Leptin receptor+ cells promote bone marrow innervation and regeneration by synthesizing nerve growth factor

The bone marrow contains peripheral nerves that promote haematopoietic regeneration after irradiation or chemotherapy (myeloablation), but little is known about how this is regulated. Here we found that nerve growth factor (NGF) produced by leptin receptor-expressing (LepR+) stromal cells is required to maintain nerve fibres in adult bone marrow. In nerveless bone marrow, steady-state haematopoiesis was normal but haematopoietic and vascular regeneration were impaired after myeloablation. LepR+ cells, and the adipocytes they gave rise to, increased NGF production after myeloablation, promoting nerve sprouting in the bone marrow and haematopoietic and vascular regeneration. Nerves promoted regeneration by activating β2 and β3 adrenergic receptor signalling in LepR+ cells, and potentially in adipocytes, increasing their production of multiple haematopoietic and vascular regeneration growth factors. Peripheral nerves and LepR+ cells thus promote bone marrow regeneration through a reciprocal relationship in which LepR+ cells sustain nerves by synthesizing NGF and nerves increase regeneration by promoting the production of growth factors by LepR+ cells.

The bone marrow contains peripheral nerves that promote haematopoietic regeneration after irradiation or chemotherapy (myeloablation), but little is known about how this is regulated.Here we found that nerve growth factor (NGF) produced by leptin receptor-expressing (LepR + ) stromal cells is required to maintain nerve fibres in adult bone marrow.In nerveless bone marrow, steady-state haematopoiesis was normal but haematopoietic and vascular regeneration were impaired after myeloablation.LepR + cells, and the adipocytes they gave rise to, increased NGF production after myeloablation, promoting nerve sprouting in the bone marrow and haematopoietic and vascular regeneration.Nerves promoted regeneration by activating β2 and β3 adrenergic receptor signalling in LepR + cells, and potentially in adipocytes, increasing their production of multiple haematopoietic and vascular regeneration growth factors.Peripheral nerves and LepR + cells thus promote bone marrow regeneration through a reciprocal relationship in which LepR + cells sustain nerves by synthesizing NGF and nerves increase regeneration by promoting the production of growth factors by LepR + cells.
To identify the location of Ngf-expressing cells in adult bone marrow, we generated an Ngf-mScarlet (Ngf mScarlet ) knock-in reporter allele (Extended Data Fig. 1a-c).Confocal imaging 40 of cleared femurs from adult Ngf mScarlet/+ mice showed that Ngf-mScarlet was expressed by stromal cells surrounding Endomucin low arterioles as well as Endomucin high sinusoids (Fig. 1g).While the peri-arteriolar staining appeared more prominent, the abundance of sinusoids throughout the bone marrow meant that most of the Ngf-mScarlet staining was peri-sinusoidal.The peri-arteriolar staining probably reflected Ngf-mScarlet expression by both peri-arteriolar LepR + Osteolectin + cells 36 as well as SMA + NG2 + smooth muscle cells (Fig. 1e).

NGF from LepR + cells is required for bone marrow innervation
To test if NGF is required for bone marrow innervation we generated mice with a floxed Ngf allele (Extended Data Fig. 3a-c).We conditionally deleted Ngf in LepR + cells using Lepr cre , in smooth muscle cells using NG2-creER, in osteoblasts using Col1a1-creER, and in Schwann cells using GFAP-cre.Deletion from smooth muscle cells, osteoblasts or Schwann cells had no significant effect on bone marrow NGF levels (Fig. 2a) or the number of nerve fibres in adult bone marrow (Fig. 2b and Extended Data Fig. 3d-g).Therefore, smooth muscle cells, osteoblasts and Schwann cells were not significant sources of NGF for nerve maintenance in bone marrow.
Adult Lepr cre/+ ; Ngf fl/∆ mice were born in expected numbers and did not differ from littermate controls in terms of gross appearance (Extended Data Fig. 4a), body length (Extended Data Fig. 4b) or body mass (Extended Data Fig. 4c).LepR + cells from Lepr cre/+ ; Ngf fl/∆ mice had Ngf transcript levels that were approximately 30% of control levels at 2 months of age and less than 10% of control levels at 6 months of age (Fig. 2e).Deletion of Ngf from LepR + cells profoundly depleted NGF from the bone marrow by 6 months of age (Fig. 2a).circadian mobilization of haematopoietic stem/progenitor cells into the blood 6,8,10,11 and the regeneration of haematopoiesis after myeloablation by irradiation or chemotherapy 4,7,12,13 .Nerve fibres promote haematopoietic regeneration and changes in haematopoiesis during ageing by activating β adrenergic receptors 4,14,15 , though the mechanism by which β adrenergic receptors promote haematopoietic regeneration, and the cells in which they act, are unknown.
LepR + cells also include skeletal stem and progenitor cells that form the adipocytes and osteoblasts that arise in adult bone marrow [33][34][35] .The osteoblasts formed by LepR + cells contribute to the maintenance and repair of the adult skeleton 33,34,36 and secrete factors that promote osteogenesis 22,23 .The adipocytes that arise from LepR + cells in adult bone marrow promote the regeneration of HSCs and haematopoiesis after myeloablation by synthesizing SCF 35 .LepR + cells and adipocytes also promote HSC maintenance and quiescence by secreting adiponectin, which suppresses inflammation 37 .In this article, bone marrow nerve fibers were found to be maintained by NGF synthesized by LepR + cells and, in turn, nerves promote haematopoietic and vascular regeneration by secreting adrenergic neurotransmitters that activate β2/β3 adrenergic receptor signaling in LepR + cells.

Nerve growth factor is mainly synthesized by LepR + cells
Peripheral nerves require neurotrophic factors for their maintenance 38 , but the source of such factors in the bone marrow is unknown.Analysis of published microarray data 16 (National Center for Biotechnology Information (NCBI) accession number GSE33158) suggested that nerve growth factor (Ngf) was the only neurotrophic factor detected in adult bone marrow (Fig. 1a).Ngf expression was detected in Scf-GFP + CD45 − Ter119 − CD31 − stromal cells, nearly all of which are LepR + (ref.33), but little or no Ngf was detected in osteoblasts, endothelial cells or unfractionated whole bone marrow (WBM) cells (Fig. 1a).Similar results were obtained by RNA sequencing 22 (NCBI accession number PRJNA914703), which detected Ngf in PDGFRα + CD45 − Ter119 − CD31 − stromal cells, nearly all of which are LepR + (ref.33), but not in endothelial cells or WBM cells (Fig. 1b).
Single-cell RNA sequencing of enzymatically dissociated cells from the femurs and tibias of 8-week-old mice showed that most Ngf-expressing cells in adult bone marrow were LepR + cells (Fig. 1c,d; NCBI accession number PRJNA835050) 39 .Ngf was also expressed by a much smaller number of SMA + NG2 + smooth muscle cells, and by rare  16 (a) and RNA sequencing 22 (b) in bone marrow stromal cells (isolated on the basis of expression of Scf-GFP (a) or PDGFRα (b) staining, both of which are nearly completely overlapping with LepR expression 33 ), VE-cadherin + bone marrow endothelial cells, Col2.3-GFP + CD45 − Ter119 − CD31 − osteoblasts, and WBM cells (three mice in a and two mice in b, from three or two independent experiments, respectively).c, Uniform manifold approximation and projection (UMAP) plot showing clustering of single-cell RNA sequencing analysis of 4,209 non-haematopoietic cells from enzymatically dissociated bones/bone marrow in 8-week-old mice 39 .d, Ngf is mainly expressed by Lepr + stromal cells (cell cluster 11 in c) and smooth muscle cells (cell cluster 12).e, Ngf expression by all cell clusters shown in c (cells were obtained from four mice and analysed in three independent experiments).f, Ngf expression by qRT-PCR in LepR + CD45 − Ter119 − CD31 − stromal cells, NG2-DsRed + smooth muscle cells, Col1a1-GFP + osteoblasts, VE-cadherin + endothelial cells and unfractionated cells from the bone marrow of 2-monthold mice (three mice from three independent experiments).g, Deep imaging of femur bone marrow from adult Ngf mScarlet/+ mouse: the Ngf-mScarlet + cells were found around endomucin high sinusoids (arrowhead) as well as around endomucin low arterioles (arrow; the images are representative of five mice).h,i, Flow cytometric analysis of enzymatically dissociated bone marrow from Ngf mScarlet/+ mice: 89% of Ngf-mScarlet + cells were LepR + , and most LepR + cells were Ngf-mScarlet + (four mice from four independent experiments).SSC-A, side scatter area.All data represent mean ± standard deviation.

Article
https://doi.org/10.1038/s41556-023-01284-9 No bone marrow innervation defect was apparent in Lepr cre/+ ; Ngf fl/∆ mice during development as the number of nerve fibres in the bone marrow was normal in 2-month-old Lepr cre/+ ; Ngf fl/∆ mice (Fig. 2d).However, by 6 months of age, when recombination in LepR + cells was nearly complete, we observed virtually no nerve fibres in the bone marrow of Lepr cre/+ ; Ngf fl/∆ mice (Fig. 2b-d).Peripheral nerves appeared to be present in normal numbers in the quadriceps of 6-month-old Lepr cre/+ ; Ngf fl/∆ mice (Extended Data Fig. 4d-f).Nerve fibres thus grew into the bone marrow normally during development in Lepr cre/+ ; Ngf fl/∆ mice but became depleted within the bone marrow, but not outside of the bone marrow, by 6 months of age, when NGF was depleted to less than 10% of control levels in the bone marrow.
Consistent with prior studies 4,6,11 , loss of nerve fibres from the bone marrow did not have any gross effect on steady-state haematopoiesis.Six-month-old Lepr cre/+ ; Ngf fl/∆ mice did not differ from littermate controls in terms of bone marrow or spleen cellularity (Fig. 2f), or the frequencies of HSCs, multipotent haematopoietic progenitors (MPPs), granulocyte-macrophage progenitors (GMPs), megakaryocyte-erythroid progenitors (MEPs), common myeloid progenitors (CMPs) or common lymphoid progenitors (CLPs) in the bone marrow (Fig. 2g).There were also no differences in blood cell counts (Extended Data Fig. 4g-i) or in the frequencies of B220 +  B cells, CD3 + T cells, Gr1 + Mac1 + myeloid cells, CD41 + megakaryocyte lineage cells or CD71 + /Ter119 + erythroid lineage cells in the bone marrow or spleen (Extended Data Fig. 4j-n).Finally, WBM cells from 6-month-old Lepr cre/+ ; Ngf fl/∆ mice and littermate controls did not differ in their capacity to reconstitute myeloid, B or T cells upon competitive transplantation into irradiated mice (Extended Data Fig. 4o-r).Bone marrow nerve fibres thus appear to be dispensable for normal adult haematopoiesis.

Bone marrow innervation promotes haematopoietic regeneration
To test for haematopoietic regeneration defects, we lethally irradiated (1,080 rads) and transplanted a radioprotective dose of 1,000,000 WBM cells into 6-month-old Lepr Cre/+ ; Ngf fl/∆ mice and littermate controls.While all control mice survived, 41% (9 of 22) of Lepr Cre/+ ; Ngf fl/∆ mice died between 10 and 18 days after irradiation, consistent with haematopoietic failure (Fig. 3a).The Lepr Cre/+ ; Ngf fl/∆ mice exhibited significantly lower white blood cell (WBC, Fig. 3b), red blood cell (RBC, Fig. 3c) and platelet (PLT) counts (Fig. 3d) as well as bone marrow cellularity (Fig. 3e) and LSK cell numbers (Fig. 3f) at 14 and 28 days after irradiation.At 28 days after irradiation, HSC numbers were much lower in the bone marrow of Lepr Cre/+ ; Ngf fl/∆ as compared with littermate control mice (Fig. 3g).We thus observed broad reductions in blood and bone marrow cell counts as well as the numbers of haematopoietic stem and progenitor cells in Lepr Cre/+ ; Ngf fl/∆ mice at 14-28 days after irradiation.
We also assessed vascular and stromal cell regeneration in Lepr Cre/+ ; Ngf fl/∆ and littermate control mice.At 10 days after lethal irradiation and transplantation, we observed significantly reduced numbers of bone marrow cells (Fig. 3h), LSK cells (Fig. 3i), LepR + stromal cells (Fig. 3j) and endothelial cells (Fig. 3k), as well as increased vascular leakage (Fig. 3l) in the bone marrow of Lepr Cre/+ ; Ngf fl/∆ mice as compared with littermate controls.At 28 days after irradiation, blood vessels were patent in control mice but remained leaky (Fig. 3m) and morphologically abnormal (Fig. 3n) in Lepr Cre/+ ; Ngf fl/∆ mice.Lepr Cre/+ ; Ngf fl/∆ mice had significantly less proliferation by LepR + cells at 14 days after irradiation (Fig. 3o) and by endothelial cells at 14 and 28 days after irradiation (Fig. 3p).In this experiment we observed trends towards reduced numbers of LepR + cells and endothelial cells at 14 days after irradiation and significant reductions in the numbers of these stromal cells at 28 days after irradiation (Fig. 3q,r).The loss of nerve fibres from the bone marrow in Lepr Cre/+ ; Ngf fl/∆ mice was thus associated with broad defects in the regeneration of haematopoietic, stromal and vascular cells at 10-28 days after irradiation.
To test if defects in haematopoietic regeneration were also evident after sublethal irradiation, we administered 650 rads to 6-month-old Lepr Cre/+ ; Ngf fl/∆ and littermate control mice.The Lepr Cre/+ ; Ngf fl/∆ mice exhibited significantly reduced survival from 12 to 15 days after irradiation (Extended Data Fig. 5a) as well as reduced bone marrow cellularity (Extended Data Fig. 5b), HSC numbers (Extended Data Fig. 5c) and LSK cell numbers (Extended Data Fig. 5d) as compared with littermate controls at 28 days after irradiation.
To test if defects in haematopoietic, vascular and stromal cell regeneration were evident after chemotherapy, we treated Lepr Cre/+ ; Ngf fl/∆ and littermate control mice with 5-fluorouracil (5-FU).The Lepr Cre/+ ; Ngf fl/∆ mice exhibited significantly reduced survival from 12 to 15 days after irradiation (Fig. 3s) as well as reduced numbers of bone marrow cells (Fig. 3t), HSCs (Fig. 3u) and LSK cells (Fig. 3v) as compared with littermate controls at 12 days after 5-FU treatment.Impaired haematopoietic regeneration was thus observed in Lepr Cre/+ ; Ngf fl/∆ mice irrespective of whether myeloablation was induced by 5-FU treatment, sublethal irradiation or lethal radiation.

NGF acts locally to promote nerve maintenance in bone marrow
Neurotrophic factors act locally to promote the survival of innervating neurons 41,42 .To test if NGF acts locally within the bone marrow to promote nerve fibre maintenance, we deleted Ngf using Prx1-cre.Prx1-cre recombines in limb mesenchymal cells, including in LepR + cells that form in the bone marrow of limb bones, but not within the axial skeleton 33,39,43 .Two-month-old Prx1-cre; Ngf fl/fl mice exhibited a lack of nerve fibres in femur bone marrow but normal innervation of vertebral bone marrow (Fig. 4a-c).This demonstrates that NGF acts locally within the bone marrow to promote nerve fibre maintenance.
Consistent with the phenotype observed in Lepr Cre/+ ; Ngf fl/∆ mice, Prx1-cre; Ngf fl/fl mice exhibited normal bone marrow haematopoiesis under steady-state conditions but impaired haematopoietic and vascular regeneration in limb bones.Compared with littermate controls, Prx1-cre; Ngf fl/fl mice exhibited normal femur bone marrow, vertebral bone marrow and spleen cellularity (Fig. 4d), and normal frequencies of HSCs and restricted haematopoietic progenitors (Fig. 4e) in femur bone marrow.WBM cells from the femurs of Prx1-cre; Ngf fl/fl mice and littermate controls did not differ in their capacity to reconstitute myeloid, B or T cells upon competitive transplantation into irradiated mice (Fig. 4f and Extended Data Fig. 5e-g).
To assess the regeneration of haematopoiesis after irradiation, we lethally irradiated (1,080 rads) and transplanted a radioprotective dose of 1,000,000 WBM cells into 2-month-old Prx1-cre; Ngf fl/fl mice and littermate controls.At 28 days after irradiation, the regeneration of bone marrow cellularity (Fig. 4g), HSCs (Fig. 4h), LSK cells (Fig. 4i), LepR + cells (Fig. 4j) and endothelial cells (Fig. 4k) were all significantly impaired in femur, but not vertebral, bone marrow in Prx1-cre; Ngf fl/fl mice.Consistent with this, femur, but not vertebral, bone marrow blood vessels in Prx1-cre; Ngf fl/fl mice were leaky at 28 days after irradiation (Fig. 4l).Blood cell counts did not significantly differ between Prx1-cre; Ngf fl/fl and littermate control mice before or after irradiation (Extended Data Fig. 5h-j), consistent with the observation that haematopoietic regeneration was impaired only in limb bones.NGF thus acts locally within the bone marrow to promote haematopoietic, stromal and vascular cell regeneration.

Nerve sprouting increases regeneration factor expression
When compared with non-irradiated bone marrow, we observed a significant increase in NGF levels at 14 days after irradiation in 6-month-old control mice but not in 6-month-old Lepr Cre/+ ; Ngf fl/∆ mice (Fig. 5a).In the bone marrow, Ngf-mScarlet was mainly expressed by adipocytes (Fig. 5b) and LepR + cells (Fig. 5c and Extended Data Fig. 6a) at 14 days after irradiation.Little or no Ngf-mScarlet expression was observed among haematopoietic/endothelial cells or LepR negative stromal cells in the bone marrow (Fig. 5c).By qRT-PCR, Ngf levels were similar in adipocytes and in LepR + cells (Fig. 5d).
We observed significantly increased nerve fibre density in control bone marrow after irradiation but not in the bone marrow of 6-month-old Lepr Cre/+ ; Ngf fl/∆ mice (Fig. 5e).We irradiated Wnt1-cre; Rosa26-tdTomato mice, which express Tomato in neural crest-derived cells, including nerve fibres and Schwann cells 44 .Nerve fibres were much more abundant in the bone marrow at 14 days after irradiation as compared with non-irradiated controls (Fig. 5f,g).Nerve fibres were closely associated with arterioles under steady-state conditions and after irradiation (Extended Data Fig. 6b).By 28 days after irradiation, when NGF levels in the bone marrow returned nearly to normal (Fig. 5a), the density of nerve fibres also returned nearly to normal (Fig. 5g).
To test if the increase in NGF levels in the bone marrow after irradiation caused nerve fibre sprouting, we examined 4-5-month-old   from five donor mice were transplanted into a total of five recipients per donor per genotype in five independent experiments).g-k, At 28 days after irradiation, Prx1-cre; Ngf fl/fl and littermate control mice did not significantly differ in terms of bone marrow cellularity (g) and the numbers of HSCs (h), LSK cells (i), LepR + cells (j) or endothelial cells (k) in the vertebrae, but all of these parameters were significantly lower in femur bone marrow (six mice from six independent experiments).l, Leakage of intravenously injected Evans blue dye into femur and vertebra bone marrow 28 days after irradiation (five mice from five independent experiments).All data represent mean ± standard deviation.The statistical significance of differences among treatments was assessed using Mann-Whitney tests (c), Student's t-tests (d, e and l) or Welch's t-tests (i) followed by the Holm-Šidák's multiple comparisons adjustment, or matched samples two-way ANOVAs (f-h, j and k) followed by the Šidák's multiple comparisons adjustment.All the statistical tests were two-sided.Not significant (NS): P > 0.05.

Article
https://doi.org/10.1038/s41556-023-01284-9 Lepr Cre/+ ; Ngf fl/∆ mice.These mice had lower levels of NGF in the bone marrow as compared with littermate controls (Extended Data Fig. 6c).They had a normal density of nerve fibres in the bone marrow before irradiation but, unlike control mice, did not exhibit an increase in nerve fibres 14 days after irradiation (Extended Data Fig. 6d).These 4-5-month-old Lepr Cre/+ ; Ngf fl/∆ mice exhibited delayed regeneration of bone marrow cellularity (Extended Data Fig. 6e), HSCs (Extended Data Fig. 6f) and LSK cells (Extended Data Fig. 6g) as compared with littermate controls.Therefore, the sprouting of nerve fibres in the bone marrow after irradiation occurs in response to increased NGF production by LepR + cells, and the adipocytes they give rise to, and this accelerates haematopoietic regeneration.We hypothesized that bone marrow nerve fibres increased the production of growth factors that promote haematopoietic and vascular regeneration.We found by enzyme-linked immunosorbent assay analysis that SCF (Fig. 5h), VEGF (Fig. 5i) and Ang2 (Fig. 5j) levels increased significantly in the bone marrow of control mice at 14 days after irradiation but to a significantly lesser extent in the bone marrow of 6-month-old Lepr Cre/+ ; Ngf fl/∆ mice.By 28 days after irradiation, when NGF levels and nerve fibre density had returned to normal in control mice (Fig. 5a,e), SCF, VEGF and Ang2 levels had also returned to normal (Fig. 5h-j).Each of these factors is necessary for normal haematopoietic 35 or vascular regeneration 45,46 .In contrast to what we observed in the bone marrow, the levels of NGF, SCF, VEGF and Ang2 in the blood did not significantly differ before versus after irradiation, or between Lepr Cre/+ ; Ngf fl/∆ and littermate control mice (Extended Data Fig. 6h-k).
To test if stromal cells regenerated immediately adjacent to nerve fibres or throughout the bone marrow, we assessed the distances of LepR + cells, Scf-GFP + stromal cells and Scf-GFP + adipocytes to nerve fibres in non-irradiated mice and mice 14 days after irradiation.In both cases, most LepR + cells, Scf-GFP + stromal cells and Scf-GFP + adipocytes were distant from nerve fibres and the percentages of cells in each cell population that were at least 20 µm from nerve fibres did not significantly change between non-irradiated and irradiated mice (Extended Data Fig. 6l-n).This suggested that nerve fibres promoted regeneration throughout the bone marrow, not just immediately adjacent to nerve fibres.On the other hand, the percentages of LepR + cells and Scf-GFP + stromal cells that were within 10 µm of nerve fibres were significantly higher in irradiated as compared with non-irradiated mice.Thus, regeneration may have been somewhat enhanced immediately adjacent to nerve fibres.
We also used Adiponectin-creER to delete Ngf from LepR + cells and the adipocytes they gave rise to after irradiation.Nearly all LepR + cells and adipocytes express Adiponectin and recombine with Adiponectin-creER, including the skeletal stem cells in adult bone marrow 34 .Tamoxifen was administered to Adiponectin-creER; Ngf fl/∆ and littermate control mice at 2-3 months of age, then 2 weeks later the mice were irradiated and transplanted with a radioprotective dose of 1,000,000 WBM cells.At this early timepoint, these mice still had normal numbers of nerve fibres in the bone marrow but they did not exhibit the increase in nerve fibres after irradiation that was observed in control bone marrow (Extended Data Fig. 7a).The Adiponectin-creER; Ngf fl/∆ mice also exhibited impaired regeneration of haematopoietic cells (Extended Data Fig. 7b-d), increased vascular leakiness (Extended Data Fig. 7e), reduced numbers of LepR + cells and endothelial cells (Extended Data Fig. 7f,g) and lower levels of bone marrow SCF, VEGF and Ang2 (Extended Data Fig. 7h-j) after irradiation.Thus, 2-3-month-old Adiponectin-creER; Ngf fl/∆ mice phenocopied 4-5-month-old Lepr Cre/+ ; Ngf fl/∆ mice with reduced nerve fibre sprouting as well as impaired haematopoietic and vascular regeneration as compared with control mice.
β adrenergic receptors signal through protein kinase A (PKA) to increase the expression of VEGF by cancer cells 47 .Consistent with this, LepR + cells from Lepr Cre/+ ; Ngf fl/∆ mice had lower levels of phosphorylated PKA (Fig. 5o) and reduced levels of SCF, VEGF and Ang2 as compared with LepR + cells from control mice at 14 days after irradiation (Extended Data Fig. 7m-o).Treatment of irradiated mice with salbutamol rescued PKA phosphorylation in LepR + cells from Lepr Cre/+ ; Ngf fl/∆ mice (Fig. 5o) as well as SCF, VEGF and Ang2 levels in the bone marrow (Extended Data Fig. 7m-o).These data suggest that β adrenergic receptors in LepR + cells increase the expression of growth factors by promoting PKA signalling.
Single-cell RNA sequencing 29 showed that Adrb1 was not expressed by bone marrow stromal cells (Extended Data Fig. 8a).Adrb2 and Adrb3 were mainly expressed by LepR + cells in the bone marrow (Extended Data Fig. 8b,c).By qRT-PCR, we did not detect Adrb2 and Adrb3 expression in unfractionated bone marrow cells but found that Adrb2 and Adrb3 were expressed at similar levels in LepR + cells and adipocytes (Extended Data Fig. 8d,e).Deficiency for either of these receptors did not significantly impair haematopoietic regeneration (Fig. 6a-c); however, deficiency for Adrb2 and Adrb3 did significantly impair haematopoietic regeneration (Fig. 6a-c).Therefore, β2 and β3 adrenergic receptors both promote haematopoietic regeneration while β1 adrenergic receptor is dispensable.

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 Ngfexpressing 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-monthold 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.

Article
https://doi.org/10.1038/s41556-023-01284-9 To identify the cells in which β2/β3 adrenergic receptors signal to promote haematopoietic regeneration, we made floxed alleles of Adrb2 and Adrb3 (Extended Data Fig. 8i-l) and conditionally deleted Adrb2 and Adrb3 from LepR + cells using Lepr Cre .Two-month-old Lepr Cre/+ ; Adrb2 fl/fl ; Adrb3 fl/fl mice had normal vasculature and haematopoiesis under steady-state conditions, including normal blood cell counts (Fig. 6d-f), and frequencies of HSCs and restricted progenitors (Fig. 6g) in the bone marrow.However, when these mice were lethally irradiated and transplanted with radioprotective wild-type bone marrow cells, they were less likely to survive (Fig. 6h), and exhibited impaired regeneration of bone marrow cellularity (Fig. 6i), HSCs (Fig. 6j), LSK cells (Fig. 6k), vasculature (Fig. 6l), LepR + cells (Fig. 6m) and endothelial cells (Fig.     m,n, Numbers of LepR + cells (m) and endothelial cells (n) in the bone marrow 28 days after irradiation.o-q, SCF (o), VEGF (p) and Ang2 (q) in bone marrow serum from 2-month-old Lepr cre/+ ; Adrb2 fl/fl , Adrb3 fl/fl mice and littermate controls before (D0) or 14 or 28 days after irradiation (a total of six mice per genotype per timepoint from six independent experiments in a-c, g and i-q).All data represent mean ± standard deviation.r, Western blot of protein from LepR + cells isolated from Lepr cre/+ ; Adrb2 fl/fl , Adrb3 fl/fl and littermate control mice 14 days after irradiation (representative of three independent experiments).All data represent mean ± standard deviation.The statistical significance of differences among treatments was assessed using two-way ANOVAs (a-c and i-n) or matched samples two-way ANOVAs (d-f) followed by Šidák's multiple comparisons adjustment, Student's t-tests followed by Holm-Šidák's multiple comparisons adjustments for comparisons among genotypes (g and q), a log-rank test (h), two-way ANOVAs (o and p) followed by Tukey's multiple comparisons adjustment, or a one-way ANOVA followed by Sidak's multiple comparisons adjustment for comparisons among timepoints (q).All statistical tests were twosided.Not significant (NS): P > 0.05.

Article
https://doi.org/10.1038/s41556-023-01284-9 6n) as compared with littermate controls.Lepr Cre/+ ; Adrb2 fl/fl ; Adrb3 fl/fl mice also exhibited significantly reduced levels of SCF (Fig. 6o), VEGF (Fig. 6p) and Ang2 (Fig. 6q) in the bone marrow as compared to littermate controls, as well as reduced PKA phosphorylation in LepR + cells (Fig. 6r) at 14 days after irradiation.Adipocytes in the bone marrow 14 days after irradiation and transplantation expressed levels of Scf, Vegf and Ang2 that were comparable to LepR + cells (Extended Data Fig. 8f-h).Bone marrow nerve fibres thus promote regeneration by activating β2/β3 adrenergic receptors in LepR + cells, and potentially their adipocyte progeny, increasing the production of growth factors by these cells.

Discussion
Nerve fibres in the bone marrow are known to promote haematopoietic regeneration after myeloablation 4 , but little is known about the mechanism by which nerve fibres are maintained in the bone marrow or how they promote regeneration.Our results reveal a reciprocal relationship between LepR + stromal cells and nerve fibres in which nerve fibres are maintained by NGF produced by LepR + cells and, in turn, promote haematopoietic and vascular regeneration by secreting adrenergic neurotransmitters that activate β2/β3 adrenergic receptors in LepR + cells (see model in Extended Data Fig. 9).Adrenergic receptor activation in LepR + cells increases the production of multiple growth factors by LepR + cells, and the adipocytes they give rise to, that promote haematopoietic and vascular regeneration.LepR + cells, and the adipocytes they give rise to after myeloablation, are the major sources of SCF and VEGF for haematopoietic and vascular regeneration in the bone marrow 24,35 .Some prior studies of the effects of peripheral nerves on haematopoiesis depended on systemic ablation of sympathetic nerve fibres, such as with 6-hydroxydopamine, raising the question of whether the observed effects reflected local loss of nerve fibres within the bone marrow or more systemic effects.These studies showed that sympathetic nerves regulate circadian variation in HSC mobilization into the blood, as well as the effects of G-CSF on mobilization, by influencing the expression of CXCL12 by stromal cells 6,11 .Nociceptive nerve fibres promote HSC mobilization by releasing calcitonin gene-related peptide, which activates receptors expressed by HSCs 8 .In agreement with these studies 6,11 , we observed that circadian mobilization of haematopoietic progenitors is dependent on bone marrow innervation (Fig. 2h,i), suggesting that this reflects a local effect within the bone marrow.
Nerve fibres localize exclusively around arterioles in both irradiated and non-irradiated mice (Extended Data Fig. 6b) but appear to promote the regeneration of LepR + cells throughout the bone marrow.The ability of nerve fibres to promote regeneration throughout the bone marrow may be enhanced by the sprouting of nerve fibres after myeloablation.Peripheral nerves also release adrenergic neurotransmitters through non-synaptic volume transmission in which neurotransmitters can diffuse considerable distances away from nerve fibres 48,49 .LepR + cells have long processes that allow them to interact with cells that are not adjacent to the LepR + cell body.Thus, nerve sprouting, volume transmission, long LepR + cell processes and perhaps other mechanisms that propagate signals among LepR + cells may enable nerve fibres around arterioles to promote regeneration throughout the bone marrow.
Peripheral nerve fibres are depleted by chemotherapy 50 and in diabetes mellitus 51 .Our results raise the question of whether peripheral neuropathy undermines engraftment in people who receive bone marrow, or other forms of HSC, transplants.Moreover, many people take drugs that block the signalling of β2/β3 adrenergic receptors, such as for heart conditions.Our results raise the question of whether β blockers delay haematopoietic regeneration after transplantation.Another interesting question for future studies is whether nerve fibres also promote the regeneration of non-haematopoietic tissues by sprouting after injury and by promoting the β adrenergic receptor-mediated expression of regeneration factors.

Mice
All mouse experiments complied with all relevant ethical regulations and were performed according to protocols approved by the Institutional Animal Care and Use Committee at UT Southwestern Medical Center (protocol 2017-101896) and the National Institute of Biological Sciences, Beijing (NIBS2022M0024).All mice were maintained on a C57BL/6 background, including Lepr cre (ref.To generate Ngf mScarlet mice, CleanCap Cas9 messenger RNA (Tri-Link) and single guide RNAs (transcribed using MEGAshortscript Kit (Ambion) and purified using the MEGAclear Kit (Ambion)), and recombineering plasmids were microinjected into C57BL/Ka zygotes.The coding sequence for the monomeric red fluorescent protein (mScarlet) was as described 64 .Chimeric mice were genotyped by restriction fragment length polymorphism analysis and insertion of the mScarlet sequence into the correct locus was confirmed by Southern blotting and sequencing of the targeted allele.Founders were mated with C57BL/Ka mice to obtain germline transmission then backcrossed with wild-type C57BL/Ka mice for at least three generations before analysis.
To generate the Ngf floxed allele, the targeting vector was obtained from The European Conditional Mouse Mutagenesis Program, linearized, and electroporated into C57BL-derived Bruce4 ES cells.Successfully targeted clones were expanded in culture then injected into C57BL/6-Tyrc-2J blastocysts.Chimeric mice were bred with C57BL/Ka mice to obtain germline transmission.The LacZ and neocassette was removed by mating with Flpe mice 65 and backcrossed for five generations onto a C57BL/Ka background before analysis.
For flow cytometric analysis of stromal cells, WBM was flushed using HBSS-free with 2% bovine serum then enzymatically dissociated with type I collagenase (3 mg ml −1 ), dispase (4 mg ml −1 ) and DNase I (1 U ml −1 ) at 37 °C for 30 min as described previously 23 .Samples were then stained with antibodies and analysed by flow cytometry.Goat-anti-LepR-biotin (AF497), BV421 streptavidin (used at 1:500), anti-CD45 (30F-11), anti-CD31 (clone 390) and anti-TER119 antibodies were used to isolate LepR + stromal cells that were negative for haematopoietic and endothelial markers.For analysis of bone marrow endothelial cells, mice were intravenously injected with 10 µg per mouse of eFluor660-conjugated anti-VE-cadherin antibody (BV13, eBiosciences).Ten minutes later, the long bones were removed and bone marrow was flushed, digested and stained as above.Samples were analysed using FACSAria Fusion or FACSCanto II flow cytometers and FACSDiva (BD) or FlowJo v10.6.1 (Tree Star) software.The flow cytometry gating strategy used for the isolation of haematopoietic stem and progenitor cell populations, LepR + cells and endothelial cells is shown in Extended Data Fig. 2.

Irradiation and competitive reconstitution assays
Adult recipient mice were irradiated using an XRAD 320 X-ray irradiator (Precision X-Ray) or Cesium-137 Gammacell 1000 irradiator (Best Theratronics) with two doses of 540 rad at least 4 h apart (1,080 rads total).C57BL/Ka (CD45.1/CD45.2heterozygous) mice were used as recipients.A total of 500,000 unfractionated bone marrow cells from donor (CD45.2) and competitor (CD45.1)mice were mixed and injected intravenously through the retro-orbital venous sinus.Recipient mice were bled from 4 to 16 weeks after transplantation to examine the levels of donor-derived myeloid, B and T cells in their blood.RBCs were lysed with ammonium chloride potassium buffer before antibody staining.The antibodies used to analyse donor chimerism in the blood were Mice were backcrossed at least three times onto a C57BL/Ka background before analysis (representative of three independent experiments).(d) Flow cytometric analysis showed that 12% of Ngf-mScarlet + bone marrow stromal cells were NG2 + smooth muscle cells in enzymatically dissociated bone marrow cells.(e) When bones were crushed and enzymatically dissociated, 19% of osteoblasts were Ngf-mScarlet + (4 mice from 4 independent experiments).(f-h).Deep imaging of femur bone marrow from 2-8 month-old Ngf mScarlet/+ mice.The Ngf-mScarlet + cells included SMA + periarteriolar smooth muscle cells (arrow, f), a subset of Col1a1-GFP + osteoblasts associated with trabecular bone in the metaphysis (arrow, g), and S100 + Schwann cells associated with nerve fibers in the bone marrow (arrow, h) (each panel reflects data from three mice from three independent experiments).As in Fig. 1, most of the Ngf-mScarlet + cells in these images were LepR + perisinusoidal stromal cells.(i, j) Flow cytometric analysis showed that only rare macrophages in the bone marrow (i) or blood ( j) were Ngf-mScarlet + (3 mice from 3 independent experiments).Extended Data Fig. 4 | Lepr cre/+ ; Ngf fl/∆ mice developed normally, had normal numbers of peripheral nerves outside of bones, and bone marrow innervation was dispensable for hematopoiesis in adult bone marrow.(a-c) Six month-old Lepr cre/+ ; Ngf fl/∆ mice were grossly normal in size and appearance as compared to Ngf fl/∆ littermate controls (a), with similar body length (b) and body mass (c) (a total of 8 mice per genotype from 8 independent experiments).(d-f) Immunofluorescence analysis of nerve fibers in longitudinal femur sections from 6 month-old Lepr cre/+ ; Ngf fl/∆ (d) and Ngf fl/∆ littermate control mice (e), showing the presence of nerve fibers outside bone marrow in both Lepr cre/+ ; Ngf fl/∆ and control mice but nerve fibers were only present inside the bone marrow of control mice (d).(f) Nerves in the quadriceps of 6-8 month-old Lepr cre/+ ; Ngf fl/∆ and littermate control mice appeared to be comparable in numbers (3 mice per genotype from 3 independent experiments).(g-i) Blood cell counts in 6 month-old Lepr cre/+ ; Ngf fl/∆ mice and littermate controls.(j-n) 6 month-old Lepr cre/+ ; Ngf fl/∆ and littermate controls exhibited no significant differences in the frequencies of B220 + B cells (j), CD3 + T cells (k), Gr-1 + Mac-1 + myeloid cells (l), CD41 + megakaryocyte lineage cells (m), or CD71 + /Ter119 + erythroid lineage cells (n) in the bone marrow and spleen (panels g-n reflect a total of 6 mice per genotype in 6 independent experiments).(o-r) Bone marrow cells from 6-8 month-old Lepr cre/+ ; Ngf fl/∆ and littermate control mice gave similar levels of donor cell reconstitution upon competitive transplantation into irradiated mice (bone marrow cells from 5 donor mice per genotype were transplanted into a total of 5 recipients per donor in 5 independent experiments).All data represent mean ± standard deviation.Statistical significance was assessed using Student's t-tests (b, c, and f), t-tests followed by the Holm-Sidak's multiple comparisons adjustment (g-n), or matched samples two-way ANOVAs followed by the Sidak's multiple comparisons adjustment (o-r).All statistical tests were two-sided.Not significant (NS): P > 0.05.(a) LepR + cells were positive for Ngf-mScarlet in sections from Ngf mScarlet/+ femur bone marrow at 14 days after irradiation and bone marrow transplantation (representative of 3 experiments).(b) The percentage of nerve fibers in the bone marrow that were within 10 µm of arterioles in Ngf fl/∆ control mice before (D0), or 14 or 28 days after irradiation and transplantation (7 mice from 7 independent experiments per time point).(c) NGF levels in bone marrow serum from 4-5 month-old Lepr cre/+ ; Ngf fl/∆ and Ngf fl/∆ littermate control mice before, or 14 or 28 days after irradiation and transplantation (4 mice per genotype from 4 independent experiments).(d) The area occupied by peripherin + nerve fibers in bone marrow sections from 4-5 month-old Lepr cre/+ ; Ngf fl/∆ and littermate controls before, or 14 or 28 days after irradiation and transplantation.(e-g) Cellularity (e), number of HSCs (f) and LSK cells (g) in bone marrow (one tibia and one femur) from 4-5 month-old Lepr cre/+ ; Ngf fl/∆ mice and littermate controls 28 days after irradiation and transplantation (a total of 5 mice per genotype from 5 independent experiments in panels d-g).(h-k) Blood serum levels of NGF (n = 5) (h), SCF (n = 6) (i), VEGF (n = 6) (j), and Ang2 (n = 6) (k) from 6 month-old Lepr cre/+ ; Ngf fl/∆ mice and littermate controls before, or 14 or 28 days after irradiation and transplantation (5-6 independent experiments).(l-n) The distance from LepR + cells (n = 14 mice per treatment) (l), Scf-GFP + stromal cells (n = 14) (m), and Scf-GFP + adipocytes (n = 6) (n) to the nearest nerve fiber before or 14 days after irradiation and transplantation (6-14 independent experiments).All data represent mean ± standard deviation.The statistical significance of differences among treatments was assessed using a one-way ANOVA followed by the Sidak's multiple comparisons adjustment (b and c), Student's t-tests (c, i-m) or Mann-Whitney tests (n) followed by the Holm-Sidak's multiple comparisons adjustment, or two-way ANOVAs followed by the Tukey's (d) or Sidak's (e-g) multiple comparisons adjustment.All the statistical tests were two-sided.Not significant (NS): P > 0.05.

Extended Data
Extended Data Fig. 7 | Adiponectin-creER; Ngf fl/∆ mice exhibit defects in nerve fiber sprouting and hematopoietic and vascular regeneration after irradiation.(a) The area occupied by peripherin + nerve fibers in bone marrow sections from 2-3 month-old Adiponectin-creER; Ngf fl/∆ and Ngf fl/∆ littermate controls before, or 28 days after irradiation and transplantation (7 mice per genotype per time point from 7 independent experiments).(b-d) Cellularity (b), numbers of HSCs (c) and LSK cells (d) in bone marrow (one tibia and one femur) from Adiponectin-creER; Ngf fl/∆ and littermate control mice before and 28 days after irradiation and transplantation.(e) Leakage of intravenouslyinjected Evans blue dye into femur bone marrow at the indicated time points after irradiation and bone marrow transplantation.(f, g) Numbers of LepR + cells (f) and endothelial cells (g) in the bone marrow before, or 14 or 28 days after irradiation and bone marrow transplantation (5 mice per genotype from 5 independent experiments in panels b-g).(h -j) SCF (h), VEGF (i) and Ang2 (j) in bone marrow serum from 2-3 month-old Adiponectin-creER; Ngf fl/∆ and littermate control mice before, or 14 or 28 days after irradiation and transplantation (a total of 8 mice per genotype per time point from 8 independent experiments).(k-o) When administered to Lepr cre/+ ; Ngf fl/∆ mice after irradiation, the b2 agonist salbutamol rescued the regeneration of bone marrow LepR + cells (k) and endothelial cells (l) at 28 days after irradiation, as well as bone marrow SCF (m), VEGF (n), and Ang2 (o) levels 14 days after irradiation (6 mice per genotype per treatment from 6 independent experiments).All data represent mean ± standard deviation.Statistical significance was assessed using matched samples two-way ANOVAs followed by the Tukey's (a, h, and i) or Sidak's (b, d, f, and k-o) multiple comparisons adjustment, Student's t-tests (c, e, and g) or Mann-Whitney tests (for genotype comparisons of j) followed by the Holm-Sidak's multiple comparisons adjustment, or Friedman tests followed by the Dunn's multiple comparisons adjustment (for time-point comparisons of j).All statistical tests were two-sided.Not significant (NS): P > 0.05.

Fig. 1 |
Fig. 1 | Ngf is mainly expressed in the bone marrow by LepR + stromal cells.a,b, The expression of neurotrophic factors by microarray analysis 16 (a) and RNA sequencing 22 (b) in bone marrow stromal cells (isolated on the basis of expression of Scf-GFP (a) or PDGFRα (b) staining, both of which are nearly completely overlapping with LepR expression 33 ), VE-cadherin + bone marrow endothelial cells, Col2.3-GFP + CD45 − Ter119 − CD31 − osteoblasts, and WBM cells (three mice in a and two mice in b, from three or two independent experiments, respectively).c, Uniform manifold approximation and projection (UMAP) plot showing clustering of single-cell RNA sequencing analysis of 4,209 non-haematopoietic cells from enzymatically dissociated bones/bone marrow in 8-week-old mice 39 .d, Ngf is mainly expressed by Lepr + stromal cells (cell cluster 11 in c) and smooth muscle cells (cell cluster 12).e, Ngf expression by all cell clusters shown in c /doi.org/10.1038/s41556-023-01284-9

9 aFig. 3 |
Fig. 3 | Lepr cre/+ ; Ngf fl/∆ mice exhibit defects in haematopoietic and vascular regeneration after irradiation.a, Survival of Lepr cre/+ ; Ngf fl/∆ and Ngf fl/∆ littermate control mice after irradiation and transplantation of wild-type bone marrow cells (22 mice per genotype from 3 independent experiments).b-d, WBC (b), RBC (c)and PLT (d) counts from Lepr cre/+ ; Ngf fl/∆ and littermate control mice before (D0) and 7, 14 and 28 days after irradiation.e,f, Bone marrow cellularity (e) and LSK cell numbers (f) from 6-month-old Lepr cre/+ ; Ngf fl/∆ and littermate control mice on day (D)0, D7, D14 and D28 after irradiation.g, Numbers of HSCs in bone marrow (always one tibia and one femur) from Lepr cre/+ ; Ngf fl/∆ and littermate control mice before and 28 days after irradiation.h-k, Cellularity (h) and numbers of LSK cells, (i) LepR + cells (j) and endothelial cells (k) in the bone marrow of Lepr cre/+ ; Ngf fl/∆ and littermate control mice on D10 after irradiation (a total of 12 mice per genotype from 3 independent experiments).l,m, Leakage of intravenously injected Evans blue dye into femur bone marrow at D10 (l), D14 and D28 (m) after irradiation of Lepr cre/+ ; Ngf fl/∆ and littermate control mice.n, Endomucin staining of the vasculature in the bone marrow of Lepr cre/+ ; Ngf fl/∆ and littermate control mice 28 days after irradiation (representative of three experiments).

Fig. 4 |
Fig. 4 | Prx1-cre; Ngf fl/fl mice exhibit a loss of nerve fibres as well as defects in haematopoietic and vascular regeneration in long bones but not in vertebrae.a,b, Nerve fibres (green) were visible in femur (a) and vertebra (b) bone marrow from Ngf fl/fl control mice and in vertebra bone marrow (b) from 2-month-old Prx1-cre; Ngf fl/fl mice but not in femur bone marrow (a) from Prx1-cre; Ngf fl/fl mice (representative of three independent experiments).c, The area occupied by peripherin + nerve fibres in bone marrow sections from 2-month-old Prx1-cre; Ngf fl/fl and littermate control mice.d-f, Under steady-state conditions, 2-month-old Prx1-cre; Ngf fl/fl mice did not significantly differ from littermate control mice in terms of spleen, femur bone marrow or vertebral bone marrow cellularity (d), the frequencies of haematopoietic stem and progenitor cell populations in femur bone marrow (e) (six mice per genotype from six independent experiments in c-e), or the levels of donor cell reconstitution upon competitive transplantation into irradiated mice (f, femur bone marrow cells

Fig. 6 |
Fig. 6 | Nerve sprouting after irradiation increases the expression of regeneration factors by activating β adrenergic receptors in LepR + cells and their progeny.a-c, Two-to 4-month-old mice, with adrb1, adrb2 and/ or adrb3 deficiency, were irradiated and transplanted with radioprotective wild-type bone marrow cells; then the cellularity (a) and numbers of HSCs (b) and LSK cells (c) in the bone marrow (always one tibia and one femur) were analysed 28 days later.d-f, White blood cell (d), red blood cell (e) and platelet (f) counts from non-irradiated 2-month-old Lepr cre/+ ; Adrb2 fl/fl ; Adrb3 fl/fl mice and Adrb2 fl/fl ; Adrb3 fl/fl littermate controls (a total of five mice per genotype from five independent experiments).g, Haematopoietic stem and progenitor cell frequencies in the bone marrow of non-irradiated 2-month-old Lepr cre/+ ; Adrb2 fl/fl ; Adrb3 fl/fl and littermate controls.h, Survival of 2-month-old Lepr cre/+ ; Adrb2 fl/fl ; Adrb3 fl/fl and littermate controls after irradiation and transplantation (22 mice per genotype in 3 independent experiments).i-l, Cellularity (i) and numbers of HSCs (j) and LSK cells (k) in the bone marrow and leakage of intravenously injected Evans blue dye into femur bone marrow (l) of 2-month-old Lepr cre/+ ; Adrb2 fl/fl , Adrb3 fl/fl and littermate controls at 14 or 28 days after irradiation.D, day.

Extended Data Fig. 1 |
Generation and characterization of the Ngf mScarlet mouse reporter allele.(a-c) The mouse Ngf gene was modified by inserting an mScarlet-WPRE-pA cassette after an alternative ATG start codon in exon 4, replacing most of the coding sequence in exon 4. Open boxes indicate untranslated regions and black boxes indicate translated regions of Ngf.The correctly targeted founder mouse (F0) was identified by southern blotting (b) using 3' and WPRE probes (black bars in a) (representative of three independent experiments).(c) PCR genotyping of genomic DNA confirmed germline transmission of the Ngf mScarlet allele.

Fig. 2 |
Flow cytometry gating strategy for the isolation of hematopoietic stem and progenitor cell populations, LepR + cells and endothelial cells.(a) Representative flow cytometry gates used to isolate hematopoietic stem and progenitor cell populations from bone marrow.(b) Representative flow cytometry gates used to isolate LepR + cells and endothelial cells from bone marrow.Extended Data Fig. 3 | Generation of the Ngf flox mouse allele.(a) LoxP elements were inserted on either side of exon 3 Ngf such that Cre-mediated recombination eliminates exon 3 and introduces a frameshift.Open boxes indicate untranslated exon sequences and black boxes indicate translated sequences.The insertion sites were selected to avoid disrupting conserved intron sequences.The Ngf LacZ-Neo-flox targeting vector was obtained from the European Conditional Mouse Mutagenesis Program (EUCOMM), linearized, and electroporated into C57BL-derived Bruce4 ES cells.Chimeric mice were generated by injecting ES clones into blastomeres and were bred with C57BL/Ka mice to obtain germline transmission of the Ngf LacZ-Neo-flox allele.(b) Ngf LacZ-Neo-flox mice were bred with Flpe mice 55 to remove the LacZ-Neo cassette.Successful removal of the LacZ-Neo cassette was confirmed by PCR primers spanning the FRT sites, as shown by arrows on the targeting vector in panel a.(c) PCR genotyping of genomic DNA confirmed germline transmission of the Ngf flox allele using the genotyping primers shown in panel a.Mice were backcrossed at least five times onto a C57BL/Ka background before analysis (images in panels b and c are representative of three independent experiments).(d-g) Deep imaging of peripherin + nerve fibers in the bone marrow of 6-8 month-old Ngf fl/∆ control (d), NG2-CreER; Ngf fl/∆ (e), Col1a1-CreER; Ngf fl/∆ (f), and GFAP-Cre; Ngf fl/∆ (g) mice.Images are representative of a total of 4 mice per genotype from 4 experiments.

Fig. 5 |
Nerve fibers promote hematopoietic and vascular regeneration after lethal and sublethal irradiation but are not required under steady-state conditions.(a) Survival of 6-8 month-old Lepr cre/+ ; Ngf fl/∆ and Ngf fl/∆ littermate control mice after sublethal irradiation.(b-d) Cellularity (b), numbers of HSCs (c) and LSK cells (d) in bone marrow (one tibia and one femur) from of 6 month-old Lepr cre/+ ; Ngf fl/∆ and Ngf fl/∆ littermate control mice at 28 days after sublethal irradiation (5 mice per genotype from 5 independent experiments per time point).(e-g) Bone marrow cells from femurs of non-irradiated 2 monthold Prx1-cre; Ngf fl/fl mice and Ngf fl/fl littermate controls gave similar levels of myeloid, T cell and B cell reconstitution upon competitive transplantation into irradiated recipients (bone marrow cells from 5 donor mice were transplanted into a total of 5 recipients per donor in 5 independent experiments).(h-j) White blood cell (h), red blood cell (i), and platelet (j) counts from 2 month-old Prx1cre; Ngf fl/fl and Ngf fl/fl littermate control mice before (D0) and 7, 14, and 28 days after irradiation and transplantation (5 mice per genotype from 5 independent experiments per time point).All data represent mean ± standard deviation.The statistical significance of differences among treatments was assessed using longrank test (a), Student's t-test (b), t-tests followed by the Holm-Sidak's multiple comparisons adjustment (c, d, and h-j), or matched samples two-way ANOVAs followed by the Sidak's multiple comparisons adjustment (e-g).All statistical tests were two-sided.Not significant (NS): P > 0.05.Extended Data Fig. 6 | Bone marrow nerve sprouting after myeloablation increases hematopoietic regeneration and growth factor production.

Fig. 9 |
Schematic illustrating a reciprocal relationship between LepR + stromal cells and nerve fibers in the bone marrow.The nerve fibers are maintained by NGF produced by LepR + cells and, in turn, promote hematopoietic and vascular regeneration by secreting adrenergic neurotransmitters that activate β2/β3 adrenergic receptors in LepR + cells and the adipocytes they give rise to after myeloablation.