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Maternal and Zygotic Control
of Zebrafish Dorsoventral
Axial Patterning
Yvette G. Langdon and Mary C. Mullins∗
Department of Cell and Developmental Biology, University of Pennsylvania School of
Medicine, Philadelphia, Pennsylvania 19104; email: [email protected],
[email protected]
Annu. Rev. Genet. 2011. 45:357–77
Keywords
First published online as a Review in Advance on
September 13, 2011
animal-vegetal polarity, axis formation, bone morphogenetic protein
(BMP), fibroblast growth factor (FGF), organizer, Wnt
The Annual Review of Genetics is online at
genet.annualreviews.org
This article’s doi:
10.1146/annurev-genet-110410-132517
c 2011 by Annual Reviews.
Copyright All rights reserved
0066-4197/11/1201-0357$20.00
∗
Corresponding author.
Abstract
Vertebrate development begins with precise molecular, cellular, and
morphogenetic controls to establish the basic body plan of the embryo.
In zebrafish, these tightly regulated processes begin during oogenesis
and proceed through gastrulation to establish and pattern the axes of the
embryo. During oogenesis a maternal factor is localized to the vegetal
pole of the oocyte that is a determinant of dorsal tissues. Following fertilization this vegetally localized dorsal determinant is asymmetrically
translocated in the egg and initiates formation of the dorsoventral axis.
Dorsoventral axis formation and patterning is then mediated by maternal and zygotic factors acting through Wnt, BMP (bone morphogenetic
protein), Nodal, and FGF (fibroblast growth factor) signaling pathways,
each of which is required to establish and/or pattern the dorsoventral
axis. This review addresses recent advances in our understanding of
the molecular factors and mechanisms that establish and pattern the
dorsoventral axis of the zebrafish embryo, including establishment of
the animal-vegetal axis as it relates to formation of the dorsoventral axis.
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INTRODUCTION
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Animal-vegetal axis:
the axis running from
the blastodisc to the
yolk in the zebrafish
egg
Mid-blastula
transition (MBT):
developmental point
after fertilization when
cells begin to divide
asynchronously, the
cell cycle slows, and
wholesale zygotic gene
transcription initiates
Dorsal organizer:
a signaling center that
when transplanted is
sufficient to establish a
secondary dorsal axis
Early vertebrate development involves the precise coordination and regulation of multiple
signaling pathways and morphogenetic movements to establish the body plan. These tightly
regulated processes begin during oogenesis as
the oocyte matures, becomes fertilized, and the
newly fertilized egg transitions through cellular
cleavage, the process of gastrulation, and patterning of its pluripotent embryonic cells into a
fully formed organism. Prior to gastrulation the
vertebrate embryo establishes its primary axes,
which provide the foundation for its body plan.
Axis formation in zebrafish requires molecular
cues that distinguish the animal pole marked by
the blastodisc, from the vegetal pole where the
zebrafish yolk resides. Additionally, signals are
required to form and pattern the dorsoventral
axis. In this review, we focus on the molecular factors and mechanisms that establish and
pattern the dorsoventral axis of the zebrafish
(Danio rerio) embryo, including establishment
of the animal-vegetal axis during oogenesis as
it relates to formation of the dorsoventral axis.
In addition, we highlight areas of current research and suggest areas for future research
that could provide a better understanding of zebrafish dorsoventral patterning.
EARLY ZEBRAFISH
EMBRYOGENESIS
For the purpose of this review, early zebrafish
development consists of the transition from
the single cell zygote through the completion
of gastrulation (59). Immediately following
fertilization the chorion surrounding the
egg lifts away from the egg surface, and the
cytoplasm, which was previously interspersed
with the yolk, begins streaming toward the
animal pole to form the single cell blastodisc.
The cleavage period of development begins
at 45 minutes postfertilization (mpf ) and 10
cell-cycle cleavages ensue. During the cleavage
stage of development, cells synchronously
and rapidly divide every 15 minutes with a
corresponding decrease in their mass following
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each division. Prior to the 16-cell stage, all cells
of the embryo undergo incomplete cytokinesis,
such that cell membranes are not complete at
the yolk cell interface (59). In subsequent divisions, the central-most blastomeres undergo
complete cytokinesis and only the peripheral
blastomeres adjacent to the yolk incompletely
cleave, leaving the cytoplasm of these vegetalmost blastomeres connected with the yolk.
Following the cleavage period, the midblastula transition (MBT) ensues and three
critical events are initiated: wholesale zygotic
gene expression, formation of the yolk syncytial
layer (YSL) and enveloping cell layer (EVL),
and the morphogenetic process of epiboly.
As the MBT initiates (512-cell stage), the cell
cycle lengthens and the cells begin to divide
asynchronously (53, 59). At the same time,
the marginal blastomere membranes collapse
and discharge their contents into the yolk cell,
resulting in a layer of nuclei in the yolk [yolk
synctial nuclei (YSN)] and establishment of
the YSL. Although an extraembryonic tissue,
the YSL is vital for development of the embryo
proper, as it acts in mesoderm and endoderm
induction (58, 104). The YSL also regulates
epiboly, the process by which cells of the blastoderm and the YSN spread over and completely
encapsulate the yolk (132). Epiboly movements
initiate approximately one-and-a-half hours after the MBT and continue through the gastrula
period of zebrafish development until the yolk
is fully covered (105, 120). The YSN lead the
EVL and blastoderm cells through the latter
half of epiboly and appear to be the driving
force behind their movement (5a, 11, 121, 124).
Concurrent with the formation of the YSL
is the initiation of zygotic gene expression.
Some of the earliest zygotic genes expressed
in the embryo are mediators of dorsoventral
patterning, such as bozozok, chordin, goosecoid,
vox/vent/ved, bmp2b, and bmp7 (79, 82, 113,
118, 123, 139). Several of these genes are required to mediate formation of, or regulate
the functions of, the zebrafish dorsal organizer,
which is morphologically defined by the shield,
a thickening of cell layers that mark the dorsal side of the embryo (41, 108). The zebrafish
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dorsal organizer is formed at the onset of gastrulation and functions as a source of secreted
factors and cell autonomous transcription factors that act together to establish and pattern
cell fates dorsally. Several maternal factors are
now known that induce ventralizing factors
throughout the blastula, which together with
dorsal factors generate a BMP (bone morphogenetic protein) signaling gradient that patterns
ventrolateral tissues. In conjunction with organizer formation and function, the gastrulation
movements of convergence and extension shape
the body plan of the early embryo (109).
OOCYTE FACTORS AND
ANIMAL-VEGETAL POLARITY
Establishment of the dorsoventral axis depends
on the prior establishment of the animalvegetal axis during oogenesis. The zebrafish
female gamete, the oocyte, develops through
four stages before it can be fertilized as an egg
(70, 77, 90). Oocyte asymmetry and ultimately
oocyte animal-vegetal polarity is first evident
with the establishment of the Balbiani body
during stage I of oogenesis (38, 62, 77). Until
recently, little was known regarding Balbiani
body formation or localization of its associated
mRNAs to the vegetal pole of the oocyte and
future egg. One key factor localized to the vegetal pole of the egg is a currently unidentified
dorsal determinant(s), which acts to establish
the dorsoventral axis of the embryo following
its asymmetric translocation to embryonic
marginal blastomeres. Thus, dorsoventral axis
determination is linked to the establishment of
the animal-vegetal axis.
The Balbiani body originates adjacent to the
nucleus on the future vegetal side of the oocyte
and subsequently translocates to the prospective vegetal pole, where it releases its contents and dissociates (Figure 1a) (8, 78). The
Balbiani body is composed of mitochondria,
as well as nuage, mRNAs and proteins, which
become localized to the vegetal pole of the
oocyte following Balbiani body disassembly
(Figure 1a). A recently identified maternaleffect gene bucky ball encodes a novel protein
required for formation of the Balbiani body and
animal-vegetal polarity of the oocyte and egg
(1, 23, 78). In the absence of Bucky ball, the
Balbiani body fails to form, although components of the Balbiani body are evident in the
oocyte but are no longer localized. The absence
of the Balbiani body likely causes the animalvegetal polarity defect of this mutant. Animal
and vegetal pole transcripts are expressed but
mislocalized in bucky ball mutants (8, 23, 78).
mRNAs normally vegetally restricted are instead found throughout the oocyte, whereas
transcripts normally animally restricted are localized radially at the oocyte cortex. Bucky ball
protein localizes to the Balbiani body itself (8),
making it tempting to speculate that it may
act directly to assemble this symmetry-breaking
structure.
A second newly identified maternal-effect
gene, microtubule actin crosslinking factor 1
(macf1), also regulates animal-vegetal polarity
in zebrafish (Figure 1a) (37). Macf1 belongs to
the spectraplakin family of proteins, which associate with actin and microtubules (51, 106).
In the zebrafish macf1 mutant (magellan), the
Balbiani body is enlarged, the nucleus and
Balbiani body are mislocalized, and peripheral
regions of the oocyte near the cortex are disrupted (37). This disruption likely results from
a lack of stable microtubules at the oocyte periphery, suggesting a role for Macf1 in linking stable microtubules to the cortex, possibly through its direct binding of cytoplasmic
microtubules and the oocyte actin cortex. In
magellan mutant oocytes, the Balbiani body as
well as the transcripts associated with it fail to
reach the vegetal cortex and remain in what
appears to be a persisting Balbiani body, thus
causing a defect in animal-vegetal polarity. It
is currently unknown whether Macf1 functions directly or indirectly in promoting proper
Balbiani body function and disassembly.
Although two factors acting in the formation and localization of the Balbiani body and
thus animal-vegetal polarity are now known,
a multitude of questions remain. Specifically,
what factors act upstream of Bucky ball to
initiate Balbiani body formation? How is the
www.annualreviews.org • Zebrafish Dorsoventral Patterning
Balbiani body:
a conserved structure
from insects to
mammals that carries
vegetally destined
transcripts and
proteins, and marks
the future vegetal pole
of the oocyte; also
called the
mitochondrial cloud
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c
Oogenesis
Animal mRNAs
Maternal factors promote ventral
cell fate
Caveolin-1
Nucleus
Bucky
ball
Bb
Pou2
bmp2b/4/7
Macf1
Radar
Non-Balbiani
associated
mRNAs
Stage I: Balbiani body
formation, localization,
and dissociation
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b
β-catenin
Tob1a
Nucleus
Kzp
Vegetal
mRNAs
Syntabulin
mRNA
wnt8
vox/vent/ved
Runx2bt2
Stage II/III: Localization of
animal and vegetal
pole transcripts
d
Embryogenesis
pou2
radar
runx2bt2
pou2
radar
runx2bt2
Dorsal
determinant
Syntabulin
pou2
radar
runx2bt2
Syntabulin
2o transport
mechanism?
Transport
Dorsal
determinants
β-catenin
DD released
?
Syntabulin
degraded
Kinesin I
Maternal factors promote dorsal
cell fate
Wnt target
genes
hecate
Microtubule
network
Animal
Ventral
Dorsal
Vegetal
Figure 1
Maternal factors establish both the animal-vegetal and dorsoventral axes. (a) Schematic of early stage
zebrafish oocytes. In stage I oocytes (left), the nucleus is located centrally and the Balbiani body is located
adjacent to the nucleus. Bucky ball is required for Balbiani body formation, whereas Macf1 regulates Balbiani
body size and translocation to the prospective vegetal cortex in mid- to late-stage I oocytes. Following its
localization to the prospective vegetal pole in late-stage I oocytes, the Balbiani body disassembles, and its
associated transcripts become localized to the vegetal cortex (blue, green circles). In stage II/III oocytes,
additional non-Balbiani body–associated transcripts also become localized vegetally (orange circles) and
animally (red circles). (b) Schematic of one-cell- and two-cell-stage zebrafish embryos. In the one-cell and
two-cell stage embryos, some maternal transcripts, for example, radar, pou2, and runx2bt2, are localized to
these animal pole blastomeres. The yolk cell consists of a microtubule network containing the motor protein
Kinesin I. Syntabulin protein associates with Kinesin I and possibly also with the dorsal determinant(s) to
facilitate its transport to the prospective dorsal side of the embryo. Once at the dorsal side Syntabulin is
degraded, and a second transport mechanism is postulated to translocate the dorsal determinant(s) further to
the future dorsal blastomeres. (c) Maternal factors promoting ventral cell fates. (d ) Maternal factors
promoting dorsal cell fates.
initial asymmetric localization of the Balbiani
body determined? Is it stochastically determined or does it rely on an earlier established
oocyte asymmetry? What additional factors
act in conjunction with Bucky ball and Macf1
to establish and translocate the Balbiani body
to the vegetal pole? Germline determinants
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Langdon
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are known to be translocated vegetally by the
Balbiani body (8, 61, 62, 65, 78, 142), but
what additional factors are transported in the
Balbiani body and what are their roles during
development? Importantly, what is the nature
of the vegetally localized dorsal determinant(s)
that establishes the dorsoventral axis of the
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embryo, and how is it localized? Efforts are
currently under way to identify and characterize maternal factors and mutants that may
affect these processes. Because zygotically
required genes certainly also play important
maternal roles in these processes, additional
studies are required to examine these genes for
maternal function. To address these questions,
the development of efficient methods to culture
oocytes and manipulate them via morpholino
knockdown and misexpression of gene function
is needed (8, 16, 115). Furthermore, genetic
manipulations that can efficiently generate
germline loss-of-function clones, chimeras, or
conditional mutations will greatly facilitate the
elucidation of these mechanisms (7, 12, 13, 88).
MATERNAL FACTORS
AND DORSOVENTRAL
AXIS FORMATION
Establishing the Dorsal Side
of the Embryo
Residing at the vegetal pole of the egg are
determinants that are crucial to establish the
dorsoventral axis. The nature of the determinant(s) is not known in zebrafish, nor if
it is localized vegetally via the Balbiani body
or via the late vegetal localization pathway
(Figure 1a) (78, 87, 134). Within the early
embryo, asymmetric translocation of the dorsal determinant(s) from the vegetal pole to
marginal blastomeres via microtubules establishes the dorsal side of the embryo through
activation of a Wnt signaling pathway, a mechanism conserved in Xenopus (132a). Resultant
nuclear localization of β-catenin then activates
dorsal-specific gene expression. Evidence for
a microtubule-dependent dorsal determinant
transport mechanism in zebrafish is derived
from several observations. When microtubule
networks of early cleavage stage embryos are
disrupted by UV irradiation, cold temperatures,
or drug treatment, the embryo becomes ventralized (52). Further evidence for the requirement of microtubules in this process comes
from the maternal-effect brom bones mutant,
which produces embryos with disordered microtubules at their vegetal cortex and ventralized embryos, consistent with a microtubule
network transporting a dorsal determinant(s)
from the vegetal pole to the dorsal marginal
blastomeres (81).
Further elucidation of the microtubulederived transport mechanism came with the
recent molecular identification of the zebrafish
maternal-effect mutant gene tokkaebi as the microtubule cargo linker protein gene syntabulin
(93). The syntabulin transcript is localized vegetally through the Balbiani body–dependent
localization mechanism, and loss of syntabulin
causes ventralization of the embryonic axis
(93, 94). Interestingly, the Syntabulin protein
associates with the motor protein Kinesin 1
to link cargo to the microtubule network,
and in zebrafish, Syntabulin is translocated
in a microtubule-dependent manner asymmetrically from the vegetal pole of the egg to
a lateral position. Thus, it is hypothesized
that Syntabulin mediates the asymmetric
transport of the dorsal determinant(s) toward
the prospective dorsal side of the embryo
(Figure 1b,d ). Once laterally translocated,
Syntabulin is degraded and postulated to
release the dorsal determinant(s) to be transported by alternate means to the prospective
dorsal-marginal blastomeres (93). Zebrafish
maternal tokkaebi mutants lack Syntabulin and
therefore are unable to initiate the transport
of the dorsal determinant(s), resulting in a failure to activate Wnt signaling, which is required
to initiate dorsal cell fate specification and
establish the dorsoventral axis (93, 94). It will
be interesting to determine if Syntabulin also
functions in dorsal determinant translocation in
Xenopus and how homologous the mechanisms
are between different vertebrate embryos.
Another maternal factor, hecate, still uncharacterized molecularly, acts upstream of, or
in parallel to, maternal β-catenin to promote
dorsoventral axis formation (75). The ventralized phenotype of hecate mutant embryos can
be rescued by expression of all Wnt signaling pathway components tested, and thus it
may function upstream of, or in parallel to,
www.annualreviews.org • Zebrafish Dorsoventral Patterning
Maternal-effect
mutant: the genotype
of the mother is the
cause of the embryonic
phenotype; a
maternally expressed
gene that functions in
embryogenesis
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b
Organizer formation, function, and regulation
Dorsoventral patterning zygotic factors
Bmp1
Tolloid
Admp
Chordin
Tsg
Bozozok
Bmp2b/4/7
Bozozok
Cvl2
FGF
Squint
Lnx2b
V
FGF
D
V
Goosecoid
Chordin
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Sizzled
β-catenin
Bmp2b/4/7
Vox/Vent/Ved
D
V
Bmp2b/4/7
Chordin
Goosecoid
Bmp2b/4/7
Admp X
Bozozok
Vox/Vent/Ved
FGF
Lnx2b
D
SoxB1
Follistatin-like 2
Admp
Bozozok
Wnt8
Lnx2b
Noggin1
Goosecoid
Vox/Vent/Ved
Wnt8
Figure 2
Maternal and zygotic genes that establish the dorsal organizer and pattern the dorsoventral axis. (a) Schematic of maternal and zygotic
factors required for organizer formation. Wiring diagrams represent an approximation of the onset and maintenance of proteins
required for initiation and establishment of the zebrafish organizer. (b) Schematic of the major zygotic factors acting in dorsoventral
patterning. Black names indicate proteins that promote dorsal cell fates. Blue names indicate proteins that promote ventral cell fates.
Red T-bars indicate dorsalizing proteins that inhibit ventral genes or proteins. Black T-bars indicate ventralizing proteins that inhibit
dorsal genes or proteins. Blue arrows indicate positive interactions between ventral promoting genes. Black arrows indicate positive
interactions between dorsal promoting genes. Green arrows indicate a dorsalizing or ventralizing gene that promotes genes with the
opposing activity.
Wnt/β-catenin signaling to establish the
dorsoventral axis (Figure 1d ) (75).
Maternal β-catenin in zebrafish is present
throughout the blastoderm, but it only localizes
to the nucleus of dorsal marginal cells (111).
This nuclear localization is the first indication
of dorsoventral polarity in the embryo and is
required to establish dorsal cell fates. There are
two maternally expressed β-catenin genes in
zebrafish, β-catenin 1 and 2 (4). In the absence
of maternal β-catenin 2, zebrafish embryos
are ventralized as observed in the β-catenin 2
mutant ichabod (4, 56). Recent results suggest
that maternal Caveolin-1 may be a negative
regulator of β-catenin, binding β-catenin
and inhibiting its translocation to the nucleus
(Figure 1c) (86). Once in the nucleus, a second
protein, Tob1a, can bind β-catenin and inhibit
its ability to bind LEF1 and promote dorsal cell
fate specification (137). Loss of Tob1a causes
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dorsalization of the embryonic axis, suggesting
that it functions ventrally to inhibit maternal
β-catenin-derived transcriptional activation in
ventral cells (Figure 1c). Activation of Wnt
signaling and β-catenin nuclear translocation
results in the expression of genes required for
dorsal organizer formation or function, including bozozok, chordin, and goosecoid (82, 113,
123, 139). The balance between expression
of genes required for ventral cell fates and
those promoting dorsal cell fates patterns the
dorsoventral axis in zebrafish (Figure 2b).
In conjunction with Wnt/β-catenin, Nodal
signaling is required for organizer formation
and mesendoderm formation (28). An additional maternal requirement for the zebrafish
nodal gene squint during dorsoventral axis formation has been suggested, but remains controversial. Morpholino knockdown of squint suggests a role in dorsoventral axis formation (35);
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however, other loss-of-function studies have
not found a role for squint in this process (5, 43).
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Promoting Ventral Cell Fates
Like the role of β-catenin in dorsal cell fate
specification, it has been hypothesized that
a maternally regulated pathway establishes
ventral cell fates (36, 119). It is well established
that zygotic BMP signaling is required for
ventral cell fate specification, but the upstream
factors initiating bmp expression have not been
extensively studied. Zebrafish Radar/Gdf6a,
another member of the BMP class of proteins,
was identified as the first maternal signaling
factor regulating ventral cell fate specification
(Figure 1b,c). Gain- and loss-of-function of
Radar cause embryonic ventralization and dorsalization, respectively. Radar appears to function upstream of Bmp2b, Bmp7a, and Alk8, and
a dominant negative form of Radar can block
the initiation of bmp2b expression in the embryo
(36, 119). Additional evidence for the active
promotion of ventral cell fates comes from
studies of the transcription factor pou2, which is
defective in the maternal-zygotic (MZ) mutant
spiel-ohne-grenzen (100). MZpou2 mutants lack
bmp2b and bmp4 expression, and fail to maintain
bmp7a expression. Maternal pou2 likely functions upstream of the bmp genes because their
expression can rescue the dorsalized phenotype
of MZpou2 mutants (Figure 1c) (100).
A third maternal factor, runx2b type2
(runx2bt2), is required for ventral cell fate
specification (30). Best known for its role as a
transcriptional regulator of skeletal development (63, 112), zebrafish runx2bt2 is expressed
in oocytes and eggs as well as during gastrulation. Morpholino-mediated translational
inhibition of maternal runx2bt2 strongly dorsalizes the embryo. Runx2bt2 appears to act as a
direct transcriptional activator of vox, vent, and
ved (vox/vent/ved ), which promote ventral cell
fate specification by inhibiting the ventrolateral
expression of dorsal genes, such as bozozok
(Figure 2), and maintaining ventrolateral
expression of bmp2b, bmp7a, and wnt8 during
gastrulation (30, 44, 55, 99, 118). Runx2bt2
binding sites are found in the promoters of the
vox/vent/ved genes, and Runx2bt2 regulation
of a ved reporter in the zebrafish embryo
depends on the Runx2bt2 binding site. Loss of
Runx2bt2 in the embryo results in a loss of all
ved expression and a one hour delay in expression of vox and vent. Given that Ved functions
redundantly to Vox and Vent, it is unclear if
the embryonic dorsalization caused by loss of
Runx2bt2 is due solely to the altered expression
of vox, vent, and ved, or if Runx2bt2 regulates
the expression of other ventral specifying genes.
The evidence from these studies establishes
Runx2bt2 as an independent maternal gene
regulatory mechanism to Pou2 that actively
promotes ventral cell fate specification (30).
Ventralization is actively promoted by the
embryo and thus must be repressed dorsally to
allow dorsal cell fate specification (119). The
identification of radar and pou2 as maternal
factors that initiate or activate zygotic bmp
expression and the identification of runx2bt2
as a maternal factor regulating vox/vent/ved
expression show that ventral identity is indeed
actively promoted maternally in the embryo.
Thus, both ventral cell fates and dorsal cell
fates, through establishment of the organizer,
are actively initiated and regulated during
dorsoventral axis formation and patterning.
Clearly, there are many missing components to
the maternal pathways that initiate ventral zygotic gene expression. Further maternal-effect
mutant screens and morpholino knockdown
studies of maternal mRNAs are needed to
identify these missing factors in dorsoventral
patterning.
ZYGOTIC GENE REGULATION
AND DORSOVENTRAL
PATTERNING
Organizer Formation and
Dorsoventral Patterning
The zebrafish organizer is an important
inducer of dorsal cell fate and regulator of
dorsoventral axis patterning. Among the first
zygotic genes expressed in the embryo and
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acting in organizer formation and function is
the homeodomain transcriptional repressor,
bozozok. bozozok is expressed just prior to the
MBT, when wholesale zygotic gene expression
is initiated (53, 71). Maternal β-catenin and its
DNA-binding cofactor Lef1 likely directly activate bozozok because Lef1 binds to sites within
the bozozok promoter, and regions containing
these sites are required for bozozok expression
(71, 107). Expression of bozozok is initially
observed in dorsal blastomeres prior to its
restriction to the dorsal YSL (64, 139). bozozok
is a mediator of dorsal organizer function by
repressing in dorsal regions the expression of
the ventralizing genes vox/vent/ved, bmp2b, and
wnt8 (Figure 2a) (71, 122). Bozozok directly
mediates repression of bmp2b expression by
binding to and regulating the bmp2b promoter
(71). In the absence of bozozok, the embryonic
shield fails to form, dorsal organizer function is
impaired, and embryos lack midline mesoderm
and are weakly ventralized (27). Consistent
with its function in mediating dorsal organizer
function, misexpression of bozozok causes an
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a
BMP, Nodal, Wnt, and FGF signals
just after MBT
expansion of the dorsal organizer and can
induce a secondary axis (64, 102, 139).
The TGF-β Nodal signaling pathway is also
required to establish the shield and pattern the
dorsal organizer (25, 28, 126). Nodal signaling
induces expression of dorsal mesendodermal
genes and inhibitors of ventral cell fates, such
as chordin and goosecoid (Figures 2 and 3) (21,
113). Interestingly, the organizer gene goosecoid can inhibit ventral BMP signals required for
dorsoventral patterning independently of the
BMP antagonist chordin (21).
Antagonistic to bozozok are the transcriptional repressors vox/vent/ved, which are first
expressed at mid-blastula stages. vox/vent/ved
function to repress the dorsal organizer promoting genes bozozok, chordin, and goosecoid, in
ventrolateral regions. Loss of vox/vent/ved expression results in the ventral expansion of bozozok, chordin, and goosecoid expression and of dorsal cell fates (34, 44, 82, 98). Rnx2bt2 is required
to initiate vox/vent/ved expression, whereas
Wnt8 and BMP signaling maintain their expression during gastrulation (3, 30, 98, 99).
b
BMP gradient and early Nodal, Wnt,
and FGF signals at late blastula
BMP gradient
Organizer
Nodal
V
FGF
Wnt
D
V
D
Figure 3
Bone morphogenetic protein (BMP), Nodal, Wnt, and fibroblast growth factor (FGF) signaling in
overlapping domains act to pattern dorsoventral embryonic tissues. (a) Schematic of signals just after the
mid-blastula transition that promote the establishment of the zebrafish dorsal organizer. (b) Schematic of
BMP signaling gradient and other signals acting during late blastula/early gastrula stages.
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Wnt8 maintains vox/vent/ved expression to restrict organizer size and loss of wnt8 results
in dorsalized embryos with an expanded organizer (98, 99). Double loss of wnt8 and bmp2b
function elicits a radial expansion of dorsal
gene expression identical to loss of vox/vent/ved
(98). Maternal β-catenin 1 and 2 appear to
function downstream of Wnt8 signaling as the
double knockdown of β-catenin 1 and 2 produces dorsalized embryos with an expanded organizer, whereas maternal β-catenin 2/ichabod
mutants are ventralized (4, 56). Initially, organizer gene expression is absent in both ichabod
and β-catenin 1/2 double knockdown mutant
embryos; however, by gastrulation, organizer
gene expression is restored in β-catenin double knockdown embryos, indicating that an alternate pathway during gastrulation can induce
organizer gene expression (4, 56).
Recently, SoxB1 family members (Sox3
and Sox19a/b) have been identified as transcriptional repressors that must be excluded
from dorsal-marginal blastomeres for the
organizer to form (Figure 2a) (116). Although
overexpression of Sox3/Sox19a/b is sufficient
to inhibit organizer formation, the quadruple
knockdown of Sox2/3/19a/19b results in
dorsoventral patterning defects without affecting organizer formation. These results indicate
that Sox family members have no direct role in
organizer formation, but instead are important
to maintain the balance between ventral and
dorsal cell fates (96, 116). The dorsoventral
patterning defects observed in the quadruple
knockdown appear to be due to decreases in
bmp2b and bmp7 expression (96).
The zebrafish embryo develops rapidly, undergoing dynamic changes in gene expression,
the regulation of multiple signaling pathways,
and the activation of transcription factors
to modulate dorsal organizer formation and
patterning of the dorsoventral axis. Considering the rapidity with which this patterning
occurs (during less than eight hours), protein
degradation is expected to play an important
role in regulating the dynamic changes in gene
expression and patterning in the early zebrafish
embryo. Recently, it has been found that
Bozozok protein turnover is modulated
through an E3 ubiquitin ligase pathway mediated by maternally supplied lnx2b (previously
called lnx-l ) (Figure 2a) (102, 103). Lnx2b can
bind to Bozozok, resulting in polyubiquitination and proteosomal degradation of Bozozok.
In the absence of Lnx2b, the dorsal organizer
is expanded, indicating that Lnx2b regulates
organizer size by mediating Bozozok protein
turnover (102). Identification of Lnx2b as a
mediator of Bozozok protein degradation raises
the question of how the turnover of other
proteins required for organizer formation
and function is regulated during the rapid
development of the zebrafish embryo.
BMP family members may also regulate
organizer size. Antidorsalizing morphogenetic
protein (Admp) is a BMP that is exclusively expressed in the dorsal midline of the embryo
(Figure 2a) (20, 69, 135). Loss of admp results in
a modest expansion of the organizer, and maintenance of admp expression is bozozok and nodal
dependent (69, 135). Thus, refinement of organizer size may result from precisely established
domains of bozozok and admp expression (69).
BMP Signaling
Genes encoding BMP signaling components
have been identified as zebrafish mutants defective in dorsoventral patterning, including
swirl/bmp2b, snailhouse/bmp7a, somitabun/smad5,
lost-a-fin/alk8, mini fin/tolloid, chordin/chordino,
and ogon/sizzled (14, 19, 42, 60, 66, 83, 85, 89,
110, 113). Molecular-genetic studies of these
genes have facilitated the elucidation of the
mechanisms by which BMP signaling patterns
the embryo. BMPs are members of the TGF-β
family of signaling molecules, which includes
TGF-β, Nodal, BMP, and Activin (29). BMPs
are secreted ligands that bind as a dimer to
serine-threonine kinase transmembrane receptors classified as type I and type II. Each BMP
monomer binds to two type I receptors and
one type II receptor. The other BMP monomer
binds the same two type I receptors and an additional type II receptor. In an active signaling complex, the constitutively active type II
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receptor phosphorylates and activates the type
I receptor (73). The activated type I receptor then phosphorylates the regulatory Smads
(Smads 1, 5, or 8), which in turn associate with
the co-Smad (Smad4) and translocate into the
nucleus to regulate expression of BMP target
genes (24, 73).
BMP homodimers and heterodimers form
and in several in vitro cell culture assays, heterodimers exhibit higher activity than homodimers (2, 40, 45, 46, 129, 144). Although BMP
heterodimers have been postulated to function in developing vertebrate embryos, only
recently has their requirement been demonstrated in vivo. Molecular-genetic studies together with biochemical evidence in zebrafish
demonstrate that although BMP homodimers
and heterodimers are both present in the embryo, only Bmp2b/Bmp7a heterodimer signaling leads to Smad1/5 phosphorylation and
dorsoventral patterning (74). Furthermore, this
BMP heterodimer signals through an obligate
BMP type I heteromeric complex composed of
Alk3/6 and Alk8 (the Alk2 paralog in zebrafish)
to pattern the dorsoventral axis of the embryo.
bmp2b and bmp7a expression is initiated
rapidly throughout the blastoderm following
the MBT. Expression of bmp4 is initiated
slightly later in a ventrally restricted domain,
and its expression depends on bmp2b and bmp7a.
A BMP signaling gradient begins to form in
late blastula stages and by the onset of gastrulation, BMP signaling is cleared from the dorsal side of the embryo (Figure 3). Attenuation
of BMP signaling in dorsal regions is mediated
first by Bozozok repression of bmp2b expression in the dorsal-most blastomeres (71), followed by signal repression by the BMP antagonists Chordin, Noggin 1, and Follistatin-like
2 (17). The newly established BMP signaling
gradient can be visualized by use of an antibody
specific to the phosphorylated form of Smad1/5
(128). BMPs function as a morphogen with high
levels of BMP signaling specifying ventral tissues, moderate levels specifying lateral tissues,
and little-to-no BMP activity is required to
specify dorsal tissues (22, 91). bmp expression
and ventral cell fates are maintained through
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several autoregulatory feedback loops (82, 101).
One of these loops requires the transcriptional
regulators Vox, Vent, and Ved (Figure 2).
Vox/Vent/Ved function to maintain the gradient of BMP activity during mid-to-late gastrulation through maintenance of bmp expression
and indirectly through Wnt8-mediated repression ventrally of bmp antagonist gene expression
(44, 98, 118, 130).
Although BMP signaling is initiated shortly
after the MBT in zebrafish, BMP signaling is
not required for dorsoventral patterning until
the late blastula/early gastrula stage (Figures 2
and 3) (19, 42, 60, 66, 85, 110, 128). Disruption
of BMP signaling with heat-shock-inducible
chordin expression at blastula or early gastrula
stages results in strongly dorsalized embryos,
whereas BMP signaling disrupted at progressively later gastrula stages allows progressively
larger anterior domains of ventral tissue to be
specified, and dorsalization becomes restricted
to progressively more posterior regions of the
embryo (128). These studies show that BMP
signaling functions in a progressive temporal
manner along the anterior-posterior axis in patterning dorsoventral tissues (128). Induction of
BMP signaling in an MZ-lost-a-fin (alk8) mutant that lacks BMP signaling indicates that
posterior tissues require BMP signaling at progressively later intervals, rather than requiring signaling for a longer duration (128). It is
postulated that temporal cues or competence
mediates the progressive cellular responses to
BMP signaling and thus coordinates patterning of the dorsoventral axis with patterning of
the anterior-posterior axis (128).
The BMP Signaling Gradient
A BMP signaling gradient forms along the
dorsoventral axis and patterns tissues as a
morphogen with high levels ventrally and
low levels dorsally (Figure 3). The BMP
gradient is established and maintained through
positive and negative regulators of BMP
signaling. BMP antagonists emanating from
the embryonic shield inhibit BMP signaling and bmp expression in dorsal regions
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(17, 55, 71, 118). In zebrafish, Chordin is the
only BMP antagonist acting nonredundantly
in dorsoventral patterning (17). Chordin
binds BMPs abrogating their ability to bind
BMP receptors and initiate signaling (18,
73, 84). Zebrafish chordino mutant embryos
are moderately ventralized, whereas chordin
overexpression dorsalizes embryos (39, 84,
113). Chordin function is unique among BMP
antagonists, as its transcripts extend beyond
the boundaries of the embryonic shield and
include the dorsal half of the embryo (84, 113).
Two additional BMP antagonists, Noggin1
and Follistatin-like 2, function redundantly
with Chordin to further refine the BMP
signaling domain (Figure 2b) (17, 31). noggin1
and follistatin-like 2 are first expressed at
blastula and early gastrula stages, respectively,
within the dorsal organizer, and knockdown of
each individually has no effect on dorsoventral
patterning. However, knockdown of either
noggin1 or follistatin-like 2 in chordin morphants
enhances the chordin ventralization phenotype
(17). Triple loss of chordin, noggin1, and
follistatin-like 2 caused a similar strong ventralization but with increased penetrance compared
to the double knockdown phenotypes (17).
Like Chordin, Noggin1 and Follistatin-like
2 directly bind BMP ligands and block their
association with receptors. Together Chordin,
Noggin1, and Follistatin-like 2 antagonism is
key to generating the BMP signaling gradient.
Additional extracellular factors further
modulate BMP signaling levels across the embryo. A critical secreted protein that promotes
ventral cell fate specification is the matrix metalloprotease Tolloid (Figure 2b). Loss of tolloid
results in mildly dorsalized embryos as observed
in the mini fin zebrafish mutant (14). Tolloid
binds and proteolytically cleaves the BMP antagonist Chordin to pattern ventral tail tissues
during postgastrula stages (6, 14, 15, 97). Subsequently, it was determined that zebrafish Bmp1,
a Tolloid-like protease, functions redundantly
with Tolloid to cleave Chordin during gastrulation (50). Loss of Bmp1 and Tolloid strongly
dorsalizes embryos (50). Thus, Bmp1 and
Tolloid function in concert to promote BMP
signaling ventrally through restriction of the
BMP antagonist Chordin during gastrulation.
Sizzled, a secreted Frizzled-related protein,
functions as a competitive inhibitor of Tolloid,
binding its active site, and thus abrogating its
ability to bind and cleave Chordin (18, 73).
Sizzled thus promotes dorsal cell fates and is
unique among dorsal promoting genes in that
it is exclusively expressed ventrally beginning
at late blastula stages (Figure 2b) (80, 138).
Loss of sizzled in the ogon mutant causes ventralization. Although an inhibitor of BMP signaling, sizzled expression is positively regulated
by BMP signaling (39, 83, 131, 138). Thus, Sizzled functions as a negative feedback inhibitor
to limit BMP signaling and ventral cell fate
specification.
Two additional extracellular secreted factors, Twisted gastrulation and Crossveinless2, function ventrally to promote BMP signaling in zebrafish. Twisted gastrulation can bind
Chordin and BMPs and functions to enhance
BMP signaling in zebrafish (10, 72, 95, 114,
136). Twisted gastrulation has been shown to
increase Tolloid-mediated cleavage of Chordin
but can also function independently of Chordin
and Tolloid to promote BMP signaling (72,
136). In zebrafish, morpholino knockdown of
twisted gastrulation causes dorsalization of the
embryo, which can be rescued by expression of
BMP signaling pathway components, indicating that Twisted gastrulation acts upstream of
Bmp2b/Bmp7 (72, 136). Unexpectedly, overexpression of twisted gastrulation also dorsalizes
embryos (72), suggesting that Twisted gastrulation protein levels are normally tightly regulated in the embryo.
Similar to twisted gastrulation, loss of
crossveinless-2 (cvl2) function in zebrafish indicates that it promotes BMP signaling, whereas
overexpression of Cvl2 causes inhibition of
BMP signaling (73). Morpholino knockdown
of cvl2 moderately dorsalizes the embryo,
whereas overexpression causes weak dorsalization and ventralization of embryos. Cvl2 contains cysteine-rich domains similar to those
found in Chordin family members. Its expression is initiated at mid-blastula stages and
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is restricted ventrally at early gastrula stages
through a positive BMP signaling feedback loop
(101). The protein has a cleaved and noncleaved form, and there is evidence suggesting that cleavage of Cvl2 converts the protein
from a BMP antagonist to a BMP agonist (101).
Noncleaved Cvl2 is localized to the extracellular membrane and may sequester BMP ligands
away from their receptors. However, cleaved
Cvl2 can bind both BMP ligand and Chordin
and may initiate a conformational change in
Chordin, altering its ability to inhibit BMP
signaling and thus promoting BMP activity
(101, 143).
Tolloid, Twisted gastrulation, and Cvl2
all promote BMP signaling through Chordindependent regulation (Figure 2b) (18, 141).
Considering that BMP heterodimers are
the obligate signaling ligand complex, but
that homodimers are also present in the
embryo (74), one might consider if Tolloid
preferentially cleaves Chordin bound to a
Bmp2b/Bmp7 heterodimer as opposed to
a Bmp2b or Bmp7 homodimer to promote
BMP signaling. Alternatively or additionally,
Twisted gastrulation or Cvl2 may preferentially promote Bmp2b/Bmp7 heterodimer
signaling over BMP homodimer signaling,
thus contributing to the exclusive signaling of
BMP heterodimers in the zebrafish gastrula.
The cohort of extracellular factors required
to establish, refine, and maintain the BMP
signaling gradient highlights the importance of
shaping the gradient and ensuring its robustness to natural fluctuations. In conjunction with
the multiple levels of extracellular modulation
built into the system, the gradient is reinforced
through positive and negative transcriptional
feedback loops. This self-regulating system
makes it possible, for example, to ubiquitously
express bmp pathway components in mRNA
injection experiments and rescue the corresponding bmp pathway mutant (14, 19, 92,
110, 113), although bmp component expression
is normally restricted to either ventral or
dorsal regions. Due to the tight and robust
modulation of ligand levels, the embryo can
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effectively negate the bmp component misexpression (within a range) and generate a BMP
gradient that can completely rescue mutants to
normal development. Although we understand
the basic biochemical and genetic properties
of many of the BMP pathway components
discussed here, we still do not understand how
they function together in a spatial and temporal
manner to generate the robust BMP gradient
that functions throughout gastrulation to
pattern the embryo.
ZYGOTIC WNT SIGNALING AND
DORSOVENTRAL PATTERNING
During the cleavage stage canonical Wnt signaling through maternal β-catenin 2 establishes
the dorsoventral axis by inducing dorsal tissues and initiating formation of the zebrafish
organizer (Figure 1d ). After this initial dorsal phase of Wnt signaling, zygotic marginal
Wnt signaling is required to promote ventral
cell fates (Figure 3). In ventrolateral gastrula
regions Wnt8 signaling together with BMP signaling maintain expression of the vox/vent/ved
transcriptional repressors, which restrict the expression of dorsal promoting genes, including
bozozok to the dorsal-most regions (3, 98, 99).
Loss of the bicistronic wnt8 gene dorsalizes,
as well as posteriorizes, the embryo (57, 68,
109, 117). Loss of wnt3a by morpholino knockdown appears to enhance the wnt8 loss of function defects (117), suggesting that wnt3a plays
a partially redundant modulatory role to wnt8
primarily in anterioposterior patterning. Dorsally, wnt8 expression is inhibited by Bozozok
(26). Recently, the transcription factor Kaiso
zinc finger–containing protein (Kzp) was identified as a maternal factor required for the zygotic expression of wnt8 (Figure 1c) (140). Kzp
misexpression and loss of function posteriorized and dorsalized embryos, respectively (140).
Kzp adds to the maternally expressed transcription factors of Runx2b and Pou2 that regulate
the expression of specific zygotic genes acting
in dorsoventral patterning.
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FIBROBLAST GROWTH
FACTOR SIGNALING AND
DORSOVENTRAL PATTERNING
FGF signaling is implicated in a number of
developmental processes from early patterning and axis formation to organogenesis (47–
49, 54). The roles of FGF signaling during
dorsoventral patterning are not well understood, although it is clear that it acts in part
by inhibiting BMP signaling (Figure 2b) (32,
33). Zebrafish fgf3/fgf8/fgf24 are expressed in
the dorsal margin at blastula stages and function
to repress the initial dorsal expression of bmp2b
and bmp7 independently of Chordin (Figure 3)
(32, 33). fgf8 depletion alone is insufficient to
ventralize embryos; however, its depletion enhances the ventralization of chordino mutants.
These results suggest functional redundancy of
Fgf8 with additional Fgfs and that a specific
role for fgf8 in dorsoventral patterning is normally masked by Chordin (33). Additional studies suggest an earlier role for Fgf signaling in organizer formation (Figure 2a). In the absence
of Fgf signaling, β-catenin is insufficient to rescue ichabod mutants, indicating a requirement
for Fgf signaling downstream of β-catenin to
induce organizer formation (76).
PERSPECTIVES
In recent years, a number of discoveries have
expanded our knowledge of early patterning
and axis formation in zebrafish. From recessive
maternal-effect mutagenesis screens, factors
functioning in animal-vegetal polarity, regulators of previously identified dorsoventral axis
patterning components, and a component of
an early microtubule transport network have
been identified. However, there are still many
unidentified factors involved in formation of
the vertebrate body plan; in fact, fundamental
questions remain. For example, what is the
identity of the vegetally localized dorsal
determinant(s)? In Xenopus, vegetally localized
maternal wnt11 and wnt5a appear to be the
dorsal determinants acting in conjunction with
cortical rotation to activate Wnt signaling and
β-catenin accumulation in the prospective
dorsal-most blastomeres (9, 67, 125, 133).
Thus, it is tempting to speculate that a Wnt
family member could be the elusive dorsal
determinant necessary to establish the dorsal
side of the zebrafish embryo.
What is the mechanism by which the dorsal
determinant(s) is translocated asymmetrically
to establish the dorsoventral axis? What is
the precise function of the microtubule linker
protein Syntabulin in this process? Key to
the asymmetric translocation of the dorsal
determinant(s) is the establishment of the
parallel microtubule array at the vegetal pole
of the egg following fertilization (52, 81). How
is this parallel array established, and how is its
orientation determined? In Xenopus, the position of the sperm entry point and sperm aster
determine the orientation of the vegetal microtubule array required for dorsal determinant
translocation (132a). In zebrafish, the sperm
enters the egg through the single animally
localized micropyle (39a), and it is not known if
sperm aster positioning plays a role in orienting
the vegetal microtubule array like in Xenopus.
Several maternally supplied transcription
factors have now been identified that regulate
the expression of a number of zygotic genes acting in ventral cell fate specification. Runx2bt2,
Pou2, and Kzp are required for the initiation or
maintenance of vox/vent/ved, bmp2b and bmp7a,
and wnt8, respectively. What additional factors
function with these to regulate gene expression,
and do gene regulatory networks exist?
Few studies have addressed the posttranscriptional and posttranslational regulation of
protein production and degradation. Because
zebrafish embryos develop rapidly, a mechanism to rapidly turn over proteins could be
essential. Identification of Lnx2b as a regulator of Bozozok protein degradation suggests
that such mechanisms may regulate the levels
of other proteins functioning in dorsoventral
patterning.
The BMP signaling gradient that patterns dorsoventral tissues in zebrafish functions
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exclusively via Bmp2b-Bmp7a heterodimer signaling. Why is it that only BMP heterodimers
signal in this process? Do BMP antagonists
preferentially bind BMP homodimers leaving
BMP heterodimers to do the signaling? Do
BMP heterodimers bind with higher affinity
to the heteromeric BMP receptors than BMP
homodimers? Alternatively, does another extracellular modulator facilitate the delivery of
BMP heterodimers to its receptors or abrogate the delivery of BMP homodimers? Further
studies are required to elucidate the mechanism
of BMP heterodimer signaling.
Most studies have focused on fixed developmental stages and thus do not readily
account for the coordination of patterning and
signaling systems in time and space during
development. Recent studies suggest that patterning of the dorsoventral and anteroposterior
axes is coordinated in a temporally progressive
manner from anterior to posterior (128). Is patterning of the dorsoventral and anteroposterior
axes coordinated as suggested or does each
function by distinct temporal mechanisms? If
they are temporally coordinated, what is the
mechanism? Is there a competence factor that
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allows progressively more posterior tissues to
be patterned by both the dorsoventral and anteroposterior patterning mechanisms? Is there
an inhibitory factor that must be repressed in
progressively more posterior tissues to allow
dorsoventral and anteroposterior tissues to
be patterned? Do the anteroposterior axis
signaling pathways, Wnt, FGF, and Retinoic
acid signaling, act on the dorsoventral patterning mechanism to modulate its function and
coordinate the patterning of these two axes?
Many extracellular factors have now been
identified that modulate the BMP signaling
gradient. However, how these factors function together in a spatiotemporal manner
to generate the gradient is not understood.
Mathematical modeling may be valuable at
this point to generate testable hypotheses of
the extracellular modulation of BMP signaling
and gradient formation. Interpretation of the
gradient also has yet to be elucidated. How
does the phospho-Smad1/5 gradient ultimately
generate distinct cell types? And how do
distinct phospho-Smad1/5 levels elicit distinct
expression domains? These and many other
questions remain to be elucidated.
SUMMARY POINTS
1. Studies of maternal-effect mutants have led to the identification of several previously unknown maternal factors that function in establishing the dorsoventral axis or the animalvegetal axis via regulation of the Balbiani body.
2. Maternal factors actively promote both dorsal and ventral cell fate specification in embryonic dorsoventral axis formation.
3. A protein degradation mechanism, as well as positive and negative transcriptional regulation, including autoregulatory loops restrict organizer gene expression to the dorsal-most
regions and maintain ventral-specific gene expression.
4. Multiple levels of extracellular modulation generate a robust BMP signaling gradient
that acts as a morphogen to establish distinct cellular fates along the dorsoventral axis.
5. BMP heterodimers acting through a heteromeric type I receptor complex signal exclusively in dorsoventral patterning.
6. BMP signaling acts in a temporally progressive manner along the anteroposterior axis to
pattern dorsoventral tissues.
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FUTURE ISSUES
1. Identify the dorsal determinant(s) that is asymmetrically translocated shortly after fertilization and during the cleavage stage.
2. Determine the mechanism that generates and orients the parallel microtubule array at
the vegetal pole and the mechanism by which it translocates the dorsal determinant(s).
3. Understand the role of protein degradation in dorsoventral embryonic axial patterning.
4. Determine the mechanism that restricts signaling to BMP heterodimers in dorsoventral
patterning.
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5. Understand how the BMP morphogen gradient is interpreted by cells to establish the
distinct dorsoventral cell fates of the embryo.
6. Determine the mechanism by which dorsoventral cell fates are specified in a temporally
progressive manner along the anteroposterior axis.
7. Determine how the multitudes of extracellular modulators of BMP signaling function in
space and time to generate the robust BMP signaling gradient.
DISCLOSURE STATEMENT
The authors are not aware of any affiliations, memberships, funding, or financial holdings that
might be perceived as affecting the objectivity of this review.
ACKNOWLEDGMENTS
We would like to thank James Dutko, Elliott Abrams, Megumi Hashiguchi, and Lee Kapp for
comments and careful reading of this manuscript. We would also like to acknowledge grants from
the National Institutes of Health R01- GM56326, The MORE Division of NIH, and Penn-PORT
Program for support.
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Contents
Volume 45, 2011
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Comparative Genetics and Genomics of Nematodes: Genome
Structure, Development, and Lifestyle
Ralf J. Sommer and Adrian Streit p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 1
Uncovering the Mystery of Gliding Motility in the Myxobacteria
Beiyan Nan and David R. Zusman p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p21
Genetics and Control of Tomato Fruit Ripening and Quality Attributes
Harry J. Klee and James J. Giovannoni p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p41
Toxin-Antitoxin Systems in Bacteria and Archaea
Yoshihiro Yamaguchi, Jung-Ho Park, and Masayori Inouye p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p61
Genetic and Epigenetic Networks in Intellectual Disabilities
Hans van Bokhoven p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p81
Axis Formation in Hydra
Hans Bode p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 105
The Rules of Engagement in the Legume-Rhizobial Symbiosis
Giles E.D. Oldroyd, Jeremy D. Murray, Philip S. Poole, and J. Allan Downie p p p p p p p p 119
A Genetic Approach to the Transcriptional Regulation
of Hox Gene Clusters
Patrick Tschopp and Denis Duboule p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 145
V(D)J Recombination: Mechanisms of Initiation
David G. Schatz and Patrick C. Swanson p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 167
Human Copy Number Variation and Complex Genetic Disease
Santhosh Girirajan, Catarina D. Campbell, and Evan E. Eichler p p p p p p p p p p p p p p p p p p p p p p 203
DNA Elimination in Ciliates: Transposon Domestication
and Genome Surveillance
Douglas L. Chalker and Meng-Chao Yao p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 227
Double-Strand Break End Resection and Repair Pathway Choice
Lorraine S. Symington and Jean Gautier p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 247
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11 October 2011
19:52
CRISPR-Cas Systems in Bacteria and Archaea: Versatile Small RNAs
for Adaptive Defense and Regulation
Devaki Bhaya, Michelle Davison, and Rodolphe Barrangou p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 273
Human Mitochondrial tRNAs: Biogenesis, Function,
Structural Aspects, and Diseases
Tsutomu Suzuki, Asuteka Nagao, and Takeo Suzuki p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 299
The Genetics of Hybrid Incompatibilities
Shamoni Maheshwari and Daniel A. Barbash p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 331
Annu. Rev. Genet. 2011.45:357-377. Downloaded from www.annualreviews.org
by Reed College on 07/26/13. For personal use only.
Maternal and Zygotic Control of Zebrafish Dorsoventral Axial
Patterning
Yvette G. Langdon and Mary C. Mullins p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 357
Genomic Imprinting: A Mammalian Epigenetic Discovery Model
Denise P. Barlow p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 379
Sex in Fungi
Min Ni, Marianna Feretzaki, Sheng Sun, Xuying Wang, and Joseph Heitman p p p p p p 405
Genomic Analysis at the Single-Cell Level
Tomer Kalisky, Paul Blainey, and Stephen R. Quake p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 431
Uniting Germline and Stem Cells: The Function of Piwi Proteins
and the piRNA Pathway in Diverse Organisms
Celina Juliano, Jianquan Wang, and Haifan Lin p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p p 447
Errata
An online log of corrections to Annual Review of Genetics articles may be found at http://
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Contents
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