Induction and prepatterning of the zebrafish pectoral fin bud requires axial retinoic acid signaling

Vertebrate forelimbs arise as bilateral appendages from the lateral plate mesoderm (LPM). Mutants in aldh1a2 (raldh2), an embryonically expressed gene encoding a retinoic acid (RA)-synthesizing enzyme, have been used to show that limb development and patterning of the limb bud are crucially dependent on RA signaling. However, the timing and cellular origin of RA signaling in these processes have remained poorly resolved. We have used genetics and chemical modulators of RA signaling to resolve these issues in the zebrafish. By rescuing pectoral fin induction in the aldh1a2/neckless mutant with exogenous RA and by blocking RA signaling in wild-type embryos, we find that RA acts as a permissive signal that is required during the six- to eight-somite stages for pectoral fin induction. Cell-transplantation experiments show that RA production is not only crucially required from flanking somites, but is sufficient to permit fin bud initiation when the trunk mesoderm is genetically ablated. Under the latter condition, intermediate mesoderm alone cannot induce the pectoral fin field in the LPM. We further show that induction of the fin field is directly followed by a continued requirement for somite-derived RA signaling to establish a prepattern of anteroposterior fates in the condensing fin mesenchyme. This process is mediated by the maintained expression of the transcription factor hand2, through which the fin field is continuously posteriorized, and lasts up to several hours prior to limb-budding. Thus, RA signaling from flanking somites plays a dual early role in the condensing limb bud mesenchyme.

ETV6 (TEL1) regulates embryonic hematopoiesis in zebrafish

Chromosomal translocations involving fusions of the human ETV6 (TEL1) gene occur frequently in hematologic malignancies. However, a detailed understanding of the normal function of ETV6 remains incomplete. This study has employed zebrafish as a relevant model to investigate the role of ETV6 during embryonic hematopoiesis. Zebrafish possessed a single conserved etv6 ortholog that was expressed from 12 hpf in the lateral plate mesoderm, and later in hematopoietic, vascular and other tissues. Morpholino-mediated gene knockdown of etv6 revealed the complex contribution of this gene toward embryonic hematopoiesis. During primitive hematopoiesis, etv6 knockdown resulted in reduced levels of progenitor cells, erythrocyte and macrophage populations, but increased numbers of incompletely differentiated heterophils. Definitive hematopoiesis was also perturbed, with etv6 knockdown leading to decreased erythrocytes and myeloid cells, but enhanced lymphopoiesis. This study suggests that ETV6 plays a broader and more complex role in early hematopoiesis than previously thought, impacting on the development of multiple lineages. © 2015 Ferrata Storti Foundation.

ZebRA: an overview of retinoic acid signaling during zebrafish development

Retinoic acid (RA), the main active vitamin A derivative, is crucial for embryo development, regulating cellular processes, embryo patterning and organogenesis. Many studies performed in mammalian or avian models have successfully undertaken the investigation of the role played by RA during embryogenesis. Since the early 1980s, the zebrafish (Danio rerio) has emerged as a powerful developmental model to study the in vivo role of RA during embryogenesis. Unlike mammalian models, zebrafish embryogenesis is external, not only allowing the observation of the translucent embryo from the earliest steps but also providing an easily accessible system for pharmacological treatment or genetic approaches. Therefore, zebrafish research largely participates in deciphering the role of RA during development. This review aims at illustrating different concepts of RA signaling based on the research performed on zebrafish. Indeed, RA action relies on a multitude of cross-talk with other signaling pathways and requires a coordinated, dynamic and fine-regulation of its level and activity in both temporal and spatial dimensions. This review also highlights major advances that have been discovered using zebrafish such as the observation of the RA gradient in vivo for the first time, the effects of RA signaling in brain patterning, its role in establishing left-right asymmetry and its effects on the development of a variety of organs and tissues including the heart, blood, bone and fat. This review demonstrates that the zebrafish is a convenient and powerful model to study retinoic acid signaling during vertebrate embryogenesis. This article is part of a Special Issue entitled: Nuclear receptors in animal development.

Lipid abundance in zebrafish embryos is regulated by complementary actions of the endocannabinoid system and retinoic acid pathway

The endocannabinoid system (ECS) and retinoic acid (RA) signaling have been associated with influencing lipid metabolism. We hypothesized that modulation of these pathways could modify lipid abundance in developing vertebrates and that these pathways could have a combinatorial effect on lipid levels. Zebrafish embryos were exposed to chemical treatments altering the activity of the ECS and RA pathway. Embryos were stained with the neutral lipid dye Oil-Red-O (ORO) and underwent whole-mount in situ hybridization. Mouse 3T3-L1 fibroblasts were differentiated under exposure to RA modulating chemicals and subsequently stained with ORO and analyzed for gene expression by qRT-PCR. ECS activation and RA exposure increased lipid abundance and the expression of lipoprotein lipase. Additionally, RA treatment increased expression of CCAAT/enhancer binding protein alpha. Both ECS receptors and RA receptor subtypes were separately involved in modulating lipid abundance. Finally, increased ECS or RA activity ameliorated the reduced lipid abundance caused by peroxisome proliferator-activated receptor gamma (PPARγ) inhibition. Therefore, the ECS and RA pathway influence lipid abundance in zebrafish embryos and have an additive effect when treated simultaneously. Furthermore, we demonstrated that these pathways act downstream or independently of PPARγ to influence lipid levels. Our study shows for the first time that the RA and ECS pathways have additive function in lipid abundance during vertebrate development.

Zebrafish as a model for leukemia and other hematopoietic disorders

Zebrafish is an established model for the study of vertebrate development, and is especially amenable for investigating hematopoiesis, where there is strong conservation of key lineages, genes, and developmental processes with humans. Over recent years, zebrafish has been increasingly utilized as a model for a range of human hematopoietic diseases, including malignancies. This review provides an overview of zebrafish hematopoiesis and describes its application as a model of leukemia and other hematopoietic disorders.

Expression of anti-viral shRNA molecules in transgenic zebrafish

 Development of transgenic zebrafish specifically resistant to Viral Hemorrhagic Septicemia Virus mediated by short-hairpin RNA interference. Introduction of shRNAs by Tol2 transgenisis in to zebrafish overwhelmed multiple facets of the endogenous microRNA pathway including Exportin-5 and Argonaute-2 and prevented normal zebrafish development.

Suppressor of cytokine signaling 4 and 5 in zebrafish development

This research provides insight into the evolution and function of zebrafish cell signaling proteins, Suppressor of Cytokine Signaling 4a, 4b and 5a. These signaling proteins were found to play essential roles during early and late stages of embryonic development, with specific roles in sensory processing and immune cell development.

Conserved IL-2Rγc signaling mediates lymphopoiesis in Zebrafish

The IL-2 receptor γ common (IL-2Rγc) chain is the shared subunit of the receptors for the IL-2 family of cytokines, which mediate signaling through JAK3 and various downstream pathways to regulate lymphopoiesis. Inactivating mutations in human IL-2Rγc result in SCID, a primary immunodeficiency characterized by greatly reduced numbers of lymphocytes. This study used bioinformatics, expression analysis, gene ablation, and specific pharmacologic inhibitors to investigate the function of two putative zebrafish IL-2Rγc paralogs, il-2rγc.a and il-2rγc.b, and downstream signaling components during early lymphopoiesis. Expression of il-2rγc.a commenced at 16 h post fertilization (hpf) and rose steadily from 4-6 d postfertilization (dpf) in the developing thymus, with il-2rγc.a expression also confirmed in adult T and B lymphocytes. Transcripts of il-2rγc.b were first observed from 8 hpf, but waned from 16 hpf before reaching maximal expression at 6 dpf, but this was not evident in the thymus. Knockdown of il-2rγc.a, but not il-2rγc.b, substantially reduced embryonic lymphopoiesis without affecting other aspects of hematopoiesis. Specific targeting of zebrafish Jak3 exerted a similar effect on lymphopoiesis, whereas ablation of zebrafish Stat5.1 and pharmacologic inhibition of PI3K and MEK also produced significant but smaller effects. Ablation of il-2rγc.a was further demonstrated to lead to an absence of mature T cells, but not B cells in juvenile fish. These results indicate that conserved IL-2Rγc signaling via JAK3 plays a key role during early zebrafish lymphopoiesis, which can be potentially targeted to generate a zebrafish model of human SCID.

The role of acid-sensing ion channels (ASICs) in epithelial Na+ uptake in adult zebrafish (Danio rerio)

Acid-sensing ion channels (ASICs) are epithelial Na+ channels gated by external H+. Recently, it has been demonstrated that ASICs play a role in Na+ uptake in freshwater rainbow trout. The current paper investigated the potential involvement of ASICs in Na+ transport in another freshwater fish species, the zebrafish (Danio rerio). Using molecular and histological techniques we found that asic genes and the ASIC4.2 protein are expressed in the gill of adult zebrafish. Immunohistochemistry revealed that mitochondrion-rich cells positive for ASIC4.2 do not co-localize with Na+/K+-ATPase (NKA)-rich cells, but co-localize with cells expressing vacuolar-type H+-ATPase (VHA). Furthermore, pharmacological inhibitors of ASIC and Na+/H+-exchanger (NHEs) significantly reduced uptake of Na+ in adult zebrafish exposed to low Na+ media, but did not cause the same response in individuals exposed to ultra-low Na+ water. Our results suggest that in adult zebrafish ASICs play a role in branchial Na+ uptake in media with low Na+ concentrations and that mechanisms used for Na+ uptake by zebrafish may depend on the Na+ concentration in the acclimation media.

Zebrafish embryonic lipidomic analysis reveals that the yolk cell as metabolically active in processing lipid

The role of lipids in providing energy and structural cellular components during vertebrate development is poorly understood. To elucidate these roles further, we visualized lipid deposition and examined expression of key lipid-regulating genes during zebrafish embryogenesis. We also conducted a semiquantitative analysis of lipidomic composition using liquid chromatography (LC)-mass spectrometry. Finally, we analyzed processing of boron-dipyrromethene (BODIPY) lipid analogs injected into the yolk using thin layer chromatography. Our data reveal that the most abundant lipids in the embryo are cholesterol, phosphatidylcholine, and triglyceride. Moreover, we demonstrate that lipids are processed within the yolk prior to mobilization to the embryonic body. Our data identify a metabolically active yolk and body resulting in a dynamic lipid composition. This provides a foundation for studying lipid biology during normal or pharmacologically compromised embryogenesis.

Genome editing in zebrafish: a practical overview

Zebrafish is a powerfulmodel for the study of vertebrate development, being amenable to a wide range of genetic and other manipulations to probe themolecular basis of development and its perturbation in disease. Over recent years, genome editing approaches have become increasingly used as an efficient and sophisticated approach to precisely engineer the zebrafish genome, which has further enhanced the utility of this organism. This review provides a practical overview of genome editing and its application in zebrafish research, including alternate strategies for introducing and screening for specific genetic changes.