High-throughput scNMT protocol for multiomics profiling of single cells from mouse brain and pancreatic organoids.

Single-cell nucleosome, methylome, and transcriptome (scNMT) sequencing is a recently developed method that allows multiomics profiling of single cells. In this scNMT protocol, we describe profiling of cells from mouse brain and pancreatic organoids, using liquid handling platforms to increase throughput from 96-well to 384-well plate format. Our approach miniaturizes reaction volumes and incorporates the latest Smart-seq3 protocol to obtain higher numbers of detected genes and genomic DNA (gDNA) CpGs per cell. We outline normalization steps to optimally distribute per-cell sequencing depth. For complete details on the use and execution of this protocol, please refer to Clark (2019), Clark et al. (2018), and Clark et al., 2018, Hagemann-Jensen et al., 2020a, Hagemann-Jensen et al., 2020b.

Wt1 transcription factor impairs cardiomyocyte specification and drives a phenotypic switch from myocardium to epicardium.

During development, the heart grows by addition of progenitor cells to the poles of the primordial heart tube. In the zebrafish, Wilms tumor 1 transcription factor a (wt1a) and b (wt1b) genes are expressed in the pericardium, at the venous pole of the heart. From this pericardial layer, the proepicardium emerges. Proepicardial cells are subsequently transferred to the myocardial surface and form the epicardium, covering the myocardium. We found that while wt1a and wt1b expression is maintained in proepicardial cells, it is downregulated in pericardial cells that contributes cardiomyocytes to the developing heart. Sustained wt1b expression in cardiomyocytes reduced chromatin accessibility of specific genomic loci. Strikingly, a subset of wt1a- and wt1b-expressing cardiomyocytes changed their cell-adhesion properties, delaminated from the myocardium and upregulated epicardial gene expression. Thus, wt1a and wt1b act as a break for cardiomyocyte differentiation, and ectopic wt1a and wt1b expression in cardiomyocytes can lead to their transdifferentiation into epicardial-like cells.

Ventricular Cryoinjury as a Model to Study Heart Regeneration in Zebrafish.

Zebrafish have the capacity to regenerate most of its organs upon injury, including the heart. Due to its amenability for genetic manipulation, the zebrafish is an excellent model organism to study the cellular and molecular mechanisms promoting heart regeneration. Several cardiac injury models have been developed in zebrafish, including ventricular resection, genetic ablation, and ventricular cryoinjury. This chapter provides a detailed protocol of zebrafish ventricular cryoinjury and highlights factors and critical steps to be considered when performing this method.

Recent insights into zebrafish cardiac regeneration.

In humans, myocardial infarction results in ventricular remodeling, progressing ultimately to cardiac failure, one of the leading causes of death worldwide. In contrast to the adult mammalian heart, the zebrafish model organism has a remarkable regenerative capacity, offering the possibility to research the bases of natural regeneration. Here, we summarize recent insights into the cellular and molecular mechanisms that govern cardiac regeneration in the zebrafish.

Wilms Tumor 1b Expression Defines a Pro-regenerative Macrophage Subtype and Is Required for Organ Regeneration in the Zebrafish.

Organ regeneration is preceded by the recruitment of innate immune cells, which play an active role during repair and regrowth. Here, we studied macrophage subtypes during organ regeneration in the zebrafish, an animal model with a high regenerative capacity. We identified a macrophage subpopulation expressing Wilms tumor 1b (wt1b), which accumulates within regenerating tissues. This wt1b+ macrophage population exhibited an overall pro-regenerative gene expression profile and different migratory behavior compared to the remainder of the macrophages. Functional studies showed that wt1b regulates macrophage migration and retention at the injury area. Furthermore, wt1b-null mutant zebrafish presented signs of impaired macrophage differentiation, delayed fin growth upon caudal fin amputation, and reduced cardiomyocyte proliferation following cardiac injury that correlated with altered macrophage recruitment to the regenerating areas. We describe a pro-regenerative macrophage subtype in the zebrafish and a role for wt1b in organ regeneration.

Transient fibrosis resolves via fibroblast inactivation in the regenerating zebrafish heart.

In the zebrafish (Danio rerio), regeneration and fibrosis after cardiac injury are not mutually exclusive responses. Upon cardiac cryoinjury, collagen and other extracellular matrix (ECM) proteins accumulate at the injury site. However, in contrast to the situation in mammals, fibrosis is transient in zebrafish and its regression is concomitant with regrowth of the myocardial wall. Little is known about the cells producing this fibrotic tissue or how it resolves. Using novel genetic tools to mark periostin b- and collagen 1alpha2 (col1a2)-expressing cells in combination with transcriptome analysis, we explored the sources of activated fibroblasts and traced their fate. We describe that during fibrosis regression, fibroblasts are not fully eliminated but become inactivated. Unexpectedly, limiting the fibrotic response by genetic ablation of col1a2-expressing cells impaired cardiomyocyte proliferation. We conclude that ECM-producing cells are key players in the regenerative process and suggest that antifibrotic therapies might be less efficient than strategies targeting fibroblast inactivation.

Tbx5a lineage tracing shows cardiomyocyte plasticity during zebrafish heart regeneration.

During development, mesodermal progenitors from the first heart field (FHF) form a primitive cardiac tube, to which progenitors from the second heart field (SHF) are added. The contribution of FHF and SHF progenitors to the adult zebrafish heart has not been studied to date. Here we find, using genetic tbx5a lineage tracing tools, that the ventricular myocardium in the adult zebrafish is mainly derived from tbx5a+ cells, with a small contribution from tbx5a- SHF progenitors. Notably, ablation of ventricular tbx5a+-derived cardiomyocytes in the embryo is compensated by expansion of SHF-derived cells. In the adult, tbx5a expression is restricted to the trabeculae and excluded from the outer cortical layer. tbx5a-lineage tracing revealed that trabecular cardiomyocytes can switch their fate and differentiate into cortical myocardium during adult heart regeneration. We conclude that a high degree of cardiomyocyte cell fate plasticity contributes to efficient regeneration.