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MOLECULAR GENETICS OF EMBRYOGENESIS
IN XENOPUS AND ZEBRAFISH
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Igor
B. Dawid, PhD, Head, Section on
Developmental Biology Reiko
Toyama, PhD, Staff Scientist Mizuki
Azuma, PhD, Postdoctoral Fellowa Sung-Kook
Hong, PhD, Postdoctoral Fellow Nobuhiro
Takahashi, PhD, Postdoctoral Fellow Kosuke
Tanegashima, PhD, Postdoctoral Fellow Michael
Tsang, PhD, Postdoctoral Fellowa Haiyan
Wan, PhD, Postdoctoral Fellow Hui Zhao,
PhD, Postdoctoral Fellow Martha
Rebbert, BS, Senior Technician Elizabeth
Laver, BA, Technician Reema
Kar, Student |
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The
laboratory is engaged in studies of molecular-genetic mechanisms of early
vertebrate development, using the frog Xenopus laevis and the
zebrafish Danio rerio as experimental systems. Combinatorial
action of FGF and BMP signaling in patterning the early nervous system Dawid; in
collaboration with Kudoh, Wilson Since
the discovery of the organizer and neural induction by Spemann and Mangold in
1924, the initial formation of the nervous system in the vertebrate embryo
has undergone extensive study in several model systems. Work carried out in
the past decade mostly in the frog Xenopus has suggested a model in
which the embryonic ectoderm has an innate tendency to develop into neural
tissue unless diverted into epidermal differentiation by signaling from bone
morphogenetic proteins (BMPs). The organizer carries out neural induction by
producing BMP antagonists that allow the innate neural tendency of the
ectoderm to emerge. This model proved incomplete according to the more recent
work of several laboratories, which showed that signaling by fibroblast
growth factors (FGFs) is critically involved in neural specification. At the
time of its initial induction, the neural ectoderm is patterned along the
anterior-posterior axis so that head-to-tail polarity emerges coincidentally
with the formation of the nervous system. Several studies have shown that
FGF, along with certain other factors, confers more posterior properties on
the neural ectoderm. Our studies on the subject, carried out in the zebrafish
embryo, have illuminated the interactions of BMP and FGF signaling in the
ectoderm during gastrulation that lead to the specification of the nervous
system and its patterning along the anterior-posterior axis. We found that,
in contrast to earlier views, high BMP levels are compatible with neural
differentiation, provided that FGF signaling is also active and that both FGF
and BMP act as posteriorizing factors in addition to their role in neural
specification per se. Summarizing these results simply, we conclude that the
fate of ectodermal domains is specified combinatorially by BMP and FGF
signaling in the following way: a BMP-/FGF state leads to head neural
differentiation, BMP-/FGF+ specifies trunk neural, BMP+/FGF+ specifies tail
neural, and a BMP+/FGF state specifies ectoderm as future epidermis. In the
further elaboration of the anterior-posterior pattern, other signaling
systems such as Wnt factors and retinoic acid also play an important role. Kudoh T, Concha ML, Houart C, Dawid IB, Wilson SW. Combinatorial
Fgf and Bmp signaling patterns the gastrula ectoderm into prospective neural
and epidermal domains. Development 2004;131:3581-3592. Kudoh T, Wilson SW, Dawid IB. Distinct roles for Fgf, Wnt and
retinoic acid in posteriorizing the neural ectoderm. Development 2002;129:4335-4346.
Application
of DNA microarray technology to Xenopus development Zhao, Rebbert, Dawid Developmental
processes are associated with large-scale changes in gene expression. These
changes are the result of developmental events and drive the specification
and subsequent differentiation of cells and tissues in the embryo. DNA
microarray technology has provided an opportunity to analyze these changes in
gene expression on a large scale. The Xenopus embryo has long been a
premier model system for studying vertebrate development. Recently,
microarrays for Xenopus have become available through Affymetrix, and
we have initiated a project to take advantage of this development. We
designed the initial project primarily to test the suitability of the arrays,
which had just been released. We used a well-established system in which
ectodermal explants, so called animal caps, are exposed to activin, which
induces mesoderm and endoderm in these cells. This induction is associated
with extensive gene activation, and many activin-induced genes have been
described. We found that array analysis faithfully reproduced previously established
responses such that over 20 known activin-induced genes were found by array
analysis to be activated in the treated animal caps. The experiments
validated the usefulness of the microarray chips but also provided a large
number of activin-induced genes that had not previously been studied. Some of
these genes are now under further investigation. A
zebrafish mutant that impairs ribosomal RNA maturation Azuma, Dawid A
majority of mutants studied to date in zebrafish developmental genetics
affect patterning in the embryo, and many result from disruption of
regulatory genes such as transcription factors and signaling molecules. The
analysis of a mutant we found during a genetic screen provided an opportunity
to apply the power of genetic analysis to basic cellular processes. The
mutant, named bap28, affects the brain, leading to malformations and
widespread apoptosis starting before 24 hours of development. Positional
cloning showed that the gene responsible for the mutation encodes a large
protein similar to the human protein BAP28, which had not been characterized.
However, the apparent ortholog in yeast, called UTP10, is known to be a
component of a nucleolar ribonucleoprotein complex involved in the maturation
of 18S ribosomal RNA (rRNA). Thus, we hypothesized that BAP28 may have a
similar function and studied rRNA processing in normal and mutant zebrafish.
With the mechanism of rRNA processing in zebrafish unknown, we generated a
basic outline of the processing pathway, which is similar but not identical
to that in Xenopus. We found that bap28 mutant embryos have a
defect in the accumulation of 18S rRNA precursors, supporting the view that
the mutation affects rRNA processing. It is notable that the mutation affects
only a discrete region of the embryo even though it disrupts a basic cellular
process. At least two explanations can be offered: (1) genetic redundancy
could alleviate the effect in other tissues; (2) the brain may be the first
tissue to require new ribosome production in embryogenesis and therefore
shows the effect of disruption of this pathway. The bap28 mutation
illustrates how zebrafish genetics can be applied to the analysis of basic
cellular processes in vertebrates. Studies
on FGF signaling in zebrafish development Tsang, Hong, Habas,b
Dawid; in collaboration with Friesel, Greer, Hoying, Ron, Weinberg Fibroblast
growth factors (FGFs) constitute a family of signaling molecules that
regulate many different developmental and physiological processes, and
mutations in FGF receptors are associated with several conditions in humans.
The importance of this pathway implies that it must be tightly regulated, and
indeed several molecularly distinct feedback regulators of FGF signaling are
known. Previously, we and the Thisse group reported the isolation of a novel
feedback inhibitor of the pathway named Sef. Sef is a transmembrane protein
whose expression is controlled by FGF signaling. More recent studies in
collaboration with Dina Ron and colleagues showed that human Sef has at least
two splice variants that produce different proteins with slightly different
functional properties. A
different group of feedback inhibitors of FGF signaling, the Map kinase
phosphatase (Mkp) family, had been described earlier in mammals, but the
expression and function of Mkp in the early embryo had not been studied. We
found that Mkp3 is expressed immediately after the start of zygotic
transcription in the zebrafish embryo, slightly earlier than the expression
of FGF. We conclude that it is necessary to have the control of FGF signaling
in place from the outset in order to prevent excessive FGF signaling in the
embryo. Our studies point to the need for precise regulation of signaling
pathways in development. The
importance of FGF signaling suggested to us that a global study of gene
regulation by this pathway in the embryo will be informative. Thus, we have
initiated a DNA microarray study to compare the transcriptome of normal
embryos and embryos in which the FGF pathway has been artificially enhanced
or repressed. Numerous genes are affected by these manipulations, and their
properties are currently under study. Preger E, Ziv I, Shabtay A, Sher I, Tsang M, Dawid IB, Altuvia
Y, Ron D. Alternative splicing generates an isoform of the human Sef gene
with altered subcellular localization and specificity. Proc Natl Acad Sci
USA 2004;101:1229-1234. Tsang M, Dawid IB. Promotion and attenuation of FGF signaling
through the Ras-MAPK pathway. Sci STKE 2004; pe17. Tsang M, Friesel R, Kudoh T, Dawid IB. Identification of Sef, a
novel modulator of FGF signaling. Nat Cell Biol 2002;4:165-169. Tsang M, Maegawa S, Kiang A, Habas R, Weinberg E, Dawid IB. A
role for MKP3 in axial patterning of the zebrafish embryo. Development
2004;131:2769-2779. aLeft
the group during reporting period. bRaymond
Habas, PhD, former Postdoctoral Fellow. COLLABORATORS Robert Friesel, PhD, Maine Medical Center
Research Institute, Kevin A. Greer, BS, Vascular Research
Group, James B. Hoying, PhD, Vascular Research
Group, David Klein, PhD, Laboratory of
Developmental Neurobiology, NICHD, Tetsuhiro Kudoh, PhD, Dina Ron, PhD, Technion-Israel Institute of
Technology, Eric Weinberg, PhD, Heiner Westphal, MD, Laboratory of
Mammalian Genes and Development, NICHD, Stephen Wilson, PhD,
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