Fish Development and Genetics

The following sections are included:

• Contents

• Preface

The earliest events in zebrafish development are driven by maternal factors deposited in the egg during oogenesis that become activated upon fertilization and initiate cascades of events that drive early development. This review summarizes the forward and reverse genetic methods used to identify and analyze genes coding for such maternal factors. I also discuss current knowledge on the cellular processes involved in two important developmental transitions: the redistribution and activation of maternal factors at fertilization, and the transition from maternal to zygotic genetic programs. In addition, I summarize current knowledge on the function of maternal factors, both before and after zygotic gene activation, in embryonic processes involved in general cellular functions, axis formation, and cell fate specification.

The following sections are included:

• Introduction

• Cellular Mechanisms
  • Epiboly
  • Internalization
  • Convergence and Extension
  • Future Directions

• Molecular Mechanisms
  • Wnt/PCP Pathway
  • Cell Adhesion Molecules
  • Nodal/TGFβ Signaling
  • PDGF/PI3K Pathway
  • Other Pathways

• Future Prospects
  • ‘New’ Methods
  • Remaining and Arising Questions

• Acknowledgments

• References

The major axes of a zebrafish embryo are established early in its development. One key structure, centrally involved in the specification of these axes, is the dorsal Organizer. The Organizer becomes apparent at the beginning of gastrulation and generates signals that pattern the mesoderm to generate dorsal structures and specifies the neurectoderm. In addition to providing inductive signals, the Organizer itself will eventually differentiate to form the axial mesendoderm tissues prechordal plate and notochord. These tissues provide signals to pattern surrounding tissues and, in the case of the notochord, mechanical support required for locomotion of zebrafish larvae. Thus, how the Organizer forms and functions has been a source of great interest in developmental biology. Modern molecular and genetic studies are now providing a detailed picture of the events controlling both formation of the Organizer and its activity. Focusing on these events during zebrafish development, but drawing on results from a variety of other experimental systems, we review dorsal Organizer establishment and function, differentiation of Organizer tissue to chordamesoderm and notochord and finally, the patterning and mechanical functions of the notochord.

The most ventral cells of the vertebrate neural tube, the floor plate, comprise a specialized group of cells distinct in form and function from the rest of the neural tube. These cells have been ascribed many functions, ranging from the differentiation of motor neurons that innervate specific muscle cell types in the body, to providing cues for the correct path-finding of various axons. Here, we summarize the process by which the floor plate develops in a few model vertebrate organisms, the various functions of the floor plate, and the molecular nature of signals emanating from the floor plate. Finally, we assess the prevailing models of how the cells of the floor plate are thought to arise.

The following sections are included:

• Introduction

• Development of the Nervous System: Axon Guidance

• Function of the Nervous System: Sensory Perception
  • Mechanotransduction
  • Odor Perception

• Network Function
  • The Visual System
  • The Touch Response

• Concluding Remarks

• References

The early zebrafish embryo develops a simple primary nervous system that controls motility. Similar to that of the nematode C. elegans, this primary nervous system is composed of a relatively low number of neurons and is amenable to observation and experimental manipulation at the single cell level. The primary nervous system is derived from neurogenic regions in the neural plate. Several conserved gene loci are required for the development of the primary neurons, suggesting that the molecular mechanisms underlying neurogenesis are strongly related to that of the fruitfly Drosophila melanogaster and higher vertebrates. Since inhibition of gene activity or mutations in the zebrafish emulate many human hereditary disorders, it renders the zebrafish an attractive model for the study of vertebrate nervous system development and human hereditary diseases.

The following sections are included:

• Amazing Conservation: Organization of Olfactory Sensory Systems
  • Fish as a Model System
  • Sensory Epithelium
  • Olfactory Bulb
  • Terminal Nerve

• The Olfactory Placode Arises from the Edge of the Developing Neural Plate
  • Origin
  • Gene Expression in the Olfactory Field of the Anterior Neural Plate
  • Induction
  • The Differentiating Olfactory Organ
  • Mutations Disrupting Olfactory Placode Development
  • Structural Elements of the Olfactory System Arise from Cranial Neural Crest

• The Olfactory Bulb Arises from the Anterior Neural Plate
  • Gene Expression in the Developing Olfactory Bulb
  • Axonal Input and Olfactory Bulb Development

• Axon Guidance: The Connection of the Olfactory Sensory Axons with the Developing Olfactory Bulb
  • Guidance Cues
  • Cell Surface Molecules
  • Olfactory Receptors
  • Pioneer Neurons

• Olfactory Receptors: The Interface with the Outside World
  • Main Olfactory Epithelium
  • Onset of Olfactory Receptor Expression during Development
  • Do Fish Have a Vomeronasal System?
  • Development

• Other Derivatives of the Olfactory Placode: Neuroendocrine Cells
  • The Terminal Nerve
  • Cells Appear to Exit the Olfactory Placode
  • The Neural Crest
  • The Adenohypophysis

• Olfactory Physiology and Behavior: The Key to Survival and Reproduction
  • Fishy Smells

• Evolution of the Olfactory Sensory System
  • Is there a Unifying Placode Theme?
  • Olfactory and Adenohypophysis
  • Olfactory Placode Field and Neural Crest

• A World of Questions Remain

• Acknowledgments

• References

Somite formation, a process in which reiterated epithelial structures are progressively demarcated from the mesenchymal presomitic mesoderm (PSM) in a anterior-posterior sequence, is the earliest manifestation of segmentation and is a feature shared by all vertebrate embryos. The temporal and spatial regulation of this process requires a molecular oscillator, the segmentation clock. The mechanisms driving and regulating the oscillation in PSM cells have been actively studied in zebrafish, chick and mouse. The oscillator is comprised of genetic circuit involving the Notch signaling pathway and its target genes her1 and her7 in zebrafish. Converting clock oscillation into the periodic arrangement of segment boundaries is achieved at the ‘wavefront’ located in the anterior PSM. The level of Fgf/MAPK activation (highest in the posterior PSM) serves as a positional cue within the PSM to restrict the wavefront to the anterior PSM. Once the level of Fgf/MAPK signaling declines in the anterior PSM, the wavefront activity mediated by a transcription factor, Fss/Tb×24, arrests the oscillation and leads to activation of a number of key genes required for subsequent sequences of somite formation. In the anterior PSM or wavefront, a complicated gene network centered on Mesp, a bHLH transcription factor, finally establishes a rostrocaudal subdivisions within somite primordium, which is prerequisite for formation of morphological distinct somite boundaries.

The molecular basis of somite development: the periodic generation of somites, rostrocaudal (RC) polarization in formed somites, somite furrow formation and somite differentiation has been substantially explored among different vertebrates for last few decades, enabling us to understand it from a more mechanistic way. The work on chicken c-hairy1 cycling, mouse knock-outs of Notch components and zebrafish somite mutants has demonstrated a vital role of Notch signaling in somite segmentation. A mechanism involving cyclical activation of transcription and delayed negative feedback regulation is emerging. Fgf8 and Wnt3a gradients are important in positioning somite boundaries and, probably, in coordinating tail growth and segmentation. In addition to segmentation, Notch signaling is also essential for RC polarity and boundary formation in collaboration with a variety of genes, including Mesp, Eph, ephrin, Protocadherin (Papc), Foxc and T-box genes. Zebrafish has played an indispensable role in recent progress. Studies of other species will also be discussed in a comparative and complementary way.

The formation of skeletal muscles in vertebrate embryos involves a series of events including induction, specification and differentiation. The multi-potential mesoderm cells in gastrula stage embryos are first induced to become paraxial mesoderm that subsequently form segmented somites. Somitic cells are specified into osteoblasts, myoblasts and dermal mesenchyme that ultimately differentiate into axial skeleton, skeletal muscles, and dermis of the skin, respectively. Somite formation and subsequent specification and differentiation of myoblasts are regulated by many extracellular signaling molecules and intracellular transcription factors. Signaling molecules of the Notch and FGF families are involved in somitogenesis, whereas Hedgehog (Hh), Wnt and TGF-β families play critical roles in myoblast specification and differentiation. These signaling molecules bind to their receptors and activate or repress intracellular transcription factors, such as members of the MyoD family and the paired transcription factors Pax3 and Pax7 that directly regulate muscle-specific gene expression, and muscle cell differentiation. The challenging task at present is to understand how these signaling cascades coordinate with each other and how they control the myogenic transcription network to allow for precise changes in gene expression during myoblast specification and differentiation.

The zebrafish, a species within the family Cyprinidae (minnows and carps), has emerged as an important vertebrate model for the study of development, including skeletal development, due to the availability of embryological, molecular, and genetic tools. Ichthyology has a long history, extending from Aristotle through present, fueled by a large number of species relative to most major vertebrate groups and by the considerable fossil record consisting of fish skeletons. The comparative adult osteology of fishes is a traditional area of study within the field, and many of the differences in skeletal anatomy closely track the paths of evolution within various clades. Developmental studies in zebrafish have the potential to clarify historically significant evolutionary questions pertaining to the evolution of the skeleton.

We focus on the study of skeletogenesis in zebrafish, although our emphasis is on the extent to which findings can be generalized to other vertebrates. After a review of descriptive studies, we discuss the availability of molecular markers, and the mutational analysis of skeletal development in zebrafish. Finally, we conclude with a discussion of prospects for additional, targeted mutant screens, and studies to examine lineage relationships among skeletogenic cells in the embryo and the adult.

Endoderm, the innermost embryonic germ layer, gives rise to the epithelium of the digestive tract and of the respiratory system. Contrary to ectoderm and mesoderm, endoderm formation had been poorly analyzed. These three germ layers are formed during gastrulation via specific cell movements. An in-depth understanding of endoderm formation requires the defination of the molecular basis leading to the establishment of endodermal identity but also the defination of the mechanisms driving cell migration during gastrulation. Studies in Xenopus, mouse and zebrafish have converged on one conserved signaling and transcription pathway responsible for endoderm formation: the TGF-β/Nodal pathway. These past years, zebrafish mutants analyses have allowed the isolation of several loci necessary for endoderm formation. They encode elements of the Nodal ligand/receptor complex or transcription factors that have been shown to act downstream of Nodal ligands, in the Nodal signaling pathway. Using the genetic and embryologic advantages of the zebrafish, recent studies have started linking molecular data to cellular behavior during gastrulation. The different mutant backgrounds and the manipulation of Nodal signaling have allowed researchers to start deciphering key embryological events such as fate choice decision between mesoderm and endoderm, endodermal specification and commitment as well as the mechanism triggering and controlling cell movements during gastrulation.

During gastrulation, the few thousands cells resulting from the cleavage of the fertilized egg become organized into three distinct germ layers: the ectoderm, the mesoderm and the endoderm. Endoderm, the innermost layer, will give rise to the vast majority of the digestive tract. Endoderm derivatives populate the entire epithelium of the gastrointestinal tract and form the associated organs: liver, pancreas and gall bladder. In addition, endoderm derivatives also participate in the build-up of the respiratory system by forming, respectively, the epithelium of the gills in lower vertebrates and the epithelium of the lungs in tetrapod. Last, they contribute to the thymus, the thyroid gland and the swim bladder. During early development, additional roles for endoderm include the induction, the patterning and proper morphogenesis of neighboring structures, including the heart and the head.^1–3 Therefore, endoderm development represents an attractive system to define the rules governing early patterning and differentiation of the embryo, as well as late morphogenesis.

In contrast to the ectoderm and mesoderm, the formation of which has now been studied for many years, endoderm development had been poorly addressed, probably because its deep position inside the embryo prevents easy observation. Within the past few years though, endoderm formation has become a field of intense scrutiny, and zebrafish has proven to be a fruitful model system in these studies. Zebrafish embryos are optically clear, permitting direct observation of endoderm development. Combined with techniques for the labeling of living cells, this optical clarity allows high-resolution imaging of morphogenetic movements and the construction of detailed fate maps. Fish also offer the opportunity to carry out genetic approaches and a number of mutants affecting endoderm development have been identified. Many of them have now been cloned and a first molecular pathway controlling endoderm formation can be assembled.

Derived from therapeutic tools developed for the clinic, unconventional antisense technology has emerged as a new and broadly applicable RNA-based gene inhibition approach. Its targeting mechanisms of action are both RNaseH-independent and distinct from other sequence-based tools such as small inhibitory RNAs. Among the novel classes of antisense oligonucleotides, morpholino phosphorodiamidate oligonucleotides (morpholinos) have emerged as the preferred effector molecules of targeted gene “knockdown” strategies. Morpholinos have been shown to be extremely effective, specific and convenient for elucidating gene functions in a variety of model systems. This chapter provides an overview on technical aspects of morpholino usage and some examples of the many potential biological applications of this technology ranging from human disease modeling to functional genomics.

Transgenic technology is the introduction of foreign DNA into a host organism so that the function and regulation of the inserted foreign DNA can be studied in the transgenic organism. Over the past decade, most of the key transgenic techniques have been developed in various fish models. In the present review, we focus on the transgenic studies in two experimental fish models, the zebrafish (Danio rerio) and medaka (Oryzias latipes). These include transient transgenic assays, stable transgenic lines focusing on GFP transgenic fish and their applications, conditional activation of transgene expression using inducible promoters and binary transgenic systems (GAL4-UAS and Cre-loxP), cell lineage ablation, insertional mutagenesis, gene traps and the potential of gene targeting approaches in fish. Future prospects of transgenic fish studies are also discussed.

Although the efforts started 40 years ago, resources dedicated to fish cloning and achievements made so far lag far behind those in other vertebrates, especially mammals. In this review, we summarize the fish cloning work carried out by a number of laboratories and our data of generating cloned zebrafish from long-term cultured embryonic fibroblast cells. This success lays the foundation for developing cloningbased gene-trapping and homologous recombination technologies for gene function studies in zebrafish. In addition, fish cloning should contribute to studies addressing basic issues such as development and aging of cloned animals.

Transgenic fish were first made more than 30 years ago. Since then a variety of methods and constructs have been tested for introducing genetic sequences into fish for scientific investigations as well as commercial purposes. Here we review transposable elements and their applications in fish. Transposons can be used to deliver genes to chromosomes to confer new traits or as insertional agents and traps to uncover the functions and expression patterns of natural genes in chromosomes. Two DNA transposons have been characterized for transposon-based gene transfer and insertional mutagenesis. The first is the Sleeping Beauty transposon system that was reconstituted from a Tc1/mariner-like relic in salmonid genomes after more than a 10 million year evolutionary sleep. The second is a naturally occurring transposon from medaka, the Tol2 transposon that belongs to the hAT family of mobile elements. In comparison with random integration of plasmid sequences and pseudotyped retroviral genomes, transposons have several advantages for genetic studies in fish. These include introduction of a single, defined DNA sequence into a cellular chromosome, stable expression from the integrant for multiple generations, no absolute size restrictions on the transferred gene, ease in construction and use, and safety. Early experiments have validated the versatility of the Sleeping Beauty transposon for all of these purposes. The applications of transposon systems surpass use just in fish; the Sleeping Beauty transposon system is being used in mice to discover functions of genes and is being developed for gene therapy in humans.

Because the zebrafish lineage is basally diverging among Euteleost fish, analysis of its genome can complement information from other key species to understand the origin of genomes of teleost fish, the most species-rich group of vertebrates, and to infer some history of the human genome. Analysis of gene maps shows that zebrafish chromosomes have conserved syntenies with large segments of human chromosomes, suggesting that translocations have been rather infrequent during vertebrate evolution. Nevertheless, zebrafish chromosomes consist of mosaics of segments orthologous to several human chromosomes, in part, probably because ancestral chromosomes experienced fission in the human lineage. Because the zebrafish orthologs of small regions of human chromosomes appear to have exploded over much of the zebrafish chromosome, inversions have been fixed in populations much more frequently than translocations. Genetic maps show that segments of human chromosomes are generally present in zebrafish as two orthologous copies. These data suggest a genome duplication event in the zebrafish lineage. Comparative mapping of zebrafish, pufferfish and medaka genomes shows that this event occurred before the divergence of Euteleosts, suggesting the hypothesis that this genome duplication may have contributed to the evolutionary success of teleosts. The complete sequencing of the zebrafish genome will reveal the answer to many questions, such as the fraction of genes derived from this event that are retained in duplicate copy, and the evolutionary principles that cause gene duplicates to be retained. Because of the elegant functional analyses possible with zebrafish, the sequence of the zebrafish genome will facilitate exploration of conserved gene function in vertebrate development and physiology. In addition, functional analysis of duplicated zebrafish genes can reveal ancestral gene functions sometimes obscured in mammals by pleiotropy.

Large-scale genome sequencing using several model organisms has provided a new pathway to identify new genes, the function of which can be discovered through comparative approaches. Pufferfish (Fugu rubripes and Tetraodon nigroviridis) has been sequenced by whole genome shotgun (WGS) assembly, and the zebrafish (Danio rerio) genome sequencing project, including whole genome shotgun sequencing and selective BAC shotgun sequencing, is now on going. Still, the number of model organisms is too small to understand fully how fish genomes evolved. Now, medaka (Oryzias latipes) is emerging as another important model fish that is phylogenically distant from zebrafish but closer to pufferfish. Recent additions to genetic toolkit of medaka, such as BAC resources, WGS sequences, and highly dense genome markers based on polymorphic inbred strains, can facilitate genome assembly with high quality (http://medaka.dsp.jst.go.jp/MGI/). About 1400 markers including 800 randomly selected EST markers were mapped, and all of them successfully assembled into 24 linkage groups that correspond to the medaka chromosome number. This genomic map is a powerful tool for positional cloning of mutated genes in medaka, and conserved syntenies of medaka, pufferfish and zebrafish genes to the human genome provide evidence for a whole genome duplication event that occurred after divergence of fish and tetrapods and before divergence of medaka and zebrafish. For gene regulation studies, polyploidy in model fish species might be advantageous, because regulatory elements and functional domains in each of the fish duplicates may have unique functional roles.

In this review we summarize the current status of medaka genome studies for functional genomics.

Embryonic stem (ES) cells are undifferentiated cell cultures that are derived from early developing animal embryos. ES cells retain the potential of differentiation into all cell types including germ cells and therefore provide a unique bridge linking in vitro and in vivo genetic manipulations. ES cells have been widely used in the production knockout mice. Attempts have been made to develop ES cells in fish. We used the medaka (Oryzias latipes) to develop the ES cell technology in a second vertebrate model. We have established feeder cell-free culture conditions and obtained several ES cell lines from midblastula embryos. These ES cells show all features of mouse ES cells including a diploid karyotype, the potential for differentiation into various cell types and chimera competence. This review is to use medaka ES cells to highlight the major advances and future prospects for obtaining and utilizing ES cells in model and aquaculture fish species.

The following sections are included:

• Subject Index