Early postnatal interactions between beige adipocytes and sympathetic neurites regulate innervation of subcutaneous fat

Figure 1.. Sympathetic innervation of iWAT is established during early postnatal development. (A) Schematic view of iWAT. Red lines represent main blood vessels. Using the lymph node and blood vessels as landmarks, iWAT depot is divided into the inguinal and dorsolumbar regions. Dorsolumbar region is further divided into medial and lateral subregions, hereafter referred to as the dorsomedial and dorsolateral regions. Dotted lines indicate boundaries of each region. (B–F) Representative images of iWAT from a P6 C57BL6/J mouse immunolabeled with TH (green) and CD31 (magenta). (B) Maximum intensity projection (MIP) from a 1000 μm z-stack. Arrows indicate convergence points where nerve fibers deviate from nerve bundles to establish blood vessel innervation. (C) High-magnification image of the indicated (*) convergence point in (B). Arrows indicate distinct features of sympathetic innervation in iWAT: (a) nerve fascicle, (b) blood vessel innervation, (c) a nerve fiber departing from a nerve fascicle to join blood vessel innervation. (D) High-magnification image showing sympathetic innervation from main blood vessel extending to arterioles or venules. (E–F) High-magnification images showing…

Figure 1—figure supplement 1.. Organization of sympathetic nervous system in iWAT at P2. (A–B) Representative whole-tissue maximum intensity projections (MIPs) of 50 μm z-stacks of iWAT from a P2 mouse. (A) TH (green) and CD31 (magenta). (B) TH only. (C–F) High-magnification images showing distinct sympathetic innervation patterns: (a) fibers wrapping around blood vessel, (b) nerve fascicle, and (c) fibers reaching lobules. (C) TH (green), CD31 (magenta). (D) TH (green), tissue autofluorescence (magenta). (E) TH. (F) CD31. Lymph nodes are indicated as LN. Scale bars are indicated.

Figure 1—figure supplement 2.. Organization of sympathetic nervous system in iWAT at P6. (A–B) Single channel images corresponding to Figure 1B: (A) TH, (B) CD31. (C–D) Single channel images corresponding to Figure 1C: (C) TH, (D) CD31. (E–F) Single channel images corresponding to Figure 1D: (E) TH, (F) CD31. (G–H) Single channel images of Figure 1E: (G) TH, (H) CD31. (I–J) Single channel images of Figure 1F: (I) TH, (J) CD31. Scale bars are indicated. (K–L) Photographs of iWAT with various regions labeled. Dotted lines correspond to region boundaries shown in Figure 1A and cutting sites to isolate various regions for molecular analysis as described in Materials and methods. Lymph nodes are indicated as LN.

Figure 1—figure supplement 3.. Development of sympathetic parenchymal innervation in iWAT. High-magnification images corresponding to Figure 1G,H,I & J. MIPs of 50 μm z-stacks from iWAT of (A–B) P6, (C–D) P10, (E–F) P14, (G–H) P21, and (I–J) P28 immunolabeled with TH. (A, C, E, G, and I) Images of the dorsolumbar region. (B, D, F, H, and J) Images of the inguinal region. Scale bars are indicated.

Figure 2.. UCP1+ beige adipocytes emerge during early postnatal development. (A–J) Representative optical sections of iWAT from P6, P10, P14, and P21 C57BL6/J mice immunolabeled with UCP1. (A) Whole-tissue optical section of a P6 iWAT. (B) High-magnification view of the boxed inguinal region in (A). (C) Whole-tissue optical section of a P10 iWAT. (D) High-magnification view of the boxed inguinal region in (C). (E) Whole-tissue optical section of a P14 iWAT. (F) High-magnification view of the boxed dorsolumbar region in (E). (G) High-magnification view of the boxed inguinal region in (E). (H) Whole-tissue optical section of a P21 iWAT. (I) High-magnification view of the boxed dorsolumbar region in (H). (J) High-magnification view of the boxed inguinal region in (H). Lymph nodes are indicated as LN. Dotted lines indicate tissue boundaries based on tissue autofluorescence signals shown in Figure 2—figure supplement 1D–H. Scale bars are indicated. All imaging studies were performed in at least three independent animals, and representative images are shown.

Figure 2—figure supplement 1.. UCP1+ beige adipocytes emerge during early postnatal development. (A–C) Representative optical sections of iWAT from a P28 C57BL6/J mouse immunolabeled with UCP1. (A) Whole-tissue optical section of a P28 iWAT. (B) High-magnification view of the boxed dorsolumbar region in (A). (C) High-magnification view of the boxed inguinal region in (A). (D–H) Two-channel images corresponding to Figure 2A,C,E,H, and (A). Tissue autofluorescence (magenta) and UCP1 (green) are shown. Lymph node is indicated as LN. Scale bars are indicated. Imaging was performed in three independent animals, and representative images are shown.

Figure 2—figure supplement 2.. UCP1+ beige adipocytes emerge during early postnatal development. (A–C) Normalized gene expression of dorsolumbar vs. inguinal regions in iWAT from P6, P8, P10, P12, P14, P16, P21, and P28 C57BL6/J mice, n = 4. Representative genes involved in (A) the thermogenic program, (B) transcriptional regulation of beige adipocyte development and function, and (C) the adipogenic program. Data are presented as mean ± SEM and analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. * denotes p<0.05 dorsolumbar vs. inguinal region at each time point. # denotes p<0.05 inguinal region at P14 in comparison to inguinal regions at other time points.

Figure 2—figure supplement 3.. UCP1+ beige adipocytes and dense sympathetic parenchymal innervation emerge together during early postnatal development. (A–J) High-magnification MIPs of 50 μm z-stacks from iWAT of (A–B) P6, (C–D) P10, (E–F) P14, (G–H) P21, and (I–J) P28 immunolabeled with TH (green) and UCP1 (magenta), corresponding to Figure 1—figure supplement 3. (K–L) High-magnification MIPs of 50 μm z-stacks from iWAT of a P14 C57BL6/J mouse housed at thermoneutrality (30°C), immunolabeled with TH (green) and UCP1 (magenta). (A, C, E, G, I and K) Images of the dorsolumbar region. (B, D, F, H, J and L) Images of the inguinal region. Scale bars are indicated.

Figure 3.. Prdm16 regulates the emergence of early postnatal beige adipocytes and dense sympathetic parenchymal innervation. (A–D) Representative images of iWAT from (A–B) control and (C–D) cPrdm16KO mice at P14, immunolabeled with TH. MIPs from 50 μm z-stacks are shown. (E–H) Representative images of iWAT from (E–F) control and (G–H) cPrdm16KO mice at P21, immunolabeled with TH. MIPs from 50 μm z-stacks are shown. (A, C, E and G) Images of the dorsolumbar region. (B, D, F and H) Images of the inguinal region. Scale bars are indicated. Imaging was performed in at least three independent animals per genotype, and representative images are shown. (I) Normalized gene expression of dorsolumbar vs. inguinal regions in iWAT from control and cPrdm16KO mice at P6, P14, and P21, n = 2–3. Representative genes involved in the thermogenic program are shown. Data are presented as mean ± SEM and analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. * denotes p<0.05 dorsolumbar vs. inguinal regions of control samples at each time point. # denotes p<0.05 inguinal regions of control vs. cPrdm16KO samples at each time point. & denotes p<0.05 inguinal region at P14 in comparison to…

Figure 3—figure supplement 1.. Prdm16 regulates the emergence of early postnatal beige adipocytes and dense sympathetic parenchymal innervation. (A–D) Representative images of iWAT from (A–B) control and (C–D) cPrdm16KO mice at P6, immunolabeled with TH. MIPs from 50 μm z-stacks are shown. (E–H) Overlay of TH (green) and UCP1 (magenta), corresponding to (A–D). (A, C, E, and G) Images of the dorsolumbar region. (B, D, F, and H) Images of the inguinal region. Scale bars are indicated. Imaging was performed in at least three independent animals per genotype, and representative images are shown. (I) Normalized gene expression of dorsolumbar vs. inguinal regions in iWAT from control and cPrdm16KO mice at P6, P14, and P21, n = 2–3. Representative genes involved in the adipogenic program are shown. Data are presented as mean ± SEM and analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test.

Figure 3—figure supplement 2.. Prdm16 regulates the emergence of early postnatal beige adipocytes and dense sympathetic parenchymal innervation. (A–D) Overlay of TH (green) and UCP1 (magenta), corresponding to Figure 3A–D. (E–H) Overlay of TH (green) and UCP1 (magenta), corresponding to Figure 3E–H. (I) qPCR analysis of Prdm16 mRNA levels in inguinal and dorsolateral regions of control and cPrdm16KO mice at P14, n = 4. Data are normalized to cPrdm16KO dorsolateral group, presented as mean + SEM, and analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. **** denotes p<0.0001 control inguinal vs. the rest of the groups. (J) Western blot of PRDM16 and lamin A/C of various fat depots from WT and cPrdm16KO 8-week-old mice. (K–L) Representative images of iBAT sections from (K) control and (L) cPrdm16KO 8-week-old mice, immunolabeled with TH. MIPs of 5 μm z-stacks are shown. Scale bars are indicated.

Figure 4.. Prdm16 deletion during early development causes decreased sympathetic parenchymal innervation. (A) Schematic representation of the genetic components of the control and iPrdm16KO mice. iPrdm16KO mice carry floxed Prdm16 alleles (Prdm16lox/lox), two copies of AdipoqrtTA transgene, and one copy of TRE-Cre transgene. Littermates carrying only Prdm16lox/lox and AdipoqrtTA (i.e. Cre-) were used as the control animals. (B) Control (Cre-) and iPrdm16KO (Cre+) mice housed at RT (23°C) were kept on a doxycycline-containing chow diet from E14 until P21 before being switched to a regular chow diet for another 2 weeks. Control and iPrdm16KO mice were either maintained at RT (23°C) or exposed to cold (8°C) for 2 days at P35. (C) Normalized gene expression of inguinal regions from control and iPrdm16KO (perinatal, 5 weeks) mice exposed to RT or cold, n = 3–5. Data are presented as mean + SEM and analyzed by two-way ANOVA followed by Bonferroni’s multiple comparisons test. * denotes p<0.05 iKO (perinatal, 5 weeks) vs. control samples at RT. # denotes p<0.05 iKO (perinatal, 5 weeks) vs. control samples at cold. & denotes p<0.05 cold- vs. RT-exposed control samples. (D–I) Representative optical…