Fig 2: Differentiation of splenic white pulp reticular cell populations. a Schematic depiction of the triple transgenic Ccl19-iEYFP mouse model. b Spleen cross-sections from adult Ccl19-iEYFP mice without doxycycline treatment analyzed by confocal microscopy after staining with the indicated antibodies (scale bar = 500 µm). Boxed areas in (b) indicate the splenic white pulp EYFP+ reticular cells analyzed by confocal microscopy after staining with the indicated antibodies in (c, d). Boxed areas in (c) show region of higher magnification in the images (Scale bar = 50 µm and 10 µm in the boxes). Boxed regions in (d) indicate marginal zone reticular cells (MRC), follicular dendritic cells (FDC), T-cell zone reticular cells (TRC), and perivascular reticular cells (PRC), shown at higher magnification in lower panels (scale bars = 5 µm/10 µm). e Spleen section from adult Ccl19-iEYFP Ltbrfl/fl mice without doxycycline treatment stained with the indicated antibodies and analyzed by confocal microscopy. Box indicates magnified areas in (f) (scale bar in (e) = 200 µm, scale bars in (f) = 20 µm). g Representative flow cytometric analysis of viable CD45-depleted cells from Ccl19-iEYFP and Ccl19-iEYFP Ltbrfl/fl spleens without doxycycline treatment, gated on CD45-TER119-CD31-EYFP+ cells and analyzed for the indicated markers. Values indicate percentage of the respective population. h Percentage of EYFP+ cells from spleens of the indicated mouse strains, based on gating in (g) (n = 7–8 mice per group from 2 to 3 independent experiments, mean ± SEM). Statistical analysis was performed using Mann–Whitney test for (h). Microscopy data are representative for two or more independent experiments (n = 6 mice). Source data are provided as Source Data file

Fig 3: EcsitPrx1 mice show defects in skeletogenesis, fracture healing, and the bone marrow niche.a Immunostaining of ECSIT in 2-month-old wild-type femurs. tb: trabecular bone, cb: cortical bone. b Immunofluorescence showing the localization of ECSIT in P21 mouse femurs. Arrows indicate co-localization of ECSIT and NDUFS3. c. Immunoblot analysis showing mitochondrial fractionation of ECSIT in human BMSCs. d, e X-radiography (d, left) and microCT showing P21 whole body and hindlimbs (d, middle) and quantification of femoral bone mass (e, n = 5). Alizarin red/alcian blue staining of P1 neonates (d, right). Arrows indicate spontaneous fractures. Tra. BV/TV: Trabecular bone volume/total volume. f H&E staining of E16.5 and P0 femurs (left, middle). Immunofluorescence showing osteocalcin (OCN) expression in the femurs (right). Arrows indicate femoral trabecular bone. g Skeletal progenitors isolated by FACS from P0 Ecsit;Prx1Rosa26mTmG (cKO) and Prx1;Rosa26mTmG (WT) limbs using GFP+CD45−Ter119−Tie2− markers. Osteogenic gene expression (n = 4) and alkaline phosphatase activity (ALP, n = 3) were assessed for osteogenic differentiation. h Flow cytometry analysis showing frequency of the indicated bone marrow cell populations at P10 (right, HSCs (Ecsitfl/fl, n = 7; EcsitPrx1, n = 5); other subpopulations, n = 4−5) and total number of the isolated cells (left, Ecsitfl/fl, n = 7; EcsitPrx1, n = 6). Supplemental Fig. 2 demonstrates the gating strategy for this analysis. i GFP-expressing PRX1-lineage cells residing in the diaphyseal periosteum of P1 PRX1-cre;Rosa26mTmG mice. m: muscle, bm: bone marrow. j P21 femoral cortical bones were stained for Von Kossa (top), H&E (bottom) and immunofluorescence for OCN (middle). k H&E staining of EcsitPrx1 femurs at the age of P0, P10, and P21. l, m Flow cytometry analysis showing frequency of SSCs, pre-BCSP, and BCSP in skeletal cells (l, n = 5). RT-PCR shows expression of Ecsit in pre-BCSP (m, n = 3). Supplemental Fig. 3 demonstrates the gating strategy for this analysis. A two-tailed unpaired Student’s t-test for comparing two groups (e, g, h, l, m; error bars, data represent mean ± SD). Data are representative of three independent experiments. Scale bars = a, 100 μm; b, 50 μm; d (left, middle), 2 mm; d (right), 1 mm; f, 60 μm; i, 75 μm; j, 40 μm; k, 500 μm.

Fig 4: Nemp1 KO mice display splenomegaly, blood defects, and erythroid lineage expansion. Representative spleen size (A) and spleen/body weight ratios (B) across genotypes. Scale bar: 1 cm. (C) H&E staining of WT and Nemp1 KO spleen. (D) Wright-Giemsa staining of WT and Nemp1 KO blood smears. Red arrows point to cytoplasmic membrane spikes of Nemp1 KO RBCs. (E) Hemavet measurement of RBC, Hb content, RDW, MCH, and MCV of Nemp1 KO and WT blood from P4 to P150. N: number of biological replicates for each time point and genotype. Data are shown as mean ± SD. Student t test. ns: not significant, *p < 0.05.**p < 0.01. ***p < 0.001. ****p < 0.0001. (F) Percentage of immature nucleated erythroblasts in PB of indicated genotypes. (G) H&E staining and quantification of cellularity of WT and Nemp1 KO BM. (H, I) Percentage of Ter119+ (H) and Ter119+/CD71+ (I) cells in WT and Nemp1 KO BM assessed by FACS. (J) Percentage of nucleated Ter119+/Hoechst+ cells in WT and Nemp1 KO BM measured by flow imaging. (K, L) Percentage of Ter119+ (H) and Ter119+/CD71+ (I) cells in WT and Nemp1 KO spleen assessed by FACS. (M) Percentage of nucleated Ter119+/Hoechst+ cells in WT and Nemp1 KO spleen measured by flow imaging. Data are shown as mean ± SD. Student t test. ns: not significant, *p < 0.05. **p < 0.01. ***p < 0.001. ****p < 0.0001. Data underlying the graphs shown can be found in S1 Data. Hb, hemoglobin; KO, knockout; MCH, mean corpuscular hemoglobin; MCV, mean corpuscular volume; PB, peripheral blood; RBC, red blood cell; RDW, red cell distribution width; WT, wild type.

Fig 5: Nemp1 supports erythroblasts enucleation.(A) Immunolocalization of Nemp1 at different stages of enucleation. Scale bars: 5 μm. (B) DNA and Ter119 FACS analysis of lineage-negative cells purified from WT and KO BM and subsequently cultured in erythropoietin-containing medium for 2 days. Note the higher nucleated/enucleated ratio in Nemp1 KO cells. Duplicate experiments are shown for WT and KO BM. (C) Quantification of nucleated and enucleated Ter119+ cells in WT and Nemp1 KO BM measured by flow imaging. Right: representative flow imaging plots of nucleated (Hoechst+Ter119+) and enucleated (Hoechst-Ter119+) in WT and Nemp1 KO BM in flow imaging. (D) Graphical depiction of Nemp1 phenotypes and functions during erythropoiesis. Green and red arrows respectively depict increased or decreased populations in Nemp1 KO by comparison to WT cells or biological processes affected by the lack of expression of NEMP1. Blue arrows depict increased apoptosis. See text for details. Data underlying the graphs shown can be found in S4 Data. BM, bone marrow; KO, knockout; WT, wild type.