Liver Disintegration in the Mouse Embryo Caused by Deficiency in the RNA-editing Enzyme ADAR1
ADAR1 (adenosine deaminase acting on RNA-1) is widely expressed in mammals, but its biological role is unknown. We show here by gene targeting that ADAR1 selectively edits in vivo two of five closely spaced adenosines in the serotonin 5-hydroxytryptamine subtype 2C receptor pre-mRNA of nervous tissue; and hence, site-selective adenosine-to-inosine editing is indeed a function of ADAR1. Remarkably, homozygosity for two different null alleles of ADAR1 caused a consistent embryonic phenotype appearing early at embryonic day 11 and leading to death between embryonic days 11.5 and 12.5. This phenotype manifests a rapidly disintegrating liver structure, along with severe defects in definitive hematopoiesis, encompassing both erythroid and myeloid/granuloid progenitors as well as spleen colony-forming activity from the aorta-gonad-mesonephros region and fetal liver. Probably as a consequence of these developmental impairments, ADAR1-deficient embryonic stem cells failed to contribute to liver, bone marrow, spleen, thymus, and blood in adult chimeric mice. Thus, ADAR1 subserves critical steps in developing non-nervous tissue, which are likely to include transcript editing. ADAR1 (adenosine deaminase acting on RNA-1) is widely expressed in mammals, but its biological role is unknown. We show here by gene targeting that ADAR1 selectively edits in vivo two of five closely spaced adenosines in the serotonin 5-hydroxytryptamine subtype 2C receptor pre-mRNA of nervous tissue; and hence, site-selective adenosine-to-inosine editing is indeed a function of ADAR1. Remarkably, homozygosity for two different null alleles of ADAR1 caused a consistent embryonic phenotype appearing early at embryonic day 11 and leading to death between embryonic days 11.5 and 12.5. This phenotype manifests a rapidly disintegrating liver structure, along with severe defects in definitive hematopoiesis, encompassing both erythroid and myeloid/granuloid progenitors as well as spleen colony-forming activity from the aorta-gonad-mesonephros region and fetal liver. Probably as a consequence of these developmental impairments, ADAR1-deficient embryonic stem cells failed to contribute to liver, bone marrow, spleen, thymus, and blood in adult chimeric mice. Thus, ADAR1 subserves critical steps in developing non-nervous tissue, which are likely to include transcript editing. From Caenorhabditis elegans to humans, adenosine-to-inosine (A-to-I) editing of primary transcripts in the nucleus recodes exonic information and can lead to structural and functional changes in the encoded protein, thereby enlarging the protein space encoded by the transcriptome (1Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (977) Google Scholar). In mammals, only few edits have been identified, all by serendipity and all in brain-expressed transcripts. Two sequence-related genes encode candidate enzymes for A-to-I editing, termed ADAR1 and ADAR2, each endowed with regions for binding double-stranded RNA (dsRNA) 1The abbreviations used are: dsRNAdouble-stranded RNA5-HT2C5-hydroxytryptamine subtype 2CESembryonic stemEembryonic day(s)CFU-Ecolony-forming unit-erythroid, BFU-E, burst-forming unit-erythroidCFU-M/Gcolony-forming unit-myeloid/granuloidCFU-Scolony-forming unit(s)-spleenAGMaorta-gonad-mesonephrosSNPsingle nucleotide polymorphismTUNELterminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. and an enzyme domain distantly related to bacterial cytidine deaminase (1Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (977) Google Scholar). A third protein, RED2 (dsRNA-specific editase-2), which meets the sequence requirements for a candidate A-to-I editing enzyme, has no editing activity on synthetic dsRNA (2Melcher T. Maas S. Herb A. Sprengel R. Higuchi M. Seeburg P.H. J. Biol. Chem. 1996; 271: 31795-31798Abstract Full Text Full Text PDF PubMed Scopus (243) Google Scholar), rendering its candidate status questionable. double-stranded RNA 5-hydroxytryptamine subtype 2C embryonic stem embryonic day(s) colony-forming unit-erythroid, BFU-E, burst-forming unit-erythroid colony-forming unit-myeloid/granuloid colony-forming unit(s)-spleen aorta-gonad-mesonephros single nucleotide polymorphism terminal deoxynucleotidyltransferase-mediated dUTP nick end labeling. The single instance in which an ADAR protein could be assigned to a particular edit concerns ADAR2 and the transcript for the glutamate receptor subunit GluR-B, which is edited to nearly 100% in a particular codon for a critical ion channel determinant (3Higuchi M. Maas S. Single F.N. Hartner J. Rozov A. Burnashev N. Feldmeyer D. Sprengel R. Seeburg P.H. Nature. 2000; 406: 78-81Crossref PubMed Scopus (747) Google Scholar). ADAR2-deficient mice die early from a neurological syndrome, but appear normal when the edited codon (CGG) replaces the unedited CAG version in both gluR-B alleles (4Kask K. Zamanillo D. Rozov A. Burnashev N. Sprengel R. Seeburg P.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13777-13782Crossref PubMed Scopus (74) Google Scholar). This is the only example where editing is necessary for survival, whereas for the other known edited transcripts, site-selective A-to-I conversion may serve to fine-tune the function of the encoded proteins (5Seeburg P.H. Neuron. 2002; 35: 17-20Abstract Full Text Full Text PDF PubMed Scopus (128) Google Scholar). The biological function of ADAR1 and its involvement in transcript editing are still unresolved and have become even enigmatic with a report that haploinsufficiency of ADAR1 leads to defects in the hematopoietic system and an embryonic lethal phenotype (6Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar). ADAR1 exhibits several features that set it apart from the other two, more closely related ADAR proteins. These comprise two putative Z-DNA-binding domains (7Herbert A. Alfken J. Kim Y.G. Mian I.S. Nishikura K. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8421-8426Crossref PubMed Scopus (264) Google Scholar), a third dsRNA-binding region, more widespread expression (8Kim U. Wang Y. Sanford T. Zeng Y. Nishikura K. Proc. Natl. Acad. Sci. U. S. A. 1994; 91: 11457-11461Crossref PubMed Scopus (372) Google Scholar, 9O'Connell M.A. Krause S. Higuchi M. Hsuan J.J. Totty N.F. Jenny A. Keller W. Mol. Cell. Biol. 1995; 15: 1389-1397Crossref PubMed Google Scholar), and an additional promoter for interferon-inducible transcripts encoding an ADAR1 version with altered amino-terminal sequence (10George C.X. Samuel C.E. Proc. Natl. Acad. Sci. U. S. A. 1999; 96: 4621-4626Crossref PubMed Scopus (234) Google Scholar). These features might indicate that the biological functions of ADAR1 exceed transcript editing. However, as of today, no molecular process with ADAR1 participation has been delineated in mammals, nor has ADAR1 been convincingly shown to edit any of the known sites in brain-expressed transcripts, although two observations provide suggestive evidence for at least one transcript. In ADAR2-deficient mice, two sites in the serotonin 5-HT2C receptor transcript change little in editing status (3Higuchi M. Maas S. Single F.N. Hartner J. Rozov A. Burnashev N. Feldmeyer D. Sprengel R. Seeburg P.H. Nature. 2000; 406: 78-81Crossref PubMed Scopus (747) Google Scholar), but show decreased editing in adar1+/- embryonic stem (ES) cell-derived teratomas (6Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar). To gain better insight into the biological role of ADAR1, we generated mice with adar1 null alleles. These alleles, in combination with an adar2 null allele, permitted us to use a genetic approach in the mouse to assign different, closely spaced edits in 5-HT2C receptor transcripts unambiguously to either ADAR1 or ADAR2 activity, thus demonstrating that site-selective A-to-I editing is indeed a biological function of ADAR1. Heterozygosity in mice for differently configured adar1 null alleles is compatible with survival and a normal appearance, in stark contrast to the embryonic lethality claimed for haploinsufficiency of ADAR1 (6Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar). With homozygosity for our adar1 null alleles, however, embryos at mid-gestation developed a severe liver defect and died between E11.5 and E12.5. Remarkably, the defect involved cells of both the hematopoietic and hepatic lineages, as further suggested by analysis of mouse chimeras derived from ADAR1-deficient ES cells. Hence, ADAR1 has a critical role in non-nervous tissue, but edited transcripts remain to be identified. adar1 Gene Targeting—The targeting vector for Cre/loxP-mediated functional ablation of the murine adar1 gene contained 6.9 kb of 5′-homology sequence including exons 3-6 and a pgk-neo gene flanked by two loxP511 elements (11Hoess R.H. Wierzbicki A. Abremski K. Nucleic Acids Res. 1986; 14: 2287-2300Crossref PubMed Scopus (233) Google Scholar, 12Lee G. Saito I. Gene (Amst.). 1998; 216: 55-65Crossref PubMed Scopus (258) Google Scholar) cloned into an SmaI site and 3.3 kb including exons 7-11 as 3′-homology sequence with a third loxP511 element inserted into an MscI site in intron 9 (Fig. 1). The linearized targeting vector was electroporated in R1 ES cells (13Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8424-8428Crossref PubMed Scopus (1992) Google Scholar), and PCR-mediated screening of 540 G418 (aminoglycoside antibiotic)-resistant clones yielded one clone with a correctly targeted adar1 allele, as confirmed by Southern analyses. Chimeric mice generated by injection of the targeted ES cell clone into C57BL/6-derived blastocysts transmitted the the The was generated by mice with mice U. K. Nucleic Acids Res. 1995; PubMed Scopus Google Scholar). The loxP511 elements used to in the of an adar2 with The of loxP511 in mice was with that of the adar2 mice to the and of the adar1 gene was by Southern and of from mice. The was by exons of the adar1 gene with a pgk-neo kb of intron generated by as 5′-homology and a cloned exons and as 3′-homology of the linearized targeting vector into R1 ES cells and screening of clones for in the adar1 yielded clones with a correctly targeted allele, as confirmed by Southern analyses. Two clones into C57BL/6-derived blastocysts and to chimeric chimeric mice to either or mice transmitted the the and derived from of or mice, with of the day as and by in combination with The of Scholar). by analysis of from or with adar1 and in cell of to from embryos for each on and for in and cells with and RNA was with and into the five editing sites generated with 5-HT2C receptor and and with a The of editing was derived from the different for the two in the five editing in sequence and as R. the A Scholar). of embryos in for and with and for with to or and with the primary a was with cell of fetal liver and blood in in with and to the of erythroid or with murine spleen to myeloid/granuloid days unit-erythroid days unit-erythroid and 9 days unit-myeloid/granuloid of cell of from two two and E11.5 embryos with in a and with cell of aorta-gonad-mesonephros regions and fetal from embryos and of regions and fetal from embryos into the of adult and mice. Single from the 11 days and was and ADAR1 by analysis with adar1 and and ES cell clone was and in G418 for cells with two adar1 null alleles. screening of with adar1 yielded as confirmed by Southern analyses. mice generated by injection of two ES cell clones into blastocysts by in mice. was from of adult day chimeric mice, and the of ES cells to these was by two analyses. adar1 are the of and null alleles. The of by the ADAR1-deficient ES cells was from the of a particular single nucleotide polymorphism the particular in the of the of on to the mouse for and for and the generated with and and with a adar1 null alleles the a targeting vector for Cre/loxP-mediated functional ablation of the murine adar1 gene by a pgk-neo gene flanked by two elements into intron and a third element into intron 9 of cloned adar1 sequence (Fig. 1). The sequence of the interferon-inducible of ADAR1 including regions for editing activity in (Fig. R. Nishikura K. J. Biol. Chem. 1995; Full Text Full Text PDF PubMed Scopus Google Scholar, S. T. Herb A. Seeburg P.H. Keller W. Krause S. Higuchi M. M.A. J. Biol. Chem. 1996; 271: Full Text Full Text PDF PubMed Scopus Google Scholar, Y. A. Rich A. Samuel C.E. 1998; 15: PubMed Scopus Google Scholar). Chimeric mice generated by injection of a targeted R1 ES cell clone (13Nagy A. Rossant J. Nagy R. Abramow-Newerly W. Roder J.C. Proc. Natl. Acad. Sci. U. S. A. 1993; 90: 8424-8428Crossref PubMed Scopus (1992) Google Scholar) into C57BL/6-derived blastocysts transmitted the the and mice with mice U. K. Nucleic Acids Res. 1995; PubMed Scopus Google Scholar) for of the gene mice but no and of ADAR1 with embryonic The normal of mice was on a report on lethality in chimeric embryos caused by a of cells for a particular adar1 null (6Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar), which as we may have a the the of embryos to the chimeric embryos (6Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar) might indicate functional by our To the for we a null (Fig. in which exons encoding which comprise all dsRNA-binding two putative Z-DNA-binding domains (7Herbert A. Alfken J. Kim Y.G. Mian I.S. Nishikura K. Rich A. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: 8421-8426Crossref PubMed Scopus (264) Google Scholar), and of the domain of ADAR1, with a pgk-neo chimeric mice derived from two ES cell clones transmitted the and normal mice, in with mice. In further no from whereas and at Hence, differently configured adar1 null alleles embryonic death but of RNA by ADAR1 ADAR1 is to be involved in site-selective A-to-I editing (1Bass B.L. Annu. Rev. Biochem. 2002; 71: 817-846Crossref PubMed Scopus (977) Google Scholar), we the of ADAR1 on the editing status of the 5-HT2C receptor for which editing been suggested (3Higuchi M. Maas S. Single F.N. Hartner J. Rozov A. Burnashev N. Feldmeyer D. Sprengel R. Seeburg P.H. Nature. 2000; 406: 78-81Crossref PubMed Scopus (747) Google Scholar, Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar). We that at when embryos die transcript was that the editing sites Q. C.X. Nishikura K. J. 2000; PubMed Scopus Google Scholar) was but was correctly from E11.5 for of was by our that from embryos of editing at the known sites that of for (3Higuchi M. Maas S. Single F.N. Hartner J. Rozov A. Burnashev N. Feldmeyer D. Sprengel R. Seeburg P.H. Nature. 2000; 406: 78-81Crossref PubMed Scopus (747) Google Scholar). This further that the transcripts editing in the correctly edited of derived from mid-gestation 5-HT2C receptor transcripts are edited to different at five a of five involved in protein (Fig. Nature. 1997; PubMed Scopus Google Scholar). ADAR1 a at sites A and at which editing from in the to that these sites are edited by ADAR1. only five of site was by of ADAR1. at site which is the least edited site in vivo Q. C.X. Nishikura K. J. 2000; PubMed Scopus Google Scholar), and site was to the which may by ADAR2 in the of ADAR1. these site-selective A-to-I conversion in pre-mRNA as an in vivo function of ADAR1, only shown for ADAR2 (3Higuchi M. Maas S. Single F.N. Hartner J. Rozov A. Burnashev N. Feldmeyer D. Sprengel R. Seeburg P.H. Nature. 2000; 406: 78-81Crossref PubMed Scopus (747) Google Scholar). further indicate that the two ADAR proteins edit of closely spaced We the of ADAR on the editing sites in 5-HT2C receptor transcripts, from E11.5 of the ADAR2-deficient mice die a few (3Higuchi M. Maas S. Single F.N. Hartner J. Rozov A. Burnashev N. Feldmeyer D. Sprengel R. Seeburg P.H. Nature. 2000; 406: 78-81Crossref PubMed Scopus (747) Google Scholar), we used mice in which lethality was by gluR-B alleles (4Kask K. Zamanillo D. Rozov A. Burnashev N. Sprengel R. Seeburg P.H. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: 13777-13782Crossref PubMed Scopus (74) Google Scholar). ADAR2 the of editing least at sites A and that these are edited by ADAR1. we failed to any A-to-I conversion in 5-HT2C receptor transcripts in with ADAR (Fig. and hence, the of ADAR1 and ADAR2 all known A-to-I editing in transcripts for ADAR1-deficient the and of the embryonic death from of ADAR1, we and embryos derived from both and at different developmental We for both ADAR1-deficient mouse in two different and to as mice. as with at the of at between and However, whereas embryos at E12.5. We that ADAR1-deficient embryos die in between E11.5 and derived from in a at and as by embryos a in fetal liver which a and an with embryos (Fig. The of fetal was normal but whereas and adar1+/- in between E11.5 and E12.5. ADAR1-deficient fetal liver an but embryos in as shown by and with although normal (Fig. of embryos (Fig. in fetal liver from E11.5 a cell and blood in from cell The failed to in other including the and even when blood and of cells in with to cell A. M. A. W. A. J. Biol. 1994; Google Scholar) normal of the and the the and We in embryos at any of into as and in embryos was with that in embryos at including fetal liver, as by at of cells of the in ADAR1-deficient fetal although cells in other of in the fetal liver the hematopoietic tissue, to the blood cell in the adult to spleen and bone A. 1995; Full Text PDF PubMed Scopus Google Scholar). E11.5 at cell in ADAR1-deficient embryonic hematopoietic in the fetal liver and blood with adar1+/- and ADAR1-deficient fetal from a of hematopoietic cells with which in in liver (Fig. cells with (Fig. of cell as by The of hematopoietic progenitors in fetal liver into erythroid cells M.A. Nature. PubMed Scopus Google Scholar). we in liver, at a in the of erythroid cells (Fig. In only a few erythroid of as in ADAR1-deficient fetal at (Fig. in as by normal of at (Fig. analysis by that the expression of hepatic genes 2000; PubMed Scopus Google Scholar, Rev. 2002; PubMed Scopus Google Scholar), including the and and was with that in embryos at However, the of the ADAR1-deficient fetal liver at E11.5 and our analysis that cell of from E11.5 embryos with from adar1+/- and hematopoietic progenitors with expression of the receptor M. S. K. S. T. J. T. T. 1993; Google Scholar), as by (Fig. We the of hematopoietic progenitors in the fetal and blood from E11.5 an in colony-forming In all we in embryos a in of BFU-E, and progenitors with embryos (Fig. a defect in definitive in the which the blood cells of the developing on ADAR1, as as in the from embryos with from embryos ADAR1 the of definitive hematopoietic it to as from of cells in from ADAR1-deficient fetal at with ADAR1-deficient and at and E11.5 expression by of hematopoietic genes T. M. N. N. K. S. Y. T. M. 1998; PubMed Scopus Google Scholar), including embryonic and erythroid and of an in of progenitors for erythroid as well as myeloid/granuloid cells in ADAR1-deficient hematopoietic that which can along both J. Cell. Scopus Google Scholar), may ADAR1. These progenitors in the spleen several days injection into mice Res. 14: PubMed Scopus Google Scholar). mouse the at in the region, at and the fetal liver Nature. 1993; PubMed Scopus Google Scholar). Thus, we single cell of regions and fetal from and embryos into spleen 11 days and by We in regions from and embryos and in fetal from In of regions and fetal from embryos in spleen These a for ADAR1 in progenitors in both regions and fetal liver. that ADAR1 we generated chimeric mice by injection of ADAR1-deficient ES cell clones into blastocysts of adult chimeras day and the of ES cells to different was by two analyses. adar1 the of and null alleles (Fig. the of by the ADAR1-deficient ES cells was from the of an to the mouse for and for and (Fig. by these cells to of including and and in However, we failed to a of cells to all hematopoietic marrow, spleen, thymus, and in with our analysis of ADAR1-deficient hematopoietic Remarkably, cells in the and hence, the for ADAR1 might hematopoietic ADAR1, ADAR2, in genetic analysis of A-to-I editing in 5-HT2C receptor transcripts for the that ADAR1 A-to-I conversion in further a ADAR1 and ADAR2 edit different adenosines Hence, ADAR1 and ADAR2 appear to This is by our that the phenotype of embryos in both ADAR proteins was from that caused by ADAR1 even these enzymes are expressed early in with transcripts in ES cells (6Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar). M. and P. early expression to have no as severe of ADAR1 become only and of ADAR2 (3Higuchi M. Maas S. Single F.N. Hartner J. Rozov A. Burnashev N. Feldmeyer D. Sprengel R. Seeburg P.H. Nature. 2000; 406: 78-81Crossref PubMed Scopus (747) Google Scholar). for the role of ADAR1 in mice and that homozygosity for differently configured null alleles caused embryonic lethality between E11.5 and along with fetal liver structure, and definitive Thus, the of haploinsufficiency for ADAR1 (6Wang Q. Khillan J. Gadue P. Nishikura K. Science. 2000; 290: 1765-1768Crossref PubMed Scopus (347) Google Scholar) could be and to have from expression of the adar1 allele, leading to ADAR1 in the ADAR proteins edit as W. R. Nishikura K. J. Biol. Chem. Full Text Full Text PDF PubMed Scopus Google Scholar, A. M.A. J. PubMed Scopus Google Scholar), of ADAR1 functional in a for ADAR1 in indicate a for ADAR1 in region and fetal liver hematopoiesis, as by the of spleen colony-forming activity of ADAR1-deficient blood cell progenitors in both The of ES cells to contribute to any hematopoietic in the adult mouse chimeras generated by us may well that hematopoietic progenitors ADAR1 in a ADAR1 is in the survival of these as by in colony-forming in combination with and in fetal However, the of the hematopoietic progenitors to to be by of ADAR1, for example from the of cells in of fetal liver. in the ADAR1, with embryos blood and normal of Thus, on ADAR1 an additional of and definitive ADAR1 is to ADAR1 with other definitive 1996; PubMed Google Scholar, I. A. Rev. 2002; PubMed Scopus Google Scholar). no to the phenotype of however, by two in for definitive hematopoiesis, but for These are K. Cell. Full Text PDF PubMed Scopus Google Scholar) and with its T. J. G. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Q. T. M. M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar) and T. M. N. N. K. S. Y. T. M. 1998; PubMed Scopus Google Scholar, K. K. T. K. Y. T. T. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar, Q. T. J.C. G. A. M. M. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, M. J. K. S. Y. M. T. Proc. Natl. Acad. Sci. U. S. A. 1997; 94: PubMed Scopus Google Scholar). the subunit of is for the of all definitive hematopoietic cells and to be involved in the of early progenitors from the T. T. Wang Q. M. M. 1999; PubMed Google Scholar, M. T. 2000; Full Text Full Text PDF PubMed Scopus Google Scholar). embryos at mid-gestation all erythroid and progenitors T. J. G. Cell. 1996; Full Text Full Text PDF PubMed Scopus Google Scholar, Q. T. M. M. Proc. Natl. Acad. Sci. U. S. A. 1996; PubMed Scopus Google Scholar), whereas in ADAR1-deficient embryos at erythroid and myeloid/granuloid progenitors are still at progenitors are at in embryos as a of survival in fetal liver R. A. M. J. 2000; PubMed Scopus Google Scholar). This phenotype that of although lethality in and in contrast to ADAR1 is known to the fetal liver K. Cell. Full Text PDF PubMed Scopus Google Scholar). ADAR1 and fetal liver at a cell in its A of no with in mid-gestation embryos in the P. M. T. K. T. T. 1999; PubMed Google Scholar, S. Y. A. T. M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar) and in embryos the A. I. A. J. A. 2000; 14: Google Scholar). it has been embryos that can in the of definitive hematopoietic progenitors T. K. S. D. G. J. S. N. T. J. T. M. M. Y. T. T. Biol. 2002; PubMed Google Scholar). may ADAR1, a by our in which of ADAR1-deficient ES cells in adult liver, but in other of and the to contribute to adult liver is a consequence of fetal liver hematopoiesis, as ES cells in the which is for fetal liver and hematopoiesis, contribute to adult liver Keller G. M. J. M. Nature. 1994; PubMed Scopus Google Scholar). these and a function of ADAR1 in the hepatic by ADAR1 lethality at caused by definitive is embryos all definitive blood cells to and embryos of definitive hematopoietic progenitors die at K. Cell. Full Text PDF PubMed Scopus Google Scholar). Thus, definitive the death of the of the fetal liver as in which die at with P. M. T. K. T. T. 1999; PubMed Google Scholar, S. Y. A. T. M.A. Proc. Natl. Acad. Sci. U. S. A. 1998; 95: PubMed Scopus Google Scholar), leads to of the blood in and we in embryos an of blood in fetal liver which might the of and defects in our might the embryonic death caused by ADAR1 as in mice, which die of A. M. A. Y. J. P. J.C. M. J.C. G. Nature. PubMed Scopus Google Scholar), from the by the molecular for the that the developing as a of ADAR1 the of ADAR1 as an enzyme in vivo has been no transcripts that A-to-I editing are known in the fetal liver. In the editing status of known edits in brain-expressed transcripts, the an unedited in a transcript in at the of the mid-gestation however, as by the single edit critical for (3Higuchi M. Maas S. Single F.N. Hartner J. Rozov A. Burnashev N. Feldmeyer D. Sprengel R. Seeburg P.H. Nature. 2000; 406: 78-81Crossref PubMed Scopus (747) Google Scholar). that additional functional of ADAR1, as a more involvement in RNA are even more that is by ADAR1 We N. Single for with gene targeting and ES cell for with for of and for and for and and for the