assessing-metabolic-activity-whole-regenerating-zebrafish-ventricles

Assessing the Metabolic Activity of Whole Regenerating Zebrafish Ventricles <em>Ex Vivo</em> Using an Extracellular Flux Assay

The ability of zebrafish to regenerate their hearts throughout adulthood is partially attributed to metabolic adaptations. Although it has been ...

University of Oxford

Congenital heart disease (CHD) is a common birth defect affecting approximately 1% of newborns. Great progress has been made in elucidating the genetic aetiology of CHD with advances in genomic technology, which we leveraged in recovering a new pathway affecting heart development in humans previously known to affect heart development in an animal model.

Loss of function in  is associated with tetralogy of Fallot and septal defects.

The Slit ligands and their Robo receptors are well-known for their roles during axon guidance in the central nervous system but are still relatively unknown in the cardiac field. However, data from different animal models suggest a broad involvement of the pathway in many aspects of heart development, from cardiac cell migration and alignment, lumen formation, chamber formation, to the formation of the ventricular septum, semilunar and atrioventricular valves, caval veins, and pericardium. Absence of one or more of the genes in the pathway results in defects ranging from bicuspid aortic valves to ventricular septal defects and abnormal venous connections to the heart. Congenital heart defects are the most common congenital malformations found in life new-born babies and progress in methods for large scale human genetic testing has significantly enhanced the identification of new causative genes involved in human congenital heart disease. Recently, loss of function variants in ROBO1 have also been linked to ventricular septal defects and tetralogy of Fallot in patients. Here, we will give an overview of the role of the Slit-Robo signalling pathway in Drosophila, zebrafish, and mouse heart development. The extent of these data warrant further attention on the SLIT-ROBO signalling pathway as a candidate for an array of human congenital heart defects.

Slit-Robo signalling in heart development.

Regulation of heart size and shape is one of the least understood processes in developmental biology. We have for the first time analysed the hearts of Astyanax mexicanus and identified several differences in heart morphology between the surface (epigean morph) and cave-dwelling (troglomorph) morphs. Examination of the adult revealed that the troglomorph possesses a smaller heart with a rounder ventricle in comparison to the epigean morph. The size differences identified appear to arise early in development, as early as 24 h post-fertilisation (hpf), while shape differences begin to appear at 2 days post-fertilisation. The heart of the first-generation cross between the cave-dwelling and river-dwelling morph shows uncoupling of different phenotypes observed in the parental populations and indicates that the cardiac differences have become embedded in the genome during evolution. The differences in heart morphology are accompanied by functional changes between the two morphs, with the cave-dwelling morph exhibiting a slower heart rate than the river-dwelling morph. The identification of morphological and functional differences in the A. mexicanus heart could allow us to gain more insight into how such parameters are regulated during cardiac development, with potential relevance to cardiac pathologies in humans.

The developmental origin of heart size and shape differences in Astyanax mexicanus populations.

A small network of spontaneously active Tbx3 cardiomyocytes forms the cardiac conduction system (CCS) in adults. Understanding the origin and mechanism of development of the CCS network are important steps towards disease modeling and the development of biological pacemakers to treat arrhythmias. We found that Tbx3 expression in the embryonic mouse heart is associated with automaticity. Genetic inducible fate mapping revealed that Tbx3 cells in the early heart tube are fated to form the definitive CCS components, except the Purkinje fiber network. At mid-fetal stages, contribution of Tbx3 cells was restricted to the definitive CCS. We identified a Tbx3 population in the outflow tract of the early heart tube that formed the atrioventricular bundle. Whereas Tbx3 cardiomyocytes also contributed to the adjacent Gja5 atrial and ventricular chamber myocardium, embryonic Gja5 chamber cardiomyocytes did not contribute to the Tbx3 sinus node or to atrioventricular ring bundles. In conclusion, the CCS is established by progressive fate restriction of a Tbx3 cell population in the early developing heart, which implicates  as a useful tool for developing strategies to study and treat CCS diseases.

Embryonic Tbx3 cardiomyocytes form the mature cardiac conduction system by progressive fate restriction.

Although Astyanax mexicanus surface fish regenerate their hearts after injury, their Pachón cave-dwelling counterparts cannot and, instead, form a permanent fibrotic scar, similar to the human heart. Myocardial proliferation peaks at similar levels in both surface fish and Pachón 1 week after injury. However, in Pachón, this peak coincides with a strong scarring and immune response, and ultimately, cavefish cardiomyocytes fail to replace the scar. We identified lrrc10 to be upregulated in surface fish compared with Pachón after injury. Similar to cavefish, knockout of lrrc10 in zebrafish impairs heart regeneration without affecting wound cardiomyocyte proliferation. Furthermore, using quantitative trait locus (QTL) analysis, we have linked the degree of heart regeneration to three loci in the genome, identifying candidate genes fundamental to the difference between scarring and regeneration. Our study provides evidence that successful heart regeneration entails a delicate interplay between cardiomyocyte proliferation and scarring.

Heart Regeneration in the Mexican Cavefish.

Runx1 is a transcription factor that plays a key role in determining the proliferative and differential state of multiple cell types, during both development and adulthood. Here, we report how Runx1 is specifically upregulated at the injury site during zebrafish heart regeneration, and that absence of  results in increased myocardial survival and proliferation, and overall heart regeneration, accompanied by decreased fibrosis. Using single cell sequencing, we found that the wild-type injury site consists of Runx1-positive endocardial cells and thrombocytes that induce expression of smooth muscle and collagen genes. Both these populations cannot be identified in  mutant wounds that contain less collagen and fibrin. The reduction in fibrin in the mutant is further explained by reduced myofibroblast formation and upregulation of components of the fibrin degradation pathway, including plasminogen receptor annexin 2A as well as downregulation of plasminogen activator inhibitor  in myocardium and endocardium, resulting in increased levels of plasminogen. Our findings suggest that Runx1 controls the regenerative response of multiple cardiac cell types and that targeting Runx1 is a novel therapeutic strategy for inducing endogenous heart repair.

Runx1 promotes scar deposition and inhibits myocardial proliferation and survival during zebrafish heart regeneration.

Genome-wide association studies have identified noncoding variants near  that are associated with PR interval and QRS duration, suggesting that subtle changes in  expression affect atrioventricular conduction system function. To explore whether and to what extent the atrioventricular conduction system is affected by Tbx3 dose reduction, we first characterized electrophysiological properties and morphology of heterozygous  mutant () mouse hearts. We found PR interval shortening and prolonged QRS duration, as well as atrioventricular bundle hypoplasia after birth in heterozygous mice. The atrioventricular node size was unaffected. Transcriptomic analysis of atrioventricular nodes isolated by laser capture microdissection revealed hundreds of deregulated genes in  mutants. Notably,  atrioventricular nodes showed increased expression of working myocardial gene programs (mitochondrial and metabolic processes, muscle contractility) and reduced expression of pacemaker gene programs (neuronal, Wnt signaling, calcium/ion channel activity). By integrating chromatin accessibility profiles (ATAC sequencing) of atrioventricular tissue and other epigenetic data, we identified Tbx3-dependent atrioventricular regulatory DNA elements (REs) on a genome-wide scale. We used transgenic reporter assays to determine the functionality of candidate REs near  an up-regulated chamber-enriched gene, and in  a down-regulated conduction system-specific gene. Using genome editing to delete candidate REs, we showed that a strong intronic bipartite RE selectively governs  expression in the conduction system in vivo. Our data provide insights into the multifactorial Tbx3-dependent transcriptional network that regulates the structure and function of the cardiac conduction system, which may underlie the differences in PR duration and QRS interval between individuals carrying variants in the  locus.

T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system.

The adult human heart cannot repair itself after injury and, instead, forms a permanent fibrotic scar that impairs cardiac function and can lead to incurable heart failure. The zebrafish, amongst other organisms, has been extensively studied for its innate capacity to repair its heart after injury. Understanding the signals that govern successful regeneration in models such as the zebrafish will lead to the development of effective therapies that can stimulate endogenous repair in humans. To date, many studies have investigated cardiac regeneration using a reverse genetics candidate gene approach. However, this approach is limited in its ability to unbiasedly identify novel genes and signalling pathways that are essential to successful regeneration. In contrast, drawing comparisons between different models of regeneration enables unbiased screens to be performed, identifying signals that have not previously been linked to regeneration. Here, we will review in detail what has been learnt from the comparative approach, highlighting the techniques used and how these studies have influenced the field. We will also discuss what further comparisons would enhance our knowledge of successful regeneration and scarring. Finally, we focus on the , an intraspecies comparative fish model that holds great promise for revealing the secrets of the regenerating heart.

Unlocking the Secrets of the Regenerating Fish Heart: Comparing Regenerative Models to Shed Light on Successful Regeneration.

Identifying the genetic factors that underlie complex traits is central to understanding the mechanistic underpinnings of evolution. Cave-dwelling Astyanax mexicanus populations are well adapted to subterranean life and many populations appear to have evolved troglomorphic traits independently, while the surface-dwelling populations can be used as a proxy for the ancestral form. Here we present a high-resolution, chromosome-level surface fish genome, enabling the first genome-wide comparison between surface fish and cavefish populations. Using this resource, we performed quantitative trait locus (QTL) mapping analyses and found new candidate genes for eye loss such as dusp26. We used CRISPR gene editing in A. mexicanus to confirm the essential role of a gene within an eye size QTL, rx3, in eye formation. We also generated the first genome-wide evaluation of deletion variability across cavefish populations to gain insight into this potential source of cave adaptation. The surface fish genome reference now provides a more complete resource for comparative, functional and genetic studies of drastic trait differences within a species.

A chromosome-level genome of Astyanax mexicanus surface fish for comparing population-specific genetic differences contributing to trait evolution.

Advances in sequencing and assembly technology have led to the creation of genome assemblies for a wide variety of non-model organisms. The rapid production and proliferation of updated, novel assembly versions can create vexing problems for researchers when multiple-genome assembly versions are available at once, requiring researchers to work with more than one reference genome. Multiple-genome assemblies are especially problematic for researchers studying the genetic makeup of individual cells, as single-cell RNA sequencing (scRNAseq) requires sequenced reads to be mapped and aligned to a single reference genome. Using the , this study highlights how the interpretation of a single-cell dataset from the same sample changes when aligned to its two different available genome assemblies. We found that the number of cells and expressed genes detected were drastically different when aligning to the different assemblies. When the genome assemblies were used in isolation with their respective annotations, cell-type identification was confounded, as some classic cell-type markers were assembly-specific, whilst other genes showed differential patterns of expression between the two assemblies. To overcome the problems posed by multiple-genome assemblies, we propose that researchers align to each available assembly and then integrate the resultant datasets to produce a final dataset in which all genome alignments can be used simultaneously. We found that this approach increased the accuracy of cell-type identification and maximised the amount of data that could be extracted from our single-cell sample by capturing all possible cells and transcripts. As scRNAseq becomes more widely available, it is imperative that the single-cell community is aware of how genome assembly alignment can alter single-cell data and their interpretation, especially when reviewing studies on non-model organisms.

Discordant Genome Assemblies Drastically Alter the Interpretation of Single-Cell RNA Sequencing Data Which Can Be Mitigated by a Novel Integration Method.

Background Binding of Slit ligands to their Robo receptors regulates signaling pathways that are important for heart development. Genetic variants in and  have been linked to congenital heart defects in humans. These defects are recapitulated in mouse models with ubiquitous deletions of the Slit ligands or Robo receptors and include additional heart defects not currently linked to SLIT or ROBO mutations in humans. Given the broad expression patterns of these genes, the question remains open which tissue-specific ligand-receptor interactions are important for the correct development of different cardiac structures. Methods and Results We used tissue-specific knockout mouse models of /, ,  and and scored cardiac developmental defects in perinatal mice. Knockout of  in either the whole heart, endocardium and its derivatives, or the neural crest in ubiquitous  knockout background resulted in ventricular septal defects. Neural crest-specific removal of  in  knockouts showed fully penetrant bicuspid aortic valves (BAV). Endocardial knock-out of either or  caused low penetrant BAV. In contrast, endocardial knockout of  using a newly generated line resulted in fully penetrant BAV, while removal from smooth muscle cells also resulted in BAV. Caval vein and diaphragm defects observed in ubiquitous  mutants were recapitulated in the tissue-specific knockouts. Conclusions Our data will help understand defects observed in patients with variants in  and . The results strongly indicate interaction between endocardial and neural crest  in the development of BAV, highlighting the need for further studies of this connection.

Tissue-Specific Roles for the Slit-Robo Pathway During Heart, Caval Vein, and Diaphragm Development.

Regeneration is the remarkable phenomenon through which an organism can regrow lost or damaged parts with fully functional replacements, including complex anatomical structures, such as limbs. In 2019, Development launched its 'Model systems for regeneration' collection, a series of articles introducing some of the most popular model organisms for studying regeneration in vivo. To expand this topic further, this Perspective conveys the voices of five expert biologists from the field of regenerative biology, each of whom showcases some less well-known, but equally extraordinary, species for studying regeneration.

Extraordinary model systems for regeneration.

In contrast to mammals, adult zebrafish achieve complete heart regeneration via proliferation of cardiomyocytes. Surprisingly, we found that regenerating cardiomyocytes experience DNA replication stress, which represents one reason for declining tissue regeneration during aging in mammals. Pharmacological inhibition of ATM and ATR kinases revealed that DNA damage response signaling is essential for zebrafish heart regeneration. Manipulation of Bone Morphogenetic Protein (BMP)-Smad signaling using transgenics and mutants showed that BMP signaling alleviates cardiomyocyte replication stress. BMP signaling also rescues neonatal mouse cardiomyocytes, human fibroblasts and human hematopoietic stem and progenitor cells (HSPCs) from replication stress. DNA fiber spreading assays indicate that BMP signaling facilitates re-start of replication forks after replication stress-induced stalling. Our results identify the ability to overcome replication stress as key factor for the elevated zebrafish heart regeneration capacity and reveal a conserved role for BMP signaling in promotion of stress-free DNA replication.

BMP signaling promotes zebrafish heart regeneration via alleviation of replication stress.

In contrast to humans, fish can fully regenerate their hearts after cardiac injury. However, not all fish have the same regenerative potential, allowing comparative inter-species and intra-species analysis to identify the mechanisms controlling successful heart regeneration. Here we report a differential regenerative response to cardiac cryo-injury among different wild-type zebrafish strains. Correlating these data with single-cell and bulk RNA sequencing data, we identify oxidative phosphorylation (OXPHOS) as a positive regulator of long-term regenerative outcome. OXPHOS levels, driven by glycolysis through the malate-aspartate shuttle, increase as soon as cardiomyocyte proliferation decreases, and this increase is required for cardiomyocyte re-differentiation and successful long-term regeneration. Reduced upregulation of OXPHOS in Astyanax mexicanus cavefish results in the absence of a dynamic temporal sarcomere gene expression program during cardiomyocyte re-differentiation. These findings challenge the assumption that OXPHOS inhibits regeneration and reveal targetable pathways to enhance heart repair in humans after myocardial infarction.

Article	Year
Loss of function in is associated with tetralogy of Fallot and septal defects. Journal of medical genetics\| PubMed ID: 28592524	2017
Slit-Robo signalling in heart development. Cardiovascular research\| PubMed ID: 29538649	2018
The developmental origin of heart size and shape differences in Astyanax mexicanus populations. Developmental biology\| PubMed ID: 29940142	2018
Embryonic Tbx3 cardiomyocytes form the mature cardiac conduction system by progressive fate restriction. Development (Cambridge, England)\| PubMed ID: 30042181	2018
Heart Regeneration in the Mexican Cavefish. Cell reports\| PubMed ID: 30462998	2018
Runx1 promotes scar deposition and inhibits myocardial proliferation and survival during zebrafish heart regeneration. Development (Cambridge, England)\| PubMed ID: 32341028	2020
T-box transcription factor 3 governs a transcriptional program for the function of the mouse atrioventricular conduction system. Proceedings of the National Academy of Sciences of the United States of America\| PubMed ID: 32675240	2020
Unlocking the Secrets of the Regenerating Fish Heart: Comparing Regenerative Models to Shed Light on Successful Regeneration. Journal of cardiovascular development and disease\| PubMed ID: 33467137	2021
A chromosome-level genome of Astyanax mexicanus surface fish for comparing population-specific genetic differences contributing to trait evolution. Nature communications\| PubMed ID: 33664263	2021
Discordant Genome Assemblies Drastically Alter the Interpretation of Single-Cell RNA Sequencing Data Which Can Be Mitigated by a Novel Integration Method. Cells\| PubMed ID: 35203259	2022
Tissue-Specific Roles for the Slit-Robo Pathway During Heart, Caval Vein, and Diaphragm Development. Journal of the American Heart Association\| PubMed ID: 35343246	2022
Extraordinary model systems for regeneration. Development (Cambridge, England)\| PubMed ID: 39012059	2024
BMP signaling promotes zebrafish heart regeneration via alleviation of replication stress. Nature communications\| PubMed ID: 39962064	2025
Oxidative phosphorylation is required for cardiomyocyte re-differentiation and long-term fish heart regeneration. Nature cardiovascular research\| PubMed ID: 41034455	2025

Mathilda T.M. Mommersteeg

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