Hematopoietic Defects in Cited2 Mutant Fetal Liver.

Cited2 [cAMP-responsive element-binding protein (CBP)/p300-interacting transactivators with glutamic acid (E) and aspartic acid (D)-rich tail 2] is a newly identified transcriptional modulator. Knockout of Cited2 gene is embryonic lethal because of heart and neural tube defects. Cited2 binds directly to CBP and p300, which have been shown to be crucial for hematopoietic stem cell self-renewal and proper hematopoietic differentiation, respectively. Cited2 also induces the expression of a polycomb-group gene, Bmi-1, which is essential for self-renewal of adult hematopoietic stem cells. These connections provided rationale to study the potential role of Cited2 in hematopoiesis. Mouse fetal liver is the major hematopoietic organ from day 10 postcoitus until right before birth. The smaller sized Cited2^−/− fetal liver and significantly decreased fetal liver cellularity strongly suggest the potential defect in hematopoiesis. In vitro colony formation assay in methycellulose-based medium was used to characterize the hematopoietic progenitors. We found that fetal liver cells from E13.5, 14.5 and E15.5 Cited2^−/− embryos gave rise to much less colonies, which reflects the decreased number and proliferative ability of hematopoietic progenitors due to Cited2 deficiency. Immunostaining of lineage-specific cell surface markers followed by flow cytometry was performed to characterize different hematopoietic populations in E14.5 and E15.5 fetal liver of wild type and Cited2^−/− embryos. Cited2^−/− fetal liver cells displayed a significant reduction in numbers throughout the hematopoietic hierarchy including hematopoietic stem cells (Lin⁻ c-Kit⁺ Sca-1⁺), progenitor cells (Lin⁻ c-Kit⁺), and differentiated cells of different lineages (CD45⁺, Ter119⁺, Mac-1⁺, Gr-1⁺), thus revealing a multi-level hematopoietic deficiency of Cited2^−/− embryos. Long-term reconstitution experiment was then carried out to measure the ability of hematopoietic stem cells from Cited2^−/− fetal liver cells to engraft and reconstitute hematopoietic system of congenic recipient mice. Mice transplanted with Cited2^−/− fetal liver cells showed reconstitution of T cells whereas a 2-fold decrease in the reconstitution of B cell and myeloid lineages was observed, indicating a compromised ability of Cited2^−/− fetal liver hematopoietic stem cells to maintain hematopoiesis. The results suggest an important role of Cited2 in hematopoietic differentiation and a selective function of Cited2 in B lymphoid &myeloid induction. The underlying mechanisms responsible for these defects will be pursued by microarray analysis of gene expression profile of Cited2^−/− fetal liver cells, followed by more detailed phenotypic analyses of B and myeloid lineage markers plus in vitro and in vivo functional assays.