Project description:Lung function is closely coupled to its structural anatomy, which varies greatly across vertebrates. Although architecturally simple, a complex pattern of airflow is thought to be achieved in the lizard lung due to its cavernous central lumen and honeycomb-shaped wall. We find that the wall of the lizard lung is generated from an initially smooth epithelial sheet, which is pushed through holes in a hexagonal smooth muscle meshwork by forces from fluid pressure, similar to a stress ball. By combining next-generation sequencing with timelapse imaging, we reveal that the hexagonal smooth muscle geometry self-assembles in response to circumferential and axial stresses downstream of pressure. A quantitative computational model predicts the pressure-driven changes in epithelial topology, which we replicate using a 3D-printed engineered tissue model of optogenetically-driven smooth muscle contraction. These results reveal the physical principles used to sculpt the unusual architecture of the lizard lung, which could be exploited as a novel strategy to engineer tissues.
Project description:Expansion of neoplastic lesions generates the initial signal that instigates the creation of a tumor niche. Nontransformed cell types within the microenvironment continuously coevolve with tumor cells to promote tumorigenesis. Here, we identify p38MAPK as a key component of human lung cancer, and specifically stromal interactomes, which provides an early, protumorigenic signal in the tissue microenvironment. We found that lung cancer growth depends on short-distance cues produced by the cancer niche in a p38-dependent manner. We identified fibroblast-specific hyaluronan synthesis at the center of p38-driven tumorigenesis, which regulates early stromal fibroblast activation, the conversion to carcinoma-associated fibroblasts (CAFs), and cancer cell proliferation. Systemic down-regulation of p38MAPK signaling in a knock-in model with substitution of activating Tyr182 to phenylalanine or conditional ablation of p38 in fibroblasts has a significant tumor-suppressive effect on K-ras lung tumorigenesis. Furthermore, both Kras-driven mouse lung tumors and orthotopically grown primary human lung cancers show a significant sensitivity to both a chemical p38 inhibitor and an over-the-counter inhibitor of hyaluronan synthesis. We propose that p38MAPK-hyaluronan-dependent reprogramming of the tumor microenvironment plays a critical role in driving lung tumorigenesis, while blocking this process could have far-reaching therapeutic implications.
Project description:Blood vessels are essential for blood circulation but also control organ growth, homeostasis, and regeneration, which has been attributed to the release of paracrine signals by endothelial cells. Endothelial tubules are associated with specialised mesenchymal cells, termed pericytes, which help to maintain vessel wall integrity. Here we identify pericytes as regulators of epithelial and endothelial morphogenesis in postnatal lung. Mice lacking expression of the Hippo pathway components YAP and TAZ in pericytes show defective alveologenesis. Mutant pericytes are present in normal numbers but display strongly reduced expression of hepatocyte growth factor leading to impaired activation of the c-Met receptor, which is expressed by alveolar epithelial cells. YAP and TAZ are also required for expression of angiopoietin-1 by pulmonary pericytes, which also controls hepatocyte growth factor expression and thereby alveologenesis in an autocrine fashion. These findings establish that pericytes have important, organ-specific signalling properties and coordinate the behavior of epithelial and vascular cells during lung morphogenesis.
Project description:With over 9000 species, squamates, which include lizards and snakes, are the largest group of reptiles and second-largest order of vertebrates, spanning a vast array of appendicular skeletal morphology. As such, they provide a promising system for examining developmental and molecular processes underlying limb morphology. Using the central bearded dragon (Pogona vitticeps) as the primary study model, we examined limb morphometry throughout embryonic development and characterized the expression of three known developmental genes (GHR, Pitx1 and Shh) from early embryonic stage through to hatchling stage via reverse transcription quantitative polymerase chain reaction (RT-qPCR) and immunohistochemistry (IHC). In this study, all genes were found to be transcribed in both the forelimbs and hindlimbs of P. vitticeps. While the highest level of GHR expression occurred at the hatchling stage, Pitx1 and Shh expression was greatest earlier during embryogenesis, which coincides with the onset of the differentiation between forelimb and hindlimb length. We compared our finding of Pitx1 expression-a hindlimb-determining gene-in the forelimbs of P. vitticeps to that in a closely related Australian agamid lizard, Ctenophorus pictus, where we found Pitx1 expression to be more highly expressed in the hindlimb compared with the forelimb during early and late morphogenesis-a result consistent with that found across other tetrapods. Expression of Pitx1 in forelimbs has only rarely been documented, including via in situ hybridization in a chicken and a frog. Our findings from both RT-qPCR and IHC indicate that further research across a wider range of tetrapods is needed to more fully understand evolutionary variation in molecular processes underlying limb morphology.
Project description:The molecular mechanisms that govern the choreographed timing of organ development remain poorly understood. Our investigation of the role of the Lin28a and Lin28b paralogs during the developmental process of branching morphogenesis establishes that dysregulation of Lin28a/b leads to abnormal branching morphogenesis in the lung and other tissues. Additionally, we find that the Lin28 paralogs, which regulate post-transcriptional processing of both mRNAs and microRNAs (miRNAs), predominantly control mRNAs during the initial phases of lung organogenesis. Target mRNAs include Sox2, Sox9, and Etv5, which coordinate lung development and differentiation. Moreover, we find that functional interactions between Lin28a and Sox9 are capable of bypassing branching defects in Lin28a/b mutant lungs. Here, we identify Lin28a and Lin28b as regulators of early embryonic lung development, highlighting the importance of the timing of post-transcriptional regulation of both miRNAs and mRNAs at distinct stages of organogenesis.
Project description:The role of regulated cell death in organ development, particularly the impact of non-apoptotic cell death, remains largely uncharted. Ferroptosis, a non-apoptotic cell death pathway known for its iron dependence and lethal lipid peroxidation, is currently being rigorously investigated for its pathological functions. The balance between ferroptotic stress (iron and iron-dependent lipid peroxidation) and ferroptosis supervising pathways (anti-lipid peroxidation systems) serves as the key mechanism regulating the activation of ferroptosis. Compared with other forms of regulated necrotic cell death, ferroptosis is critically related to the metabolism of lipid and iron which are also important in organ development. In our study, we examined the role of ferroptosis in organogenesis using an ex vivo tooth germ culture model, investigating the presence and impact of ferroptotic stress on tooth germ development. Our findings revealed that ferroptotic stress increased during tooth development, while the expression of glutathione peroxidase 4 (Gpx4), a crucial anti-lipid peroxidation enzyme, also escalated in dental epithelium/mesenchyme cells. The inhibition of ferroptosis was found to partially rescue erastin-impaired tooth morphogenesis. Our results suggest that while ferroptotic stress is present during tooth organogenesis, its effects are efficaciously controlled by the subsequent upregulation of Gpx4. Notably, an overabundance of ferroptotic stress, as induced by erastin, suppresses tooth morphogenesis.
Project description:Drosophila trachealess (Trl), master regulator of tracheogenesis, has no known functional mammalian homolog. We hypothesized that genes similar to trachealess regulate lung development. Quantitative (Q)RT-PCR and immunostaining were used to determine spatial and temporal patterns of npas1 gene expression in developing murine lung. Immunostaining for alpha-smooth muscle actin demonstrated myofibroblasts, and protein gene product (PGP)9.5 identified neuroendocrine cells. Branching morphogenesis of embryonic lung buds was analyzed in the presence of antisense or sense oligodeoxynucleotides (ODN). Microarray analyses were performed to screen for changes in gene expression in antisense-treated lungs. QRT-PCR was used to validate the altered expression of key genes identified on the microarrays. We demonstrate that npas1 is expressed in murine embryonic lung. npas1 mRNA peaks early at Embryonic Day (E)10.5-E11.5, then drops to low levels. Sequencing verifies the identity of npas1 transcripts in embryonic lung. NPAS1 immunostaining occurs in nuclei of parabronchial mesenchymal cells, especially at the tracheal bifurcation. Arnt, the murine homolog of Tango (the heterodimerization partner for Trl) is also expressed in developing lung but at constant levels. npas1- or arnt-antisense ODN inhibit lung branching morphogenesis, with altered myofibroblast development and increased pulmonary neuroendocrine cells. On microarrays, we identify > 50 known genes down-regulated by npas1-antisense, including multiple genes regulating cell migration and cell differentiation. QRT-PCR confirms significantly decreased expression of the neurogenic genes RBP-Jk and Tle, and three genes involved in muscle development: beta-ig-h3, claudin-11, and myocardin. Npas1 can regulate myofibroblast distribution, branching morphogenesis, and neuroendocrine cell differentiation in murine embryonic lung.
Project description:The arborescent architecture of mammalian conductive airways results from the repeated branching of lung endoderm into surrounding mesoderm. Subsequent lung's striking geometrical features have long raised the question of developmental mechanisms involved in morphogenesis. Many molecular actors have been identified, and several studies demonstrated the central role of Fgf10 and Shh in growth and branching. However, the actual branching mechanism and the way branching events are organized at the organ scale to achieve a self-avoiding tree remain to be understood through a model compatible with evidenced signaling. In this paper we show that the mere diffusion of FGF10 from distal mesenchyme involves differential epithelial proliferation that spontaneously leads to branching. Modeling FGF10 diffusion from sub-mesothelial mesenchyme where Fgf10 is known to be expressed and computing epithelial and mesenchymal growth in a coupled manner, we found that the resulting laplacian dynamics precisely accounts for the patterning of FGF10-induced genes, and that it spontaneously involves differential proliferation leading to a self-avoiding and space-filling tree, through mechanisms that we detail. The tree's fine morphological features depend on the epithelial growth response to FGF10, underlain by the lung's complex regulatory network. Notably, our results suggest that no branching information has to be encoded and that no master routine is required to organize branching events at the organ scale. Despite its simplicity, this model identifies key mechanisms of lung development, from branching to organ-scale organization, and could prove relevant to the development of other branched organs relying on similar pathways.