The Daley laboratory maintains active projects in basic molecular embryology, the specification of blood-forming or hematopoietic tissue, germ cell specification, genomic imprinting, and the hemangioblast. These projects also come to bear upon problems of abnormal development in malignancy as well as rare congenital diseases.
Lu
CW, Yabuuchi A, Chen L, Viswanathan S, Kim K, and Daley GQ.
Ras-MAPK signaling promotes trophectoderm formation from embryonic stem
cells and mouse embryos.
Nat Genet. 2008 Jul;40(7):921-6. (go
to PubMed)
Abstract:
In blastocyst chimeras, embryonic stem (ES) cells contribute to embryonic tissues but not extraembryonic trophectoderm. Conditional activation of HRas1(Q61L) in ES cells in vitro induces the trophectoderm marker Cdx2 and enables derivation of trophoblast stem (TS) cell lines that, when injected into blastocysts, chimerize placental tissues. Erk2, the downstream effector of Ras-mitogen-activated protein kinase (MAPK) signaling, is asymmetrically expressed in the apical membranes of the 8-cell-stage embryo just before morula compaction. Inhibition of MAPK signaling in cultured mouse embryos compromises Cdx2 expression, delays blastocyst development and reduces trophectoderm outgrowth from embryo explants. These data show that ectopic Ras activation can divert ES cells toward extraembryonic trophoblastic fates and implicate Ras-MAPK signaling in promoting trophectoderm formation from mouse embryos.
(a) Schema for generating inducible Hras1Q61L ES cells. Ainv15 mouse embryonic stem cells have the rtTA integrated at the Rosa26 locus. HRas1Q61L was inserted by Cre-mediated recombination of a targeting vector (plox) into the region of the Hprt locus, so it is expressed from the tetracycline response element (tetOP). Successful recombination regenerates an ATG-truncated neomycin (G418) resistance gene (Neo) driven by the pgk promoter (PGK-ATG). (b) Ras activation assay (Upstate Bioscience). Upper blot: co-precipitation with Raf indicates expression of active GTP-bound Ras. Lower blot: expression of total Ras can be detected by immunoblot in doxycycline-induced cells with antibody to Ras. Lane 1, uninduced (no doxycycline); lane 2, induced with doxycycline. (c–h) Tumors obtained from iRasES cells implanted in Rag2-/-;gamma c-/- mice. Scale bars in c and e, 1 cm. Magnification is indicated in each panel. (c) Cross-section of teratoma from control mice (no doxycycline). (d) Histology of teratoma in c showing tissue complexity (H&E staining). (e) Hemorrhagic tumors isolated from mice fed doxycycline. (f,g) Histology of tumors in e showing clusters of giant cells (H&E staining). (h) Periodic acid–Schiff staining of sections of tumors from doxycycline-induced animals, showing glycogen-rich granules. (i) RT-PCR analysis of gene expression of two teratomas from control animals (uninduced) and hemorrhagic tumors from two mice fed doxycycline (induced). Dihydrofolate reductase (Dhfr) is used as a loading control for this analysis. Afp (alpha-fetoprotein), Actc1 (cardiac alpha-actin) and Pax6 are markers of differentiation toward endoderm, mesoderm and ectoderm, respectively. Markers for trophectoderm are Prl3d1 (placenta lactogen 1) and Plac1l (placenta alkaline phosphatase) for trophoblastic giant cells; and Tpbpa (trophoblastic specific protein alpha) and Prl2c2 (proliferin) for spongiotrophoblasts.
Viswanathan
SR, Daley GQ and Gregory RI.
Selective blockade of microRNA processing by Lin28.
Science. 2008 (320): 97-100. (go
to PubMed)
Abstract:
MicroRNAs (miRNAs) play critical roles in development, and dysregulation of miRNA expression has been observed in human malignancies. Recent evidence suggests that the processing of several primary miRNA transcripts (pri-miRNAs) is blocked posttranscriptionally in embryonic stem cells, embryonal carcinoma cells, and primary tumors. Here we show that Lin28, a developmentally regulated RNA binding protein, selectively blocks the processing of pri-let-7 miRNAs in embryonic cells. Using in vitro and in vivo studies, we found that Lin28 is necessary and sufficient for blocking Microprocessor-mediated cleavage of pri-let-7 miRNAs. Our results identify Lin28 as a negative regulator of miRNA biogenesis and suggest that Lin28 may play a central role in blocking miRNA-mediated differentiation in stem cells and in certain cancers.
Ectopic expression of Lin28 selectively inhibits pri-miRNA processing in vivo. (A) In each panel, 293T cells were untransfected (lane 1), cotransfected with the indicated pri-miRNA and 0.5 mg of pCMV-Flag empty vector (lane 2), or cotransfected with the indicated pri-miRNA and 0.5 mg of Flag-Lin28 cDNA (lane 3). Total RNA was collected 40 hours after transfection and subjected to Northern blotting for the indicated miRNA. (B) Quantitative PCR analysis of pri-let-7g levels [average of experimental triplicates performed on the RNA samples from upper right panel of (A)]. (C) Mature let-7g levels upon cotransfection of 293T cells with pri-let-7g and pCMV-Flag, Flag-Lin28, FlaghnRNPA1, Flag-hnRNPL, Flag-YBX-1, or Flag-Msi-2 cDNAs, as measured by quantitative PCR. First, the amount of mature let-7g in each sample was calculated relative to untransfected control cells; then, Flag-protein cotransfected samples were normalized to the corresponding pCMV-Flag cotransfected samples. (D) Quantitative PCR showing changes in levels of endogenousmature miRNAs upon transfection of Flag-Lin28 in 293T cells. (E) Quantitative PCR showing accumulation of endogenous pri-let-7g upon transfection of Flag-Lin28 in 293T cells. For (C) to (E), values are given as average ± SEM from two or more independent transfections.
Geijsen
N, Horoschak M, Kim K, Gribnau J, Eggan K, and Daley GQ.
Derivation of embryonic germ cells and male gametes from embryonic stem
cells.
Nature. 2004 (427): 148-54. (go
to PubMed)
Abstract:
Egg and sperm cells (gametes) of the mouse are derived from a founder population of primordial germ cells that are set aside early in embryogenesis. Primordial germ cells arise from the proximal epiblast, a region of the early mouse embryo that also contributes to the first blood lineages of the embryonic yolk sac. Embryonic stem cells differentiate in vitro into cystic structures called embryoid bodies consisting of tissue lineages typical of the early mouse embryo. Because embryoid bodies sustain blood development, we reasoned that they might also support primordial germ cell formation. Here we isolate primordial germ cells from embryoid bodies, and derive continuously growing lines of embryonic germ cells. Embryonic germ cells show erasure of the methylation markers (imprints) of the Igf2r and H19 genes, a property characteristic of the germ lineage. We show that embryoid bodies support maturation of the primordial germ cells into haploid male gametes, which when injected into oocytes restore the somatic diploid chromosome complement and develop into blastocysts. Our ability to derive germ cells from embryonic stem cells provides an accessible in vitro model system for studies of germline epigenetic modification and mammalian gametogenesis.
Fluorescence image of blastocysts derived from intracytoplasmic injection of oocytes with EB-derived FE-J1+ haploid cells.
Wu
X*, Lensch MW*, Wylie-Sears J, Daley GQ, and Bischoff J.
Hemogenic endothelial progenitor cells isolated from human umbilical cord
blood.
Stem Cells. 2007 (25): 2770-6. (go
to PubMed)
Abstract:
Hemogenic endothelium has been identified in embryonic dorsal aorta and in tissues generated from mouse embryonic stem cells, but to date there is no evidence for such bipotential cells in postnatal tissues or blood. Here we identify a cell population from human umbilical cord blood that gives rise to both endothelial cells and hematopoietic progenitors in vitro. Cord blood CD34+/CD133+ cells plated at high density in an endothelial basal medium formed an endothelial monolayer and a nonadherent cell population after 14-21 days. AML-1, a factor required for definitive hematopoiesis, was detected at low levels in adherent cells and at high levels in nonadherent cells. Nonadherent cells coexpressed the endothelial marker vascular endothelial (VE)-cadherin and the hematopoietic marker CD45, whereas adherent cells were composed primarily of VE-cadherin+/CD45- cells and a smaller fraction of VE-cadherin+/CD45+ cells. Both nonadherent and adherent cells produced hematopoietic colonies in methylcellulose, with the adherent cells yielding more colony-forming units (CFU)-GEMM compared with the nonadherent cells. To determine whether the adherent endothelial cells were producing hematopoietic progenitors, single cells from the adherent population were expanded in 96-well dishes for 14 days. The clonal populations expressed VE-cadherin, and a subset expressed AML-1, epsilon-globin, and gamma-globin. Three of 17 clonal cell populations gave rise to early CFU-GEMM hematopoietic progenitors and burst-forming unit-erythroid progenitors. These results provide evidence for hemogenic endothelial cells in human umbilical cord blood.
Clonal cells from the adherent monolayer express endothelial and hematopoietic markers and produce budding cells and hematopoietic progenitors. (A): Clonal populations were analyzed for expression of AML-1, embryonic (e), fetal (g), and adult (b) globins, VE-cadherin, and ribosomal S9 by reverse transcriptase-polymerase chain reaction (PCR). Hematopoietic cells (HL-60 for AML-1 and K562 cells for globins) and human ECs served as positive and negative controls. Ribosomal S9 served as an internal control for the PCR and gel loading. (B, C): Phase-contrast micrographs of a clone 6 days after plating as a single cell, photographed at x40 (B) and x100 (C). Arrows in (C) indicate budding cells. (D): Colony forming units-GEMM colony formed from clonal cells harvested after 14 days of growth and plated in methylcellulose for an additional 14 days. Abbreviations: AML-1, acute myeloid leukemia-1; dH2O, distilled water; VE, vascular endothelial.