Sive Lab Research Summary
The questions of how an embryo decides where
to place it's organs ("positional information") and
how these organs are correctly organized into functional
three dimensional structures ("morphogenesis") are of
fundamental importance. We study these processes in
the frog, Xenopus, and in the zebrafish, Danio.
We have two major areas of interest: the nervous system,
including very early patterning events as well as later
events that build the three dimensional structure of
the brain, and the extreme anterior of the embryo that
forms the primary mouth, and is an evolutionarily conserved
and important region. Frog and fish embryos are ideal
for these studies, since the events we analyze take
place very early in development, when mammalian embryos
are tiny and inaccessible. Genes that are important
for frog and fish embryogenesis are conserved in mammals,
and our research is therefore relevant for understanding
normal and abnormal human development.
Mechanism by which inhibitors of BMP signaling
activate neural determination. We have shown
that in fish and frogs, the embryo decides to make a
nervous system by the onset of gastrulation. This is
a very early decision, corresponding to a two and one
half week old human embryo. Expression of the transcription
factor zic1 at the onset of gastrulation is
one of the earliest molecular indicators of neural fate
determination in Xenopus. Inhibition of bone
morphogenetic protein (BMP) signaling is critical for
activation of zic1 expression and fundamental
for establishing neural identity in both vertebrates
and invertebrates. The mechanism by which interruption
of BMP signaling activates neural-specific gene expression
is not understood. We have identified of a 215 bp genomic
module that is both necessary and sufficient to activate
Xenopus zic1 transcription upon interruption
of BMP signaling. Transgenic analyses demonstrate that
this BMP inhibitory response module (BIRM) is required
for appropriate spatial and temporal expression in the
whole embryo. Multiple consensus binding sites for specific
transcription factor families within the BIRM are required
for its activity and some of these regions are phylogenetically
conserved between orthologous vertebrate zic1
genes. These data suggest that interruption of BMP signaling
facilitates neural determination via a complex mechanism,
involving multiple regulatory factors that cooperate
to control zic1 expression.
Hindbrain patterning. The embryonic
hindbrain gives rise to the cerebellum and medulla.
In the embryonic zebrafish hindbrain, we have shown
that cells in the posterior hindbrain acquire unique
anterior/posterior (A/P) identity when they both express
the homeodomain transcription factor vhnf1
and are exposed to fibroblast growth factor (FGF) signals.
Each of these factors alone is required for formation
of other tissues, but together they regulate a distinct
set of genes that specify rhombomeres 5 + 6 (r5+6),
including valentino (val), krox20
and hoxB3. In order to understand better the
genetic program that is initiated by the combination
of vhnf1 and FGF signals, we have tried to
recapitulate the determination of rhombomeres 5+6 in
explants of zebrafish embryonic ectoderm. We have obtained
the exciting result that expression of vhnf1
+ FGF is sufficient to activate val and krox20
expression in this simple explant system. We have performed
expression microarray experiments in which we compare
genes regulated by the two factors together to the targets
of each factor alone. From these assays, we have identified
two new genes that are targets of vhnf1 expression
and may play a pivotal role in regulation of posterior
hindbrain determination.
Brain ventricle morphogenesis. A unique
and conserved feature of the vertebrate nervous system
is that it is tubular. Why is this so? One answer may
be that a tubular structure allows exposure of the nervous
system to different "inside" and "outside" environments.
The inside of the neural tube forms a system of cavities,
the brain ventricles. Brain ventricles are required
for normal brain development and function, and ventricular
abnormalities are apparent in several neurodevelopmental
disorders, including autism, indicating that this system
is essential for normal mental health. Several functions
have been assigned to the adult brain ventricular system
and the cerebrospinal fluid it contains, however, embryonic
development and function of this system are poorly studied.
We have begun to analyze brain ventricle development,
using the zebrafish as a model, and taking a forward
genetic approach. The zebrafish is an excellent system
for this study as imaging the brain in live embryos
is feasible, and as we have collected more than thirty
brain ventricle mutants, identified in several mutagenesis
screens, but not generally studied further. We have
shown that the neural tube expands into primary forebrain,
midbrain, and hindbrain ventricles rapidly, over a four-hour
period during mid-somitogenesis. Two mutants that do
not develop brain ventricles are nagie oko
and snakehead. Mutants in nagie oko,
which encodes a MAGUK family protein, fail to undergo
ventricle morphogenesis. This correlates with an abnormal
brain neuroepithelium, with no clear midline and disrupted
junctional protein expression. In contrast, the snakehead
neural tube undergoes normal ventricle morphogenesis,
however the ventricles do not inflate, likely due to
impaired ion transport and disrupted osmotic gradient
formation. We have shown that snakehead is
allelic to small heart, which has a mutation
in the Na+K+ ATPase gene atp1a1a.1. We are
using transplantation techniques to generate mosaic
snakehead embryos to examine the tissue requirement
for Atp1a1a.1 function during brain ventricle
formation. This study defines three steps required for
brain ventricle development and that occur independently
of circulation: 1) morphogenesis of the neural tube,
requiring nok function; 2) lumen inflation
requiring atp1a1a.1 function, and 3) localized
cell proliferation. We suggest that mechanisms of brain
ventricle development are conserved throughout the vertebrates.
The extreme anterior is a conserved region.
In most animals, the three first cell types to form,
the ectoderm, mesoderm and endoderm, are present in
all parts of the embryo, with ectoderm on the outside,
endoderm on the inside and mesoderm sandwiched between
them. However, at the extreme front (anterior) of deuterostome
embryos, such as sea urchins and all vertebrates including
humans, there is a mesoderm-free region, where the ectoderm
and endoderm are directly juxtaposed. It is our hypothesis
that this region is an evolutionary relic of the diploblasts,
animals with only ectodermal and endodermal germ layers.
This anterior region forms the first opening between
the gut and the outside, the primary mouth, which may
be the most evolutionarily conserved part of the head.
In amphibians, this region also forms the mucus-secreting
cement gland.
Development of the primary mouth.
The extreme anterior of the deuterostome embryo is unique
in that ectoderm and endoderm are directly juxtaposed,
without intervening mesoderm. From this region the initial
opening between the gut and outside of the embryo breaks
through. We have termed this the primary mouth, which
is essential for eating and therefore life. In vertebrates,
the neural crest grows around the primary mouth to form
the face and a secondary mouth opening must therefore
form. The primary mouth then becomes the pharyngeal
opening. In order to establish a molecular understanding
of this important process we have examined primary mouth
formation during Xenopus development. We find
that multiple steps are involved. An early step involves
dissolution of the basal lamina separating ectoderm
and endoderm. A subsequent step requires cell death
in the ectoderm. Later, the ectodermal and endodermal
layers overcome their normally separate identities and
intercalate to generate a single cell layer. The final
step is perforation, where the primary mouth breaks
through. From fate mapping, we have defined the ectodermal
and endodermal regions that will form the primary mouth.
Extirpations and transplants indicate that, surprisingly,
ectoderm from any region of the embryo can be used to
generate the primary mouth. In contrast, endoderm specifically
in the presumptive primary mouth region provides essential
signals. We have performed expression microarray analysis
to define genes expressed during primary mouth formation,
and have defined at least two that are required for
this process.
The cement gland as an anterior paradigm.
The amphibian cement gland is a mucus-secreting epithelium
that forms from extreme anterior ectoderm, and that
we have used to analyze positional cues. We have proposed
that the overlap of anterodorsal identity (AD), ventrolateral
identity (VL), and ectodermal outer layer identity (EO)
results in determination of the cement gland primordium,
formulated in the equation A+VL+EO=CG. We are constructing
the hierarchy of transcription factors involved in cement
gland determination. Anterior identity is conferred
by the transcription factor Otx2, which is necessary
and sufficient to activate the cement gland program.
Using hormone-inducible fusion proteins, we have shown
that the homeodomain genes pitx1 and pitx2C
lie immediately downstream of Otx2. Neither Otx2, not
Pitx proteins are able to directly activate cement gland-specific
gene expression, but require expression of intervening
or additional genes. In order to identify these, we
have examined the cis-acting elements by which transcription
of two related genes is activated specifically in the
cement gland. These are Xag1 and gob4,
which are members of the conserved agr gene
family we have defined. Ets and ATF/CREB sites are required
for expression of Xag1 in the cement gland.
We have also defined a 140bp region of the gob4
promoter that is necessary and sufficient for robust
cement gland specific expression. Linker scan and point
mutagenesis implicate AP1 sites as crucial for gob4
regulation. Binding sites for AP1 and ATF/CREB factors
are likely to bind related bZip family members. Thus,
comparison of these promoters suggests that the AP1
superfamily is pivotal in directing gene expression
specifically to the cement gland.
Research in the Sive Lab is supported in part through grants from the National Institutes of Health and the National Science Foundation. |
