Probes for INS

Short stature is a major skeletal phenotype in osteogenesis imperfecta (OI), a genetic disorder mainly caused by mutations in genes encoding type I collagen. However, the underlying mechanism is poorly understood and no effective treatment is available. In OI mice that carry a G610C mutation in COL1A2, we previously found that mature hypertrophic chondrocytes (HCs) are exposed to cell stress due to accumulation of misfolded mutant type I procollagen in the endoplasmic reticulum (ER). By fate mapping analysis of HCs in G610C OI mice, we found that HCs stagnate in the growth plate, inhibiting translocation of HC descendants to the trabecular area and their differentiation to osteoblasts. Treatment with 4-phenylbutyric acid (4PBA), a chemical chaperone, restored HC ER structure and rescued this inhibition, resulting in enhanced longitudinal bone growth in G610C OI mice. Interestingly, the effects of 4PBA on ER dilation were limited in osteoblasts and the bone fragility was not ameliorated. These results highlight the importance of targeting HCs to treat growth deficiency in OI. Our findings demonstrate that HC dysfunction induced by ER disruption plays a critical role in the pathogenesis of OI growth deficiency, which lays the foundation for developing new therapies for OI.

(1) Background: Lizard tail regeneration provides a unique model of blastema-based tissue regeneration for large-scale appendage replacement in amniotes. Green anole lizard (Anolis carolinensis) blastemas contain fibroblastic connective tissue cells (FCTCs), which respond to hedgehog signaling to create cartilage in vivo. However, an in vitro model of the blastema has not previously been achieved in culture. (2) Methods: By testing two adapted tissue dissociation protocols and two optimized media formulations, lizard tail FCTCs were pelleted in vitro and grown in a micromass blastema organoid culture. Pellets were analyzed by histology and in situ hybridization for FCTC and cartilage markers alongside staged original and regenerating lizard tails. (3) Results: Using an optimized serum-free media and a trypsin- and collagenase II-based dissociation protocol, micromass blastema organoids were formed. Organoid cultures expressed FCTC marker CDH11 and produced cartilage in response to hedgehog signaling in vitro, mimicking in vivo blastema and tail regeneration. (4) Conclusions: Lizard tail blastema regeneration can be modeled in vitro using micromass organoid culture, recapitulating in vivo FCTC marker expression patterns and chondrogenic potential.

Fibroblast growth factor (FGF) signaling is known to play an important role in lung organogenesis. However, we recently demonstrated that FGF10 fails to induce branching in human fetal lungs as is observed in mouse. Our previous human fetal lung RNA sequencing data exhibited increased FGF18 during the pseudoglandular stage of development, suggestive of its importance in human lung branching morphogenesis. Whereas it has been previously reported that FGF18 is critical during alveologenesis, few studies have described its implication in lung branching, specifically in human. Therefore, we aimed to determine the role of FGF18 in human lung branching morphogenesis. Human fetal lung explants within the pseudoglandular stage of development were treated with recombinant human FGF18 in air-liquid interface culture. Explants were analyzed grossly to assess differences in branching pattern, as well as at the cellular and molecular levels. FGF18 treatment promoted branching in explant cultures and demonstrated increased epithelial proliferation as well as maintenance of the double positive SOX2/SOX9 distal bud progenitor cells, confirming its role in human lung branching morphogenesis. In addition, FGF18 treated explants displayed increased expression of SOX9, FN1, and COL2A1 within the mesenchyme, all factors that are important to chondrocyte differentiation. In humans, cartilaginous airways extend deep into the lung up to the 12th generation of branching whereas in mouse these are restricted to the trachea and main bronchi. Therefore, our data suggest that FGF18 promotes human lung branching morphogenesis through regulating mesenchymal progenitor cells.

Activation of the hypoxia-signalling pathway induced by deletion of the ubiquitin-ligase von Hippel-Lindau protein causes an endocrine shift of renin-producing cells to erythropoietin (EPO)-expressing cells. However, the underlying mechanisms have not yet been investigated. Since oxygen-regulated stability of hypoxia-inducible transcription factors relevant for EPO expression is dependent on the activity of prolyl-4-hydroxylases (PHD) 2 and 3, this study aimed to determine the relevance of different PHD isoforms for the EPO expression in renin-producing cells in vivo. For this purpose, mice with inducible renin cell-specific deletions of different PHD isoforms were analysed. Our study shows that there are two subgroups of renal renin-expressing cells, juxtaglomerular renin+ cells and platelet-derived growth factor receptor-β+ interstitial renin+ cells. These interstitial renin+ cells belong to the cell pool of native EPO-producing cells and are able to express EPO and renin in parallel. In contrast, co-deletion of PHD2 and PHD3, but not PHD2 deletion alone, induces EPO expression in juxtaglomerular and hyperplastic renin+ cells and downregulates renin expression. A strong basal PHD3 expression in juxtaglomerular renin+ cells seems to prevent the hypoxia-inducible transcription factor-2-dependent phenotype shift into EPO cells. In summary, PHDs seem important for the stabilization of the juxtaglomerular renin cell phenotype. Moreover, these findings reveal tubulointerstitial cells as a novel site of renal renin expression and suggest a high endocrine plasticity of these cells. Our data concerning the distinct expression patterns and functions of PHD2 and PHD3 provide new insights into the regulation of renin-producing cells and highlight the need for selective PHD inhibitors. KEY POINTS: Renal renin-expressing cells can be clearly distinguished into two subgroups, the typical juxtaglomerular renin-producing cells and interstitial renin+ cells. Interstitial renin+ cells belong to the cell pool of native erythropoietin (EPO)-producing cells, show a fast EPO response to acute hypoxia-inducible factor-2 (HIF-2) stabilization and are able to express EPO and renin in parallel. Only co-deletion of the prolyl-4-hydroxylases (PHD) 2 and 3, but not PHD2 deletion alone, induces EPO expression in juxtaglomerular renin+ cells. Chronic HIF-2 stabilization in juxtaglomerular renin-expressing cells leads to their phenotypic shift into EPO-producing cells. A strong basal PHD3 expression in juxtaglomerular renin+ cells seems to prevent a HIF-2-dependent phenotype shift into EPO cells suggesting PHD3 fulfils a stabilizer function for the juxtaglomerular renin cell phenotype.

In endochondral bone development, bone-forming osteoblasts and bone marrow stromal cells have dual origins in the fetal cartilage and its surrounding perichondrium. However, how early perichondrial cells distinctively contribute to developing bones remain unidentified. Here we show using in vivo cell-lineage analyses that Dlx5+ fetal perichondrial cells marked by Dlx5-creER do not generate cartilage but sustainably contribute to cortical bone and marrow stromal compartments in a manner complementary to fetal chondrocyte derivatives under the regulation of Hedgehog signaling. Postnatally, Dlx5+ fetal perichondrial cell derivatives preferentially populate the diaphyseal marrow stroma with a dormant adipocyte-biased state and are refractory to parathyroid hormone-induced bone anabolism. Therefore, early perichondrial cells of the fetal cartilage are destined to become an adipogenic subset of stromal cells in postnatal diaphyseal bone marrow, supporting the theory that the adult bone marrow stromal compartments are developmentally prescribed within the two distinct cells-of-origins of the fetal bone anlage.

While prior work has established that articular cartilage arises from Prg4-expressing perichondrial cells, it is not clear how this process is specifically restricted to the perichondrium of synovial joints. We document that the transcription factor Creb5 is necessary to initiate the expression of signaling molecules that both direct the formation of synovial joints and guide perichondrial tissue to form articular cartilage instead of bone. Creb5 promotes the generation of articular chondrocytes from perichondrial precursors in part by inducing expression of signaling molecules that block a Wnt5a autoregulatory loop in the perichondrium. Postnatal deletion of Creb5 in the articular cartilage leads to loss of both flat superficial zone articular chondrocytes coupled with a loss of both Prg4 and Wif1 expression in the articular cartilage; and a non-cell autonomous up-regulation of Ctgf. Our findings indicate that Creb5 promotes joint formation and the subsequent development of articular chondrocytes by driving the expression of signaling molecules that both specify the joint interzone and simultaneously inhibit a Wnt5a positive-feedback loop in the perichondrium.

Atherosclerotic plaques consist mostly of smooth muscle cells (SMCs), and genes that influence SMC phenotype can modulate coronary artery disease (CAD) risk. Allelic variation at 15q22.33 has been identified by genome-wide association studies to modify the risk of CAD and is associated with the expression of _SMAD3_ in SMCs. However, the mechanism by which this gene modifies CAD risk remains poorly understood. Here we show that SMC-specific deletion of _Smad3_ in a murine atherosclerosis model resulted in greater plaque burden, more outward remodeling and increased vascular calcification. Single-cell transcriptomic analyses revealed that loss of _Smad3_ altered SMC transition cell state toward two fates: an SMC phenotype that governs both vascular remodeling and recruitment of inflammatory cells as well as a chondromyocyte fate. Together, the findings reveal that _Smad3_ expression in SMCs inhibits the emergence of specific SMC phenotypic transition cells that mediate adverse plaque features, including outward remodeling, monocyte recruitment and vascular calcification.

Lung organogenesis requires precise timing and coordination to effect spatial organization and function of the parenchymal cells. To provide a systematic broad-based view of the mechanisms governing the dynamic alterations in parenchymal cells over crucial periods of development, we performed a single-cell RNA-sequencing time-series yielding 102,571 epithelial, endothelial and mesenchymal cells across nine time points from embryonic day 12 to postnatal day 14 in mice. Combining computational fate-likelihood prediction with RNA in situ hybridization and immunofluorescence, we explore lineage relationships during the saccular to alveolar stage transition. The utility of this publicly searchable atlas resource (www.sucrelab.org/lungcells) is exemplified by discoveries of the complexity of type 1 pneumocyte function and characterization of mesenchymal Wnt expression patterns during the saccular and alveolar stages - wherein major expansion of the gas-exchange surface occurs. We provide an integrated view of cellular dynamics in epithelial, endothelial and mesenchymal cell populations during lung organogenesis.

Relevance of mineralized nodules in two-dimensional (2D) osteoblast/osteocyte cultures to bone biology, pathology, and engineering is a decades old question, but a comprehensive answer appears to be still wanting. Bone-like cells, extracellular matrix (ECM), and mineral were all reported but so were non-bone-like ones. Many studies described seemingly bone-like cell-ECM structures based on similarity to few select bone features _in vivo_, yet no studies examined multiple bone features simultaneously and none systematically studied all types of structures coexisting in the same culture. Here, we report such comprehensive analysis of 2D cultures based on light and electron microscopies, Raman microspectroscopy, gene expression, and _in situ_ mRNA hybridization. We demonstrate that 2D cultures of primary cells from mouse calvaria do form _bona fide_ bone. Cells, ECM, and mineral within it exhibit morphology, structure, ultrastructure, composition, spatial-temporal gene expression pattern, and growth consistent with intramembranous ossification. However, this bone is just one of at least five different types of cell-ECM structures coexisting in the same 2D culture, which vary widely in their resemblance to bone and ability to mineralize. We show that the other two mineralizing structures may represent abnormal (disrupted) bone and cartilage-like formation with chondrocyte-to-osteoblast trans differentiation. The two non-mineralizing cell-ECM structures may mimic periosteal cambium and pathological, non-mineralizing osteoid. Importantly, the most commonly used culture conditions (10 mM β-glycerophosphate) induce artificial mineralization of all cell-ECM structures, which then become barely distinguishable. We therefore discuss conditions and approaches promoting formation of _bona fide_ bone and simple means for distinguishing it from the other cell-ECM structures. Our findings may improve osteoblast differentiation and function analyses based on 2D cultures and extend applications of these cultures to general bone biology and tissue engineering research.

It has been proposed that smooth muscle differentiation may physically sculpt airway epithelial branches in mammalian lungs. Serum response factor (SRF) acts with its co-factor myocardin to activate the expression of contractile smooth muscle markers. In the adult, however, smooth muscle exhibits a variety of phenotypes beyond contractile, and these are independent of SRF/myocardin-induced transcription. To determine whether a similar phenotypic plasticity is exhibited during development, we deleted Srf from the mouse embryonic pulmonary mesenchyme. Srf-mutant lungs branch normally, and the mesenchyme displays mechanical properties indistinguishable from controls. scRNA-seq identified an Srf-null smooth muscle cluster, wrapping the airways of mutant lungs, which lacks contractile smooth muscle markers but retains many features of control smooth muscle. Srf-null embryonic airway smooth muscle exhibits a synthetic phenotype, compared with the contractile phenotype of mature wild-type airway smooth muscle. Our findings identify plasticity in embryonic airway smooth muscle and demonstrate that a synthetic smooth muscle layer promotes airway branching morphogenesis.

There is a growing amount of data uncovering the cellular diversity of the pulmonary circulation and mechanisms governing vascular repair after injury. However, the molecular and cellular mechanisms contributing to the morphogenesis and growth of the pulmonary vasculature during embryonic development are less clear. Importantly, deficits in vascular development lead to significant pediatric lung diseases, indicating a need to uncover fetal programs promoting vascular growth. To address this, we used a transgenic mouse reporter for expression of Cxcl12, an arterial endothelial hallmark gene, and performed single-cell RNA sequencing on isolated Cxcl12-DsRed+ endothelium to assess cellular heterogeneity within pulmonary endothelium. Combining cell annotation with gene ontology and histological analysis allowed us to segregate the developing artery endothelium into functionally and spatially distinct subpopulations. Expression of Cxcl12 is highest in the distal arterial endothelial subpopulation, a compartment enriched in genes for vascular development. Accordingly, disruption of CXCL12 signaling led to, not only abnormal branching, but also distal vascular hypoplasia. These data provide evidence for arterial endothelial functional heterogeneity and reveal conserved signaling mechanisms essential for pulmonary vascular development.

Organs consist of the parenchyma and stroma, the latter of which coordinates the generation of organotypic structures. Despite recent advances in organoid technology, induction of organ-specific stroma and recapitulation of complex organ configurations from pluripotent stem cells (PSCs) have remained challenging. By elucidating the in vivo molecular features of the renal stromal lineage at a single-cell resolution level, we herein establish an in vitro induction protocol for stromal progenitors (SPs) from mouse PSCs. When the induced SPs are assembled with two differentially induced parenchymal progenitors (nephron progenitors and ureteric buds), the completely PSC-derived organoids reproduce the complex kidney structure, with multiple types of stromal cells distributed along differentiating nephrons and branching ureteric buds. Thus, integration of PSC-derived lineage-specific stroma into parenchymal organoids will pave the way toward recapitulation of the organotypic architecture and functions.