de Bruijn, MFTR

Dzierzak, Elaine

Luteijn, TK

Peeters, MCE

Speck, NA

Visser, Petra

Undetermined

EUR Research Repository

Brief reportCFU-S11 activity does not localize solely with the aorta in theaorta-gonad-mesonephros regionMarella F. T. R. de Bruijn, Marian C. E. Peeters, Tanya Luteijn, Pim Visser, Nancy A. Speck, and Elaine DzierzakThe aorta-gonad-mesonephros (AGM) re-gion is a potent hematopoietic site in themidgestation mouse conceptus and firstcontains colony-forming units–spleen day11 (CFU-S11) at embryonic day 10 (E10).Because CFU-S 11 activity is present in theAGM region before the onset of hemato-poietic stem cell (HSC) activity, CFU-S 11activity in the complex developing vascu-lar and urogenital regions of the AGM waslocalized. From E10 onward, CFU-S 11 ac-tivity is associated with the aortic vascu-lature, and is found also in the urogenitalridges (UGRs). Together with data ob-tained from organ explant cultures, inwhich up to a 16-fold increase in CFU-S 11activity was observed, it was determinedthat CFU-S 11 can be increased autono-mously both in vascular sites and inUGRs. Furthermore, CFU-S 11 activity ispresent in vitelline and umbilical vessels.This, together with the presence of CFU-S 11in the UGRs 2 days before HSC activity,suggests both temporally and spatially dis-tinct emergent sources of CFU-S 11. (Blood.2000;96:2902-2904)© 2000 by The American Society of HematologyIntroductionDuring mouse embryonic development, different hematopoieticcell types are successively generated at several locations within theconceptus. At embryonic day 7.5 (E7.5), primitive erythrocytes andtheir direct progenitors are found in the yolk sac (YS).1 Shortlythereafter, the first definitive, in vitro clonable hematopoieticprogenitors are formed in the YS and embryo body.2-4 Cells capableof in vivo hematopoietic activity are found slightly later in both theYS and the embryo: at E9 colony-forming units–spleen day 8(CFU-S8)5 and neonatal repopulating cells6 and at E10 CFU-S115and adult repopulating cells.7 We previously showed that long-termadult repopulating hematopoietic stem cells (HSCs) autonomouslyemerge first in the intrabody aorta-gonad-mesonephros (AGM)region (more than 34 somite pair [sp]).8 More recently wesubdissected the AGM region into separate pieces containing eitherthe aorta and surrounding mesenchyme or urogenital ridges(UGRs) and demonstrated that adult repopulating HSCs are firstfound exclusively in the area of the dorsal aorta and not in theflanking UGRs.9 B-cell progenitors in E10 AGM were also foundpredominantly in the aorta/mesentery subregion.3 However, sublo-calization has not been performed on CFU-S11, a myeloid-restrictedstem cell subset that is slightly downstream from HSCs in the adulthematopoietic differentiation hierarchy.We previously showed that CFU-S11 can be detected in theAGM region slightly earlier (32-33 sp) than adult repopulatingHSCs (at least 34 sp).7,8 Spatial and temporal mapping of CFU-S11activity in the AGM should help delineate the relationship betweenthe functionally distinct CFU-S11 and HSCs and contribute to ourinsight into the complex process of the onset of definitivehematopoiesis. Here we show that unlike HSCs, which are initiallylocalized to the area of the dorsal aorta, CFU-S11 are found in boththe aorta and UGRs of E10 to E12 embryos. The number ofCFU-S11 in the aorta and in UGRs increases over developmentaltime. Furthermore, our data demonstrate that the aorta and UGRsare capable of autonomously increasing CFU-S11 activity inexplant cultures, suggesting that CFU-S11 are generated in situeither from direct precursors or from pre-existing HSCs andexpand during the in vitro culture period.Study designEmbryo generationC57BL/103 CBA F1 female mice were mated with C57BL/103 CBA F1or ln7210 male mice to generate embryos of appropriate ages. The day ofvaginal plug discovery was considered E0. At day 10, 11, or 12 ofpregnancy, the females were killed by cervical dislocation. The mice werehoused according to institutional guidelines, and animal procedures werecarried out in compliance with the standards for Humane Care and Use ofLaboratory Animals.Cell preparationThe embryos were taken out of the uterus, and AGMs were dissected.11Subsequently, AGMs were subdissected into the aorta with its surroundingmesenchyme and the UGRs using 27-gauge insulin syringes (BectonDickinson, San Jose, CA). The tissues were either cultured in 3-day explantcultures8 or directly processed for transplantation into irradiated adult mice.Cell suspensions of freshly isolated or cultured tissues were obtained byFrom the Department of Cell Biology and Genetics and the Department ofClinical Genetics, Erasmus University, Rotterdam, The Netherlands, and theDepartment of Biochemistry, Dartmouth Medical School, Hanover, NH.Submitted March 21, 2000; accepted June 12, 2000.Supported by grant 901-09-090 (M.dB. and E.D.) from the The NetherlandsOrganization for Scientific Research (NWO), The Hague, The Netherlands;grant EUR 99-1965 (M.P. and E.D.) from the Dutch Cancer Society,Amsterdam, The Netherlands; the Leukemia Society of America (New York,NY) award no. 103-94 (E.D.); the Fogarty Award no. FO6TW03300 (N.S.); andgrants ROCA58343 (N.S.) and R01 DK51077-01 (E.D.) from the NationalInstitutes of Health, Bethesda, MD.Reprints: Elaine Dzierzak, Department of Cell Biology and Genetics, ErasmusUniversity, PO Box 1738, 3000 DR Rotterdam, The Netherlands; e-mail:dzierzak@ch1.fgg.eur.nl.The publication costs of this article were defrayed in part by page chargepayment. Therefore, and solely to indicate this fact, this article is herebymarked ‘‘advertisement’’ in accordance with 18 U.S.C. section 1734.© 2000 by The American Society of Hematology2902 BLOOD, 15 OCTOBER 2000 z VOLUME 96, NUMBER 8collagenase digestion as previously described.11 Viable cells were countedusing trypan blue (Sigma Chemical, St Louis, MO).CFU-S11 assayCell suspensions of AGM, aorta, or UGRs were injected intravenously intothe tail vein of adult (8-14 weeks old) C57BL/103 CBA F1 mice (male orfemale). The recipients were conditioned by 1000 rads ofg-irradiation,which was administered in a split dose, with 3 hours between doses. Themice were killed 11 days after transplantation by cervical dislocation. Thespleens were removed and fixed in Tellesniczky’s fixative, and the colonieswere counted by macroscopic observation.Scanning electron microscopyIsolated AGMs were fixed in 2.5% glutaraldehyde (Sigma), post-fixed in1% osmium tetroxide (OsO4), dehydrated in ethanol, and critically point-dried. The tissues were mounted on stubs, coated with gold/paladium, andexamined with a JEOL (Tokyo, Japan) JSM-25 electron microscope.Results and discussionThe spatial and temporal distribution of CFU-S11 in the E10 to E12AGM region was examined after subdissecting this tissue into partscontaining the aorta with its surrounding mesenchyme and theUGRs. As shown in scanning electron micrographs, the AGMdoubles in size from E10 to E12 (Figure 1A). During this time theFigure 1. Changes in morphology and cell counts of the E10, E11, and E12 AGMregion. (A) Scanning electron micrographs of the E10 (left), E11 (middle), and E12(right) AGM region in which the excretory and reproductive systems are undergoingrapid growth and morphogenic changes. A indicates aorta; G, genital ridge/gonad; M,pro/mesonephros; Mn, metanephros; Me, mesenchyme; and Mt, mesentery (originalmagnification 3 40). (B) The ongoing organogenesis results in an increase in the cellnumber within the AGM from E10 to E12. Viable cell numbers for the subdissectedaorta, UGRs, and entire AGM after collagenase treatment are given for eachembryonic day (the mean 6 SD). UGRs comprised pro/mesonephroi and genitalridges/gonads. Cell counts of E12 UGRs were obtained from counts of whole UGRs(n 5 2) and in part from counts of UGRs further subdissected into pro/mesonephroiand gonads (n 5 3). At E12 the emerging metanephroi were not included in the cellcounts of the total AGM region or UGRs. After 3 days of organ explant culture, the cellcounts (given as 3 104 cells) for the different tissues were (1) E10: aorta, 5.6; UGRs,7.1; and AGM, 13.0; (2) E11: aorta, 5.4 6 2.5; UGRs, 8.2 6 1.9; and AGM,22.0 6 2.7; and (3) E12: aorta, 6.0; UGRs, 18.0; and AGM, 30.8.Figure 2. Distribution of CFU-S 11 in the aorta and UGRs. (A, B) The aorta withsurrounding mesenchyme and the UGRs were dissected from E10 to E12 embryos,and cell suspensions of these tissues were directly injected into irradiated adult miceto assay for CFU-S11. (C) Organ explant cultures (3 days) of the subdissected tissueswere performed prior to transplantation. The total number of CFU-S11 per tissue isgiven in panel A, the frequency in panel B, and the total number after organ explantculture in panel C (fold increase compared with uncultured tissues are shown inparentheses). The data are the mean of results obtained from individually trans-planted mice in a total of 14 independent experiments. CFU-S11 values (given astimes 104 cells) for the aorta and UGRs ranged from (A) E10: aorta, 0-0.3, and UGRs,0-0.2; E11: aorta, 0-0.7, and UGRs, 0-0.35; E12: aorta, 0.42-1.0, and UGRs,0.42-0.75; (B) E10: aorta, 0-4.2, and UGRs, 0-3.17; E11: aorta, 0-5.1, and UGRs,0-3.2; E12: aorta, 2.8-6.6, and UGRs, 3.2-7.2; and (C) E10: aorta, 0.2-0.71, andUGRs, 0-0.27; E11: aorta, 0.22-3.14, and UGRs, 1.5-3.33; and E12, aorta 0.67-1.1,and UGRs, 0.6-0.9. In panels A and C, the number of CFU-S11 per UGRs at E12 wascalculated from the number of CFU-S11 obtained from whole UGRs and partly fromthe number obtained from isolated gonads and pro/mesonephroi. The number ofCFU-S11 obtained per embryo equivalent (ee) of whole E12 UGRs was 0.6 in panel Aand 0.75 in panel C; the number obtained from gonads and pro/mesonephroi was 0.1and 0.4, respectively, in panel A and 0 and 0.7, respectively, in panel C. Thefrequency of CFU-S11 (panel B) was calculated from the number of colonies per eeand the cell counts per tissue. For direct transplantations (panels A and B) thefollowing total numbers of tissues were injected: E10: both aorta and UGRs, 40 ee;E11: aorta, 53 ee, and UGRs, 47.5 ee; and E12: aorta, 22 ee; UGRs, 8 ee; gonads, 14ee; and mesonephroi, 14 ee. For the cultured data (panel C): E10, aorta, 26 ee, andUGRs, 27 ee; E11: both aorta and UGRs, 22 ee; and E12: aorta, 26 ee; UGRs, 20 ee;gonad and mesonephri, 6 ee. (D) A summary of the distribution of HSCs in the E10 toE12 aorta and UGRs is given (from de Bruijn et al9). ND indicates not determined.DISTINCT EMERGENT SOURCES OF CFU-S11 2903BLOOD, 15 OCTOBER 2000 z VOLUME 96, NUMBER 8distinctive features of the maturing excretory and reproductivesystems allow for the precise separation of the UGRs from thedorsal aorta and mesenchyme. Cell counts were performed on thewhole AGM, subdissected aorta, and UGRs. All tissues showed a2-fold increase in cell number from E10 to E12 (Figure 1B). AfterE12, following further organogenesis, the AGM region as suchrecedes, and hematopoiesis takes place in the fetal liver.12CFU-S11 were present in both the area of the aorta and UGRs atE10, E11, and E12 (Figure 2A). This was in agreement with thesublocalization of CFU-S8 that was found at E10.13 A significantincrease in CFU-S11 was observed between E10 and E12, ie,4.8-fold for the aorta and 11.2-fold for the UGRs (P # .003, asdetermined by linear regression analysis). At all time points therewere more CFU-S11 in the aorta than in the UGRs (75%, 70%, and56% of the recovered CFU-S11 for E10, E11, and E12, respec-tively). Also, the frequency of CFU-S11 was highest in the aorta atE10 and E11 (Figure 2B). Although the aorta appears to harbormore CFU-S11 than the UGRs, the precise site of CFU-S11 originremains uncertain. Similar to adult repopulating HSCs,9 CFU-S11may find their origin in association with the intra-embryonicvasculature. To examine this possibility, we also transplanted cellsof the vitelline and umbilical vessels. At E10.5 and E11, wefound 0.3 and 1.6 CFU-S11, respectively, per embryo equivalentof transplanted vitelline and umbilical cells, suggesting thatCFU-S11 may be generated and/or expanded in association withvascular tissue.The temporal increase in CFU-S11 activity in the aorta and alsoin the UGRs may reflect either the autonomous generation and/orexpansion of these cells in the aorta and UGRs or the influx ofCFU-S11 into these tissues. To examine these possibilities, organexplants of the E10 to E12 aorta and UGRs were cultured prior toassaying CFU-S11 activity. At E10, CFU-S11 activity was main-tained during culture (Figure 2C) and at best showed an average2.5-fold increase compared to directly transplanted cells. Incontrast, CFU-S11 activity significantly increased in cultures of theE11 aorta (5-fold) and UGRs (16.6-fold) (P , .05 andP , .005,respectively, as determined by the Mann-Whitney U test). At E12,CFU-S activity was maintained in these cultured tissues. Thesedata indicate that both the E11 aorta and UGRs are capable ofsignificantly increasing CFU-S11 in a manner that is independent ofinflux from other embryonic sites. This suggests that the increasedCFU-S11 activity between E11 and E12 (P , .02, as determined bythe t test) reflects in situ generation and/or expansion of CFU-S11.Interestingly, the culture of E11 UGR explants results in thegreatest increase in CFU-S11 numbers. This result is reminiscent ofthe dramatic increase in HSCs in the E11 UGR explant cultures wer cently observed9 and leads us to speculate that this increase inCFU-S11 results from their differentiation from newly formedHSCs in the UGR cultures. Indeed, CFU-S11 were found in all thesame sites where the first HSCs were found. However, theobservation that CFU-S11 were detected in the UGRs before HSCsemerged in this site (Figure 2D) leaves open the question of howthese 2 hematopoietic cell types are related to each other duringearly embryogenesis. A possibility is that CFU-S11 and HSCs arederived separately from different precursors, such as putativehemangioblasts. This would be in agreement with data showingmultiple sites of hematopoietic birth in avian embryos.14 Thus, theCFU-S we detected may be derived by 2 different means.In the present study we report the temporal and spatialmergence of CFU-S11 activity within the mouse AGM. We showthat from mid-E10 onward, CFU-S11 are increasingly found both inthe area of the aorta and in the UGRs, and that CFU-S11 can bencreased autonomously in both sites. It is of great interest tofurther examine the cells and factors involved in the increase ofCFU-S11 activity as well as the possible lineage relationshipsbetween putative hemangioblasts, CFU-S11 and HSCs. Such stud-ies are likely to provide additional insight in the origin and ex vivomanipulation of the clinically important HSCs.AcknowledgmentsWe would like to thank Alexander Medvinsky and all the membersof the laboratory for their helpful discussions. We also thank RobPloemacher and Sjaak Philipsen for critical comments on thismanuscript and Jeroen Essers for help with the figures.References1. Russell ES, Bernstein SE. Blood and blood for-mation. In: Green EL, ed. Biology of the Labora-tory Mouse. New York, McGraw-Hill; 1966:351-372.2. Palis J, Robertson S, Kennedy M, Wall C, KellerG. Development of erythroid and myeloid pro-genitors in the yolk sac and embryo proper of themouse. Development. 1999;126:5073-5084.3. Godin I, Garcia-Porrero JA, Dieterlen-Lievre F,Cumano A. Stem cell emergence and hemopoi-etic activity are incompatible in mouse intraem-bryonic sites. J Exp Med. 1999;190:43-52.4. Cumano A, Dieterlen-Lievre F, Godin I. Lymphoidpotential, probed before circulation in mouse, isrestricted to caudal intraembryonic splanchno-pleura. Cell. 1996;86:907-916.5. Medvinsky AL, Samoylina NL, Muller AM, Dzier-zak EA. An early pre-liver intraembryonic sourceof CFU-S in the developing mouse. Nature. 1993;364:64-67.6. Yoder MC, Hiatt K, Dutt P, Mukherjee P, BodineDM, Orlic D. Characterization of definitive lym-phohematopoietic stem cells in the day 9 murineyolk sac. Immunity. 1997;7:335-344.7. Muller AM, Medvinsky A, Strouboulis J, GrosveldF, Dzierzak E. Development of hematopoieticstem cell activity in the mouse embryo. Immunity.1994;1:291-301.8. Medvinsky A, Dzierzak E. Definitive hematopoi-esis is autonomously initiated by the AGM region.Cell. 1996;86:897-906.9. de Bruijn MFTR, Speck NA, Peeters MCE, Dzier-zak E. Definitive hematopoietic stem cells firstdevelop within the major arterial regions of themouse embryo. EMBO J. 2000;19:2465-2474.10. Strouboulis J, Dillon N, Grosveld F. Developmen-tal regulation of a complete 70-kb human beta-globin locus in transgenic mice. Genes Dev.1992;6:1857-1864.11. Dzierzak E, de Bruijn M. Isolation and analysis ofhematopoietic stem cells of mouse embryos. In:Klug CA, Jordan CT, eds. Methods in MolecularMedicine: Hematopoietic Stem Cell Protocols.Totowa, NJ: The Humana Press, Inc. In press.12. Dzierzak E, Medvinsky A, de Bruijn M. Qualitativeand quantitative aspects of haematopoietic celldevelopment in the mammalian embryo. ImmunolToday. 1998;19:228-236.13. Medvinsky AL, Gan OI, Semenova ML,Samoylina NL. Development of day-8 colony-forming unit-spleen hematopoietic progenitorsduring early murine embryogenesis: spatial andtemporal mapping. Blood. 1996;87:557-566.14. Dieterlen-Lievre F. Hematopoiesis: progenitorsand their genetic program. Curr Biol. 1998;8:R727–R730.2904 de BRUIJN et al BLOOD, 15 OCTOBER 2000 z VOLUME 96, NUMBER 8

CFU-S11 activity does not localize solely with the aorta in the aorta-gonad-mesonephros region

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CFU-S11 activity does not localize solely with the aorta in the aorta-gonad-mesonephros region

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