Pyrenocyte clearance is impaired in Vav1 ^iCre Xpr1 ^fl/fl FLs. (A) Microscopic images of peripheral blood smears and quantification of RBC enucleation (mean ± SEM) from E15.5 Vav1^iCreXpr1^fl/fl and control littermate (Xpr1^fl/fl and Xpr1^fl/+) embryos stained with modified Giemsa stain. Scale bars are 40 µm. Red arrows point to nucleated RBCs. n = 7–8 per genotype pooled from two independent experiments. Statistical analysis was performed using the Student’s t test. (B) Analysis of peripheral blood parameters from Vav1^iCreXpr1^fl/fl and control littermates (Xpr1^fl/fl and Xpr1^fl/+) using a scil ABC plus blood counter (mean ± SD). Data shown are n = 8–11 pooled from three independent experiments. Statistical analysis was performed using the Student’s t test. (C) Immunohistochemistry of E15.5 Vav1^iCreXpr1^fl/fl and control littermate (Xpr1^fl/fl and Xpr1^fl/+) livers and corresponding quantification of pyrenocyte clusters. The overview image shows DAPI (white) and the insets show magnification of the outlined regions and expression of DAPI (white) and CD64 (green). Yellow arrows point to CD64⁺ cells containing pyrenocytes. n = 5 per genotype, pooled from three independent experiments. Scale bars are 500 µm (main images) and 20 µm (insets). Statistical analysis was performed using the Student’s t test. (D) UMAP plots and violin plots of scRNA-seq data (Fig. 3 B) show the expression of Dnase2a among the eight cell clusters. (E) Violin plots of scRNA-seq data (Fig. 3 B) showing expression of the cathepsin genes Ctsd, Ctsl, and Ctsb among the eight cell clusters. ****P < 0.0001. ns, not significant; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit; MCV, mean corpuscular volume; MCH, mean corpuscular hemoglobin; RDW, red cell distribution width.