Zfp423 expression identifies committed preadipocytes and localizes to adipose endothelial and perivascular cells

Abstract

Progress has been made in elucidating the cell-surface phenotype of primary adipose progenitors; however, specific functional markers and distinct molecular signatures of fat depot-specific preadipocytes have remained elusive. In this study, we label committed murine adipose progenitors through expression of GFP from the genetic locus for Zfp423, a gene controlling preadipocyte determination. Selection of GFP-expressing fibroblasts from either subcutaneous or visceral adipose-derived stromal vascular cultures isolates stably committed preadipocytes that undergo robust adipogenesis. Immunohistochemistry for Zfp423-driven GFP expression in vivo confirms a perivascular origin of preadipocytes within both white and brown adipose tissues. Interestingly, a small subset of capillary endothelial cells within white and brown fat also express this marker, suggesting a contribution of specialized endothelial cells to the adipose lineage. Zfp423(GFP) mice represent a simple tool for the specific localization and isolation of molecularly defined preadipocytes from distinct adipose tissue depots.

Figure 1. Derivation and characterization of Zfp423^GFP transgenic mice

(A) Genomic structure of Zfp423^GFP transgene modified from bacterial artificial chromosome (BAC) RP23-102G4. Coding sequence of enhanced GFP (eGFP) followed by a polyadenylation signal (polyA) was inserted into the initiation codon of Zfp423 located in exon 1. Proper targeting of the BAC was verified by sequencing and PCR analysis using primers as shown (P1–P4). (B) Quantitation of GFP+ cells in cultures of epididymal SV cells passaged 3–4 times after initial isolation. Cells were defined as GFP+ (shown in green) if fluorescent intensity was clearly greater than background fluorescence levels in wild-type (WT) cultures (shown in red) (See Supplementary Figure 1) ~50% of the cells within the SV cultures from this depot are GFP⁺. (C) Quantitation of GFP+ cells in cultures of inguinal SV cells passaged 3–4 times after initial isolation. ~85% of the cells within the SV cultures from this depot are GFP⁺ (D) Relative mRNA levels of GFP and Zfp423 in purified GFP+ and GFP− cells from epididymal SV cultures. (E) Relative mRNA levels of GFP and Zfp423 in purified GFP+ and GFP− cells from inguinal SV cultures. n=3 replicates

Figure 2. GFP-expressing cells from both subcutaneous and visceral SV cultures are functional preadipocytes

(A) Oil red-O staining of purified GFP− and GFP+ cells from epididymal SV cultures 6 days following the induction of adipocyte differentiation. (B) Expression of adipocyte selective genes in the differentiated cultures shown in (A). (C) Oil red-O staining of purified GFP− and GFP+ cells from inguinal SV cultures 6 days following the induction of adipocyte differentiation. (D) Expression of adipocyte selective genes in the differentiated cultures shown in (C). n=3 replicates.

Figure 3. Elucidation of a core preadipocyte gene program

(A, B) M vs. A plot of gene expression data obtained from microarray analysis of GFP+ and GFP− SV cells from inguinal or epididymal depots. “M” represents intensity ratio [log₂ GFP⁺ − log₂ GFP⁻] and “A” represents average intensity value of the gene across all samples [1/2 × (log₂ GFP⁺ + log₂ GFP⁻)]. Red spots represents genes significantly differentially expressed greater than 2-fold (p< 0.05). (C) Venn diagram illustrating overlap between gene signatures derived from expression analysis of epididymal and inguinal GFP+ and GFP− cultures. (D) Real-time PCR confirmation of the 12 genes depicted in (B) as being preadipocyte enriched genes. (E) Real-time PCR confirmation of the 10 genes depicted in (B) as being enriched in non-adipogenic GFP− SV cells. n=3 replicates.

Figure 4. GFP+ preadipocytes reside in the adipose vasculature as a subset of both pericytes and capillary endothelial cells

(A,B) Confocal images of adult inguinal WAT stained with antibodies recognizing GFP (red) and the endothelial cell protein CD31 (green), with nuclei counterstained with DAPI. In (A) note the expression of GFP in mature adipocytes and in some blood vessels (*). In (B) note the expression of GFP in a subset of perivascular cells (arrow) and in a subset of endothelial cells (arrowhead) of the blood vessel highlighted in (A). (C) Confocal image of developing inguinal WAT from postnatal day 4 mice stained with antibodies recognizing GFP (red) and the endothelial cell protein CD31 (green). Note the expression of GFP in a subset of perivascular cells (arrow) and in a strong subset of endothelial cells (arrowhead) even before the full development of mature adipocytes at this stage. (D,E) Confocal images of embryonic day 18.5 interscapular BAT stained with the same antibodies shown in (A–C). In (D) note the expression of GFP in mature adipocytes and in numerous perivascular cells (arrows). In (E) note the expression of GFP in a subset of endothelial cells (arrowhead). (F) Confocal image of skeletal muscle directly adjacent to the interscapular BAT shown in (D–E). Note the absence of GFP+ cells in the vasculature of this tissue.