Fancd2 and p21 function independently in maintaining the size of hematopoietic stem and progenitor cell pool in mice

Abstract

Fanconi anemia patients suffer from progressive bone marrow failure. An overactive p53 response to DNA damage contributes to the progressive elimination of Fanconi anemia hematopoietic stem and progenitor cells (HSPC), and hence presents a potential target for therapeutic intervention. To investigate whether the cell cycle regulatory protein p21 is the primary mediator of the p53-dependent stem cell loss, p21/Fancd2 double-knockout mice were generated. Surprisingly double mutant mice displayed even more severe loss of HSPCs than Fancd2(-/-) single mutants. p21 deletion did not rescue the abnormal cell cycle profile and had no impact on the long-term repopulating potential of Fancd2(-/-) bone marrow cells. Collectively, our data indicate that p21 has an indispensable role in maintaining a normal HSPC pool and suggest that other p53-targeted factors, not p21, mediate the progressive elimination of HSPC in Fanconi anemia.

Keywords: CBC; FA; FITC; Fanconi anemia; H&E; HSPC; KSL; cKit(+)Sca1(+)Lin(−); complete blood count; fluorescein isothiocyanate; hematopoietic stem and progenitor cells; hematoxylin and eosin; qPCR; quantitative real-time PCR.

Figure 1. p21 deletion reduced KSL pool but did not change stem cell function

(A) Quantification of KSL hematopoietic stem and progenitor cell frequencies in the bone marrow of Fancd2^−/−/Cdkn1a^−/− mice and controls. The percentage of the KSL gate refers to the proportion of KSL cells in the whole nucleated bone marrow. n=8 for Fancd2^+/+/Cdkn1^+/+ mice, n=11 for Fancd2^+/+/Cdkn1a^−/− mice, n=8 for Fancd2^−/− /Cdkn1^+/+ mice, and n=10 for Fancd2^−/−/Cdkn1a^−/− mice. (B, C) Cell cycle profiles of bone marrow KSL cells. n=9 for Fancd2^+/+/Cdkn1^+/+ mice, n=9 for Fancd2^+/+/Cdkn1a^−/− mice, n=7 for Fancd2^−/−/Cdkn1^+/+ mice, and n=9 for Fancd2^−/−/Cdkn1a^−/− mice. NS denotes not significant. (D) Upper panel: Strategy used in the competitive repopulation experiment. BMCs denotes bone marrow cells. Lower panel: In vivo competitive repopulation of Fancd2^−/−/Cdkn1a^−/− (or control) donor and ROSA26^Tg/O bone marrow cells. qPCR analyses were performed to evaluate donor contribution to the peripheral blood cells from each donor. Three independent qPCR analyses were performed for each sample and results from 5 animals were pooled together for each experimental group. Data are presented as mean ± SD. NS denotes not significant.

Figure 2. Representative flow cytometry profile of cell cycle analysis

p21 deletion did not change the abnormal cell cycle status of Fancd2^−/− mice. Hoechst 33342 and FITC-conjugated anti-mouse Ki67 (BD Sciences) were used in combination to distinguish cells in G0, G1, and S-G2-M phases of the cell cycle.

Figure 3. Representative pictures after H&E staining of testis sections

p21 deletion did not correct the germ-cell-loss phenotype of Fancd2^−/− mice. The mice used were 4 months old. The arrows indicate tubules with vacuolated Sertoli cell cytoplasm. Original magnification x100.

Figure 4. p21 deletion did not affect hematologic parameters of peripheral blood

Comprehensive CBC tests of peripheral blood from Fancd2^−/−/Cdkn1a^−/− mice and controls. n=11 for Fancd2^+/+/Cdkn1^+/+ mice, n=15 for Fancd2^+/+/Cdkn1a^−/− mice, n=9 for Fancd2^−/−/Cdkn1^+/+ mice, and n=11 for Fancd2^−/−/Cdkn1a^−/− mice. WBC denotes white blood cells; RBC, red blood cells; HGB, hemoglobin; and NS, not significant. Unless specified otherwise on the figure, P values were not significant.