Cdan^ΔEry embryos die in-utero between E12.5 and E13.5 from severe anemia. (A) Cdan^ΔEry embryos were extracted and photographed at different dpc. From E10.5 and on, Cdan^ΔEry embryos and Yolk Sacs (right column) are very pale with no visible vasculature or Fetal Liver. (B) Benzidine staining of E10.5 Cdan^ΔEry embryo (right) Vs. littermate control (left). Mutant embryo and Yolk sac stained very lightly and are notably smaller.

Erythroid-specific knockout of Cdan1 recapitulates the CDA-I phenotype. (A) Morphology of Cdan^ΔEry circulating erythroblasts, compared to littermate controls. Cytospins were prepared from blood samples collected from E9.5 to E11.5 yolk sacs and embryos. The arrows mark primitive enucleated cells. Scale bar = 10 μm. (B) Cdan^ΔEry erythroblasts show aberrant morphology including binuclear cells (arrowhead) and basophilic stippling (asterisk). Scale bar = 10 μm.

Cdan^ΔEry erythroblasts display the pathognomonic spongy heterochromatin of CDA type I. (A) TEM of E9.5 (upper panel) and E11.5 (lower panel) Cdan^ΔEry erythroblasts compared to littermate controls. Magnification: X6000-9900. Scale bar: 1–2 μm. (B) High power magnification (X20,000-43,000) of E9.5 erythroblasts in A, showing the normal appearance of heterochromatin and the intact nuclear membrane in control erythroblast (left), compared to Cdan^ΔEry erythroblast with spongy heterochromatin, dilated membrane pore (arrow) and ribosomes (R) inside the nucleus. Scale bar: 500 nm.

Cdan1 depletion impairs cell cycle progression and increases cell death. (A) Number of circulating primitive erythroid cells collected from E9.5 to E11.5 control and CdanΔEry embryos. Values represent mean ± Standard error. CdanΔEry: n = 3; Control: n = 9. *p < 0.002 compared to controls. (B) CdanΔEry E9.5 erythroblasts show aberrant G1-to-S progression. The percentage of cells in G0/G1, S and G2/M phases of the cell cycle in wild-type (control) and CdanΔEry E9.5 erythroblasts gated for the CD71+/Ter119+ population. CdanΔEry n = 3; control n = 12. Error bars indicate standard error. *p < 0.02 compared to wild-type. (C) Morphology of E12.5 CdanΔEry circulating erythroblasts, compared to littermate controls. (D) E10.5 peripheral blood cells were gated for CD71+/Ter119+ erythroblasts, and analyzed for AnnexinV/7AAD staining to assess for apoptosis (Representative flow plots are shown in upper panel). Early apoptotic cells are AnnexinV-positive/7-AAD-negative; Late apoptotic cells are double positive. Data presented as mean ± SEM (n = 3-4 for each genotype). *p < 0.005. (E) Cdan1 ablation decreases Bcl-xl expression and increases p53 levels. qRT-PCR was performed with RNA extracted from peripheral blood cells at E11.5 and normalized to Rplp0. Relative expression data are presented as mean ± SEM (n = 4–6 for each genotype). Student’s t-test was applied for statistical analysis. Asterisks indicate significant difference from the Control. *p < 0.05.

Cdan1 is required for primitive erythroid differentiation. (A) Cdan^ΔEry erythroblasts do not demonstrate the semi-synchronous maturation characteristic of primitive erythroblasts. Quantification of erythroid sub-populations represented as mean ± SEM. Sub-population were grouped by CD71/Ter119 surface expression according to the following: I – Ter119loCD71+; II – Ter119⁺CD71⁺; III – Ter119+CD71lo. (B) Representative FACS profile of Cdan^ΔEry peripheral blood erythroblasts compared to littermate controls.

Dysregulated embryonal globin expression in Cdan^ΔEry erythroblasts. qRT-PCR was performed with peripheral blood at E9.5 and E11.5, and normalized to Rplp0. The relative expression of embryonic β-like globins (A) and embryonic α-like globin (B) are presented as mean ± SEM (n = 4–6 for each genotype). The Student’s t-test was applied for statistical analysis. Asterisks indicate significant difference from the Control. *p < 0.01; **p < 0.001. (C) α-globin switch is delayed in Cdan^ΔEry erythroblasts. Expression of ζ and α was determined by RT-qPCR. The data are displayed as the fraction of total α-like globin (mζ+mα) expression. Embryonic day is indicated. (D) Expression of εy, and βh1 was determined by RT-qPCR. The data are displayed as the fraction of total embryonal β-like globin (mεy+mβh1) expression.

Cdan1 ablation disrupts expression of master erythroid transcription factors. qRT-PCR was performed with RNA extracted from peripheral blood cells at E9.5 and E11.5, and normalized to Rplp0. Relative expression data are presented as mean ± SEM (n = 4–7 for each genotype). Student’s t-test was applied for statistical analysis. Asterisks indicate significant difference from the control. ^∗p < 0.01 (A) Relative expression in E9.5 Cdan^ΔEry circulating erythroblasts compared to littermate controls. (B) Relative expression in E11.5 Cdan^ΔEry circulating erythroblasts compared to littermate controls.

Loss of cdan1 impairs erythropoiesis in zebrafish embryos. (A) Expression of zebrafish cdan1 at 1 dpf (top) and 2 dpf (bottom). The arrows indicate the expression of cdna1 in the embryonic hematopoietic site. (B) Top, schematic diagram of zebrafish cdan1 gene organization. The binding sites of two morpholinos (MO) are highlighted by red and orange lines. Note zebrafish cdan1 gene contains 28 exons, however, only the first 5 exons are illustrated. Bottom, relative cdan1 expression in wild type (WT) and two cdan1 morphants illustrating that the expression level of cdan1 was strongly reduced upon MO1 or MO2 injection. N indicates the number of biological replicates. (C) Expression pattern of hemoglobin alpha embryonic 1.1. (hbae1.1.) in WT and cdan1 morphants at 1 dpf. The left panel depicts representative images of the hematopoietic tissue. The right depicts the frequency of embryos that show phenotype. Note that injection of control (Ctrl) MO showed a minor effect on hbae1.1. expression. (D)o-dianisidine staining in WT and cdan1 morphants. The left panel depicts representative images of stained embryos 2 dpf. The right panel depicts the frequency of embryos showing phenotype. (E–G) Expression pattern of gata1(E), gata2a(F), and gata2b(G) in the WT and cdan1 morphants. The left panel depicts representative images of the hematopoietic tissue. The right panel depicts the frequency of embryos that show phenotype. N indicates the number of biological samples.