Biology subjects

Gonzalez-Rosa, J. M.

Publications and source records attributed to Gonzalez-Rosa, J. M..

6 recordsLinked to original sources

bioRxiv2026

High levels of the cardiomyocyte-specific kinase Tnni3k impair zebrafish heart regeneration by driving chronic myocardial inflammation

Background: Zebrafish regenerate their hearts after injury, and defining the barriers that block this capacity in mammals may reveal targets for heart failure treatment. Elevated levels of the cardiomyocyte-specific kinase TNNI3K are associated with human cardiomyopathy, and its overexpression drives adverse remodeling in mice. Recent work has linked elevated TNNI3K to cardiomyocyte polyploidization and loss of regenerative competence, but whether this cell-cycle effect accounts for the pathology seen in patients has not been established. Because TNNI3K is restricted to cardiomyocytes, whether it also acts non-cell-autonomously is unknown. Methods: We generated an allelic series of zebrafish lines comprising cardiomyocyte-specific Tnni3k overexpression, an expression-matched kinase-dead variant, a full-locus deletion, and a line overexpressing mouse Tnni3k. Regeneration, cardiomyocyte proliferation, and ploidy were assessed in uninjured hearts and after cryoinjury. We combined ventricular RNA-sequencing, macrophage depletion before or after injury, tamoxifen-inducible Cre-lox switches, and cardiomyocyte-restricted CRISPR mutagenesis. Hearts from mice overexpressing human TNNI3K were analyzed in parallel. Results: Elevated Tnni3k impaired heart regeneration in a kinase-dependent manner. Cardiomyocyte proliferation, redifferentiation, and productive cell division were preserved, and polyploidization remained well below the threshold compatible with robust regeneration, arguing against a cell-cycle defect. Instead, Tnni3k overexpression established an inflammatory and fibrotic state in the uninjured myocardium, marked by interferon, NF-{kappa}B, and antigen-presentation programs and by leukocyte and macrophage accumulation. This state amplified after injury, and these animals retained substantially more scar at 60 days post-injury. Depleting macrophages before injury, but not after, prevented the excess fibrosis. Switching Tnni3k off after injury attenuated both inflammation and fibrosis. Cardiomyocyte-specific deletion of pkmb, a glycolytic gene strongly repressed in Tnni3k-overexpressing animals and previously associated with inflammation, reproduced both phenotypes. Mice overexpressing TNNI3K showed comparable fibrosis and macrophage accumulation. Conclusions: Elevated Tnni3k impairs cardiac regeneration by sustaining a chronic inflammatory and fibrotic state rather than by driving polyploidization. Because this state requires continued kinase activity and remains reversible after injury, TNNI3K inhibition may warrant exploration as a strategy to interrupt the inflammation-fibrosis loop in inflammation-driven cardiac disease.

developmental biology↗

bioRxiv2025

A spatially localised cryoinjury model to study liver regeneration in zebrafish

This Protocol Extension describes adapting an existing cryoinjury protocol to study heart regeneration in zebrafish1. We recently characterized a novel hepatic cryoinjury model that induces localized necrosis, inflammation, and fibrosis, which resolves as the liver regenerates. Here, we present a detailed description of the hepatic cryoinjury procedure. One of the key advantages of this model is that it allows direct comparison of healthy and injured liver parenchyma within individual animals. Our hepatic cryoinjury model provides a rapid and reproducible platform to investigate the molecular and cellular mechanisms underlying fibrosis and liver regeneration. This Protocol Extension aims to extend the reach of the cryoinjury approach for deployment in studying liver regeneration.

cell biology↗

bioRxiv2024

Optimization of methods for rapid and robust generation of cardiomyocyte-specific crispants in zebrafish using the cardiodeleter system

CRISPR/Cas9 has massively accelerated the generation of gene loss-of-function models in zebrafish. However, establishing tissue-specific mutant lines remains a laborious and time-consuming process. Although a few dozen tissue-specific Cas9 zebrafish lines have been developed, the lack of standardization of some key methods, including gRNA delivery, has limited the implementation of these approaches in the zebrafish community. To tackle these limitations, we have established a cardiomyocyte-specific Cas9 line, the cardiodeleter, which efficiently generates biallelic mutations in combination with gene-specific gRNAs. We have also optimized the development of transposon-based guide shuttles that carry gRNAs targeting a gene of interest and permanently label the cells susceptible to becoming mutant. We validated this modular approach by deleting five genes (ect2, tnnt2a, cmlc2, amhc, and erbb2), all resulting in the loss of the corresponding protein or phenocopying established mutants. Additionally, we provide detailed protocols describing how to generate guide shuttles, which will facilitate the dissemination of these techniques in the zebrafish community. Our approach enables the rapid generation of tissue-specific crispants and analysis of mosaic phenotypes, bypassing limitations such as embryonic lethality, making it a valuable tool for cell-autonomous studies and genetic screenings.

developmental biology↗

bioRxiv2023

Development of a hepatic cryoinjury model to study liver regeneration

The liver is a remarkable organ that can regenerate in response to injury. Depending on the extent of injury, the liver can undergo compensatory hyperplasia or fibrosis. Despite decades of research, the molecular mechanisms underlying these processes are poorly understood. Here, we developed a new model to study liver regeneration based on cryoinjury. To visualise liver regeneration at cellular resolution, we adapted the CUBIC tissue-clearing approach. Hepatic cryoinjury induced a localised necrotic and apoptotic lesion characterised by inflammation and infiltration of innate immune cells. Following this initial phase, we observed fibrosis, which resolved as regeneration re-established homeostasis in 30 days. Importantly, this approach enables the comparison of healthy and injured parenchyma with an individual animal, providing unique advantages to previous models. In summary, the hepatic cryoinjury model provides a fast and reproducible method for studying the cellular and molecular pathways underpinning fibrosis and liver regeneration. O_FIG O_LINKSMALLFIG WIDTH=197 HEIGHT=200 SRC="FIGDIR/small/550437v2_ufig1.gif" ALT="Figure 1"> View larger version (76K): org.highwire.dtl.DTLVardef@173e431org.highwire.dtl.DTLVardef@18a75b7org.highwire.dtl.DTLVardef@13662feorg.highwire.dtl.DTLVardef@19d47a8_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2023

Dynamics of Collagen Oxidation and Cross Linking in Regenerating and Irreversibly Infarcted Myocardium

In mammalian hearts myocardial infarction produces a permanent collagen-rich scar. Conversely, in zebrafish a collagen-rich scar forms but is completely resorbed as the myocardium regenerates. The formation of cross-links in collagen hinders its degradation but cross-linking has not been well characterized in zebrafish hearts. Here, a library of fluorescent probes to quantify collagen oxidation, the first step in collagen cross-link (CCL) formation, was developed. Myocardial injury in mice or zebrafish resulted in similar dynamics of collagen oxidation in the myocardium in the first month after injury. However, during this time, mature CCLs such as pyridinoline and deoxypyridinoline developed in the murine infarcts but not in the zebrafish hearts. High levels of newly oxidized collagen were still seen in murine scars with mature CCLs. These data suggest that fibrogenesis remains dynamic, even in mature scars, and that the absence of mature CCLs in zebrafish hearts may facilitate their ability to regenerate.

biochemistry↗

bioRxiv2018

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 anti-fibrotic therapies might be less efficient than strategies targeting fibroblast inactivation.\n\nSCIENTIFIC STATEMENTAfter myocardial infarction in the mammalian heart, millions of cardiomyocytes are lost and replaced by fibrotic scar tissue. While fibrosis is persistent in adult mammals, there are some vertebrates, including zebrafish, with the capacity for regeneration. This process does not occur in the absence of fibrosis. Here we studied subpopulations of collagen producing cells and analyzed their fate after complete regeneration of the zebrafish myocardium. Our data show that fibroblasts persisted in the regenerated heart, but shut down the profibrotic program. While fibrosis could be considered as detrimental to the regeneration process, our study reveals a positive effect on cardiomyocyte proliferation. Accordingly, a fibrotic response can be beneficial for heart regeneration.

developmental biology↗