Supplementary MaterialsS1 Fig: Generation of the loss-of-function allele of in hematopoietic cells. ID1 within the fetal liver organ. (A) Representative movement cytometric information of hematopoietic progenitor cell Collagen proline hydroxylase inhibitor-1 populations (LSK; best -panel, CLP; middle -panel, CMP, MEP and GMP; bottom -panel) through the fetal liver organ of and embryos (E 14.5). Amounts reveal percentage of gated cells among total fetal liver organ mononuclear cells. Pub graphs on the proper depict Collagen proline hydroxylase inhibitor-1 absolute amounts of the indicated cell populations altogether fetal liver organ mononuclear cells from (solid pubs) and control (open up pubs) embryos (mean and SD; = 4) n. (B) Representative movement cytometric information of Collagen proline hydroxylase inhibitor-1 lineage-committed cell populations within the fetal liver organ. Pub graphs on the proper depict absolute amounts of the indicated cell populations altogether fetal liver organ mononuclear cells from (solid bars) and control (open bars) embryos (mean and SD; n = 4). B-lineage cells (CD19+ Gr-1-), granulocytes (Gr-1+ CD11b+), monocytes (Gr-1- CD11b+), proerythroblasts (I; Ter119low CD71high), basophilic erythroblast (II; Ter119high CD71high) and late erythroblasts (III; Ter119high CD71int and IV; Ter119high CD71low). (TIF)(TIF) pone.0136107.s002.tif (1.0M) GUID:?F4BF8F5F-5957-4083-871B-EAF0E68EAEC6 S3 Fig: Differentiation of megakaryocytic-lineage cells in the bone marrow of mice. Representative flow cytometric profiles of megakaryocytic-lineage cell populations from mice and littermate controls. Numbers indicate percentage of gated cells among the total cells analyzed; pro-megakaryocytes (c-Kit+ CD41+) and megakaryocytes (c-Kit- CD41+). Bar graphs on the right depict absolute numbers of the indicated cell populations in the BM of two femurs from (solid bars) and control littermate (open bars) mice (mean and SD; n = 3). (TIF)(TIF) pone.0136107.s003.tif (294K) GUID:?C4F3074D-5599-45CB-A83F-57BCC2725E49 S4 Fig: Flow cytometry gating strategy for hematopoietic stem/progenitor cells in the bone marrow. (A) Flow gating schemes for CD34+/- and Flt3+ LSK and CLP in the BM of mice (CKO) and littermates (control). Lineage markers (Lin) include CD11b, CD3, B220, Ter119, Gr-1 and 7-AAD. (B) Flow gating schemes for CMP, GMP and MEP in the BM of mice (CKO) and littermates (control). Lineage markers (Lin) include CD11b, CD3, B220, Ter119, Gr-1, IL-7R and Sca-1. (C) Flow gating schemes for cell cycle analyses of CD150+ CD48- CD41- SP cells in the BM of mice (CKO) and littermates (control). (TIF)(TIF) pone.0136107.s004.tif (640K) GUID:?39F651B9-BDCE-4BB1-8019-F279C9BA4F78 S1 Table: List of primers for PCR (doc). (DOC) pone.0136107.s005.doc (40K) GUID:?76A10F8D-FEC7-43EC-8F1B-AA90D630949F Data Availability StatementAll relevant data are within the paper and its Supporting Information files. Abstract Prep1, a TALE-family homeodomain transcription factor, has been demonstrated to play a critical role in embryonic hematopoiesis, as its insufficiency caused late embryonic lethality associated with defective hematopoiesis and angiogenesis. In the present study, we generated hematopoietic- and endothelial cell-specific Prep1-deficient mice and demonstrated that expression of Prep1 in the hematopoietic cell compartment is not essential for either embryonic or adult hematopoiesis, although its absence causes significant hematopoietic abnormalities in the adult bone marrow. Loss of Prep1 promotes cell cycling of hematopoietic stem/progenitor cells (HSPC), leading to the expansion of the HSPC pool. Prep1 deficiency also results in the accumulation of lineage-committed Collagen proline hydroxylase inhibitor-1 progenitors, increased monocyte/macrophage differentiation and arrested erythroid maturation. Maturation of T cells and B cells is also perturbed in Prep-deficient mice. These findings provide novel insight into the pleiotropic jobs of Prep1 in adult hematopoiesis which were unrecognized in earlier research using germline hypomorphic mice. Intro Many diverse features have been referred to for the three-amino-acid-loop-extension (TALE) course of homeodomain transcription elements during embryonic and postnatal advancement in vertebrates [1]. These transcription elements, such as the Meis, Pbx and Prep families, share a conserved atypical homeodomain made up of a three-amino acid loop extension between the first two -helices, through which they can bind to the target DNA as well as interact with Hox proteins [2]. In addition, Prep and Meis family members have two additional conserved domains in their N-terminal region, the MEIS-A and MEIS-B domains (also termed HM1 and HM2), which function as an interface for hetero-dimerization with Pbx family members [3C6], promoting nuclear translocation and also affecting DNA-binding choice by the Pbx proteins. Thus, the MEIS-A/B domain name of Meis/Prep family proteins is a key regulatory domain that can mediate both positive and negative effects on their biological functions. The Prep family consists of Prep1 and Prep2 in Collagen proline hydroxylase inhibitor-1 mice and humans [7C10], and Prep1 is usually relatively ubiquitously expressed in most tissues.