1. Protein O-fucosyltransferase 1 (Pofut1) regulates lymphoid and myeloid homeostasis through modulation of Notch receptor ligand interactions
by David Yao, Yuanshuai Huang, Xiaoran Huang, Weihuan Wang, Quanjian Yan, Lebing Wei, Wei Xin, Stanton Gerson, Pamela Stanley, John B. Lowe, and Lan Zhou
Blood
Volume 117(21):
May 26, 2011
©2011 by American Society of Hematology

2. Myeloid hyperplasia and reduced lymphopoiesis in Mx-Cre/Pofut1F/F mice.
Myeloid hyperplasia and reduced lymphopoiesis in Mx-Cre/Pofut1F/F mice. (A) PB neutrophil counts in control mice (Mx-Cre/Pofut1F/+ or Pofut1F/F) or Mx-Cre/Pofut1F/F mice 3 to 5 months after pIpC injection. Data are mean ± SD of 3 independent experiments with 2 or 3 mice of each genotype per experiment. Statistical analysis was by the Student t test. (B-C) FACS analysis of the percentage of granulocytes (Gr-1+), B lymphocytes (B220+), and T lymphocytes (CD4+/CD8+) in the periphery (B) and bone marrow (C). (D) FACS analysis of bone marrow myeloid progenitors (CMP: Lin−c-kit+Sca-1−IL-7R−CD34+FcγRIIlow; GMP: Lin−c-kit+Sca-1−IL-7R−CD34+FcγRII+; megakaryocyte-erythroid progenitor: Lin−c-kit+Sca-1−IL-7R−CD34lowFcγRIIlow). Populations of CMP or GMP are shown as percentages of Lin−Sca-1−IL-7R−c-kit+ cells. (E) Representative gross anatomy and hematoxylin and eosin staining of pIpC-treated control or Mx-Cre/Pofut1F/F mice spleen. The hematoxylin and eosin slides of mice were photographed using Olympus BX41 microscope (numeric aperture of the objective lens 20×/0.50) and the Spot software Version 4.7 (Diagnostic Instruments Inc). (F) FACS analysis of granulocytes, B and T lymphocytes in spleens from pIpC-treated control or Mx-Cre/Pofut1F/F mice. (B-F) Data are representative of at least 4 independent experiments with 2 mice of each genotype per experiment.
David Yao et al. Blood 2011;117:
©2011 by American Society of Hematology

3. Impaired T- and MZB-cell development in Mx-Cre/Pofut1F/F mice.
Impaired T- and MZB-cell development in Mx-Cre/Pofut1F/F mice. (A-B) Representative gross anatomy of thymus (A) and total thymocyte numbers per lobe from 16-week-old, pIpC-treated control or Mx-Cre/Pofut1F/F mice (n = 6) (B). (C) FACS analysis of CD4 versus CD8 on total thymocytes, as well as histograms for TCR-αβ and TCR-γδ expression on DN4 thymocytes, from pIpC-treated control and Mx-Cre/Pofut1F/F mice. Percentages of CD4+, CD8+, CD4+CD8+ (DP), and CD4−CD8− (DN) are indicated in the quadrants. (D) Absolute numbers of thymocyte subsets per lobe described in panel C and intrathymic B220+ cells were calculated and shown as bar diagrams (n = 5). (B,D) Bar graphs represent the mean ± SD. Student t test was performed to compare the absolute number of cells from Mx-Cre/Pofut1F/F mice with those of control mice. *P < .05. **P < .01. (E-F) FACS analysis of spleen IgM versus IgD expression on B220+ cells (E), and CD21 versus CD23 expression on B220+ cells (F) in control and Mx-Cre/Pofut1F/F mice. (C,E-F) Data are representative of 4 independent experiments with 1 or 2 mice of each genotype per experiment.
David Yao et al. Blood 2011;117:
©2011 by American Society of Hematology

4. Lymphoid and myeloid defects in Pofut1-null mice were caused by both cell-autonomous mechanism(s) and environmental cues.
Lymphoid and myeloid defects in Pofut1-null mice were caused by both cell-autonomous mechanism(s) and environmental cues. A total of 2 million donor bone marrow cells isolated from either WT (Ly5.2) or pIpC-treated Mx-Cre/PofutF/F (Ly 5.2) were delivered via tail vein to lethally irradiated (9.5 Gy) WT (Ly5.1) or Mx-Cre/Pofut1F/F (Mx-Cre/PF/F) recipients. (A) Peripheral neutrophil numbers were enumerated 3 months after transplantation. (B) FACS analysis of recipient marrow myeloid progenitors derived from WT or Mx-Cre/PF/F marrow cells in WT or Mx-Cre/PF/F recipients by gating on the Ly5.2+ cells. Populations of CMP or GMP are shown as percentages of Lin−Sca-1−IL-7R−c-kit+ cells. (C-E) FACS analysis of recipient spleens for Gr-1 expression (C), CD23 versus CD21 expression on B220+ cells (D), and plots of absolute numbers of splenic MZB cells (E). (F) Enumeration of total thymocytes derived from donors in recipients 3 months after transplantation. (G-H) FACS analysis of donor-derived thymocyte CD8 versus CD4 expression (G) and DN1-DN4 development defined by expression of CD44 and CD25 on CD4−CD8− (DN) cells (H). Percentages of CD44+CD25− (DN1), CD44+CD25+ (DN2), CD44−CD25+ (DN3), and CD44−CD25− (DN4) are indicated in the quadrants. (B-D,G-H) Data are representative of 3 independent experiments with 1 to 3 mice of each donor-recipient pair per experiment. (A,F) Histograms represent the mean ± SD of 4 to 6 mice for each donor-recipient pair. Student t test was performed to compare the numbers (A,E-F) among different donor-recipient pairs. P values that are significantly different are shown as indicated.
David Yao et al. Blood 2011;117:
©2011 by American Society of Hematology

5. Mx-Cre/Pofut1F/F (Mx-Cre/PF/F) marrow LSKs have reduced T lymphopoiesis but enhanced myelopoiesis in vitro in response to Dll4 activation.
Mx-Cre/Pofut1F/F (Mx-Cre/PF/F) marrow LSKs have reduced T lymphopoiesis but enhanced myelopoiesis in vitro in response to Dll4 activation. (A-B) A representative FACS profile of 3 similar experiments on day 20 culture of Mx-Cre/PF/F or control marrow LSK cells from 12-week-old mice (8 weeks after pIpC injection), cocultured with OP9-control cells (no ligand; Ret10) or OP9-Dll4 (Dll4) in the presence of dimethyl sulfoxide (DMSO) (A) or 10μM γ-secretase inhibitor (GSI) (B), for CD8 and Gr-1 expression. (C) A representative FACS profile of Mx-Cre/PF/F and control LSKs from 12-week-old mice (8 weeks after pIpC injection) expressing Notch receptors detected by PE-conjugated antibodies against mouse Notch 1, Notch2, Notch3, or isotype control. Mean fluorescence intensity (MFI, mean ± SD) of each genotype is included in parentheses. Student t test was performed to compare the MFI from Mx-Cre/PF/F LSKs to that from the control LSKs. * P < .05. (D) A representative FACS analysis from 3 experiments to determine recombinant Notch ligand Dll1 and Dll4 binding to Mx-Cre/PF/F and control LSKs in the presence of 0.01 g/L of Ca++, or in the presence of 10mM ethylenediaminetetraacetic acid.
David Yao et al. Blood 2011;117:
©2011 by American Society of Hematology

6. Defective T-cell development and myeloid hyperplasia are rescued by activated Notch1 (ICN1).
Defective T-cell development and myeloid hyperplasia are rescued by activated Notch1 (ICN1). Forty-eight hours after infection of 5-fluorouracil-treated Mx-Cre/PF/F marrow cells with pMig-EGFP-vector or pMig-EGFP-ICN1, 2 to 5 × 105 infected cells (Ly5.2) along with 2 × 105 WT (Lt5.1) protective cells were transferred into lethally irradiated WT (Ly5.1) recipients. Peripheral granulocytes (Gr-1+) and T cells (CD4+/CD8+) were analyzed by FACS 3 to 4 weeks after transplantation in mice receiving Mx-Cre/PF/F BM progenitors transduced with ICN1 or pMig vector (ICN1 > WT, Mig > WT). Peripheral T (CD4+ or CD8+; A) and granulocytes (Gr1+) (B) were enumerated in transduced GFP+ populations and nontransduced GFP− populations (C-D). Data are the pool of 3 independent experiments with 1 or 2 mice of each donor-recipient pair per experiment.
David Yao et al. Blood 2011;117:
©2011 by American Society of Hematology

7. Pofut1+/−/FX−/− mice have an impaired response to exogenous fucose.
Pofut1+/−/FX−/− mice have an impaired response to exogenous fucose. (A) Peripheral blood T cells, B cells, and granulocytes were analyzed by FACS in 8- to 12-week-old Pofut1+/+FX+/+ (Pofut1+/+) (n = 10) and Pofut1+/−FX+/+ (Pofut1+/−) mice (n = 11). (B-F) Pofut1+/−/FX−/− or Pofut1+/+/FX−/− mice were maintained on fucose-supplemented chow (0.5% fucose weight/weight) until 8 weeks old and then maintained on standard chow without fucose supplementation for 1 month. Pofut1+/−/FX−/− or Pofut1+/+/FX−/− mice were then fed water containing various concentrations of fucose. Four weeks later, PB and marrow were analyzed by FACS. Control group mice (no fucose) (n = 4) were maintained on standard chow for 4 weeks. (B) Absolute neutrophil numbers in the periphery 4 weeks after mice were fed fucose water or maintained on standard chow. (C-E) Absolute neutrophil numbers in Pofut1+/−/FX−/− or Pofut1+/+/FX−/− mice at different times after mice were fed 1mM (C; n = 5), 2.5mM (D; n = 5), or 5mM (E) fucose water (n = 6). (F) Marrow myeloid progenitor profiles were obtained from Pofut1+/−/FX−/− or Pofut1+/+/FX−/− mice 4 weeks after fucose supplementation and compared with those in mice receiving no-fucose chow, or in mice with Pofut1+/−/FX+/+ or Pofut1+/+/FX+/+ genotype. Shown is 1 representative profile of at least 4 similar analyses. The percentages of CMP or GMP cells of Lin−Sca-1−IL-7R−c-kit+ compartment and the ratios of GMP/CMP are summarized in Table 1. Student t test was performed to compare the cell numbers from Pofut1+/− (FX−/−) with those from Pofut1+/+ (FX−/−) mice, or from Pofut1+/−/FX−/− mice with those from Pofut1+/+/FX−/− (A-E). Data are mean ± SD. P values that are significantly different are shown as indicated.
David Yao et al. Blood 2011;117:
©2011 by American Society of Hematology