The Unmixing Problem: A Guide to Applying Single‐Cell RNA Sequencing to Bone

Bone is composed of a complex mixture of many dynamic cell types. Flow cytometry and in vivo lineage tracing have offered early progress toward deconvoluting this heterogeneous mixture of cells into functionally well‐defined populations suitable for further studies. Single‐cell sequencing is poised as a key complementary technique to better understand the cellular basis of bone metabolism and development. However, single‐cell sequencing approaches still have important limitations, including transcriptional effects of cell isolation and sparse sampling of the transcriptome, that must be considered during experimental design and analysis to harness the power of this approach. Accounting for these limitations requires a deep knowledge of the tissue under study. Therefore, with the emergence of accessible tools for conducting and analyzing single‐cell RNA sequencing (scRNA‐seq) experiments, bone biologists will be ideal leaders in the application of scRNA‐seq to the skeleton. Here we provide an overview of the steps involved with a single‐cell sequencing analysis of bone, focusing on practical considerations needed for a successful study. © 2019 American Society for Bone and Mineral Research.Peer Reviewedhttps://deepblue.lib.umich.edu/bitstream/2027.42/150567/1/jbmr3802_am.pdfhttps://deepblue.lib.umich.edu/bitstream/2027.42/150567/2/jbmr3802.pd

Distinct molecular pathways mediate Mycn and Myc-regulated miR-17-92 microRNA action in Feingold syndrome mouse models

Feingold syndrome is a skeletal dysplasia caused by loss-of-function mutations of either MYCN (type 1) or MIR17HG that encodes miR-17-92 microRNAs (type 2). Since miR-17-92 expression is transcriptionally regulated by MYC transcription factors, it has been postulated that Feingold syndrome type 1 and 2 may be caused by a common molecular mechanism. Here we show that Mir17-92 deficiency upregulates TGF-β signaling, whereas Mycn-deficiency downregulates PI3K signaling in limb mesenchymal cells. Genetic or pharmacological inhibition of TGF-β signaling efficiently rescues the skeletal defects caused by Mir17-92 deficiency, suggesting that upregulation of TGF-β signaling is responsible for the skeletal defect of Feingold syndrome type 2. By contrast, the skeletal phenotype of Mycn-deficiency is partially rescued by Pten heterozygosity, but not by TGF-β inhibition. These results strongly suggest that despite the phenotypical similarity, distinct molecular mechanisms underlie the pathoetiology for Feingold syndrome type 1 and 2

SHP2 regulates chondrocyte terminal differentiation, growth plate architecture and skeletal cell fates.

Loss of PTPN11/SHP2 in mice or in human metachondromatosis (MC) patients causes benign cartilage tumors on the bone surface (exostoses) and within bones (enchondromas). To elucidate the mechanisms underlying cartilage tumor formation, we investigated the role of SHP2 in the specification, maturation and organization of chondrocytes. Firstly, we studied chondrocyte maturation by performing RNA-seq on primary chondrocyte pellet cultures. We found that SHP2 depletion, or inhibition of the ERK1/2 pathway, delays the terminal differentiation of chondrocytes from the early-hypertrophic to the late-hypertrophic stage. Secondly, we studied chondrocyte maturation and organization in mice with a mosaic postnatal inactivation of Ptpn11 in chondrocytes. We found that the vertebral growth plates of these mice have expanded domains of early-hypertrophic chondrocytes that have not yet terminally differentiated, and their enchondroma-like lesions arise from chondrocytes displaced from the growth plate due to a disruption in the organization of maturation and ossification zones. Furthermore, we observed that lesions from human MC patients also display disorganized chondrocyte maturation zones. Next, we found that inactivation of Ptpn11 in Fsp1-Cre-expressing fibroblasts induces exostosis-like outgrowths, suggesting that loss of SHP2 in cells on the bone surface and at bone-ligament attachment sites induces ectopic chondrogenesis. Finally, we performed lineage tracing to show that exostoses and enchondromas in mice likely contain mixtures of wild-type and SHP2-deficient chondrocytes. Together, these data indicate that in patients with MC, who are heterozygous for inherited PTPN11 loss-of-function mutations, second-hit mutations in PTPN11 can induce enchondromas by disrupting the organization and delaying the terminal differentiation of growth plate chondrocytes, and can induce exostoses by causing ectopic chondrogenesis of cells on the bone surface. Furthermore, the data are consistent with paracrine signaling from SHP2-deficient cells causing SHP2-sufficient cells to be incorporated into the lesions

SHP2 depletion or MEK1/2 inhibition increases the abundance of PZ, pre-HZ and early-HZ transcripts, and decreases the abundance of late-HZ transcripts.

<p>A: Heat map showing the fold change in abundance (treated vs. control pellets at w3) of selected transcripts that are associated with a specific stage of chondrocyte maturation. B: Heat maps showing the fold change in abundance (treated vs. control pellets at w3) for all transcripts that we included in the “upper zones” and “lower zones” transcript sets. The graph below the heat map indicates the percentage of transcripts for which the increase (red) or decrease (blue) in abundance was significant (p<0.05). The number of “upper zones” transcripts that increased in abundance in treated pellets (red), and the number of “lower zones” transcripts that decreased in abundance in treated pellets (blue), is indicated below. For <i>Ptpn11</i> cKO pellets, of the 108 “lower zones” transcripts that significantly decreased in abundance, 54 did not significantly change in wild-type pellets treated with 4-OHT.</p

The role of Meteorin-like in skeletal development and bone fracture healing

Meteorin‐like protein (Metrnl), homologous to the initially identified neurotrophic factor Meteorin, is a secreted, multifunctional protein. Here we used mouse models to investigate Metrnl's role in skeletal development and bone fracture healing. During development Metrnl was expressed in the perichondrium and primary ossification center. In neonates, single cell RNA‐seq of diaphyseal bone demonstrated strongest expression of Metrnl transcript by osteoblasts. In vitro, Metrnl was osteoinductive, increasing osteoblast differentiation and mineralization in tissue culture models. In vivo, loss of Metrnl expression resulted in no change in skeletal metrics in utero, at birth, or during postnatal growth. Six‐week‐old Metrnl‐null mice displayed similar body length, body weight, tibial length, femoral length, BV/TV, trabecular number, trabecular thickness, and cortical thickness as littermate controls. In 4‐month‐old mice, lack of Metrnl expression did not change structural stiffness, ultimate force, or energy to fracture of femora under 3‐point‐bending. Last, we investigated the role of Metrnl in bone fracture healing. Metrnl expression increased in response to tibial injury, however, loss of Metrnl expression did not affect the amount of bone deposited within the healing tissue nor did it change the structural parameters of healing tissue. This work identifies Metrnl as a dispensable molecule for skeletal development. However, the osteoinductive capabilities of Metrnl may be utilized to modulate osteoblast differentiation in cell‐based orthopedic therapies

Identifying transcripts that change in abundance following SHP2 depletion or MEK1/2 inhibition in pellet cultures.

<p>A: Immunodetection of SHP2, phospho-ERK1/2, total ERK1/2, and phospho-AKT in chondrocyte pellet cultures. Top: pellets from <i>Ptpn11^fl/–</i> or <i>Tg</i>(<i>CMV-CreERT2); Ptpn11^fl/–</i> mice were treated daily with 4-OHT or vehicle control (EtOH, -) and harvested after 1 and 3 weeks. Bottom: pellets from wild-type mice were treated daily with a MEK1/2 inhibitor (U0126), or vehicle control (DMSO, -), and harvested after 1 and 3 weeks. B: Number of transcripts that had significantly lower (blue) or higher (red) abundance in treated pellets compared to vehicle treated pellets, either at w1 or w3. Transcripts that changed in abundance in <i>Ptpn11</i> cKO pellets were excluded if they exhibited similar changes in wild-type pellets treated with 4-OHT.</p

Lineage tracing of SHP2-depleted chondrocytes in mouse vertebral growth plates following mosaic postnatal <i>Ptpn11</i> inactivation.

<p>Tissue sections of lumbar vertebral growth plates (GP) and intervertebral disks (IVD) from <i>Tg</i>(<i>Col2a1-CreER); Rosa26^mTmG/+</i>;<i>Ptpn11^fl</i>^/<i>fl</i> (Col2a1-cKO) or <i>Tg</i>(<i>Col2a1-CreER); Rosa26^mTmG/+</i>;<i>Ptpn11^fl/+</i> (Ctrl) mice that had been administered tamoxifen for 5 days, starting at w1, and sacrificed at w7. A-C: Tissue sections stained with Alcian Blue and Nuclear Fast Red. In Col2a1-cKO mice, the vertebral growth plates are enlarged and disorganized, with ectopic areas of ossification inside the growth plate (red arrow in B) and enchondroma-like lesions below the growth plate (C). D-G: Merged fluorescent images showing cells in which the <i>mTmG</i> reporter allele has (green fluorescence) or has not (red fluorescence) been recombined by Cre recombinase. Note that approximately 50% of growth plate chondrocytes fluoresce green, and in Col2a1-cKO mice, the expanded regions of the growth plate (E), lateral outgrowths (F) and enchondroma-like lesions (G) contain both green and red fluorescing cells. Dashed-white lines outline the cartilage. Scale bars  =  100 µm.</p

MEK1/2 inhibition or SHP2 depletion alters the expression of transcription factors with known roles in chondrocyte maturation.

<p>Schematic diagrams of genes encoding transcription factors (solid boxes) or their known target genes (empty boxes) that significantly decreased in abundance (blue) or increased in abundance (red) in U0126-treated pellets at w3. All transcription factors also showed similar increases or decreases in <i>Ptpn11</i> cKO pellets at w3, but not all reached statistical significance. A: Transcription factors with known roles in promoting chondrocyte hypertrophy (upper green arrow), or in promoting the terminal differentiation of chondrocytes and/or their replacement by bone (lower green arrow). B: Transcription factors whose known target genes also significantly changed in abundance in U0126-treated pellets. Arrows indicate transcriptional activation while bar-headed lines indicate transcriptional repression.</p

Disordered maturation zones and delayed terminal differentiation in mouse vertebral growth plates following mosaic postnatal inactivation of <i>Ptpn11</i> in chondrocytes.

<p>Immunohistochemistry performed on tissue sections from the vertebral growth plates of Ctrl mice (A,D) or the growth plates (B,E) or enchondroma-like lesions (C) in Col2a1-cKO mice. A: Ctrl growth plate showing COLX, p-ERK1/2 and FRA1 immunoreactivity in the hypertrophic zone. COLX immunoreactivity is also observed in the calcifying region of the annulus fibrosus that lies directly above the growth plate. B: Col2a1-cKO growth plate showing ectopic COLX-positive clusters at the top of the growth plate (a) and ectopic COLX-negative clusters at the bottom of the growth plate (b). Furthermore, hypertrophic chondrocytes within the center of the growth plate display disorganized, scattered p-ERK1/2 and FRA1 immunoreactivity (c). C: Enchondroma-like lesions contain both COLX-positive and COLX-negative chondrocytes. D: Ctrl growth plate showing SPP1 immunoreactivity at the bottom of the hypertrophic zone and in mineralizing annulus fibrosus cells. E: In Col2a1-cKO growth plates, expanded regions of SPP1-negative hypertrophic chondrocytes are observed (red arrows) and only a few scattered hypertrophic chondrocytes are SPP1-positive (black arrows). IHC tissue sections were counterstained with hematoxylin. Scale bars  =  25 µm.</p