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The ability to translate a single genome into multiple phenotypes, or developmental plasticity, defines how phenotype derives from more than just genes. However, to study the evolutionary targets of plasticity and their evolutionary fates, we need to understand how genetic regulators of plasticity control downstream gene expression. Here, we have identified a transcriptional response specific to polyphenism (i.e., discrete plasticity) in the nematode Pristionchus pacificus. This species produces alternative resource-use morphs—microbivorous and predatory forms, differing in the form of their teeth, a morphological novelty—as influenced by resource availability. Transcriptional profiles common to multiple polyphenism-controlling genes in P. pacificus reveal a suite of environmentally sensitive loci, or ultimate target genes, that make up an induced developmental response. Additionally, in vitro assays show that one polyphenism regulator, the nuclear receptor NHR-40, physically binds to promoters with putative HNF4α (the nuclear receptor class including NHR-40) binding sites, suggesting this receptor may directly regulate genes that describe alternative morphs. Among differentially expressed genes were morph-limited genes, highlighting factors with putative “on–off” function in plasticity regulation. Further, predatory morph-biased genes included candidates—namely, all four P. pacificus homologs of Hsp70, which have HNF4α motifs—whose natural variation in expression matches phenotypic differences among P. pacificus wild isolates. In summary, our study links polyphenism regulatory loci to the transcription producing alternative forms of a morphological novelty. Consequently, our findings establish a platform for determining how specific regulators of morph-biased genes may influence selection on plastic phenotypes.

Polyphenism, the extreme form of developmental plasticity, is the ability of a genotype to produce discrete morphologies matched to alternative environments. Because polyphenism is likely to be under switch-like molecular control, a comparative genetic approach could reveal the molecular targets of plasticity evolution. Here we report that the lineage-specific sulfotransferase SEUD-1, which responds to environmental cues, dosage-dependently regulates polyphenism of mouthparts in the nematode Pristionchus pacificus . SEUD-1 is expressed in cells producing dimorphic morphologies, thereby integrating an intercellular signalling mechanism at its ultimate target. Additionally, multiple alterations of seud-1 support it as a potential target for plasticity evolution. First, a recent duplication of seud-1 in a sister species reveals a direct correlation between genomic dosage and polyphenism threshold. Second, inbreeding to produce divergent polyphenism thresholds resulted in changes in transcriptional dosage of seud-1 . Our study thus offers a genetic explanation for how plastic responses evolve. Certain genotypes define developmental plasticity and discrete morphologies, but a mechanism as to how this arises is unclear. Here, the authors show that dosage-specific sulfotransferase (SEUD-1) expression specifies which mouthparts are expressed in the nematode Pristionchus pacificus , dependent on the environment.

The original version of this Article contained errors in Figure 4. In panel a, the x axis labels of bars 6-11 were incorrect, as depicted in the associated Author Correction. These errors have been corrected in both the PDF and HTML versions of the Article.

Background Ferns, being vascular yet seedless, present unparalleled opportunities to investigate important questions regarding the evolution and development of land plants. Ceratopteris richardii, a diploid, homosporous fern has been advanced as a model fern system; however, the tenuous ability to transform the genome of this fern greatly limited its usefulness as a model organism. Here we report a simple and reliable Agrobacterium -mediated method for generating transient and stable transformants of mature C. richardii gametophytes. Results Transformation success was achieved by enzyme treatment that partially digested the cell walls of mature gametophytes to facilitate Agrobacteria infection. Co-incubation of Agrobacteria with enzymatically treated gametophytes was sufficient to generate transient transformants at a frequency of nearly 90% under optimal conditions. Stable transformation was achieved at a rate of nearly 3% by regenerating entire gametophytes from single transformed cells from T 0 gametophytes on selective media. Conclusions This transformation method will allow for the immediate observation of phenotypes in the haploid gametophytes of transformed plants, as well as the generation of stably transformed C. richardii lines for further analysis. Transformation capability will greatly facilitate gene functional studies in C. richardii , more fully realizing the potential of this model fern species. These protocols may be adapted to other plant species that are recalcitrant to Agrobacterium -mediated transformation.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyze the transcriptomes of 611,398 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observe a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD AT2 cells express higher levels of genes linked directly to the efficiency of viral replication and the innate immune response. Additionally, we identify basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection. Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 and poor outcomes. Here the authors compare the transcriptomes of single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in these patients.

Patients with chronic lung disease (CLD) have an increased risk for severe coronavirus disease-19 (COVID-19) and poor outcomes. Here, we analyzed the transcriptomes of 605,904 single cells isolated from healthy and CLD lungs to identify molecular characteristics of lung cells that may account for worse COVID-19 outcomes in patients with chronic lung diseases. We observed a similar cellular distribution and relative expression of SARS-CoV-2 entry factors in control and CLD lungs. CLD epithelial cells expressed higher levels of genes linked directly to the efficiency of viral replication and innate immune response. Additionally, we identified basal differences in inflammatory gene expression programs that highlight how CLD alters the inflammatory microenvironment encountered upon viral exposure to the peripheral lung. Our study indicates that CLD is accompanied by changes in cell-type-specific gene expression programs that prime the lung epithelium for and influence the innate and adaptive immune responses to SARS-CoV-2 infection.

Polyphenism, the extreme form of developmental plasticity, is the ability of a genotype to produce discrete morphologies matched to alternative environments. Because polyphenism is likely to be under switch-like molecular control, a comparative genetic approach could reveal the molecular targets of plasticity evolution. In the nematode Pristionchus pacificus, which form two alternative feeding-morphs, the polyphenism threshold is set by relative dosage of two lineage-specific enzymes that respond to morph-inducing cues. One enzyme, the sulfotransferase SEUD-1, integrates an intercellular signalling mechanism at its ultimate target, the cells producing dimorphic mouthparts. Additionally, multiple alterations of seud-1 support it as a potential target for plasticity evolution. First, a recent duplication of seud-1 in a sister species reveals a direct correlation between genomic dosage and the polyphenism threshold. Second, laboratory selection on the polyphenism threshold resulted in changes in relative transcriptional dosage. Our study thus offers a genetic explanation for how plastic responses evolve. Footnotes * Annotations for tables and figure legends.

Abstract Background Despite its importance in health and disease, the cellular diversity of the lung endothelium has not been systematically characterized in humans. Here we provide a reference atlas of human lung endothelial cells (ECs), to facilitate a better understanding of the phenotypic diversity and composition of cells comprising the lung endothelium, both in health and disease. Methods We reprocessed control single cell RNA sequencing (scRNAseq) data from five datasets of whole lungs that were used for the analysis of pan-endothelial markers, we later included a sixth dataset of sorted control EC for the vascular subpopulation analysis. EC populations were characterized through iterative clustering with subsequent differential expression analysis. Marker genes were validated by immunohistochemistry and in situ hybridization. Signaling network between different lung cell types was studied using connectomic analysis. For cross species analysis we applied the same methods to scRNAseq data obtained from mouse lungs. Results The six lung scRNAseq datasets were reanalyzed and annotated to identify over 15,000 vascular EC cells from 73 individuals. Differential expression analysis of EC revealed signatures corresponding to endothelial lineage, including pan-endothelial, pan-vascular and subpopulation-specific marker gene sets. Beyond the broad cellular categories of lymphatic, capillary, arterial and venous ECs we found previously indistinguishable subpopulations; among venous EC we identified two previously indistinguishable populations, pulmonary-venous ECs (COL15A1neg) localized to the lung parenchyma and systemic-venous ECs (COL15A1pos) localized to the airways and the visceral pleura; among capillary EC we confirmed their subclassification into recently discovered aerocytes characterized by EDNRB, SOSTDC1 and TBX2 and general capillary EC. We confirmed that all six endothelial cell types, including the systemic-venous EC and aerocytes are present in mice and identified endothelial marker genes conserved in humans and mice. Ligand-Receptor connectome analysis revealed important homeostatic crosstalk of EC with other lung resident cell types. Our manuscript is accompanied by an online data mining tool (www.LungEndothelialCellAtlas.com). Conclusion Our integrated analysis provides the comprehensive and well-crafted reference atlas of lung endothelial cells in the normal lung and confirms and describes in detail previously unrecognized endothelial populations across a large number of humans and mice. Competing Interest Statement NK served as a consultant to Biogen Idec, Boehringer Ingelheim, Third Rock, Pliant, Samumed, NuMedii, Theravance, LifeMax, Three Lake Partners, Optikira, Astra Zeneca over the last 3 years, reports Equity in Pliant and a grant from Veracyte and non-financial support from MiRagen and Astra Zeneca. NK as IP on novel biomarkers and therapeutics in IPF licensed to Biotech.

Pulmonary fibrosis is a form of chronic lung disease characterized by pathologic epithelial remodeling and accumulation of extracellular matrix. In order to comprehensively define the cell types, mechanisms and mediators driving fibrotic remodeling in lungs with pulmonary fibrosis, we performed single-cell RNA-sequencing of single-cell suspensions from 10 non-fibrotic control and 20 PF lungs. Analysis of 114,396 cells identified 31 distinct cell types. We report a remarkable shift in epithelial cell phenotypes occurs in the peripheral lung in PF, and identify several previously unrecognized epithelial cell phenotypes including a KRT5-/KRT17+, pathologic ECM-producing epithelial cell population that was highly enriched in PF lungs. Multiple fibroblast subtypes were observed to contribute to ECM expansion in a spatially-discrete manner. Together these data provide high-resolution insights into the complexity and plasticity of the distal lung epithelium in human disease, and indicate a diversity of epithelial and mesenchymal cells contribute to pathologic lung fibrosis. Footnotes * https://github.com/tgen/banovichlab/

Appendiceal neoplasms include a heterogeneous group of epithelial and non-epithelial tumors with varying malignant potential. Despite the rise in incidence of appendiceal neoplasms in recent years, limited progress has been made in the understanding, management and therapeutic treatment. To comprehensively characterize the cell types and molecular mechanisms driving cellular remodeling in epithelial appendiceal neoplasms, we performed an integrated scRNA-seq study. We analyzed 126,998 cells from 16 appendix samples (11 peritoneal metastases samples, 5 healthy controls) and identified 33 distinct cell types/cell states with seven being cancer-specific. Highlights of our study include the characterization of tumor cells across the histologic spectrum, the identification of a novel cancer-associated-fibroblast (CAF) subtypes (fiCAFs) and the identification of pathologic-specific cellular crosstalk between tumor cells and the tumor microenvironment (TME). Together, our study provides a high-resolution insight into the complexity and heterogeneity of epithelial appendiceal neoplasms and a valuable resource for therapeutic strategies.