Transcriptome-based identification of DAPK2 in erythroid progenitor cells, and its stage- and lineage- restricted expression. (A) Preparation, purification and initial characterization of developmentally staged erythroblasts. Splenic erythroblasts from pA2gata1-EE-Y343 mice were prepared at 80, 100 and 120 hours after thiamphenicol treatment and withdrawal (stages I-III), and were purified via MACS. Purity and stagedness were assessed by flow cytometric analyses of EE-positive cells (see Figure S1A), and by morphologic analyses of cytospin preparations. Following staining with HEMA-3 (Fisher Scientific, Hampton, NH), micrographs were acquired using a Zeiss Axioskop 2 microscope (Carl Zeiss, Thornwood, NY) equipped with a 100×/1.3 numeric aperture oil-immersion objective, and a Spotflex model 15.2 camera (Diagnostic Instruments, Sterling Heights, MI). Spot advance version 4.6 (Diagnostic Instruments) and Photoshop version CS2 software (Adobe Systems, San Jose, CA) were used. Stage I cells are Kit^highCD71⁺Ter119⁻ high-FALS (forward-angle light scatter) proerythroblasts; stage II cells are Kit^lowCD71⁺Ter119^pos high-FALS polychromatophilic erythroblasts; and stage III cells are Kit⁻CD71⁺Ter119^high low-FALS orthochromatic normoblasts. Stage I, II and III cells were also initially characterized based on relative expression levels of Epo receptor (EpoR) transcripts (assayed via Northern blotting and phosphoimaging; data not shown). (B) Profiling of erythroblast stage-modulated survival factors. For survival factors, profiling outcomes are illustrated as stage I-to-II, and II-to-III modulated sets. Top panels (■) are for transcripts with increased expression at stages I-to-II, while transcripts with decreased stage I-to-II expression (□) are graphed in the bottom subpanel. (C) RT-qPCR results for Dapk2 transcript levels in stage I-III erythroblasts also are shown (beta-actin transcript levels were used as an internal control). DAPK2 and its major structural features also are diagrammed. Stage-wise increases in DAPK2 expression also were observed among developmentally staged splenic and bone marrow (BM) erythroblasts as expanded ex vivo,^17-19  fractionated as indexed, and analyzed by Western blotting (anti-DAPK2 antibody, Chemicon, Temecula, CA). (D) DAPK2 is expressed predominantly in the erythroid lineage. Dapk2 and Dapk1 transcript levels in mouse cell lines, primary bone marrow–derived hematopoietic progenitors, and select tissues were assayed by reverse-transcription, and quantitative PCR.^17,18 Beta-Actin transcript levels were used as internal controls. For Dapk2 and Dapk1, levels were normalized to maximums.