Epicardium-derived cells organize through tight junctions to replenish cardiac muscle in salamanders

Eroğlu, Elif; Yen, Christopher; Tsoi, Yat Long; Witman, Nevin; Elewa, Ahmed; Araus, Alberto Joven; Wang, Heng; Szattler, Tamara; Umeano, Chimezie H; Sohlmér, Jesper; Goedel, Alexander; Simon, András; Chien, Kenneth R.

doi:10.1038/s41556-022-00902-2

Cited by 21 publications

(19 citation statements)

References 108 publications

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“…The Spanish ribbed newt, Pleurodeles waltl , is an emerging regenerative model organism, which has a high quality de novo transcriptome (Matsunami et al 2019) but no available common SNPs VCF file. We first set out to assess souporcell demux assignments on pooled splenocytes from three transgenic Pleurodeles newts, which express different fluorescent proteins under the same ubiquitous promoter (CAG)(Joven et al 2018; Eroglu et al 2022). We designed this experiment to only contain non-erythroid spleen cells from one individual of each transgenic newt line (Supplemental Figure 5), making it technically feasible to benchmark souporcell cell assignments for individuals through comparisons to fluorescent-based assignments (Figure 3A).…”

Section: Resultsmentioning

confidence: 99%

“…Pleurodeles newts, which express different fluorescent proteins under the same ubiquitous promoter (CAG) (Joven et al 2018;Eroglu et al 2022). We designed this experiment to only contain non-erythroid spleen cells from one individual of each transgenic newt line (Supplemental Figure 5), making it technically feasible to benchmark souporcell cell assignments for individuals through comparisons to fluorescent-based assignments (Figure 3A).…”

Section: Successful Snp-based Demuxing Of Pooled Fluorescent Pleurode...mentioning

confidence: 99%

“…Spleens were harvested from three separate Pleurodeles waltl and processed as individual samples in parallel. All animals were post-metamophic newts from established transgenic lines close to sexual maturity: one female tgTol2(CAG:Nucbow CAG:Cytbow) Simon (5.67g weight, 10.8cm snout-to-tail length) (Joven et al 2018), one male tgSceI(CAG:loxP-GFP-loxP-Cherry) Simon (5.36g,11.1cm) (Joven et al 2018), and female tgTol2(CAG:loxP-Cherry-loxP-H2B:YFP) Simon (6.25g, 10.8cm) (Eroglu et al 2022). Forceps and iridectomy scissors were used to remove the spleen in one piece, making sure that the forceps did not tear the spleen.…”

Section: Fluorescent Pooling Experimentsmentioning

confidence: 99%

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Accurate genotype-based demultiplexing of single cell RNA sequencing samples from non-human animals

Cardiello

Araus

Giatrellis

et al. 2022

Preprint

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Single cell sequencing technologies (scRNA-seq, scATAC-seq, etc.) have revolutionized the study of complex tissues and unique organisms, providing researchers with a much needed species agnostic tool to study biological processes at the cellular level. To date, scRNA-seq technologies are expensive, require sufficient cell quantities, and need biological replicates to avoid batch effects or artifactual results. Pooling cells from multiple individuals into a single scRNA-seq library can address these problems. However, sample labeling protocols for facilitating the computational separation of pooled scRNA-seq samples, termed demultiplexing, have undesirable limitations, particularly in resource-limited organisms. One promising solution developed for use in humans exploits the genetic diversity between individuals (i.e., single nucleotide polymorphisms (SNP)) to demultiplex pooled scRNA-seq samples. The use of SNP-based demultiplexing methods has not been validated for use in non-human species, but the widespread use of SNP-based demuxers would greatly facilitate research in commonly used, emerging, and more obscure species. In this study we applied SNP-based demultiplexing algorithms to pooled scRNA-seq datasets from numerous species and applied diverse ground truth confirmation assays to validate genetic demultiplexing results. SNP-based demultiplexers were found to accurately demultiplex pooled scRNA-seq data from species including zebrafish, African green monkey, Xenopus laevis, axolotl, Pleurodeles waltl, and Notophthalmus viridescens. Our results demonstrate that SNP-based demultiplexing of unlabeled, pooled scRNA-seq samples can be used with confidence in all of the species studied in this work. Further, we show that the only genomic resource required for this approach is the single-cell sequencing data and a de novo transcriptome. The incorporation of pooling and SNP-demultiplexing into scRNA-seq study designs will greatly increase the reproducibility and experimental options for studying species previously limited by technical uncertainties, computational hurdles, or limited tissue or cell quantities.

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Section: Resultsmentioning

confidence: 99%

Section: Successful Snp-based Demuxing Of Pooled Fluorescent Pleurode...mentioning

confidence: 99%

Section: Fluorescent Pooling Experimentsmentioning

confidence: 99%

See 1 more Smart Citation

Accurate genotype-based demultiplexing of single cell RNA sequencing samples from non-human animals

Cardiello

Araus

Giatrellis

et al. 2022

Preprint

Self Cite

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“…These EpiPCs are multipotent cardiac progenitors (the only widely accepted source) that contribute to multiple different cell lineages in the heart and can aid in the healing and repair process (Martínez-Estrada et al, 2010;Kikuchi et al, 2011a;Smart et al, 2011;Van Wijk et al, 2012;Streef & Smits, 2021). While there is substantial evidence to suggest that EpiPCs can differentiate into cardiomyocytes during development (Cai et al, 2008;Zhou et al, 2008) and in injured adult hearts (Smart et al, 2011;Van Wijk et al, 2012;Eroglu et al, 2022), this subject is still a matter of ongoing debate (Christoffels et al, 2009;Zhou et al, 2012). Notably, adult epicardial cells also secrete paracrine factors to modulate heart repair and regeneration post-MI (Zhou et al, 2011;Wei et al, 2015).…”

Section: Introductionmentioning

confidence: 99%

Oxytocin promotes epicardial cell activation and heart regeneration after cardiac injury

Wasserman

Huang

Lewis-Israeli

et al. 2022

Front. Cell Dev. Biol.

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Cardiovascular disease (CVD) is one of the leading causes of mortality worldwide, and frequently leads to massive heart injury and the loss of billions of cardiac muscle cells and associated vasculature. Critical work in the last 2 decades demonstrated that these lost cells can be partially regenerated by the epicardium, the outermost mesothelial layer of the heart, in a process that highly recapitulates its role in heart development. Upon cardiac injury, mature epicardial cells activate and undergo an epithelial-mesenchymal transition (EMT) to form epicardium-derived progenitor cells (EpiPCs), multipotent progenitors that can differentiate into several important cardiac lineages, including cardiomyocytes and vascular cells. In mammals, this process alone is insufficient for significant regeneration, but it might be possible to prime it by administering specific reprogramming factors, leading to enhanced EpiPC function. Here, we show that oxytocin (OXT), a hypothalamic neuroendocrine peptide, induces epicardial cell proliferation, EMT, and transcriptional activity in a model of human induced pluripotent stem cell (hiPSC)-derived epicardial cells. In addition, we demonstrate that OXT is produced after cardiac cryoinjury in zebrafish, and that it elicits significant epicardial activation promoting heart regeneration. Oxytocin signaling is also critical for proper epicardium development in zebrafish embryos. The above processes are significantly impaired when OXT signaling is inhibited chemically or genetically through RNA interference. RNA sequencing data suggests that the transforming growth factor beta (TGF-β) pathway is the primary mediator of OXT-induced epicardial activation. Our research reveals for the first time an evolutionary conserved brain-controlled mechanism inducing cellular reprogramming and regeneration of the injured mammalian and zebrafish heart, a finding that could contribute to translational advances for the treatment of cardiac injuries.

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“…The salamander is a unique animal model for studying tissue development and regeneration. 1 Many of its organs have perfect regeneration capabilities and have been extensively studied, such as the limb, 2 , 3 brain, 4 heart, 5 , 6 spinal cord, 7 and lens. 8 , 9 Most of the regeneration processes occur via the formation of a blastema‐like structure and the subsequent regrowth of a morphological replica of the lost tissue, which is also called epimorphic regeneration.…”

Section: Introductionmentioning

confidence: 99%

Developmental switch from morphological replication to compensatory growth for salamander lung regeneration

Yin

Zhang

et al. 2022

Cell Proliferation

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Salamanders possess a pair of lungs for active air breathing, but the lung respiration is fully operational only during the late stage of development, particularly after metamorphosis. Larval salamanders mainly exchange air through the gills and skin, thus sparing the developing lungs. Salamanders can repair their lungs after injury, but a comparative analysis of regenerative responses between the lungs of young and adult animals is lacking. In this study, lung resections were performed in both larval and adult newts (Pleurodeles waltl). The cellular dynamics, tissue morphology and organ function during lung regeneration were examined and the Yap mutants were produced with CRISPR tools. We found that salamander switches the regenerative strategies from morphological replication through the blastema formation to compensatory growth via resident epithelial cells proliferation upon pulmonary resection injury as it transitions beyond metamorphosis. The larval animals achieve lung regeneration by forming a transient blastema‐like structure and regrowing full‐sized developing lungs, albeit unventilated. The adults repair injured lungs via massive proliferating epithelial cells and by expanding the existing alveolar epithelium without neo‐alveolarization. Yap signalling promotes epithelial cell proliferation and prevents epithelial‐to‐mesenchymal transition to restore functional respiration. The salamanders have evolved distinct regenerative strategies for lung repair during different phases of life. Our results demonstrate a novel strategy for functional lung recovery by inducing epithelial cell proliferation to strengthen the remaining alveoli without rebuilding new alveoli.

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Epicardium-derived cells organize through tight junctions to replenish cardiac muscle in salamanders

Cited by 21 publications

References 108 publications

Accurate genotype-based demultiplexing of single cell RNA sequencing samples from non-human animals

Accurate genotype-based demultiplexing of single cell RNA sequencing samples from non-human animals

Oxytocin promotes epicardial cell activation and heart regeneration after cardiac injury

Developmental switch from morphological replication to compensatory growth for salamander lung regeneration

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