Fig. 1: Severe skeletal defects in Sp7^OcyKO mice.

a Sp7 immunohistochemistry was performed on tibiae from 8-week-old control (Dmp1-Cre; Sp7^+/+) and Sp7^OcyKO (Dmp1-Cre; Sp7^f/f) mice, quantification of Sp7-positive osteocytes is shown in e. b Cross-sectional µ-CT images from the femoral midshaft diaphysis reveal increased cortical porosity and reduced mineralization in Sp7^OcyKO mice, quantified in f and g. c 8-week-old mice were labeled with calcein (green) and demeclocycline (red) 7 and 2 days prior to sacrifice, respectively. Non-decalcified sections from the cortical bone in the tibia were analyzed. Control mice show orderly endosteal bone formation. In contrast, Sp7^OcyKO animals show abnormal intracortical bone formation. d TRAP-stained (red) paraffin-embedded sections from the tibia show an increased number of intracortical osteoclasts Sp7^OcyKO in mice. h Cortical bone RANKL expression was measured by RT-qPCR. i Cryosections from 8-week-old control (Dmp1-Cre; Sp7^+/+) and Sp7^OcyKO (Dmp1-Cre; Sp7^f/f) tibia were stained with phalloidin to visualize actin filaments, m shows quantification indicating reduced filament density (percent of acellular bone matrix occupied by phalloidin-positive filaments) in Sp7^OcyKO mice. j In vivo third-generation harmonic imaging of the skull was performed to visualize osteocyte cell bodies and canaliculi in the skull. Quantification of defects in canaliculi/surface area and osteocyte–osteocyte connection is shown in n–o. k–l Apoptosis in situ was analyzed on tibia sections by TUNEL staining and activated caspase-3 immunohistochemistry; both methods demonstrate increased osteocyte apoptosis in Sp7^OcyKO mice versus controls. p, u show the quantification. q Hematoxylin and eosin-stained paraffin-embedded sections from the tibia show abnormal cortical porosity and empty osteocyte lacunae, quantified in s–t. r, v TUNEL staining (green) was performed on control (Dmp1-Cre; Sp7^+/+, Ai14) and Sp7^OcyKO (Dmp1-Cre; Sp7^f/f, Ai14) mice. Thereafter, osteocyte filaments were visualized by tdTomato fluorescence within dendritic projections. Dendrite number was counted in TUNEL-positive (rare in control mice) and TUNEL-negative osteocytes. Reduced dendrite numbers are noted in non-apoptotic (TUNEL-negative) osteocytes lacking Sp7. *p < 0.05, **p < 0.01. ***p < 0.001, ****p < 0.0001. In graphs in e–h, m–p, and s–v, each data point represents a biologically independent animal. Data are presented as mean values ±SEM. Two-sided student’s t tests were used without adjustment for multiple comparisons for all panels except u where two-way ANOVA was performed followed by Tukey’s multiple comparisons test. Exact p values for comparisons are as follows: e <0.0001, f <0.0001, g <0.0001, h 0.0384, m 0.0015, p 0.0118, s 0.0055, u 0.0014, v 0.0002. Formal statistical analysis was not performed for n, o where two mice per genotype were analyzed.