Figure 1.
Generation of mice carrying conditional Cxxc1 alleles.
(A) Schematic illustrating structures of the endogenous murine Cxxc1 allele, targeting vector, targeted Cxxc1 allele (Cxxc1-T), conditional Cxxc1 allele (Cxxc1-flox), and disrupted Cxxc1 allele (ΔCxxc1). (B) Schematic illustrating the PCR primers used to detect the presence of loxP elements within Cxxc1 alleles. (C) Southern blot analysis performed with EcoRI digested genomic DNA and a probe for a region of the Cxxc1 locus downstream of the targeting construct detects a 7 kb fragment from the targeted allele (Cxxc1-T) and a 13 kb fragment from the wild type allele. The presence of the targeted allele is indicated by asterisks. (D) PCR using primers CXXC1F and LOXP1R demonstrates the presence of a loxP element within intron 1 of the Cxxc1 allele in a subset of ES clones (lanes 1 and 5). (E) Mice carrying the targeted Cxxc1 allele were bred with mice carrying the EIIa-Cre transgene. Tail DNA isolated from offspring was analyzed by PCR, using primers LOXP1F and CXXC1R, to detect Cre-mediated recombination events that removed the Neo cassette from intron 1 of the targeted Cxxc1 allele, to produce the conditional Cxxc1-flox allele (indicated by the presence of the upper band of the doublet) (lanes 2, 4, and 9). (F) Following germline transmission of the putative conditional Cxxc1-flox allele, tail DNA of offspring was analyzed by PCR to confirm the presence of loxP elements within introns 1 and 14. Primers LOXP1F and CXXC1R were used to detect the intron 1 loxP element, and primers LOXP3F and LOXP3R were used to detect the loxP element within intron 14. In both cases the presence of the loxP element leads to the production of a band slightly larger than that produced from the wild type Cxxc1 allele. In addition, the PCR fragment containing LoxP1 was purified for DNA sequence analysis to confirm its presence and the absence of the NeoR cassette (data not shown). (G) PCR analysis, using primers LOXP1F and CXXC1R, was performed to identify mice lacking (+/+), heterozygous (+/flox), or homozygous (flox/flox) for the conditional Cxxc1 allele.
Figure 2.
Ablation of the Cxxc1 gene in adult mice causes death.
Mice homozygous for the conditional Cxxc1 allele and carrying or lacking the Mx1-Cre transgene were injected with poly(I:C) at days 0, 2, and 4. (A) Indicated tissues were collected at day 4 or day 7 following the initiation of Cre induction. Genomic DNA was isolated and PCR analysis was performed to assess the relative abundance of the conditional Cxxc1 allele (Cxxc1-flox) and recombined Cxxc1 (ΔCxxc1) allele, as well as the presence of the Mx1-Cre transgene (Cre). (B) Bone marrow cells were collected 48 hours after a single poly(I:C) injection and cultured ex vivo for 2 days. Cellular extracts were analyzed for Cfp1 protein levels by western analysis. Actin levels were determined as a loading control. (C) Animals were observed for survival following initiation of Cre induction. (N = 12 for each genotype) (D) Light microscopy of femur, spleen, and liver collected 11 days after initiation of Cre induction. There is a marked depletion of hematopoietic cells in the bone marrow and reduction of red pulp in the spleen of mutant mice. The liver appears normal.
Figure 3.
Peripheral blood cells are depleted following ablation of the Cxxc1 gene.
Mice homozygous for the conditional Cxxc1 allele and carrying or lacking the Mx1-Cre transgene were injected with poly(I:C) at days 0, 2, and 4. Peripheral blood cell counts were determined at days 0, 5, 8 and 11 following the initiation of Cre induction for (A) neutrophils, (B) monocytes, (C and D) lymphocytes, (E) red blood cells (hematocrit), and (F) platelets. Open triangles, Cxxc1f/f Cre− control mice; filled triangles, Cxxc1f/f Cre+ mice. Asterisks denote a statistically significant reduction in cell numbers following ablation of the Cxxc1 gene (P<0.02). N = 3–7 for each of two independent experiments.
Figure 4.
Hematopoiesis failure following Cxxc1 gene ablation is intrinsic to bone marrow cells.
Bone marrow was harvested from mice homozygous for the conditional Cxxc1 allele and either carrying or lacking the Mx1-Cre transgene and used to transplant lethally-irradiated wild type recipient mice. Following a several month period for engraftment, transplanted mice were injected with poly(I:C) at days 0, 2, and 4, and peripheral blood cell counts were determined at the indicated days following initiation of Cre induction as described for Figure 3. Open triangles, Cxxc1f/f Cre− control mice; filled triangles, Cxxc1f/f Cre+ mice. Asterisks denote a statistically significant reduction in cell numbers following ablation of the Cxxc1 gene (p≤0.02). N = 5 for recipients of mutant bone marrow and N = 6 for recipients of control bone marrow for each of two independent experiments.
Figure 5.
Bone marrow exhibits decreased cellularity and increased apoptosis, but no change in global cytosine methylation, following ablation of the Cxxc1 gene.
(A) Following transplantation of control or mutant bone marrow (N = 2 and N = 3 recipients, respectively), poly(I:C) was injected on days 0, 2, and 4, and on day 9 following initiation of Cre induction total bone marrow cells were isolated and analyzed (P = 0.04). Similarly, control or mutant mice were induced with poly(I:C), and on day 8 following initiation of Cre induction, total bone marrow was analyzed; N = 11 controls and N = 16 mutant mice (P<0.0001). (B) Bone marrow cells were collected from mice of the indicated genotypes on day 8 after day 0, 2, and 4 poly(I:C) injections, and cells were analyzed for apoptosis by flow cytometry using Annexin V and PI staining (“in vivo BM”); N = 6 for Cre- and N = 5 for Cre+ for each of two independent experiments. Alternatively, after a poly(I:C) injection on day 0, bone marrow was collected on day 2 from mice of the indicated genotypes, cultured ex vivo, and then analyzed for apoptosis by Annexin V and PI staining at days 3, 4, and 5 following initiation of Cre induction (“in vitro cultured BM”); for each genotype, N = 4 for day 3 and N = 7 for days 4 and 5 (P≤0.0003) for each of two independent experiments. Asterisks denote statistically significant differences in cell number or frequency in total LDBMCs. (C) Genomic DNA was isolated from ex vivo cultured bone marrow cells as described in (B). Global genomic cytosine methylation levels were determined using a methyl-acceptance assay, as previously described [9]. Similar analysis was done using DNA isolated from wild type or Cfp1-null ES cells as controls. Asterisk denotes a statistically significant difference (P<0.001).
Figure 6.
Hematopoietic progenitor cell populations are sensitive to Cfp1 depletion.
Mutant and control mice were induced with poly(I:C) injections at days 0, 2, and 4. Tissues were collected on day 8 following initiation of Cre induction for all analyses. (A) Bone marrow and spleen cells were each collected and analyzed by colony forming assay for myeloid hematopoietic progenitors. For both tissues, N = 3 for Cre- controls and N = 8 for Cre+ mutants. (B–D) Bone marrow cells were collected, stained for the indicated cell surface markers, and analyzed by flow cytometry. (B) Scatter plot showing a representative experiment. (C) Summary for frequency per total LDBMCs (percent) of CMP (Lin- Sca1- Kit+ IL7Ra- FcgRII/III lo CD34+), GMP (Lin- Sca1- Kit+ IL7Ra- FcgRII/III hi CD34+), MEP (Lin- Sca1- Kit+ IL7Ra- FcgRII/III lo CD34-), and CLP (Lin- Sca1 lo Kit lo IL7Ra+). (D) Summary for number of stained cells per femur. (For C–D, N = 11 for Cre- controls and N = 16 for Cre+ mutants for three independent experiments.) Asterisks denote statistically significant differences in cell number or frequency (P≤0.03).
Figure 7.
Hematopoietic cells enriched for stem cells are tolerant of Cfp1 depletion.
Mutant and control mice were induced with poly(I:C) injections at days 0, 2, and 4. Bone marrow cells were collected at day 8 following initiation of Cre induction and analyzed by flow cytometry for the indicated cell surface markers. (A) Scatter plot showing a representative experiment. (B–C) Summary for enumeration of LSK (Lin- Sca1+ Kit+), LT-HSC (Lin- Sca1+ Kit+ CD34- Flt3-), ST-HSC (Lin- Sca1+ Kit+ CD34+ Flt3+), and more primitive ST-HSC (Lin- Sca1+ Kit+ CD34+ Flt3-). (A) and (B) present the data in terms of percent of total LDBMCs, while (C) presents cell number per femur. (For B–C, N = 11 for Cre- controls and N = 16 for Cre+ mutants for each of three independent experiments.) (D) Total RNA was isolated from purified LSK cells derived from the bone marrow of mice of the indicated genotypes (day 8 after poly(I:C) injections on days 0, 2, and 4), and semi-quantitative RT-PCR was performed to determine the level of Cxxc1 transcript. RNA from wild type or Cxxc1-null ES cells was used as positive and negative controls, respectively [9]. The conditions for all PCR reactions were confirmed to be within the linear range (data not shown). (E) Mutant and control mice were induced with poly(I:C) injections on days 0, 2, and 4, and BrdU was injected on day 7. On day 8 following initiation of Cre induction, bone marrow was isolated and analyzed for the frequency of BrdU incorporation into LSK cells and CD34/Flt3 subpopulations. Asterisks denote statistically significant differences (P≤0.02). (F) LSK cells were recovered from the bone marrow of mutant or control mice 8 days following Cre induction and examined for apoptosis by Annexin V and PI staining.
Figure 8.
The accumulation of LSK CD150+ cells following Cxxc1 deletion is distinct from transient interferon effects.
Mutant and control mice were induced with poly(I:C) injections on days 0, 2, and 4, and bone marrow was analyzed by flow cytometry for LSK-CD150+ frequency on days 4, 6, or 8 following initiation of Cre induction. N = 4 or 5 for each genotype and each time point. Asterisks denote statistically significant differences (P≤0.01).