Myelin protein zero (P0)- and Wnt1-Cre marked muscle resident neural crest-derived mesenchymal progenitor cells give rise to heterotopic ossification in mouse models
概要
Genes & Diseases (2023) 10, 731e734
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Myelin protein zero (P0)- and Wnt1-Cre
marked muscle resident neural
crest-derived mesenchymal progenitor
cells give rise to heterotopic ossification in
mouse models
Heterotopic ossification (HO) describes bone formation at
non-skeletal sites and results from traumatic injury, surgery, or genetic disease such as fibrodysplasia ossificans
progressiva (FOP).1,2 Although it is known that BMP
signaling regulates HO, knowledge about the developmental origin of the osteogenic progenitors responsible for
the BMP-associated metamorphosis is comparably less.
With the use of transgenic mice and labelled neural crestderived cell,3 we found myelin protein zero (P0, or MPZ )and Wnt1-lineage cells give rise to BMP-7 induced adult
ectopic cartilage and bone. In addition, the induced
expression of ACVR1(R206H), which is the major mutation
found in FOP patients, in P0-lineage cells formed ectopic
bone after cardiotoxin-induced muscle injury. We also
found that the majority of muscle-resident fibro-adipogenic progenitors (FAPs), essential for muscle homeostasis
and responsible for HO in skeletal muscle,4,5 are derived
from P0- and Wnt1-lineage cells. The data collectively
suggest that muscle-resident neural crest-derived progenitor cells account for both nonhereditary and genetic
type HO.
To determine whether neural crest-derived cells
contribute to BMP-induced HO, we performed a lineagetracing experiment. Transgenic mice containing P0-Cre
and floxed LacZ reporter alleles were used to label neural
crest-derived cells in vivo (Fig. 1A). Mice were injected
with BMP-7 into the right gastrocnemius muscle to induce
HO. Fourteen days after the injection, HO formation in
P0-Cre/floxed LacZ mice was confirmed by X-ray and
micro-computed tomography (mCT) analysis (Fig. 1B).
Peer review under responsibility of Chongqing Medical
University.
Histological analyses for Hematoxylin and Eosin (H&E),
Safranin O (acidic proteoglycan, an extracellular matrix
protein of chondrocytes), and von Kossa (calcium deposition) staining indicated that the ectopic bones were
formed through endochondral ossification (Fig. S1).
Importantly, BMP-7-induced HO tissue was ubiquitously
positive for X-gal staining (Fig. 1C). BMP-7-injected tissue
expressed the osteoblast-specific factor SP7 (Fig. 1D),
cartilage marker collagen II (COL2) (Fig. 1F), and bone
marker collagen I (COL1) (Fig. 1G). Immunostaining Creþ
mice (P0-Creþ) and Cre- littermate (LacZ ) controls
revealed the considerable co-staining of b-galactosidase
positive (LacZþ) cells with SP7 (81.28% 4.05%) (Fig. 1D,
E) and co-localization with the COL2 or COL1 positive site
(Fig. 1F, G) only in Creþ mice, suggesting that neural
crest-lineage cells contributed to the formed ectopic
bone. However, there was a small portion of LacZ cells
positive for SP7, COL2 and COL1, indicating the existence
of cartilage/bone-forming progenitors derived from
another source. Similar results were obtained when
transgenic mice containing a Wnt1-Cre reporter allele,
which has been widely used to label neural crest-derived
cells, were used (Fig. 1AeG; Fig. S1).
Next, to investigate the role of neural crest-lineage cells
in HO formation in adult FOP model mice, Cre transgene
driven by P0 promoter was combined with a Rosa26-loxPstop-loxP (LSL)-rtTA3 allele and Col1a1-tetO-FOP-ACVR1
allele, in which FOP-ACVR1 (ACVR1R206H) can be induced in
P0-lineage cells upon doxycycline (Dox) treatment
(Fig. 1H). Transgenic mice were treated with Dox in the
drinking water to induce FOP-ACVR1 and treated with CTX
in the right gastrocnemius muscle to initiate skeletal muscle injury and subsequent HO. Notably, HO was observed
https://doi.org/10.1016/j.gendis.2022.09.002
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Figure 1 P0- and Wnt1-Cre marked PDGFRaþ mesenchymal progenitor cells give rise to adult ectopic bone in both BMP-induced
and FOP mutant mice. (A) Schematic representation of the lineage-tracing experiment using P0 or Wnt1-Cre and floxed LacZ reporter alleles. (B) Representative X-ray and mCT images of BMP-7-induced HO, n Z 5. Scale bars: 5 mm. (C) Representative image of
whole mount X-gal staining for HO tissue, n Z 3. Scale bars: 2 mm. (DeG) Immunostaining of P0 or Wnt1-Creþ mice and Cre
littermate control (LacZ ) for b-galactosidase (green) and SP7 (D), COL2 (F) and COL1 (G) (red). Scale bars: 100 mm. (E) Percentage
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only in mutant mice (P0-Cre;Rosa26-LSL-rtTA3;Col1a1tetO-FOP-ACVR1, hereafter referred to as FOP-ACVR1(P0Cre(þ)), 14 days after the CTX treatment, while P0-Crenegative controls (Rosa26-LSL-rtTA3; Col1a1-tetO-FOPACVR1; designated as wild type(P0-Cre()) never developed HO, suggesting that neural crest-derived cells are a
cell-of-origin for the CTX-triggered HO in FOP model mice
(Fig. 1I, J). H&E, Safranin O and von Kossa staining of FOPACVR1(P0-Cre(þ)) mutants illustrated that ectopic cartilage and bone were formed extensively throughout the
CTX-injected region (Fig. 1K). The ectopic cartilage and
bone robustly expressed SP7, COL2, and COL1 (Fig. 1L, N,
O) in the tissue of FOP-ACVR1(P0-Cre(þ)) mutants but not
of wild-type(P0-Cre()) mice. In the FOP-ACVR1(P0Cre(þ)) mutants, a cluster of Venus positive cells (marked
by GFP antibody) detected in the HO region was co-stained
with SP7 (79.13% 7.33%) (Fig. 1L, M) and colocalized with
COL2 or COL1 (Fig. 1N, O), showing FOP-ACVR1 in P0-lineage cells lead to ectopic cartilage and bone.
Previous studies have found that PDGFRaþ FAPs are
likely to be the major precursor of ectopic bone,5 while
their cell-of-origin remains to be fully understood. Therefore, muscle tissues from P0-Cre/floxed-LacZ mice were
examined to confirm whether FAPs were derived from P0lineages. Vimentin- (Fig. 1P) and PDGFRa- (Fig. 1S)
expressing mesenchymal progenitors resided in the muscle
interstitium, which was marked by laminin and collagen VI
(COL6) in the untreated state. These cells were co-stained
with b-galactosidase, indicating their origin of P0-expressing cells. Fourteen days after the BMP-7 treatment, majority of Vimentin- (91.86% 7.97%) and PDGFRaexpressing cells co-stained with b-galactosidase in the HO
tissue of P0-Cre/floxed-LacZ mice were observed (Fig. 1P,
R, S). Similar results were also observed in Wnt1-Cre/
floxed-LacZ mice (Fig. 1Q, R, T). These data suggested that
neural crest-lineage cells act as a cell-of-origin of FAPs,
which contribute to BMP-7-induced HO.
Histological evaluation of the stages of the lesion formation in FOP patients has shown that a phase of tissue
destruction precedes the phase of fibroblast proliferation
and ectopic cartilage and bone formation.2 To pursue the
fate of neural crest-derived cells in the stages of FOP HO in
mice, tissue from the CTX-injected site was collected
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serially and subjected to H&E staining and immunohistochemistry. In both FOP-ACVR1(P0-Cre(þ)) mutants and
wild-type(P0-Cre()) tissue, muscle fiber disruption, more
inflammatory cells and spindle-shaped fibroblastic cells
were observed in the interstitial space 3 days after the CTX
injection (Fig. 1U). Highly proliferating fibroblastic cells
formed the FOP lesion 7 days after the injection. Subsequently, chondrogenesis and osteogenesis occurred at 14
days in FOP-ACVR1(P0-Cre(þ)) mutants. We confirmed that
FAPs residing in the muscle interstitium (marked by COL6)
were increased according to the proliferation of fibroblastic cells in the injected FOP-ACVR1(P0-Cre(þ)) muscle
and contributed to the ectopic bone formation (Fig. 1V). In
P0-Cre() tissue, the population of PDGFRaþ cells
decreased as muscle regeneration proceeded. These results suggested that neural crest-derived PDGFRaþ cells
were a cell-of-origin for the proliferating cells and cartilage- and bone-forming cells that contribute to ectopic
bone in FOP.
In this study, we identified PDGFRa-expressing mesenchymal progenitor cells in the P0 and Wnt1-lineages as a
cell type that gives rise to adult ectopic bone. Our results
also showed that the expression of FOP mutated R206H
ACVR1 (FOP-ACVR1) in P0-lineage cells gives rise to ectopic
bone induced by CTX injection. Although it is also known
that P0 or Wnt1 alone is not exclusive to neural crest cells,
our observation strongly suggests that ectopic bones were
neural crest cell-origin since ectopic bone was marked in
both Wnt1-cre and P0-cre lineages, which mark different
neural crest populations (P0-cre: migrating neural crest
cells vs Wnt1-cre: the dorsal neural tube prior to the
emigration of the neural crest).
In skeletal muscle, muscle-resident FAPs are essential
for muscle homeostasis and regeneration. Our study highlights that the PDGFRaþ FAPs responsible for BMP-7-induced
HO originate from the neural crest. In FOP tissue, P0-lineage PDGFRaþ cells contributed to the assembly of mesenchymal progenitors, which caused the FOP lesion and
ectopic cartilage/bone tissue in damaged muscle. These
findings will contribute the study of FAP behavior in HO.
In conclusion, our results strongly suggest that neural
crest-lineage cells give rise to adult ectopic bone, targeting
neural crest-lineage progenitors may open new
of LacZþ cells in SP7þ cells. Data represent the mean SD of representative sections from three independent experiments.
***P < 0.001, by Student’s t test compared with the LacZ group. (H) Schematic illustration of the genetic construct for Doxinducible FOP-ACVR1 in neural crest derivatives. (I) Representative X-ray and mCT images of CTX-induced HO. (J) Average ectopic
bone volume induced by CTX. Data represent the mean SD of n Z 6/group. ...