Fig. 1: Csf1r^iCre-mediated deletion of Wnk1 results in rapid mortality, MΦ deficiency, and neutrophilia.

a Csf1r^iCre-mediated deletion of WNK1 results in early mortality. Survival curve of Csf1r^Cre+; Wnk1^fl/fl (Cre + , magenta) vs. Csf1^Cre–; Wnk1^fl/fl (Cre-, blue) mice. Litters were monitored from birth and sacrificed humanely when failure to thrive criteria were met. Data are from n = 74 Cre- mice and n = 48 Cre+ mice. Differences in survival were determined using the Mantel-Cox test. ****p < .0001. b Analysis of gross morphology reveals severe underdevelopment. Representative images of three- to four-week-old Cre- and Cre+ mice body (top) and teeth (bottom). Images are representative of n = 74 Cre− mice and n = 48 Cre+ mice. (Right) Representative images of organs from Cre− to Cre+ mice. Shown are (clockwise from top left) brain, spleen, colon, kidneys, bones, liver, heart, and lungs. Images are representative of organ analysis from six Cre− mice and six Cre+ mice. See Supplementary Fig. 1 for additional gross morphology and organ size analysis. c Flow cytometric analysis of macrophage subsets in major organs. Flow cytometry analysis of CD45+ tissue-resident macrophages in Cre+ (n = 6, magenta dots) and Cre− (n = 6, blue dots) 3-to-4-week-old mice. Subsets were first gated on live CD45+ CD11c− CD11b+ (except for lung macrophages; see Supplementary Fig. 2a for flow cytometry gating strategy). Tissue-resident macrophages were gated as follows: alveolar macrophage (CD11c^high CD11b^low SiglecF+), Kupffer cells (Ly6C− Ly6G− CLEC4F+ F4/80+), splenic red pulp macrophages (Ly6C− Ly6G− CD163+ F4/80+), and kidney macrophages (Ly6C− Ly6G− CX₃CR1+ F4/80+). Images (top) and summary plots (bottom) of absolute numbers per milligram of tissue are from four independent experiments. Data are shown as mean ± SEM. Statistical significance was determined via two-tailed independent samples t-test. *p < .05, **p < .01. See also Supplementary Fig. 2b, c for analysis of additional tissues and Supplementary Fig. 3 for immunofluorescence (IF) analysis. d Flow cytometric analysis of neutrophilia in major organs. Flow cytometry analysis of neutrophils (Ly6G+) in major tissues from Cre+ (n = 6, magenta dots) and Cre− (n = 6, blue dots) 3-to-4-week-old mice (see Supplementary Fig. 2a for flow cytometry gating strategy). Shown are summary plots of absolute numbers per milligram of tissue from four independent experiments. Statistical significance was determined via two-tailed independent samples t-test. *p < .05, **p < .01, ***p < .001. See also Supplementary Fig. 2e–h for analysis of additional tissues, Supplementary Fig. 3 for IF analysis, and Supplementary Fig. 4 for morphological and functional analysis of Cre− and Cre+ neutrophils. e, f Flow cytometric analysis of embryonic macrophages and neutrophils in E9.5 embryos. Eight pregnant dams were euthanized at E9.5, and embryos were isolated, genotyped, and analyzed. Shown are summary plots of the absolute counts from flow cytometry analysis of CD11b^Low F4/80+ macrophages (e) and CD11b^High Ly6C+ Ly6G+ fetal neutrophils (f) in Cre− (n = 18, blue dots) and Cre+ (n = 8, magenta dots) E9.5 embryos. Yolk sac (left), head (middle), and trunk (right) were separated to delineate yolk sac-derived and brain-derived macrophages from tissue-resident macrophages in other organs. Pooled data are from eight independent experiments with between two and six biological replicates per experiment. Data are shown as mean ± SEM. Statistical significance was determined via two-tailed independent samples t-test. **p < .01, ****p < .0001, ns = not significant. See also Supplementary Figs. 2a and 6a, b for flow cytometry gating strategy and Supplementary Fig. 6c–h for analysis of E9.5 erythromyeloid progenitors and monocytes. Source data are provided in a Source Data file included with this manuscript.