Introduction

The conservation and management of peatlands by practitioners is often assumed to work best when guided by science (e.g. Maltby 1997). However, there are also many excellent peatland management and restoration projects, which have built upon years of practical experience (sometimes through trial and error), undertaken by organisations involved in hands-on peatland conservation. Parry, Holden and Chapman (2014) provide many examples of techniques developed through common sense and ingenuity on the part of practitioners, often with little input from the science community. Often restoration projects have to make progress well before the science is fully understood. Significant investment is being poured into peatland management projects across the world (Parish et al. 2008), and it is important for those investing resources in peatland environments that there is some evaluation of the impacts of such investment. Evaluating the success of peatland management projects may involve the scientific community (e.g. taking measurements of carbon fluxes). In many instances, however, practitioners may involve less stringent measures with success measured by recording some simple visible changes to the landscape. The evaluation of success may indeed be an economic one (Kent 2000) based on cost–benefit analyses (Christie et al. 2011) of, for example, money spent on restoration that has been or will be saved elsewhere through, for instance, improved water quality entering water company treatment works. The observations for measuring peatland conservation success may depend on spatial and temporal scale, geographic settings and project targets, as well as available expertise and funding. There are therefore questions about how we measure success and how scientists, practitioners and policy makers can work closely together to deliver the best outcomes for peatland ecosystem services. Careful attention should be given to the mechanisms for science knowledge exchange between science and practical application so that practical experience and knowledge by those managing peatlands is transferred into the scientific understanding of peatlands. Scientists value the opinions and ideas of the restoration community and there have been recent attempts to move towards improved co-design of research and co-production of knowledge of science and practitioner communities in peatland restoration environments (Reed 2008; Reed et al. 2009).

Taking an ecosystem services approach to peatland conservation means that scientists, practitioners and policy makers have to understand the wider interconnectedness of peatland processes that lead to the provision of goods and services to society.

Liver

The liver forms as an evagination from the developing intestine. It lies ventral to the esophagus and intestinal bulb–3. It is not a discrete organ as it is in mammals but rather fills the abdominal cavity as a large U-shaped structure (Figure 7.1), surrounding the intestine. The liver is intimately associated with pancreatic tissue and the gallbladder,. Blood flow to the liver is similar to that in mammals. Blood enters the liver from the portal vein and the hepatic artery. After entering the liver, the blood flows into smaller and smaller tributaries, which end with a series of anastomosing capillaries referred to as sinusoids. The sinusoids are lined by endothelial cells. Kupffer cells, which line the sinusoids in the mammalian liver and have phagocytic abilities, probably do not exist in zebrafish. From the sinusoids, blood collects into the central veins and then flows to the heart through the hepatic vein.

The distinct liver lobules present in mammals with a central vein and surrounding portal triads are not present in the zebrafish. Many of the same types of cells seen in mammalian livers are, however, present in zebrafish. The hepatocytes are the most abundant cell type and are recognized as epithelioid, polygonal-shaped cells with a central nucleus (Figure 7.2). They surround a sinusoid and form cords, rather than the plates as seen in higher vertebrates. In some fishes, the hepatocyte cytoplasm contains abundant glycogen easily demonstrated with a PAS stain. As in higher vertebrates, hepatocytes function in storage of lipids, glycogen, and iron. They produce a variety of proteins and amino acids and aid in the detoxification of a variety of compounds. Historically, oil prepared from cod liver was an important source of vitamins A and D. Veins are clearly visible in the zebrafish liver as they contain nucleated red cells. Because no distinct liver lobules are present, it is not possible to distinguish between portal and hepatic vein branches on routine histologic sections.

Skeletal system

Zebrafish are bony fish whose skeleton is composed of bone and cartilage. Embryonically, most bone initially forms from a cartilaginous model and the skeleton is composed of both cartilage and ossified cartilage. Cartilage in its non-ossified state is firm but flexible as opposed to bone, which is hard and inflexible. Microscopically, cartilage is composed of a pale blue to purple stromal material (chondroitin sulfate) in which are dispersed large cells (chondrocytes) with pale or clear cytoplasm surrounding small central dark nuclei (Figure 15.1). Bone is composed of a pink, often lamellar, substance with small dark nuclei in lacunae and distributed along the edge (Figure 15.1).

In zebrafish, the skeletal system is most readily divided into the head and post-cranial skeletons. The components of the head skeleton are functionally interrelated and include the chondrocranium, the visceral skeleton, and the dermal bones of the integumentary skeleton. A predominant function of the head skeleton is to protect the brain and form an effective jaw. The integumentary skeleton is composed of bony scales that form just beneath the skin. The chondrocranium is part of the axial somatic system. The somatic skeleton is divided into the axial and appendicular components. The chondrocranium of the skull, the vertebral column, and the ribs, along with the median fins and sternum, are all components of the axial skeleton. The paired appendages and their component girdles (lateral fins) are portions of the appendicular skeleton. In fish as opposed to mammals, a visceral skeleton exists in the gut wall and forms skeletal arches associated with the pharyngeal pouches. Hence, mandibular arches, hyoid arch, and branchial arches all exist in fishes.

In higher vertebrates (reptiles, birds, and mammals) development of the kidney proceeds in three stages. The first kidney to form in the embryo is the pronephros. It consists of paired glomeruli and two separate pronephric ducts that drain waste products into the cloaca. The cloaca in teleosts is a small embryologic structure, which is lost in the adult. Later on in embryogenesis the pronephros is replaced by the mesonephros, which in turn is replaced by the metenephros or the adult kidney. In teleosts, kidney development ends with the mesonephros, no metenephric kidney forms.

In zebrafish, the pronephros consists of two nephrons emanating from a centrally fused paired glomerulus. In further development, the nephrons of the pronephros degenerate but the structure remains as the head kidney and becomes the site for the immune system and for the steroidogenic and chromaffin cells, which are homologous to the adrenal gland of mammals. The mesonephros becomes the adult kidney in the zebrafish and contains the nephrons responsible for filtering blood wastes and for salt and water uptake.

The kidney extends the length of the body cavity (Figures 2.3, 2.4, and 10.1). The anterior or head kidney contains lymphoid, hematopoietic, steroidogenic, and endocrine cells. The head (cranial) kidney is the dominant site of hematopoiesis and is composed predominately of hematopoietic elements. The immature hematopoietic cells lie between the nephron tubules. Distinct areas of lymphopoiesis and red blood cell formation are seen. The posterior or tail kidney contains nephrons with surrounding lymphoid tissue (Figure 10.2). The kidney can be seen occupying space between the vertebral column and the gas bladder. The head kidney contains both a left and right side but they become fused in the tail kidney.

Olfactory sac

The olfactory sacs are paired organs, anterior to the eyes and lying above and dorsal to the oropharynx. They are continuous with external nostrils and consist of sensory receptor cells admixed with goblet and ciliated cells (Figure 12.1). Processes from the receptor cells lead to the olfactory bulbs in the brain. The olfactory sacs consist of folded lamella to increase surface area (Figure 12.2). The paired organs receive water from an inflow tract through the nostrils. Odorants interact with receptor cells and signal through the olfactory tracts leading to the olfactory lobe (Figure 12.3) of the brain. The olfactory epithelium contains two types of receptors, ciliated and microvillous. The crypt neuron axons lead into the olfactory tracts and to the olfactory bulb where they terminate on structures termed glomeruli.

Eye

The eyes of fish lie anterior and inferior to the brain and are structurally similar to the eyes of other vertebrates including mammals (Figure 12.4). Light enters the eye through the transparent cornea (Figure 12.5). Because of the aqueous environment in which fishes live, the cornea requires little refractive power to bend incoming light waves. Thus the cornea in fish is relatively thin. As in mammals, the iris controls the amount of light passing through the pupil. The lens of the fish is nearly spherical and focuses light onto the retina (Figure 12.6). Focusing is achieved by varying the distance between the lens and the retina rather than changing the shape of the lens.

The retina is composed of five layers. The order from outer- to innermost layer is: (1) pigment epithelium; (2) photoreceptor layer; (3) bi-polar layer (synapses present); (4) ganglion layer; and (5) nerve fiber layer (Figure 12.7). Fish retinas contain two types of cells: rods and cones. The rods are sensitive to low levels of light. The pigmented layer helps protect the rods in high light levels. The cones are responsible for vision in bright light. Four types of cones exist, each characterized by sensitivity to a particular wavelength of light.

Fish possess a complex endocrine system that is less well understood than that of mammals. Wendelaar Bonga has reviewed the major fish hormones with fairly well defined functions.

Thyroid

The thyroid follicles arise from the pharyngeal endoderm. As with the pancreas, there is not a distinct thyroid gland as seen in higher vertebrates,. Instead, the zebrafish thyroid is composed of loosely scattered follicles present in soft tissue,. They are best observed in the ventral pharynx in the vicinity of the ventral aorta (Figure 9.1). The histology is similar to that of other species,. The follicles are composed of cuboidal to flat follicular cells surrounding an eosinophilic colloid material composed mostly of thyroglobulin (Figure 9.2). The size of the thyroid follicles ranges from 14 to 140 µm in diameter. As in higher vertebrates, the follicular cells produce thyroglobulin, which is then released into the follicle. In the thyroid follicle, thyroglobulin undergoes iodination of its tyrosine residues to produce thyroxine (T4) and tri-iodothyronine (T3), which are stored in the thyroid follicle. These hormones are important regulators of growth, metabolism, and development.

Ultimobranchial gland

The ultimobranchial gland is derived from the last pharyngeal pouch. It is homologous to the medullary C cells of the mammalian thyroid. In mammals, the C cells fuse with the thyroid glandandbecome known as the parafollicular cells. They produce calcitonin, a hypocalcemic hormone important in regulating serum calcium levels. In zebrafish, fusion with the C cells does not take place and these cells remain as a separate gland; the ultimobranchial gland. The ultimobranchial gland produces calcitonin. The gland is best observed lying ventral to the esophagus (Figure 9.3). It has follicles arranged around a central lumen.

The zebrafish pancreas is similar to the pancreas in higher vertebrates. It serves both as an exocrine and endocrine organ. The endocrine function resides in cells composing the islets of Langerhans. Pancreatic neuroendocrine cells in the islets include the alpha, beta, and delta cells, which produce the hormones glucagon, insulin, and somatostatin, respectively. The exocrine function of the pancreas is a responsibility of the acinar cells. These cells synthesize digestive enzymes that collect in the centrally located acinar ducts, then flow into the pancreatic duct and finally into the intestine. Interestingly, recent data suggest that the zebrafish pancreas may also be the site for the developing B cells of the immune system.

In routine histologic sections, unlike in higher vertebrates, the zebrafish pancreas is seen as a diffuse collection of acini and islets scattered in fatty tissue and located around the liver and intestine (Figure 8.1). Sometimes pancreatic tissue appears embedded in the liver, which has given rise to the term hepatopancreas. The pancreas develops from dorsal and ventral buds. The dorsal bud appears to be strictly endocrine while the ventral is predominately exocrine.

At least two types of islets, Brockmann body/principal islet (Figure 8.2) and the diffuse islets (Figure 8.3), can be recognized histologically. The Brockman or principal islet is a large collection of pancreatic neuroendocrine cells surrounded by a small amount of acinar tissue. The neuroendocrine cells are arranged in ribbons and the islet is highly vascularized (Figure 8.4). The smaller, diffuse islets are scattered throughout pancreatic acinar tissue. A third type, the single beta cell, can be detected only with immunofluorescent techniques.

Respiration in fish is achieved by bringing water through the lips into the buccal cavity and hence, into the pharynx and over the gills (Figure 5.1). Gill respiration only functions effectively when water moves in a unilateral direction over the gill membranes,. Most fishes pump water over their gills by alternately relaxing and contracting the buccal chamber (anterior to the gills) and the opercular chamber (posterior to the gills),. A second method for achieving water flow is by swimming with the mouth open (ram ventilation) forcing water through the pharynx, over the gills, and out the opercular chamber and gill slits,.

Gills

Like terrestrial organisms, oxygen is required for ATP production and basic body metabolism in fish. Unlike terrestrial organisms, fish live in an oxygen poor environment. The gills are the structures that allow fish to efficiently extract oxygen from the water for use in metabolic reactions.

The zebrafish gills are composed of four bilateral gill arches and can be seen in the ventral oropharynx (Figure 5.1). The gill arches are supported by bony and cartilaginous tissue and contain skeletal muscle. They are richly innervated by the facial, glossopharyngeal, and vagus cranial nerves. They are covered by a mucinous epithelium continuous with that of the oropharynx. Extending from each gill arch are two paired primary gill filaments (primary lamella) (Figure 5.2). The primary gill filaments are supported by cartilage: sometimes referred to as a cartilaginous ray (Figures 5.3 and 5.4). Easily seen in the primary gill filaments are the large central venous sinuses (Figures 5.3 and 5.4). Perpendicular from each primary filament are many thin-walled secondary gill filaments (secondary lamella) (Figure 5.2). The secondary gill filaments provide a large surface area and are the site for oxygen uptake and the release of carbon dioxide and ammonia. The secondary gill filaments are lined by thin squamous cells referred to as pavement cells (Figures 5.3, 5.6, and 5.7). The pavement cells are epithelial in nature and stain positively for keratins by immunohistochemistry (Figure 5.5). Red blood cells are easily seen in the secondary gill filament.

Utility of zebrafish as an animal model for study of oncogenesis and developmental defects

The development of animal models for a variety of neoplasms has greatly facilitated our understanding of oncogenesis and the relationship between somatic and germ line mutations with tumor growth and development. While mammalian models for developmental abnormalities and neoplasia would appear most appropriate, they are often hindered by issues of cost, latency to expression of phenotype, and animal care issues. Hence, non-mammalian but vertebrate organisms have a number of advantages. The zebrafish (Danio rerio) has emerged as a useful model system for the study of cancer biology because it has a reduced latency to expression of phenotype, a relatively low cost, a susceptibility to many tractable techniques for analysis of gene function, and the species amenability to oncogenic and chemical modifiers. In addition, early zebrafish embryos are optically clear, allowing observation of tumor development and organogenesis. This allows in vivo examination of cell and tissue behavior. A number of zebrafish models of neoplasia have been developed for both inactivating mutations and for the expression of human oncogenes including C-MYC, BRAF, and N-ras, which are known to be associated with a variety of human neoplasms.

The majority of living fishes including the zebrafish are members of the division Teleostei. This division represents the most advanced of the living bony fishes accounting for 96% of all fish species. Teleosts occur in both fresh and marine water habitats. The order Cypriniformes includes the zebrafish and other popular aquarium fishes including the goldfish and koi. Because these fishes are relatively easy to raise and the maintenance of colonies of these fishes is straightforward, members of this order have become popular for hobbyists and researchers alike. The zebrafish is the standard research animal for developmental genetics as well as being a popular species for the aquarium enthusiast.

Preface

The present atlas is designed to aid basic and translational scientists who require a fundamental understanding of zebrafish macro- and micro-anatomy. Many investigators with interests in the molecular features of oncogenesis or molecular genetics and organ development have found zebrafish to be a valuable animal model for their studies. However, these investigators often lack basic training in the fundamentals of fish histology and anatomy. This book is intended to address that gap.

The present atlas makes use of H&E stained longitudinal and cross sections to demonstrate the macro- and micro-anatomy of the zebrafish. Unlike many other atlases, all the photographs in the present book are obtained from zebrafish. This is important because many species of bony fishes show considerable anatomic variation. While some bony fishes possess a tongue and a true stomach, these are absent in zebrafish resulting in significant anatomic differences. The text concentrates on elucidating the actual microscopic appearance of zebrafish. An initial chapter uses longitudinal and cross sections photographed at low or no magnification to illustrate the relationships of zebrafish anatomy. Later chapters follow an organ systems approach in which the histology at both low and high power is addressed in detail. It is hoped that this combined approach will allow investigators to quickly and accurately identify specific organs and tissues involved by neoplasms or developmental abnormalities induced by molecular genetic changes. To this end, the text accompanying the photomicrographs has been made relatively brief while a large number of photomicrographs have been included. The photomicrographs have been generously labeled for easy identification of structures within the tissue sections. Correlation of photomicrographs present in the organ-based chapters with those present in the orientation chapter should allow rapid identification of tissue structures observed in study preparations.

Spleen

In fishes, hematopoiesis occurs within both the spleen and kidney. The spleen lies between the intestinal bulb and the liver (Figure 14.1). On gross and microscopic exam, the spleen appears to be composed predominantly of nucleated red blood cells with fewer numbers of lymphocytes. White and red pulp are not well demarcated in most histologic sections of fish splenic tissue (Figure 14.2). Most of the spleen is composed of red pulp formed by sinusoids containing nucleated red blood cells. The white pulp is composed of lymphocytes aggregating around vessels.

Thymus

The thymus appears as a lymphoid organ seen in the gill cavity just ventral to the ear (Figure 14.3). The thymus lies in the same plane as the heart and is characterized by a high density of small cells with large nuclei (Figure 14.4). This pattern results in dark staining of the organ. The zebrafish thymus lies within the same position and has the same appearance as the thymus of other teleosts,. The epithelium associated with the thymus is continuous with the pharyngeal epithelium. At high magnification, thymocytes appear as groups of packed cells (epithelial cells) between lymphoid cells (Figure 14.4),.

The zebrafish digestive system can be divided into anterior and posterior portions. The anterior portion is composed of the mouth, buccal cavity, and oropharynx. The esophagus, intestinal bulb (proximal intestine), mid intestine, posterior intestine, and anus comprise the posterior portion–4. The oropharynx is the portion of the digestive system running from the mouth to just posterior to the pharyngeal teeth (Figure 4.1). The esophagus continues caudally and empties into the intestinal bulb, or proximal intestine. Zebrafish are not thought to have true stomachs and the intestinal bulb is a dilated portion of the intestine, located between the esophagus and mid intestine (Figure 4.1). Pyloric caeca, blind-ended finger-like pouches arising from the anterior intestine, are not present in zebrafish. They are only present in fishes with true stomachs. From the intestinal bulb, the digestive tract continues to the mid intestine, then to the posterior intestine, and ends at the anus. Absorption of fats is believed to occur in the intestinal bulb, while absorption of proteins occurs in the mid intestine. The posterior intestine may play a role in osmoregulation.

Oropharynx

The lining of the oropharynx is continuous with the epidermis and begins after the keratinizing squamous epithelium of the lips. The squamous epithelium continues into the buccal cavity. The buccal cavity lacks salivary glands but contains mucous cells and taste buds (Figures 4.2 and 4.3). The oropharyngeal epithelium, like the epidermis, is a non-keratinizing squamous epithelium (Figures 4.2 and 4.3). In the oropharyngeal epithelium, the alarm cells that were so common in the epidermis (Figure 3.2) are no longer present, but many more mucous cells are found (Figure 4.3). No true tongue exists in zebrafish. As in the epidermis, taste buds are present in the epithelium (Figure 4.3). It has been estimated that zebrafish may contain at least 2200 oropharyngeal taste buds. Like the epidermis, the oropharyngeal taste buds are seen to be composed of both light and dark cells (Figure 4.4). The oropharynx begins to acquire a surrounding layer of skeletal muscle distally. Unlike mammals, no salivary gland tissue is present.