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

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.

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.

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.

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
Using Zebrafish to Analyze the Genetic and Environmental Etiologies of Congenital Heart Defects. Advances in experimental medicine and biology\| PubMed ID: 32304074	2020
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
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
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

Rabina Shrestha

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