University of Zurich
Zurich Open Repository and Archive
Winterthurerstr. 190
CH-8057 Zurich
http://www.zora.unizh.ch
Year: 1996
Mutations affecting the formation and function of the
cardiovascular system in the zebrafish embryo
Stainier, D Y; Fouquet, B; Chen, J N; Warren, K S; Weinstein, B M; Meiler, S E;
Mohideen, M A; Neuhauss, S C; Solnica-Krezel, L; Schier, A F; Zwartkruis, F;
Stemple, D L; Malicki, J; Driever, W; Fishman, M C
Stainier, D Y; Fouquet, B; Chen, J N; Warren, K S; Weinstein, B M; Meiler, S E; Mohideen, M A; Neuhauss, S C;
Solnica-Krezel, L; Schier, A F; Zwartkruis, F; Stemple, D L; Malicki, J; Driever, W; Fishman, M C. Mutations
affecting the formation and function of the cardiovascular system in the zebrafish embryo. Development 1996,
123:285-92.
Postprint available at:
http://www.zora.unizh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.
http://www.zora.unizh.ch
Originally published at:
Development 1996, 123:285-92
Stainier, D Y; Fouquet, B; Chen, J N; Warren, K S; Weinstein, B M; Meiler, S E; Mohideen, M A; Neuhauss, S C;
Solnica-Krezel, L; Schier, A F; Zwartkruis, F; Stemple, D L; Malicki, J; Driever, W; Fishman, M C. Mutations
affecting the formation and function of the cardiovascular system in the zebrafish embryo. Development 1996,
123:285-92.
Postprint available at:
http://www.zora.unizh.ch
Posted at the Zurich Open Repository and Archive, University of Zurich.
http://www.zora.unizh.ch
Originally published at:
Development 1996, 123:285-92
Mutations affecting the formation and function of the
cardiovascular system in the zebrafish embryo
Abstract
As part of a large-scale mutagenesis screen of the zebrafish genome, we have identified 58 mutations
that affect the formation and function of the cardiovascular system. The cardiovascular system is
particularly amenable for screening in the transparent zebrafish embryo because the heart and blood
vessels are prominent and their function easily examined. We have classified the mutations affecting the
heart into those that affect primarily either morphogenesis or function. Nine mutations clearly disrupt
the formation of the heart. cloche deletes the endocardium. In cloche mutants, the myocardial layer
forms in the absence of the endocardium but is dysmorphic and exhibits a weak contractility. Two loci,
miles apart and bonnie and clyde, play a critical role in the fusion of the bilateral tubular primordia.
Three mutations lead to an abnormally large heart and one to the formation of a diminutive, dysmorphic
heart. We have found no mutation that deletes the myocardial cells altogether, but one, pandora, appears
to eliminate the ventricle selectively. Seven mutations interfere with vascular integrity, as indicated by
hemorrhage at particular sites. In terms of cardiac function, one large group exhibits a weak beat. In this
group, five loci affect both chambers and seven a specific chamber (the atrium or ventricle). For
example, the weak atrium mutation exhibits an atrium that becomes silent but has a normally beating
ventricle. Seven mutations affect the rhythm of the heart causing, for example, a slow rate, a fibrillating
pattern or an apparent block to conduction. In several other mutants, regurgitation of blood flow from
ventricle to atrium is the most prominent abnormality, due either to the absence of valves or to poor
coordination between the chambers with regard to the timing of contraction. The mutations identified in
this screen point to discrete and critical steps in the formation and function of the heart and vasculature.
INTRODUCTION
Organogenesis is the process by which cells of different
embryonic origins assemble to form discrete structures. As
these cells aggregate, a number of organotypic decisions give
the organ its final shape and structure. In the case of the heart,
the definitive heart tube, once formed, is first patterned in the
anteroposterior (A-P) axis to form the different chambers. It
then undergoes looping morphogenesis, and specific endocar-
dial cells go through an epithelial to mesenchymal transition
to form the prevalvular mesenchyme.
The heart has several advantages for the study of the
processes of organ formation. It is a relatively simple structure,
consisting of two concentric epithelial tubes, the inner, endo-
cardial and outer, myocardial. It is also the first organ to form
and function during vertebrate embryogenesis, and cardiac
function can be assessed by simple visual inspection, at least
in the optically transparent zebrafish embryo.
Heart development has been well described morphologically
in several species including chick (DeHaan, 1965; Viragh et
al., 1989), mouse (DeRuiter et al., 1992), and zebrafish (Stainier
et al., 1993; Lee et al., 1994). In all vertebrates, the post-gas-
trulation cardiogenic cells migrate medially as part of the
lateral plate mesoderm. They form two primitive myocardial
tubes on either side of the midline. These tubes then fuse to
enclose the endocardial cells and form the definitive heart tube.
Subsequently, this tube is patterned along the A-P axis to form
the different chambers, the main ones being the atrium and
ventricle, and valves form at chamber boundaries. The heart
starts beating at the time when the primitive heart tubes fuse
285
Development 123, 285-292
Printed in Great Britain © The Company of Biologists Limited 1996
DEV3321
As part of a large-scale mutagenesis screen of the zebrafish
genome, we have identified 58 mutations that affect the
formation and function of the cardiovascular system. The
cardiovascular system is particularly amenable for
screening in the transparent zebrafish embryo because the
heart and blood vessels are prominent and their function
easily examined. We have classified the mutations affecting
the heart into those that affect primarily either morpho-
genesis or function.
Nine mutations clearly disrupt the formation of the
heart. cloche deletes the endocardium. In cloche mutants,
the myocardial layer forms in the absence of the endo-
cardium but is dysmorphic and exhibits a weak contractil-
ity. Two loci, miles apart and bonnie and clyde, play a
critical role in the fusion of the bilateral tubular primordia.
Three mutations lead to an abnormally large heart and one
to the formation of a diminutive, dysmorphic heart. We
have found no mutation that deletes the myocardial cells
altogether, but one, pandora, appears to eliminate the
ventricle selectively.
Seven mutations interfere with vascular integrity, as
indicated by hemorrhage at particular sites.
In terms of cardiac function, one large group exhibits a
weak beat. In this group, five loci affect both chambers and
seven a specific chamber (the atrium or ventricle). For
example, the weak atrium mutation exhibits an atrium that
becomes silent but has a normally beating ventricle. Seven
mutations affect the rhythm of the heart causing, for
example, a slow rate, a fibrillating pattern or an apparent
block to conduction. In several other mutants, regurgita-
tion of blood flow from ventricle to atrium is the most
prominent abnormality, due either to the absence of valves
or to poor coordination between the chambers with regard
to the timing of contraction.
The mutations identified in this screen point to discrete
and critical steps in the formation and function of the heart
and vasculature.
Key words: heart, vasculature, zebrafish
SUMMARY
Mutations affecting the formation and function of the cardiovascular system
in the zebrafish embryo
Didier Y. R. Stainier*, Bernadette Fouquet, Jau-Nian Chen, Kerri S. Warren, Brant M. Weinstein,
Steffen E. Meiler, Manzoor-Ali P. K. Mohideen, Stephan C. F. Neuhauss, Liliana Solnica-Krezel,
Alexander F. Schier, Fried Zwartkruis
†
, Derek L. Stemple, Jarema Malicki, Wolfgang Driever
and Mark C. Fishman
‡
Cardiovascular Research Center, Massachusetts General Hospital, 149 13th Street, Charlestown, MA 02129, USA and
Department of Medicine, Harvard Medical School, Boston, MA 02115, USA
*Present address: Department of Biochemistry and Biophysics and Program in Developmental Biology, University of California San Francisco, San Francisco,
CA 94143-0554, USA
†
Present address: Laboratory for Physiological Chemistry, Utrecht University, Universiteitsweg 100, 3584 CG Utrecht, The Netherlands
‡
Author for correspondence
286
and once the definitive heart is formed, contraction proceeds
in a coordinated and characteristic manner: first the atrium
beats, and then the ventricle, with the valves opening and
closing to prevent the retrograde flow of blood.
In the zebrafish, cardiogenic progenitors are concentrated in
a marginal zone that extends from 90° to 180° longitude
(Stainier et al., 1993). Precardiac cells involute during early
gastrulation and migrate towards the embryonic axis as part of
the lateral plate mesoderm. They form two myocardial tubular
primordia on either side of the midline, with a distinct group
of cells, the endocardial progenitor cells, sitting medially
between them. The myocardial tubes then fuse to enclose the
endocardial cells and form the definitive heart tube. By 22
hours post-fertilization (hpf), the heart tube is clearly beating
and around 24 hpf, circulation begins. The regionalization of
cardiac myosin heavy chain expression distinguishes the
cardiac chambers at this stage, although they are not morpho-
logically delineated until 36 hpf. By 36 hpf, the heart tube has
looped, is beating at about 140 beats/minute at 28.5°C, and
provides a strong circulation to the trunk and head.
The molecular events underlying vertebrate heart formation
are poorly understood. A murine homeobox-containing gene,
Nkx-2.5, has been isolated, based on its homology with the fly
gene tinman (Komuro and Izumo, 1993; Lints et al., 1993). In
the fly, tinman is expressed in the heart as well as in the visceral
mesoderm and tinman mutants do not form a heart (Azpiazu
and Frasch, 1993; Bodmer, 1993). In the mouse, Nkx-2.5 tran-
scripts are first detected at the end of gastrulation in myocar-
dial progenitors as well as in a few other tissues. This gene is
the earliest known marker for myocardial progenitor cells. The
heart of Nkx2.5 mutant mouse embryos does not loop, nor
develop endocardial cushions nor trabeculae (Lyons et al.,
1995). Other transcription factors expressed in early myocar-
dial cells that may interact with Nkx-2.5 include MEF-2, SP-
1, TEF-1 and GATA-4 (Lyons, 1994).
A genetic approach to vertebrate heart formation provides
several advantages. Firstly, it makes no preconceived
judgement about the role of specific genes in this process.
Indeed, recent experiments in the mouse have revealed both
unexpected roles for certain genes in heart formation, for
example in the case of the retinoid X receptor α gene (Sucov
et al., 1994), and phenotypes disappointing in their informa-
tiveness about cardiac development, as in the case of TGFβ
1
(Shull et al., 1992). Secondly, the mutant phenotypes can point
to critical steps of heart formation. Thirdly, the mutations
themselves provide relevant entry points into these processes.
In this paper, we categorize and briefly describe the cardio-
vascular mutations identified in a large-scale mutagenesis
screen of the zebrafish genome (Driever et al., 1996). These
mutations specifically affect distinct aspects of heart formation
and function as well as the integrity of the vasculature.
MATERIALS AND METHODS
Zebrafish were raised and handled as described by Westerfield (1993).
Developmental time at 28.5°C was determined from the morpho-
logical features of the embryo. The design of the ENU screen and
screening methods are as described in Solnica-Krezel et al. (1994) and
Driever et al. (1996). Screening for cardiovascular mutations was at
48 hpf, although effects of some mutations were first noted earlier.
Complementation analyses between all members of each group
(Tables 1 and 2) were performed by pairwise matings of heterozy-
gous fish bearing different mutations. Different group assignments
were made by dint of reproducibly and obviously different visible
D. Y. R. Stainier and others
Table 1. Mutations affecting cardiovascular morphogenesis
Genetic loci Alleles Phenotype Other phenotypes Refs
Group I. No endocardium
cloche (clo) m39,m378 No endocardium Blood, vascular
Group II. Large heart
valentine (vtn) m201 Large, distended heart
heart of glass (heg) m552 Large, distended heart
santa (san) m775 Large, distended heart a
Group III. Small heart
heart and soul (has) m129,m567,m781 Small, dense heart Brain, eye, body b,c
Group IV. Ventricle defect
pandora (pan) m313 Reduced ventricle Eye, ear, somite, body shape c,d
Group V. Bifid heart
miles apart (mil) m93 Cardia bifida Tail e
bonnie and clyde (bon) m425 Cardia bifida
Group VI. No valve
jekyll (jek) m151,m310 No valve Branchial arch, jaw reduced f
(−) m27 No valve
Group VII. Vascular integrity
mush for brains (mfb) m75,m381,m508 Anterior hemorrhage Degeneration, brain
bubble head (bbh) m292 Hemorrhage (brain)
leaky heart (leh) m166 Hemorrhage (pericardial area)
gridlock (gdl) m145 Cranial/anterior hemorrhage Caudal circulation defect
migraine (mig) m247 Hemorrhage (brain) Degeneration, starting from brain
(−) m521 Hemorrhage (brain) Degeneration, brain
(−) m413 Hemorrhage (brain)
References: (a) san
ty219c
, Tubingen allele; Chen et al. (1996); (b) Schier et al. (1996); (c) Malicki et al. (1996); (d) Abdelilah et al. (1996); (e) mil
te273
,
Tubingen allele; Chen et al. (1996); (f) Neuhauss et al. (1996).
287Zebrafish cardiovascular mutations
phenotypes. Mutants with incomplete complementation analysis were
not given locus names, but rather are referred to by m number alone.
Histological analysis was performed as described in Stainier and
Fishman (1992). Contractility was assessed visually by examination
for both global and regional wall motion abnormalities, with regard
to rate and distance of systolic contraction.
Tables 1 and 2 list all the mutations and provide the locus names
and abbreviations, and the alleles determined by complementation
analysis.
RESULTS
Mutations affecting cardiac form
The wild-type heart is composed of two concentric epithelial
tubes, the inner endocardium and outer myocardium (Fig.
1A,B). cloche (clo) deletes the endocardial cells. In this mutant,
the myocardial layer forms in the absence of the endocardial
cells but is dysmorphic: the ventricle is reduced in size and the
walls of the atrium are distended. The clo heart also exhibits a
reduced contraction. The original allele, clo
m39
(Stainier et al.,
1995) was identified in an Indonesian fish background and sub-
sequently an ENU allele, clo
m378
, was identified with the same
phenotype (data not shown). The clo mutation also affects blood
cell differentiation, as assessed morphologically and by the
expression patterns of GATA-1 and GATA-2, two transcription
factor genes expressed very early during differentiation of the
hematopoietic lineages (Stainier et al., 1995). clo mutants, like
most of the heart mutants described in this study, exhibit pro-
gressively more severe edema, first of the pericardial sac and
then around the eyes and yolk sac. They die around day 7, as
do most other heart mutants.
In the valentine (vtn), heart of glass (heg) and santa (san)
mutations, the walls of the heart are grossly distended, yet they
have a full complement of endocardium (shown for vtn in Fig.
1C,D). Blood is also present in these mutants. In the heart and
soul (has) mutation, the heart is much smaller than normal and
no distinctive chambers are evident (Fig. 1E,F). In pandora
(pan), there is mainly one chamber, which histologically
appears to be atrium (Fig. 2A,B). The ventricle is markedly
reduced.
The fusion of the primitive myocardial tubes results in the
formation of the definitive heart tube. The cellular and
molecular events underlying this process are not understood.
Two mutations, miles apart (mil) and bonnie and clyde (bon),
disrupt this fusion process, resulting in the differentiation of
two hearts, one on either side of the midline, a situation
commonly known as cardia bifida. Fig. 3A,B shows a wild-
type and mil
m93
embryo, respectively, at 36 hpf. Transverse
sections in the heart region of mil
m93
show two complete heart
tubes on either side of the midline (Fig. 3C). In some mutants,
the two hearts actually contact each other, yet they do not fuse.
The two bilateral hearts have endocardial cells lining their
lumen and are composed of two chambers; they also beat inde-
pendently of each other. There is no blood flow in these
mutants, presumably because there is no connection to the
dorsal aorta.
Table 2. Mutations affecting cardiovascular function
Genetic loci Alleles Phenotype Other phenotypes Refs
Group I. Atrial contractility
weak atrium (wea) m58,m229,m448 Silent atrium a
Group II. Ventricular contractility
sisyphus (sis) m351,m644 Ventricle
main squeeze (msq) m347 Ventricle Curved body, pigment
hal (hal) m235 Weak ventricle
weiches herz (whz) m245 Weak ventricle
low octane (loc) m543 Weak ventricle
dead beat (ded) m582 Weak ventricle Curved body
Group III. Both chamber contractility
pickwick (pik) m171,m186,m242 Weak beat
m740
lazy susan (laz) m647 Weak beat
pipe dream (ppd) m301 Weak beat
beach bum (bem) m281 Weak beat
pipe line (ppl) m340 Weak beat
Group IV. Heart rate
slow mo (smo) m51 Slow beat
Group V. Heart rhythm
tremblor (tre) m116,m139,m158, Fibrillation b
m276,m736
Group VI. Conduction
island beat (isl) m231,m379,m458 Isolated twitches
reggae (reg) m230 Spasmodic
silent partner (sil) m656 Silent ventricle
ginger (gin) m47,m155,m739 Ventricle becomes silent
tell tale heart (tel) m225 Nearly silent heart
Group VII. Retrograde flow
ping pong (png) m683 Regurgitation of blood
tennis match (ten) m686 Regurgitation of blood
yoyo (yyo) m721 Regurgitation of blood
References: (a) wea
tw220a
, Tubingen allele; Chen et al. (1996); (b) tre
tc318d
, tre
te381
, Tubingen alleles; Chen et al. (1996).
