Our laboratory has a keen interest in understanding the process of hematopoiesis at all levels including the specification of blood tissue in the developing embryo and mechanisms contributing to efficient transplantation of blood-forming activity. Our work in hematopoiesis bridges our interests in pluripotent cell biology and cancer therapeutics with projects in the derivation and engraftment of lympho/myeloid repopulating activity from a pluripotent cell starting point as well as the study of blood formation in disease-specific contexts.
Cdx gene deficiency compromises embryonic hematopoiesis in the mouse.
Proc Natl Acad Sci U S A. 2008 Jun 3;105(22):7756-61. (go to PubMed)
Abstract:
Cdx genes (Cdx1, Cdx2, and Cdx4) encode a family of caudal-related transcription factors that mediate anterior-posterior patterning during embryogenesis through Hox gene regulation. Homologues in the zebrafish have been shown to play key roles in blood formation. To define the role of Cdx genes during embryonic hematopoiesis in a mammalian system, we examined the hematopoietic potential of Cdx-deficient mouse embryonic stem cells (ESCs) in vitro and in vivo. Individual Cdx-deficient ESCs exhibited impaired embryonic hematopoietic progenitor formation and altered Hox gene expression, most notably for Cdx2 deficiency. A more severe hematopoietic defect was observed with compound Cdx deficiency than loss of function of any single Cdx gene. Reduced hematopoietic progenitor formation of ESCs deficient in multiple Cdx genes could be rescued by ectopic expression of Cdx4, concomitant with partially restored Hox gene expression. These results reveal an essential and partially redundant role for multiple Cdx genes during embryonic hematopoiesis in the mouse.
Cdx2 deficiency results in severe blood defects during embryonic hematopoiesis. (A) Hematopoietic colony formation of day 6 EB cells from Cdx2 +/+ (WT) or two Cdx2 -/- ESC lines, KO1 and KO2. P value <0.01 for all comparison of Cdx2 -/- to WT ESC lines. (B) Surface antigens analyzed by flow cytometry at different time points during EB development. *, P value >0.1; **, P value = 0.01–0.04; ***, P value <0.01. (C) Hematopoietic colony formation of day 6 EB cells from mixed population of either Cdx2 +/+ (W) or two Cdx2 -/- ESC lines (1 and 2) with normal GFP+ ESCs. Percentage of GFP+ ESC composition is indicated below the x axis. Data are averaged CFU from duplicates in one representative experiment and reproduced from two independent experiments. (D) Relative expression level of Cdx2 in CD41+ or whole day 6 EBs, measured by real-time RT-PCR. (A, B, and D) Data are averaged fold changes (±1 SD) relative to WT control from replicates in two or three independent experiments. Each data point denotes six to eight biological replicates. (E) Progenitor colony activity of YS from chimeric embryos at 8.5 dpc generated with either Cdx2 -/- or Cdx2 +/+ ESCs. Donor blastocysts are lacZ+. The percentage of lacZ- (ESC-contributed)/total CFU was measured. Data represent average ± 1 SD. * P values <0.001.
Lengerke
C, Schmitt S, Bowman TV, Jang IH, Maouche-Chretien L, McKinney-Freeman
S, Davidson AJ, Hammerschmidt M, Rentzsch F, Green JB, Zon LI, and Daley
GQ.
BMP and Wnt specify hematopoietic fate by activation of the Cdx-Hox pathway.
Cell Stem Cell. 2008 Jan 10;2(1):72-82. (go
to PubMed)
Abstract:
The formation of blood in the embryo is dependent on bone morphogenetic protein (BMP), but how BMP signaling intersects with other regulators of hematopoietic development is unclear. Using embryonic stem (ES) cells, we show that BMP4 first induces ventral-posterior (V-P) mesoderm and subsequently directs mesodermal cells toward blood fate by activating Wnt3a and upregulating Cdx and Hox genes. When BMP signaling is blocked during this latter phase, enforced expression of either Cdx1 or Cdx4 rescues hematopoietic development, thereby placing BMP4 signaling upstream of the Cdx-Hox pathway. Wnt signaling cooperates in BMP-induced hemogenesis, and the Wnt effector LEF1 mediates BMP4 activation of Cdx genes. Our data suggest that BMP signaling plays two distinct and sequential roles during blood formation, initially as an inducer of mesoderm, and later to specify blood via activation of Wnt signaling and the Cdx-Hox pathway.
BMP4 and Wnt3a Activate Cdx and Posterior Hox Genes (A) EBs differentiated in SCM for 2.25 days were then transferred to SFM + BMP4 or SFM + Noggin. RNA was harvested and analyzed by quantitative PCR. Numbers represent fold change in relative expression levels as compared to undifferentiated ES cells, after normalization to ß-actin. (B) EBs were differentiated in SCM, and proteins were added at days 2–2.25 (unless otherwise indicated). RNA harvested on days 3 and 4 was analyzed by quantitative PCR. Numbers represent fold change in relative expression levels as compared to expression in SCM, after normalization to ß-actin.Data are from one representative time-course experiment (A) or averages from three or more independent experiments (B). Error bars show SEM. (C) cdx4 expression during zebrafish embryogenesis (11.5 hpf) in wild-type (i) and Tg(hsp70:bmp2b; ii) embryos. Embryos were heat shocked at 75% epiboly.
Kyba
M, Perlingeiro RC, and Daley GQ.
HoxB4 confers definitive lymphoid-myeloid engraftment potential on embryonic
stem cell and yolk sac hematopoietic progenitors.
Cell. 2002 (109): 29-37. (go
to PubMed)
Abstract:
The extent to which primitive embryonic blood progenitors contribute to definitive lymphoid-myeloid hematopoiesis in the adult remains uncertain. In an effort to characterize factors that distinguish the definitive adult hematopoietic stem cell (HSC) and primitive progenitors derived from yolk sac or embryonic stem (ES) cells, we examined the effect of ectopic expression of HoxB4, a homeotic selector gene implicated in self-renewal of definitive HSCs. Expression of HoxB4 in primitive progenitors combined with culture on hematopoietic stroma induces a switch to the definitive HSC phenotype. These progenitors engraft lethally irradiated adults and contribute to long-term, multilineage hematopoiesis in primary and secondary recipients. Our results suggest that primitive HSCs are poised to become definitive HSCs and that this transition can be promoted by HoxB4 expression. This strategy for blood engraftment enables modeling of hematopoietic transplantation from ES cells.
Effect of Doxycycline Induction of HoxB4 on EB Cells in vitro (A) Colony formation by cells from day 6 EB controls, and day 6 EBs treated with doxycycline from day 4 to day 6, in hematopoietic methylcellulose suspension culture. HPP-GEMM refers to dense GEMMs not normally seen at day 6 of EB development. (B) Morphology of a typical day 6 GEMM. (C) Morphology of a doxycycline-induced HPP-GEMM at the same magnification as in (B). (D) A colony of semiadherent cells from HoxB4-induced day 6 EB cells plated on OP9 in the presence of doxycycline. (E) Cytospin preparation of these cells growing on OP9. (F) Colony-forming activity of doxycycline-induced cells grown on OP9 in methylcellulose with myeloid cytokines. Control indicates doxycycline not added to methylcellulose cultures; +dox indicates doxycycline induction maintained during methylcellulose culture. (G) Cytospin preparation from a GEMM obtained from cultures described in (F). Open arrowheads show erythroblasts; filled arrowhead shows megakaryocyte.
McKinney-Freeman
SL*, Lengerke C*, Jang I-H, Schmitt S, Wang Y, Philitas M, Shea J, and
Daley GQ.
Modulation of murine embryonic stem cell-derived CD41+c-kit+ hematopoietic
progenitors by ectopic expression of Cdx genes.
Blood. 2008 (111): 4944-4953. (go
to PubMed)
Abstract:
Cdx1, Cdx2, and Cdx4 comprise the caudal-like Cdx gene family in mammals, whose homologues regulate hematopoietic development in zebrafish. Previously, we reported that overexpression of Cdx4 enhances hematopoietic potential from murine embryonic stem cells (ESCs). Here we compare the effect of ectopic Cdx1, Cdx2, and Cdx4 on the differentiation of murine ESC-derived hematopoietic progenitors. The 3 Cdx genes differentially influence the formation and differentiation of hematopoietic progenitors within a CD41+c-kit+ population of embryoid body (EB)-derived cells. Cdx1 and Cdx4 enhance, whereas Cdx2 strongly inhibits, the hematopoietic potential of CD41+ckit+ EB-derived cells, changes that are reflected by effects on hematopoietic lineage-specific and Hox gene expression. When we subject stromal cell and colony assay cultures of EB-derived hematopoietic progenitors to ectopic expression of Cdx genes, Cdx4 dramatically enhances, whereas Cdx1 and Cdx2 both inhibit hematopoietic activity, probably by blocking progenitor differentiation. These data demonstrate distinct effects of Cdx genes on hematopoietic progenitor formation and differentiation, insights that we are using to facilitate efforts at in vitro culture of hematopoietic progenitors from ESC. The behavior of Cdx genes in vitro suggests how derangement of these developmental regulators might contribute to leukemogenesis.
Summary model of the differential effects of Cdx genes on ESC-derived hematopoiesis. Our data support a model in which Cdx genes differentially affect both the formation (ie, induction of Cdx gene expression during EB differentiation) and the hematopoietic activity (ie, induction of Cdx genes expression in purified CD41c-kit hematopoietic progenitors) of already specified hematopoietic progenitor cells. Cdx1 and Cdx4 both promote the specification of hematopoietic progenitors, whereas Cdx2 inhibits this process. However, only Cdx4 can enhance the expansion/differentiation of hematopoietic progenitors; Cdx1 and Cdx2 both suppress these functions.
Wang
Y, Yates F, Naveiras O, and Daley GQ.
Embryonic stem cell-derived hematopoietic stem cells.
Proc Natl Acad Sci U S A. 2005 (102): 19081-6. (go
to PubMed)
Abstract:
Despite two decades of studies documenting the in vitro blood-forming potential of murine embryonic stem cells (ESCs), achieving stable long-term blood engraftment of ESC-derived hematopoietic stem cells in irradiated mice has proven difficult. We have exploited the Cdx-Hox pathway, a genetic program important for blood development, to enhance the differentiation of ESCs along the hematopoietic lineage. Using an embryonic stem cell line engineered with tetracycline-inducible Cdx4, we demonstrate that ectopic Cdx4 expression promotes hematopoietic mesoderm specification, increases hematopoietic progenitor formation, and, together with HoxB4, enhances multilineage hematopoietic engraftment of lethally irradiated adult mice. Clonal analysis of retroviral integration sites confirms a common stem cell origin of lymphoid and myeloid populations in engrafted primary and secondary mice. These data document the cardinal stem cell features of self-renewal and multilineage differentiation of ESC-derived hematopoietic stem cells.
Clonal analysis of hematopoietic populations of mice engrafted with ESC-derived HSCs, as determined by Southern hybridization analysis of retroviral integration sites. (A) Structure of the retroviral vector MSCV-HoxB4-ires-GFP. Probes used in Southern hybridization analysis are indicated. (B Left) Southern analysis of fractionated myeloid and lymphoid populations from primary (1ry) and unrelated secondary (2ry) engrafted mice, showing multiple comigrating fragments. (B Right) Bone marrow and spleen cells from two primary engrafted animals and comparable tissue from the corresponding secondary animals, showing comigrating fragments. (C) Southern analysis of hematopoietic tissues from one primary and two corresponding secondary recipients engrafted with ESC-HSCs: spleen (S), BM (B), Gr1+ BM cells (B/G), Gr1+ splenocytes (S/G), and CD3+ or B220+ splenic lymphocytes (S/L). Mye/Lym represents the ratio of Gr-1+ cells to CD3+ and B220+ populations in corresponding sample, as determined by flow cytometry. Relative DNA level was calculated by comparing endogenous HoxB4 (endog) with control (DNA isolated from Ainv15 ES cells). Proviral copy number was calculated by comparing the level of proviral HoxB4 (Rv-HoxB4) with endogenous HoxB4 level. Samples reflect Cdx4/HoxB4-engrafted cells, except the third and fourth lanes in B Left, which represent HoxB4-treated cells. #, fragments detected only in primary recipients; *, fragments unique to secondary engrafted animals; ^, fragments detected predominantly in one lineage.