1. Available online at www.sciencedirect.com
Early zebrafish development: It’s in the maternal genes
Elliott W Abrams and Mary C Mullins
The earliest stages of embryonic development in all animals
examined rely on maternal gene products that are generated
during oogenesis and supplied to the egg. The period of
maternal control of embryonic development varies among
animals according to the onset of zygotic transcription and the
persistence of maternal gene products. This maternal
regulation has been little studied in vertebrates, owing to the
difficulty in manipulating maternal gene function and lack of
basic molecular information. However, recent maternal-effect
screens in the zebrafish have generated more than 40 unique
mutants that are providing new molecular entry points to the
maternal control of early vertebrate development. Here we
discuss recent studies of 12 zebrafish mutant genes that
illuminate the maternal molecular controls on embryonic
development, including advances in the regulation of animal–
vegetal polarity, egg activation, cleavage development, body
plan formation, tissue morphogenesis, microRNA function and
germ cell development.
Address
University of Pennsylvania School of Medicine, Department of Cell and
Developmental Biology, 1211 BRB II/III, 421 Curie Blvd, Philadelphia,
PA 19104-6058, United States
Corresponding author: Mullins, Mary C (mullins@mail.med.upenn.edu)
Current Opinion in Genetics & Development 2009, 19:396–403
This review comes from a themed issue on
Pattern formation and developmental mechanisms
Edited by Kathryn Anderson and Kenneth Irvine
Available online 14th July 2009
0959-437X/$ – see front matter
# 2009 Elsevier Ltd. All rights reserved.
DOI 10.1016/j.gde.2009.06.002
Introduction
The zebrafish has emerged as a premiere genetic tool for
studying vertebrate development. In the 1990s forward
genetic screens identified numerous zygotic mutants
defective in key molecules important in early embryonic
development [1–7]. Recently, a major focus has shifted
towards more specialized screens, including the identi-
fication of maternal-effect mutations in adult screens in
the zebrafish. Since the earliest stages of development
are driven primarily by maternal gene products, the
identification of corresponding mutants is crucial to
provide genetic entry points to known maternally con-
trolled processes, which are still poorly understood in
vertebrates.
Maternal-effect screens have identified over 40 mutants
affecting many early developmental processes (Figure 1).
Collectively, these mutants have defects in oocyte de-
velopment [8], egg activation [8,9], embryonic cleavage
[8–11], patterning and morphogenesis [9–11]. New stu-
dies have focused on the molecular identification of the
corresponding mutant genes, a key step to understand-
ing the molecular mechanisms governing the very
earliest stages of embryogenesis. The results have
unveiled novel and known genes with unanticipated
roles in early development. Mutants generated by
reverse genetic TILLING methods in the zebrafish
have also recently revealed important roles for small
noncoding RNA molecules, miRNAs [12] and piRNAs
[13,14], in the regulation of maternal processes in zebra-
fish development.
In this review, we highlight recent contributions to the
molecular regulation of animal–vegetal polarity in the
oocyte and egg, maternal gene regulation of early embryo-
nic patterning, tissue morphogenesis and small noncoding
RNA molecules, which are emerging as important players
in germ line development.
Molecular insights into zebrafish animal–
vegetal polarity
In frogs and fish, the first developmental asymmetry of
the embryo is the animal–vegetal axis, which predicts the
anterior–posterior axis of the embryo. This asymmetry is
established during early stages of oogenesis and is first
marked by formation of the Balbiani body (Bb; also
referred to as the mitochondrial cloud) adjacent to the
germinal vesicle (the oocyte nucleus) in stage I oocytes.
The Bb position predicts the location of the vegetal pole,
but its presence is only transient, as it disassembles by
stage II of oogenesis. The Bb is composed of a collection
of mitochondria, ER, germinal granules and several germ
plasm mRNAs (reviewed in [15]). Recently, a thorough
study recapitulated in zebrafish transgenic constructs the
localization pattern of three Bb-localized transcripts. The
30
UTRs of nanos, vasa and dazl directed their localization
initially to the Bb and then to the vegetal cortex of the
oocyte [16
], probably via the METRO pathway
described in Xenopus [17]. The ensuing distribution at
the vegetal cortex differs among these transcripts: dazl
persists at the vegetal cortex, vasa extends around the
cortex, and nanos becomes unlocalized (Figure 2),
suggesting, unexpectedly, that coordinate localization
of germ plasm components is followed by their redistri-
bution to distinct locations. However, after fertilization of
the egg, these germ plasm RNAs reunite in the blastodisc
at the animal pole, where they localize to the germ plasm
Current Opinion in Genetics Development 2009, 19:396–403 www.sciencedirect.com

2. Maternal Zebrafish Development Abrams and Mullins 397
Figure 1
Maternal-effect mutant genes in zebrafish disrupt development at distinct stages. Mutants discussed here are indicated in red. acytokinesis [11],
atomos [9], aura [9], barrette [9], bo peep [9], bedazzled [10], blistered [10], claustro [9], cobblestone [9], emulsion [8], golden gate [9], indivisible [8],
irreducible [8], jumpstart [8], kwai [9], misson impossible [9], nebel [9], over easy [8], poky [10], pollywog [10], pug [10], slow [10], ruehrei [8], souffle [8],
screeching halt [10], sunny side up [8], under repair [9], waldo [9], weeble [9].
Figure 2
mRNA localization during oocyte development. During stage I of oogenesis buc, nanos, vasa and dazl transcripts localize to the Balibani body (Bb,
pink), while cyclinB begins to be localized to the animal pole. By stage II of oogenesis the Bb has disassembled, leaving buc, vasa and dazl mRNAs at
the vegetal cortex, whereas nanos becomes unlocalized, and pou2 becomes localized to the animal pole. Note vasa has a broad vegetal cortical
domain at stage II. By stage III bruno-like and mago nashi become vegetally localized (late pathway); now buc, and Vg1 are localized to the animal pole
and vasa is localized radially at the cortex. Animal (An) pole is to top and vegetal (Ve) to bottom.
www.sciencedirect.com Current Opinion in Genetics Development 2009, 19:396–403

3. at the cleavage furrows of the four-cell stage embryo
[16
,18–21].
Three regions within the dazl 30
UTR are crucial for (1)
localization to the Bb, (2) anchoring to the vegetal cortex,
and (3) localization to the cleavage furrows in the early
embryo [16
]. Interestingly, although the 30
UTR of the
nanos-related gene Xcat2 in Xenopus, also localizes it to
the Bb and vegetal cortex in zebrafish, little sequence
similarity is evident in the 30
UTRs of Xcat2, and zebrafish
nanos, dazl and vasa [16
], suggesting either small motifs,
or secondary/tertiary structure of the UTRs are con-
served. Thus, germ plasm mRNA localization occurs in
a stepwise, spatiotemporal fashion, with conserved fea-
tures to the transport systems in Xenopus [16
].
Recent studies have provided genetic access into the
mechanisms of Bb formation and germ plasm assembly
in the oocyte. The maternal-effect mutant bucky ball (buc),
identified through its animal–vegetal polarity egg pheno-
type [8], is defective in Bb formation and early oocyte
polarity, as animal pole markers are expanded radially and
vegetal pole markers are unlocalized [22
,23
]. Buc is
required for early vegetal pole mRNA localization that
occurs via the Bb, as well as late vegetal pole localization
that occurs after Bb dissociation, which is postulated to
depend on the early pathway [24,25]. Thus, the failure of
late vegetal pole mRNA localization in buc mutant
oocytes may be secondary to the lack of Bb formation.
Buc functions to promote vegetal and exclude animal
pole identity also in the surrounding follicle cell layer,
preventing the formation of multiple micropylar cells, an
animal pole-specific follicle cell fate. Thus, patterning of
the oocyte and surrounding somatic follicle cell layer
appears to be coordinated through an as yet unknown
signaling mechanism [22
].
Recently the molecular identity of buc was determined to
be a novel 639 amino acid protein [23
], first identified as
XVelo1 in Xenopus [26]. The predicted Buc protein
contains no known functional motifs. The localization
of buc mRNA is dynamic during oocyte development,
initially localizing to the Bb, moving vegetally and then
ultimately localizing to the animal pole during late
oogenesis [23
] (Figure 2). A Buc-GFP fusion also loca-
lizes to the Bb and, interestingly, to the germ plasm of the
embryo and can induce ectopic germ cells in the embryo
[23
]. Buc homologues in other vertebrates have low
predicted amino acid conservation, indicating that it
has evolved at a relatively accelerated rate. Interestingly,
the human version lacks a complete open reading frame,
suggesting that it has lost its function all together in
humans or functions as an RNA. In zebrafish Buc clearly
functions as a protein, since a nonsense mutation trun-
cating it by just 30 amino acids causes a failure in Bb
formation [23
]. However, the buc RNA could have
additional functions in Bb formation and animal–vegetal
polarity, similar to oskar in Drosophila [27], also a germ
plasm component. Future structure-function exper-
iments of the buc RNA/protein, together with the iso-
lation of interacting partners will unravel mechanistically
how this novel gene functions.
Egg activation molecular genetics
In zebrafish egg activation is marked by cortical granule
exocytosis (CGE), chorion elevation and the segregation
of cytoplasm from the yolk to the animal pole to form the
single cell blastodisc. Embryos derived from brom bones
(brb) mutant females are defective in this process [28].
The egg activation defect is a result of failure of inositol
1,4,5-triphosphate (IP3) signaling, which induces a Ca2+
wave crucial for normal egg activation in all animals
examined. The reinstatement of either IP3 or Ca2+
in
brb mutant eggs can rescue the egg activation defect [28].
brb was shown to encode heterogeneous nuclear ribonu-
cleoprotein I (hnRNP I), probably regulating during
oogenesis the production of an egg activation signaling
component. hnRNP I has been previously shown to be
important in a variety of developmental processes in-
cluding translational control in oogenesis [29], spermato-
genesis [30] and RNA localization [31–34]. Thus, brb
mutants reveal yet another developmental function for
hnRNP I, that is, a role in egg activation.
Cleavage stage molecular genetics
Maternal-effect screens have yielded a significant num-
ber of mutations affecting cleavage development [8–11],
although most of the mutant genes have yet to be cloned.
cellular atoll (cea) mutants fail to undergo cleavages at the
second cell division and beyond [8,35], and was recently
shown to encode the centriolar component sas-6 [35].
Interestingly, as sperm normally provide the centriole to
the zygote, cea also has a paternal-effect function,
whereby wild-type eggs fertilized with cea mutant sperm
result in inviable tetraploid embryos. Since the cleavage
stage of development is primarily under maternal control,
the eventual molecular identification of other maternal-
effect mutant genes functioning during this period will
enhance our understanding of this important stage of
development.
The maternal-effect cellular island (cei) mutant displays an
early defect in cleavage furrow formation [8]. cei encodes a
hypomorphic allele of Aurora B Kinase [36], a protein
previously shown to be important in several aspects of cell
division in other systems (reviewed in [37]). Aurora B
Kinase function is vital zygotically later in zebrafish
embryogenesis, as a null retroviral insertional allele
reveals furrow defects during this period [36,38]. Inter-
estingly, the maternal-effect cei allele causes a specific
defect in distal furrow formation during the early cleavage
stage, while medially positioned furrows can form, prob-
ably owing to intact mitotic spindle-derived signals med-
iating medial furrow formation [36]. The ability of the cei
398 Pattern formation and developmental mechanisms
Current Opinion in Genetics Development 2009, 19:396–403 www.sciencedirect.com

4. mutant Aurora B Kinase protein to mediate all its cyto-
kinesis functions zygotically, but its inability to mediate
distal furrow formation in the cleavage embryo may be
related to unique requirements for astral microtubules
and Aurora B Kinase to divide the large cells of the very
early embryo [36].
Molecular genetic advances in maternal
regulated patterning and morphogenesis
In fish and amphibians, the dorsoventral embryonic axis is
established through a maternally regulated Wnt/bcatenin
pathway. Several zebrafish maternal-effect mutants with
defects in dorsoventral axis formation have been ident-
ified. The maternal-effect ventralized mutants hecate and
tokkaebe produce embryos lacking a dorsal organizer and
consequently are radially ventralized. Although hecate and
tokkaebe have not yet been molecular defined, injection/
rescue experiments with Wnt signaling pathway com-
ponents indicates that they act upstream of, or within a
Wnt/bcatenin signaling pathway, respectively, to induce
the dorsal organizer [39–41]. tokkaebe probably corre-
sponds to a novel component of the Wnt pathway, since
no known Wnt components are found within the 0.5 cM
interval containing the tokkaebe mutation [41].
The maternal-effect ventralized mutant ichabod is caused
by a specific loss of maternal bcatenin2 function [42].
Two bcatenin genes have been identified in the zebrafish
genome; however, only bcatenin2 is essential maternally
to induce the dorsal organizer through a Wnt signaling
pathway. Interestingly, the maternal, dorsal organizer
Wnt/bcatenin2 pathway also functions to repress expres-
sion of a later Wnt8/bcatenin pathway that opposes dorsal
specification ventrally [42] (Figure 3). In zebrafish this
late blastula Wnt pathway depends on both bcatenin1 and
bcatenin2 function and is mediated by zygotic Wnt8
signaling ventrally [42–45]. Thus in the absence of both
bcatenin1 and bcatenin2 function, early dorsal organizer
formation fails; however, at later stages the ventral Wnt8
pathway also fails to oppose dorsal fate specification
ventrally. The result is the formation of dorsal tissues
circumferentially. These results show, surprisingly, that
at a late blastula stage circumferential organizer-like
tissue can form in the absence of a maternal Wnt/bcatenin
pathway and independently of Wnt/bcatenin signaling
entirely.
The transcription factor pou2/oct4 also acts maternally in
dorsoventral patterning and morphogenesis [46,47],
revealing additional roles to its described maternal func-
tion in endoderm specification [48,49]. Loss of both
maternal and zygotic (MZ) Pou2 causes severe dorsaliza-
tion owing to failure to induce bmp ligand gene expres-
sion, which functions in ventrolateral tissue specification
[46]. These results demonstrate that ventral specification
via the BMP signaling pathway is not a default pathway,
as previously thought, but instead is initiated by maternal
Pou2/Oct4 in the early embryo [46] (Figure 3). MZPou2
Maternal Zebrafish Development Abrams and Mullins 399
Figure 3
The role of BMP and early and late Wnt signaling in dorsoventral patterning. (a) Maternal Pou2 induces early zygotic bmp ligand expression. bmp
expression is initially present throughout the blastoderm (grey). An early maternal Wnt signal from the yolk cell promotes future dorsal organizer (DO)
formation. (b) By the late blastula stage (50% epiboly), a BMP activity gradient (highest ventrally (black)) is established that promotes ventral fates. At
this stage Wnt 8 signaling opposes dorsal fate specification ventrally. Ventral is positioned to the left and dorsal to the right.
www.sciencedirect.com Current Opinion in Genetics Development 2009, 19:396–403

5. also regulates the morphogenic process of epiboly, the
thinning and spreading of the blastoderm over the yolk
cell, through functions in yolk cell microtubule formation,
cell adhesive properties and blastoderm cell movements
via a cell non-autonomous mechanism [46,47]. Together,
these studies reveal that the renowned pou2/oct4 stem cell
gene in mammals is a key maternal regulator of early
zebrafish development.
The betty boop (bbp) mutant, identified in a maternal-
effect screen, is a strictly, maternally acting gene reg-
ulating the morphogenic process of epiboly [10,50
].
Embryos from bbp mothers develop normally until they
reach 50% epiboly at which point the embryo abruptly
bursts via a presumptive premature constriction of the
actin cytoskeleton in the yolk cell [50
]. Interestingly,
bbp was recently shown to encode the zebrafish homol-
ogue of Mitogen Activated Protein Kinase Activated
Protein Kinase 2 (MAPKAPK2), a target of p38 MAP
kinase (MAPK) in cell culture systems [51]. During
zebrafish epiboly, p38 MAPK also appears to activate
MAPKAPK2, as a dominant-negative p38 MAPK causes
the same epiboly defect as loss of bbp [50
]. Neither p38
MAPK, nor MAPKAPK2 have been previously impli-
cated in tissue morphogenesis. Thus, the identification
and cloning of the bbp gene is a model genetic case for an
unexpected pathway being placed in a developmental
process, in this case epiboly, which may not have
been considered in a candidate-gene reverse genetic
approach.
Small RNA molecules in early development
Small noncoding RNAs are emerging as important
players in early zebrafish development. Maternal-zygo-
tic (MZ) mutant embryos of the microRNA (miRNA)-
processing enzyme Dicer exhibit early embryonic
defects in gastrulation, somitogenesis, brain morpho-
genesis and heart development [52]. By removing the
strong maternal component of dicer, MZ-dicer mutant
embryos are completely devoid of miRNA processing
and therefore, devoid of all miRNA function. miRNAs
negatively regulate target genes by binding to their
30
UTR, promoting deadenylation, translational repres-
sion and/or ultimately degradation of the transcript
(reviewed in [53]). In zebrafish, the predominantly
expressed miRNA during early embryogenesis is miR-
430, which is first expressed at the mid-blastula tran-
sition (MBT), and is not expressed maternally [54]. A
central finding revealed by the loss of miR-430 through
MZ-dicer is its role in the clearance of maternal mRNAs
at the MBT. In MZ-dicer mutant embryos, maternal
mRNAs abnormally persist beyond the MBT [12]. To
date, no maternal miRNAs have been reported in the
zebrafish embryo [54]. Thus, it is unclear whether the
maternal role of dicer is to primarily process miR-430 at
the MBT or in addition, also process unidentified
maternal miRNA(s).
Inhibition of miRNA function plays a role in germ line
development. nanos1 is expressed early in germ line
development and a mutant, generated through TIL-
LING methods, demonstrates its maternal requirement
for PGC survival and a function in the adult in maintain-
ing oocyte production [55]. nanos1 is resistant to miRNA
repression in the germ line, but not in the soma, promot-
ing its specific expression in the germ line [56]. This
resistance is conferred, at least partly, by the maternally
expressed germ cell-localized RNA binding protein Dead
End (Dnd), which interacts with the nanos1 30
UTR,
presumably blocking the binding of miR-430 [57
]. Inter-
estingly, this resistance to miRNA repression is also found
in another germ plasm mRNA, Tudor-domain-contain-
ing-7 (Tdrd-7) [56], suggesting a general mechanism
involving Dnd in influencing germ cell-specific gene
expression (Figure 4).
Germ plasm mRNA resistance to miRNA regulation is
not universal, however, as the dnd 30
UTR itself lacks a
miR-430 site and therefore is presumably not repressed in
the soma by miRNAs [58]. Since dnd maternal transcripts
are dramatically eliminated in the soma about one hour
after the MBT [59], independently of miR-430 regula-
tion, it suggests the existence of additional mode(s) of
modulating maternal transcripts at this crucial develop-
400 Pattern formation and developmental mechanisms
Figure 4
miRNA regulation of germ line development. In the soma miR-430
inhibits expression of germ line specific genes (GLSGs). In the germ line
Dnd blocks miR-430-mediated repression of GLSGs. Note that an
unidentified factor X is required in the soma to silence GLSGs that are
not regulated by miR-430. Soma shown in grey; germ line in purple.
Current Opinion in Genetics Development 2009, 19:396–403 www.sciencedirect.com

6. mental transition. Likewise, vasa removal from the soma
is also independent of miR-430 regulation [56]. Thus, an
unknown, possibly common additional mechanism elim-
inates dnd and vasa transcripts from the soma.
A Dicer-independent class of small noncoding RNAs,
known as piRNAs or Piwi associated RNAs, appear to
be germ line specific [13,14,60,61]. Piwi proteins are
important for target gene silencing [62]. Presumptive
null, zygotic mutations in either of two piwi homologues
in zebrafish, ziwi and zili, cause the progressive loss of
germ cells between three and seven weeks of age [13,14].
Mutant adults are phenotypically male, consistent with
recent studies demonstrating that zebrafish develop phe-
notypically as male when eliminating the germ line
[13,63,64]. Interestingly, there is a maternal-effect meio-
tic progression defect in zili hypomorphic mutants.
Although mutant eggs can be fertilized, they fail to
undergo meiosis I and II [13]. Piwi proteins in the mouse
function to repress transposon activity in the germ line
[65,66]. The meiotic defects in zili hypomorphs mutants,
however, are not linked to increased transposon activity as
measured by quantitative RT-PCR and in situ hybridiz-
ation [13]. These findings reveal a novel function for
piRNAs, whose function in vertebrates was previously
thought confined to regulating transposon activity in the
germ line [13].
Future outlook
A major hindrance to the molecular cloning of chemically
induced mutant genes in zebrafish is the incomplete
assembly of the genomic sequence. This obstacle is
becoming less of an issue with recent improvements to
the assembly (currently Zv8; www.sanger.ac.uk). As exist-
ing gaps are eliminated and the genome sequence is
completed, the molecular cloning of maternal-effect
mutant genes will be greatly accelerated.
The investigation of the maternal functions of essential
zygotic genes will be more difficult to study. Although
germ line chimeric analysis is a successful method to
examine the maternal function of zygotic lethal genes in
zebrafish [67], it is quite labour intensive and would be
impractical for large throughput analysis of such maternal
function. This problem may be overcome by employing
techniques that utilize mitotic recombination, a principle
heavily relied upon in Drosophila genetics [68]. RecQ
helicases are known to prevent recombination during
replication [69]. Induced mitotic recombination through
the suppression of RecQ helicases was recently demon-
strated in zebrafish [70]. This approach can generate
mutant clones from heterozygous cells at a significant
frequency (1.7–3.4%) [70]. With further improvements
in this technology, mitotic recombination could poten-
tially be used to generate homozygous mutant germ line
clones of zygotic lethal mutations from heterozygous
individuals. Such germ line mosaic females would gen-
erate maternally deficient eggs and embryos, allowing the
study of the maternal gene function.
Reverse genetic techniques are very valuable comp-
lements to forward genetic approaches in the zebrafish.
Antisense morpholino oligos are widely used to block
translation or disrupt splicing [71,72]. However, the func-
tion of maternal protein already present in the egg cannot
be blocked by morpholino injection into the egg,
although advances in oocyte cell culture methods may
make it possible to use this method in the future to
examine maternal gene functions. Reverse genetic
approaches that rely on induction of genomic sequence
alterations are considerably more laborious, but generate
robust loss-of-function reagents. Recently, the TILLING
(Target Induced Local Lesions in Genomes) approach
[13,14,55], as well as the zinc finger nuclease approach
designed to mutate a specific sequence of the genome
[73–75], have been very successful in generating mutants
in zebrafish. Hence, TILLING and zinc finger nuclease
strategies look very promising for eliminating gene func-
tion of suspected maternal-effect genes in zebrafish in the
future.
Acknowledgements
We thank Lee Kapp for comments on the manuscript, and Eric Weinberg
and Mate Varga for helpful discussion. Funding was provided by NIH grant
HD050901 to MCM.
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