Studies in an Early Development Window Unveils a Severe HSC Defect in both Murine and Human Fanconi Anemia

Abstract

Fanconi anemia (FA) causes bone marrow failure early during childhood, and recent studies indicate that a hematopoietic defect could begin in utero. We performed a unique kinetics study of hematopoiesis in Fancg^-/- mouse embryos, between the early embryonic day 11.5 (E11.5) to E12.5 developmental window (when the highest level of hematopoietic stem cells [HSC] amplification takes place) and E14.5. This study reveals a deep HSC defect with exhaustion of proliferative and self-renewal capacities very early during development, together with severe FA clinical and biological manifestations, which are mitigated at E14.5 due to compensatory mechanisms that help to ensure survival of Fancg^-/- embryos. It also reports that a deep HSC defect is also observed during human FA development, and that human FA fetal liver (FL) HSCs present a transcriptome profile similar to that of mouse E12.5 Fancg^-/- FL HSCs. Altogether, our results highlight that early mouse FL could represent a good alternative model for studying Fanconi pathology.

Keywords: Fanconi anemia; HSC; fetal liver; human embryonic development; mouse embryonic development; placenta; transcriptome.

Figure 1

Dysmorphology of Fancg^−/− Embryos Accompanied with Cellular Defect and Abnormal Hematopoietic Content in the FL and Pl (A) smaller size and microphthalmia (blue asterisk) of Fancg^−/− embryos between E11.5 and E14.5, accompanied with a smaller size of FL (red arrowhead). Respective numbers of Fancg^−/− and WT embryos studied was n = 20 and n = 43 at E11.5, n = 186 and n = 254 at E12.5, and n = 52 and n = 53 at E14.5. (B) Total cellularity of FL and Pl at E11.5, E12.5, and E14.5. (C) Total number of erythroid (TER119), myeloid (GR1), and B lymphoid (B220) cells per FL and Pl at E12.5 and E14.5. Black bars, WT; white bars, Fancg^−/−. Error bars correspond to standard deviation. ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05; NS, not significant.

Figure 2

Defect of HSPC-Enriched Populations and CFUs in the Pl and FL of Fancg^−/− Embryos Is Observed as Soon as E11.5 (A) Total number of HSPC-enriched cells in the Pl and FL at E11.5 and E12.5, and in the FL at E14.5. At E11.5 in the AGM (n = 4), Pl (n = 3), and FL (n = 3), and at E12.5 in the Pl (n = 9) the HSPC-enriched population is 34K, while in the FL at E12.5 (n = 7) and E14.5 (n = 6) it is LSA. (B) Total CFUs and CFU-GM, BFU-E, and CFU-mixed content per FL at E11.5, E12.5, and E14.5 and per Pl at E11.5 and E12.5. Results shown represent the mean ± SE and correspond to 3–5 independent experiments, with two independent Petri dishes scored for each experiment. (C) In vitro comparison of LTC-IC frequency in Fancg^−/− and WT FL at E12.5 (n = 3) and E14.5 (n = 3). n represents distinct experiments. Error bars correspond to standard deviation. Black bars, WT; white bars, Fancg^−/−. ^∗∗∗∗ p<0.0001, ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05; NS, not significant.

Figure 3

High LTR Ability Defect and Impaired Myeloid Potential in E12.5 Fancg^−/− Pl and FL (A) CD45.2 chimerism in the blood of primary recipients 20–24 weeks post injection of 5 × 10⁵ (circles) or 10⁶ (squares) WT (black) or Fancg^−/− (white) total FL cells. (B) CD45.2 chimerism in primary recipients' blood 20–24 weeks post injection of 5 × 10⁵ (circles) or 10⁶ (squares) WT (black) or Fancg^−/− (white) Pl cells. (C and D) Analysis of BM hematopoietic content of primary recipients transplanted with FL (C) or Pl (D), expressed as a percentage of CD45.2⁺ erythroid (TER119), myeloid (GR1), B lymphoid (B220), and T-lymphoid (CD4) cells. Circles, 5 × 10⁵ cells injected; squares, 1 × 10⁶ cells injected; black, WT FL or Pl cells; white, Fancg^−/− FL or Pl cells. ^∗∗∗p < 0.001, ^∗∗p < 0.01, ^∗p < 0.05; NS, not significant.

Figure 4

Induced DNA Damage in E12.5 and E14.5 Fancg^−/− HCs and HSCs without Apoptosis or Cell-Cycle Changes (A) MFI of γH2AX^pos cells in total, HC (CD45), and HSC (34K or SA) in E12.5 PL (n = 3), FL (n = 4), and E14.5 FL (n = 3, and each dot represents an individual mouse), measured by flow cytometry. (B) Histograms of G₀, G₁, S, and G₂/M phases of the cell cycle determined after Hoechst staining, for Fancg^−/− and WT HC, and HSCs of E12.5 FL and Pl and E14.5 FL. Dots of same colors are related to the same experiment. Error bars correspond to standard deviation. Gray bars, WT; white bars, Fancg^−/−. ^∗p < 0.05; NS, not significant.

Figure 5

Important Frequency and LTC-IC Defect in the CD34⁺CD38⁻CD45⁺CD117^hi HSC-Enriched Population of a Human FA 13 WG FL (A) Comparison of the frequency of CD34⁺CD38⁻CD45⁺CD117^hi HSC-enriched population from an FA (FANCB) FL or from an age-matched control FL (13 WG). (B) Comparison of LTC-IC frequency of CD34⁺CD38⁻CD45⁺CD117^hi cells sorted from an FA (FANCB) FL (13 WG) and from two age-matched control FL (13 and 17.7 WG). Error bars correspond to standard deviation. Black bar, control FL; white bar, FA FL.

Figure 6

Highly Conserved GO Biological Processes between E12.5 Fancg^−/− Pl and FL and Human FA FL HSCs (A) Enriched GO biological process (BP) terms for downregulated (white bars) and upregulated (black bars) genes in E12.5 Fancg^−/− FL HSCs and in human FA FL HSCs. GO BPs determined using DAVID annotation tool are expressed as –log₁₀ (p value). NES, normalized enrichment score. (B) GSEA plots for up- and downregulated genes, specifically expressed in E12.5 and E14.5 Fancg^−/− FL HSCs. (C) Anticorrelation study. Plot of log₂(FC) between Fancg^−/− and WT HSCs at E12.5 against E14.5. (D) GSEA plots for up- and downregulated genes, specifically expressed in human FA FL HSCs.