BackgroundNotophthalmus viridescens, an urodelian amphibian, represents an excellent model organism to study regenerative processes, but mechanistic insights into molecular processes driving regeneration have been hindered by a paucity and poor annotation of coding nucleotide sequences. The enormous genome size and the lack of a closely related reference genome have so far prevented assembly of the urodelian genome.ResultsWe describe the de novo assembly of the transcriptome of the newt Notophthalmus viridescens and its experimental validation. RNA pools covering embryonic and larval development, different stages of heart, appendage and lens regeneration, as well as a collection of different undamaged tissues were used to generate sequencing datasets on Sanger, Illumina and 454 platforms. Through a sequential de novo assembly strategy, hybrid datasets were converged into one comprehensive transcriptome comprising 120,922 non-redundant transcripts with a N50 of 975. From this, 38,384 putative transcripts were annotated and around 15,000 transcripts were experimentally validated as protein coding by mass spectrometry-based proteomics. Bioinformatical analysis of coding transcripts identified 826 proteins specific for urodeles. Several newly identified proteins establish novel protein families based on the presence of new sequence motifs without counterparts in public databases, while others containing known protein domains extend already existing families and also constitute new ones.ConclusionsWe demonstrate that our multistep assembly approach allows de novo assembly of the newt transcriptome with an annotation grade comparable to well characterized organisms. Our data provide the groundwork for mechanistic experiments to answer the question whether urodeles utilize proprietary sets of genes for tissue regeneration.

Bronchopulmonary dysplasia (BPD) is the most common cause of morbidity and mortality in preterm infants. A key histopathological feature of BPD is stunted late lung development, where the process of alveolarization—the generation of alveolar gas exchange units—is impeded, through mechanisms that remain largely unclear. As such, there is interest in the clarification both of the pathomechanisms at play in affected lungs, and the mechanisms of de novo alveoli generation in healthy, developing lungs. A better understanding of normal and pathological alveolarization might reveal opportunities for improved medical management of affected infants. Furthermore, disturbances to the alveolar architecture are a key histopathological feature of several adult chronic lung diseases, including emphysema and fibrosis, and it is envisaged that knowledge about the mechanisms of alveologenesis might facilitate regeneration of healthy lung parenchyma in affected patients. To this end, recent efforts have interrogated clinical data, developed new—and refined existing—in vivo and in vitro models of BPD, have applied new microscopic and radiographic approaches, and have developed advanced cell-culture approaches, including organoid generation. Advances have also been made in the development of other methodologies, including single-cell analysis, metabolomics, lipidomics, and proteomics, as well as the generation and use of complex mouse genetics tools. The objective of this review is to present advances made in our understanding of the mechanisms of lung alveolarization and BPD over the period 1 January 2017–30 June 2019, a period that spans the 50th anniversary of the original clinical description of BPD in preterm infants.

Bronchopulmonary dysplasia (BPD) is a common complication of preterm birth characterized by arrested lung alveolarization, which generates lungs that are incompetent for effective gas exchange. We report here deregulated expression of miR‐34a in a hyperoxia‐based mouse model of BPD, where miR‐34a expression was markedly increased in platelet‐derived growth factor receptor (PDGFR)α‐expressing myofibroblasts, a cell type critical for proper lung alveolarization. Global deletion of miR‐34a; and inducible, conditional deletion of miR‐34a in PDGFRα+ cells afforded partial protection to the developing lung against hyperoxia‐induced perturbations to lung architecture. Pdgfra mRNA was identified as the relevant miR‐34a target, and using a target site blocker in vivo, the miR‐34a/Pdgfra interaction was validated as a causal actor in arrested lung development. An antimiR directed against miR‐34a partially restored PDGFRα+ myofibroblast abundance and improved lung alveolarization in newborn mice in an experimental BPD model. We present here the first identification of a pathology‐relevant microRNA/mRNA target interaction in aberrant lung alveolarization and highlight the translational potential of targeting the miR‐34a/Pdgfra interaction to manage arrested lung development associated with preterm birth.

The lysyl hydroxylases (procollagen-lysine 5-dioxygenases) PLOD1, PLOD2, and PLOD3 have been proposed as pathogenic mediators of stunted lung development in bronchopulmonary dysplasia (BPD), a common complication of preterm birth. In affected infants, pulmonary oxygen toxicity stunts lung development. Mice lacking Plod1 exhibit 15% mortality, and mice lacking Plod2 or Plod3 exhibit embryonic lethality. Therefore, to address any pathogenic role of lysyl hydroxylases in stunted lung development associated with BPD, minoxidil was administered to newborn mice in an oxygen-toxicity based BPD animal model. Minoxidil, which has attracted much interest in the management of systemic hypertension and androgenetic alopecia, can also be used to reduce lysyl hydroxylase activity in cultured cells. An in vivo pilot dosing study established 50 mg.kg-1 .day-1 as the maximum possible minoxidil dose for intraperitoneal administration in newborn mouse pups. When administered at 50 mg.kg-1 .day-1 to newborn mouse pups, minoxidil was detected in the lungs, but did not impact lysine hydroxylation, collagen cross-linking, or lysyl hydroxylase expression in the lungs. Consistent with no impact on mouse lung extracellular matrix structures, minoxidil administration did not alter the course of normal or stunted lung development in newborn mice. At doses of up to 50 mg.kg.day-1 , pharmacologically active concentrations of minoxidil were not achieved in neonatal mouse lung tissue; thus, minoxidil cannot be used to attenuate lysyl hydroxylase expression or activity during mouse lung development. These data also highlight the need for new and specific lysyl hydroxylase inhibitors. Significance statement Extracellular matrix cross-linking is mediated by lysyl hydroxylases, which generate hydroxylated lysyl residues in procollagen peptides. Deregulated collagen cross-linking is a pathogenic component of a spectrum of diseases, and thus, there is interest in validating lysyl This article has not been copyedited and formatted. The final version may differ from this version.

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