Geminin deletion from hematopoietic cells causes anemia and thrombocytosis in mice
Kathryn M. Shinnick, Elizabeth A. Eklund, Thomas J. McGarry
Kathryn M. Shinnick, Elizabeth A. Eklund, Thomas J. McGarry
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Research Article Hematology

Geminin deletion from hematopoietic cells causes anemia and thrombocytosis in mice

Abstract

HSCs maintain the circulating blood cell population. Defects in the orderly pattern of hematopoietic cell division and differentiation can lead to leukemia, myeloproliferative disorders, or marrow failure; however, the factors that control this pattern are incompletely understood. Geminin is an unstable regulatory protein that regulates the extent of DNA replication and is thought to coordinate cell division with cell differentiation. Here, we set out to determine the function of Geminin in hematopoiesis by deleting the Geminin gene (Gmnn) from mouse bone marrow cells. This severely perturbed the pattern of blood cell production in all 3 hematopoietic lineages (erythrocyte, megakaryocyte, and leukocyte). Red cell production was virtually abolished, while megakaryocyte production was greatly enhanced. Leukocyte production transiently decreased and then recovered. Stem and progenitor cell numbers were preserved, and Gmnn–/– HSCs successfully reconstituted hematopoiesis in irradiated mice. CD34+Gmnn–/– leukocyte precursors displayed DNA overreplication and formed extremely small granulocyte and monocyte colonies in methylcellulose. While cultured Gmnn–/– megakaryocyte-erythrocyte precursors did not form erythroid colonies, they did form greater than normal numbers of megakaryocyte colonies. Gmnn–/– megakaryocytes and erythroblasts had normal DNA content. These data led us to postulate that Geminin regulates the relative production of erythrocytes and megakaryocytes from megakaryocyte-erythrocyte precursors by a replication-independent mechanism.

Authors

Kathryn M. Shinnick, Elizabeth A. Eklund, Thomas J. McGarry

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Figure 4

Geminin is not required for HSC self renewal.

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Geminin is not required for HSC self renewal. (A) Experimental protocol....
(A) Experimental protocol. Recipient CD45.1 mice were irradiated and transplanted with CD45.2 marrow from R26R-GFP/Mx1-Cre/Gemfl/fl or R26R-GFP/Mx1-Cre/Gemfl/+ donors. After engraftment, the mice were either left untreated or treated with pIpC. (B) Typical flow profile quantifying CD45.1+ and CD45.2+ populations. (C–G) Time-dependent changes in peripheral blood GFP+ cells (C), CD45.2+cells (D), wbc count (E), rbc count (F), and platelet count (G) in transplanted mice after pIpC injection. Black, uninjected control mice; green, plpC-injected control mice; blue, uninjected Mx1-Cre/Gemfl/f mice; red, plpC-injected Mx1-Cre/Gemfl/f mice. Average of 6 measurements (pIpC-treated mice) or 4 measurements (untreated mice). *P < 0.05; **P < 0.01. (H) Geminin mRNA levels in CD45.1 and CD45.2 cells recovered from transplanted mice. Number of measurements is shown above each column. Black bar, uninjected control mice; blue bar, uninjected Mx1-Cre/Gemfl/f mice; red bar, plpC-injected Mx1-Cre/Gemfl/f mice. (I) Absolute numbers of recovered CD45.2+ marrow cells by flow cytometry. Color code is the same as in (H). (J) Colony types grown from CD45.1 or CD45.2 cells recovered from transplanted mice. Number of mice is shown above each column. Red, erythroid colonies (E); yellow, granulcoyte colonies (G); green, monocyte colonies (M); blue, granulocyte-monocyte colonies (GM); lavender, granulocyte-erythrocyte-megakaryocyte colonies (MK). (K) Recovered marrow cells (CD45.1+CD45.2) were retransplanted into irradiated CD45.1 recipients. Time-dependent changes in CD45.2 peripheral blood cells in individual mice transplanted with cells from pIpC-treated control mice (green curves) or with cells from pIpC-treated Mx1-Cre/Gemfl/fl mice (red curves). Cross symbol indicates death. Green lines, donor cells from plpC-injected control mice; red lines, donor cells from plpC-injected Mx1-Cre/Gemfl/fl mice. (L) Right, experimental protocol. Mice were treated with pIpC and 4 weeks later marrow cells were transplanted into irradiated recipients. Left, number of CD45.1+ and CD45.2+ cells in the peripheral blood 2 weeks after transplantation. Black, percentage of CD45.1+ cells; red, percentage of CD45.2+ cells. n = 6 for both groups. P values calculated by ANOVA.