk sacs E, and In some there to be yolk blood at these indicate that Dicer may be required for blood vessel development during in dicerex1/2 Embryos and Yolk vascular yolk sacs from embryos were stained with to the endothelial cell. The blood in yolk sacs from wild type embryos at this stage were well into vascular and an of blood with large and in a to In the blood in dicerex1/2 yolk sacs were and the of a vascular structure that and the of angiogenesis in the these that in the of a Dicer, angiogenesis to to that the be of embryos also defects in the angiogenesis of mutant embryos The blood vessel in the wild type embryos were in mutant embryos there were vascular the of vascular suggests that Dicer is required for and of the endothelial cells. These suggest that Dicer is required for the angiogenesis of both the embryos and their yolk of vegf, flt1, kdr, and tie1 in vascular defects in the dicerex1/2 embryos to the expression of involved in embryonic angiogenesis. Messenger RNA was isolated from wild type and dicerex1/2 embryos and to analysis. Interestingly, the mRNA levels of and the of its and kdr, in dicerex1/2 embryos were than those in wild type embryos. the the mRNA of a was in the mutant embryos These results were confirmed by analysis of embryos at the same stage The of the protein in dicerex1/2 embryos was confirmed by Western blotting analysis Thus, these results indicate that the angiogenesis in dicerex1/2 embryos is probably to the altered expression levels of angiogenesis regulators in the cell. These results also indicate that Dicer probably exerts its function on embryonic angiogenesis through the processing of miRNA that control the expression levels of angiogenesis We have to the dicer gene via homologous recombination in embryonic stem cells. 1 the initiation we that an of the first two exons of the mouse dicer gene with a neo expression the expression of a Dicer to we a protein with a of in dicerex1/2 embryos. This protein is probably by using the in this is with a As one of the roles for Dicer is to generate and of miRNA have been identified I. Bartel D.P. Cell. Full Text Full Text PDF Scopus Google we sought to the Dicer protein function by the RNA and protein levels of the miRNA and in the mutant and wild type embryos. that the levels of both gene were in the mutant embryos than in the wild type embryos but that their levels were similar indicate that Dicer function is severely in mutant embryos. Recently, Bernstein also the embryonic for their dicer E. H. Li A.A. Hannon G.J. Nat. Genet. PubMed Scopus Google Scholar). In their the embryos died which is earlier than the for dicerex1/2 embryos obtained in this Therefore, these results show that dicerex1/2 is a hypomorphic The of of dicerex1/2 that Dicer is essential for normal development. This finding is consistent with the that Dicer a developmental probably through its to process miRNAs. of embryos between to that the dicerex1/2 embryos die at with defects in vascular The of blood during embryonic development consists of two in which and in to form the vascular W. I. Rev. Cell Dev. Biol. PubMed Scopus Google Scholar, W. Nature. PubMed Scopus Google and a process that the vessel through and vessel to form the of the mature S. J. Nature. 2000; PubMed Scopus Google Scholar). In the of Dicer, in both the yolk sacs and the embryos to Therefore, Dicer may be required for the and of endothelial cells. The initiation of angiogenesis is probably by the of Dicer the first of yolk defects at and a of dicerex1/2 yolk sacs to be also be found in yolk sacs of some dicerex1/2 embryos. in the mutant embryos the the and the to be These indicate that Dicer is required for the of blood vessel during of the expression of important regulators for embryonic and angiogenesis an in their expression Interestingly, the of dicerex1/2 embryos are similar to those of the embryos. Both mutant embryos vascular and die days and 14.5 of gestation. The been shown to be required cell for the and of vascular endothelial cells during J. Bernstein A. J. EMBO J. PubMed Scopus Google Scholar). Chimeric indicate that is required for the of endothelial is required for the of angiogenic vessel functions to endothelial cell J. Fischer Bernstein A. J. PubMed Google Scholar). Recently, was also shown to and to Cell. Biol. 2002; PubMed Scopus Google Scholar). In dicerex1/2 the mRNA of tie1 was to of that in the wild type embryos. As a result, the protein also in the mutant embryos. Therefore, this expression of to the of endothelial the of blood in the mutant embryos. is one of the important growth for blood vessel as is required for both and angiogenic mouse embryos are to the of in the during vessel vascular to embryonic For of a of in mice results in vascular abnormalities and embryonic P. V. G. S. M. M. A. C. C. J. D. W. A. Nature. PubMed Scopus Google Scholar, N. H. M. Nature. PubMed Scopus Google an of results in the of and J. J. Full Text PDF PubMed Scopus Google Scholar, L.P. H. N. N. J. PubMed Scopus Google Scholar). functions through its binding to the and It been shown that plays an important role in the regulation of angiogenesis. The of the gene is via under expression in a vessel the to be D. A. J. Biol. 2002; Full Text Full Text PDF PubMed Scopus Google Scholar). also and in cells F. J. N. J. Biol. Full Text Full Text PDF PubMed Scopus Google Scholar, J. S. V. PubMed Scopus Google Scholar). In dicerex1/2 vegf, and levels are than those in wild type embryos. This expression results from the vessel which and in these mutant embryos. with this a in the of a known gene and a was in the dicerex1/2 embryos the function of Dicer in vivo is to process results suggest that embryonic angiogenesis also be by miRNAs. miRNAs to as post-transcriptional of their a Dicer is to a in the processing of which to of their in the mutant embryos. This been the expression levels of and were in the mutant embryos than those in the wild type embryos. Thus, the dicerex1/2 embryos be a for the of miRNA in miRNAs from the have been in the control of developmental cell cell Ambros V. Science. PubMed Scopus Google Scholar). It is possible that miRNAs to the also regulate endothelial cell in mice during development. We for the neo expression and for with PCR analysis. We for and for

Specific pathways linking heterotrimeric G proteins and Fcgamma receptors to the actin-based cytoskeleton are poorly understood. To test a requirement for Rho family members in cytoskeletal events mediated by structurally diverse receptors in leukocytes, we transfected the full-length human chemotactic peptide receptor in RAW 264.7 cells and examined cytoskeletal alterations in response to the chemotactic peptide formyl-methionyl-leucyl-phenylalanine (FMLP), colony stimulating factor-1 (CSF-1), IgG-coated particles, and phorbol 12-myristate 13-acetate (PMA). Expression of Rac1 N17, Cdc42 N17, or the GAP domain of n-chimaerin inhibited cytoskeletal responses to FMLP and CSF-1, and blocked phagocytosis. Accumulation of F-actin- rich "phagocytic cups" was partially inhibited by expression of Rac1 N17 or Cdc42 N17. In contrast, PMA-induced ruffling was not inhibited by expression of Rac1 N17, but was blocked by expression of Cdc42 N17, indicating that cytoskeletal inhibition by these constructs was nonoverlapping. These results demonstrate differential requirements for Rho family GTPases in leukocyte motility, and indicate that both Rac1 and Cdc42 are required for Fcgamma receptor- mediated phagocytosis and for membrane ruffling mediated by structurally distinct receptors in macrophages.