Fig. 5: NGF from LepR⁺ cells and adipocytes promotes nerve sprouting after irradiation, increasing the expression of regeneration factors.

a, NGF in bone marrow serum from 6–8-month-old Lepr^cre/+; Ngf^fl/∆ and Ngf^fl/∆ littermate controls before (n = 3 mice per genotype), or 14 (n = 4) or 28 (n = 4) days after irradiation and transplantation of radioprotective wild-type bone marrow cells (three to four independent experiments per timepoint). D, day. b, Perilipin⁺ adipocytes in a 30-µm-thick section from Ngf^mScarlet/+ femur bone marrow were positive for Ngf–mScarlet (representative of three experiments). There are also Ngf-expressing LepR⁺ cells in this image (Extended Data Fig. 6a). c, Flow cytometric analysis of enzymatically dissociated bone marrow from Ngf^mScarlet/+ mice 14 days after irradiation (four mice from four independent experiments). d, Ngf expression by qRT–PCR (three mice (WBM or LepR⁺ cells) or five mice (adipocytes) from three independent experiments). e, The area occupied by peripherin⁺ nerve fibres in bone marrow sections from 6–8-month-old Lepr^cre/+; Ngf^fl/∆ and littermate control mice (five mice per genotype per timepoint from five independent experiments). f,g, Representative images (f) and quantification (g) showing that nerve fibres (red) in femur bone marrow sections from 6–8-month-old Wnt1–Cre; Rosa26^tdTomato mice before or at 14 or 28 days after irradiation (five mice per timepoint from five independent experiments). h–j, SCF (h), VEGF (i) and Ang2 (j) in bone marrow serum from 6–8-month-old Lepr^cre/+; Ngf^fl/∆ and littermate control mice (eight mice per genotype per timepoint from eight independent experiments). k–n, The β2 agonist salbutamol (Salb.) rescued the regeneration of bone marrow cellularity (n = 6) (k) and the numbers of HSCs (n = 6 mice per treatment) (l) and LSK cells (n = 6) (m) as well as the patency of the vasculature (n = 5) (n) in 6–8-month-old Lepr^cre/+; Ngf^fl/∆ mice at 28 days after irradiation (five to six independent experiments). o, Western blot of protein from LepR⁺ cells isolated from Lepr^cre/+; Ngf^fl/∆ and littermate control mice at 14 days after irradiation and transplantation (representative of three independent experiments). All data represent mean ± standard deviation. Statistical significance was assessed using two-way ANOVAs followed by Tukey’s (a and h) or Šidák’s (k–n) multiple comparisons adjustments, Mann–Whitney (e) or Student’s t-tests (i and j) followed by Holm–Šidák’s multiple comparisons adjustments for comparisons between mutants and controls, or one-way ANOVAs (e, g, i and j) followed by Šidák’s multiple comparisons adjustments for comparisons between timepoints. All statistical tests were two-sided. Not significant (NS): P > 0.05.

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