RESEARCH ARTICLE
PDGFRα controls the balance of stromal and adipogeni c cells
during adipose tissue organogenesis
Chengyi Sun
1,2
, William L. Berry
2
and Lorin E. Olson
1,2,
*
ABSTRACT
Adipose tissue is distributed in depots throughout the body with
specialized roles in energy storage and thermogenesis. PDGFRα is
a marker of adipocyte precursors, and increased PDGFRα activity
causes adipose tissue fibrosis in adult mice. However, the function
of PDGFRα during adipose tissue organogenesis is unknown. Here,
by analyzing mice with juxtamembrane or kinase domain point
mutations that increase PDGFR α activity (V561D or D842V), we
found that PDGFRα activation inhibits embryonic white adipose
tissue organogenesis in a tissue-autonomous manner. By lineage
tracing analysis, we also found that collagen-expressing precursor
fibroblasts differentiate into white adipocytes in the embryo. PDGFRα
inhibited the formation of adipocytes from these precursors while
favoring the formation of stromal fibroblasts. This imbalance between
adipocytes and stromal cells was accompanied by overexpression of
the cell fate regulator Zfp521. PDGFRα activation also inhibited the
formation of juvenile beige adipocytes in the inguinal fat pad. Our data
highlight the importance of balancing stromal versus adipogenic cell
expansion during white adipose tissue development, with PDGFRα
activity coordinating this crucial process in the embryo.
KEY WORDS: Platelet-derived growth factor, Adipocyte,
Lipodystrophy, Myf5, Cell fate, Mouse
INTRODUCTION
There are two main types of adipose tissue in mammals: white
adipose tissue (WAT) and brown adipose tissue (BAT). WAT
contains unilocular adipocytes with a large, single lipid droplet for
energy storage (Rosen and MacDougald, 2006; Rosen and
Spiegelman, 2014). BAT contains multilocular adipocytes that
dissipate the electrochemical gradient in mitochondria to generate
heat (Harms and Seale, 2013; Sidossis and Kajimura, 2015; Schulz
and Tseng, 2013). Recently, a third kind of adipocyte – the beige
adipocyte – has been identified within WAT depots (Wu et al.,
2012, 2013). Beige adipocytes have low basal levels of thermogenic
activity, which can be increased by low temperature or beta-
adrenergic stimulation. The adipose tissue is composed of mature
adipocytes and a heterogeneous population of stromal-vascular cells
that includes preadipocytes, stromal fibroblasts, vascular cells and
immune cells. Fibroblasts and preadipocytes are closely related
mesenchymal cells, although the former are more specialized
for collagen secretion and the latter are specialized for generating
new adipocytes. Importantly, the extracellular matrix (ECM)
composition of adipose tissue must be carefully controlled to
allow hypertrophic expansion of the lipid-storing compartment.
Improper ECM remodeling leads to fibrosis, which is associated
with metabolic dysfunction as excess ECM impairs adipocyte lipid-
handling mechanisms (Sun et al., 2013).
Obesity is the accumulation of excess adipose tissue to the extent
that it creates a risk fo r insulin resistance, type 2 diabetes and other
diseases. Lipodystrophy – the absence or degeneration of body fat –
is also a cause of severe metabolic dysfunction. Understanding how
adipose tissue develops and changes over time is essential to
identifying new biomarkers and therapeutic targets to address these
diseases. In the adult, adipose tissue mass increases by two
mechanisms: adipocyte hypertrophy, which adds lipid into existing
adipocytes, and adipocyte hyperplasia, which is the differentiation
of new adipocytes (Jo et al., 2009). However, adipose tissue
organogenesis is still poorly understood and the exact mechanisms
remain to be discovered (Gesta et al., 2007; Han et al., 2011; Hong
et al., 2015; Hudak et al., 2014; Jiang et al., 2014). A specific
question that has not been explored is how mesenchymal precursor
cells adopt an adipocyte fate versus a stromal fibroblast fate to
achieve the correct balance between lipid-storing and stromal
compartments during organogenesis.
Platelet-derived growth factor (PDGF) is an important
extracellular signal for developing mesenchymal cells. PDGF
binds the receptor tyrosine kinases PDGFRα and PDGFRβ, which
are tightly regulated by an autoinhibitory allosteric conformation.
PDGF binding induces receptor dimerization, which relieves this
autoinhibition and initiates tyrosine kinase activity to induce
downstream signal transduction pathways (Tallquist and
Kazlauskas, 2004). Knockout of either receptor has revealed
crucial roles for PDGFRα and PDGFRβ in the development of
vasculature, craniofacial structures, skeleton and other organs
(Andrae et al., 2008; French et al., 2008; Hoch and Soriano,
2003; Soriano, 1994, 1997). PDGFRα is commonly used as a
marker for fibroblasts and undifferentiated mesenchymal cells, and
it is not expressed by differentiated adipocytes. Recent studies have
shown that all adipocytes of juvenile and adult mice are derived
from PDGFRα-expressing precursors (Berry and Rodeheffer, 2013;
Joe et al., 2010; Lee et al., 2012). However, most of our knowledge
about PDGF signaling and adipogenesis comes from cell culture
experiments (Artemenko et al., 2005; Fitter et al., 2012; Vaziri and
Faller, 1996), and the function of PDGFRα in adipose tissue
organogenesis remains to be elucidated in vivo.
We recently found that PDGFRα activation in specific nestin
+
fibro-adipogenic progenitor cells causes adult WAT fibrosis by
converting progenitor cells into ECM-producing fibroblasts.
PDGFRα activation also blocks the differentiation of nestin
+
progenitors into adipocytes in vitro (Iwayama et al., 2015),
suggesting that PDGFRα regulates the balance between
adipogenic and non-adipogenic mesenchymal cell populations.
Received 30 January 2016; Accepted 9 November 2016
1
Cardiovascular Biology Program, Oklahoma Medical Research Foundation,
Oklahoma City, OK 73104, USA.
2
Department of Cell Biology, University of
Oklahoma Health Sciences Center, Oklahoma City, OK 73104, USA.
*Author for correspondence (lorin-olson@omrf.org)
L.E.O., 0000-0003-2168-7836
83
© 2017. Published by The Company of Biologists Ltd
|
Development (2017) 144, 83-94 doi:10.1242/dev.135962
DEVELOPMENT
However, nestin
+
precursors do not contribute to embryonic
adipogenesis that generates newborn fat depots. Homeostatic
adipogenesis in the adult and developmental adipogenesis in the
embryo are likely to be regulated by different mechanisms. To
address the role of PDGFRα in embryonic adipogenesis, we have
now analyzed mouse embryos and pups with constitutive activating
mutations in PDGFRα during the time period of adipose tissue
organogenesis. We found that PDGFRα regulates the balanced
formation of stromal and lipid-storing compartments durin g WAT
organogenesis. PDGFRα activation promoted the accumulation of
ECM as the result of an enlarged stromal fibroblast population,
resulting in lipodystrophy. Furthermore, preadipocyte commitment
was disrupted in accordance with overexpression of the anti-
adipocyte commitment factor Zfp521 and loss of downstream pro-
adipogenic transcription factors. These findings identify PDGFRα
as a regulator of cell commitment within the early fibroblast-
adipocyte lineage that promotes a stromal fibroblast fate at the
expense of generating adipocytes.
RESULTS
Strong PDGFRα activation ablates WAT but not BAT in Myf5-
D842V mutants
PDGFRα
D842V
is a strongly hyperactivated isoform associated with
human gastrointestinal stromal tumors and inflammatory fibroid
polyps (Corless et al., 2004; Schildhaus et al., 2008). Here, we
generated mice expressing this mutation in a tissue-specific manner
by crossing Myf5-Cre mice with Cre/lox-inducible lox-stop-lox-
PDGFRα
D842V
knockin mice. These knockin mice express
PDGFRα
D842V
from the endogenous Pdgfra gene after Cre/lox
recombination removes an intervening stop cassette (Olson and
Soriano, 2009). It is well established that Myf5-Cre targets cells that
give rise to skeletal muscle and interscapular BAT (iBAT) (Seale
et al., 2008). Myf5-Cre also targets inte rscapular WAT (iWAT) and
retroperitoneal WAT (rWAT) but not inguinal WAT (ingWAT) or
perigonadal WAT (pWAT) (Sanchez-Gurmaches and Guertin,
2014; Sanchez-Gurmaches et al., 2012). Myf5-Cre;PDGFRα
+/D842V
mutants (hereafter referred to as Myf5-D842V) were viable,
although an overall smaller body size became apparent around
postnatal day (P)5. Upon dissection at P18 it was very clear that
Myf5
+
iWAT and rWAT were missing in Myf5-D842V mutants
(Fig. 1A,C), but the Myf5
neg
ingWAT and pWAT in Myf5-D842V
mutants appeared similar in volume to those of control littermates
(Fig. 1B,D). The absolute weight of ingWAT was slightly decreased
in Myf5-D842V mutants, but the relative weight normalized to total
body weight was the same as for control mice (Fig. 1E,F).
Morphological analysis of these depots revealed that iWAT and
rWAT in Myf5-D842V mutants were replaced by a tiny remnant of
stromal tissue (Fig. 1G,I, arrowheads). By contrast, ingWAT and
pWAT were fully expanded and indistinguishable between mutants
and controls (Fig. 1H,J). There was also no difference in the
expression of the adipocyte marker genes Ap2 (also known as
Fabp4) and adipsin (Adn; also known as Cfd) in mutant versus
control ingWAT (Fig. S1A). Therefore, the effects of specific
ablation of iWAT and rWAT of the Myf5
+
lineage, without affecting
ingWAT or pWAT of the Myf5
neg
lineage, strongly suggest that
PDGFRα activation disrupts WAT organogenesis in a tissue-
autonomous manner. We could not perform gene expression
analysis on the interscapular and retroperitoneal stromal remnant
of Myf5-D842V mice owing to the exceedingly small tissue size.
In contrast to iWAT and rWAT, iBAT was not ablated in Myf5-
D842V mutants (Fig. 1A). The absolute weight of Myf5-D842V
iBAT was slightly decreased, but the relative weight normalized to
total body weight was the same as for control mice (Fig. 1E,F).
There was also no difference in the expression of Ap2 and Adn or in
the brown adipocyte marker Ucp1 in mutant versus control iBAT
(Fig. S1B). We expected PDGFRα
D842V
to be expressed in iBAT
because its expression is under the control of the endogenous
Pdgfra gene, which is expressed in iBAT. To confirm this, we
compared PDGFRα phosphorylation in cultured iBAT and iWAT
stromal vascular cells isolated from Myf5-D842V and control mice.
We found that cells from iBAT have a lower basal level of total
PDGFRα expression compared with cells isolated from iWAT.
However, in Myf5-D842V mutants, cells from both iBAT and
iWAT exhibited constitutively phosphorylated PDGFRα, reflecting
expression of the activated PDGFRα
D842V
isoform (Fig. S1C).
Therefore, it is poss ible that the difference in phenotype between
iBAT and iWAT is related to the lower expression of PDGFRα in
iBAT. Skeletal muscle was not analyzed in our study.
Weak PDGFRα activation causes lipodystrophy and fibrosis
As the WAT phenotype in Myf5-D842V mutants was not amenable
to gene expression analysis owing to a lack of tissue, we undertook a
detailed examination of a less severe WAT phenotype.
PDGFRα
V561D
is a weakly hyperactivated isoform seen in human
tumors and polyps at a lower frequency than PDGF Rα
D842V
.In
previous work we showed that PDGFRα
V561D
generates weaker
phenotypes than PDGFRα
D842V
when activated in adult mice via a
tamoxifen-inducible Cre or in development via an epiblast-specific
Cre (Olson and Soriano, 2009). Here, we generated mice expressing
the V561D mutation in all adipose tissues by crossing Sox2-Cre
with lox-stop-lox-PDGFRα
V561D
knockin mice, where
PDGFRα
V561D
is expressed from the endogenous Pdgfra gene
after Cre/lox recombination. Sox2-Cre targets the epiblast and
therefore creates a germline Pdgfra mutation in all tissues of the
embryo proper (Hayashi et al., 2002). Similar to previous work
using Meox2-Cre to target the epiblast (Olson and Soriano, 2009),
Sox2-Cre;PDGFRα
+/V561D
mice (hereafter referred to as Sox2-
V561D) were viable for ∼3 weeks.
At P18, Sox2 -V561D mutants exhibited smaller WAT depots
than Sox2-Cre;PDGFRα
+/+
control mice. In controls, iWAT
covered and partially concealed the underlying iBAT, but Sox2-
V561D iBAT was left exposed by smaller and thinner iWAT
(Fig. 2A). In Sox2-V561D mutants ingWAT was also reduced and
lacked the beige color normally exhibited by control ingWAT at this
time point (Fig. 2B). rWAT and pWAT were absent or nearly absent
when assessed by anatomical examination in mutant mice (Fig. 2C,
D). iWAT and ingWAT in mutant mice weighed ∼90% less than
littermate control tissue at P18, both in absolute weight and when
normalized to total body weight (Fig. 2E,F). However, consistent
with observations in Myf5-D842V mutants, iBAT weight was not
diminished by PDGFRα activation.
We performed Hematoxylin and Eosin (H&E) staining of
paraffin sections to characterize adipose tissue morphology at
P18. The smaller iWAT, ingWAT and rWAT in Sox2-V561D
mutants contained adipocytes that were smaller than con trol
adipocytes (Fig. 3A,B,E). The stromal margin of the iWAT and
ingWAT depots appeared more ECM-rich by H&E staining
(Fig. 3A,B, arrows), as did the interstitial space between
adipocytes. Mutant rWAT was replaced by a stromal remnant
(Fig. 3C). Adipocytes in mu tant pWAT were difficult to identify
morphologically (Fig. 3D). Immunofluorescence staining for the
adipocyte marker perilipin 1 (Plin1) identified Plin1
+
cells within
the remnant of mutant tissue; however, most cells in the mutant did
not express detectable Plin1 (Fig. S2A). Therefore, all WAT depots
84
RESEARCH ARTICLE Development (2017) 144, 83-94 doi:10.1242/dev.135962
DEVELOPMENT
exhibited an imbalance between the stromal and lipid-storing
compartments. Mutant iBAT appeared similar between control and
Sox2-V561D mic e (Fig. 3A, Fig. S3A), except for more prominent
connective tissue septa between the lobules of iBAT, as noted
below. Western blotting verified the activation of PDGFRα
signaling in Sox2-V561D iBAT stromal vascular cells (Fig. S3B).
The increased stromal area seen by H&E staining of WAT was
suggestive of fibrosis. Indeed, Picrosirius Red staining and imaging
with polarized light demon strated an increased density of collagen
fibers surrounding adipocytes and in septa-like tracts through the
ingWAT (Fig. 3F,G). In mutant iBAT, the septa were more collagen-
rich than in controls, but unlike WAT there was no increase in
collagen deposition surrounding individual brown adipocytes
(Fig. S3C).
W e also generated Myf5-V561D mutants and e xamined fat depots a t
P18. Once again, the Myf5
+
lineage-derived iWAT and rWA T display ed
small adipocytes and an increase in stromal area, where as other tissues
appeared normal, includ ing Myf5
+
lineage-derived iBAT and ingW AT/
pWA T derived from Myf5
neg
sources (Fig. S2B-D). Tak en together,
these results demonstra te that WA T develops in Sox2-V561D mice, but
tissue expansion is apparently disrupted by hyperplasia of the s tr omal
compartment and dysplasia of the adipocyte compartment. By contr as t,
iBA T resis ts the effects of PDGFRα
V561D
.
Emergence of stromal fibroblasts and adipo cytes during
organogenesis
Plin1 expression has been shown to identify early adipocytes in the
ingWAT anlage at embryonic day (E)16.5, several days before the
Fig. 1. PDGFRα activation inhibits WAT but not BAT development in Myf5-D842V mutants. (A-D) Gross morphology of the indicated adipose tissue depots of a
Myf5-D842V mouse and littermate control a t P18. Orange outlined areas show the region of iBAT. Yellow outlined ar eas sho w the region of WAT. (E) Mass of the
indicated tis sues at P18. n=3 littermate tissue samples per data point. (F) Mass of the indicated tissues at P18 rela tive to whole body mass. n=3 littermates per da ta point.
(E,F) Mean±s.e.m. ND, non-detected. (G-J) H&E-stained images of adipose depots derived fr om Myf5-Cre
+
lineage (iWAT, rWAT) or Myf5-Cre
neg
lineage (ingWAT,
pWAT) tissues at P21 from a Myf5-D842V mouse and littermate control. Arrows indicate the remnant of str omal tissue that remains in pla ce of W AT. Scale bars: 100 μm.
85
RESEARCH ARTICLE Development (2017) 144, 83-94 doi:10.1242/dev.135962
DEVELOPMENT
cells acquire mature, lipid-filled adipocyte morphology after birth
(Hong et al., 2015). To explore the coordinated development of
the stromal and lipid-storing compartments in normal ingWAT
organogenesis, which has not been described previously, we
examined Plin1
+
cells in mouse embryos where stromal
fibroblasts are marked by EGFP under the control of the collagen
type 1, α1(Col1a1) promoter. At E14.5, the vast majority of the
cells in the prospective ingWAT were labeled with EGFP, albeit
Fig. 2. PDGFRα activation causes
lipodystrophy in Sox2-V561D
mutants. (A-D) Gross morphology of
the indicated adipose tissue depots of a
Sox2-V561D mouse and littermate
control at P18. Orange outlined areas
show the region of iBAT. Yellow outlined
areas show the region of WAT. (E) Mass
of the indicated tissues at P18. n=3
tissue samples per data point. (F) Mass
of the indicated tissues at P18 relative to
whole body mass. n=3 mice per data
point. All data are presented as mean±
s.e.m. *P<0.05, **P<0.01, ***P<0.001,
Student’s t-test.
Fig. 3. PDGFRα activation inhibits adipose tissue organogenesis and causes fibrosis in Sox2-V561D mutants. (A-D) H&E-stained images of tissue
sections from a Sox2-V561D mouse and littermate control at P18. Arrows indicate the tissue margin enriched for stromal fibroblasts. (E) Quantification of iWAT
and ingWAT adipocyte size at P18. n=30 adipocytes per data point. (F) Picrosirius Red-stained images of ingWAT at P18, with polarized light microscopy to
visualize collagen fibers. (G) Quantification of collagen fiber area as a percentage of total area. n=3 polarized light images per genotype. All data are presented as
mean±s.e.m. **P<0.01, ****P<0.0001, Student’s t-test. Scale bars: 100 μm.
86
RESEARCH ARTICLE Development (2017) 144, 83-94 doi:10.1242/dev.135962
DEVELOPMENT
with variable intensity (Fig. 4A). Among this population were
scattered Plin1
+
cells colabeled with medium or low EGFP
fluorescence (Fig. 4B, arrows indicate double labeling). At E16.5,
Plin1
+
cells increased in number and formed clusters that expanded
in size and cell number through P0 (Fig. 4A), confirming
observations from earlier work (Hong et al., 2015). During this
morphogenic process between E16.5 and E18.5, most of the
remaining cells with high EGFP became restricted to the periphery
of the clusters, while cells inside the clusters lost EGFP and
developed intense Plin1 staining (Fig. 4B). Plin1/EGFP colabeling
dropped from 100% at E14.5 to ∼40% at E16.5 and ∼3% at E18.5
and P0 (Fig. 4C). A few intensely EGFP
+
cells remained within the
clusters, possibly representing a minor population of interstitial
fibroblasts or precursors. Because adipocytes quickly fill with lipid
after birth, the distinct morphology of adipocyte clusters surrounded
by stromal fibroblasts is not seen in postnatal ingWAT. However, at
P4 many Col1a1-EGFP
+
cells persisted in a layer along the outer
margin of the depot and in the septa dividing tissue lobules
(Fig. S4A). We also checked Col1a1-EGFP expression in
developing BAT. Unlike the expression in WAT, where ∼30% of
Fig. 4. Emergence of stromal fibroblasts and adipocytes during embryogenesis. (A) Analysis of ingWAT organogenesis in Col1a1-EGFP mice at the
indicated embryonic time points, with immunofluorescence staining for Plin1. (B) Higher magnification of A. Arrows indicate colocalization of Col1a1-EGFP
reporter and Plin1. (C) Quantification of the percentage of Plin1
+
cells colabeled with EGFP. n=3. (D) Lineage tracing analysis by breeding Col1a2-CreER mice
and R26-tdTomato reporter mice, as shown in E,F. (E) Immunofluorescence of Col1a2-CreER;R26-tdTomato lineage tracing in ingWAT, iWAT and iBAT at E18.5,
with staining for Plin1. Arrows indicate colocalization of Tomato and Plin1. (F) Quantification of percentage of Tomato
+
cells colabeled with Plin1. n=4 embryos. All
data are presented as mean±s.e.m. **P<0.01, ****P<0.0001, Student’s t-test. Scale bars: 100 μminA;50μm in B,E.
87
RESEARCH ARTICLE Development (2017) 144, 83-94 doi:10.1242/dev.135962
DEVELOPMENT