7/27/2012
Zebrafish:
Anatomy, Histology,
and Physiology
Jeffrey C. Wolf, DVM, DACVP
Experimental Pathology
Laboratories (EPL®), Inc.
Sterling, VA
Introduction
• Zebrafish have become a
premier research model
– Prolific females produce
non-adhesive eggs
– Particularly suited to embryology
• Embryos are small (fewer cells)
• Embryos are transparent therefore easily
viewed and easily manipulated
– Embryos are being used for highthroughput drug screening
– Wealth of genetic information
– A variety of genetically modified
and mutant strains are commercially
available
– Only commercial SPF research fish
– > 750 zebrafish facilities worldwide
Large zebrafish facility
GFP transgenic
transparent zebrafish larva
Introduction
• The zebrafish Danio rerio is a prototypical fish species
zebrafish anatomy/physiology ≈ fish anatomy/physiology
• Highlight:
– Salient differences between fish and mammals
– Salient differences between zebrafish and other fishes
• Topics:
– Small fish histology
– Zebrafish anatomy and physiology
Scanning EM of Zebrafish Heart
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Histologic Preparation of Whole Laboratory Fish
• Euthanasia and Necropsy
–
–
–
–
–
Tricaine methanesulfonate (MS-222), buffered
Fish examined for gross lesions
Incision along abdominal ventral midline
Removal of dorsal cranium if brain to be examined
Abdomen and gills flushed with fixative prior to
immersion
• Fixation and Decalcification
– Modified Davidson’s or Bouin’s solution for 24 hours or
less (depending upon size and age of fish)
– Rinse and switch to 10% neutral buffered formalin
– Decalcification in formic acid/EDTA solution for several
hours if necessary (not always necessary with these
fixatives)
Histologic Preparation of Whole Laboratory Fish
Davidson’s Fixative (Fournie et al., 2000)
Formaldehyde (37-40%)
Glycerol
Glacial acetic acid
Absolute alcohol
Distilled water
200 ml
100 ml
100 ml
300 ml
300 ml
Modified Davidson’s Fixative
Formaldehyde (37-40%)
Glacial acetic acid
95% Ethyl alcohol
Distilled water
220 ml
115 ml
330 ml
335 ml
Gills: Effect of Fixative Type
10% Neutral Buffered Formalin
Modified Davidson’s Solution
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Kidneys: Effect of Fixative Type
10% Neutral Buffered Formalin Modified Davidson’s Solution
Testis: Effect of Fixative Type
10% Neutral Buffered Formalin
Bouin’s Solution
Histologic Preparation of Whole Laboratory Fish
• Gross Trimming
– Fish examined for gross lesions
– Caudal fin removed at caudal
peduncle and discarded
– Fish placed in cassette
left side down
– Gross lesions on the skin or eyes
may prompt an alternate sectioning method
• Processing and Embedding
– Automatic tissue processor (~10 hr. cycle)
– Paraffin embedding of whole fish,
left side down
– Position of fish in the block critical to
consistency in sectioning
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Plane of Section
Slide courtesy of J. Fournie and L. Courtney
Histologic Preparation of Whole Laboratory Fish
• Microtomy
– Five step sections are cut in a
parasagittal plane through the
whole body
– Steps include two from the
left side of the body, one
midline, and two from the
right side
– Two serials of each step are
cut for a total of 10 sections
per fish
– Two or three slides
accommodate the sections
Tissues Evaluated in 5 Sagittal / Parasagittal Sections










Bone
Brain
Chromaffin Tissue
Corpuscle of
Stannius
Esophagus
Eye
Gallbladder
Gill
Heart
Hematopoietic
Tissue











Interrenal Tissue
Intestine
Kidney
Liver
Nares
Oral Cavity
Ovary
Pancreas
Peripheral Nerve
Pineal Organ
Pituitary










Pseudobranch
Skeletal Muscle
Skin
Spinal Cord
Spleen
Stato-acoustic
Organ
Swim Bladder
Testis
Thymus
Thyroid Tissue
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Zebrafish Anatomy and Physiology by System
•
•
•
•
•
•
•
•
•
•
Integumentary: scaled skin
Musculoskeletal: skull, axial skeleton, fins, musculature
Cardiovascular: heart, blood and lymph vessels
Hematologic: renal hematopoietic tissue, spleen, thymus
Respiratory: gills, pseudobranch
Digestive: oral and pharyngeal cavities, esophagus,
intestines, liver, gallbladder, pancreas, swim bladder
Urinary: opisthonephric kidney and ducts
Reproductive: testes, ovaries, efferent ducts, genital pore
Endocrine: pituitary gland, pineal gland, corpuscle of
Stannius, ultimobranchial body, islets of Langherhans,
interrenal and chromaffin tissue, gonads
Neurologic and Sensory: brain, spinal cord, peripheral
nerves, eyes, statoacoustic organ, olfactory organ, lateral line
Tissues Not Found in Zebrafish
•
•
•
•
•
•
•
•
•
•
•
Adrenal Glands
Bone Marrow
Glandular Stomach
Hair Follicles
Lungs
Lymph Nodes
Mammary Glands
Salivary Glands
Thyroid Gland C-cells
Urinary Bladder
Uterus
Anatomic Orientation During Presentation
dorsal
rostral
caudal
ventral
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Integumentary System: Anatomy
• Zebrafish skin not particularly unique
• Layers:
– Epidermis
• Non-keratinized
• Ridged surface for cuticle adherence
– Dermis -- contains the scale pocket
– Hypodermis (subcutis) -- minimal
Yonkos et al 2000
Epidermis
Scale pocket
Dermis stratum compactum
Skeletal muscle
Integumentary System: Anatomy
• Scales are membranous bones
embedded in pockets within the
epidermis and dermis
• Zebrafish: elasmoid ctenoid scales
• Scales form relatively late in
embryonic development
Biology of Fishes,
Bond,1979
– Formation occurs caudal to cranial
Sire and
Akimeko
2004
Sire and
Akimeko
2004
Integumentary System: Anatomy
• Specialized structures:
Yonkos et al 2000
– Alarm substance cells
taste bud
alarm cell
• = “club cells”
• Dermal injury causes
cells to release
hypoxanthine-3N-oxide
(Schreckstoff)
pheromone
– Goblet mucous cells
– Taste buds (face only)
– Chromatophores
(dermal pigment cells):
• Iridophores: silver
• Melanophores: brown
• Xanthophores: yellow
– brown + yellow = blue
Taste Bud
Schematic
and
Distribution
Hansen et al 2002
Hirata et al 2003
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Integumentary System: Physiology
• Pathogen barrier and defense system
– Fish mucus contains antimicrobial substances:
• Antibodies, peptides, lysozyme, lectins, and
proteases
• Maintenance of ionic balance
– Freshwater fish are hypertonic relative to their
surroundings, therefore skin breaches can lead to
generalized edema
• Skin also shown to excrete
excess acid in larval zebrafish
– Maintains pH homeostasis
before gills and kidneys are
fully developed
Generalized Edema in Betta splendens
Musculoskeletal System: Anatomy
• Components of skeleton:
ZEBRAFISH SKELETON
Menke et al 2011
– Axial
•
•
•
•
Skull
Spine
Ribs
Pectoral and pelvic girdles
– Appendicular
•
•
•
•
•
Pectoral fins (paired)
Pelvic fins (paired)
Dorsal fin
Anal fin
Caudal fin
ZEBRAFISH SPINE
Notochord
• Types of bone:
– Endochondral: most bones
– Membranous: skull, scales
• Fish bones vary from poorly
cellular to acellular
• Types of cartilage:
– Hyaline
– Fibrocartilage
– Elastic: two sets of paired barbels
Musculoskeletal System: Anatomy
• Skeletal Muscle
Zebrafish
muscle
labeled with
anti-myosin
– Membranous skeleton:
• Connective tissue that divides muscle into
four quadrants
http://www.bio.umass.e
du/biology/research/gbi/
zebrafish-muscle-lateral
– Vertical septum: left vs. right
– Horizontal septum: epaxial vs. hypaxial
– Myosepta:
• Connective tissue that
divide myomeres
CHANNEL CATFISH MUSCULATURE
– Myomere:
• Functional unit of trunk
musculature
• Muscles angled to
create comparable
distance from spine and
allow for synchronous
contraction
Courtesy of Al Camus
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Musculoskeletal System: Anatomy & Physiology
• Skeletal Muscle
– White muscle
• Majority of trunk musculature
• Large diameter, fast twitch fibers, anaerobic
• Poor vascular supply with little myoglobin
and few mitochondria
• Rapidly fatigues, useful for short bursts
white
muscle
red muscle
– Red muscle
• Narrow, superficial band at lateral midline
• Small diameter, slow twitch fibers, aerobic
• Extensive vascular supply with little
myoglobin and few mitochondria
• Fatigue resistant for continuous swimming
Cardiovascular System: Anatomy
• Heart
– Situated between left and right branchial cavities, anterior to
transverse septum and below esophagus
Zebrafish, Midline Sagittal Section
Posterior
Kidney
Anterior
Kidney
Brain
Pharynx
Transverse
Septum
Testis
Liver
Proximal Intestine
Liver
HEART
Cardiovascular System: Anatomy
•
Heart: 2 chambers, 4 compartments
1. Sinus venosus
• Analogous to cranial vena cava
• Sinoatrial valve
2. Atrium
• Thin-walled
• Atrioventricular valve
3. Ventricle
Hirata et al 2003
A
4. Bulbus arteriosis
SV
BA
V
ZEBRAFISH HEART
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Cardiovascular System: Anatomy
•
Heart: 2 chambers, 4 compartments
1. Sinus venosus
• Analogous to cranial vena cava
• Sinoatrial valve
2. Atrium
• Thin-walled
• Atrioventricular valve
3. Ventricle
• Trabecular meshwork
• Spongy outer compact layer
• Coronary arteries
• Bulboventricular valve
4. Bulbus arteriosis
Hirata et al 2003
A
SV
BA
V
ZEBRAFISH HEART
Cardiovascular System: Anatomy
•
Heart: 2 chambers, 4 compartments
1. Sinus venosus
• Analogous to cranial vena cava
• Sinoatrial valve
2. Atrium
• Thin-walled
• Atrioventricular valve
3. Ventricle
• Trabecular meshwork
• Spongy outer compact layer
• Coronary arteries
• Bulboventricular valve
4. Bulbus arteriosis
• Analogous to ascending aorta
• Fibroelastic tissue, smooth muscle
• Functions as a capacitor to
maintain continuous level of
blood supply to the gills
Hirata et al 2003
A
SV
BA
V
ZEBRAFISH HEART
Cardiovascular System: Anatomy & Physiology
• Arteries and Veins
– Ventral and dorsal aortas
• Pre- and post-gills, respectively
• Hepatic Portal System
– Venous blood from intestines
enters liver
– Part of enterohepatic circulation
• Renal Portal System
– Venous blood from caudal
musculature enters kidney
– Consideration for substances
injected in caudal half of fish
• Lymphatic System
– Only recently demonstrated in
zebrafish
– No lymph nodes
Diagram courtesy of Susan Knowles
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Hematologic System: Anatomy
• Hematopoiesis
–
–
–
–
Primarily within renal interstitium
Spleen to lesser extent
No bone marrow
Thymus  T-lymphocyte maturation
• Hematologic Cell Types:
– Erythrocytes
• Nucleated
• Aerobic metabolism
– Thrombocytes
• Nucleated
– Leukocytes:
•
•
•
•
Lymphocyte
Monocyte / macrophage
Neutrophilic granulocyte
Eosinophilic granulocyte
–
Combined eosinophil / mast cell?
Carradice D , Lieschke G J Blood 2008;111:3331-3342
Hematologic System: Anatomy
• Renal Hematopoietic Tissue
– Sinusoidal reticuloendothelial tissue
– Systemic inflammation  leukocyte precursors become more
abundant and expand the renal interstitium
Zebrafish, Midline Sagittal Section
Anterior
Kidney
Posterior
Kidney
Hematologic System: Anatomy
• Renal Hematopoietic Tissue
– Sinusoidal reticuloendothelial tissue
– Systemic inflammation  leukocyte precursors become more
abundant and expand the renal interstitium
Zebrafish, Midline Sagittal Section
Anterior
Kidney
Posterior
Kidney
T
T
T
T
T
T
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Hematologic System: Anatomy
• Spleen
–
–
–
–
–
Located left mid-abdomen below swim bladder
Very thin capsule
Ellipsoids (periarteriolar macrophages sheaths)
Red pulp >> white pulp
Lymphoid follicles rare
Anterior
Kidney
Swim Bladder
Esophagus
SPLEEN
Ovary
Heart
Liver
Intestines
Zebrafish, Left Parasagittal Section
Hematologic System: Anatomy
• Spleen
–
–
–
–
–
Located left mid-abdomen below swim bladder
Very thin capsule
Ellipsoids (periarteriolar macrophages sheaths)
Red pulp >> white pulp
Lymphoid follicles rare
Anterior
Kidney
Swim Bladder
Esophagus
ellipsoids
Ovary
Heart
Liver
Intestines
Zebrafish, Left Parasagittal Section
Hematologic System: Anatomy
• Thymus
–
–
–
–
Paired, located in dorsomedial branchial cavity
No obvious cortex and medulla in adult zebrafish
Doesn’t usually involute
Lymphocytes, macrophages, epithelial cells
THYMUS
Brain
Eye
Liver
Gills
Zebrafish, Left Parasagittal Section
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Hematologic System: Anatomy
• Thymus
–
–
–
–
Paired, located in dorsomedial branchial cavity
No obvious cortex and medulla in adult zebrafish
Doesn’t usually involute
Lymphocytes, macrophages, epithelial cells
Brain
Eye
Liver
Gills
Zebrafish, Left Parasagittal Section
Hematologic System: Anatomy
• Thymus
–
–
–
–
Paired, located in dorsomedial branchial cavity
No obvious cortex and medulla in adult zebrafish
Doesn’t usually involute
Lymphocytes, macrophages, epithelial cells
Brain
Eye
Liver
Gills
Respiratory System: Anatomy
• Gills
–
–
–
–
Bilateral branchial chambers
Each chamber contains four sets of cartilaginous gill arches
Filaments (primary lamellae) branch off arches at right angles
Lamellae (secondary lamellae) branch of filaments at right angles
Brain
Eye
Liver
GILLS
Zebrafish, Left Parasagittal Section
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Respiratory System: Anatomy
• Gills
–
–
–
–
Gill Arch
Bilateral branchial chambers
Each chamber contains four sets of cartilagenous gill arches
Filaments (primary lamellae) branch
off arches at right angle
Filaments
Lamellae (secondary lamellae) branch of filaments at right angle
Brain
Lamellae
Eye
Zebrafish, Left Parasagittal Section
Respiratory System: Anatomy
• Lamellar Cells:
–
–
–
–
–
Pavement (epithelial) cells: single cell layer
Endothelial cells: line capillaries
Pillar cells: support walls of capillaries
Chloride cells: maintain ionic balance
Mucous cells: increase with irritation
Respiratory System: Anatomy
• Pseudobranch
– Reduced first branchial arches
– Bilateral, located craniodorsal to branchial chamber
– Not respiratory per se, no direct contact with external
environment
– Countercurrent system for supplying and regulating oxygenated
blood to the eyes
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Respiratory System: Anatomy
• Pseudobranch
– Reduced first branchial arch
– Bilateral, located craniodorsal to branchial chamber
– Not respiratory per se, no direct contact with external
environment
– Countercurrent system for supplying and regulating oxygenated
blood to the eyes
Respiratory System: Physiology
• Gill Functions:
– Gas exchange
– Excretion of nitrogenous wastes
• Responsible for 80% of
total nitrogen elimination
– Osmoregulation
– Acid-base balance
Evans et al 1999
Digestive System: Anatomy
• Components:
–
–
–
–
–
–
Oral and Pharyngeal Cavities
Esophagus
Intestines
Liver and Gallbladder
Pancreas
Swim Bladder
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Digestive System: Anatomy
• Oral and Pharyngeal
Cavities
– No oral teeth
– Pharyngeal teeth and pad
Zebrafish, Midline Sagittal Section
Digestive System: Anatomy
• Oral and Pharyngeal
Cavities
– No oral teeth
– Pharyngeal teeth and pad
Zebrafish, Midline Sagittal Section
Digestive System: Anatomy
• Esophagus
– Abundant mucous cells
– Highly distensible
Esophagus
Proximal
Intestine
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Digestive System: Anatomy
• Esophagus
– Abundant mucous cells
– Highly distensible
Esophagus
Proximal
Intestine
Digestive System: Anatomy
• Intestines
–
–
–
–
–
Morphology changes little along its length
No glandular stomach
Lumen and folds become smaller caudally
Columnar enterocytes and goblet mucous cells
Relatively thin muscularis, submucosa barely evident
with few resident leukocytes in healthy gut
Digestive System: Anatomy
• Intestines
–
–
–
–
–
Morphology changes little along its length
No glandular stomach
Lumen and folds become smaller caudally
Columnar enterocytes and goblet mucous cells
Relatively thin muscularis, submucosa barely evident
with few resident leukocytes in healthy gut
Zebrafish, Midline Sagittal Section
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Digestive System: Anatomy
• Intestines
–
–
–
–
–
Morphology changes little along its length
No glandular stomach
Lumen and folds become smaller caudally
Columnar enterocytes and goblet mucous cells
Relatively thin muscularis, submucosa barely evident
in healthy gut
Zebrafish, Midline Sagittal Section
Digestive System: Anatomy
Glandular Stomach of Winter Flounder
Digestive System: Anatomy
• Liver
– Multi-lobed in cyprinids: 3 lobes in zebrafish
Anterior
Kidney
Swim Bladder
Esophagus
Ovary
Heart
LIVER
Intestines
Zebrafish, Midline Sagittal Section
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Digestive System: Anatomy
• Liver
–
–
–
–
–
–
Multi-lobed in cyprinids: 3 lobes in zebrafish
Fish livers lack defined lobular architecture
Relatively few hepatic arterioles
Indistinct portal triads
No lymphatic vessels
No Kupffer cells
pt
Lobules not apparent in fish liver
Lobules in pig liver
Digestive System: Anatomy
• Liver
– Fish livers have tubules rather than hepatic plates
Schematic adapted from
Hinton et al., 2001
Digestive System: Anatomy
• Liver
– Fish livers have tubules rather than hepatic plates
Schematic adapted from
Hinton et al., 2001
Sinusoids
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Digestive System: Anatomy
• Liver
– Fish livers have tubules rather than hepatic plates
Schematic adapted from
Hinton et al., 2001
Bile
Ductules
Sinusoids
Digestive System: Anatomy
• Liver
– Fish livers have tubules rather than hepatic plates
Schematic adapted from
Hinton et al., 2001
Bile
Ductules
Canaliculi
Sinusoids
Digestive System: Anatomy
• Liver
– Fish livers have tubules rather than hepatic plates
Schematic adapted from
Hinton et al., 2001
Bile
Ductules
Canaliculi
Space of Disse
Sinusoids
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Digestive System: Anatomy & Physiology
• Liver
– Appearance of the liver varies by sex
• Reproductively active females have basophilic livers due to
increased production of vitellogenin
(a phospholipoglycoprotein required for egg yolk formation)
Adult Male Zebrafish
Adult Female Zebrafish
Digestive System: Physiology
• Liver
– Basic liver functions same as mammals
• Uptake, metabolism, storage, synthesis, and redistribution of
nutrients and other endogenous molecules
• Formation and excretion of bile
• Metabolism of xenobiotics
– Fish also have similar enzymes and metabolic
pathways for xenobiotics
• Phase I and Phase II biotransformation reactions
• Microsomal enzymes (mixed function oxidases)
– Unlike mammals
• Homogeneous distribution of biotransforming enzymes
• Therefore, no zonal patterns of toxicity
Digestive System: Anatomy
• Gallbladder
– Non-descript sac in mid abdomen
– Common bile duct is extrahepatic
Swim Bladder
Gallbladder
Ovary
Heart
Liver
Liver
Intestine
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Digestive System: Anatomy
• Gallbladder
– Non-descript sac in mid abdomen
– Common bile duct is extrahepatic
Swim Bladder
Gallbladder
Ovary
Ciliated
transitional epithelium
Liver
Heart
Liver
Intestine
Digestive System: Anatomy
• Exocrine Pancreas
– Not a discrete organ
• Islands of acinar tissue scattered throughout the
abdominal mesentery
– Otherwise, pancreas looks like pancreas
Digestive System: Anatomy
• Swim Bladder
– Located dorsal to abdominal viscera and ventral to
spine and kidneys
– Double-chambered in cyprinids like zebrafish
1
1
2
Zebrafish Swim Bladder
Medaka Swim Bladder
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Digestive System: Anatomy
• Swim Bladder
– Located dorsal to abdominal viscera and ventral to
spine and kidneys
– Double-chambered in cyprinids like zebrafish
– Two types of swim bladder configuration in fish:
• Physoclist (kleisein = to close)
– No connection between
esophagus and swim bladder
– Cichlids, killifish, seahorses,
most marine species
• Physostome – retained pneumatic duct
– Catfishes, characins, salmonids, cyprinids
Digestive System: Anatomy
• Swim Bladder
– Located dorsal to abdominal viscera and ventral to
spine and kidneys
– Double-chambered in cyprinids like zebrafish
2
1
1
2
Pneumatic
Duct
Goldfish
Swim Bladder
Zebrafish Swim Bladder
Digestive System: Anatomy
• Swim Bladder
– Pneumatic duct:
• Tortuous tube-like structure that connects esophagus and
swim bladder
Swim Bladder
Swim Bladder
Esophagus
Pneumatic
Duct
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Digestive System: Physiology
• Swim Bladder Functions (species dependent):
– Neutral buoyancy
– Generation of sounds
– Oxygen respiration
– Detection of sound waves or pressure changes
• Weberian Ossicles
Bang et al 2002
– Series of four tiny bones
facilitate transmission
of sound waves from swim
bladder to the inner ear
– Fish that have this apparatus
are classified as otophyseans
that
Urinary System: Anatomy
Ontogeny of the Urinary System
•
•
Pronephros
•
•
Mesonephros
•
•
Opisthonephros
•
Metanephros
•
Regresses in higher
vertebrates and most fish
Present in juvenile fish
and adult guppies
One nephron
Regresses in higher
vertebrates
Adult fish kidney
10 – 50 nephrons
Adult kidney in higher
vertebrates (amniotes)
One million nephrons
Urinary System: Anatomy
• Kidneys
– Retroperitoneal, ventral to vertebrae and dorsal to swim bladder
– Left and right conjoined; anterior, posterior and tail segments
Gerlach et al 2011
– Anterior kidney has urinary components
• Not purely hematopoietic as in some
other fishes
– Renal portal system
– No cortex and medulla, no loop of Henle
Vertebrae
Posterior Kidney
Anterior Kidney
Swim Bladder
Swim
Bladder
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Urinary System: Anatomy
• Kidneys
– Glomeruli are variably-sized and sparse
– Distinctive vacuolated tubular epithelium
Glomeruli
Vacuolated Tubules
Urinary System: Anatomy
• Vacuolated renal tubular epithelium
– Proximal tubule vs. collecting duct?
PTII
CD
Diep et al., 2011
Urinary System: Anatomy
• Vacuolated renal tubular epithelium
– Proximal tubule vs. collecting duct?
Vacuolated Tubules
Collecting Duct
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Urinary System: Anatomy
• Vacuolated renal tubular epithelium
– Do not diagnose this as a lesion!
Vacuolated Tubules
Collecting Duct
Urinary System: Anatomy
• Opisthonephric duct (ureter)
– Ciliated columnar epithelium
– No urinary bladder
Posterior Kidney
Opisthonephric Duct
Swim Bladder
Vent
Urinary System: Anatomy
• Opisthonephric duct (ureter)
– Ciliated columnar epithelium
– No urinary bladder
Posterior Kidney
Swim Bladder
Vent
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Urinary System: Physiology
• The gill is the principal site for
excretion of nitrogenous wastes
• The primary task of the adult fish
urinary system is:
osmoregulation
• Freshwater fish such as
zebrafish are hypertonic
relative to their surroundings
• Consequently, they must continuously
excrete dilute urine in order to avoid
systemic edema
– Active uptake of NaCl ions by gills
– Resorption of 95% of NaCl by renal
tubules
Reproductive System: Anatomy
• Functional unit of the ovary is the follicle
– Follicle = oocyte + granulosa and theca cells
• Ovulation occurs into an ovarian cavity
• Ovarian wall contiguous with oviduct
– No true uterus
• Oogonia continue to
divide in adults
Oocyte
Follicle
Reproductive System: Anatomy
• Follicles become larger with maturity
• Follicular phases:
– Perinucleolar
• Small, dense
cytoplasm
– Cortical alveolar
• Lipid vacuoles
– Vitellogenic
• Red globules
= yolk
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Reproductive System: Anatomy
• Follicles become larger with maturity
• Follicular phases:
– Perinucleolar
• Small, dense
cytoplasm
– Cortical alveolar
• Lipid vacuoles
– Vitellogenic
• Red globules
= yolk
Reproductive System: Anatomy
• Follicles become larger with maturity
• Follicular phases:
– Perinucleolar
• Small, dense
cytoplasm
– Cortical alveolar
• Lipid vacuoles
– Vitellogenic
• Red globules
= yolk
Reproductive System: Anatomy
• Functional unit of the testis is the spermatocyst
– Clonal group of germ cells surrounded by cytoplasmic
arms of a Sertoli cell
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Reproductive System: Anatomy
• Functional unit of the testis is the spermatocyst
– Clonal group of germ cells surrounded by cytoplasmic
arms of a Sertoli cell
• Two types of seminiferous tubule organization:
– Restricted Type
• Spermatocysts form only at one
end of the tubule, and migrate
toward the efferent duct
• Medaka, e.g.
– Unrestricted Type
• Spermatocysts form all along the
length of the tubule, and sperm
are discharged into a lumen
• Zebrafish, e.g.
Reproductive System: Anatomy
• Testis
– Paired structures, mid to caudal abdomen beneath swim bladder
Posterior Kidney
Swim
Bladder
Swim
Bladder
TESTIS
Liver
Liver
Reproductive System: Anatomy
• Testis
– Paired structures,
Sertoli mid to caudal abdomen beneath swim bladder
cell Kidney
Posterior
spermatocytes
spermatogonia
Swim
Bladder
Leydig
cell
Liver
Seminiferous
Tubule
spermatids
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Reproductive System: Anatomy
Ductus Deferens
Reproductive System: Physiology
• Sex Determination
– Zebrafish are gonochoristic
HPGL Axis
• Maintain same gender throughout adult
in Teleosts
life
• However, all zebrafish are phenotypically
female until ~3 wks post-fertilization
• Trigger for male phenotype may be
multigenic (i.e. no single chromosome)
• Phenotypic sex in larval fish can be
affected by temperature, pH, or exposure
to exogenous hormones
• HPGL Axis
– Hormonal control of reproduction in
adult fish is similar to that of mammals
• GTH-I is homologous to FSH
• GTH-II is homologous to LH
Endocrine System: Anatomy
•
•
•
•
•
•
•
•
•
Pituitary Gland
Pineal Gland
Urophysis
Corpuscle of Stannius
Ultimobranchial Body
Islets of Langerhans
Interrenal Tissue
Chromaffin Tissue
Gonads
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Endocrine System: Anatomy
• Pituitary Gland
– Floor of diencephalon, beneath hypothalamus
– Almost entirely encased in bone in zebrafish
Zebrafish, Midline Sagittal Section
Endocrine System: Anatomy
• Pituitary Gland
– Floor of diencephalon, beneath hypothalamus
– Almost entirely encased in bone in zebrafish
PITUITARY GLAND
Zebrafish, Midline Sagittal Section
Endocrine System: Anatomy
Medaka Pituitary Gland
Neurohypophysis
Optic nerves
Adenohypophysis
Adenohypophysis (prolactin cells)
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Endocrine System: Anatomy
Zebrafish Pituitary Gland
Anterior Pars Nervosa
Posterior Pars Nervosa
Pars Distalis
Pars Intermedia
Endocrine System: Physiology
• Pituitary Gland
– “The Master Gland”
– Produces a variety of hormones
that control a number of other
endocrine glands
• H-P-Thyroid Axis
• H-P-Interrenal (adrenal) Axis
• H-P-Gonadal Axis
– Positive and negative feedback loops
– No hypothalamic-hypophyseal portal
vascular system in fish
• Pituitary receives messages from
hypothalamus via neurons
Endocrine System: Physiology
• Pituitary Gland
– Major hormones produced:
• Thyroid Stimulating Hormone (TSH)
– Stimulates synthesis and release of thyroid hormones, and
iodine uptake by thyroid follicular cells
• Gonadotropic Hormones (GTH-I, GTH-II)
– Analogous to mammalian FSH and LH, respectively
– Regulates gonadal development and cyclicity
• Growth Hormone (GH)
– Somatic growth and many other functions
• Prolactin
AquaBounty salmon
– Many functions, but predominant role is osmoregulatory
• Somatolactin
– Released from pars intermedia, many reported functions
• Adrenocorticotropin Hormone (ACTH)
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Pituitary Tumor Excision
in Fish
Pituitary Tumor Excision in
Humans
Endocrine System: Anatomy
• Pineal Gland
– Part of the dorsal (upper)
diencephalon (forebrain) area
called the “epithalamus”
– “Pineal Complex” = pineal gland
trout pineal transverse section
plus parapineal organ
– Pineal gland located along the midline,
parapineal is asymmetrical on left side
– Extends from the brain to the dorsal skull
– Anatomy varies markedly among fish
species
– Part gland, part stalk (tube-like structure),
Concha and Wilson, 2001
par sac
po = preoptic area
par = paraphysis
– Lumen lined by ependymal-like cells, may
sd = dorsal sac
communicate with 3rd ventricle of the brain
pc = posterior commisure
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Endocrine System: Physiology
• Pineal Gland
– Neurosensory component
• Pinealocytes are photoreceptor cells that
detect light transmitted through
translucent areas of the dorsal skull
GFP Labeling of
Zebrafish
Pineal Gland
– Hormonal component: Melatonin
• Indole amine secreted mainly during dark
phase of day
• Involved with synchronization of
physiological and behavioral activities
with photoperiod and temporal factors
(i.e., time of day and season)
• Affects growth, reproduction, migration,
metabolic rate, skin pigmentation
Gothilf et al., 2002
Endocrine System: Anatomy
• Urophysis
–
–
–
–
–
Organ unique to jawed fishes
Ventral swelling at the posterior end of the spinal cord
Structurally similar to the pituitary neurohypophysis
Forms a “neuro-hemal complex”
Not often examined histologically (not by me, anyway)
Endocrine System: Physiology
• Urophysis
– “Caudal Neurosecretory System”
– Major hormones produced:
• Urotensin I
– Structurally similar to corticotropin-releasing hormone
•
Urotensin II
– Structurally similar to somatostatin
– Primary functions of urotensins:
•
•
•
•
Stress response
Osmoregulation
Neural transmitters in brain?
Urotensin II present in mammals, and has vasoactive
effects when administered experimentally
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Endocrine System: Anatomy
• Corpuscle of Stannius
– Organ unique to teleost and holostean fishes
– Embedded in the tail region of the posterior kidney
– Paired, discrete, encapsulated structures consisting of folded
cords of cells
– Do not diagnose these as adenomas!
Ostrander, 2000
Vertebrae
Kidney
Swim
Bladder
Kidney
Intestine
Zebrafish, Midline Sagittal Section
Endocrine System: Anatomy
• Corpuscle of Stannius
– Organ unique to teleost and holostean fishes
– Embedded in the tail region of the posterior kidney
– Paired, discrete, encapsulated structures consisting of folded
cords of cells
– Do not diagnose these as adenomas!
Ostrander, 2000
Vertebrae
Kidney
Swim
Bladder
Kidney
Intestine
Zebrafish, Midline Sagittal Section
Endocrine System: Physiology
• Calcium regulation in fishes
– Fish have no parathyroid gland, no thyroid C-cells
– Corpuscles of Stannius
• Stanniocalcin, Teleocalcin: reduce plasma calcium
by decreasing Ca++ absorption via gills and
intestine
– Ultimobranchial body
• Calcitonin: reduces plasma calcium via increased
renal excretion and sequestration into bone (?)
– Hypophysis
• Parathormone: increases plasma calcium
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Endocrine System: Anatomy & Physiology
• Ultimobranchial Body
– Single midline organ, located within the transverse membrane
that separates the pericardial cavity from the abdominal cavity
– Very small, often difficult to capture in histologic sections
– Pseudorosettes with basally-oriented nuclei
Anterior
Kidney
Brain
Pharynx
Heart
Liver
Zebrafish, Midline Sagittal Section
Endocrine System: Anatomy & Physiology
• Ultimobranchial Body
– Single midline organ, located within the transverse membrane
that separates the pericardial cavity from the abdominal cavity
– Very small, often difficult to capture in histologic sections
– Pseudorosettes with basally-oriented nuclei
Anterior
Kidney
Brain
Pharynx
Heart
Liver
Zebrafish, Midline Sagittal Section
Endocrine System: Anatomy & Physiology
• Ultimobranchial Body
– Single midline organ, located within the transverse membrane
that separates the pericardial cavity from the abdominal cavity
– Very small, often difficult to capture in histologic sections
– Pseudorosettes with basally-oriented nuclei
– Analogous to the
Anterior
C-cells of the
Kidney
mammalian thyroid
gland
Brain
– Major hormone
produced: Calcitonin
Pharynx
Heart
Liver
Zebrafish, Midline Sagittal Section
• Reduces plasma
calcium
• May also have a role
in regulation of fluids,
electrolytes, and other
minerals
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Endocrine System: Anatomy
• Interrenal and Chromaffin Tissue
–
–
–
–
Analogs of adrenal cortex and adrenal medulla, respectively
Cellular aggregates located within anterior kidney
Surround branches of the dorsal posterior cardinal veins
Interrenal and chromaffin tissues intermingled in zebrafish
Anterior
Kidney
Cardinal
Vein
Ostrander, 2000
Endocrine System: Anatomy
• Interrenal and Chromaffin Tissue
–
–
–
–
Analogs of adrenal cortex and adrenal medulla, respectively
Cellular aggregates located within anterior kidney
Surround branches of the dorsal posterior cardinal veins
Interrenal and chromaffin tissues intermingled in zebrafish
Ostrander, 2000
Endocrine System: Anatomy
• Interrenal and Chromaffin Tissue
–
–
–
–
Analogs of adrenal cortex and medulla, respectively
Cellular aggregates located within anterior kidney
Surround branches of the dorsal posterior cardinal veins
Interrenal and chromaffin tissues intermingled in zebrafish
– H&E sections:
• Chromaffin cells
have pale or clear
cytoplasm
• Interrenal cells
have pink cytoplasm
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Endocrine System: Physiology
• Interrenal Tissue (adrenal cortex analogue)
–
Major hormones produced: Corticosteroids
• Cortisol primarily, but also corticosterone,
deoxycorticosterone, and aldosterone
• Cortisol also functions as mineralocorticoid in fish
• Functions in stress response similar to other
vertebrates
STRESS
RESPONSE
• Chromaffin Tissue (medulla analogue)
–
Major hormones produced: Catecholamines
• Epinephrine (adrenaline) and norepinephrine, and
lesser amounts of dopamine
• Also part of stress response
• Role in regulating gill blood flow and oxygen uptake
Ostrander,
2000
Endocrine System: Anatomy
• Pancreatic Islets of Langerhans
– Located within patches of exocrine (acinar) pancreas, which
are scattered throughout the abdominal mesentery
– Discrete organs with fine capsules, consisting of sheets or
tightly folded cords of cells
– Single, large islets are called “Brockman Bodies”
Zebrafish, Midline Sagittal Section
Endocrine System: Anatomy
• Pancreatic Islets of Langerhans
– Located within patches of exocrine (acinar) pancreas, which
are scattered throughout the abdominal mesentery
– Discrete organs with fine capsules, consisting of Liver
sheets or
tightly folded cords of cells
– Single, large islets are called “Brockman Bodies”
Endocrine
Pancreas Exocrine
Pancreas
Intestine
Zebrafish, Midline Sagittal Section
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7/27/2012
Endocrine System: Physiology
• Pancreatic Islets of Langerhans
– Roles in amino acid metabolism in fish, and possibly
carbohydrate metabolism
– Major hormones produced:
• Alpha cells: Glucagon-like Peptide  promotes
hyperglycemia and gluconeogenesis
• Beta cells: Insulin  promotes hypoglycemia
• Delta cells: Somatostatin (SRIF)  inhibits release
of insulin and glucagon
• Pancreastatin  inhibits insulin
Endocrine System: Anatomy
• Thyroid Glands
– Higher vertebrates: paired, discrete, encapsulated structures in the neck
region (also in elasmobranchs and some teleosts)
– Zebrafish and most other fish: loosely distributed follicles located
ventral and caudal to the gill chamber, around the ventral aorta
– However, cells with the potential to form thyroid follicles can be
found almost anywhere in the fish
– Each roughly spherical follicle consists of a single layer of cuboidal
epithelial cells that surround a colloid-filled lumen
Endocrine System: Anatomy
• Thyroid Glands
– Higher vertebrates: paired, discrete, encapsulated structures in the
neck region (also in elasmobranchs and some teleosts)
– Zebrafish and most other fish: loosely distributed follicles located
ventral and caudal to the gill chamber, around the ventral aorta
– However, cells with the potential to form thyroid follicles can be
found almost anywhere in the fish
– Each roughly spherical follicle consists of a single layer of cuboidal
epithelial cells that surround a colloid-filled lumen
38
7/27/2012
Endocrine System: Anatomy
• Thyroid Glands
– Higher vertebrates: paired, discrete, encapsulated structures in the
neck region (also in elasmobranchs and some teleosts)
– Zebrafish and most other fish: loosely distributed follicles located
ventral and caudal to the gill chamber, around the ventral aorta
– However, cells with the potential to form thyroid follicles can be
found almost anywhere in the fish
– Each roughly spherical follicle consists of a single layer of cuboidal
epithelial cells that surround a colloid-filled lumen
Endocrine System: Physiology
• Thyroid Glands
– Functionally similar to thyroids of
higher vertebrates
– Major hormone produced: T4
• Converted peripherally to T3
• Colloid formation is iodine dependent
• Thyroid hormone receptors act as
transcription factors
– T4 in fish has roles in:
• Growth and metabolism
• Gonadal effects
• Pigment deposition
– Permissive for expression of
growth hormone activity
Ostrander, 2000
Endocrine System: Physiology
• Non-classical Endocrine Tissues
– Ovaries and Testes: Estrogens,
Androgens, and Progestins
– Gastrointestinal Tract: Gastrin,
Secretin
– Heart: Atrial Natriuretic Peptide
– Kidney: Erythropoietin, Renin
– Liver: Insulin-like Growth Factor,
Angiotensin II
– Lymphocytes: Cytokines,
Interleukins
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Neurologic and Sensory Systems: Anatomy
• Brain
– Brain Regions:
•
•
•
•
•
•
Olfactory Lobe
Telencephalon: cerebrum
Mesencephalon: optic tectum
Metencephalon: cerebellum
Myelencephalon: medulla
Diencephalon: hypothalamus
– Fish compared to mammals
• Dominance of optic cortex
• Reduction of cerebrum
• Gray and white matter tracts less distinctly separated
Neurologic and Sensory Systems: Anatomy
Dr Hideo Otsuna, University of Utah Medical Center,
Department of Neurobiology and Anatomy, Salt Lake City, Utah, USA.
5-day old zebrafish head, (20x). Confocal
Neurologic and Sensory Systems: Anatomy
• Spinal Cord
– Spinal cord white matter tends to be more loosely
fibrillar in fish than in mammals
Image courtesy of S. Frasca
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7/27/2012
Neurologic and Sensory Systems: Anatomy
• Spinal Cord
– Spinal cord white matter tends to be more loosely
fibrillar in fish than in mammals
– Especially true in zebrafish that are infected with
microsporidia
Neurologic and Sensory Systems: Anatomy
Gray et al 2009
• Eyes
– Cornea thicker and flatter in fish
• Doesn’t refract much
– Minimal anterior chamber
– Spherical lens
• Maximum refractility, little elasticity, no ability to accommodate
– Zebrafish sclera has disc-shaped bony ossicles
Zebrafish Lens
Zebrafish, Left Parasagittal Section
Neurologic and Sensory Systems: Anatomy
• Eyes
– Retina
• Comparable layers
• Color & UV vision
• Bi- and tri-nucleate
cone cells
Optic Nerve
Ganglion
Cells
Zebrafish, Left Parasagittal Section
Rods &
Cones
Bipolar
Cells
Choroid
Rete
Pigmented
Epithelium
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7/27/2012
Neurologic and Sensory Systems: Anatomy
•
Statoacoustic Organ (Inner Ear)
-- 3 Semicircular Canals:
1.
2.
3.
Anterior
Posterior
Horizontal/Lateral
Pars Superior  Spatial Orientation
-- 3 Otolith Organs:
1.
2.
3.
Utricle
Saccule
Lagena
Pars Inferior  Hearing (no cochlea)
(no external or middle ear either)
Zebrafish, Left Parasagittal Section
Neurologic and Sensory Systems: Anatomy
•
Statoacoustic Organ (Inner Ear)
-- 3 Semicircular Canals:
1.
2.
3.
Anterior
Posterior
Horizontal/Lateral
Pars Superior  Spatial Orientation
-- 3 Otolith Organs:
1.
2.
3.
Utricle
Saccule
Lagena
Pars Inferior  Hearing (no cochlea)
(no external or middle ear either)
Zebrafish, Left Parasagittal Section
Neurologic and Sensory Systems: Anatomy
•
Statoacoustic Organ (Inner Ear)
-- 3 Semicircular Canals:
1.
2.
3.
Anterior
Posterior
Horizontal/Lateral
-- 3 Otolith Organs:
1.
2.
3.
Utricle
Saccule
Lagena
Pars Superior  Spatial Orientation
otolith
Pars Inferior  Hearing (no cochlea)
hair cells
macula
Zebrafish, Left Parasagittal Section
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Neurologic and Sensory Systems: Anatomy
• Olfactory Tissues
– Nares
• Parasagittal location
• Squamous and ciliated
columnar epithelium
Zebrafish, Left Parasagittal Section
Neurologic and Sensory Systems: Anatomy
• Olfactory Tissues
– Nares
• Parasagittal location
• Squamous and ciliated
columnar epithelium
Zebrafish, Left Parasagittal Section
Neurologic and Sensory Systems: Anatomy
• Olfactory Tissues
– Olfactory Organ
• Paired, parasagittal
• Olfactory epithelium
• Specialized bipolar cells
Zebrafish, Left Parasagittal Section
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7/27/2012
Neurologic and Sensory Systems: Anatomy
• Lateral Line System
– Linear dermal canals
encased in bone
– Extend along left and right
sides
– Pores allow contact with
external water
– Neuromasts: sensory
hair cell receptors that
synapse with afferent
nerve fibers
• Sensitive to water
movement
Images courtesy of S. Frasca
References
Acknowledgments (unwittingly donated images)
•
•
•
•
•
•
•
Al Camus
Jack Fournie
Sal Frasca
Dave Hinton
Susan Knowles
Mac Law
Marilyn Wolfe
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Questions?
45