The Principle of Inverse Correlation between Variance of FP% and precursor numbers.

(A) Schematic of the Confetti cassette at the mouse Rosa26 locus. Sequences of four fluorescence protein are interspaced by loxP sites. Upon Cre-expression, the Confetti cassette will recombine and express one of the four fluorescence proteins.

(B) The inverse relationship between variance of markers and precursor numbers. The distribution of FP% is determined in the progeny to estimate the number of precursors.

(C) Workflow to validate the correlation formula between variance of FP% and precursor numbers by a two-color cell system.

(D) BFP frequency in wells seeded with 1 to 100,000 HL-60. Each dot represents a well. Two replicates were shown. Each replicate consists of at least 24 wells per seeded number. Error bars represent mean ± SD.

(E) The standard deviation of BFP% in wells seeded with various numbers of HL-60. Error bars represent mean ± SD. N = 2 replicates.

(F) The correlation between seeded numbers and standard deviation of BFP%. N = 2 replicates.

(G) The correlation between seeded numbers and the numbers estimated with standard deviation of BFP% and equation 2. N = 2 replicates.

For (A-C), images were created with Biorender.

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Transplantation of Defined Number of Precursors

(A) Representative flow plots showing Confetti induction by HSC-Scl-CreERT in the LSK population.

(B) Schematic for transplantation of defined number of precursors. Briefly, 4x106 Confetti-induced WBM for “high precursor number” groups, or 0.25x106 WBM for “low precursor number” groups were transplanted into lethally irradiated recipient mice. Image was created with Biorender.

(C) RFPConf% distribution in PB myeloid cells. Each dot represents one animal. Recipient sample size = 7-14 mice per replicate, N = 3 replicates per group. Error bars represent mean ± SD.

(D) The precursor numbers of B cells, T cells, and myeloid cells in recipient mice. Dash lines mark the level of historically estimated transplantable clone numbers. Each dot represents a precursor number calculated from multiple mice. Error bars represent mean ± SD.

(E) The correlation of precursor numbers calculated from RFPConf%, YFPConf% or CFPConf%. Each dot represents a precursor number calculated from multiple mice.

(F) The precursor number of BM MP and HSPC in recipient mice. Dash lines represent historically estimated transplantable clone numbers. Each dot represents a precursor number calculated from multiple mice. Error bars represent mean ± SD.

(G) The donor chimerism of recipient mice, and the CD45.2+ chimerism differences between high- and low-precursor number groups. Each solid dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. Recipient sample size = 7-14 mice per replicate, N = 3 replicates for each precursor number group. Two-way ANOVA for paired samples was performed.

(H) The frequency of myeloid cells in the peripheral blood of recipients. Each dot represents one animal. Recipient sample size = 7-14 mice per replicate, N = 3 replicates for each precursor number group. Two-way ANOVA for paired samples was performed.

ns, non-significant, * p < 0.05, ** p < 0.001.

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Quantification of active hematopoietic precursor at steady-state

(A) Experiment schematic for Confetti induction in adult-induced animals. Image was created with Biorender.

(B) Confetti labeling in B cells, myeloid cells and BM HSPCs. Statistics for comparisons between labeling of month seven and other months were shown. One-way ANOVA for paired samples was performed.

(C) RFPConf% in PB myeloid cells and BM HSPCs. Statistics for comparisons between labeling of month seven and other months were shown. One-way ANOVA for paired samples was performed.

(D) The number of myeloid precursors and the average precursor number from month five to seven.

(E) The number of BM precursors. Paired permutation was performed.

(F) The frequency-to-clone ratio of bone marrow HSPCs. Paired permutation test was performed. Each solid dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. Error bars represent mean ± SD. PB sample size = 14-22 mice per replicate; BM sample size = 8-10 mice per replicate; N = 4 replicates. ns, non-significant, * p < 0.05, ** p < 0.001.

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Number of precursor post-5-FU treatment and along developmental ontogeny

(A) Experiment schematic for one-dose 5-FU treatment. Image was created with Biorender.

(B) The number of BM precursors post-5FU treatment. Permutation test was performed. For UT, sample size = 8-10 mice per replicate; N = 4 replicates; for 5-FU, sample size = 6-8 mice per replicate; N = 5 replicates.

(C) Experiment schematic for Confetti induction at fetal stages. Image was created with Biorender.

(D) The Confetti labeling of B cells, T cells and myeloid cells. Each dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. Sample size = 4-11 mice per replicate; N = 5 replicates.

(E) The number of precursors in PB myeloid cells, B cells and T cells. Permutation test was performed. For E14.5 and adult-induction, N = 4 replicates; for E11.5, n = 3 replicates.

(F) The number of BM precursors. Each dots represent one precursor number. Permutation test was performed. For E14.5 and adult-induction, N = 4 replicates; for E11.5, N = 3 replicates.

(G) The relative fold change increase of precursor numbers from fetal-to adult-stage, as well as fold change of CRU and cell counts.

Error bars represent mean ± SD. ns, non-significant, * p < 0.05, ** p < 0.001.

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Precursor numbers in a mouse model of FA

(A) Experimental workflow to generate Fancc+/+ and Fancc-/- mice, induce Confetti expression and track precursor number dynamics. Image was created with Biorender.

(B) Confetti labeling in PB B cells and myeloid cells. Each dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. Dashed lines connect the values for the same replicate.

(C) The number of PB myeloid precursor.

(D) The number of BM precursor.

(E) Experiment schematic for competitive transplantation. Image was created with Biorender.

(F) PB donor chimerism in recipient mice of young FA donors. Each dot represents one animal. Dashed lines connect the average donor chimerism of three replicates. For Fancc+/+ or Fancc+/-, n = 10 per replicate, N = 3 replicates; for Fancc-/-, N = 7-9 per replicate, N = 3 replicates. Two-way ANOVA was performed.

(G) The number of PB myeloid precursor post-transplantation of young FA donors. Dashed lines connect the average precursor numbers. Permutation test was performed. N = 3 for each group.

(H) PB donor chimerism recipient mice of aging FA donor cells. Each dot represents one animal. Dashed lines connect the average donor chimerism of three replicates. For both genotype, N = 10 per replicate, N = 3 replicates. Two-way ANOVA was performed.

(I) The number of PB B precursor post-transplantation of aging FA donors. Dashed lines connect the average precursor numbers. Permutation test was performed. N = 3 for each genotype.

For (A-C), Fancc+/+, sample size = 11-17 mice per replicate, N =5 replicates; Fancc+/-, sample size = 10-22 mice per replicate, N =7 replicates; Fancc-/-, sample size = 9-17 mice per replicate, N =4 replicates. For (D), Fancc+/+, sample size = 5-9 mice per replicate, N =5 replicates; Fancc+/-, sample size = 6-12 mice per replicate, N =5 replicates; Fancc-/-, sample size = 6-11 mice per replicate, N =4 replicates. Error bars represent mean ± SD. nd, not determined; ns, non-significant; * p < 0.05, ** p < 0.001. # represents lowest p value possible for permutation test.

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Establishing the correlation between variance of FP and precursor numbers.

(A) The correlation between precursor numbers and CVs of individual FP or all CVs (original correlation formula), replotted from Ganuza et al., 2017.

(B) FPmean% in a previously published dataset, replotted from Ganuza et al., 2017.

(C) The correlation between precursor numbers and standard deviation (σ) of individual FP, replotted from Ganuza et al., 2017.

(D) The BFP% in HL-60 mix during two weeks of growth.

(E) The effect of outliers on precursor number estimation. Left, BFP% in samples with/without outliers. Quartiles by Tukey method was shown. The outlier is highlighted by circle. Middle, standard deviation of BFP% with/without outliers. Right, estimated precursor numbers with/without outliers.

(F) The changes of standard deviation when recorded events varied. “&” denotes the potential overestimated standard deviation due to low flow recorded events.

(G) The variance of estimated precursor numbers when varying sample sizes were used.

(H) The relative error of estimation as sample sizes increased. Each dot represents data from one seeded number. Error bars represent mean ± SD.

The induction specificity of HSC-Scl-CreERT

(A) Experiment schematic to examine the induction specificity of HSC-Scl-CreERT. N =10 for induced animals, N =3 for non-induced animals. Image was created with Biorender.

(B) Flow gating strategy for BM HSPC and MPs.

(C) Confetti labeling in BM HSPCs. Error bars represent mean ± SD.

(D) Flow gating strategy for BM lineage cells.

(E) Confetti labeling in BM lineage cells. Error bars represent mean ± SD.

(F) Flow gating strategy to examine spleen cells.

(G) Confetti labeling in spleen cells. Error bars represent mean ± SD.

See Methods and Table S1 for the detailed antibody, fluorophore and marker combination used for each cell type.

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Analysis of HSC-Scl-CreERT background Cre activity

(A) Representative flow plot showing Confetti expression in PB T cells in 48-50-week-old HSC-Scl-CreERT/Confetti animals compared to an animal induced with one-day tamoxifen treatment.

(B) Quantification of RFP and YFP% in PB T cells. N =8 for non-induced 48-50-week-old animals.

(C) Representative flow plot for Confetti labeling in PB T cells post HSC-Scl-CreERT-mediated induction.

(H) The level of CFP+ and RFP+ double positive cells in PB T cells of animals induced by HSC-Scl-CreERT compared to Vav-Cre/Confetti animals analyzed at the same age. N =3 for Vav-Cre animals, N= 8 for HSC-Scl-CreERT animals.

Variance of FP% inversely correlates with precursor numbers in vivo

(A) The correlation between expected numbers and the estimated numbers calculated from FP% of precursors following Poisson distribution. See Methods for simulation details.

(B) Flow gating strategy for PB B cell, T cell and myeloid cell. See Methods and Table S1 for the detailed antibody, fluorophore and marker combination used for each cell type.

(C) RFPConf% distribution in B cell and T cell.

(D) YFPConf% distribution in myeloid cell.

(E) CFPConf% distribution in myeloid cell.

(F) RFPPB%, CFPPB%, YFPPB% and Confetti% distribution in myeloid cells.

(G) Linear correlation of PB precursor numbers calculated from RFPConf%, RFPPB%, CFPPB%, YFPPB%, Confetti% or RFPRFP+YFP%, Each dot represents a precursor number calculated from multiple mice.

For (C-F), each dot represents one animal. Recipient sample size = 7-14 mice per replicate, N =3 replicates per group. Error bars represent mean ± SD.

BM analysis for high- and low-precursor number groups

(A) RFPConf% distribution in BM HSPCs. Each dot represents one animal

(B) BM donor chimerism.

(C) BM nucleated cell numbers. N =15 mice for high precursor number group, n= 14 for low precursor number group. Unpaired t-test was performed.

(D) BM HSPC and MP frequency in donor CD45.2+ population. Two-way ANOVA for paired samples was performed.

For (B) and (D), each solid dot represents one animal; each outlined dot represents the average value of a replicate containing multiple animals. Except for (C), recipient sample size = 7-14 mice per replicate, N =3 replicates per group. For all graphs, Error bars represent mean ± SD. ns, non-significant, * p < 0.05.

Analysis of FP induction in adult-induced animals

(A) Confetti labeling in LSK after 14days of tamoxifen treatment. N =5 animals. Error bars represent mean ± SD.

(B) CFPConf% in PB myeloid cells.

(C) CFPPB% in PB myeloid cells.

(D) RFPRFP+YFP% in PB myeloid cells.

(E) RFPPB% and YFPPB% in PB myeloid cells.

(F) CFPConf% in PB myeloid cells of fetal-induced animals

(G) BM HSPC frequencies. Error bars represent mean ± SD.

For (B-E) sample size = 14-22 mice per replicate; for (F), sample size = 4-11 mice per replicate; for

(G) sample size = 8-10 mice per replicate. For (B-E) and (G), N =4 replicates. For (F), N =5 replicates.

Analysis related to 5-FU treatment and Confetti fetal induction

(A) PB myeloid cells frequencies before 5-FU treatment and ten days post-5-FU treatment. Dashed lines connect the values for the same replicate. Sample size = 6-8 mice per replicate, N =5 replicates for 5-FU. Paired t test was performed.

(B) The BM HSPC frequencies of UT and 5-FU treated groups ten days post-5-FU treatment. For UT, sample size = 8-10 mice per replicate, N =4 replicates; for 5-FU, sample size = 6-8 mice per replicate, N =5 replicates.

(C) Confetti% in PB in E11.5-labeled animals. Dashed lines connect values for the same replicate.

(D) The distribution of FPs in Confetti+ population of B cells. Data showing average from all mice. N =24-34 for E14.5, N =7-24 for E11.5. Error bars represent mean ± SD.

For (A-C), each solid dot represents one animal; each outlined dot represents the average value of a replicate containing multiple animals. ns, non-significant; * p < 0.05, ** p < 0.001.

Analysis of FA mice at steady-state

(A) PB myeloid cell frequencies. Each dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals.

(B) PB B cell frequencies. Each dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals.

(C) Complete blood count analysis. Each dot represents one animal. N =13 for Fancc+/+, N =16 for Fancc-/- mice. Unpaired t test was performed.

(D) BM HSPC frequencies. Each dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. One-way ANOVA was performed.

(E) BM nucleated cells counts. Each dot represents one animal. Unpaired t test was performed. N =12 for Fancc+/+and Fancc-/- mice.

(F) RFPRFP+YFP% in PB myeloid cells and B cells. Each dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals.

For (A-B) and (F), Fancc+/+, sample size = 11-17 mice per replicate, N =5 replicates; Fancc+/-, sample size = 10-22 mice per replicate, N =7 replicates; Fancc-/-, sample size = 9-17 mice per replicate, N =4 replicates. For (D), Fancc+/+, sample size = 5-9 mice per replicate, N =5 replicates; Fancc+/-, sample size = 6-12 mice per replicate, N =5 replicates; Fancc-/-, sample size = 6-11 mice per replicate, N =4 replicates. Error bars represent mean ± SD. ns, non-significant; * p < 0.05.

Analysis of FA recipient mice

(A) Young Fancc donor BM HSPC frequency analysis. N = 5 for Fancc+/+ or Fancc+/-, N = 2 for Fancc-/-.

(B) Donor chimerism in recipient BM of young Fancc donor. Each dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. Two-way ANOVA was performed. Recipient sample size = 7-10 mice per replicate, N =3 replicates per group.

(C) Number of PB B cell precursor in recipients of young FA donor.

(D) Number of PB T cell precursor in recipients of young FA donor.

(E) Number of BM HSPC precursor in recipients of young FA donor.

(F) Ageing Fancc donor BM HSPC frequency analysis. N = 3 for Fancc+/+, N = 3 for Fancc-/-.

(G) Number of PB myeloid cell precursor in recipients of aging FA donor.

(H) Number of PB T cell precursor in recipients of aging FA donor.

(I) Donor chimerism in recipient BM of ageing Fancc donor. Each dot represents one animal. Each outlined dot represents the average value of a replicate containing multiple animals. Two-way ANOVA was performed. Recipient sample size = 7-10 mice per replicate, N =3 replicates per group.

(J) Number of BM HSPC precursor in recipients of aging FA donor.

For (C-E), (G-H) and (J), N =3 replicates per group; permutation test was performed; dashed lines connect the average precursor numbers. Error bars represent mean ± SD. nd, not determined; ns, non-significant; * p < 0.05, ** p < 0.001. # represents lowest p value possible for permutation test.