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invited review
‘‘Physiological genomics’’: mutant screens in zebrafish
KERRI S. WARREN AND MARK C. FISHMAN
Cardiovascular Research Center, Massachusetts General Hospital, Charlestown 02129;
and Harvard Medical School, Boston, Massachusetts 02115
mutagenesis; embryonic physiology; embryonic cardiovasculature; vertebrate genetic screen
BY THE YEAR 2000, we will have the sequences of most of
the transcribed regions in the human and mouse
genomes, but we still will lack a grasp of their functions. One approach to the investigation of function
might be to selectively eliminate genes in mice one by
one. However, such grand-scale targeted mutagenesis
programs would be inordinately expensive. In addition,
mouse embryos with severe cardiac malfunction die
very early (embryonic days 9–14, see Ref. 56), making
functional analysis difficult, if not impossible. An alternative is large-scale mutagenesis screening in a species
less dependent on cardiovascular development for early
embryonic survival, using screens focused on phenotypes of interest followed by identification of the mutant gene. The key to success in such studies is a
sophisticated eye and a clever assay, exemplified by the
genetic screens in Drosophila, which leveraged an
entire field of developmental biology focused on generation of body plan and pattern (60). In principal, assays
of physiology or behavior might serve as the phenotypic
targets. This has been successful as part of the hunt for
genes involved in circadian rhythmicity (18, 41, 69).
Hence, mutagenesis screens start with a biologically
significant and specifically interesting defect and move
to the hunt for the affected gene, without making
assumptions about molecule, cell, or tissue type.
Thanks to considerable evolutionary conservation,
the studies in Drosophila have shed light on homologous genes with essential roles in vertebrate development. But when questions are asked about the physiology of a process particular to vertebrates, information
gleaned from the fly, worm, and yeast will be indirect.
Mutagenesis screens in vertebrates are needed to resolve determinants and modulators of vertebrate-
specific structure, function, and behavior. Recently, this
has been accomplished in the tropical freshwater fish,
the zebrafish Danio rerio (19, 20, 31). Here, we review
this system, focusing on the set of mutations discovered
that reveal unitary aspects of the function of the
cardiovascular system.
ADVANTAGES OF ZEBRAFISH
Recent technological advances have made the measure of many cardiovascular functions feasible even in
minute animals and embryos (6). Pulsed Doppler technology is being used for blood flow velocity, impedance
measurement for heart rate or change in blood flow,
high-resolution videomicroscopy for stroke volume calculations, and servo-null micropressure techniques for
blood pressure in many animal models, including chick,
rat, and mouse embryos (37, 38, 48), as well as frog and
fish larvae (32, 33, 51, 52). Details on these and other
attributes of the developing cardiovascular system of
amphibians, fish, reptiles, birds, and mammals are well
documented (7).
There are many advantages for using zebrafish for
assaying cardiovascular function in the embryo. Their
embryos are transparent and can be monitored without
being removed from their natural environment. Heart
rate, oxygen consumption, and blood pressure all have
been assayed in developing wild-type embryos (Fig. 1;
see Ref. 52). In the zebrafish, the cardiovasculature is
functional at 24 h postfertilization, but it is not essential for survival of the early embryo, which obtains
adequate oxygen by simple diffusion (8, 52, 62, 63).
Thus zebrafish mutants that completely lack a circulation still develop relatively normally for a day or two.
This distinguishes fish embryos from mammalian em-
0363-6135/98 $5.00 Copyright r 1998 the American Physiological Society
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Warren, Kerri S., and Mark C. Fishman. ‘‘Physiological genomics’’:
mutant screens in zebrafish. Am. J. Physiol. 275 (Heart Circ. Physiol. 44):
H1–H7, 1998.—Large-scale mutagenesis screens have proved essential
in the search for genes that are important to development in the fly,
worm, and yeast. Here we present the power of large-scale screening in a
vertebrate, the zebrafish Danio rerio, and propose the use of this genetic
system to address fundamental questions of vertebrate developmental
physiology. As an example, we focus on zebrafish mutations that reveal
single genes essential for normal development of the cardiovascular
system. These single gene mutations disrupt specific aspects of rate,
rhythm, conduction, or contractility of the developing heart.
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INVITED REVIEW
All the stages of zebrafish heart and vasculature
development mentioned above can be appreciated when
viewed through a dissecting microscope and observed
at the single cell level with a compound microscope. The
zebrafish heart and vasculature are thus well suited for
screens aimed at identifying developmental disorders.
THE BIG SCREENS
bryos, whose survival depends on an intact circulation,
so that mutations with cardiovascular effects are inevitably complicated by secondary degeneration or death.
NORMAL ZEBRAFISH
CARDIOVASCULAR DEVELOPMENT
The earliest stages of heart formation in zebrafish
are quite similar to those of the human heart during
the first 3 wk of gestation, from cardiac specification to
the formation of a two-chambered looped heart (22).
The zebrafish heart forms by fusion of bilateral precardiac populations of the lateral plate mesoderm. Myocardial primitive heart tubes fuse and enclose the endocardial cells to create the bilayered definitive heart tube at
the midline (64). The atrial end of this tube juts out
from under the neural tube on the animal’s left side as
unidirectional peristaltic waves propel blood through
the first elements of the circulatory system by 24 h of
development. The vasculature at this time encloses a
simple circulatory loop; the blood travels from the heart
to the trunk and tail via the first aortic arches and the
paired dorsal aortas, and it returns by way of the axial
vein and the Ducts of Cuvier (for review see Ref. 74).
During the next few hours, the heart swings around to
the midline of the embryo over the yolk, and the
ventricle loops rightward as cardiac activity gradually
changes from a peristaltic wave to sequential contractions of the atrium and ventricle. By 36 h, cardiac
looping is complete, each chamber has a distinct contractile pulse, and the circulation now extends to the head.
Valve morphogenesis follows and cushions become obvious between the chambers by 48 h of development (63).
Fig. 2. Schematic outline of zebrafish F2 mutagenesis screens.
N-Ethyl-N-nitrosourea (ENU) was used to mutagenize spermatogonia of G0 males. Outcrosses were performed with wild-type females to
produce F1 generation, each F1 fish possessing a unique set of
mutations. Sibling F1 matings created the F2 generation, and the
mutations were driven to homozygosity in the F3 embryos. See text
and Refs. 20 and 31 for details.
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Fig. 1. Blood pressure recorded in the day 3.5 to day 4 zebrafish
embryo using a servo-null micropressure system. [Reproduced from
Pelster and Burggren (52) with permission from Circ. Res. 79:
358–362. Copyright 1996, American Heart Association.]
The real advantage of zebrafish for genetic research
is the ease with which one can carry out large-scale
screens. Two classic diploid F2 screens were recently
completed in Boston and Tübingen (20, 31) (Fig. 2).
N-Ethyl-N-nitrosourea, which creates point mutations
in the spermatogonia of adult G0 males, was titered to
generate one to two recessive lethal mutations per
haploid genome. Mutagenized males were bred with
wild-type females, and their offspring (F1 ) interbred to
yield heterozygotic carriers of the mutations (F2 ). The
phenotypes of the recessive mutations were revealed in
the F3 generation embryos, in which the mutated gene
was driven to homozygosity. The F3 egg clutches were
examined at five stages of development (during the first
6–12 h and on the first, second, third, and fifth days
after fertilization) for any signs of abnormal development and deemed a single gene, recessive mutation if
evident in 25% of the growing embryos and larvae.
Approximately one-half of the mutations from the
screens were discarded as ‘‘uninformative’’ because
they caused a general retardation, deterioration, or
brain necrosis. A total of 1,858 mutations were kept for
further characterization; 1,225 were analyzed for
complementation and, although complementation test-
INVITED REVIEW
ing between the two groups has not been completed,
these represent more than 500 genetic loci. The mutations affect an impressively large range of targets: eye,
pigment, kidney, notochord, muscle, brain, and fins, to
name a few. Although many pleiotropic mutations with
early-onset phenotypes also perturb heart shape, size,
and function, a combined total of 156 mutations were
identified, which primarily affect the heart, vasculature, and blood (10, 55, 63, 75).
GENES AFFECTING DEVELOPMENT
OF CARDIOVASCULAR FORM
WHAT ARE THE KEY STEPS IN THE MATURATION
OF CARDIOVASCULAR FUNCTION?
One opportunity created by these screens is to use
zebrafish to understand the development of normal
physiological control. What are the ‘‘units’’ of physiological function? One might be global heart size. It is
known that heart size, rate, and cardiac output match
the size of the vertebrate embryo (34, 38, 47). Increases
in body mass during development are also closely
correlated with increases in blood pressure (8, 13, 47,
51, 66). What genes are involved in matching the size of
the heart to general metabolic needs and overall body
size? Injection of the transcription factor Nkx2.5 into
embryonic zebrafish (9) or frogs (14) causes development of a larger-than-normal heart. Nkx2.5 is ex-
pressed in the premyocardial and myocardial cells, and
its homolog tinman is necessary for heart formation in
Drosophila (4), suggesting that it may be part of a
pathway whereby allocation of cells to the myocardium
is determined. It is of interest, along these lines, that
the zebrafish mutations santa, valentine, and heart of
glass all cause a globally enlarged heart (10, 63).
Hence, this essential attribute of organ development
may be amenable to analysis in zebrafish.
Another set of programs might serve to set up the
genetic components of homeostatic loops, such as those
that control blood pressure, osmolarity, and PO2. Hemodynamic flow in the embryo is tightly controlled before
innervation of the heart or vessels (12, 37, 48). Are
there ‘‘receptor’’ genes and ‘‘feedback’’ genes? How is
compensation achieved if one element is abnormal?
Preliminary evidence suggests that zebrafish mutations could affect some of these processes and provide
some of these answers.
The nature of the chamber contraction sequence of
the embryonic vertebrate heart is another carefully
controlled function. It begins as a slow peristaltic wave
and then changes with maturation to sequential atrial
and ventricular beats. At that time, the cells of each
chamber must contract simultaneously, a coordination
imparted by the development of gap junction-mediated
rapid conduction of the electrical impulse between cells
(16, 29, 68). The cells of the inflow region, the outflow
tract, and the atrioventricular junction maintain their
slow conducting properties (17, 45), causing a pause
between chamber contractions. One consequence of
this functional patterning is that unidirectional flow
can be maintained even before development of valves or
nodes.
The molecular basis of this functional patterning is
likely to be complex and due to changes in expression
and function of channels and pumps in the plasma
membrane and the sarcoplasmic reticulum. Studies in
several species have revealed region-specific and developmental changes in calcium regulatory mechanisms
(30, 40, 43, 67), potassium channel expression patterns
(15, 35, 50, 72, 73), and the differential appearance of
the fast sodium current (25, 59), all of which affect the
characteristics of the action potential. In addition,
some components of the contractile apparatus are also
specifically restricted to a chamber or junctional regions (28, 42, 46, 49, 71). A genetic dissection would,
presumably, reveal not only the function of these elements but, perhaps, genes that serve to coordinate
function in a global manner.
PHYSIOLOGICAL MUTANTS
A localized pacemaker, nodes to delay the beat between atrium and ventricle, and rapid unidirectional
impulse propagation are vertebrate-specific innovations (22). The first zebrafish screens have revealed
several mutations that perturb cardiac rhythm and
contractility. For example, pacemaker function is perturbed in reggae and slow mo mutations (2, 63). In
reggae, the local twitching in the sinoatrial (SA) region
eventually ‘‘escapes’’ to the rest of the atrium and is
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One unexpected conclusion from the screens is that
the cardiovascular assembly appears to be ‘‘modular’’
(23). In other words, discrete components or attributes
are eliminated by single gene mutations as though the
vertebrate heart is the sum total of sets of independent
structures. This is important because if all cardiovascular developments were to have been interdependent, all
mutations might have had indistinguishable or uninformative phenotypes. Instead, for example, the cloche
mutation eliminates endocardium but does not block
assembly of the primitive heart tube or its division into
chambers; the pandora and lonely atrium mutations
truncate the ventricle, although atrial formation is
relatively unimpaired; the jekyll mutation blocks valve
formation, but the atrioventricular junction is properly
placed (10, 61, 63). Of course, ‘‘modularity’’ does not
imply autonomy. Tissue-to-tissue interactions are quite
important for heart formation and function.
The assembly of a seamless vascular system also is
selectively perturbed by different mutations, which
speak to regional identity of otherwise apparently
homogeneous endothelium. For example, the notochord
appears to be required for assembly of the aorta but not
of the caudal vein, which may be dependent on endoderm (B. M. Weinstein and M. C. Fishman, unpublished results and Ref. 24). The gridlock mutation
perturbs one particular vascular branch point where
the two dorsal aortas merge (63, 76). A similar defect is
found in the kurzschluss mutant (10). The mutations
bubblehead, migraine, and mush for brains lead to
localized cranial hemorrhage, whereas the leaky heart
mutation causes pooling of blood in the pericardial
cavity (63).
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INVITED REVIEW
followed by normal beat propagation, suggesting that
there is a block in the region of the SA node, perhaps
similar to the human disorder known as sinus exit
block. In slow mo, the heartbeat is rhythmic but slow
(Fig. 3A and Ref. 2). Dissociated slow mo cardiomyocytes beat at one-half the rate of wild-type cardiomyocytes. All currents contributing to diastolic depolarization (the pacemaker potential, Ih ) are normal in slow
mo cardiomyocytes except Ih, which is greatly diminished (Fig. 3B and Ref. 2). This strongly implicates Ih as
crucial to normal cardiac pacemaking. The slow mo
defect may be in the Ih channel (which has not been
CLONING THE MUTANT GENES
The zebrafish genome size is ,2,700 centimorgans
(cM) (53) and at 1.7 3 109 bp, it is a little more than
one-half the physical size of the human genome. The
positional cloning of a mutant gene is a task of similar
magnitude in the zebrafish, mouse, or human. One
current disadvantage in using zebrafish for genetic
studies is that the infrastructure for cloning, while
currently usable, is still under construction. However,
Table 1. Zebrafish mutations for which the disrupted gene has been identified
Cloning Approach
Candidate gene
Mutant Name
no tail (ntl)
floating head (flh)
no isthmus (noi)
Affected Gene
ntl/brachyury
flh/Not-1
pax-b
Mutant Phenotype
Notochord fails to differentiate fully, body shortened
Notochord absent, floorplate absent, body shortened
Midbrain-hindbrain boundary, cerebellum, optic tectum,
pronephric ducts all missing
chordino (dino)
chordin homolog
Ventral fates expanded, tail enlarged, head reduced
swirl (swr)
BMP2
Dorsal fates expanded, affecting notochord and somites;
ventral fates deleted, affecting blood and nephros
valentino (val)
kreisler
Hindbrain segregation for rhombomeres 5 and 6 absent
Proviral tag recovery pescadillo (pes)
Conserved, novel
Eye and head reduced, failure of gut and liver expansion
dead eye (dye)
NIC96-like
Necrosis of eye and optic tectum
no arches (nar)
clipper homolog
Eye and head reduced, near absence of pharyngeal arches
Positional cloning
one-eyed pinhead (oep) EGF-related ligand Cyclopia, with defects in ventral neurectoderm, prechordal
plate, and endoderm
Reference No.
58
65
5
57
39
44
1
1
27
77
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Fig. 3. A: slow mo mutation causes a slow heart rate. Representative
tracings of cardiac activity from slow mo (smo) and wild-type 72-h
embryos. Heart motion was recorded at 22°C, using a patch-clamp
electrode positioned close to the heart on external surface of each
embryo. Heart rates shown were 96 beats/min for wild type, and 48
beats/min for slow mo embryos (K. Baker, K. S. Warren, G. Yellen,
and M. C. Fishman, unpublished results). B: slow mo causes a
reduction in pacemaker current (Ih ). Whole cell voltage-clamp recordings of Ih from smo and wild-type embryonic cardiomyocytes show
that reduction of Ih in smo is due to absence of a fast component
normally seen in wild type. [Reproduced from Baker et al. (2) with
permission from Proc. Natl. Acad. Sci USA 94: 4554–4559. Copyright
1997, The National Academy of Sciences, USA.]
cloned in any species) or in a regulator of the channel.
Because slow mo mutant embryos also show an inability to elevate heart rate with increased temperature,
slow mo may also help define an important component
of intrinsic heart rate variability and response to
temperature.
Other zebrafish mutations perturb impulse conduction or atrioventricular nodal function. Cell-to-cell comunication appears blocked in island beat and polka
mutant genes, where individual cardiomyocytes contract but fail to excite neighboring cells. Atrioventricular junction block develops in ginger, breakdance, and
hiphop mutants, so that atrial beats outnumber ventricular beats. Uncoordinated fibrillating hearts are
found in tremblor, apparent from the first visible
motions of the heart, and in legong and slip jig mutant
genes, which progress from weak contractions to fibrillation on day 2. These mutant genes may lie in the
pathway to proper cell coupling, ionic means of orchestrating membrane potential, or differentiation of specialized cells at chamber borders.
There are also mutants that perturb normal development of contractility, without affecting heart rate or
rhythm. For example, weak atrium is a chamberspecific defect affecting the atrium. Sisyphus, main
squeeze, hal, weiches herz, low octane, dead beat, pipe
heart, and tango mutants show ventricle-specific contractility defects. The remaining contractility mutants
exhibit weakness in both chambers. These mutants
progress to different end points, including some with
dilated thin walls and others with an apparently stiff
and hypertrophic myocardium (10, 63). These may
provide systems in which to study heart failure in an
intact organism. Once cloned, the genes will become
candidates for heart failure propensity genes.
INVITED REVIEW
MEDICAL RELEVANCE
The constellation of zebrafish mutations speak to all
of the most common cardiovascular disorders. For
example, some mutations disturb the essential structure or pattern of the heart, including abnormalities in
left-right looping (11), problems that underlie many
congenital heart malformations. In others, the overall
structure of the heart is patterned correctly, but there is
markedly diminished cardiac output. In some, the
heart is dilated and in others, the heart is thick walled,
the former similar to dilated and the latter to hypertrophic cardiomyopathies. Single gene mutations cause
disturbances in rate (slow mo and reggae), rhythm
(tremblor, slip jig, legong), and conduction (island beat,
tell tale heart, polka). Mutations in these genes, which
likely have human homologs, are themselves candidates for genes that contribute propensity to clinical
disorders and provide handles on genetic pathways
involving other genes that may also be critical in the
establishment of normal function. Additionally, mutant
fish, in principal, could serve as targets for development of new, more effective pharmaceuticals.
FUTURE DIRECTIONS
Future screens can be designed to examine specific
physiological parameters or to follow, using in situ
hybridization or immunohistochemical techniques, the
expression of particular genes. The task of cloning will
be expedited by denser maps and, hopefully, strategies
for genetic complementation in which phenotypes may
be rescued by injection of DNA. Additionally, insertional mutagenesis, using pseudotyped retroviruses (1,
26, 27) or transposable elements (36), will facilitate
cloning by tagging the region affected by the insertion.
Address reprint requests to M. Fishman, Cardiovascular Research
Center, Massachusetts General Hospital, 149 13th St., Charlestown,
MA 02129.
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the zebrafish has a major advantage: the ability to
generate thousands of mutant progeny and thereby
increase genetic resolution. For example, 2,000 mutant
fish from map crosses (4,000 meioses) provide genetic
resolution of 0.025 cM, ,15.6 kb in zebrafish.
The field of zebrafish genomics has grown markedly
in the last few years, with the construction of framework genetic maps and the implementation of mapping
tools and technology for polymorphic marker generation. The standard genetic map in zebrafish, as in other
species, uses microsatellite repeats as polymorphic
markers between strains. The current map contains
over 700 of these simple sequence repeat (SSR)-based
markers, with an average interval of 5 cM between map
positions (E. W. Knapik, N. Shimoda, and M. C. Fishman, personal communication). This map has been
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current total of mapped markers .1,300.
Some of the zebrafish mutations have mapped markers close enough to begin the ‘‘walk.’’ For others, closer
markers can be generated using auxilliary mapping
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for chromosome walking, and physical mapping reagents include zebrafish-mouse somatic cell hybrids
(21) and radiation hybrids. Two recent reviews cover
the details of these resources and techniques (3, 54); the
latter providing Internet addresses for several pertinent zebrafish resource sites.
To date, a handful of zebrafish mutant genes have
been identified and published. As detailed in Table 1,
most of these disrupted genes have been identified
using a candidate gene approach, although a few were
found by tracking proviral DNA from a retroviral
insertion event. Importantly, the recent cloning of the
one-eyed pinhead locus (77) marks the first reported
mutant locus identified with a positional cloning strategy.
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