custom-3d-printed-molds-for-zebrafish-imaging-and-cardiac-development

Custom 3D-Printed Molds for Zebrafish Imaging and Cardiac Development

Embryo mounting is one of the technical challenges researchers encounter when undertaking an imaging project. Embryos need to be oriented in a ...

University of Mississippi

Pacemaker cardiomyocytes that create the sinoatrial node are essential for the initiation and maintenance of proper heart rhythm. However, illuminating developmental cues that direct their differentiation has remained particularly challenging due to the unclear cellular origins of these specialized cardiomyocytes. By discovering the origins of pacemaker cardiomyocytes, we reveal an evolutionarily conserved Wnt signaling mechanism that coordinates gene regulatory changes directing mesoderm cell fate decisions, which lead to the differentiation of pacemaker cardiomyocytes. We show that in zebrafish, pacemaker cardiomyocytes derive from a subset of Nkx2.5+ mesoderm that responds to canonical Wnt5b signaling to initiate the cardiac pacemaker program, including activation of pacemaker cell differentiation transcription factors Isl1 and Tbx18 and silencing of Nkx2.5. Moreover, applying these developmental findings to human pluripotent stem cells (hPSCs) notably results in the creation of hPSC-pacemaker cardiomyocytes, which successfully pace three-dimensional bioprinted hPSC-cardiomyocytes, thus providing potential strategies for biological cardiac pacemaker therapy.

Canonical Wnt5b Signaling Directs Outlying Nkx2.5+ Mesoderm into Pacemaker Cardiomyocytes.

Congenital heart defects (CHDs) are among the most common human birth defects. However, the etiology of a large proportion of CHDs remains undefined. Studies identifying the molecular and cellular mechanisms that underlie cardiac development have been critical to elucidating the origin of CHDs. Building upon this knowledge to understand the pathogenesis of CHDs requires examining how genetic or environmental stress changes normal cardiac development. Due to strong molecular conservation to humans and unique technical advantages, studies using zebrafish have elucidated both fundamental principles of cardiac development and have been used to create cardiac disease models. In this chapter we examine the unique toolset available to zebrafish researchers and how those tools are used to interrogate the genetic and environmental contributions to CHDs.

Using Zebrafish to Analyze the Genetic and Environmental Etiologies of Congenital Heart Defects.

Organ maturation entails the reshaping of simple tissues into more complex structures critical for function. In a recent issue of Nature, Priya et al. show how tension heterogeneity between developing cardiomyocytes can coordinate the cell behaviors that remodel the architecture of the cardiac chamber wall.

Cardiac Morphogenesis: Crowding and Tension Resolved through Social Distancing.

Unveiling Complexity and Multipotentiality of Early Heart Fields.

Visualizing dynamic cellular behaviors using live imaging is critical to the study of cell movement and to the study of cellular and embryonic polarity. Similarly, live imaging can be vital to elucidating the pathology of genetic disorders and diseases. Model systems such as zebrafish, whose in vivo development is accessible to both the microscope and genetic manipulation, are particularly well-suited to the use of live imaging. Here we describe an overall approach to conducting live-imaging experiments with a specific emphasis on investigating cell movements during the early stages of heart development in zebrafish.

Using Live Imaging to Examine Early Cardiac Development in Zebrafish.

Coordinated cell movement is a fundamental process in organ formation. During heart development, bilateral myocardial precursors collectively move towards the midline (cardiac fusion) to form the primitive heart tube. Along with extrinsic influences such as the adjacent anterior endoderm which are known to be required for cardiac fusion, we previously showed that the platelet-derived growth factor receptor alpha (Pdgfra) is also required. However, an intrinsic mechanism that regulates myocardial movement remains to be elucidated. Here, we uncover an essential intrinsic role in the myocardium for the phosphoinositide 3-kinase (PI3K) intracellular signaling pathway in directing myocardial movement towards the midline.  imaging reveals that in PI3K-inhibited zebrafish embryos myocardial movements are misdirected and slower, while midline-oriented dynamic myocardial membrane protrusions become unpolarized. Moreover, PI3K activity is dependent on and genetically interacts with Pdgfra to regulate myocardial movement. Together our findings reveal an intrinsic myocardial steering mechanism that responds to extrinsic cues during the initiation of cardiac development.

The myocardium utilizes Pdgfra-PI3K signaling to steer towards the midline during heart tube formation.

The heart, a vital organ which is first to develop, has adapted its size, structure and function in order to accommodate the circulatory demands for a broad range of animals. Although heart development is controlled by a relatively conserved network of transcriptional/chromatin regulators, how the human heart has evolved species-specific features to maintain adequate cardiac output and function remains to be defined. Here, we show through comparative epigenomic analysis the identification of enhancers and promoters that have gained activity in humans during cardiogenesis. These cis-regulatory elements (CREs) are associated with genes involved in heart development and function, and may account for species-specific differences between human and mouse hearts. Supporting these findings, genetic variants that are associated with human cardiac phenotypic/disease traits, particularly those differing between human and mouse, are enriched in human-gained CREs. During early stages of human cardiogenesis, these CREs are also gained within genomic loci of transcriptional regulators, potentially expanding their role in human heart development. In particular, we discovered that gained enhancers in the locus of the early human developmental regulator  are selectively accessible within a subpopulation of mesoderm cells which exhibits cardiogenic potential, thus possibly extending the function of  beyond its conserved left-right asymmetry role. Genetic deletion of these enhancers identified a human gained enhancer that was required for not only  and early cardiac gene expression at the mesoderm stage but also cardiomyocyte differentiation. Overall, our results illuminate how human gained CREs may contribute to human-specific cardiac attributes, and provide insight into how transcriptional regulators may gain cardiac developmental roles through the evolutionary acquisition of enhancers.

Human-gained heart enhancers are associated with species-specific cardiac attributes.

Coordinated cell movement is a fundamental process in organ formation. During heart development, bilateral myocardial precursors collectively move toward the midline (cardiac fusion) to form the primitive heart tube. Extrinsic influences such as the adjacent anterior endoderm are known to be required for cardiac fusion. We previously showed however, that the platelet-derived growth factor receptor alpha (Pdgfra) is also required for cardiac fusion (Bloomekatz et al., 2017). Nevertheless, an intrinsic mechanism that regulates myocardial movement has not been elucidated. Here, we show that the phosphoinositide 3-kinase (PI3K) intracellular signaling pathway has an essential intrinsic role in the myocardium directing movement toward the midline. In vivo imaging further reveals midline-oriented dynamic myocardial membrane protrusions that become unpolarized in PI3K-inhibited zebrafish embryos where myocardial movements are misdirected and slower. Moreover, we find that PI3K activity is dependent on and interacts with Pdgfra to regulate myocardial movement. Together our findings reveal an intrinsic myocardial steering mechanism that responds to extrinsic cues during the initiation of cardiac development.

The myocardium utilizes a platelet-derived growth factor receptor alpha (Pdgfra)-phosphoinositide 3-kinase (PI3K) signaling cascade to steer toward the midline during zebrafish heart tube formation.

Author Correction: Human-gained heart enhancers are associated with species-specific cardiac attributes.

The basic helix-loop-helix transcription factor HAND2 has multiple roles during vertebrate organogenesis, including cardiogenesis. However, much remains to be uncovered about its mechanism of action. Here, we show the generation of several hand2 mutant alleles in zebrafish and demonstrate that dimerization-deficient mutants display the null phenotype but DNA-binding-deficient mutants do not. Rescue experiments with Hand2 variants using a newly identified hand2 enhancer confirmed these observations. To identify Hand2 effectors critical for cardiogenesis, we analyzed the transcriptomes of hand2 loss- and gain-of-function embryonic cardiomyocytes and tested the function of eight candidate genes in vivo; pdgfra was most effective in rescuing myocardial migration in hand2 mutants. Accordingly, we identified a putative Hand2-binding region in the zebrafish pdgfra locus that is important for its expression. In addition, Hand2 loss- and gain-of-function experiments in mouse embryonic stem cell-derived cardiac cells decreased and increased Pdgfra expression, respectively. Altogether, these results further our mechanistic understanding of HAND2 function during early cardiogenesis.

PDGFRA is a conserved HAND2 effector during early cardiac development.

Embryo mounting is one of the technical challenges researchers encounter when undertaking an imaging project. Embryos need to be oriented in a reproducible manner such that the tissue of interest is accessible to a microscope objective for the entire imaging period. To overcome this challenge researchers can embed embryos in viscous media or create specialized dishes and casts to hold embryos in a desired orientation during imaging. Here we describe a method for using a cheap stereolithographic (SLA) 3D-printer to manufacture re-usable molds that create agarose wells in which embryos can be mounted for imaging. These agarose wells provide a reliable means for orienting multiple embryos for imaging. This method includes a design framework that can be easily customized for a variety of tissues, organisms and imaging challenges. Using this method we have created molds for imaging cardiac development in zebrafish for both upright and inverted microscopes. By utilizing materials and equipment that are accessible this method allows researchers to easily create molds specific to their mounting needs.

Article	Year
Canonical Wnt5b Signaling Directs Outlying Nkx2.5+ Mesoderm into Pacemaker Cardiomyocytes. Developmental cell\| PubMed ID: 31402282	2019
Using Zebrafish to Analyze the Genetic and Environmental Etiologies of Congenital Heart Defects. Advances in experimental medicine and biology\| PubMed ID: 32304074	2020
Cardiac Morphogenesis: Crowding and Tension Resolved through Social Distancing. Developmental cell\| PubMed ID: 33497621	2021
Unveiling Complexity and Multipotentiality of Early Heart Fields. Circulation research\| PubMed ID: 34162224	2021
Using Live Imaging to Examine Early Cardiac Development in Zebrafish. Methods in molecular biology (Clifton, N.J.)\| PubMed ID: 35147940	2022
The myocardium utilizes Pdgfra-PI3K signaling to steer towards the midline during heart tube formation. bioRxiv : the preprint server for biology\| PubMed ID: 36712046	2023
Human-gained heart enhancers are associated with species-specific cardiac attributes. Nature cardiovascular research\| PubMed ID: 36817700	2022
The myocardium utilizes a platelet-derived growth factor receptor alpha (Pdgfra)-phosphoinositide 3-kinase (PI3K) signaling cascade to steer toward the midline during zebrafish heart tube fo... eLife\| PubMed ID: 37921445	2023
Author Correction: Human-gained heart enhancers are associated with species-specific cardiac attributes. Nature cardiovascular research\| PubMed ID: 39196171	2022
PDGFRA is a conserved HAND2 effector during early cardiac development. Nature cardiovascular research\| PubMed ID: 39658721	2024
A method for creating custom 3D-printed molds to facilitate zebrafish imaging studies, including of cardiac development. bioRxiv : the preprint server for biology\| PubMed ID: 40501848	2025

Joshua Bloomekatz

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