A model 450 million years in the making: zebrafish and vertebrate immunity

Since its first splash 30 years ago, the use of the zebrafish model has been extended from a tool for genetic dissection of early vertebrate development to the functional interrogation of organogenesis and disease processes such as infection and cancer. In particular, there is recent and growing attention in the scientific community directed at the immune systems of zebrafish. This development is based on the ability to image cell movements and organogenesis in an entire vertebrate organism, complemented by increasing recognition that zebrafish and vertebrate immunity have many aspects in common. Here, we review zebrafish immunity with a particular focus on recent studies that exploit the unique genetic and in vivo imaging advantages available for this organism. These unique advantages are driving forward our study of vertebrate immunity in general, with important consequences for the understanding of mammalian immune function and its role in disease pathogenesis.     

HYPERMUTABILITY OF HOXA13A AND FUNCTIONAL DIVERGENCE FROM ITS PARALOG ARE ASSOCIATED WITH THE ORIGIN OF A NOVEL DEVELOPMENTAL FEATURE IN ZEBRAFISH AND RELATED TAXA (CYPRINIFORMES)

Gene duplication is widely regarded as the predominant mechanism by which genes with new functions and associated phenotypic novelties arise. A whole genome duplication occurred shortly before the most recent common ancestor of teleosts, the most diverse chordate group, resulting in duplication and retention of many Hox cluster genes. Because they play a key role in determination of body plan morphology, it has been widely assumed that Hox genes play a key role in the evolution of diverse metazoan body plans. However, it is not clear whether certain aspects of molecular evolution, such as asymmetric divergence and neofunctionalization, contribute to the initial retention of paralogs. We investigate the molecular evolution and functional divergence of the duplicated HoxA13 paralogs in zebrafish to determine when asymmetric divergence and functional divergence occurred after the duplication event. Our findings demonstrate the contribution of gene duplication to the evolution of novel features through evolutionary mechanisms other than those traditionally investigated, such as positive selection occurring immediately after gene duplication. Rather, we find a latent build up of molecular changes in a gene associated with the development of a novel feature in a very diverse group of fishes.     

It's time to swim! Zebrafish and the circadian clock

The zebrafish represents a fascinating model for studying key aspects of the vertebrate circadian timing system. Easy access to early embryonic development has made this species ideal for investigating how the clock is first established during embryogenesis. In particular, the molecular basis for the functional development of the zebrafish pineal gland has received much attention. In addition to this dedicated clock and photoreceptor organ, and unlike the situation in mammals, the clocks in zebrafish peripheral tissues and even cell lines are entrainable by direct exposure to light thus providing unique insight into the function and evolution of the light input pathway. Finally, the small size, low maintenance costs and high fecundity of this fish together with the availability of genetic tools make this an attractive model for forward genetic analysis of the circadian clock. Here, we review the work that has established the zebrafish as a valuable clock model organism and highlight the key questions that will shape the future direction of research.     

Expression of zebrafish aldh1a3 (raldh3) and absence of aldh1a1 in teleosts

The vitamin A-derived morphogen retinoic acid (RA) plays important roles during the development of chordate animals. The Aldh1a-family of RA-synthesizing enzymes consists of three members, Aldh1a1-3 (Raldh1-3), that are dynamically expressed throughout development. We have searched the known teleost genomes for the presence of Raldh family members and have found that teleost fish possess orthologs of Aldh1a2 and Aldh1a3 only. Here we describe the expression of aldh1a3 in the zebrafish, Danio rerio. Whole mount in situ hybridization shows that aldh1a3 is expressed during eye development in the retina flanking the optic stalks and later is expressed ventrally, opposite the expression domain of aldh1a2. During inner ear morphogenesis, aldh1a3 is expressed in developing sensory epithelia of the cristae and utricular macula and is specifically up-regulated in epithelial projections throughout the formation of the walls of the semicircular canals and endolymphatic duct. In contrast to the mouse inner ear, which expresses all three Raldhs, we find that only aldh1a3 is expressed in the zebrafish otocyst, while aldh1a2 is present in the periotic mesenchyme. During larval stages, additional expression domains of aldh1a3 appear in the anterior pituitary and the swim bladder. Our analyses provide a starting point for genetic studies to examine the role of RA in these organs and emphasize the suitability of the zebrafish inner ear in dissecting the contribution of RA signaling to the development of the vestibular system.     

Identification of differentially expressed genes in the zebrafish hypothalamic–pituitary axis

The vertebrate hypothalamic–pituitary axis (HP) is the main link between the central nervous system and endocrine system. Although several signal pathways and regulatory genes have been implicated in adenohypophysis ontogenesis, little is known about hypothalamic–neurohypophysial development or when the HP matures and becomes functional. To identify markers of the HP, we constructed subtractive cDNA libraries between adult zebrafish hypothalamus and pituitary. We identified previously published genes, ESTs and novel zebrafish genes, some of which were predicted by genomic database analysis. We also analyzed expression patterns of these genes and found that several are expressed in the embryonic and larval hypothalamus, neurohypophysis, and/or adenohypophysis. Expression at these stages makes these genes useful markers to study HP maturation and function.     

Breaking symmetry: the zebrafish as a model for understanding left-right asymmetry in the developing brain

How does left-right asymmetry develop in the brain and how does the resultant asymmetric circuitry impact on brain function and lateralized behaviors? By enabling scientists to address these questions at the levels of genes, neurons, circuitry and behavior,the zebrafish model system provides a route to resolve the complexity of brain lateralization. In this review, we present the progress made towards characterizing the nature of the gene networks and the sequence of morphogenetic events involved in the asymmetric development of zebrafish epithalamus. In an attempt to integrate the recent extensive knowledge into a working model and to identify the future challenges,we discuss how insights gained at a cellular/developmental level can be linked to the data obtained at a molecular/genetic level. Finally, we present some evolutionary thoughts and discuss how significant discoveries made in zebrafish should provide entry points to better understand the evolutionary origins of brain lateralization.     

Ciona intestinalis as a model for cardiac development

The primitive chordate Ciona intestinalis has emerged as a significant model system for the study of heart development. The Ciona embryo employs a conserved heart gene network in the context of extremely low cell numbers and reduced genetic redundancy. Here, I review recent studies on the molecular genetics of Ciona cardiogenesis as well as classic work on heart anatomy and physiology. I also discuss the potential of employing Ciona to decipher a comprehensive chordate gene network and to determine how this network controls heart morphogenesis.     

Light reaches the very heart of the zebrafish clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.     

Light Reaches the Very Heart of the Zebrafish Clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early-stage zebrafish embryos contain clocks that are directly light-responsive. We describe recent experiments using single-cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light-responsiveness of early-stage zebrafish embryos.     

Homoploid hybrid speciation in animals

Among animals, evidence for homoploid hybrid speciation (HHS, i.e. the creation of a hybrid lineage without a change in chromosome number) was limited until recently to the virgin chub, Gila seminuda, and some controversial data in support of hybrid status for the red wolf, Canis rufus. This scarcity of evidence, together with pessimistic attitudes among zoologists about the evolutionary importance of hybridisation, prompted the view that HHS is extremely rare among animals, especially as compared with plants. However, in recent years, the literature on animal HHS has expanded to include several new putative examples in butterflies, ants, flies and fishes. We argue that this evidence suggests that HHS is far more common than previously thought and use it to provide insights into some of the genetic and ecological aspects associated with this type of speciation among animals.     

Genetic evidence for an evolutionarily conserved role of IL-7 signaling in T cell development of zebrafish

In mammals, the cytokine IL-7 is a key regulator of various aspects of lymphocyte differentiation and homeostasis. Because of the difficulty of identifying cytokine homologs in lower vertebrates and the paucity of assay systems and reagents, the degree of functional conservation of cytokine signaling pathways, particularly those pertaining to lymphocyte development, is unclear. In this article, we report on the analysis and characterization of three zebrafish mutants with severely impaired thymopoiesis. The identification of affected genes by positional cloning revealed components of the IL-7 signaling pathway. A presumptive null allele of the zebrafish homolog of the IL-7Rα-chain causes substantially reduced cellularity of the thymus but spares B cell development in the kidney. Likewise, nonsense mutations in the zebrafish homologs of janus kinases JAK1 and JAK3 preferentially affect T cell development. The functional interactions of the cytokine receptor components were examined in the three groups of fish hetero- or homozygous for either il7r and jak1, il7r and jak3, or jak1 and jak3 mutations. The differential effects on T cell development arising from the different genotypes could be explained on the basis of the known structure of the mammalian IL-7R complex. Because IL-7 signaling appears to be a universal requirement for T cell development in vertebrates, the mutants described in this article represent alternative animal models of human immunodeficiency syndromes amenable to large-scale genetic and chemical screens.     

Using zebrafish to study podocyte genesis during kidney development and regeneration

During development, vertebrates form a progression of up to three different kidneys that are comprised of functional units termed nephrons. Nephron composition is highly conserved across species, and an increasing appreciation of the similarities between zebrafish and mammalian nephron cell types has positioned the zebrafish as a relevant genetic system for nephrogenesis studies. A key component of the nephron blood filter is a specialized epithelial cell known as the podocyte. Podocyte research is of the utmost importance as a vast majority of renal diseases initiate with the dysfunction or loss of podocytes, resulting in a condition known as proteinuria that causes nephron degeneration and eventually leads to kidney failure. Understanding how podocytes develop during organogenesis may elucidate new ways to promote nephron health by stimulating podocyte replacement in kidney disease patients. In this review, we discuss how the zebrafish model can be used to study kidney development, and how zebrafish research has provided new insights into podocyte lineage specification and differentiation. Further, we discuss the recent discovery of podocyte regeneration in adult zebrafish, and explore how continued basic research using zebrafish can provide important knowledge about podocyte genesis in embryonic and adult environments. genesis 52:771–792, 2014. © 2014 Wiley Periodicals, Inc.     

Zebrafish models in cardiac development and congenital heart birth defects

The zebrafish has become an ideal vertebrate animal system for investigating cardiac development due to its genetic tractability, external fertilization, early optical clarity and ability to survive without a functional cardiovascular system during development. In particular, recent advances in imaging techniques and the creation of zebrafish transgenics now permit the in vivo analysis of the dynamic cellular events that transpire during cardiac morphogenesis. As a result, the combination of these salient features provides detailed insight as to how specific genes may influence cardiac development at the cellular level. In this review, we will highlight how the zebrafish has been utilized to elucidate not only the underlying mechanisms of cardiac development and human congenital heart diseases (CHDs), but also potential pathways that may modulate cardiac regeneration. Thus, we have organized this review based on the major categories of CHDs-structural heart, functional heart, and vascular/great vessel defects, and will conclude with how the zebrafish may be further used to contribute to our understanding of specific human CHDs in the future.     

Ontogeny of the collar cord: Neurulation in the hemichordate Saccoglossus kowalevskii

The chordate body plan is characterized by a central notochord, a pharynx perforated by gill pores, and a dorsal central nervous system. Despite progress in recent years, the evolutionary origin of each of theses characters remains controversial. In the case of the nervous system, two contradictory hypotheses exist. In the first, the chordate nervous system is derived directly from a diffuse nerve net; whereas, the second proposes that a centralized nervous system is found in hemichordates and, therefore, predates chordate evolution. Here, we document the ontogeny of the collar cord of the enteropneust Saccoglossus kowalevskii using transmission electron microscopy and 3D‐reconstruction based on completely serially sectioned stages. We demonstrate that the collar cord develops from a middorsal neural plate that is closed in a posterior to anterior direction. Transversely oriented ependymal cells possessing myofilaments mediate this morphogenetic process and surround the remnants of the neural canal in juveniles. A mid‐dorsal glandular complex is present in the collar. The collar cord in juveniles is clearly separated into a dorsal saddle‐like region of somata and a ventral neuropil. We characterize two cell types in the somata region, giant neurons and ependymal cells. Giant neurons connect via a peculiar cell junction that seems to function in intercellular communication. Synaptic junctions containing different vesicle types are present in the neuropil. These findings support the hypotheses that the collar cord constitutes a centralized element of the nervous system and that the morphogenetic process in the ontogeny of the collar cord is homologous to neurulation in chordates. Moreover, we suggest that these similarities are indicative of a close phylogenetic relationship between enteropneusts and chordates. J. Morphol., 2010. ©2010 Wiley‐Liss, Inc.     

Light Reaches the Very Heart of the Zebrafish Clock

Zebrafish are typically used as a model system to study various aspects of developmental biology, largely as a consequence of their ex vivo development, high degree of transparency, and, of course, ability to perform forward genetic mutant screens. More recently, zebrafish have been developed as a model system with which to study circadian clocks. Cell lines generated from early‐stage zebrafish embryos contain clocks that are directly light‐responsive. We describe recent experiments using single‐cell luminescent imaging approaches to study clock function in this novel cell line system. Furthermore, studies examining the process of entrainment to light pulses within this cell population are described in this review, as are experiments examining light‐responsiveness of early‐stage zebrafish embryos.     

The other side of phenotypic plasticity: a developmental system that generates an invariant phenotype despite environmental variation

Understanding how the environment impacts development is of central interest in developmental and evolutionary biology. On the one hand, we would like to understand how the environment induces phenotypic changes (the study of phenotypic plasticity). On the other hand, we may ask how a development system maintains a stable and precise phenotypic output despite the presence of environmental variation. We study such developmental robustness to environmental variation using vulval cell fate patterning in the nematode Caenorhabditis elegans as a study system. Here we review both mechanistic and evolutionary aspects of these studies, focusing on recently obtained experimental results. First, we present evidence indicating that vulval formation is under stabilizing selection. Second, we discusss quantitative data on the precision and variability in the output of the vulval developmental system in different environments and different genetic backgrounds. Third, we illustrate how environmental and genetic variation modulate the cellular and molecular processes underlying the formation of the vulva. Fourth, we discuss the evolutionary significance of environmental sensitivity of this developmental system.     

Notch-responsive cells initiate the secondary transition in larval zebrafish pancreas

Zebrafish provide a highly versatile model in which to study vertebrate development. Many recent studies have elucidated early events in the organogenesis of the zebrafish pancreas; however, several aspects of early endocrine pancreas formation in the zebrafish are not homologous to the mammalian system. To better identify mechanisms of islet formation in the zebrafish, with true homology to those observed in mammals, we have temporally and spatially characterized zebrafish secondary islet formation. As is the case in the mouse, we show that Notch inhibition leads to precocious differentiation of endocrine tissues. Furthermore, we have used transgenic fish expressing fluorescent markers under the control of a Notch-responsive element to observe the precursors of these induced endocrine cells. These pancreatic Notch-responsive cells represent a novel population of putative progenitors that are associated with larval pancreatic ductal epithelium, suggesting functional homology between secondary islet formation in zebrafish and the secondary transition in mammals. We also show that Notch-responsive cells persist in the adult pancreas and possess the classical characteristics of centroacinar cells, a cell type believed to be a multipotent progenitor cell in adult mammalian pancreas.