Border-zone cardiomyocytes and macrophages contribute to remodeling of the extracellular matrix to promote cardiomyocyte invasion during zebrafish cardiac regeneration.

Despite numerous advances in our understanding of zebrafish cardiac regeneration, an aspect that remains less studied is how regenerating cardiomyocytes invade, and eventually replace, the collagen-containing fibrotic tissue following injury. Here, we provide an in-depth analysis of the process of cardiomyocyte invasion using live-imaging and histological approaches. We observed close interactions between protruding cardiomyocytes and macrophages at the wound border zone, and macrophage-deficient irf8 mutant zebrafish exhibited defects in extracellular matrix (ECM) remodeling and cardiomyocyte protrusion into the injured area. Using a resident macrophage ablation model, we show that defects in ECM remodeling at the border zone and subsequent cardiomyocyte protrusion can be partly attributed to a population of resident macrophages. Single-cell RNA-sequencing analysis of cells at the wound border revealed a population of cardiomyocytes and macrophages with fibroblast-like gene expression signatures, including the expression of genes encoding ECM structural proteins and ECM-remodeling proteins. The expression of mmp14b , which encodes a membrane-anchored matrix metalloproteinase, was restricted to cells in the border zone, including cardiomyocytes, macrophages, fibroblasts, and endocardial/endothelial cells. Genetic deletion of mmp14b led to a decrease in 1) macrophage recruitment to the border zone, 2) collagen degradation at the border zone, and 3) subsequent cardiomyocyte invasion. Furthermore, cardiomyocyte-specific overexpression of mmp14b was sufficient to enhance cardiomyocyte invasion into the injured tissue and along the apical surface of the wound. Altogether, our data shed important insights into the process of cardiomyocyte invasion of the collagen-containing injured tissue during cardiac regeneration. They further suggest that cardiomyocytes and resident macrophages contribute to ECM remodeling at the border zone to promote cardiomyocyte replenishment of the fibrotic injured tissue.

The developing epicardium regulates cardiac chamber morphogenesis by promoting cardiomyocyte growth.

The epicardium, the outermost layer of the heart, is an important regulator of cardiac regeneration. However, a detailed understanding of the crosstalk between the epicardium and myocardium during development requires further investigation. Here, we generated three models of epicardial impairment in zebrafish by mutating the transcription factor genes tcf21 and wt1a, and ablating tcf21+ epicardial cells. Notably, all three epicardial impairment models exhibited smaller ventricles. We identified the initial cause of this phenotype as defective cardiomyocyte growth, resulting in reduced cell surface and volume. This failure of cardiomyocyte growth was followed by decreased proliferation and increased abluminal extrusion. By temporally manipulating its ablation, we show that the epicardium is required to support cardiomyocyte growth mainly during early cardiac morphogenesis. By transcriptomic profiling of sorted epicardial cells, we identified reduced expression of FGF and VEGF ligand genes in tcf21-/- hearts, and pharmacological inhibition of these signaling pathways in wild type partially recapitulated the ventricular growth defects. Taken together, these data reveal distinct roles of the epicardium during cardiac morphogenesis and signaling pathways underlying epicardial-myocardial crosstalk.

Apelin signaling dependent endocardial protrusions promote cardiac trabeculation in zebrafish.

During cardiac development, endocardial cells (EdCs) produce growth factors to promote myocardial morphogenesis and growth. In particular, EdCs produce neuregulin which is required for ventricular cardiomyocytes (CMs) to seed the multicellular ridges known as trabeculae. Defects in neuregulin signaling, or in endocardial sprouting toward CMs, cause hypotrabeculation. However, the mechanisms underlying endocardial sprouting remain largely unknown. Here, we first show by live imaging in zebrafish embryos that EdCs interact with CMs via dynamic membrane protrusions. After touching CMs, these protrusions remain in close contact with their target despite the vigorous cardiac contractions. Loss of the CM-derived peptide Apelin, or of the Apelin receptor, which is expressed in EdCs, leads to reduced endocardial sprouting and hypotrabeculation. Mechanistically, neuregulin signaling requires endocardial protrusions to induce extracellular signal-regulated kinase (Erk) activity in CMs and trigger their delamination. Altogether, these data show that Apelin signaling-dependent endocardial protrusions modulate CM behavior during trabeculation.

Global DNA methylation profile at LINE-1 repeats and promoter methylation of genes involved in DNA damage response and repair pathways in human peripheral blood mononuclear cells in response to γ-radiation.

DNA methylation is an epigenetic mechanism, which plays an important role in gene regulation. The present study evaluated DNA methylation profile of LINE1 repeats and promoter methylation of DNA damage response (DDR) and DNA repair (DR) genes (PARP1, ATM, BRCA1, MLH1, XPC, RAD23B, APC, TNFα, DNMT3A, MRE11A, MGMT, CDKN2A, MTHFR) in human peripheral blood mononuclear cells (PBMCs) of healthy donors in response to γ-radiation. Methylation level was correlated with gene expression profile of selected DDR and DR genes (APC, MLH1, PARP1, MRE11A, TNFα, MGMT) to understand their role in gene regulation. Blood samples were collected from 15 random healthy donors, PBMCs were isolated, exposed to 0.1 Gy (low) and 2.0 Gy (high) doses of γ-radiation and proliferated for 48 h and 72 h. Genomic DNA and total RNA were isolated from irradiated PBMCs along with un-irradiated control. Methylation profile was determined from bisulphite converted DNA and amplified by methylation sensitive high resolution melting (MS-HRM) method. Total RNA was converted to cDNA and relative expression was analysed using real time quantitative-PCR. Our results revealed that at 0.1 Gy, MRE11A and TNFα showed significant (P < 0.05) increase in methylation at 72 h. At 2.0 Gy, significant increase (P < 0.05) in methylation profile was observed at LINE1, MRE11A, PARP1, BRCA1, DNMT3A and RAD23B at 48 h and 72 h. PARP1 showed significant positive correlation of methylation status with gene expression. In conclusion, low and high doses of γ-radiation have significant influence on DNA methylation status of LINE1, DDR and DR genes suggesting their potential role as epigenetic signatures in human PBMCs, which can be further explored in human populations.

The zebrafish issue: 25†years on.

In the 1990s, labs on both sides of the Atlantic performed the largest genetic mutagenesis screen at that time using an emerging model organism: the zebrafish. Led by Christiane Nüsslein-Volhard in Tübingen, Germany, and Wolfgang Driever in Boston, USA, these colossal screens culminated in 1996 with the publication of 37 articles in a special issue of Development, which remains the journal's largest issue to this day. To celebrate the anniversary of the zebrafish issue and reflect on the 25 years since its publication, five zebrafish researchers share what the issue means to them, how it has contributed to their career and its impact on the zebrafish community.

The EMT transcription factor Snai1 maintains myocardial wall integrity by repressing intermediate filament gene expression.

The transcription factor Snai1, a well-known regulator of epithelial-to-mesenchymal transition, has been implicated in early cardiac morphogenesis as well as in cardiac valve formation. However, a role for Snai1 in regulating other aspects of cardiac morphogenesis has not been reported. Using genetic, transcriptomic, and chimeric analyses in zebrafish, we find that Snai1b is required in cardiomyocytes for myocardial wall integrity. Loss of snai1b increases the frequency of cardiomyocyte extrusion away from the cardiac lumen. Extruding cardiomyocytes exhibit increased actomyosin contractility basally as revealed by enrichment of p-myosin and α-catenin epitope α-18, as well as disrupted intercellular junctions. Transcriptomic analysis of wild-type and snai1b mutant hearts revealed the dysregulation of intermediate filament genes, including desmin b (desmb) upregulation. Cardiomyocyte-specific desmb overexpression caused increased cardiomyocyte extrusion, recapitulating the snai1b mutant phenotype. Altogether, these results indicate that Snai1 maintains the integrity of the myocardial epithelium, at least in part by repressing desmb expression.

Sculpting the heart: Cellular mechanisms shaping valves and trabeculae.

The transformation of the heart from a simple tube to a complex organ requires the orchestration of several morphogenetic processes. Two structures critical for cardiac function, the cardiac valves and the trabecular network, are formed through extensive tissue morphogenesis-endocardial cell migration, deadhesion and differentiation into fibroblast-like cells during valve formation, and cardiomyocyte delamination and apico-basal depolarization during trabeculation. Here, we review current knowledge of how these specialized structures acquire their shape by focusing on the underlying cellular behaviors and molecular mechanisms, highlighting findings from in vivo models and briefly discussing the recent advances in cardiac cell culture and organoids.

Tension heterogeneity directs form and fate to pattern the myocardial wall.

How diverse cell fates and complex forms emerge and feed back to each other to sculpt functional organs remains unclear. In the developing heart, the myocardium transitions from a simple epithelium to an intricate tissue that consists of distinct layers: the outer compact and inner trabecular layers. Defects in this process, which is known as cardiac trabeculation, cause cardiomyopathies and embryonic lethality, yet how tissue symmetry is broken to specify trabecular cardiomyocytes is unknown. Here we show that local tension heterogeneity drives organ-scale patterning and cell-fate decisions during cardiac trabeculation in zebrafish. Proliferation-induced cellular crowding at the tissue scale triggers tension heterogeneity among cardiomyocytes of the compact layer and drives those with higher contractility to delaminate and seed the trabecular layer. Experimentally, increasing crowding within the compact layer cardiomyocytes augments delamination, whereas decreasing it abrogates delamination. Using genetic mosaics in trabeculation-deficient zebrafish models-that is, in the absence of critical upstream signals such as Nrg-Erbb2 or blood flow-we find that inducing actomyosin contractility rescues cardiomyocyte delamination and is sufficient to drive cardiomyocyte fate specification, as assessed by Notch reporter expression in compact layer cardiomyocytes. Furthermore, Notch signalling perturbs the actomyosin machinery in cardiomyocytes to restrict excessive delamination, thereby preserving the architecture of the myocardial wall. Thus, tissue-scale forces converge on local cellular mechanics to generate complex forms and modulate cell-fate choices, and these multiscale regulatory interactions ensure robust self-organized organ patterning.

Induction of different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells treated with various doses of 177Lu-Nimotuzumab.

INTRODUCTION: Radioimmunotherapy (RIT) is a major anti-cancer therapy in cancer management multimodalities. 177Lu-Nimotuzumab has been in the use for radioimmunotherapy of EGFR expressing tumors. This study focuses on understanding the differential cellular and molecular mechanisms of anti-tumor effects of 177Lu-Nimotuzumab on low EGFR expressing A549 and high EGFR expressing A431 tumor cells. MATERIALS AND METHODS: Nimotuzumab labeled with 177Lu was characterized by SE-HPLC. Specificity of 177Lu-Nimotuzumab to EGFR expressed on A549 and A431 cells was confirmed by competitive assay using increasing amounts of unlabeled Nimotuzumab. Cellular responses of A549 (low EGFR) and A431 (high EGFR) in response to different doses of 177Lu-Nimotuzumab were determined by Viable count assay for cellular viability, cell-cycle analysis by DNA staining, apoptotic assay for cell death, and CFSE dilution assay for cellular proliferation capacity. The number of DNA DSBs formed was determined using γ-H2AX assay with PI staining. Transcription of genes involved in DNA damage response and repair (DRR) pathways was monitored by RT-qPCR. RESULTS: 177Lu-Nimotuzumab characterized by SE-HPLC exhibited a radiochemical purity of 99.1 ± 0.6%. Cell binding competition studies with 177Lu-Nimotuzumab showed specific binding of 34.3 ± 1.7% with A431 cells and 18.4 ± 1.9% with A549 cells which decreased when co-incubated with unlabeled Nimotuzumab. Cytotoxicity and DNA damage (DNA DSBs) increased with an increase in the dose of 177Lu-Nimotuzumab. A549 displayed G2/M arrest while A431 showed G1 arrest. Apoptotic death was determined to be one of the modes of death of arrested A549 and A431 cells which increases with the increase in the dose of 177Lu-Nimotuzumab. Loss of proliferation capacity was higher in A431 showed by CFSE staining at different doses of 177Lu-Nimotuzumab. Transcription profile of most DRR genes in A431 and A549 showed a decrease in the transcription at 4 h followed by recovery at 16 h post-treatment. The degree of transcription of most DRR genes was similar, irrespective of 177Lu-Nimotuzumab dose. CONCLUSION: 177Lu-Nimotuzumab induces different cellular arrest and molecular responses in low EGFR expressing A549 and high EGFR expressing A431 tumor cells. This study would enable the development of integrative novel treatment strategies for radioimmunotherapy in low and high EGFR expressing tumors by 177Lu-Nimotuzumab with therapeutic gains.

Spatio-Temporal Regulation of RhoGTPases Signaling by Myosin II.

RhoGTPase activation of non-muscle myosin II regulates cell division, extrusion, adhesion, migration, and tissue morphogenesis. However, the regulation of myosin II and mechanotransduction is not straightforward. Increasingly, the role of myosin II on the feedback regulation of RhoGTPase signaling is emerging. Indeed, myosin II controls RhoGTPase signaling through multiple mechanisms, namely contractility driven advection, scaffolding, and sequestration of signaling molecules. Here we discuss these mechanisms by which myosin II regulates RhoGTPase signaling in cell adhesion, migration, and tissue morphogenesis.

Bistable front dynamics in a contractile medium: Travelling wave fronts and cortical advection define stable zones of RhoA signaling at epithelial adherens junctions.

Mechanical coherence of cell layers is essential for epithelia to function as tissue barriers and to control active tissue dynamics during morphogenesis. RhoA signaling at adherens junctions plays a key role in this process by coupling cadherin-based cell-cell adhesion together with actomyosin contractility. Here we propose and analyze a mathematical model representing core interactions involved in the spatial localization of junctional RhoA signaling. We demonstrate how the interplay between biochemical signaling through positive feedback, combined with diffusion on the cell membrane and mechanical forces generated in the cortex, can determine the spatial distribution of RhoA signaling at cell-cell junctions. This dynamical mechanism relies on the balance between a propagating bistable signal that is opposed by an advective flow generated by an actomyosin stress gradient. Experimental observations on the behavior of the system when contractility is inhibited are in qualitative agreement with the predictions of the model.

ROCK1 but not ROCK2 contributes to RhoA signaling and NMIIA-mediated contractility at the epithelial zonula adherens.

Rho kinases (ROCK1 and ROCK2) function downstream of the small GTPase RhoA to drive actomyosin cytoskeletal remodeling. It has often been believed that ROCK1 and ROCK2 may be functionally redundant, as they share a highly conserved kinase domain. However, in this study, we report differential functional effects for these ROCKs at the epithelial zonula adherens (ZA). Using specific siRNA, we found that ROCK1 depletion disrupted cadherin organization at the ZA, accompanied by loss of F-actin and NMIIA, whereas ROCK2 knockdown had no significant effect. Further, ROCK1, but not ROCK2, was necessary to stabilize GTP-RhoA at the ZA, thereby sustaining junctional tension and inhibiting intraepithelial cell movement. We also found that nonmuscle myosin IIA is a major determinant of ROCK1 cortical stability. Thus, despite sharing the catalytic domain with ROCK2, ROCK1 appears to be the dominant kinase essential for junctional integrity and contractile tension at epithelial ZA.

Coronin 1B supports RhoA signaling at cell-cell junctions through Myosin II.

Non-muscle myosin II (NMII) motor proteins are responsible for generating contractile forces inside eukaryotic cells. There is also a growing interest in the capacity for these motor proteins to influence cell signaling through scaffolding, especially in the context of RhoA GTPase signaling. We previously showed that NMIIA accumulation and stability within specific regions of the cell cortex, such as the zonula adherens (ZA), allows the formation of a stable RhoA signaling zone. Now we demonstrate a key role for Coronin 1B in maintaining this junctional pool of NMIIA, as depletion of Coronin 1B significantly compromised myosin accumulation and stability at junctions. The loss of junctional NMIIA, upon Coronin 1B knockdown, perturbed RhoA signaling due to enhanced junctional recruitment of the RhoA antagonist, p190B Rho GAP. This effect was blocked by the expression of phosphomimetic MRLC-DD, thus reinforcing the central role of NMII in regulating RhoA signaling.

Contact inhibition of locomotion and mechanical cross-talk between cell-cell and cell-substrate adhesion determine the pattern of junctional tension in epithelial cell aggregates.

We used a computational approach to analyze the biomechanics of epithelial cell aggregates-islands, stripes, or entire monolayers-that combines both vertex and contact-inhibition-of-locomotion models to include cell-cell and cell-substrate adhesion. Examination of the distribution of cell protrusions (adhesion to the substrate) in the model predicted high-order profiles of cell organization that agree with those previously seen experimentally. Cells acquired an asymmetric distribution of basal protrusions, traction forces, and apical aspect ratios that decreased when moving from the edge to the island center. Our in silico analysis also showed that tension on cell-cell junctions and apical stress is not homogeneous across the island. Instead, these parameters are higher at the island center and scale up with island size, which we confirmed experimentally using laser ablation assays and immunofluorescence. Without formally being a three-dimensional model, our approach has the minimal elements necessary to reproduce the distribution of cellular forces and mechanical cross-talk, as well as the distribution of principal stress in cells within epithelial cell aggregates. By making experimentally testable predictions, our approach can aid in mechanical analysis of epithelial tissues, especially when local changes in cell-cell and/or cell-substrate adhesion drive collective cell behavior.

Transcriptional Regulation of Atp-Dependent Chromatin Remodeling Factors: Smarcal1 and Brg1 Mutually Co-Regulate Each Other.

The ATP-dependent chromatin remodeling factors regulate gene expression. However, it is not known whether these factors regulate each other. Given the ability of these factors to regulate the accessibility of DNA to transcription factors, we postulate that one ATP-dependent chromatin remodeling factor should be able to regulate the transcription of another ATP-dependent chromatin remodeling factor. In this paper, we show that BRG1 and SMARCAL1, both members of the ATP-dependent chromatin remodeling protein family, regulate each other. BRG1 binds to the SMARCAL1 promoter, while SMARCAL1 binds to the brg1 promoter. During DNA damage, the occupancy of SMARCAL1 on the brg1 promoter increases coinciding with an increase in BRG1 occupancy on the SMARCAL1 promoter, leading to increased brg1 and SMARCAL1 transcripts respectively. This is the first report of two ATP-dependent chromatin remodeling factors regulating each other.

Feedback regulation through myosin II confers robustness on RhoA signalling at E-cadherin junctions.

Actomyosin at the epithelial zonula adherens (ZA) generates junctional tension for tissue integrity and morphogenesis. This requires the RhoA GTPase, which establishes a strikingly stable active zone at the ZA. Mechanisms must then exist to confer robustness on junctional RhoA signalling at the population level. We now identify a feedback network that generates a stable mesoscopic RhoA zone out of dynamic elements. The key is scaffolding of ROCK1 to the ZA by myosin II. ROCK1 protects junctional RhoA by phosphorylating Rnd3 to prevent the cortical recruitment of the Rho suppressor, p190B RhoGAP. Combining predictive modelling and experimentation, we show that this network constitutes a bistable dynamical system that is realized at the population level of the ZA. Thus, stability of the RhoA zone is an emergent consequence of the network of interactions that allow myosin II to feedback to RhoA.

Making a Choice: How Cadherin Switching Controls Cell Migration.

Cells that undergo epithelial-to-mesenchymal transitions (EMTs) commonly switch from expressing E-cadherin to N-cadherin. But why this occurs is not well understood. In the current issue of Developmental Cell, Scarpa et al. (2015) identify a reason: cadherin switching controls Rac signaling to determine how cell locomotion is regulated by contact.

Active tension: the role of cadherin adhesion and signaling in generating junctional contractility.

In this chapter, we discuss the cell biology of contractility at cell-cell junctions. As discussed elsewhere in this volume, contractile forces play key roles in development and tissue homeostasis. Here, we review our understanding of the cellular mechanisms that functionally and physically link cadherin adhesion to the actomyosin contractile apparatus of the cell. Focusing on epithelia, we argue that E-cadherin junctions can be considered as active mechanical agents, which contribute to the assembly of actomyosin at the junctional cortex itself. This reflects cortical signaling, notably that regulated by the Rho GTPase, coordinated with actin regulation at junctions. The product, contractile tension at junctions, can then be regarded as an emergent property of a complex dynamical system that integrates adhesion with the cytoskeleton.

An RPTPα/Src family kinase/Rap1 signaling module recruits myosin IIB to support contractile tension at apical E-cadherin junctions.

Cell-cell adhesion couples the contractile cortices of epithelial cells together, generating tension to support a range of morphogenetic processes. E-cadherin adhesion plays an active role in generating junctional tension by promoting actin assembly and cortical signaling pathways that regulate myosin II. Multiple myosin II paralogues accumulate at mammalian epithelial cell-cell junctions. Earlier, we found that myosin IIA responds to Rho-ROCK signaling to support junctional tension in MCF-7 cells. Although myosin IIB is also found at the zonula adherens (ZA) in these cells, its role in junctional contractility and its mode of regulation are less well understood. We now demonstrate that myosin IIB contributes to tension at the epithelial ZA. Further, we identify a receptor type-protein tyrosine phosphatase alpha-Src family kinase-Rap1 pathway as responsible for recruiting myosin IIB to the ZA and supporting contractile tension. Overall these findings reinforce the concept that orthogonal E-cadherin-based signaling pathways recruit distinct myosin II paralogues to generate the contractile apparatus at apical epithelial junctions.