PCB and TCDD derived embryonic cardiac defects result from a novel AhR pathway.

Polychlorinated biphenyls (PCBs) and 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) are environmental contaminants known to impact cardiac development, a key step in the embryonic development of most animals. To date, little is understood of the molecular mechanism driving the observed cardiac defects in exposed fishes. The literature shows PCB & TCDD derived cardiac defects are concurrent with, but not caused by, expression of cyp1A, due to activation of the aryl hydrocarbon receptor (AhR) gene activation pathway. However, in this study, detailed visualization of fish hearts exposed to PCBs and TCDD show that, in addition to a failure of cardiac looping in early heart development, the inner endocardial lining of the heart fails to maintain proper cell adhesion and tissue integrity. The resulting gap between the endocardium and myocardium in both zebrafish and Atlantic sturgeon suggested functional faults in endothelial adherens junction formation. Thus, we explored the molecular mechanism triggering cardiac defects using immunohistochemistry to identify the location and phosphorylation state of key regulatory and adhesion molecules. We hypothesized that PCB and TCDD activates AhR, phosphorylating Src, which then phosphorylates the endothelial adherens junction protein, VEcadherin. When phosphorylated, VEcadherin dimers, found in the endocardium and vasculature, separate, reducing tissue integrity. In zebrafish, treatment with PCB and TCDD contaminants leads to higher phosphorylation of VEcadherin in cardiac tissue suggesting that these cells have reduced connectivity. Small molecule inhibition of Src phosphorylation prevents contaminant stimulated phosphorylation of VEcadherin and rescues both cardiac function and gross morphology. Atlantic sturgeon hearts show parallels to contaminant exposed zebrafish cardiac phenotype at the tissue level. These data suggest that the mechanism for PCB and TCDD action in the heart is, in part, distinct from the canonical mechanism described in the literature and that cardiac defects are impacted by this nongenomic mechanism.

Toxic Effects of Polychlorinated Biphenyl Congeners and Aroclors on Embryonic Growth and Development.

Polychlorinated biphenyls (PCBs) cause significant health and reproductive problems in many vertebrates. Exposure during embryogenesis likely leads to defects in organ development, compromising survival and growth through adulthood. The present study identifies the impact of PCBs on the embryonic development of key organs and resulting consequences on survival and growth. Zebrafish embryos were treated with individual PCB congeners (126 or 104) or one of 4 Aroclor mixtures (1016, 1242, 1254, or 1260) and analyzed for changes in gross embryonic morphology. Specific organs were assessed for defects during embryonic development, using a variety of transgenic zebrafish to improve organ visualization. Resulting larvae were grown to adulthood while survival and growth were assayed. Embryonic gross development on PCB treatment was abnormal, with defects presenting in a concentration-dependent manner in the liver, pancreas, heart, and blood vessel organization. Polychlorinated biphenyl 126 treatment resulted in the most consistently severe and fatal phenotypes, whereas treatments with PCB 104 and Aroclors resulted in a range of more subtle organ defects. Survival of fish was highly variable although the growth rates of surviving fish were relatively normal, suggesting that maturing PCB-treated fish that survive develop compensatory strategies needed to reach adulthood. Life span analyses of fish from embryogenesis through adulthood, as in the present study, are scarce but important for the field because they help identify foci for further studies. Environ Toxicol Chem 2021;40:187-201. © 2020 SETAC.

Learning to Fish with Genetics: A Primer on the Vertebrate Model Danio rerio.

In the last 30 years, the zebrafish has become a widely used model organism for research on vertebrate development and disease. Through a powerful combination of genetics and experimental embryology, significant inroads have been made into the regulation of embryonic axis formation, organogenesis, and the development of neural networks. Research with this model has also expanded into other areas, including the genetic regulation of aging, regeneration, and animal behavior. Zebrafish are a popular model because of the ease with which they can be maintained, their small size and low cost, the ability to obtain hundreds of embryos on a daily basis, and the accessibility, translucency, and rapidity of early developmental stages. This primer describes the swift progress of genetic approaches in zebrafish and highlights recent advances that have led to new insights into vertebrate biology.

LRRK2 knockdown in zebrafish causes developmental defects, neuronal loss, and synuclein aggregation.

Although mutations in the leucine-rich repeat kinase 2 (LRRK2) gene are the most common cause of genetic Parkinson's disease, their function is largely unknown. LRRK2 is pleiotropic in nature, shown to be involved in neurodegeneration and in more peripheral processes, including kidney functions, in rats and mice. Recent studies in zebrafish have shown conflicting evidence that removal of the LRRK2 WD40 domain may or may not affect dopaminergic neurons and/or locomotion. This study shows that ∼50% LRRK2 knockdown in zebrafish causes not only neuronal loss but also developmental perturbations such as axis curvature defects, ocular abnormalities, and edema in the eyes, lens, and otic vesicles. We further show that LRRK2 knockdown results in significant neuronal loss, including a reduction of dopaminergic neurons. Immunofluorescence demonstrates that endogenous LRRK2 is expressed in the lens, brain, heart, spinal cord, and kidney (pronephros), which mirror the LRRK2 morphant phenotypes observed. LRRK2 knockdown results further in the concomitant upregulation of ß-synuclein, PARK13, and SOD1 and causes ß-synuclein aggregation in the diencephalon, midbrain, hindbrain, and postoptic commissure. LRRK2 knockdown causes mislocalization of the Na(+) /K(+) ATPase protein in the pronephric ducts, suggesting that the edema might be linked to renal malfunction and that LRRK2 might be associated with pronephric duct epithelial cell differentiation. Combined, our study shows that LRRK2 has multifaceted roles in zebrafish and that zebrafish represent a complementary model to further our understanding of this central protein. © 2016 Wiley Periodicals, Inc.

Function and regulation of TRPP2 ion channel revealed by a gain-of-function mutant.

Mutations in polycystin-1 and transient receptor potential polycystin 2 (TRPP2) account for almost all clinically identified cases of autosomal dominant polycystic kidney disease (ADPKD), one of the most common human genetic diseases. TRPP2 functions as a cation channel in its homomeric complex and in the TRPP2/polycystin-1 receptor/ion channel complex. The activation mechanism of TRPP2 is unknown, which significantly limits the study of its function and regulation. Here, we generated a constitutively active gain-of-function (GOF) mutant of TRPP2 by applying a mutagenesis scan on the S4-S5 linker and the S5 transmembrane domain, and studied functional properties of the GOF TRPP2 channel. We found that extracellular divalent ions, including Ca(2+), inhibit the permeation of monovalent ions by directly blocking the TRPP2 channel pore. We also found that D643, a negatively charged amino acid in the pore, is crucial for channel permeability. By introducing single-point ADPKD pathogenic mutations into the GOF TRPP2, we showed that different mutations could have completely different effects on channel activity. The in vivo function of the GOF TRPP2 was investigated in zebrafish embryos. The results indicate that, compared with wild type (WT), GOF TRPP2 more efficiently rescued morphological abnormalities, including curly tail and cyst formation in the pronephric kidney, caused by down-regulation of endogenous TRPP2 expression. Thus, we established a GOF TRPP2 channel that can serve as a powerful tool for studying the function and regulation of TRPP2. The GOF channel may also have potential application for developing new therapeutic strategies for ADPKD.

Growth and maturation in the zebrafish, Danio rerio: a staging tool for teaching and research.

Zebrafish have been increasingly used as a teaching tool to enhance the learning of many biological concepts from genetics, development, and behavior to the understanding of the local watershed. Traditionally, in both research and teaching, zebrafish work has focused on embryonic stages; however, later stages, from larval through adulthood, are increasingly being examined. Defining developmental stages based on age is a problematic way to assess maturity, because many environmental factors, such as temperature, population density, and water quality, impact growth and maturation. Fish length and characterization of key external morphological traits are considered better markers for maturation state. While a number of staging series exist for zebrafish, here we present a simplified normalization table of post-embryonic maturation well suited to both educational and research use. Specifically, we utilize fish size and four easily identified external morphological traits (pigment pattern, tail fin, anal fin, and dorsal fin morphology) to describe three larval stages, a juvenile stage, and an adult stage. These simplified maturation standards will be a useful tool for both educational and research protocols.

Integration of nodal and BMP signals in the heart requires FoxH1 to create left-right differences in cell migration rates that direct cardiac asymmetry.

Failure to properly establish the left-right (L/R) axis is a major cause of congenital heart defects in humans, but how L/R patterning of the embryo leads to asymmetric cardiac morphogenesis is still unclear. We find that asymmetric Nodal signaling on the left and Bmp signaling act in parallel to establish zebrafish cardiac laterality by modulating cell migration velocities across the L/R axis. Moreover, we demonstrate that Nodal plays the crucial role in generating asymmetry in the heart and that Bmp signaling via Bmp4 is dispensable in the presence of asymmetric Nodal signaling. In addition, we identify a previously unappreciated role for the Nodal-transcription factor FoxH1 in mediating cell responsiveness to Bmp, further linking the control of these two pathways in the heart. The interplay between these TGFß pathways is complex, with Nodal signaling potentially acting to limit the response to Bmp pathway activation and the dosage of Bmp signals being critical to limit migration rates. These findings have implications for understanding the complex genetic interactions that lead to congenital heart disease in humans.

Analysis of postembryonic heart development and maturation in the zebrafish, Danio rerio.

BACKGROUND: Cardiac maturation is vital for animal survival and must occur throughout the animal's life. Zebrafish are increasingly used to model cardiac disease; however, little is known about how the cardiovascular system matures. We conducted a systematic analysis of cardiac maturation from larvae through to adulthood and assessed cardiac features influenced by genetic and environmental factors. RESULTS: We identified a novel step in cardiac maturation, termed cardiac rotation, where the larval heart rotates into its final orientation within the thoracic cavity with the atrium placed behind the ventricle. This rotation is followed by linear ventricle growth and an increase in the angle between bulbous arteriosus and the ventricle. The ventricle transitions from a rectangle, to a triangle and ultimately a sphere that is significantly enveloped by the atrium. In addition, trabeculae are similarly patterned in the zebrafish and humans, both with muscular fingerlike projections and muscle bands that span the cardiac chamber. Of interest, partial loss of atrial contraction in myosin heavy chain 6 (myh6/wea(hu423/+)) mutants result in the adult maintaining a larval cardiac form. CONCLUSIONS: These findings serve as a foundation for the study of defects in cardiovascular development from both genetic and environmental factors.

Heart dissection in larval, juvenile and adult zebrafish, Danio rerio.

Zebrafish have become a beneficial and practical model organism for the study of embryonic heart development, however, work examining post-embryonic through adult cardiac development has been limited. Examining the changing morphology of the maturing and aging heart are restricted by the lack of techniques available for staging and isolating juvenile and adult hearts. In order to analyze heart development over the fish's lifespan, we dissect zebrafish hearts at numerous stages and photograph them for further analysis. The morphological features of the heart can easily be quantified and individual hearts can be further analyzed by a host of standard methods. Zebrafish grow at variable rates and maturation correlates better with fish size than age, thus, post-fixation, we photograph and measure fish length as a gauge of fish maturation. This protocol explains two distinct, size dependent dissection techniques for zebrafish, ranging from larvae 3.5 mm standard length (SL) with hearts of 100 µm ventricle length (VL), to adults, with SL of 30 mm and VL 1mm or larger. Larval and adult fish have quite distinct body and organ morphology. Larvae are not only significantly smaller, they have less pigment and each organ is visually very difficult to identify. For this reason, we use distinct dissection techniques. We used pre-dissection fixation procedures, as we discovered that hearts dissected directly after euthanization have a more variable morphology, with very loose and balloon like atria compared with hearts removed following fixation. The fish fixed prior to dissection, retain in vivo morphology and chamber position (data not shown). In addition, for demonstration purposes, we take advantage of the heart (myocardial) specific GFP transgenic Tg(myl7:GFP)(twu34), which allows us to visualize the entire heart and is particularly useful at early stages in development when the cardiac morphology is less distinct from surrounding tissues. Dissection of the heart makes further analysis of the cell and molecular biology underlying heart development and maturation using in situ hybridization, immunohistochemistry, RNA extraction or other analytical methods easier in post-embryonic zebrafish. This protocol will provide a valuable technique for the study of cardiac development maturation and aging.

Direct and indirect roles for Nodal signaling in two axis conversions during asymmetric morphogenesis of the zebrafish heart.

The Nodal signaling pathway plays a conserved role in determining left-sided identity in vertebrates with this early left-right (L/R) patterning influencing the asymmetric development and placement of visceral organs. We have studied the role of Nodal signaling in asymmetric cardiac morphogenesis in zebrafish and describe two distinct rotations occurring within the heart. The first is driven by an asymmetric migration of myocardial cells during cardiac jogging, resulting in the conversion of the L/R axis to the dorsal-ventral (D/V) axis of the linear heart. This first rotation is directly influenced by the laterality of asymmetric gene expression. The second rotation occurs before cardiac looping and positions the original left cells exposed to Nodal signaling back to the left of the wild-type (WT) heart by 48 hours postfertilization (hpf). The direction of this second rotation is determined by the laterality of cardiac jogging and is not directly influenced by asymmetric gene expression. Finally, we have identified a role for Nodal signaling in biasing the location of the inner ventricular and outer atrial curvature formations. These results suggest that Nodal signaling directs asymmetric cardiac morphogenesis through establishing and subsequently reinforcing laterality information over the course of cardiac development.