STAT1 modulates tissue wasting or overgrowth downstream from PDGFRß.

Platelet-derived growth factor (PDGF) acts through two conserved receptor tyrosine kinases: PDGFRα and PDGFRß. Gain-of-function mutations in human PDGFRB have been linked recently to genetic diseases characterized by connective tissue wasting (Penttinen syndrome) or overgrowth (Kosaki overgrowth syndrome), but it is unclear whether PDGFRB mutations alone are responsible. Mice with constitutive PDGFRß signaling caused by a kinase domain mutation (D849V) develop lethal autoinflammation. Here we used a genetic approach to investigate the mechanism of autoinflammation in Pdgfrb+/D849V mice and test the hypothesis that signal transducer and activator of transcription 1 (STAT1) mediates this phenotype. We show that Pdgfrb+/D849V mice with Stat1 knockout (Stat1-/-Pdgfrb+/D849V ) are rescued from autoinflammation and have improved life span compared with Stat1+/-Pdgfrb+/D849V mice. Furthermore, PDGFRß-STAT1 signaling suppresses PDGFRß itself. Thus, Stat1-/-Pdgfrb+/D849V fibroblasts exhibit increased PDGFRß signaling, and mice develop progressive overgrowth, a distinct phenotype from the wasting seen in Stat1+/-Pdgfrb+/D849V mice. Deletion of interferon receptors (Ifnar1 or Ifngr1) does not rescue wasting in Pdgfrb+/D849V mice, indicating that interferons are not required for autoinflammation. These results provide functional evidence that elevated PDGFRß signaling causes tissue wasting or overgrowth reminiscent of human genetic syndromes and that the STAT1 pathway is a crucial modulator of this phenotypic spectrum.

Skeletal stem cell fate defects caused by Pdgfrb activating mutation.

Autosomal dominant PDGFRß gain-of-function mutations in mice and humans cause a spectrum of wasting and overgrowth disorders afflicting the skeleton and other connective tissues, but the cellular origin of these disorders remains unknown. We demonstrate that skeletal stem cells (SSCs) isolated from mice with a gain-of-function D849V point mutation in PDGFRß exhibit colony formation defects that parallel the wasting or overgrowth phenotypes of the mice. Single-cell RNA transcriptomics with SSC-derived polyclonal colonies demonstrates alterations in osteogenic and chondrogenic precursors caused by PDGFRßD849V. Mutant cells undergo poor osteogenesis in vitro with increased expression of Sox9 and other chondrogenic markers. Mice with PDGFRßD849V exhibit osteopenia. Increased STAT5 phosphorylation and overexpression of Igf1 and Socs2 in PDGFRßD849V cells suggests that overgrowth in mice involves PDGFRßD849V activating the STAT5-IGF1 axis locally in the skeleton. Our study establishes that PDGFRßD849V causes osteopenic skeletal phenotypes that are associated with intrinsic changes in SSCs, promoting chondrogenesis over osteogenesis.

Endothelial cell regulation of salivary gland epithelial patterning.

Perfusion-independent regulation of epithelial pattern formation by the vasculature during organ development and regeneration is of considerable interest for application in restoring organ function. During murine submandibular salivary gland development, the vasculature co-develops with the epithelium during branching morphogenesis; however, it is not known whether the vasculature has instructive effects on the epithelium. Using pharmacological inhibitors and siRNA knockdown in embryonic organ explants, we determined that VEGFR2-dependent signaling is required for salivary gland epithelial patterning. To test directly for a requirement for endothelial cells in instructive epithelial patterning, we developed a novel ex vivo cell fractionation/reconstitution assay. Immuno-depletion of CD31+ endothelial cells in this assay confirmed a requirement for endothelial cells in epithelial patterning of the gland. Depletion of endothelial cells or inhibition of VEGFR2 signaling in organ explants caused an aberrant increase in cells expressing the ductal proteins K19 and K7, with a reduction in Kit+ progenitor cells in the endbuds of reconstituted glands. Addition of exogenous endothelial cells to reconstituted glands restored epithelial patterning, as did supplementation with the endothelial cell-regulated mesenchymal factors IGFBP2 and IGFBP3. Our results demonstrate that endothelial cells promote expansion of Kit+ progenitor cells and suppress premature ductal differentiation in early developing embryonic submandibular salivary gland buds.

Regulation of adipocyte dedifferentiation at the skin wound edge.

Adipocytes have diverse roles in energy storage and metabolism, inflammation, and tissue repair. Mature adipocytes have been assumed to be terminally differentiated cells. However, recent evidence suggests that adipocytes retain substantial phenotypic plasticity, with potential to dedifferentiate into fibroblast-like cells under physiological and pathological conditions. Here, we develop a two-step lineage tracing approach based on the observation that fibroblasts express platelet-derived growth factor receptor alpha ( Pdgfra ) while adipocytes express Adiponectin ( Adipoq ) but not Pdgfra . Our approach specifically traces Pdgfra + cells that originate from Adipoq + adipocytes. We find many traced adipocytes and fibroblast-like cells surrounding skin wounds, but only a few traced cells localize to the wound center. In agreement with adipocyte plasticity, traced adipocytes incorporate EdU, downregulate Plin1 and PPARγ, and upregulate αSMA. We also investigate the role of potential dedifferentiation signals using constitutively active PDGFRα mutation, Pdgfra knockout, or Tgfbr2 knockout models. We find that PDGF and TGFß signaling both promote dedifferentiation, and PDGFRα does so independently of TGFßR2. These results demonstrate an intersectional genetic approach to trace the hybrid cell phenotype of Pdgfra + adipocytes, which may be important for wound repair, regeneration and fibrosis.

Temporal control of PDGFRα regulates the fibroblast-to-myofibroblast transition in wound healing.

Fibroblasts differentiate into myofibroblasts by acquiring new contractile function. This is important for tissue repair, but it also contributes to organ fibrosis. Platelet-derived growth factor (PDGF) promotes tissue repair and fibrosis, but the relationship between PDGF and myofibroblasts is unclear. Using mice with lineage tracing linked to PDGF receptor α (PDGFRα) gene mutations, we examine cell fates during skin wound healing. Elevated PDGFRα signaling increases proliferation but unexpectedly delays the fibroblast-to-myofibroblast transition, suggesting that PDGFRα must be downregulated for myofibroblast differentiation. In contrast, deletion of PDGFRα decreases proliferation and myofibroblast differentiation by reducing serum response factor (SRF) nuclear localization. Consequences of SRF deletion resemble PDGFRα deletion, but deletion of two SRF coactivators, MRTFA and MRTFB, specifically eliminates myofibroblasts. Our findings suggest a scenario where PDGFRα signaling initially supports proliferation of fibroblast progenitors to expand their number during early wound healing but, later, PDGFRα downregulation facilitates fibroblast differentiation into myofibroblasts.

Nucleotide sequence and secondary structure of 5S rRNA from Sphingobium chungbukense DJ77.

The 5S rRNA gene from Sphingobium chungbukense DJ77 was identified. The secondary structure of the 199-base-long RNA was proposed. The two-base-long D loop was the shortest among all of the known 5S rRNAs. The U19-U64 non-canonical pair in the helix II region was uniquely found in strain DJ77 among all of the sphingomonads.

The contribution of specific cell subpopulations to submandibular salivary gland branching morphogenesis.

Branching morphogenesis is the developmental program responsible for generating a large surface to volume ratio in many secretory and absorptive organs. To accomplish branching morphogenesis, spatiotemporal regulation of specific cell subpopulations is required. Here, we review recent studies that define the contributions of distinct cell subpopulations to specific cellular processes during branching morphogenesis in the mammalian submandibular salivary gland, including the initiation of the gland, the coordination of cleft formation, and the contribution of stem/progenitor cells to morphogenesis. In conclusion, we provide an overview of technological advances that have opened opportunities to further probe the contributions of specific cell subpopulations and to define the integration of events required for branching morphogenesis.

TGFß signaling promotes matrix assembly during mechanosensitive embryonic salivary gland restoration.

Mechanical properties of the microenvironment regulate cell morphology and differentiation within complex organs. However, methods to restore morphogenesis and differentiation in organs in which compliance is suboptimal are poorly understood. We used mechanosensitive mouse salivary gland organ explants grown at different compliance levels together with deoxycholate extraction and immunocytochemistry of the intact, assembled matrices to examine the compliance-dependent assembly and distribution of the extracellular matrix and basement membrane in explants grown at permissive or non-permissive compliance. Extracellular matrix and basement membrane assembly were disrupted in the glands grown at low compliance compared to those grown at high compliance, correlating with defective morphogenesis and decreased myoepithelial cell differentiation. Extracellular matrix and basement membrane assembly as well as myoepithelial differentiation were restored by addition of TGFß1 and by mechanical rescue, and mechanical rescue was prevented by inhibition of TGFß signaling during the rescue. We detected a basal accumulation of active integrin ß1 in the differentiating myoepithelial cells that formed a continuous peripheral localization around the proacini and in clefts within active sites of morphogenesis in explants that were grown at high compliance. The pattern and levels of integrin ß1 activation together with myoepithelial differentiation were interrupted in explants grown at low compliance but were restored upon mechanical rescue or with application of exogenous TGFß1. These data suggest that therapeutic application of TGFß1 to tissues disrupted by mechanical signaling should be examined as a method to promote organ remodeling and regeneration.