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Blood, 15 July 2006, Vol. 108, No. 2, pp. 501-509. Prepublished online as a Blood First Edition Paper on March 21, 2006; DOI 10.1182/blood-2005-10-4209. This Article Abstract Full Text (PDF) Supplemental Tables All Versions of this Article: 2005-10-4209v1 108/2/501    most recent Alert me when this article is cited Alert me if a correction is posted Citation Map Services Email this article to a friend Similar articles in this journal Similar articles in PubMed Alert me to new issues of the journal Download to citation manager Rights and Permissions Citing Articles Citing Articles via HighWire Citing Articles via CrossRef Citing Articles via Google Scholar Google Scholar Articles by Gribi, R. Articles by Medvinsky, A. Search for Related Content PubMed PubMed Citation Articles by Gribi, R. Articles by Medvinsky, A. Related Collections Hematopoiesis and Stem Cells Cell Adhesion and Motility Stem Cells in Hematology Social Bookmarking                   What's this?  Previous Article  |  Table of Contents  |  Next Article  HEMATOPOIESIS The differentiation program of embryonic definitive hematopoietic stem cells is largely 4 integrin independent Ruby Gribi, Lilian Hook, Janice Ure, and Alexander Medvinsky From the Medical Research Council (MRC) Centre Development in Stem Cell Biology, Institute for Stem Cell Research, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh, United Kingdom.    Abstract Top Abstract Introduction Materials and methods Results Discussion References   Previous analyses of the roles of 4 integrins in hematopoiesis by other groups have led to conflicting evidence. 4 integrin mutant cells developing in [4 integrin–/–: wt] chimeric mice are not capable of completing lymphomyeloid differentiation, whereas conditional inactivation of 4 integrin in adult mice has only subtle effects. We show here that circumventing the fetal stage of hematopoietic stem cell (HSC) development by transplantation of embryonic 4 integrin–/– cells into the adult microenvironment results in robust and stable long-term generation of 4 integrin–/– lymphoid and myeloid cells, although colonization of Peyer patches and the peritoneal cavity is significantly impaired. We argue here that collectively, our data and the data from other groups suggest a specific requirement for 4 integrin during the fetal/neonatal stages of HSC development that is essential for normal execution of the lymphomyeloid differentiation program.    Introduction Top Abstract Introduction Materials and methods Results Discussion References   The first emergence of highly repopulating definitive hematopoietic stem cells (HSCs) occurs at E10.5-E11.5 in the aorta-gonadmesonephros (AGM) region and placenta and, with a slight delay, in the yolk sac (YS).1-7 The emergence of HSCs is closely followed by their expansion and transit via the circulation into the fetal liver (FL) from E11.5 and subsequently to the bone marrow (BM) shortly after birth.4,6,8 Adhesion molecules, including integrins, play important roles in the development, homing, and functioning of HSCs and progenitor cells.9 The study of 1 integrin knockout mice established a strict dependence of HSC homing to the FL and adult hematopoietic organs on this molecule,10,11 which corroborated earlier observations that infusion with anti–1 integrin blocking antibodies inhibited the formation of myeloid spleen colonies and medullary hematopoiesis in vivo.12 Among the many 1 integrin heterodimeric complexes involving integrin subunits, 41 integrin (VLA-4) is one of the important players in hematopoiesis.9,13 Associations between VLA-4 and VCAM-114,15 and VLA-4 and fibronectin (via the Cs-1 sequence)16-19 mediate the interactions of progenitor cells with hematopoietic stroma. Addition of blocking anti–4 integrin antibodies to long-term BM cultures completely inhibits lymphopoiesis and retards myelopoiesis in long-term BM cultures.20 Anti–4 integrin (but not anti–5 integrin) and anti–VCAM-1 antibodies suppress formation of blast colony-forming cells on stroma in vitro.21 The in vivo injection of cells incubated with anti–4 integrin or infusion of anti–VCAM-1 antibodies results in a decreased homing efficiency of progenitor spleen colony-forming unit and colony-forming unit culture cells to the BM.22 In addition, administration of blocking antibodies to 4 integrin and VCAM-1 causes stem/progenitor cell mobilization in vivo and potentiates the mobilization effects of granulocyte colony-stimulating factor and stem cell factor.23-27 Binding of 4 integrin to fibronectin also has been shown to be important for recruiting erythroid progenitors in the BM.12,28 Another heterodimeric complex involving 4 integrin, 47, and its ligand MAdCAM-1 have been shown to be important for the homing of hematopoietic progenitors to the BM,29 as well as the homing of lymphocytes to Peyer patches.30-33 Generation of 4 integrin knockout mice by Arroyo et al34,35 enabled further analysis of the role of 4 integrin in hematopoiesis. 4 integrin–/– embryos have smaller and paler livers and usually die by E12.5 due to heart and, perhaps, placental defects.36 Consequently, the role of 4 integrins in the later stages of development has been studied in [4 integrin–/–: wt] and [4 integrin–/–: Rag–/–] chimeric animals generated by injection of 4 integrin–/– embryonic stem cells into wild-type or Rag–/– blastocysts.34,35 4 integrin–/– erythroid, myeloid, and B-lymphoid progenitors are present in the FL of chimeric embryos, but progressively lose their capacity to complete differentiation. In adult mice, 4 integrin–/– progenitors migrate to the spleen and BM but are unable to substantially contribute to the erythroid, myeloid, and B-lymphoid lineages.34,35 This differentiation block can be released in vitro, and it has been proposed that interaction with stroma via 4 integrin in vivo is essential for hematopoietic differentiation. The T-lymphoid lineage also shows signs of compromised development in the embryonic thymus.34,35 After birth, the development of 4 integrin–/– T lymphocytes in the adult thymus is blocked prior to the CD4/CD8 double-positive stage, with thymi becoming progressively atrophic in [4 integrin–/–: Rag–/–] chimeras. These data confirm a fundamental role for 4 integrin in hematopoietic development and differentiation, as suggested previously by antibody-blocking experiments. In contrast, conditional deletion of 4 integrin in adult mice by Scott et al37 resulted in only minor perturbations in hematopoiesis, such as an efflux of hematopoietic progenitors from the BM, with a consequential increase in progenitors in the spleen and blood. Lymphoid (CD4+ T cells and B220+ B cells), but not myeloid, populations of the BM also were slightly compromised.37 These minor effects are in marked contrast with the dramatic inability of 4 integrin–/– hematopoietic cells to be maintained and differentiated in the [4 integrin–/–: wt] chimeric environment. This discrepancy can be explained either by (1) a competitive disadvantage of 4 integrin–/– cells in the presence of wild-type counterparts in chimeric animals, or (2) an essential role that 4 integrins play during embryonic and/or fetal/neonatal development of HSCs (but not in the adult), implying that in their absence, the development/differentiation program of HSCs becomes corrupt. To discriminate between these possibilities, we investigated the developmental potential of early (embryonic) definitive 4 integrin–/– HSCs. We have found that HSCs express 4 integrin throughout development, and in its absence the initial pattern of HSC allocation in the embryo is only slightly perturbed. Early embryonic 4 integrin–/– HSCs that have bypassed fetal/neonatal development via transplantation directly into the adult differentiate normally and contribute stably and robustly to adult hematopoiesis (although the level of donor contribution tends to be lower than for mice receiving wild-type transplants). Furthermore, the differentiation of donor-derived 4 integrin–/– hematopoietic cells is not compromised by the presence of cotransplanted wild-type BM cells, and major cell subsets home normally to adult tissues, with the exception of the Peyer patches and the peritoneal cavity. We conclude that 4 integrin is critically important for HSCs to be able to develop through the fetal/neonatal stage and maintain their normal differentiation potential.    Materials and methods Top Abstract Introduction Materials and methods Results Discussion References   Animals and tissues CBA, C57BL/6, and (CBA x C57BL/6) F1 mice were bred in the animal breeding unit of the University of Edinburgh in compliance with Home Office regulations. Cell suspensions were prepared from E11.5-E13.5 embryonic tissues by treating them with 0.1% collagenase-dispase (Roche Diagnostics, Mannheim, Germany) in phosphate-buffered saline (PBS) at 37°C. Peripheral circulation was collected in PBS and put on ice within 2 to 3 minutes of collection. Wild-type and 4 integrin–/– embryo donor tissues isolated from (CBA x C57BL/6) F1 embryos were transplanted into congenic (CBA x C57BL/6) F1 Ly-5.1/1 or Ly-5.1/2 recipients. In some experiments, (CBA x C57BL/6) F1 wild-type GFP+ and 4 integrin–/– GFP+ donor tissues were used for transplantation into (CBA x C57BL/6) F1 wild-type recipients.38 In all cases, 4 integrin–/– embryos were genotyped by flow cytometry and polymerase chain reaction analysis. Additionally, upon the analysis of recipient peripheral blood (PB) after transplantation, donor cells were confirmed via flow cytometry to be lacking 4 integrin expression (Figure 4A). Irradiation of recipients and transplantations, including co-injection of 20 000 BM cells (containing on average 2 HSCs) per mouse, was performed as described previously.4 At time points of 6, 12 to 19, and more than 19 weeks after transplantation, the donor cell contribution was assessed by flow cytometry using Ly-5.1 and Ly-5.2 antibodies, or in some cases by green fluorescent protein expression. Recipients were considered reconstituted if the donor chimerism in the peripheral blood was at least 5%. Organ explant culture E11.5 AGM regions isolated from (CBA x C57BL/6) F1 embryos were cultured on Durapore 0.65 µm filters (Millipore, Watford, United Kingdom) as described previously.4 Flow cytometry Flow cytometry was performed using a FACSCalibur (Becton Dickinson, San Jose, CA) and evaluated with FlowJo software (Tree Star, Ashland, OR). Cell suspensions from embryonic tissues were obtained following 0.1% collagenase-dispase treatment, from adult BM mechanically using a syringe, and from other adult tissues by grinding in PBS. For peritoneal lavage, room-temperature PBS was used, and cells were put on ice directly following collection. Except where indicated, adult peripheral blood, BM, and spleen red blood cells were lysed using PharMLyse (BD Biosciences, Oxford, United Kingdom). Staining was performed with directly conjugated antibodies (Ly-5.1 FITC [A20], Ly-5.2 FITC [104], CD41 FITC [MWReg30], CD11b FITC [M1/70], Sca-1 FITC [E13-161.7], IgM FITC [R6-60.2], CD45 FITC [30-F11], CD34 FITC [RAM34], Ly-5.1 PE [A20], Ly-5.2 PE [104], CD3 PE [145-2C11], CD49d PE [9C10], CD11b PE [M1/70], Ter119 PE [TER-119], CD8 PE [53-6.7], CD19 PE [1D3], CD44 PE [IM7], Sca-1 PE [D7], IgD [11-26], Ly-5.1 APC [A20], c-Kit APC [2B8], AA4.1 APC [AA4.1], Gr1 biotin [RB6-8C5], B220 biotin [RA3-6B2], CD49d biotin [9C10], CD41 biotin [MWReg30], Ter119 biotin [TER-119], CD3 biotin [145-2C11], and CD11b Biotin [M1/70]). Streptavidin APC and streptavidin PE-Cy5.5 were used for detection of primary biotinylated antibodies. Dead-cell exclusion was performed using 7-AAD in all instances. All reagents were purchased from BD Biosciences (Oxford, United Kingdom) and eBiosciences (San Diego, CA). All gates were set based on appropriate isotype control stainings. Statistical analysis Fisher exact test with contingency tables was used to compute statistical significance of repopulation assay results. All other statistics were performed using unpaired Student 2-tailed t test.    Results Top Abstract Introduction Materials and methods Results Discussion References   Phenotypic characterization of 4 integrin in the developing hematopoietic system We have flow-cytometrically characterized the expression of 4 integrin in hematopoietic cell subsets during development in the E11.5 AGM region, E13.5 FL, and adult BM. To this end, cells from these tissues were stained with antibodies against CD45 (pan-leukocyte), CD41 (early progenitors; megakaryocytes), CD11b (monocytes; embryonic/fetal HSCs), Ter119 (erythroid cells), c-Kit, and CD34 and analyzed by flow cytometry (Figure 1). In the E11.5 AGM region, most, if not all, CD45+, CD41+, and CD11b+ cells express 4 integrin. In contrast, erythroid cells (Ter119+ subset) lack 4 integrin expression. Approximately 30% of c-Kit+ cells in the E11.5 AGM region coexpress 4 integrin (Figure 1). According to the previous studies, the remaining 70% of c-Kit+ cells include erythroid39 and nonhematopoietic cells.40 In contrast, expression of CD34 and 4 integrin is largely mutually exclusive, which is likely related to the fact that many CD34+ cells are endothelial41 (most VE-cadherin+ endothelial cells are 4 integrin–42). However, a rare population of CD34+4 integrin+ cells can be detected in the E11.5 AGM region (Figure 1). Of note, HSCs in the AGM region are c-Kit+CD34+.43 View larger version (42K): [in this window] [in a new window]   Figure 1.. 4 integrin expression in embryonic tissues and adult BM. Flow cytometrical analysis of 4 integrin coexpression with hematopoietic markers at key developmental stages. Dead cells were excluded using 7-AAD. Red blood cells were gated out for all adult bone marrow samples using forward scatter/side scatter plots, except where indicated (; all BM samples were unlyzed). Quadrants were placed according to appropriate isotype controls. (Collagenase-dispase treatment of BM produces similar staining patterns/intensities [data not shown].)   At a later point in development, in the E13.5 FL, nonerythroid hematopoietic CD45+ cells remain largely 4 integrin+; however, a significant portion of the erythroid population also becomes 4 integrin+ (Figure 1). All c-Kit+, CD11b+, and CD41+ cells also express 4 integrin. In the adult BM, the leukocytic (CD45+, CD41+, CD11b+) compartments are almost entirely 4 integrin+, whereas about half of the erythroid (Ter119+) population is 4 integrin–. As in the E13.5 FL, the majority of c-Kit+ cells express 4 integrin. Most, if not all, CD34+ cells in the BM become 4 integrin+. Embryonic and adult definitive HSCs express 4 integrin We have made further investigations into the expression of 4 integrin on HSC-enriched populations during embryonic development and in the adult. The first highly repopulating definitive HSCs emerging in the E11.5 AGM region reside within the CD45+VE-cadherin+ population.42,44 Most cells in this double-positive population are 4 integrin+ (Figure 2Ai, and Taoudi et al42). To functionally test whether HSCs in the AGM express 4 integrin, 4 integrin+ and 4 integrin– cells sorted from the E11.5 AGM region were injected into irradiated recipients. As shown in Figure 2Bi, only the mice that received transplants of 4 integrin+ cells were reconstituted. View larger version (30K): [in this window] [in a new window]   Figure 2.. Phenotypic and functional evidence that embryonic and adult HSCs express 4 integrin. (A) Adult and embryonic tissues have been analyzed for 4 integrin expression in their respective HSC-enriched populations. (Isotype control = filled curve; 4 integrin = empty curve.) (Ai) The CD45+VE-cadherin+ population, enriched with HSCs in the E11.5 AGM region,42,44 is 4 integrin+. (Aii) The lineage– c-Kit+Sca-1+CD11b+ population, enriched with HSCs in the fetal liver,45 is 4 integrin+. (Aiii) The lineage– c-Kit+Sca-1+ fraction, enriched with HSCs in the adult BM,29 is 4 integrin+. (B) 4 integrin+ () and 4 integrin– () fractions were isolated by flow sorting based on staining with anti–4 integrin antibody and transplanted into irradiated recipient mice. Dead cells were excluded using 7-AAD. The percentage of mice repopulated out of the total number receiving transplants is indicated (for actual numbers of mice, see Table S1). ee indicates embryo equivalent; HSCe, bone marrow hematopoietic stem cell equivalent. (Bi) Uncultured embryonic tissues. (Bii) Cultured E11.5 AGM region. (Biii) Adult BM (unlysed erythrocytes were gated out).   We then analyzed 4 integrin expression in enriched populations of definitive HSCs in E13.5 FL45 and adult BM.46 We found that the HSC-enriched lineage– c-Kit+Sca-1+CD11b+ cell fraction of the E13.5 FL expresses 4 integrin (Figure 2Aii). Similarly, the lineage– c-Kit+Sca-1+ fraction of adult BM, which contains HSCs, is entirely 4 integrin+ (Figure 2Aiii, and Katayama et al29). To functionally confirm the expression of 4 integrin on HSCs, in a separate experiment we sorted the 4 integrin+ and the 4 integrin– populations from these tissues and E13.5 circulation and transplanted them into irradiated recipients. Only the 4 integrin+ fractions demonstrated long-term repopulation capacity (Figure 2Bi,iii). The data in Figure 2 demonstrate the association of 4 integrin expression with HSCs throughout development. We also tested whether HSCs that have undergone expansion in ex vivo AGM organ culture maintain 4 integrin expression.2,4 In line with ubiquitous expression of 4 integrin on HSCs throughout development and in adult life, HSCs expanded in vitro also were found to reside exclusively within the 4 integrin+ cell fraction (Figure 2Bii). Early generation of definitive HSCs in the embryo is only slightly perturbed in the absence of 4 integrin Given that HSCs throughout development express 4 integrin, using 4 integrin knockout mice, we were able to investigate the specific role of 4 integrin in embryonic HSC emergence and distribution. Although 4 integrin–/– hematopoietic cells are able to develop and reside within the adult BM (as shown by Arroyo et al34,35 in [4 integrin–/–: wt] chimeric mice), their differentiation capacity is severely compromised. Initially, we considered the possibility that this could be the result of a severe HSC deficiency in the early embryo. Therefore, we assessed the distribution and differentiation capacity of HSCs in 4 sites that play important roles in HSC development.4,6 Cell suspensions were prepared from E12.5 AGM region, PB, YS, and livers of wild-type and 4 integrin–/– embryos and transplanted as fractions of embryo equivalents (ee) into irradiated recipients. Analysis was carried out on recipients to assess the proportion of mice reconstituted with 4 integrin–/– cells. Comparisons also were made between the levels of donor contribution in the peripheral blood of 4 integrin–/– and wild-type recipients of transplants. Additionally, we assessed the homing and differentiation capacity of 4 integrin–/– HSCs in principle hematopoietic tissues (see Figure 3 for experimental design). The proportion of mice reconstituted with 4 integrin–/– cells was in general slightly lower but statistically comparable to recipients that received transplants of wild-type embryonic tissues (Figure 4B and Table S2, which is available on the Blood website; see the Supplemental Tables link at the top of the online article). The level of donor cell contribution for mice that received transplants of 4 integrin–/– embryonic tissues also tended to be lower (except with the circulation) as compared to the wild-type recipients of transplants (Figure 4B). However, this trend is statistically significant only with YS-derived HSCs. We then tested whether HSC expansion can occur in the AGM organ culture assay in the absence of 4 integrin.2,4 To this end, E11.5 AGM regions from 4 integrin–/– and wild-type embryos were cultured for 4 days and transplanted into irradiated recipients (0.25 ee/recipient) (Figure 4C). All 5 animals in each experimental group were reconstituted (Figure 4C). Since it is likely that only one mouse would be reconstituted with 0.25 ee of fresh AGM prior to culture, this result indicates that HSCs are able to expand in the absence of 4 integrin. However, the average level of contribution per recipient was statistically lower in the absence of 4 integrin. The significance of the observation that the level of reconstitution tends to be slightly lower with 4 integrin–/– cells is unclear since 4 integrin–/– embryos die by E12.5 due to heart and placenta defects.36 This may have nonspecific impact(s) on hematopoietic development. In any case, the slight underperformance of transplanted 4 integrin–/– HSCs is not comparable to the marked failure of 4 integrin–/– cells continuing their development in the [4 integrin–/–: wt] embryo.34,35 View larger version (20K): [in this window] [in a new window]   Figure 3.. Experimental design. Cell suspensions prepared from embryonic (wild-type or 4 integrin–/–) tissues were cotransplanted with competitor wild-type BM cells intravenously into adult irradiated recipients. Primary recipients were analyzed using various approaches. Repopulation activity was assessed by 2 different (but complementary) criteria: (1) the number of recipient mice repopulated, and (2) the level of repopulation of individual mice ( 5%). Colonization capacity was assessed by donor repopulation of different recipient tissues. Differentiation capacity was assessed by the analysis of lymphoid and myeloid cell subsets in recipient hematopoietic tissues. Serial transplantations of 4 integrin–/– BM cells also were carried out and the peripheral blood of secondary recipients analyzed for donor contribution.   View larger version (37K): [in this window] [in a new window]   Figure 4.. Long-term repopulation assay with 4 integrin–/– and wild-type embryonic tissues. (A) A representative plot demonstrates repopulation with embryonic E12.5 yolk sac (4 integrin–/–) plus 20 000 competitor carrier (wild-type) bone marrow cells using the Ly-5.1/Ly-5.2 system. 4 integrin–/– repopulating yolk sac–derived cells show an absence of 4 integrin expression (right plot). The intermediate plot demonstrates that recipients of wild-type embryonic transplants express 4 integrin. (B) Transplantations with E12.5 tissues are plotted as the percentage of donor contribution in the peripheral blood of each mouse. (C) Transplantations with 4-day cultured 4 integrin–/– () and wild-type () E11.5 AGM region. Plotted horizontal lines represent the mean percentage level of reconstitution in the peripheral blood for each set of recipient mice. (*P < .05, comparison between wild-type and 4 integrin–/– reconstitution levels.) Note that by using another method for assessment of reconstitution, which compares the number of reconstituted mice out of the total that received transplants of wild-type and 4 integrin–/– cells, there is no significant difference between these 2 groups for all tissues transplanted. (For statistics, see Table S2.)   Embryonic 4 integrin–/– definitive HSCs show stable long-term levels of engraftment and differentiate normally in adult recipients Having established a possible defect in 4 integrin–/– HSCs, as evidenced by the trend for lower mean chimerism levels in the peripheral blood as compared to wild-type mice receiving transplants, we examined the reconstitution stability and long-term differentiation capacity of 4 integrin–/– HSCs in the presence of wild-type competitor cells. Importantly, we found that 4 integrin–/– donor-derived cells are not outcompeted by cotransplanted wild-type BM competitor cells. Both embryonic tissues (YS, AGM, and PB) and the cotransplanted 20 000 BM cells contain approximately 1 to 2 HSCs.4 As illustrated in Table 1, 4 integrin–/– embryonic cells and competitor BM cells coexist long-term in the peripheral blood. Furthermore, contribution of 4 integrin–/– cells to the peripheral blood was found to be stable up to 500 days after transplantation (Figure 5A). View this table: [in this window] [in a new window]   Table 1.. Coexistence of donor embryonic cells with competitor BM cells   View larger version (39K): [in this window] [in a new window]   Figure 5.. Contribution of 4 integrin–/– and wild-type embryonic cells to lymphohematopoietic tissues. (A) The level of reconstitution in the peripheral blood of recipient mice is maintained over time, up to 500 days after transplantation. Each symbol and connecting line represents an individual mouse that has been reconstituted with 4 integrin–/– E12.5 fetal liver (n = 5), yolk sac (n = 2), AGM region (n = 1), or peripheral blood (n = 4). (B) Mice that received transplants of 4 integrin–/– () or wild-type () E12.5 embryonic tissues (fetal liver, AGM region, yolk sac, and peripheral blood) were analyzed for donor contribution in various tissues using flow cytometry. Note the deficiency in colonization of the peritoneal cavity and the Peyer patches with 4 integrin–/– cells. The mean for relative contribution, as compared to the percent reconstitution in peripheral blood, is plotted. Bars represent standard error (*P < .01; wild-type n = 7, 4 integrin–/– n = 7).   To investigate the homing efficiency of 4 integrin–/– HSCs/progenitors to principle hematopoietic organs, we assessed the donor contribution of 4 integrin–/– cells in individual tissues. To account for variation in PB chimerism, results are reported quantitatively in relation to the contribution in the peripheral blood. In the BM, spleen, thymus, and lymph nodes we obtained statistically similar results to those acquired with wild-type HSCs (Figure 5B). We also analyzed the multilineage differentiation of 4 integrin–/– cells and found populations of myeloid and lymphoid cells in peripheral blood, BM, and spleen to be comparable to wild-type embryonic cells (Figure 6A-C, Table 2). In these tissues, no obvious difference was observed in the percentage of CD11b+Gr1+ and CD11b+Gr1– myeloid populations within the 4 integrin–/– donor population as compared to mice reconstituted with wild-type cells. Similarly, donor B cells (B220+) and T cells (CD3+) in these tissues were comparable in mice reconstituted with 4 integrin–/– or wild-type embryonic HSCs. Further analysis showed that 4 integrin–/– HSCs differentiate normally into mature B220+IgD+ B cells in the recipient bone marrow (representative plots and statistics shown in Figure 6B and Table 2). CD19/AA4.1 co-staining in the spleen (8.8% ± 1.0% for wild-type and 10.3% ± 0.7% for 4 integrin–/–) demonstrates a normal capacity for 4 integrin–/– immature B cells to populate the spleen (Figure 6C and Table 2). Furthermore, maturation of B cells in the spleen does not depend on 4 integrin: we show that wild-type donor-derived CD19+IgM+ cells constitute 57% ± 5% in the spleen, and likewise 4 integrin–/– donor cells give rise to a comparable population of these cells (55.8% ± 5.3%) (Figure 6C). View this table: [in this window] [in a new window]   Table 2.. Statistical analysis of 4 integrin–/– and wild-type donor cell subsets in recipient tissues   In the thymus, staining with anti-CD4 and anti-CD8 antibodies demonstrates normal development of 4 integrin–/– double-positive (77.0% ± 7.2% for wild-type and 67.2% ± 6.8% for 4 integrin–/–) and single-positive (SP) T-cell fractions of recipient mice (CD8 SP: 2.8% ± 0.9% for wild-type and 4.0% ± 0.6% for 4 integrin–/–; CD4 SP: 10.7% ± 1.9% for wild-type and 13.8% ± 1.9% for 4 integrin–/–) (Figure 6D). Likewise, in the lymph nodes 4 integrin–/– CD4 SP (40.2% ± 5.4%) and CD8 SP (18.4% ± 1.9%) fractions are represented equally to respective fractions of wild-type–derived donor cells (CD4 SP: 47.3% ± 3.3%; CD8 SP: 21.0% ± 1.7%) (Figure 6E and Table 2). Colonization of the Peyer patches and peritoneal cavity is hindered by lack of 4 integrin In contrast to other principal hematopoietic tissues, the contribution to both the Peyer patches and the peritoneal cavity is significantly reduced with 4 integrin–/– donor cells compared to wild-type cells (Figure 5B). This concerns both myeloid and lymphoid 4 integrin–/– populations (Figure 6F-G and Table 2). In the Peyer patches, the B220+ population is most affected, with its relative contribution being half as much as that observed from wild-type cells (69.7% ± 5.5% for wild-type; 25.5% ± 5.3% for 4 integrin–/–) (Figure 6F and Table 2). Mature B220+IgM+ 4 integrin–/– cells are substantially underrepresented (Figure 6F and Table 2), yet the B-1 (B220+CD5+) population (13.1% ± 1.8%) is comparable to wild-type B-1 percentages (15.0% ± 0.7%) (Figure 6F). By contrast, CD4 SP 4 integrin–/– cells are overrepresented (23.9% ± 4.9% for wild-type versus 45.4% ± 7.4% for 4 integrin–/–) (Figure 6F and Table 2). This, however, is only a relative value, since the overall colonization of Peyer patches is significantly reduced (Figure 5B). The percentage of CD8 SP 4 integrin–/– cells (7.8% ± 1.4%) is shown to be equivalent to wild-type donor cells (7.7% ± 1.6%) in this tissue (Figure 6F and Table 2). View larger version (56K): [in this window] [in a new window]   Figure 6.. Distribution of 4 integrin–/– and wild-type donor cell subsets in recipient tissues. Adult hematopoietic tissues of mice reconstituted with E12.5 embryonic tissues (AGM region, yolk sac, peripheral circulation, and fetal liver) of wild-type and 4 integrin–/– origin were analyzed by flow cytometry, and sample plots are given (see Table 2 for corresponding statistics. (A-C) Lymphoid (B220+ B-cell and CD3+ T-cell) and myeloid (CD11b/Gr1) populations for wild-type and 4 integrin–/– donor fractions of the peripheral blood (A), BM (B), and spleen (C) are illustrated. Mature B220+IgD+ and CD19+IgM+ B cells were produced by 4 integrin–/– HSCs in BM and spleen (B, C). Early 4 integrin–/– B cells expressing AA4.1 enter the spleen normally (C). (D,E) CD4/CD8 staining in the thymus (D) and lymph nodes (E) indicates normal T-cell development and distribution in 4 integrin–/– donor populations, as compared to wild-type T cells. (F,G) The inability of 4 integrin–/– cells to engraft the Peyer patches or peritoneal cavity is particularly apparent in the B-cell populations of these tissues. This is most noticeable in the mature B220+IgM+ population of the Peyer patches (F) and the B220+CD5+ B-1 cells of the peritoneal cavity (G).   In the peritoneal cavity, where 4 integrin–/– cell colonization also is severely lacking (Figure 5B), the CD11b+ population is substantially underrepresented (80.8% ± 4.4% for wild-type; 44.1% ± 8.1% for 4 integrin–/–) (Figure 6G and Table 2). Meanwhile, 4 integrin–/– CD3+ T cells (21.1% ± 4.1%) and B220+ B cells (33.7% ± 9.0%) are represented similarly to wild-type (CD3+: 19.2% ± 5.8%; B220+: 47.3% ± 2.7%). In contrast to B-cell distribution in the Peyer patches, 4 integrin–/– B220+CD5+ B-1 cells are scarce in the peritoneal cavity (only 3.9% ± 1.7% compared to 12.5% ± 1.4% for wild-type) (Figure 6G and Table 2). 4 integrin–/– HSCs are able to reconstitute secondary recipients We further assessed the properties of 4 integrin–/– HSCs by performing secondary transplantations. BM cells isolated from primary recipients were transplanted into secondary irradiated recipients (Figure 3). We found that 4 integrin–/– HSCs maintain their ability to home to and engraft principal hematopoietic organs and reconstitute the peripheral circulation of secondary recipients (Table 3). All mice (7 of 7) that received transplants of BM from primary recipients (E12.5 AGM and E12.5 FL primary donor tissues) were successfully reconstituted. Multilineage engraftment was confirmed in peripheral blood, BM, spleen, and thymus of secondary recipients using principal lymphoid and myeloid markers (data not shown). View this table: [in this window] [in a new window]   Table 3.. Long-term repopulation of secondary irradiated recipients with 4 integrin–/– HSCs      Discussion Top Abstract Introduction Materials and methods Results Discussion References   Over many years a large body of evidence has accumulated that suggests important roles for VLA-4 (41) and LPAM-1 (47) receptor complexes in myelopoiesis and lymphopoiesis. Antibody blockade experiments in vitro and in vivo demonstrated the importance of 4 integrins in hematopoiesis, particularly in homing and differentiation. Targeted ablation of the 4 integrin genetic locus further confirmed the involvement of 4 integrins in hematopoietic development and differentiation. Although 4 integrin–/– myeloid and lymphoid progenitors are present and differentiate in the embryo, their differentiation becomes progressively corrupt through fetal into adult life,34,35 with 4 integrin–/– myeloid and B-lymphoid progenitors failing to complete differentiation unless they are cultured in vitro. It was proposed that these differentiation defects observed in the absence of 4 integrin are a result of restricted interaction of 4 integrin–/– cells with the hematopoietic stroma.34,35 In contrast, more recent studies on the conditional inactivation of 4 integrin in adult mice resulted in only minor changes in the BM, although increased long-term release of mutant progenitor cells into the peripheral blood was observed.37 These seemingly contradictory reports may have 2 alternative explanations: (1) the normal long-term execution of the myeloid and lymphoid differentiation program critically depends on 4 integrin-mediated interactions at some point during development prior to the adult stage, or (2) 4 integrin deficiency causes a competitive disadvantage of mutant versus wild-type cells co-developing in the [4 integrin–/–: wt] chimeric animals used for analysis of differentiation of 4 integrin–/– cells. To explore these possibilities, our experiments were devised to allow us to test the differentiation capacity of 4 integrin–/– HSCs from the embryo when the fetal/neonatal period of development is bypassed, and in the presence of wild-type BM competitor cells (Figures 3, 7). In initial analyses, we established that in general the proportion of recipients repopulated with 4 integrin–/– embryonic hematopoietic tissues tended to be slightly, but not significantly, lower as compared to the wild-type controls. This suggests that the number of HSCs developing in the early embryo in the absence of 4 integrin does not dramatically deviate from the wild-type pattern of HSC development. In accord with this, in vitro analysis also has demonstrated successful expansion of HSCs in the AGM region in the absence of 4 integrin. Although the overall level of hematopoietic contribution in individual recipients that received transplants of 4 integrin–/– tissues tended to be lower (especially in the yolk sac), repopulation was robust and stable over 1.5 years. The significance of the observed slightly substandard development and performance of 4 integrin–/– HSCs, as compared to wild-type HSCs, is unclear due to possible nonspecific effects caused by early embryonic lethality related to heart and placenta defects. Additionally, because we have not performed full-scale limiting dilution analyses, we are unable to conclude whether this minor defect is related to stem cell number or to a proliferative deficiency of HSCs. Whatever the reason for this minor underperformance, it is incomparable with the progressive and more dramatic failure of 4 integrin–/– cells in [4 integrin–/–: wt] chimeric mice.34,35 We found also that the differentiation of transplanted 4 integrin–/– hematopoietic cells was not inhibited by the persistent presence of competitor wild-type cells. Except in the Peyer patches and peritoneal cavity, all tested donor-derived myeloid and lymphoid subsets were represented similarly to wild-type counterparts. We observed no quantitative difference in donor-derived myeloid CD11b/Gr1 subsets or lymphoid CD3+ and B220+ cells in the peripheral blood, BM, and spleens of animals that received 4 integrin–/– cells compared to those that received wild-type cells. The analysis of CD19+AA4.1+, CD19+IgM+, and B220+IgD+ subsets in recipient spleen and BM showed no difference in early or terminal B-cell differentiation (in [4 integrin–/–: Rag–/–] chimeras, 4 integrin–/– mature B220+IgM+ cells in these tissues were severely deficient35,47). Similarly, on the basis of CD4 and CD8 staining in the thymus and lymph nodes, no aberrant T-cell differentiation patterns were observed in the absence of 4 integrin. View larger version (38K): [in this window] [in a new window]   Figure 7.. Requirements for 4 integrins throughout embryonic and adult hematopoiesis. A collective summary of works illustrates the requirements for 4 integrin, depending on developmental stage and context. When HSCs develop through the fetal stage into adulthood in the absence of 4 integrin, their differentiation program becomes progressively corrupt (pathway "X").34,35 However, if the fetal stage is bypassed by transplanting early embryonic 4 integrin–/– HSCs directly into the adult, the hematopoietic deficiency is avoided (pathway ""). If normal hematopoiesis is established in the BM, 4 integrin ablation results in only minor defects (not shown).37 Cumulatively, these data suggest that during the fetal/neonatal stage(s) HSC development is strongly 4 integrin dependent.   Thus, the reason for the dramatic failure of 4 integrin–/– cells in chimeric [4 integrin–/–: wt] mice reported previously34,35 is unlikely to be a result of competition with wild-type cells. It is more likely that during fetal/neonatal development essential HSC/stroma 4 integrin-dependent interactions take place that may affect their survival, self-renewal, proliferative potential, and/or developmental homing. In contrast to the BM, spleen, thymus, and lymph nodes, the relative repopulation of the Peyer patches by 4 integrin–/– cells was significantly compromised. This dramatic reduction in colonization of the Peyer patches with 4 integrin–/– cells is in line with previous reports suggesting a critical role for 47 integrins in recruiting lymphocytes to the Peyer patches.30-32 The development of Peyer patches in [4 integrin–/–: wt] chimeric mice is completely disrupted, and Peyer patches are entirely absent in [4 integrin–/–: Rag–/–] chimeras.35 Although we observed an overall dramatic reduction of 4 integrin–/– cells in the Peyer patches, the deficiency is most pronounced in the IgM+ B-cell compartment. The colonization of the peritoneal cavity with 4 integrin–/– cells also is significantly reduced. In this case, CD11b+ cells, representing the population of peritoneal macrophages, and B-1 (B220+CD5+) cells are most affected. Arroyo et al35 also reported a block in B-1 cells in the peritoneal cavity of [4 integrin–/–: Rag–/–] chimeras. Since both 4 integrin–/– B cells and macrophages are present in normal numbers in other organs of reconstituted animals, the most likely explanation for their deficiency in Peyer patches and the peritoneal cavity is a poor ability for 4 integrin–/– cells to colonize these sites. Collectively, the picture emerging from the present study and reports from other groups34,35,37 suggests differential requirements for 4 integrins throughout development of the hematopoietic system (summarized in Figure 7). It is proposed here that the differentiation potential of HSCs in the absence of 4 integrins dramatically deteriorates after the embryonic stage of development as a result of deficient interactions with the fetal/neonatal hematopoietic microenvironment.    Acknowledgements   We are grateful to Carolyn Manson, John Verth, Yvonne Gibson, and other members of the University of Edinburgh Animal House staff for taking care of our animals and for irradiations. We also would like to thank Jan Vrana for flow sorting and Antonius Rolink for informative discussion. We thank Samir Taoudi for helpful comments on the manuscript.    Footnotes   Submitted October 24, 2005; accepted March 4, 2006. Prepublished online as Blood First Edition Paper, March 21, 2006; DOI 10.1182/blood-2005-10-4209. Supported by an MRC Senior Fellowship (A.M.), an MRC Stem Cell Centre Development grant, the Leukaemia Research Fund, the Association for International Cancer Research, and the European Union Framework Programme VI (FPVI) integrated project EuroStemCell. R.G. and L.H. contributed equally to this study. The online version of this article contains a data supplement. An Inside Blood analysis of this article appears at the front of this issue. The publication costs of this article were defrayed in part by page charge payment. Therefore, and solely to indicate this fact, this article is hereby marked "advertisement" in accordance with 18 U.S.C. section 1734. Reprints: Alexander Medvinsky, MRC Centre Development in Stem Cell Biology, Institute for Stem Cell Research, University of Edinburgh, King's Buildings, West Mains Road, Edinburgh, United Kingdom EH9 3JQ; e-mail: a.medvinsky{at}ed.ac.uk.    References Top Abstract Introduction Materials and methods Results Discussion References   Jaffredo T, Nottingham W, Liddiard K, Bollerot K, Pouget C, de Bruijn M. From hemangioblast to hematopoietic stem cell: an endothelial connection? Exp Hematol. 2005;33: 1029-1040. Medvinsky A, Dzierzak E. Definitive hematopoiesis is autonomously initiated by the AGM region. Cell. 1996;86: 897-906. Dzierzak E, Medvinsky A. Mouse embryonic hematopoiesis. Trends in Genetics. 1995;11: 359-366. Kumaravelu P, Hook L, Morrison AM, et al. Quantitative developmental anatomy of definitive haematopoietic stem cells/long-term repopulating units (HSC/RUs): role of the aorta-gonad-mesonephros (AGM) region and the yolk sac in colonisation of the mouse embryonic liver. Development. 2002;129: 4891-4899. Ottersbach K, Dzierzak E. The murine placenta contains hematopoietic stem cells within the vascular labyrinth region. Dev Cell. 2005;8: 377-387. Gekas C, Dieterlen-Lievre F, Orkin SH, Mikkola HK. The placenta is a niche for hematopoietic stem cells. Dev Cell. 2005;8: 365-375. Muller AM, Medvinsky A, Strouboulis J, Grosveld F, Dzierzak E. Development of hematopoietic stem cell activity in the mouse embryo. Immunity. 1994;1: 291-301. Ema H, Nakauchi H. Expansion of hematopoietic stem cells in the developing liver of a mouse embryo. Blood. 2000;95: 2284-2288. Papayannopoulou T. Bone marrow homing: the players, the playfield, and their evolving roles. Curr Opin Hematol. 2003;10: 214-219. Hirsch E, Iglesias A, Potocnik AJ, Hartmann U, Fassler R. Impaired migration but not differentiation of haematopoietic stem cells in the absence of beta1 integrins. Nature. 1996;380: 171-175. Potocnik AJ, Brakebusch C, Fassler R. Fetal and adult hematopoietic stem cells require beta1 integrin function for colonizing fetal liver, spleen, and bone marrow. Immunity. 2000;12: 653-663. Williams DA, Rios M, Stephens C, Patel VP. Fibronectin and VLA-4 in haematopoietic stem cell–microenvironment interactions. Nature. 1991;352: 438-441. Papayannopoulou T. Mechanisms of stem-/progenitor-cell mobilization: the anti–VLA-4 paradigm. Semin Hematol. 2000;37: 11-18. Osborn L, Hession C, Tizard R, et al. Direct expression cloning of vascular cell adhesion molecule 1, a cytokine-induced endothelial protein that binds to lymphocytes. Cell. 1989;59: 1203-1211. Elices MJ, Osborn L, Takada Y, et al. VCAM-1 on activated endothelium interacts with the leukocyte integrin VLA-4 at a site distinct from the VLA-4/fibronectin binding site. Cell. 1990;60: 577-584. Ruegg C, Postigo AA, Sikorski EE, Butcher EC, Pytela R, Erle DJ. Role of integrin alpha 4 beta 7/alpha 4 beta P in lymphocyte adherence to fibronectin and VCAM-1 and in homotypic cell clustering. J Cell Biol. 1992;117: 179-189. van der Loo JC, Xiao X, McMillin D, Hashino K, Kato I, Williams DA. VLA-5 is expressed by mouse and human long-term repopulating hematopoietic cells and mediates adhesion to extracellular matrix protein fibronectin. J Clin Invest. 1998;102: 1051-1061. Guan JL, Hynes RO. Lymphoid cells recognize an alternatively spliced segment of fibronectin via the integrin receptor alpha 4 beta 1. Cell. 1990;60: 53-61. Wayner EA, Garcia-Pardo A, Humphries MJ, McDonald JA, Carter WG. Identification and characterization of the T lymphocyte adhesion receptor for an alternative cell attachment domain (CS-1) in plasma fibronectin. J Cell Biol. 1989;109: 1321-1330. Miyake K, Weissman IL, Greenberger JS, Kincade PW. Evidence for a role of the integrin VLA-4 in lympho-hemopoiesis. J Exp Med. 1991;173: 599-607. Oostendorp RA, Reisbach G, Spitzer E, et al. VLA-4 and VCAM-1 are the principal adhesion molecules involved in the interaction between blast colony-forming cells and bone marrow stromal cells. Br J Haematol. 1995;91: 275-284. Papayannopoulou T, Craddock C, Nakamoto B, Priestley GV, Wolf NS. The VLA4/VCAM-1 adhesion pathway defines contrasting mechanisms of lodgement of transplanted murine hemopoietic progenitors between bone marrow and spleen. Proc Natl Acad Sci U S A. 1995;92: 9647-9651. Vermeulen M, Le Pesteur F, Gagnerault MC, Mary JY, Sainteny F, Lepault F. Role of adhesion molecules in the homing and mobilization of murine hematopoietic stem and progenitor cells. Blood. 1998;92: 894-900. Papayannopoulou T, Nakamoto B. Peripheralization of hemopoietic progenitors in primates treated with anti-VLA4 integrin. Proc Natl Acad Sci U S A. 1993;90: 9374-9378. Craddock CF, Nakamoto B, Andrews RG, Priestley GV, Papayannopoulou T. Antibodies to VLA4 integrin mobilize long-term repopulating cells and augment cytokine-induced mobilization in primates and mice. Blood. 1997;90: 4779-4788. Kikuta T, Shimazaki C, Ashihara E, et al. Mobilization of hematopoietic primitive and committed progenitor cells into blood in mice by anti-vascular adhesion molecule-1 antibody alone or in combination with granulocyte colony-stimulating factor. Exp Hematol. 2000;28: 311-317. Papayannopoulou T, Priestley GV, Nakamoto B. Anti–VLA4/VCAM-1–induced mobilization requires cooperative signaling through the kit/mkit ligand pathway. Blood. 1998;91: 2231-2239. Yanai N, Sekine C, Yagita H, Obinata M. Roles for integrin very late activation antigen-4 in stroma-dependent erythropoiesis. Blood. 1994;83: 2844-2850. Katayama Y, Hidalgo A, Peired A, Frenette PS. Integrin alpha4beta7 and its counterreceptor MAdCAM-1 contribute to hematopoietic progenitor recruitment into bone marrow following transplantation. Blood. 2004;104: 2020-2026. Berlin C, Berg EL, Briskin MJ, et al. Alpha 4 beta 7 integrin mediates lymphocyte binding to the mucosal vascular addressin MAdCAM-1. Cell. 1993;74: 185-195. Holzmann B, Weissman IL. Integrin molecules involved in lymphocyte homing to Peyer's patches. Immunol Rev. 1989;108: 45-61. Holzmann B, McIntyre BW, Weissman IL. Identification of a murine Peyer's patch—specific lymphocyte homing receptor as an integrin molecule with an alpha chain homologous to human VLA-4 alpha. Cell. 1989;56: 37-46. Hu MC, Crowe DT, Weissman IL, Holzmann B. Cloning and expression of mouse integrin beta p(beta 7): a functional role in Peyer's patch–specific lymphocyte homing. Proc Natl Acad Sci U S A. 1992;89: 8254-8258. Arroyo AG, Yang JT, Rayburn H, Hynes RO. Alpha4 integrins regulate the proliferation/differentiation balance of multilineage hematopoietic progenitors in vivo. Immunity. 1999;11: 555-566. Arroyo AG, Yang JT, Rayburn H, Hynes RO. Differential requirements for alpha4 integrins during fetal and adult hematopoiesis. Cell. 1996;85: 997-1008. Yang JT, Rayburn H, Hynes RO. Cell adhesion events mediated by alpha 4 integrins are essential in placental and cardiac development. Development. 1995;121: 549-560. Scott LM, Priestley GV, Papayannopoulou T. Deletion of alpha4 integrins from adult hematopoietic cells reveals roles in homeostasis, regeneration, and homing. Mol Cell Biol. 2003;23: 9349-9360. Gilchrist DS, Ure J, Hook L, Medvinsky A. Labeling of hematopoietic stem and progenitor cells in novel activatable EGFP reporter mice. Genesis. 2003;36: 168-176. Ogawa M, Nishikawa S, Yoshinaga K, et al. Expression and function of c-Kit in fetal hemopoietic progenitor cells: transition from the early c-Kit–independent to the late c-Kit–dependent wave of hemopoiesis in the murine embryo. Development. 1993;117: 1089-1098. Bernex F, De Sepulveda P, Kress C, Elbaz C, Delouis C, Panthier JJ. Spatial and temporal patterns of c-kit–expressing cells in WlacZ/+ and WlacZ/WlacZ mouse embryos. Development. 1996;122: 3023-3033. Wood HB, May G, Healy L, Enver T, Morriss-Kay GM. CD34 expression patterns during early mouse development are related to modes of blood vessel formation and reveal additional sites of hematopoiesis. Blood. 1997;90: 2300-2311. Taoudi S, Morrison AM, Inoue H, Gribi R, Ure J, Medvinsky A. Progressive divergence of definitive haematopoietic stem cells from the endothelial compartment does not depend on contact with the foetal liver. Development. 2005;132: 4179-4191. Sanchez MJ, Holmes A, Miles C, Dzierzak E. Characterization of the first definitive hematopoietic stem cells in the AGM and liver of the mouse embryo. Immunity. 1996;5: 513-525. North TE, de Bruijn MF, Stacy T, et al. Runx1 expression marks long-term repopulating hematopoietic stem cells in the midgestation mouse embryo. Immunity. 2002;16: 661-672. Morrison SJ, Hemmati HD, Wandycz AM, Weissman IL. The purification and characterization of fetal liver hematopoietic stem cells. Proc Natl Acad Sci U S A. 1995;92: 10302-10306. Kondo M, Wagers AJ, Manz MG, et al. Biology of hematopoietic stem cells and progenitors: implications for clinical application. Annu Rev Immunol. 2003;21: 759-806. Arroyo AG, Taverna D, Whittaker CA, et al. In vivo roles of integrins during leukocyte development and traffic: insights from the analysis of mice chimeric for alpha 5, alpha v, and alpha 4 integrins. J Immunol. 2000;165: 4667-4675. CiteULike    Connotea    Del.icio.us    Digg    Reddit    Technorati    What's this? This article has been cited by other articles: H. Qian, E. Georges-Labouesse, A. Nystrom, A. Domogatskaya, K. Tryggvason, S. E. W. Jacobsen, and M. Ekblom Distinct roles of integrins {alpha}6 and {alpha}4 in homing of fetal liver hematopoietic stem and progenitor cells Blood, October 1, 2007; 110(7): 2399 - 2407. [Abstract] [Full Text] [PDF] B. M. Zeigler, D. Sugiyama, M. Chen, Y. 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