SLIT3 fragments orchestrate neurovascular expansion and thermogenesis in brown adipose tissue

Domain: biology
Published: 2026-03-25
Editorial outcome: major_revision

Abstract
Brown adipose tissue is an evolutionary innovation in placental mammals that regulates body temperature through adaptive thermogenesis. Cold exposure activates brown adipose tissue thermogenesis through coordinated induction of brown adipogenesis, angiogenesis, and sympathetic innervation; however, how these processes are coordinated remains unclear. Here, we show that fragments of Slit guidance ligand 3 (SLIT3) drive crosstalk among adipocyte progenitors, endothelial cells, and sympathetic nerves. Adipocyte progenitors secrete SLIT3, which is cleaved into functionally distinct SLIT3-N and SLIT3-C fragments that independently promote angiogenesis and sympathetic innervation. We identify PLXNA1 as a receptor for SLIT3-C and demonstrate its essential role in sympathetic innervation of brown adipose tissue. Moreover, we identify BMP1 as the first SLIT protease described in vertebrates. Coordinated neurovascular expansion mediated by distinct SLIT3 fragments provides a bifurcated yet integrated mechanism that ensures a synchronized brown adipose tissue response to environmental challenges. Finally, this study reveals a previously unrecognized role for adipocyte progenitors in regulating tissue innervation.

Introduction
The regulation of body temperature is a fundamental homeostatic process in endothermic animals. Preserving a stable internal temperature ensures the efficiency and fidelity of all cellular reactions and is essential for survival. Brown adipose tissue (BAT) is a specialized type of adipose tissue that is primarily responsible for regulating body temperature through adaptive thermogenesis. Brown adipocytes oxidize substrates and generate heat to maintain euthermia in a cold environment1.

Brown adipocytes are embedded within an intricate network of blood vessels and sympathetic nerves that support their development and thermogenic function. The high thermogenic activity of BAT requires a high rate of blood perfusion to supply O2 and substrates. The sympathetic nerves also play a key role in stimulating BAT thermogenesis by releasing norepinephrine into the tissue. Norepinephrine activates adrenergic signaling in thermogenic adipocytes, resulting in enhanced thermogenesis and lipolysis2. Chronic cold exposure increases BAT mass by de novo recruitment of brown adipocytes, as well as by expanding the network of blood vessels and sympathetic nerves in the tissue. This coordinated expansion of BAT ensures its continuous responsiveness to hormonal and neuronal stimuli and is essential for enhanced thermogenesis in cold3,4. However, how these distinct processes are spatiotemporally coordinated remains unclear. Furthermore, while significant progress has been made in understanding the molecular mechanisms of thermogenic activation in adipocytes, the equally crucial process of remodeling the thermogenic adipose niche remains less understood.

Cell–cell communication is vital for organismal development and function. Ligand-receptor interactions allow cells in complex tissues to coordinate their functions during development, homeostasis, and remodeling. To understand the mechanisms through which different cell types in BAT communicate and synchronize their response to cold to collectively enhance thermogenesis, we previously used single-cell transcriptomic data5 to construct a network of ligand-receptor interactions in BAT6. That study highlighted the central role of adipocyte progenitors, not merely as the source of adipocytes, but also as key communicators and versatile players in the adipose tissue microenvironment. These progenitors have been shown to contribute to a variety of processes, including extracellular matrix remodeling, immune modulation, and angiogenesis7.

The SLIT family of secreted proteins and their roundabout (ROBO) receptors constitute a conserved signaling pathway originally identified for its role in axon guidance during neurodevelopment8–10. SLIT guidance ligands are large extracellular glycoproteins that regulate the migration and adhesion of multiple cell types including neurons, endothelial cells, leukocytes, and epithelial cells11. Studies in Drosophila and mice revealed the role of Slit proteins in neuronal axon guidance and arborization, vascularization and angiogenesis, inflammatory cell chemotaxis, and tumor metastasis12,13. In mammals, the Slit family consists of three members: SLIT1 is predominantly expressed in the nervous system whereas SLIT2 and SLIT3 are also expressed outside the nervous system13–16. Although originally identified as axon guidance factors, SLITs regulate the migration and positioning of cells in various tissues. However, the mechanisms controlling the context-dependent outcomes of SLIT signaling are still largely unclear.

Here, we demonstrate that SLIT3 is a critical regulator of BAT neurovascular development and function. We show that SLIT3 mediates crosstalk amongst adipocyte progenitors, endothelial cells, and sympathetic nerves, regulating both angiogenesis and innervation. Using BAT- and adipocyte progenitor-specific loss-of-function models, we show that the loss of SLIT3 disrupts both angiogenesis and sympathetic innervation, ultimately blunting cold-induced BAT thermogenesis. We identify bone morphogenetic protein 1 (BMP1) as the protease responsible for SLIT3 cleavage and reveal that the resulting fragments activate distinct pathways: SLIT3-N promotes angiogenesis, while SLIT3-C, through its receptor PLXNA1, drives sympathetic innervation. This pathway demonstrates a sophisticated level of intercellular coordination whereby a single factor simultaneously drives these two distinct processes essential for thermogenic activation.

Results
Identification of SLIT3-ROBO4 signaling axis in BAT
Intercellular communication plays a critical role in coordinating tissue adaptation to external challenges. To define the role of intercellular crosstalk in cold-induced BAT thermogenesis, we previously used single-cell transcriptomic data to build a network of ligand-receptor interactions involved in the cold-induced remodeling of BAT6. This analysis revealed the significance of adipocyte progenitors as the major communication hub in the adipose niche6. This prompted us to search for ligands secreted from adipocyte progenitors that might mediate the crosstalk between adipocyte progenitors and their niche.

Ligand-receptor analysis identified SLIT3 and its putative endothelial receptor, ROBO4, as a potential crosstalk axis in BAT. In BAT, Slit3 is predominantly expressed in Pdgfra+ and Trpv1+ adipocyte progenitors, as well as in vascular smooth muscle cells, while its receptor, Robo4, is exclusively expressed in vascular and lymphatic endothelial cells (Fig. 1a). To validate these findings, we separated the stromal vascular and adipocyte fractions of mouse BAT, sorted distinct cell populations, and measured Slit3 expression. This analysis confirmed that adipocyte progenitors are the primary source of Slit3 in BAT under both basal and cold-acclimated conditions (Fig. 1b and Supplementary Fig. 1a–d), whereas Robo4 is exclusively expressed in endothelial cells (Supplementary Fig. 1e). To examine SLIT3 protein expression in BAT and its regulation by environmental temperature, we performed Western blot analysis on BAT from mice housed at different temperatures. Using an antibody that detects the N-terminal region of SLIT3 protein, we found that SLIT3-FL levels were elevated in BAT of mice exposed to cold for 2 days compared to those maintained at room temperature (Fig. 1c, d). These findings indicate that cold exposure enhances SLIT3 protein levels in BAT.

A previous study reported that SLIT3 is secreted from macrophages in white adipose tissue (WAT)17. However, our unbiased scRNA-seq data, along with targeted expression analysis in isolated macrophages, did not detect any Slit3 expression in BAT macrophages (Fig. 1a, b). Analysis of scRNA-seq datasets from mouse inguinal white adipose tissue (ingWAT) and perigonadal white adipose tissue (pgWAT), as well as human WAT18, revealed SLIT3 expression in adipocyte progenitors, mesothelial cells, and mural cells (vascular smooth muscle cells and pericytes) (Supplementary Fig. 2a–d), but no expression in macrophages in either mouse or human WAT (Supplementary Fig. 2a–d). Collectively, these findings suggest that adipocyte progenitors, rather than macrophages, are the primary source of SLIT3 in adipose tissue.

To assess the temporal pattern of Slit3 expression during adipogenesis, we measured SLIT3 protein levels in a mouse brown adipocyte progenitor cell line at multiple time points throughout in vitro adipogenic differentiation. SLIT3 protein levels sharply decreased by days 4 and 8 of differentiation, in contrast to the increasing uncoupling protein 1 (UCP1) expression (Supplementary Fig. 2e). These results indicate that SLIT3 expression is significantly higher in adipocyte progenitors than in mature adipocytes, where SLIT3 protein levels were minimal.

SLIT3 is essential for BAT thermogenesis
To investigate Slit3 function in BAT, we used AAV-mediated shRNA delivery to knockdown Slit3 expression specifically in BAT. We confirmed a significant reduction in Slit3 transcript and protein levels in BAT, but not in WAT, of mice receiving Slit3 shRNAs (Supplementary Fig. 3a–d). Mice lacking Slit3 expression in BAT exhibited severe impairments in cold-induced thermogenesis, maintaining lower core body temperature (Fig. 1e) and BAT temperature (Supplementary Fig. 3e) during cold exposure.

After 7 days of cold exposure, BAT analysis revealed pronounced whitening and increased lipid accumulation (Fig. 1f), accompanied by reduced expression of Ucp1 (Fig. 1g–i), iodothyronine deiodinase type 2 (Dio2), cell death-inducing DFFA-like effector A (Cidea), and beta-3 adrenergic receptor (Adrb3), as well as mitochondrial, and lipolysis-related genes in Slit3 knockdown mice (Fig. 1i). Furthermore, Slit3 loss resulted in a marked reduction in mitochondrial electron transport chain complex levels (Fig. 1j, k). Mice lacking Slit3 expression in BAT showed a comparable reduction in Ucp1 transcript and protein levels when housed at room temperature (Supplementary Fig. 4a–c) or under thermoneutral conditions (30°C; Supplementary Fig. 4d–f). However, the expression of other thermogenic and mitochondrial genes was only modestly reduced under these conditions (Supplementary Fig. 4a, d).

Consistent with histological and molecular analyses, Slit3 loss in BAT significantly reduced energy expenditure, oxygen consumption, and CO₂ production in mice housed at 5°C (Fig. 1l, m and Supplementary Fig. 5). Importantly, these metabolic alterations occurred without changes in body weight, body composition, BAT and WAT depot weights, respiratory exchange ratio, food intake, or locomotor activity (Supplementary Fig. 5). Together, these findings establish SLIT3 as a critical regulator of cold-induced BAT thermogenesis and energy expenditure.

Loss of SLIT3 impairs BAT neurovasculature
To investigate how the loss of SLIT3 impairs cold-induced BAT thermogenesis, we first examined the cell-autonomous effects of SLIT3 on adipocytes. We treated in vitro-differentiated brown adipocytes with recombinant SLIT3-N and SLIT3-C proteins. qPCR and immunoblot analyses showed no changes in the expression of Ucp1 or other thermogenic genes in adipocytes treated with recombinant SLIT3 fragments (Supplementary Fig. 6a, b). These results led us to hypothesize that SLIT3 loss impairs BAT thermogenesis by disrupting the brown adipocyte niche.

The expansion of BAT neurovasculature is essential for cold adaptation and enhanced thermogenesis in the cold3,4. Chronic cold exposure significantly increases the density of parenchymal sympathetic neurites and capillary blood vessels in BAT (Supplementary Fig. 6c–h). To investigate the mechanism(s) underlying impaired BAT thermogenesis in Slit3-deficient mice, we assessed vascularization and sympathetic innervation in BAT from Slit3 knockdown or control mice after 7 days of cold exposure (5°C). Analysis of BAT vasculature revealed that Slit3 knockdown significantly impaired angiogenesis, as indicated by a reduction in capillary density (Fig. 2a–c) and a decrease in endothelial-specific transcript expression (Fig. 2d).

The loss of Slit3 also resulted in a dramatic reduction in the density of tyrosine hydroxylase (TH)-expressing neurites in BAT (Fig. 2e–g). To assess the architecture and density of sympathetic innervation, we used the Adipo-Clear protocol19 to stain TH-expressing sympathetic nerves in BAT, followed by lightsheet microscopy. 3D volumetric images of TH-positive nerves revealed a marked decline in parenchymal innervation in Slit3-deficient BAT (Fig. 2h). Furthermore, total TH protein levels in BAT were significantly reduced in the absence of SLIT3 (Fig. 2i, j). Notably, even when animals were housed at room temperature or thermoneutrality, loss of SLIT3 led to a decrease in both capillary density and sympathetic innervation in BAT (Supplementary Fig. 7a–l). However, housing at cold temperatures further exacerbated these defects. These findings underscore the crucial role of SLIT3 in the development and expansion of the neurovascular network in BAT, especially during adaptation to cold environments.

To directly test whether impaired sympathetic innervation underlies the thermogenic defects in Slit3-deficient BAT, we bypassed neural input by administering the β3-adrenergic receptor agonist CL-316,243. Slit3 knockdown animals treated with CL-316,243 for 10 days exhibited fully intact induction of thermogenic, mitochondrial, and lipolytic gene programs (Supplementary Fig. 7m). These results demonstrate that the intrinsic ability of brown adipocytes to respond to adrenergic stimulation remains uncompromised in the absence of SLIT3. Thus, the primary role of SLIT3 in BAT is not to modulate adipocyte-intrinsic thermogenic capacity, but rather to drive the proper neurovascular expansion necessary for cold-induced thermogenesis.

ASPC-derived SLIT3 promotes neurovascular expansion in cold
To further confirm that adipocyte stem and progenitor cells (ASPCs) are the primary source of SLIT3 and that ASPC-derived SLIT3 is essential for cold-induced neurovascular expansion and BAT thermogenesis, we specifically deleted Slit3 in Pdgfra-expressing cells by crossing Slit3 floxed mice20 with an inducible Pdgfra-cre strain21 (Pdgfra-creERT2;Slit3flox/flox or Slit3iΔAPC). Western blotting showed a complete loss of SLIT3 protein in BAT and a marked reduction in WAT depots of Slit3iΔAPC mice (Fig. 3a–h and supplementary Fig. 8a–d). Consistent with the observed phenotypes in BAT-specific SLIT3 knockdown mice, both male and female Slit3iΔAPC mice exhibited significant impairments in BAT thermogenesis and were cold-intolerant (Fig. 3i, j), highlighting the essential role of adipocyte progenitor-derived SLIT3 in cold adaptation. H&E analysis of the BAT from Slit3iΔAPC and control littermates revealed no difference in lipid droplet size (Fig. 3k). However, consistent with our findings from Slit3 knockdown experiments, Slit3iΔAPC mice exposed to cold exhibited a significant reduction in both sympathetic neurite density and capillary density in BAT (Fig. 3l–o). Together, these results confirm that Pdgfra-expressing adipocyte progenitors are the primary source of SLIT3 in BAT, and that the loss of ASPC-derived SLIT3 disrupts cold-induced neurovascular expansion, leading to impaired thermogenic activation.

BMP1-mediated SLIT3 cleavage produces two ligands
Members of the SLIT family are proteolytically cleaved into a large N-terminal and a shorter C-terminal fragment9. However, the functional implications of SLIT cleavage and the distinct roles played by the full-length protein and its fragments remain unclear. Additionally, the SLIT proteases in vertebrates have not been identified.

To investigate whether SLIT3 is cleaved in adipocyte progenitors, we overexpressed an N- and C-terminal tagged wild-type SLIT3 (SNAP-SLIT3-HaloTag) as well as an uncleavable SLIT3 variant (SNAP-SLIT3UC-HaloTag) in a brown adipocyte progenitor cell line (Fig. 4a). Using antibodies against HaloTag and SNAP-tag, we detected the full-length SLIT3 protein (SLIT3-FL) in the total cell lysates of cells overexpressing either the wild-type or uncleavable SLIT3 constructs (Fig. 4b, c). The HaloTag antibody detected two bands in the conditioned media of cells overexpressing SNAP-SLIT3-HaloTag: a ~200 kDa band corresponding to the tagged full-length SLIT3 and a ~60 kDa band matching the expected size of the tagged SLIT3 C-terminal fragment (SLIT3-C). In contrast, the media from cells expressing the uncleavable SLIT3 only contained the SLIT3-FL (Fig. 4b). Similarly, the SNAP-tag antibody detected both the SLIT3-FL and a ~150 kDa band corresponding to the tagged SLIT3 N-terminal fragment (SLIT3-N) in the media of cells overexpressing SNAP-SLIT3-HaloTag (Fig. 4c). A minor amount of SLIT3-N, but not SLIT3-C, was also detected in the total cell lysates, which contain membrane and membrane-associated proteins (Fig. 4c). Consistent with prior reports that SLIT-N can remain membrane-associated while Slit-C is more diffusible9, we observed SLIT3-C exclusively in the media and SLIT3-N in both lysates and media (Fig. 4c), supporting a model in which fragment properties enable a differential range of action in BAT.

Moreover, analysis of a proteomics dataset of the adipocyte secretome showed the presence of SLIT3 in the conditioned media of in vitro differentiated adipocytes derived from mouse visceral (Visc), subcutaneous inguinal white (SubQ), and interscapular BAT22 (Fig. 4d). SLIT3 was significantly more abundant in the conditioned media of brown and subcutaneous white adipocytes than visceral white adipocytes. These results indicated that SLIT3 is secreted by various adipocyte types in vitro, with the highest abundance observed in the secretome of brown adipocytes.

A previous study suggested a role for the metalloprotease tolkin (TOK) in SLIT proteolysis in Drosophila melanogaster23. TOK is a member of the BMP1/Tolloid-like family of metalloproteases. In mammals, the BMP1/Tolloid-like family includes bone morphogenetic protein 1 (BMP1), tolloid (TLD), and tolloid-like 1 and 2 (TLL1 and TLL2). Given the similarity in the cleavage sites among the members of the SLIT family23, we hypothesized that the BMP1/TLD-like proteases could be involved in SLIT3 cleavage. Among the members of the BMP1/TLD-like protease family, only Bmp1 and Tll1 are expressed in BAT (Fig. 4e and Supplementary Fig. 9). Notably, Bmp1 is abundantly expressed in Pdgfra- and Trpv1-expressing adipocyte progenitors, the major cell types expressing Slit3 in BAT (Fig. 4e). BMP1 lacks growth factor activity found in other BMP family members and instead functions as a metalloprotease. To discern the role of BMP1 in SLIT3 proteolysis in adipocyte progenitors, we used a combination of pharmacological and genetic loss-of-function studies. We showed that the inhibition of BMP1 activity using a small molecule inhibitor, UK383,36724, blocked SLIT3 cleavage (Fig. 4f). Similarly, knocking down Bmp1 using siRNAs prevented SLIT3 cleavage (Fig. 4g). Thus, we concluded that BMP1 is responsible for the proteolytic processing of SLIT3, establishing BMP1 as the SLIT protease in vertebrates.

SLIT3 fragments promote angiogenesis and innervation in BAT
To investigate the specific functions of SLIT3 fragments in BAT, we generated AAV constructs for adipocyte-specific overexpression of full-length SLIT3 (SLIT3-FL), its N-terminal fragment (SLIT3-N, aa 1–1116), and its C-terminal fragment (SLIT3-C, aa 1117–1523) (Fig. 5a). Four weeks after AAV administration in the interscapular BAT, we confirmed the specific expression of SLIT3-FL, SLIT3-N, and SLIT3-C (Fig. 5b, c). Overexpression of SLIT3-FL and SLIT3-C, but not SLIT3-N, enhanced sympathetic innervation in BAT, as indicated by an increased number and relative area of TH-expressing sympathetic neurites (Fig. 5d–f). Furthermore, overexpression of SLIT3-FL and SLIT3-N led to increased capillary density in BAT (Fig. 5g, h). Mice overexpressing SLIT3-FL and SLIT3-C, but not SLIT3-N, also exhibited higher TH levels in BAT (Fig. 5i–q and Supplementary Fig. 10). Notably, SLIT3-FL and SLIT3-C overexpression enhanced BAT thermogenesis, as evidenced by increased UCP1 expression and higher BAT temperatures under cold exposure (Fig. 5k, n, q, r). These findings indicate that SLIT3 fragments directly promote the expansion of BAT’s neurovascular network, with SLIT3-C primarily driving sympathetic innervation and SLIT3-N inducing angiogenesis, collectively contributing to enhanced thermogenic function.

To determine the effects of SLIT3 overexpression in the absence of cold stimulation, we overexpressed SLIT3-FL, SLIT3-N, and SLIT3-C and assessed TH and UCP1 levels in BAT from mice housed at room temperature. Consistent with the results observed in cold, overexpression of SLIT3-FL and SLIT3-C, but not SLIT3-N, increased TH levels in BAT (Supplementary Fig. 11). These effects were, however, more modest at room temperature compared to cold exposure. Moreover, overexpression of SLIT3-FL and SLIT3-C at room temperature did not increase UCP1 protein levels, indicating that while SLIT3-FL and SLIT3-C can directly promote sympathetic innervation under basal conditions, their impact becomes more pronounced during cold exposure, when sympathetic activation and thermogenic demand are elevated (Supplementary Fig. 11).

PLXNA1 mediates SLIT3-dependent sympathetic innervation
Full-length and N-terminal SLIT fragments bind to transmembrane ROBO receptors (ROBO1-4)9, while the receptor binding and bioactivity of the C-terminal fragment (SLIT-C) remains less characterized. The C-terminal of SLIT2 has been shown to bind Plexin A1 (PLXNA1)25, but the receptor(s) for SLIT3-C have yet to be identified. To identify the receptors mediating SLIT3 signaling in BAT, we analyzed the expression of candidate SLIT receptors. scRNA-seq analysis of BAT revealed that among the four Robo family members, only Robo4 is expressed in BAT, where it is exclusively localized to vascular and lymphatic endothelial cells (Fig. 1a and Supplementary Fig. 1e). To validate this, we co-stained BAT sections with a ROBO4 antibody and Isolectin B4. ROBO4 was specifically localized to Isolectin B4-labeled capillaries (Fig. 6a). This combined with the observation that SLIT3-N increased capillary density (Fig. 5g, h), supports an endothelial SLIT3-ROBO4 axis. Furthermore, double staining of BAT with ROBO1 and TH antibodies revealed ROBO1 expression in TH-expressing sympathetic neurites (Fig. 6b). Similarly, PLXNA1 and TH were co-localized in sympathetic neurites (Fig. 6c).

Building on our finding that SLIT3-C enhances sympathetic innervation (Fig. 5d–f) and that PLXNA1 is expressed on sympathetic neurites (Fig. 6c), we hypothesized that PLXNA1 serves as the receptor for SLIT3-C. To address this, we first assessed the ability of PLXNA1 to interact with SLIT3-FL or SLIT3-C. We overexpressed the extracellular region of PLXNA1, C-terminally fused to a 6X histidine tag, along with SLIT3-FL-HaloTag or SLIT3-C-HaloTag in a mouse adipocyte progenitor cell line. Using a His-tag antibody, we immunoprecipitated PLXNA1 and its interacting proteins. We found that both SLIT3-FL and SLIT3-C co-immunoprecipitated with the extracellular region of PLXNA1 (Fig. 6d). These findings indicate that PLXNA1 specifically interacts with the C-terminal region of SLIT3 (SLIT3-C), which is distinct from the N-terminal region recognized by ROBO receptors26.

To further investigate the interaction between PLXNA1 and SLIT3, we used AlphaFold2 Multimer to model complexes involving the extracellular domain of PLXNA1 and the C-terminal region of SLIT3. Multiple structure predictions were generated based on sequence alignments and structural relaxation, with only those having average inter-protein TM (iPTM) + predicted TM (pTM) scores > 0.8 retained for further analysis. Diagnostic plots were then used to assess model quality (Supplementary Fig. 12). The top-ranked models, shown in Fig. 6e, f, reveal two distinct interaction modes: a monomeric PLXNA1-SLIT3 complex (Fig. 6e) and a dimeric PLXNA1 interacting with a monomeric SLIT3 (Fig. 6f). The predicted binding interfaces of SLIT3 and SEMA6D, another known ligand for PLXNA1, on PLXNA1’s extracellular domains suggest a partial overlap between the SLIT3 and SEMA6D binding regions (Fig. 6g, h). The AlphaFold2 diagnostic plots (Supplementary Fig. 12) further validate the structural integrity of the models, with Predicted Aligned Error (PAE) plots for both the monomeric and dimeric complexes, along with predicted Local Distance Difference Test (pLDDT) scores, demonstrating high confidence in the predicted structures.

We next examined whether PLXNA1 functions as a receptor for SLIT3-C in vivo and whether it is required for SLIT3-mediated sympathetic innervation. To test this, we overexpressed SLIT3-C in BAT with or without Plxna1 knockdown. Consistent with earlier findings (Fig. 5), SLIT3-FL overexpression significantly enhanced sympathetic innervation in BAT (Fig. 6i, j). However, this effect was diminished when PLXNA1 expression was knocked down (Fig. 6i, j). Similarly, Plxna1 knockdown markedly reduced the SLIT3-C-induced increase in sympathetic innervation (Fig. 6k, l). Notably, in the absence of PLXNA1, SLIT3-C overexpression failed to elevate BAT temperature in response to cold exposure (Fig. 6m). Mice co-expressing SLIT3-C and shPlxna1 in BAT displayed normal levels of sympathetic innervation but showed reduced BAT temperature in cold. We speculate that this effect may reflect additional roles of PLXNA1 in sympathetic neurons beyond axon growth, potentially involving pathways downstream of semaphorin ligands. Collectively, by combining biochemical assays, computational structural modeling, and in vivo studies, we provide strong and converging evidence that PLXNA1 is a direct and essential receptor for the C-terminal region of SLIT3, required for both sympathetic innervation and thermogenic activation in BAT.

PLXNA1 is essential for sympathetic innervation in BAT
Given the severe reduction in sympathetic innervation of BAT and cold tolerance in mice receiving Plxna1 shRNAs (shPlxna1 group in Fig. 6i–m), we hypothesized that PLXNA1 is crucial for the development and cold-induced expansion of sympathetic neurites in BAT. To directly test this, we injected AAVs delivering Plxna1 shRNAs into BAT. qPCR and western blot analysis confirmed a significant reduction of PLXNA1 expression in BAT (Supplementary Fig. 13a, b). Loss of PLXNA1 led to a pronounced decrease in sympathetic nerve density in mice housed at room temperature as well as after 3 and 7 days of cold exposure (Fig. 7a, b). This was accompanied by a reduction in total TH protein levels and NE concentrations (Fig. 7c–e and Supplementary Fig. 12c, d) in BAT, while capillary density remained unchanged (Fig. 7f, g). This resulted in impaired BAT thermogenesis, evidenced by reduced BAT temperature in cold conditions (Fig. 7h) and a downregulation of the thermogenic gene program both at room temperature and after 3 days of cold acclimation (Fig. 7i, j). Loss of PLXNA1 reduced expression of GAP43, a key axonal growth cone protein involved in axon outgrowth and regeneration. This was reflected by a decreased GAP43+ neurite area and a lower proportion of sympathetic neurites expressing GAP43 (Fig. 7k–m). Consistently, Gap43 mRNA levels were also reduced in BAT following Plxna1 knockdown (Fig. 7n). Together, these findings establish PLXNA1 as a key regulator of sympathetic innervation in BAT, demonstrating its essential role in both the steady-state maintenance and cold-induced expansion of sympathetic neurites in BAT (Fig. 7o).

SLIT3 expression correlates with adipose tissue health
Lastly, to assess the significance of SLIT3 in humans, we measured its expression in adipose tissue biopsies from abdominal subcutaneous WAT and omental visceral WAT, collected from two independent cohorts of the Leipzig Obesity BioBank (LOBB). In the metabolically healthy versus unhealthy obese cohort (MHUO)27, we found that SLIT3 transcript levels in human abdominal subcutaneous WAT were positively correlated with serum adiponectin concentrations (r=0.546, P=0.00176, N=31) and negatively regulated with the relative number of macrophages in omental visceral WAT (r=−0.566, P<0.001, N=31) in insulin-sensitive patients (Fig. 8a, b), but not in insulin-resistant patients (Fig. 8c, d).

Additionally, we examined SLIT3 expression in the human cross-sectional cohort composed of paired samples of omental visceral and abdominal subcutaneous WAT from 1480 individuals. In this cohort, we found SLIT3 expression in omental visceral WAT negatively correlated with Hemoglobin A1C (HbA1c) (r=-0.092, P<0.001, N=1447) (Fig. 8e). Additionally, SLIT3 expression in omental visceral and abdominal subcutaneous WAT was positively associated with circulating level of the anti-inflammatory adipokine Omentin1 (r=0.164, P<0.001, N=675 and r=0.124, P=0.00121, N=675, respectively) (Fig. 8f, g). Omentin1 has been shown to play important roles in glucose homeostasis, lipid metabolism, insulin resistance, and diabetes28,29. Collectively, these data suggest that SLIT3 may regulate adipose tissue health and inflammation in humans, potentially impacting insulin sensitivity.

Methods
Animals
All experiments were performed in compliance with all relevant ethical regulations and were approved by Research and Laboratory Safety and the Institutional Animal Care and Use Committees at New York University.

C57BL/6J mice (stock no. 000664) were purchased from Jackson Laboratory. Pdgfra-cre;Slit3flox/flox or Slit3iΔAPC were generated by crossing Slit3 floxed mice20 with Pdgfra-creERTM21 (JAX strains 018280). Mice were maintained on a 12-h light-dark cycle at 22°C and 50% relative humidity. Mice were maintained on a regular chow diet (5053-PicoLab® Rodent Diet 20; formulated with 20% protein diet and 4.5% fat) with food and water provided ad libitum unless otherwise specified. For experiments involving cold acclimation or thermoneutral housing, mice were maintained at 5°C and 30°C, respectively, with 50–60% relative humidity in a controlled diurnal environmental chamber (Caron Products & Services) and given free access to food and water. Euthanasia of the animals was accomplished by methods consistent with the guidelines of the American Veterinary Medical Association. Euthanasia was performed by CO₂ inhalation in accordance with institutional guidelines, followed by a secondary method (cervical dislocation) to ensure death.

Cold tolerance test
For cold tolerance tests, mice were single-housed and placed at 5°C in a controlled environmental diurnal chamber (Caron Products & Services). Body temperature was measured with a rectal probe (Physitemp, RET3) and a reader (Physitemp, BAT-12) or using RFID transponder temperature microchips implanted under the interscapular BAT area (Unified Information Devices).

AAV injection
6–8-week-old male mice were anesthetized with isoflurane, and an incision was made above the interscapular area to expose the underlying adipose tissue. AAV particles were injected into each BAT lobe, and the incision was closed with suture. Mice were allowed to recover for 2–3 weeks before analysis.

Slit3 and Plxna1 shRNAs
AAV8 harboring three targeting shRNA and one scramble control were purchased from VectorBuilder. The shRNA sequences are listed in Supplementary Table 1. The three shRNAs were mixed and injected into BAT at the dose of 5e+11 genomic copies per BAT lobe.

AAV constructs for the overexpression of Slit3 fragments in BAT
Mouse SLIT3-FL, SLIT3-N, and SLIT3-C sequences were cloned from a cDNA clone (Origene MR225499L4) and cloned into the pAAV-hAdiponectin-W backbone47 using NEBuilder HiFi DNA Assembly kit using the following primers:
Slit3-FL_fwd (AACTACTCGAGgccaccATGGCCCTCGGCCGGAC),
Slit3-FL_rev (tattcaGCGGCCGCTTAAACCTTATCGTCGTCATCCTT GTAATCgctgccGGAACACGCGCGGCA), Slit3-N_fwd (AACTACTCGAG gccaccATGGCCCTCGGCCGGAC), Slit3-N_rev (tattcaGCGGCCGCT TAAACCTTATCGTCGTCATCCTTGTAATCgctgccAACCATGGGTGGGG G), Slit3-C_fwd (tgctgccgccCTGCTACAAACCAGCCCC), Slit3-C_rev (ggttgattatcttctagagcTTACTTGTCGTCATCGTCTTTG), Signal peptide_fwd (tgattccataccagagggtcGCCACCATGGCCCTCGGC), and Signal peptide_rev (tttgtagcagGGCGGCAGCAGGGGGTCC).

pAAV-Adipoq-GFP construct47 was used as the control. AAV8-Adipoq-Slit3FL, AAV8-adipoq-Slit3N, AAV8-Adipoq-Slit3C, and AAV-Adipoq-GFP were packaged at VectorBuilder. 2e+10 AAV particles were injected into each BAT lobe.

Fluorescence-activated cell sorting (FACS)
Adipocytes and the Stromal Vascular Fraction (SVF) were isolated from mouse BAT following the procedure described previously5. The interscapular BAT was dissected, finely minced, and digested for 45 min using a cocktail containing type 1 collagenase (1.5 mg ml−1; Worthington Biochemical), dispase II (2.5 U ml−1; Stemcell Technologies), and fatty acid-free BSA (2%; Gemini Bio-Products) in Hanks’ balanced salt solution (Corning Hanks’ Balanced Salt Solution, with calcium and magnesium). The resulting dissociated tissue was subsequently centrifuged at 500×g and 4°C for 10 min. Adipocytes, located in the uppermost layer, were gently collected using a wide-mouthed transfer pipette and filtered through a 100 µm cell strainer. Brown adipocytes were allowed to float for 5 min at room temperature before they were centrifuged at 30g for 5 min at room temperature. This cycle was repeated three times, after which the adipocytes were immediately lysed in Trizol. For the SVF isolation, the pellet was resuspended in 10 ml of 10% FBS in DMEM, filtered through a 100 µm cell strainer into a fresh 50-ml tube, and subsequently centrifuged at 500×g for 7 min. Red blood cells were lysed in 2 ml of sterile ammonium-chloride-potassium lysis buffer (ACK Lysis Buffer, Lonza) for 5 min on ice. The cells were then filtered once more through a 40-μm cell strainer, washed with 20 ml of a solution containing 10% FBS in DMEM, and centrifuged at 500×g for 7 min. The cells were resuspended in 1 ml of Cell Staining Buffer (BioLegend) before proceeding with staining.

Cells were stained using the fluorescently conjugated antibodies as outlined in Supplementary Table 2. The cells were then incubated with the antibodies at the specified dilutions from Supplementary Table 2 for a duration of 30 min, followed by two rounds of washing in Cell Staining Buffer (BioLegend). Cells were sorted using an SH800 sorter using a 100 µm sorting chip (Sony Biotechnology). Debris and doublets were excluded based on forward and side scatter gating, and 7-AAD was used to exclude dead cells. Following sorting, cells were centrifuged at 300×g for 5 min and lysed in Trizol for subsequent RNA isolation and gene expression analysis.

RNA isolation and quantitative reverse transcription-PCR (qRT-PCR)
RNA was isolated from cells or tissues using phenol–chloroform extraction and isopropanol precipitation. qRT PCR assays were conducted using a QuantStudio™ 5 Real-Time PCR instrument (Life Technologies Corporation) and SYBR Green (Invitrogen). Relative mRNA expression was determined using the ΔCt method and normalized to the expression of housekeeping genes Rplp0 or Tbp. Primer sequences are available in Supplementary Table 3.

Western blotting
Cell sort tissues were lysed in RIPA buffer supplemented with a protease inhibitor cocktail (cOmplete™, Sigma-Aldrich, Dallas, TX). The primary antibodies are listed in Supplementary Table 2. Primary antibodies were incubated overnight at 4°C. HRP-coupled secondary antibodies were used at 1:5000 dilution for 1 hour at room temperature. Proteins were detected using the Pierce™ ECL Western Blotting Substrate (Thermo Fisher Scientific) using an Licor Odyssey M Imaging System. All the original uncropped and unprocessed scans are provided in the Source Data file. For protein detection in the conditioned media, serum-free and Phenol Red-free media were collected and centrifuged at 300×g to remove cell debris. The supernatant was concentrated using Amicon™ Ultra-4 Centrifugal Filter Units (UFC801024, Millipore Sigma™).

Immunohistochemistry
Adipose tissues were fixed in 10% formalin and embedded in paraffin. 6 µm sections were prepared and stained with the primary antibodies listed in Supplementary Table 2. Sections were deparaffinized and rehydrated, followed by an antigen retrieval step in Rodent Decloaker buffer using the Decloaking Chamber™ NxGen (BIOC Medical) in 1× rodent antigen retrieval reagent (95°C for 45 min). Sections were then incubated in Sudan Black (0.3% in 70% ethanol) to reduce the autofluorescence signal. Blocking was performed in 5% BSA in PBST (0.1% Tween-20 in PBS) for 30 minutes at room temperature, followed by incubating the sections in primary antibodies diluted in 1% BSA in PBST overnight at 4°C (Supplementary Table 2). The biotinylated isolectin B4 was diluted in 5 µg/ml in a staining buffer containing 0.1 mM CaCl2, 0.1 mM MgCl2, and 0.1 mM MnCl2. The next day, slides were washed with PBST and were incubated with appropriate fluorescently labeled secondary antibodies at a 1:200 dilution (Supplementary Table 2). Slides were stained with DAPI and mounted in a mounting medium. Images were collected on a Leica SP8 confocal microscope and processed with ImageJ.

H&E staining
Adipose tissues were fixed in 10% formalin and embedded in paraffin. 6 µm sections were prepared and stained with hematoxylin and eosin (H&E) as previously described48. Slides were imaged using an Aperio slide scanner (Leica Biosystems).

Indirect calorimetry
Mice were individually housed in metabolic cages of a TSE Phenomaster system to obtain measurements for the volume of oxygen consumption (VO2), the volume of carbon dioxide production (VCO2), respiratory exchange ratio (RER), energy expenditure, food intake, and locomotor activity. Mice had ad libitum access to drinking water and standard rodent chow. Measurements were collected first for 48 hour at room temperature, followed by the second 48 hour at 5°C. Data were analyzed using CalR49.

Dual energy X-ray absorptiometry
Lean and fat mass were quantified using a Dual Energy X-ray Absorptiometry (DEXA) scanner (Insight, Osteosys).

Tissue clearing and imaging
Whole BAT sympathetic innervation visualization was achieved using the Adipo-Clear protocol50 with some modifications. One lobe of BAT from each animal was fixed in 3% glyoxal at 4°C overnight. The fixed samples were washed in PBS three times, each time for 1 hour. Next, the samples were subjected to a series of methanol washes: 20%, 40%, 60%, 80% methanol in H2O/0.1% Triton X-100/0.3M glycine (B1N buffer), and finally 100% methanol, each for 30 minutes. Subsequently, the samples underwent a triple 30-minute delipidation process with 100% dichloromethane (DCM; Sigma-Aldrich) followed by two 30-minute washes in 100% methanol. After delipidation, the tissues were bleached overnight at 4°C with 5% H2O2 in methanol (1 volume of 30% H2O2 to 5 volumes of methanol). The rehydration process followed a reversed methanol/B1N buffer series: 80%, 60%, 40%, 20% methanol in B1N buffer, each step lasting 30 minutes. All the above steps were conducted at 4°C with continuous shaking. Subsequently, samples were washed in B1N buffer twice, each time for 30 minutes, followed by two 1-hour wash in PBS/0.1% Triton X-100/0.05% Tween-20/2 µg/ml heparin (PTwH buffer), before initiating the staining procedure. The samples were incubated with anti-tyrosine hydroxylase antibody (1:200, AB1542, Millipore Sigma) in PTxwH for 4 days. After the primary antibody incubation, the samples underwent a series of PTxwH washes lasting 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hours, 4 hours, and overnight, and then were incubated in donkey anti-sheep IgG Alexa Fluor 647 (1:200, A-21099, Thermo Fisher) in PTxwH for 4 days. Finally, the samples were washed in PTwH for 5 minutes, 10 minutes, 15 minutes, 30 minutes, 1 hour, 2 hour, 4 hour, and overnight. Finally, the samples were dehydrated using a methanol/H2O series (25%, 50%, 75%, 100%, 100%) for 30 minutes at each step at room temperature. Following dehydration, samples were incubated with 100% DCM for 30 minutes twice, followed by an overnight clearing step in dibenzyl ether (DBE; Sigma-Aldrich). The samples were then stored at room temperature in the dark until imaging. Representative samples were imaged by a blinded experimenter using a Zeiss Lightsheet Z.1 Microscope with two 1920×1920-pixel sCMOS cameras. Three-dimensional reconstructions were generated using Imaris (Bitplane).

Cloning of SNAP- and Halo-tagged Slit3 plasmids
pRP[Exp]-CMV>[SNAP-Slit3-HaloTag], pRP[Exp]-CMV>[Slit3-FL]-HaloTag, and pRP[Exp]-CMV>[Slit3-C-HaloTag] constructs were generated by VectorBuilder. The N-terminal tag was added immediately downstream of the signal peptide (aa 2-33, GCCCTCGGCCGGA CCGGGGCCGGCGCCGCTGTGCGCGCCCGCCTGGCGCTGGGCTTGGC GCTTGCGAGCATCCTGAGCGGACCCCCTGCTGCCGCC).
The ORF in the pRP[Exp]-CMV>[SNAP-Slit3UC-Halo] was generated by the deletion of a 27 bp sequence encoding the Slit cleavage site (CCCACCCATGGTTCTGCTACAAACCAG, PPPMVLLQ) from Slit3-FL cDNA.

Transient transfection
Immortalized brown preadipocytes were transfected with Slit3 overexpression plasmids, Bmp1 or scramble siRNA (Dharmacon GE) using Lipofectamine™ 3000 or Lipofectamine™ RNAiMAX Transfection Reagents (Invitrogen™) following the manufacturer’s instructions.

Bmp1 inhibitor treatment
Cells were treated with the Bmp1 inhibitor, UK-383,367 (PZ0156-5MG, Sigma Aldrich) at the final concentration of 2.5 μM.

AlphaFold multimer analysis
AlphaFold2 Multimer was used to predict the protein-protein interactions between the extracellular domains of human PLXNA1 and the C-terminal region of the Slit3 protein, as described previously51,52. Protein sequences used in this study are as follows: human PLXNA1 residues 27-1244 (UniProt ID: Q9UIW2), human Slit3 residues 1120-1523 (UniProt ID: O75094), human Semaphorin-6D residues 1-1073 (UniProt ID: Q8NFY4). Briefly, a local graphics processing unit (GPU) cluster using MMseqs (git@92deb92) was utilized for local Multiple Sequence Alignment (MSA) creation53, and ColabFold (git@7227d4c) was executed for structure prediction with 5 models per prediction, including structure relaxation54. Predictions with an average pTM + iPTM score of > 0.8 were ranked, and diagnostic plots (PAE plot, pLDDT plot, and sequence coverage) were manually inspected. The generated three-dimensional (3D) models of protein-protein complexes were analyzed using UCSF Chimera55 and the PyMOL Molecular Graphics System, Version 3.0 Schrödinger, LLC.

Norepinephrine measurement
BAT samples were lysed in buffer containing 40 mM sodium metabisulfite and 10 mM EDTA. Homogenates were centrifuged at 12,000×g for 5 min at 4°C, and the supernatants were collected for analysis. Norepinephrine levels were quantified using an ELISA kit (Biomatik, EKF577994-96T) according to the manufacturer’s instructions.

Human data
The human data used in this research were sourced from the Leipzig Obesity Biobank (LOBB, https://www.helmholtz-munich.de/en/hi-mag/cohort/leipzig-obesity-bio-bank-lobb), which comprises paired samples of abdominal subcutaneous and omental visceral adipose tissue. The metabolically healthy versus unhealthy obese cohort (MHO/MUO) comprises paired samples of omental visceral and abdominal subcutaneous tissues from 31 insulin-sensitive patients (71% women; age: 38.8±11.1 years old; BMI: 45.9±6.9 kg/m²; fasting plasma glucose: 5.2±0.2 mmol/l; fasting plasma insulin: 27.9±13.5 pmol/l) and 42 insulin-resistant patients (71.43% female; age: 47.2±7.7 years old; BMI: 47.3±8.1 kg/m²; fasting plasma glucose: 5.7±0.3 mmol/l; fasting plasma insulin: 113.7±45.7 pmol/l). The cross-sectional cohort (CSC) comprises 1,479 individuals, categorized as either normal/overweight (N=31; 52% women; age: 55.8±13.4 years; BMI: 25.7±2.7 kg/m²) or obese (N=1448; 71% women; age: 46.9±11.7 years; BMI: 49.2±8.3 kg/m²). Adipose tissue samples were collected during elective laparoscopic abdominal surgeries, following established protocols56,57. Body composition and metabolic parameters were assessed using standardized techniques as described previously58,59. The study was approved by the Ethics Committee of the University of Leipzig (approval numbers: 363-10-13122010 and 017-12-230112) and adhered to the principles outlined in the Declaration of Helsinki. All participants provided written informed consent before being included in the study. Participants received no compensation for their tissue donation to the Leipzig Obesity Biobank. Exclusion criteria included individuals under 18 years of age, those with chronic substance or alcohol abuse, smoking within the 12 months prior to surgery, acute inflammatory conditions, concurrent use of glitazones, end-stage malignancies, weight loss greater than 3% in the three months leading up to surgery, uncontrolled thyroid disorders, and Cushing’s disease.

Ribosomal RNA-depleted RNA sequencing data we generated following the SMARTseq protocol60. The libraries were sequenced as single-end reads on a Novaseq 6000 (Illumina, San Diego, CA, USA) at the Functional Genomics Center Zurich, Switzerland. The preprocessing procedures were conducted as previously described61. In brief, adapter and quality-trimmed reads were aligned to the human reference genome (assembly GRCh38.p13, GENCODE release 32), and gene-level expression quantification was performed using Kallisto62 v0.48. For samples with read counts exceeding 20 million, we downsampled them to 20 million reads utilizing the R package ezRun v3.14.1 (https://github.com/uzh/ezRun, accessed on April 27, 2023). The data normalization was carried out using a weighted trimmed mean (TMM) of the log expression ratios, with adjustments made for age, sex, and transcript integrity numbers (TINs). Analyses were conducted in R v4.3.1 (www.R-project.org). Spearman coefficient was used to assess the correlation between SLIT3 expression and metabolic parameters.

Statistics and reproducibility
No statistical method was used to predetermine sample size. No data were excluded from the analyses. Experiments were not randomized. All imaging and quantifications were performed in a blinded manner. Statistical analyses were performed using GraphPad Prism.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "In this manuscript, the authors identify Slit3 as a key factor mediating the neurovascular organization of brown adipose tissue (BAT) in mice. Using single-cell analyses and western blotting, they confirm that Slit3 is expressed in adipose tissue progenitor cells. They further demonstrate that Slit3 enhances vascularization and innervation of BAT in response to cold exposure and plays a major role in supporting thermogenesis under these conditions. The functional effects are attributed to the C-terminal fragment of Slit3, processed by BMP1 and signaling through Plxna1. Overall, the experiments are well described and the results largely support the authors’ conclusions. However, certain aspects require additional evidence or clarification.\nSpecific Points:\nThe authors report that reduction of Slit3 leads to whitening of BAT under cold conditions but do not provide an explanation for this observation. Is BAT accumulating lipids released from lipolysis in other tissues? What are the weights of the inguinal and epididymal fat pads in these mice?\nAngiogenesis is assessed via isolectin staining. However, because adipocytes enlarge under certain conditions, vessel density per field can appear reduced. This must be controlled for by normalizing to adipocyte density—for example, expressing isolectin intensity relative to LipiTox or PLIN1 staining intensity. The same normalization should apply to TH staining.\nIn shRNA-treated mice, BAT shows a marked increase in lipid droplet size. Is this phenotype recapitulated in the Pdgfra-Cre-driven Slit3 knockout (Fig. 3)? If so, isolectin and TH staining data must also be corrected for adipocyte density, as recommended for Fig. 2.\nEnforced AAV-mediated expression of the C-terminal or full-length Slit3 increases isolectin staining, suggesting enhanced angiogenesis (Fig. 5). However, adipocyte density should be reported, and the data normalized accordingly. The rationale for performing these experiments under conditions where endogenous Slit3 expression is already expected to be high (WT mice after 7 days of cold exposure) is unclear. A more compelling experiment would assess the effect of Slit3-C in non-cold-exposed mice, where any increase in innervation could be attributed directly to the construct.\nThe hypothesis that BMP1 functions as a protease for Slit3 is supported by data showing diminished proteolysis following siRNA-mediated Bmp1 knockdown or pharmacological inhibition. While these findings suggest a requirement for BMP1 in Slit3 cleavage, they do not definitively establish BMP1 as the responsible protease. Rigorous molecular characterization—including specificity and kinetic studies—would be required to confirm this. Similarly, while structural modeling and shRNA knockdown data support the role of Plxna1 as a receptor for Slit3-C, definitive evidence requires demonstration of direct receptor-ligand binding, including affinity and specificity measurements. Although these experiments may be beyond the scope of the current study, the limitations of the conclusions should be clearly acknowledged in the manuscript.", "reviewer_2": "General Assessment:\nThis study investigates the molecular mechanisms coordinating neurovascular expansion and thermogenesis in brown adipose tissue (BAT), with a focus on the role of adipocyte progenitors. Building on previous scRNA-seq findings that identified adipocyte progenitors as key communication hubs, the authors perform in-depth bioinformatics analyses to identify secreted ligands mediating intercellular crosstalk. They identify secreted axon guidance molecule Slit3 and demonstrate through functional experiments that Slit3 is essential for BAT thermogenesis, angiogenesis, and sympathetic innervation. The study further reveals that Slit3 is proteolytically processed by Bmp1 into distinct fragments (Slit3-FL, Slit3-N, and Slit3-C), each with specific roles in neurovascular remodeling. AAV-mediated gain-of-function experiments show that Slit3-C promotes sympathetic innervation, while Slit3-N enhances angiogenesis. The authors also identify Plxna1 as a receptor mediating Slit3-C–dependent sympathetic innervation. Finally, the study links Slit3 expression to adipose tissue health and inflammation in humans.\nThis is a well-structured and compelling study that provides novel mechanistic insights into how adipocyte progenitors orchestrate neurovascular remodeling in BAT. The findings are timely and significantly advance our understanding BAT biology, and the experimental design is generally robust. While there are no major concerns, the following suggestions may help further strengthen the manuscript:\nSpecific Comments:\n1. Figure 1C: The increase in Slit3-FL under cold conditions is shown, but it is unclear whether similar changes occur for Slit3-N or Slit3-C. If the antibody used is expected to recognize Slit3-N or Slit3-C, this should be clarified. Including this information as supplementary data would enhance transparency.\n2. Supplementary Figure 2a–d: To support the claim that Slit3 is not expressed in macrophages under basal or cold conditions, immunofluorescence staining could provide additional confirmation.\n3. Supplementary Figure 2e: The use of a mouse adipocyte progenitor cell line to assess Slit3 expression during adipogenesis is informative. However, staining for Slit3 in both mature adipocytes and progenitors in BAT and WAT would provide more comprehensive validation.\n4. Figure 2g: A protein loading control is missing. Including this would strengthen the interpretation of the immunoblot data.\n5. Figure 3a: Similarly, a loading control is needed. Given that Slit3 is expressed in multiple cell types in BAT, and that Slit3 expression is expected to be reduced but not absent in Slit3iDAPC tissue, it would be helpful to show Slit3 protein levels under both basal and cold conditions with appropriate controls.\n6. Figure 5g–h: Overexpression of Slit3-C also increases capillary density in BAT. This is unexpected given the fragment’s primary role in sympathetic innervation. A brief discussion of potential mechanisms would be valuable.\n7. Figure 6d: The lack of co-immunoprecipitation between Plxna1 and Slit3-N supports the specificity of Plxna1 for Slit3-C. This point could be emphasized more clearly to strengthen the conclusion.\n8. Figure 1i: The p-value for Dio2 is 0.062. While not statistically significant, the trend may still be biologically relevant and could be briefly discussed.\n9. Line 291: There appears to be a typo—“Slit3-FL” should be “Slit3-C.”\n10. Supplementary Figure 6e: The high-magnification images do not appear to align with the low-magnification views. Please verify and adjust if necessary.\n11. Literature Context: Including a brief section summarizing the Slit-Robo signaling pathway and its known roles in neurogenesis and angiogenesis in the Introduction would provide helpful context for readers less familiar with this signaling axis.", "reviewer_3": "Cold exposure leads to a marked increase in vascularization and sympathetic nervous system (SNS) innervation of thermogenic adipose tissue to meet increased energetic demands. Several independent groups implicated neurotrophins and Slit3 secreted by myeloid cells in the cold-induced expansion of sympathetic innervation in inguinal white adipose tissue (iWAT) (PMID: 33297933, PMID: 35042776; PMID: 29589323; PMID: 34782792). This manuscript identifies key molecular mediators of cold-induced neurovascular expansion in brown adipose tissue (BAT). First, the authors demonstrate that Slit3 is predominantly expressed in adipocyte progenitor cells (APCs) and vascular smooth muscle in BAT, and not myeloid cells as in iWAT. Complementary loss-of-function studies using AVV-mediated expression of shRNAs in BAT and genetic knockout in APCs provide compelling evidence that Slit3 is necessary for cold-induced increases in angiogenesis and sympathetic innervation in BAT. In vitro loss-of-function and in vivo gain of function studies support the idea that the metalloprotease BMP1 cleaves Slit3 into two fragments that regulate distinct functions. The N-terminal fragment promotes angiogenesis (consistent with PMID: 22641771), while the C-terminal fragment increases SNS innervation. The authors identified Plxna1 as a potential receptor for the C-terminal fragment of Slit3 using computational modeling. Complementary in vitro and in vivo studies support the idea that Plnxa1 is a key receptor mediating the effects of the full length or C-terminal fragment of Slit3 on SNS innervation in BAT. Expression studies identified Robo4 as a putative receptor on endothelial cells, but its requirement for cold-induced angiogenesis was not tested.\nIn summary, while Slit3 appears to regulate similar adaptive responses to cold in iWAT and BAT, these studies show that the source of Slit3 (myeloid cells vs APCs) and the specific receptors mediating effects on the SNS and vasculature are distinct (Robo1 vs Plxna1). The novelty of these studies lies in the demonstration that BMP1 cleaves Slit3 into 2 fragments that act on the SNS and vasculature via distinct receptors. The authors propose that the distinct activities of Slit3 proteolytic cleavage products provide a means to synchronize adaptive responses to cold. Overall, the complementary and rigorous approaches provide very strong support for the molecular pathways outlined here. However, there are several outstanding issues that need to be resolved, outlined below.\nSlit3 KD reduced neurovascular density at room temperature (Suppl Fig 7), which is a mild cold challenge. Analyzing Slit3 KD or cKO models at thermoneutrality would be informative as to whether Slit3’s function is restricted to cold-induced expansion or also affects baseline structure.\n2. Whereas APCS express the highest levels of Slit3, cold-induced increases in transcription are only observed in ECs (Suppl Fig 1d). Moreover, AAV-mediated Slit3 KD in BAT (Fig. 1e) produced a stronger reduction in body temperature after 5 hr at 5C than conditional KO in PdgfR+ APCs (Figure 3d) (KD = 33.5C vs KO= 36.5C). This raises the possibility that adipocyte-derived Slit3 helps to maintain the neurovascular architecture at baseline but that EC-derived Slit3 mediates cold-induced remodeling. Comparisons between adipocyte-and EC-specific Slit3 KOs at thermoneutrality and in response to a cold challenge would resolve this issue as well as issue #1.\n3. While the data supporting a role for Slit3 in cold-induced neurovascular remodeling are compelling, the relationship with BAT thermogenesis is less clear. In the Slit3 KD, BAT temperature (Suppl Fig 3e) and body temperature (Fig 1e) are lower in the first 5 hours of cold exposure – this is before any cold-induced remodeling occurs. On the other hand, after 2 days of cold exposure, when Slit3 levels are high (Figure 1d), BAT temperature of KDs is the same as controls (Suppl Fig 3e). Additionally, Plxna1 KD in conjunction with Slit3-C overexpression prevents the cold-induced increase in SNS innervation (i.e. innervation looks like controls), but the BAT temperature curve is similar to that of the Plxna1 KD alone, which has lower SNS innervation (Figure 6 l,m).\nUnless these apparent inconsistencies can be definitively resolved, I would recommend removing the body/BAT temperature data and narrowing the claims to SNS and vascular remodeling.\n4. Analyses of mice focused on the impacts of the transition from a moderate (22C) to a severe (5C) cold challenge on endpoints related to neurovascular remodeling in lean animals. In contrast, samples of abdominal subcutaneous and visceral omental WAT depots were obtained from obese people living near thermoneutrality. The most compelling associations are a positive correlation between Slit3 and adiponectin expression in abdominal subcutaneous WAT and a negative correlation with macrophages in omental visceral WAT (which could be due to the increase in adiponectin). Analysis of markers of SNS tone (i.e. TH) or capillaries would help to connect Figure 8 to the rest of the manuscript. Otherwise, the manuscript would be strengthened by saving these interesting human data for another manuscript.\nMinor Concerns:\n1. The authors posit that “the co-regulation of neurovascular expansion by distinct Slit3 fragments offers a bifurcated yet harmonized mechanism to ensure a synchronized BAT response to environmental challenges”. Neurovascular remodeling would also be “harmonized” if the same ligand acted on the vasculature and sympathetic fibers. What are the benefits of having a bifurcated system? A 1-2 sentence explanation in the Discussion would suffice.\n2. The types of quantification should be consistent across all figures to facilitate comparisons between models. For example, Figure 6j shows the number of TH+ neurites/area while Figure 6l shows % TH staining. Either method is fine.\n3. Figure 1 highlights the identification of Robo4 expression in vascular and lymphatic endothelial cells but not adipocytes. Subsequently, the authors reinforce this point by showing that brown adipocytes cannot respond to Slit3 in vitro (Suppl Fig 6a-b). It is then surprising that the authors do not show any data from Robo4 KD. The authors should consider removing Robo4-related data or at least de-emphasizing it in the initial rationale for the experiments, if they cannot report results of Robo4 KD."}, "author_response": "We thank the reviewer for their thoughtful assessment of our manuscript.\nSpecific Points:\n1. The authors report that reduction of Slit3 leads to whitening of BAT under cold conditions but do not provide an explanation for this observation. Is BAT accumulating lipids released from lipolysis in other tissues? What are the weights of the inguinal and epididymal fat pads in these mice?\nWe thank the reviewer for this insightful question. We have included the weights of the inguinal and epididymal fat pads in Slit3 knockdown and control mice in the revised manuscript. As shown in Supplementary Figure 5b, there are no differences between the groups, indicating that whitening of BAT in Slit3 knockdown mice is not due to redistribution of lipids from white adipose tissue depots.\nInstead, gene expression analysis reduced expression of the lipolytic enzymes Atgl (Pnpla2) and Hsl (Lipe) in BAT from Slit3 knockdown mice (Figure 1i). This indicates that loss of Slit3 directly reduces lipolysis within BAT itself, leading to higher lipid accumulation. In addition, BAT from Slit3 knockdown mice showed decreased expression of thermogenic and mitochondrial genes and proteins. Together, these findings indicate that the whitening phenotype arises from a combination of reduced lipolytic capacity and impaired mitochondrial oxidation in BAT of Slit3 loss-of-function mice.\n2. Angiogenesis is assessed via isolectin staining. However, because adipocytes enlarge under certain conditions, vessel density per field can appear reduced. This must be controlled for by normalizing to adipocyte density—for example, expressing isolectin intensity relative to LipiTox or PLIN1 staining intensity. The same normalization should apply to TH staining.\nWe thank the reviewer for raising this point. We agree that adipocyte hypertrophy can influence the apparent density of vessels or sympathetic fibers within a given field. To address this concern, we normalized the IB4- and TH-positive areas to the Plin1-positive area. Notably, even after this normalization, the relative densities of IB4⁺ capillary vessels and TH⁺ sympathetic fibers remained significantly reduced in shSlit3 BAT, confirming that the observed effect is not simply an artifact of increased adipocyte size (Figure 1 in the point-by-point document).\nFigure 1. Quantification of IB4+ (left) and TH+ (right) areas normalized to the Plin1+ (adipocyte) area in BAT from mice treated with AAV-shSlit3 or scramble shRNA after 7 days of cold exposure (5 °C). Data are shown as mean ± SEM and were analyzed using unpaired two-sided Student’s t-tests.\nThat said, we respectfully believe that normalizing to adipocyte area is not biologically appropriate in this context. Several lines of evidence presented in the manuscript indicate that the whitening phenotype is driven by reduced sympathetic innervation and vascular density, which secondarily influences adipocytes and leads to whitening of BAT. Normalizing to adipocyte area would obscure these causal relationships by treating change in adipocyte size as an independent confounder, when in fact it is a consequence of reduced sympathetic input. Importantly, total BAT mass does not change between the groups (Supplementary Figure 5b). For these reasons, we consider field-based quantification the most physiologically relevant approach in this case.\n3. In shRNA-treated mice, BAT shows a marked increase in lipid droplet size. Is this phenotype recapitulated in the Pdgfra-Cre-driven Slit3 knockout (Fig. 3)? If so, isolectin and TH staining data must also be corrected for adipocyte density, as recommended for Fig. 2.\nH&E analysis of the BAT from Pdgfra-creERT2;Slit3flox/flox mice did not reveal a difference in lipid droplet size. These results have been included in the revised manuscript (Figure 3k). Accordingly, capillary density and sympathetic innervation are presented as a percentage of the total tissue area.\n4. Enforced AAV-mediated expression of the C-terminal or full-length Slit3 increases isolectin staining, suggesting enhanced angiogenesis (Fig. 5). However, adipocyte density should be reported, and the data normalized accordingly. The rationale for performing these experiments under conditions where endogenous Slit3 expression is already expected to be high (WT mice after 7 days of cold exposure) is unclear. A more compelling experiment would assess the effect of Slit3-C in non-cold-exposed mice, where any increase in innervation could be attributed directly to the construct.\nWe thank the reviewer for raising these points. We would like to clarify that overexpression of full-length Slit3 or the Slit3-N fragment, but not Slit3-C, resulted in increased isolectin B4 staining and angiogenesis. H&E analysis of BAT from mice overexpressing Slit3-FL, Slit3-N, or Slit3-C revealed no differences in adipocyte size. Therefore, to ensure consistency with the loss of function studies, we quantified both TH and IB4 staining as a percentage of the total tissue area.\nTo address the reviewer’s comment regarding the effects of Slit3 overexpression at room temperature, we have included new data from mice overexpressing full-length Slit3, Slit3-N, and Slit3-C and housed at room temperature (Supplementary Figure 11). Consistent with the results observed under cold conditions, overexpression of Slit3-FL and Slit3-C, but not Slit3-N, increased TH levels in BAT (Supplementary Figure 11). These effects were, however, more modest at room temperature compared to cold condition. Moreover, overexpression of Slit3-FL and Slit3-C at room temperature did not increase UCP1 protein levels, indicating that while Slit3-FL and Slit3-C can directly promote sympathetic innervation under basal conditions, their impact becomes more pronounced during cold exposure, when sympathetic activation and thermogenic demand are elevated (Supplementary Figure 11). This observation is also consistent with our loss-of-function studies, which show that although basal sympathetic innervation is reduced in Slit3-deficient BAT at room temperature, cold exposure further amplifies this deficit and its impact on BAT thermogenesis (Figures 1-2 and Supplementary Figures 4 and 7).\n5. The hypothesis that BMP1 functions as a protease for Slit3 is supported by data showing diminished proteolysis following siRNA-mediated Bmp1 knockdown or pharmacological inhibition. While these findings suggest a requirement for BMP1 in Slit3 cleavage, they do not definitively establish BMP1 as the responsible protease. Rigorous molecular characterization—including specificity and kinetic studies—would be required to confirm this. Similarly, while structural modeling and shRNA knockdown data support the role of Plxna1 as a receptor for Slit3-C, definitive evidence requires demonstration of direct receptor-ligand binding, including affinity and specificity measurements. Although these experiments may be beyond the scope of the current study, the limitations of the conclusions should be clearly acknowledged in the manuscript.\nWe appreciate the reviewer’s thoughtful feedback. We agree that detailed molecular characterization of protease activity and affinity or specificity measurements would provide valuable mechanistic insight. However, as the reviewer points out, these analyses are beyond the scope of the present study, which focuses on defining the physiological roles of Slit3 fragments in BAT. Regarding the Slit3-Plxna1 interaction, we present three independent lines of evidence, co-immunoprecipitation, structural modeling, and in vivo data, all supporting Plxna1 as a bona fide receptor for Slit3-C.\nIn line with the reviewer’s suggestion, we have added a statement to the manuscript acknowledging these limitations and outlining potential directions for future investigation.\nWe thank the reviewer for their thoughtful assessment of our manuscript.\nSpecific Comments:\n1. Figure 1C: The increase in Slit3-FL under cold conditions is shown, but it is unclear whether similar changes occur for Slit3-N or Slit3-C. If the antibody used is expected to recognize Slit3-N or Slit3-C, this should be clarified. Including this information as supplementary data would enhance transparency.\nThe antibody used in our study (AF3629, R&D Systems) recognizes the N-terminal region of the Slit3 protein, and we have revised the manuscript text to clarify this point. However, this antibody does not reliably detect Slit3 fragments in tissue samples. We systematically evaluated all commercially available Slit3 antibodies and generated two custom antibodies; however, none demonstrated sufficient specificity for reliable use. Therefore, we are currently unable to accurately assess changes in Slit3-N or Slit3-C levels under cold conditions due to this technical limitation.\n2. Supplementary Figure 2a–d: To support the claim that Slit3 is not expressed in macrophages under basal or cold conditions, immunofluorescence staining could provide additional confirmation.\nWe agree that immunofluorescence staining would provide valuable confirmation of Slit3 expression patterns. However, to date there are no reliable antibodies available for Slit3 immunofluorescence. We have tested several commercially available antibodies and even attempted to generate new ones, but unfortunately none produced specific or reproducible immunofluorescence signal.\n3. Supplementary Figure 2e: The use of a mouse adipocyte progenitor cell line to assess Slit3 expression during adipogenesis is informative. However, staining for Slit3 in both mature adipocytes and progenitors in BAT and WAT would provide more comprehensive validation.\nUnfortunately, as noted above, the lack of reliable antibodies for Slit3 immunofluorescence has prevented us from performing such experiments. We tested multiple commercially available antibodies and attempted to generate new ones, but none were effective for IF or IHC applications. Despite this limitation, the concordance of our mRNA expression data from single-cell transcriptomics, FACS-isolated primary cells, and in vitro-differentiated adipocyte progenitors strongly supports the validity of the conclusions.\n4. Figure 2g: A protein loading control is missing. Including this would strengthen the interpretation of the immunoblot data.\nWe have added a protein loading control and quantified TH levels relative to total protein to ensure accurate normalization.\n5. Figure 3a: Similarly, a loading control is needed. Given that Slit3 is expressed in multiple cell types in BAT, and that Slit3 expression is expected to be reduced but not absent in Slit3iDAPC tissue, it would be helpful to show Slit3 protein levels under both basal and cold conditions with appropriate controls.\nWe have incorporated new data quantifying Slit3 protein levels in BAT from Pdgfra-creERT2;Slit3flox/flox mice under both room temperature and cold conditions, as well as in ingWAT and pgWAT. Loading controls and total protein stainings are included (Figure 3a-h and Supplementary Data 8). These results reveal a significant reduction in Slit3 protein levels in BAT under room temperature and cold conditions, with the decrease being more pronounced after 7 days of cold exposure (Figure 3a-d).\n6. Figure 5g–h: Overexpression of Slit3-C also increases capillary density in BAT. This is unexpected given the fragment’s primary role in sympathetic innervation. A brief discussion of potential mechanisms would be valuable.\nWe thank the reviewer for raising this point. The previous version of the manuscript had a typo in the description of the results in Figure 5g-h. As correctly shown in the Figure 5g-h, overexpression of Slit3-C does not increase capillary density in BAT (the purple vs grey bars). This typo has now been corrected in the revised version.\n7. Figure 6d: The lack of co-immunoprecipitation between Plxna1 and Slit3-N supports the specificity of Plxna1 for Slit3-C. This point could be emphasized more clearly to strengthen the conclusion.\nSince overexpression of Slit3-N did not affect sympathetic innervation in BAT, and Plxna1 expression was detected specifically on sympathetic neurites, we do not anticipate a direct or physiologically relevant interaction between Plxna1 and Slit3-N in this context. Nevertheless, to explore this possibility in silico, we employed AlphaFold2 Multimer to model complexes between the extracellular domain of Plxna1 and the N-terminal region of Slit3. The resulting model predicted potential contacts between several Leucine-rich repeat (LRR) domains of Slit3-N and Plxna1. However, this predicted interaction is unlikely to represent a bona fide signaling event and may instead arise from the characteristic solenoid architecture of LRR domains, which commonly serve as structural scaffolds mediating generic protein-protein contacts rather than specific ligand-receptor interactions1.\n8. Figure 1i: The p-value for Dio2 is 0.062. While not statistically significant, the trend may still be biologically relevant and could be briefly discussed.\nWe have now mentioned Dio2 and other thermogenic genes in the manuscript text.\n9. Line 291: There appears to be a typo—“Slit3-FL” should be “Slit3-C.”\nAs shown in Figure 5, both Slit3-FL and Slit3-C significantly enhance sympathetic innervation in BAT. The section referenced by the reviewer discusses the effect of Slit3-FL overexpression in the presence or absence of Plxna1. Similar results for Slit3-C overexpression are described later in the text.\n10. Supplementary Figure 6e: The high-magnification images do not appear to align with the low-magnification views. Please verify and adjust if necessary.\nWe thank the reviewer for identifying the error in the figure. The high-magnification images for the two groups have been corrected and appropriately swapped in the revised version.\n11. Literature Context: Including a brief section summarizing the Slit-Robo signaling pathway and its known roles in neurogenesis and angiogenesis in the Introduction would provide helpful context for readers less familiar with this signaling axis.\nWe have now added a brief description of the Slit-Robo signaling pathway and its roles in neurogenesis, angiogenesis, and other processes to the Introduction.\nWe thank the reviewer for their thoughtful assessment of our manuscript.\nSlit3 KD reduced neurovascular density at room temperature (Suppl Fig 7), which is a mild cold challenge. Analyzing Slit3 KD or cKO models at thermoneutrality would be informative as to whether Slit3’s function is restricted to cold-induced expansion or also affects baseline structure.\nWe thank the reviewer for raising this point. To address it, we conducted additional experiments in which mice injected with Slit3 shRNAs or scramble control were housed under thermoneutral conditions (30 °C) for two weeks. Mice lacking Slit3 expression in BAT showed a comparable reduction in Ucp1 protein at thermoneutrality (Supplementary Figure 4d-f). The expression of other thermogenic and mitochondrial genes was also modestly reduced under these conditions (Supplementary Figure 4d). Notably, even when animals were maintained at thermoneutrality, loss of Slit3 led to a decrease in both capillary density and sympathetic innervation in BAT (Supplementary Figure 7e-h). These findings demonstrate the essential role of Slit3 in both establishing the neurovascular network of BAT under basal conditions and driving its expansion and remodeling during cold adaptation.\n2. Whereas APCS express the highest levels of Slit3, cold-induced increases in transcription are only observed in ECs (Suppl Fig 1d). Moreover, AAV-mediated Slit3 KD in BAT (Fig. 1e) produced a stronger reduction in body temperature after 5 hr at 5C than conditional KO in PdgfR+ APCs (Figure 3d) (KD = 33.5C vs KO= 36.5C). This raises the possibility that adipocyte-derived Slit3 helps to maintain the neurovascular architecture at baseline but that EC-derived Slit3 mediates cold-induced remodeling. Comparisons between adipocyte-and EC-specific Slit3 KOs at thermoneutrality and in response to a cold challenge would resolve this issue as well as issue #1.\nWe thank the reviewer for these comments. To evaluate the contribution of Pdgfra⁺ APCs to total Slit3 protein levels in BAT under basal and cold conditions, we have now quantified Slit3 protein in BAT from Pdgfra-creERT2;Slit3flox/flox mice housed at room temperature or exposed to cold (Figure 3a-d). These data reveal a marked reduction in Slit3 protein under both conditions, with a more pronounced decrease observed following 7 days of cold exposure. This reduction, together with the significant loss of capillary density and sympathetic innervation in BAT from cold-exposed Pdgfra-creERT2;Slit3flox/flox mice, underscores the essential role of Pdgfra⁺ APC-derived Slit3 in driving cold-induced neurovascular remodeling.\nThe differences in body temperature observed between the Pdgfra-creERT2;Slit3flox/flox and Slit3 knockdown models may reflect variations in experimental design, mouse strain, and genetic background. While we agree that generating and comparing an EC-specific Slit3 knockout model would provide additional insight, this lies beyond the scope of the present study, which focuses on defining the novel role of Pdgfra⁺ APC-derived Slit3, its proteolytic processing, and the distinct functions of its cleavage fragments through different receptors in coordinating the expansion of the BAT neurovascular network.\nWe have added a limitations section to the revised manuscript, in which we discuss the study’s limitations and acknowledge the potential contribution of EC-derived Slit3 to adipose tissue function and remodeling.\n3. While the data supporting a role for Slit3 in cold-induced neurovascular remodeling are compelling, the relationship with BAT thermogenesis is less clear. In the Slit3 KD, BAT temperature (Suppl Fig 3e) and body temperature (Fig 1e) are lower in the first 5 hours of cold exposure – this is before any cold-induced remodeling occurs. On the other hand, after 2 days of cold exposure, when Slit3 levels are high (Figure 1d), BAT temperature of KDs is the same as controls (Suppl Fig 3e). Additionally, Plxna1 KD in conjunction with Slit3-C overexpression prevents the cold-induced increase in SNS innervation (i.e. innervation looks like controls), but the BAT temperature curve is similar to that of the Plxna1 KD alone, which has lower SNS innervation (Figure 6 l,m). Unless these apparent inconsistencies can be definitively resolved, I would recommend removing the body/BAT temperature data and narrowing the claims to SNS and vascular remodeling.\nWe thank the reviewer for these insightful comments. While it is true that body temperature in Slit3 KD animals eventually normalizes after the first ~12 hours of cold exposure, this likely reflects the activation of compensatory thermoregulatory mechanisms such as shivering, which allow animals to survive chronic cold stress. Importantly, our molecular analyses show that Slit3 KD animals exhibit significant impairments in the activation of thermogenic gene and protein program in BAT after 7 days of cold exposure (Figure 1f-k). They also exhibit lower energy expenditure (Figure 1l-m), oxygen consumption, and carbon dioxide production when housed chronically in cold (Supplementary Figure 5). Collectively, these findings indicate that, despite the recovery of body temperature, loss of Slit3 disrupts BAT thermogenic capacity.\nFurthermore, to directly test whether impaired sympathetic innervation underlies the thermogenic defects in Slit3-deficient BAT, we designed an experiment to bypass SNS input by administering the β3-adrenergic receptor agonist CL-316,243. Slit3 knockdown animals treated with CL-316,243 for ten days exhibited fully intact induction of thermogenic, mitochondrial, and lipolytic gene programs (Supplementary Figure 7i). These results demonstrate that the intrinsic ability of brown adipocytes to respond to adrenergic stimulation remains uncompromised in the absence of Slit3. Thus, the primary role of Slit3 in BAT is not to modulate adipocyte-intrinsic thermogenic capacity, but rather to drive the proper neurovascular expansion necessary for effective cold-induced thermogenesis.\nWe have incorporated these new data and their description into the revised manuscript.\nRegarding the data in Figure 6l-m, we agree with the reviewer about the partial disconnect between sympathetic innervation levels and BAT temperature. We speculate that this may be due to additional roles of Plxna1 in sympathetic neurons beyond axon growth, potentially involving pathways downstream of semaphorin ligands. We have expanded the discussion to address the possible contribution of non-Slit3 ligands to Plxna1 signaling and their relevance to thermogenic regulation. While delineating the relative contribution of different ligands is beyond the scope of the current study, we recognize that Plxna1 likely has Slit3-independent roles in BAT physiology. We have revised the manuscript accordingly to clarify these points.\n4. Analyses of mice focused on the impacts of the transition from a moderate (22C) to a severe (5C) cold challenge on endpoints related to neurovascular remodeling in lean animals. In contrast, samples of abdominal subcutaneous and visceral omental WAT depots were obtained from obese people living near thermoneutrality. The most compelling associations are a positive correlation between Slit3 and adiponectin expression in abdominal subcutaneous WAT and a negative correlation with macrophages in omental visceral WAT (which could be due to the increase in adiponectin). Analysis of markers of SNS tone (i.e. TH) or capillaries would help to connect Figure 8 to the rest of the manuscript. Otherwise, the manuscript would be strengthened by saving these interesting human data for another manuscript.\nWe acknowledge the limitations of the human studies, including the differences in physiological state and depot differences due to the difficulties of accessing fresh BAT biopsies, which precludes direct measurement of sympathetic innervation or capillary density. However, we believe these data provide important complementary insight. Specifically, the observed associations between Slit3 expression and markers of adipose tissue health such as increased adiponectin and decreased macrophage abundance offer the first evidence that the Slit3 pathway may be relevant in regulation of human adipose tissue function and health. While these data are correlative and do not establish causality, we feel that they are biologically meaningful and add translational relevance to the mouse findings. We have revised the manuscript to better clarify these limitations and to more explicitly frame the human data as hypothesis-generating. We hope the reviewer agrees that including these results strengthens the manuscript by broadening its scope and potential impact.\nMinor Concerns:\n1. The authors posit that “the co-regulation of neurovascular expansion by distinct Slit3 fragments offers a bifurcated yet harmonized mechanism to ensure a synchronized BAT response to environmental challenges”. Neurovascular remodeling would also be “harmonized” if the same ligand acted on the vasculature and sympathetic fibers. What are the benefits of having a bifurcated system? A 1-2 sentence explanation in the Discussion would suffice.\nWe have amended to the Discussion to describe the potential benefits of a bifurcated signaling system. Specifically, we note that a bifurcated signaling system enables independent yet coordinated regulation of vascular and neuronal components, providing greater flexibility and temporal precision in adapting to environmental stimuli. In the context of Slit3, proteolytic processing introduces an additional level of regulation, as the full-length protein and its fragments exhibit distinct cell association and diffusion properties2. Notably, Slit3-C is exclusively detected in the media and not in the cell lysate, suggesting enhanced diffusibility and a capacity to mediate long-range effects on sympathetic innervation, whereas Slit3-FL and Slit3-N are more likely to function in a localized manner (Figure 4c and reference 2).\n2. The types of quantification should be consistent across all figures to facilitate comparisons between models. For example, Figure 6j shows the number of TH+ neurites/area while Figure 6l shows % TH staining. Either method is fine.\nWe thank the reviewer for this suggestion. To ensure consistency and facilitate comparisons between models, we have revised all quantifications to present data as the percentage of TH⁺ or IB4⁺ areas across all figures.\n3. Figure 1 highlights the identification of Robo4 expression in vascular and lymphatic endothelial cells but not adipocytes. Subsequently, the authors reinforce this point by showing that brown adipocytes cannot respond to Slit3 in vitro (Suppl Fig 6a-b). It is then surprising that the authors do not show any data from Robo4 KD. The authors should consider removing Robo4-related data or at least de-emphasizing it in the initial rationale for the experiments, if they cannot report results of Robo4 KD.\nWe thank the reviewer for this comment and suggestion. Ligand-receptor analysis using single-cell transcriptomic data identified the Slit3-Robo4 pair, which provides the rationale for investigating the role of Slit3 in BAT. We believe including this rationale helps readers understand the logic of the study. As prior work has established that Robo4 mediates the angiogenic effects of Slit family members through binding to the N-terminal region, in this study we focused on elucidating the novel Slit3C-Plxna1 interaction and its specific role in regulating sympathetic innervation within BAT. Collectively, our findings, together with prior literature, reveal distinct and complementary mechanisms of Slit3 signaling that enables coordinated regulation of neurovascular expansion and remodeling.\nReferences\n1. Kobe, B. & Kajava, A. V. The leucine-rich repeat as a protein recognition motif. Curr. Opin. Struct. Biol. 11, 725–732 (2001).\n2. Brose, K. et al. Slit proteins bind Robo receptors and have an evolutionarily conserved role in repulsive axon guidance. Cell 96, 795–806 (1999)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "Overall, the authors have addressed my concerns, and the manuscript is a valuable contribution to the field of brown adipose tissue biology. However, I feel they are making an inference error by not including normalized values. The author states:\n“That said, we respectfully believe that normalizing to adipocyte area is not biologically appropriate in this context. Several lines of evidence presented in the manuscript indicate that the whitening phenotype is driven by reduced sympathetic innervation and vascular density, which secondarily influences adipocytes and leads to whitening of BAT. Normalizing to adipocyte area would obscure these causal relationships by treating change in adipocyte size as an independent confounder, when in fact it is a consequence of reduced sympathetic input.”\nThe problem here is that the authors are confusing causality with measurement validity. They are committing a causal direction fallacy, by assuming that because they understand the causal chain, they should measure in a way that reflects that chain, rather than measuring a parameter in an objective way.\nThe functional vascularization of the tissue is the measurement of vascular density per cell (or per unit of tissue cellularity). The measurement question is independent of causality. Normalizing to adipocyte area doesn't claim adipocyte size is an independent confounder. It simply accounts for the fact that you're comparing tissues with different cellular densities. If anything, not normalizing obscures whether vascular changes are proportional to cellular changes or represent an additional effect.\nThe ideal solution is to pose the hypothesis (If sympathetic denervation → reduced vascularization → adipocyte enlargement), and provide measurements to answer two questions:\n• Is vascular density per unit area reduced? (answered without normalization)\n• Is vascular density per cell reduced? (requires normalization to cell number or area)", "reviewer_2": "The authors have satisfactorily addressed my previous concerns, except for the requested immunostaining for Slit3. This could not be completed due to the lack of a specific antibody, which is entirely understandable.\nI appreciate that the authors added a concise section summarizing the Slit-Robo signaling pathway and its established roles in neurogenesis and angiogenesis in the Introduction. This addition provides valuable context for readers who may be less familiar with this signaling axis. However, citing the major original papers in this area would further strengthen the manuscript and assist readers who wish to explore the related literature in greater depth.", "reviewer_3": "The authors performed additional experiments and analyses that strengthened their central claims. Studies in KDs at thermoneutrality support critical roles for Slit3 both in establishing the BAT neurovascular network of BAT and in driving its remodeling during cold exposure. Moreover, assessment of Slit3 levels in cKOs in the cold supports the idea that APCs are the primary source of Slit3. Finally, they demonstrated that brown adipocyte responses to adrenergic stimulation do not require Slit3. These experiments, as well as additions to the discussion, fully addressed all my concerns."}, "author_response": "We thank the reviewer for their positive evaluation and for their careful consideration of our study. In response to the reviewer’s comment, we have added new graphs that explicitly shows both tissue-level and adipocyte-normalized vascular and sympathetic measurements, as suggested. Specifically, we now present sympathetic innervation and vascular density quantified in two ways: normalized to total tissue area and normalized to adipocyte area (Perilipin1-positive area). These additional analyses are included for all shSlit3 studies (Figure 2b-c, Figure 2f-g, and Supplementary Figure 7), for experiments involving Slit3 fragment overexpression (Figure 5e-f and Figure 5h), and for comparisons of vascular and innervation density in wild-type mice at different temperatures (Supplementary Figure 6). This dual normalization directly allows assessment of (i) changes in vascular and sympathetic density per unit tissue area and (ii) changes relative to adipocyte content, addressing whether vascular alterations are proportional to cellular changes or represent an additional effect.\nCollectively, these results show that the reduced sympathetic innervation and vascular density are not secondary to adipocyte enlargement.\nFor the Plxna1 knockdown studies (Figures 6-7), Perilipin1 co-staining was not performed in the original experiments. Importantly, these conditions show no changes in BAT morphology or adipocyte size. After consultation with the editor, we therefore retained the original quantification normalized to total tissue area, as additional normalization would not alter interpretation in the absence of adipocyte size differences.\nIn summary, across all conditions, normalization to adipocyte area does not change the direction or significance of the observed effects, and the conclusions of the study remain unchanged. To ensure full transparency, we also explicitly state the quantification and normalization methods used in each figure legend.\nWe thank the reviewer for their positive evaluation and for their careful consideration of our study. We have now included additional references citing key papers and discoveries related to Slit-Robo signaling and its role in tissue development and remodeling.\nWe thank the reviewer for their positive evaluation and for their careful consideration of our study."}, {"round": 3, "reviewer_comments": {"reviewer_1": "The authors have addressed all my concerns."}, "author_response": ""}]

Decision letter