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. 2022 Jun 22;11:e78496. doi: 10.7554/eLife.78496

Figure 5. BMAd-Pnpla2 deficiency causes extensive alterations to the bone marrow transcriptome only when coupled with CR.

Male control and BMAd-Pnpla2-/- mice at 24 weeks of age were either fed AL or underwent 30% CR for 6 weeks. Distal tibial cBMAT was flushed and cBMAT from two mice was pooled as one sample for RNAseq analyses (n of 3 or 4 per treatment). (A) Differential genes with our criteria (padjj <0.05 and |Log2 fold change|>1) between BMAd-Pnpla2+/+ CR and BMAd-Pnpla2+/+ad libitum (AL) were grouped into 4 clusters. (B) Genes different between BMAd-Pnpla2+/+ CR and BMAd-Pnpla2+/+ AL were ordered from maximum to minimum log2 fold change (red dots), and compared to corresponding data from BMAd-Pnpla2-/- CR versus BMAd-Pnpla2-/- AL (blue dots). Venn diagram shows the differential genes between BMAd-Pnpla2+/+ CR versus BMAd-Pnpla2+/+ AL BMAT; and BMAd-Pnpla2-/- CR versus BMAd-Pnpla2-/- AL BMAT. (C) Pathway analyses of genes significantly changed by CR in BMAd-Pnpla2+/+ mice, but not in CR mice lacking Pnpla2 (indicated by * area in panel B). Pathways further analyzed by heatmap indicated with blue arrows. (D-F) Expression Z-scores of genes related to adipogenesis (D), skeletal system development (E) and extracellular matrix organization (F) were shown as heatmap. Effects of genotype and diet, and their interactions were analyzed by three-way ANOVA.

Figure 5—source data 1. BMAd-Pnpla2 deficiency causes extensive alterations to the bone marrow transcriptome only when coupled with CR.
elife-78496-fig5-data1.xlsx (230.4KB, xlsx)

Figure 5.

Figure 5—figure supplement 1. CR causes profound changes in BMAT transcriptome.

Figure 5—figure supplement 1.

Twenty-four weeks old male mice underwent 30% CR for 6 weeks. Distal tibial cBMAT was flushed and cBMAT from two mice were pooled as one sample for RNA sample preparation. High-quality RNA samples were submitted for RNAseq analyses. (A) Principal component analysis (PCA) plot shows the distinct transcriptional characters in CR groups with (purple dots) or without (aqua dots) Pnpla2 in BMAds. (B) Volcano plots show the differential genes with padj <0.05 & |Log2 fold change|>1 in comparisons between BMAd-Pnpla2+/+ CR versus ad libitum (AL) (left), BMAd-Pnpla2-/- versus BMAd-Pnpla2+/+ at AL (middle) and BMAd-Pnpla2-/- versus BMAd-Pnpla2+/+ at CR (right). (C-F) Differential genes from comparison between BMAd-Pnpla2+/+ CR versus AL were grouped into 4 clusters according to the alteration patterns. Pathway analyses were performed on each cluster except cluster 2, which gene set was not enriched in any pathways.
Figure 5—figure supplement 2. BMAd-Pnpla2 deficiency causes extensive alterations to the bone marrow transcriptome only when coupled with CR.

Figure 5—figure supplement 2.

Twenty-four weeks old male mice underwent 30% CR for 6 weeks. Distal tibial cBMAT was flushed and cBMAT from two mice were pooled as one sample for RNA sample preparation. High-quality RNA samples were submitted for RNAseq analyses. (A) Pathway analysis of gene set that respond to CR independent of Pnpla2 deficiency in BMAds, indicated by * area. (B) qPCRs were performed to confirm the changes of adipogenesis genes in BMAT. Data are expressed as mean ± SD. * indicates p<0.05 with two-way ANOVA analysis followed by Šídák’s multiple comparisons test. (C-D) Heat maps for genes related to fatty acid biosynthesis (C) and myeloid leukocyte differentiation (D). (E) Collagen genes that upregulated by CR in BMAd-Pnpla2+/+ mice.
Figure 5—figure supplement 2—source data 1. BMAd-Pnpla2 deficiency causes extensive alterations to the bone marrow transcriptome only when coupled with CR.
Figure 5—figure supplement 3. BMAd-Pnpla2 deficiency alters gene expression in response to CR.

Figure 5—figure supplement 3.

Male mice at 24 weeks of age underwent 30% CR for 6 weeks. Distal tibial cBMAT was flushed and cBMAT from two mice were pooled as one sample for RNA sample preparation. High-quality RNA samples were submitted for RNAseq analyses. (A) Differential genes with our criteria (padj <0.05 and |Log2 fold change|>1) between BMAd-Pnpla2-/- CR and BMAd-Pnpla2+/+CR were grouped into three clusters. (B-C) Pathway analyses were performed on cluster 2 (B). Heat maps for genes related to hematopoietic progenitor cell differentiation and positive regulation of osteoblast differentiation (C). (D-E) Pathway analyses were performed on cluster 3 (D). Heat maps for genes related to regulation of lipid catabolic process and leukocyte migration (E).