Interleukin-11 signaling promotes cellular reprogramming and limits fibrotic scarring during tissue regeneration.

Damage-induced fibrotic scarring limits tissue regeneration in mammals and is a leading cause of morbidity. In contrast, species like zebrafish can regenerate damaged tissues without excessive fibrosis. However, whether specific signaling pathways can both limit fibrosis and promote regeneration is unclear. Here, we show that interleukin-11 (Il-11)/Stat3 signaling has such a dual function. Zebrafish lacking Il-11 receptor function display severely compromised heart, fin, and scale regeneration. Deep phenotyping and transcriptional analysis of adult hearts and fins show that Il-11 signaling drives cellular reprogramming to orchestrate global and tissue-specific regenerative programs and broadly antagonizes hallmarks of adult mammalian scarring. Mechanistically, our data indicate that IL-11 signaling in endothelial cells antagonizes profibrotic transforming growth factor­ß signaling and endothelial-to-mesenchymal transition, limiting scarring and promoting cardiomyocyte repopulation, after injury. Overall, our findings position damage-induced Il-11/Stat3 signaling in a key role limiting fibrosis and promoting regeneration, revealing novel targets for regenerative therapies.

The E3 ubiquitin-protein ligase Rbx1 regulates cardiac wall morphogenesis in zebrafish.

Cardiac trabeculae are muscular ridge-like structures within the ventricular wall that are crucial for cardiac function. In zebrafish, these structures first form primarily through the delamination of compact wall cardiomyocytes (CMs). Although defects in proteasomal degradation have been associated with decreased cardiac function, whether they also affect cardiac development has not been extensively analyzed. Here we report a role during cardiac wall morphogenesis in zebrafish for the E3 ubiquitin-protein ligase Rbx1, which has been shown to regulate the degradation of key signaling molecules. Although development is largely unperturbed in zebrafish rbx1 mutant larvae, they exhibit CM multi-layering. This phenotype is not affected by blocking ErbB signaling, but fails to manifest itself in the absence of blood flow/cardiac contractility. Surprisingly, rbx1 mutants display ErbB independent Notch reporter expression in the myocardium. We generated tissue-specific rbx1 overexpression lines and found that endothelial, but not myocardial, specific rbx1 expression normalizes the cardiac wall morphogenesis phenotype. In addition, we found that pharmacological activation of Hedgehog signaling ameliorates the multi-layered myocardial wall phenotype in rbx1 mutants. Collectively, our data indicate that endocardial activity of Rbx1 is essential for cardiac wall morphogenesis.

Cardiomyocyte heterogeneity during zebrafish development and regeneration.

Contrary to adult mammals, zebrafish are able to regenerate their heart after cardiac injury. This regenerative response relies, in part, on the endogenous ability of cardiomyocytes (CMs) to dedifferentiate and proliferate to replenish the lost muscle. However, CM heterogeneity and population dynamics during development and regeneration require further investigation. Through comparative transcriptomic analyses of the developing and adult zebrafish heart, we identified tnnc2 and tnni4b.3 expression as markers for CMs at early and late developmental stages, respectively. Using newly developed reporter lines for these genes, we investigated their expression dynamics during heart development and regeneration. tnnc2 reporter lines label most CMs at embryonic stages, and this labeling declines rapidly during larval stages; in adult hearts, tnnc2 reporter expression is only detectable in a small subset of CMs. Conversely, expression of a tnni4b.3 reporter is initially visible in CMs in the outer curvature of the ventricle at larval stages, and it is subsequently present in a vast majority of the CMs in adult hearts. To further characterize the adult CMs labeled by the tnnc2 (i.e., embryonic) reporter, we performed transcriptomic analyses and found that they express markers of immature CMs as well as genes encoding components of the Notch signaling pathway. In support of this finding, we observed, using two different reporters, that these CMs display higher levels of Notch signaling. Moreover, during adult heart regeneration, CMs in the injured area activate the embryonic CM reporter and downregulate the tnni4b.3 reporter, further highlighting the molecular changes in regenerating CMs. Overall, our findings provide additional evidence for CM heterogeneity in adult zebrafish.

Tie1 regulates zebrafish cardiac morphogenesis through Tolloid-like 1 expression.

Tie1 is a receptor tyrosine kinase expressed in endothelial cells, where it modulates Angiopoietin/Tie2 signaling. Previous studies have shown that mouse Tie1 mutants exhibit severe cardiovascular defects; however, much remains to be learned about the role of Tie1, especially during cardiac development. To further understand Tie1 function, we generated a zebrafish tie1 mutant line. Homozygous mutant embryos display reduced endothelial and endocardial cell numbers and reduced heart size. Live imaging and ultrastructural analyses at embryonic stages revealed increased cardiac jelly thickness as well as cardiomyocyte defects, including a loss of sarcomere organization and altered cell shape. Transcriptomic profiling of embryonic hearts uncovered the downregulation of tll1, which encodes a Tolloid-like protease, in tie1-/- compared with wild-type siblings. Using mRNA injections into one-cell stage embryos, we found that tll1 overexpression could partially rescue the tie1 mutant cardiac phenotypes including the endocardial and myocardial cell numbers as well as the cardiac jelly thickness. Altogether, our results indicate the importance of a Tie1-Tolloid-like 1 axis in paracrine signaling during cardiac development.

Prediabetes uncovers differential gene expression at fasting and in response to oral glucose load in immune cells.

BACKGROUND AND OBJECTIVE: Metabolic disorders including diabetes are associated with immune cell dysfunction. However, the effect of normal glucose metabolism or impairment thereof on immune cell gene expression is not well known. Hence, in this cross-sectional pilot study, we sought to determine the differences in gene expression in the peripheral blood mono-nuclear cells (PBMCs) of normal glucose tolerant (NGT) and prediabetic (PD) Asian Indian men, at fasting and in response to 75 g oral glucose load. METHODS: Illumina HT12 bead chip-based microarray was performed on PBMCs at fasting and 2-h post load conditions for NGT (N = 6) and PD (N = 9) subjects. Following normalization and due quality control of the raw data, differentially expressed genes (DEGs) under different conditions within and across the two groups were identified using GeneSpring GX V12.0 software. Paired and unpaired Student's t-tests were applied along with fold change cut-offs for appropriate comparisons. Validation of the microarray data was carried out through real-time qPCR analysis. Significantly regulated biological pathways were analyzed by employing DEGs and DAVID resource. Deconvolution of the DEGs between NGT and PD subjects at fasting was performed using CIBERSORT and genes involved in regulatory T-cell (Treg) function were further analyzed for biological significance. RESULTS: Glucose load specifically altered the expression of 112 genes in NGT and 356 genes in PD subjects. Biological significance analysis revealed transient up-regulation of innate and adaptive immune response related genes following oral glucose load in NGT individuals, which was not observed in PD subjects. Instead, in the PD group, glucose load led to an increase in the expression of pro-atherogenic and anti-angiogenic genes. Comparison of gene expression at fasting state in PD versus NGT revealed 21,707 differentially expressed genes. Biological significance analysis of the immune function related genes between these two groups (at fasting) revealed higher gene expression of members of the TLR signaling, MHC class II molecules, and T-cell receptor, chemotaxis and adhesion pathways in PD subjects. Expression of interferon-γ (IFN-γ) and TNFα was higher and that of type-1 interferons and TGF-ß was lower at fasting state in PD subjects compared to NGT. Additionally, expression of multiple proteasome subunits and protein arginine methyl transferase genes (PRMTs) were higher and that of Treg specific genes was significantly distinct at fasting in PD subjects compared to NGT. CONCLUSION: Prediabetes uncovers constitutive TLR activation, enhanced IFN-γ signaling, and Treg dysfunction at fasting along with altered gene expression response to oral glucose load.

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.

Tek (Tie2) is not required for cardiovascular development in zebrafish.

Angiopoietin/TIE signalling plays a major role in blood and lymphatic vessel development. In mouse, Tek (previously known as Tie2) mutants die prenatally due to a severely underdeveloped cardiovascular system. In contrast, in zebrafish, previous studies have reported that although embryos injected with tek morpholinos (MOs) exhibit severe vascular defects, tek mutants display no obvious vascular malformations. To further investigate the function of zebrafish Tek, we generated a panel of loss-of-function tek mutants, including RNA-less alleles, an allele lacking the MO-binding site, an in-frame deletion allele and a premature termination codon-containing allele. Our data show that all these mutants survive to adulthood with no obvious cardiovascular defects. MO injections into tek mutants lacking the MO-binding site or the entire tek locus cause similar vascular defects to those observed in MO-injected +/+ siblings, indicating off-target effects of the MOs. Surprisingly, comprehensive phylogenetic profiling and synteny analyses reveal that Tek was lost in the largest teleost clade, suggesting a lineage-specific shift in the function of TEK during vertebrate evolution. Altogether, these data show that Tek is dispensable for zebrafish development, and probably dispensable in most teleost species.

Analysis and modelling of septic shock microarray data using Singular Value Decomposition.

Being a high throughput technique, enormous amounts of microarray data has been generated and there arises a need for more efficient techniques of analysis, in terms of speed and accuracy. Finding the differentially expressed genes based on just fold change and p-value might not extract all the vital biological signals that occur at a lower gene expression level. Besides this, numerous mathematical models have been generated to predict the clinical outcome from microarray data, while very few, if not none, aim at predicting the vital genes that are important in a disease progression. Such models help a basic researcher narrow down and concentrate on a promising set of genes which leads to the discovery of gene-based therapies. In this article, as a first objective, we have used the lesser known and used Singular Value Decomposition (SVD) technique to build a microarray data analysis tool that works with gene expression patterns and intrinsic structure of the data in an unsupervised manner. We have re-analysed a microarray data over the clinical course of Septic shock from Cazalis et al. (2014) and have shown that our proposed analysis provides additional information compared to the conventional method. As a second objective, we developed a novel mathematical model that predicts a set of vital genes in the disease progression that works by generating samples in the continuum between health and disease, using a simple normal-distribution-based random number generator. We also verify that most of the predicted genes are indeed related to septic shock.