Krawchuk, Dayana

Yamanaka, Yojiro

Elsevier - Publisher Connector 

results in a complete failure of proximal/distal mandibular arch
patterning.
doi:10.1016/j.ydbio.2011.05.291
Program/Abstract # 335
Novel effects of folinic acid and folate supplementation on
locomotor development in embryonic Zebrafish, Danio rerio
Holly Hattway, Sarah Kosmin, T.K. Puryear, Shannon Saszik
Chicago, IL, USA
Periconceptional folate supplementation is suggested to help prevent
neural tube defects during early embryonic development as neural crest
cells migrate and differentiate. Research has shown that the developing
nervous tissue is exquisitely sensitive to endogenous and exogenous
chemical signals and that these signals must be interpreted by the
developing embryo in a specific temporal, spatial and dose dependent
manner. Thepurposeof the current studywas toexamine theeffectof folate
and folinic acid on embryonic development in Zebrafish, Danio rerio. After
fertilization (0 dpf), embryoswere exposed to either 1mMfolate or 10mM
folinic acid for a period of 48 h. After 48 h, embryos were reared using
standard procedures and examined everyday for a period of one week.
Anatomical and behavioral measures were taken at 2 dpf, 3 dpf, and 5 dpf.
Zebrafish exposed to either folate or folinic acid hatched early and showed
acceleration in the development of locomotor systems. Consistent with
advanced motor system function, exposed Zebrafish were found to have a
higher level of swimming activity compared to controls. There was also a
change in the startle response, with exposed embryos showing a
hypersensitivity to external stimuli. Thus, exposure to either folate or folinic
acid accelerates development, increases locomotor activity, and increases
anxiety. The results from this study establish the Zebrafish as an excellent
model system to examine the role of folate during embryonic development.
The effect of folate exposure on embryonic Zebrafish is similar to what has
been demonstrated in other species and adds further evidence that folate
plays an important role in embryonic development.
doi:10.1016/j.ydbio.2011.05.292
Program/Abstract # 336
Endomesoderm segregation involves cross talk between
Notch and Wnt pathways through multiple intersecting
regulatory circuits
Aditya J. Sethi
NIH/NIDCR Developmental Mechanisms Unit, Bethesda, MD, USA
Formation of primary germ layers restricts the developmental
capacities of cells in early embryos. While this has been studied
extensively, germ layer segregation is poorly understood. Using sea
urchin embryos, we show that after simultaneous activation of
endoderm and mesoderm gene regulatory networks (GRNs) in the
same blastomeres, these GRNs segregate in their progeny in response to
cross talk betweenNotch and canonicalWnt (cWnt) signaling through a
series of intersecting regulatory circuits. As in other deuterostomes,
anisotropic cWnt signaling is required in the sea urchin embryo for
endoderm specification in vegetal cells. Also in response to cWnt,
micromere descendants at the vegetal pole activate the nonskeletogenic
mesoderm (NSM) gene regulatory network (GRN) through a Notch
signal to adjacent cells, where the cWnt-dependent endoderm GRN
operates. After these endomesoderm (EM) precursors divide, their
vegetal progeny, the NSM precursors, continue to receive the Notch
signal from the micromeres, which represses operation of a key Hox11/
13b-dependent endoderm circuit in them. In contrast, each factor of this
circuit, which remains active in descendants that no longer transduce
the Notch signal, is essential for endoderm fate. Subsequently, Hox11/
13b and its target, Brachyury, maintain cWnt activity in the endoderm
throughWnt1 transcription, which reinforces the endodermGRN. Later,
Notch also promotes export of TCF out of NSM nuclei, further insulating
this territory from cWnt signals. Thus Notch initiates EM segregation by
repressing an endoderm circuit in the NSM and reinforces it by
establishing distinct cWnt signaling environments: high cWnt activity
in the endoderm and low cWnt function in the NSM.
doi:10.1016/j.ydbio.2011.05.293
Program/Abstract # 337
Elucidating the molecular mechanisms underlying cell
movements in the Visceral Endoderm
Bradley Joycea, Shankar Srinivasb
aUniversity of Oxford Dept of Physiology, Anatomy & Genetics, Oxford, UK
bUniversity of Oxford, Oxford, UK
TheAnteriorVisceral Endoderm(AVE) is a specialised groupof cells in
the simple epithelium of the Visceral Endoderm (VE). Stereotypic
migratory movement of the AVE is responsible for properly orienting
the anterior–posterior axis of the embryo. AVE cells migrate by
intercalation within the VE, which retains epithelial integrity. Using
time-lapse microscopy, we have demonstrated that regional differences
in cell behaviour in the VE regulate AVE migration. Using 3D imaging of
protein localisation patterns and various mutants with AVE migration
defects, we have shown that signalling through theWnt–PCP pathway is
required for the proper migration of AVE cells. Moreover, the TGF-β
family member Nodal is required for PCP signalling in the VE1. The
mechanisms by which Nodal controls PCP signalling and influences the
migratory behaviour of cells in the VE have yet to be established. We are
using time-lapse microscopy of various mutants with AVE migration
defects to visualise the entire VE, in order to address how these genes
might influenceAVEmigrationbyaltering the cellularbehaviourof theVE
in general.We have found thatmutants for Lefty1 (an inhibitor of Nodal)
showanabnormal reduction in the regional differences inVE cellmotility
andmorphometrics. We are also using atomic force microscopy to probe
the physical characteristics of VE cells in regions showing differences in
behaviour. To address how Nodal signalling may regulate PCP signalling
we are using confocal microscopy to assess the sub-cellular and regional
differences in localisationof keydown-streameffectors inNodal pathway
mutants andembryos cultured inpharmacological inhibitors of theNodal
receptor. Results from these experiments will be presented.
doi:10.1016/j.ydbio.2011.05.294
Program/Abstract # 338
Understanding inter-strain differences in pre-implantation
mouse development
Dayana Krawchuka, Yojiro Yamanakab
aMcGill University Human Genetics, Montreal, PQ, Canada
bMcGill University, Goodman Cancer Research Center,
Department of Human Genetics, Montréal, PQ, Canada
By implantation, the mouse embryo forms three primary lineages:
the trophectoderm, the primitive endoderm (PE) and the epiblast (EPI).
The PE and EPI are derived from the inner-cell mass (ICM) of the E3.5
blastocyst, which is used for ES cell derivation and gives rise to extra-
embryonic tissues and the embryo, respectively. While much is known
regardingwhat factors are involved in specification andmaintenance of
these three lineages, it is unknownwhether there is variability in these
processes among different mouse strains. Two lines of evidence lead us
to hypothesize that there are differences betweenmouse strains in these
Abstracts204
early stages. First, some mouse strains, such as 129 Sv, are more
permissive to ES cell derivation compared to others. Second, when
generating host/ES cell chimeras, certainmouse strain combinations are
significantlymore successful than others.Wepropose that differences in
the responsiveness to certain developmental cues may influence the
timing or manner of lineage specification, thus causing inter-strain
variance. To test this, we studymouse strains (129 Sv, C57Bl/6 and CD1)
often used for derivation of ES cell lines and generation of host-ES
chimeras. First, we grossly observe embryo division dynamics using
time-lapse imaging. Second, we compare the expression of lineage-
specific markers. Finally, we ask whether there is variable sensitivity to
FGF signaling, which is required for specification of the PE and EPI
lineages of the ICM. Our observations will not only help us understand
what processes of pre-implantation development are variable between
mouse strains, butmayalsoprovide strategies for theuseof an improved
variety of mouse strains in genetic engineering applications.
doi:10.1016/j.ydbio.2011.05.295
Program/Abstract # 339
Investigating the role of the Hippo pathway member Nf2 in
trophectoderm/inner cell mass specification
Katherine Cockburna, Robert Stephensonb, Janet Rossantb
aHospital for Sick Children Res Instit Developmental & Stem Cell Biology,
Toronto, Canada
bDepartment of Developmental & Stem Cell Biology,
The Hospital for Sick Children, Toronto, ON, Canada
The first lineage decision that takes place during mouse preimplan-
tation development is the establishment of the trophectoderm (TE) and
inner cell mass (ICM) lineages, which are distinct by the early blastocyst
stage. The kinases Lats1 and 2 (Lats1/2) and the transcriptional co-
activator Yap play a key role in TE/ICM segregation by regulating the
expression of the TE-specific transcription factor Cdx2. In outside cells of
the preimplantation embryo, Yap accumulates in the nucleus, where it
induces Cdx2 expression. In inside cells, Yap is phosphorylated by active
Lats1/2 and retained the cytoplasm,preventingCdx2expression. It is not
clear how Lats1/2 activity is regulated during preimplantation devel-
opment. Notably, Lats1/2 and Yap are members of the Hippo signaling
cascade, suggesting that upstream components of this pathway could
regulate their activity in the embryo. Here, we demonstrate that the
upstream Hippo signaling component Nf2 is expressed throughout
preimplantation development. Additionally, injection of a dominant
negative (DN) form of Nf2 cell-autonomously increases nuclear
accumulation of Yap and decreases Yap phosphorylation in inside cells
of the embryo. DN Nf2 injection also increases Cdx2 expression,
indicating that this nuclear-localized Yap is transcriptionally active.
Finally, consistent with a role upstream of Lats1/2, the effects of DN Nf2
injection can be rescued by over-expression of wild-type Lats2. This
work suggests a novel role for Nf2 in the process of TE/ICM specification
during mouse preimplantation development.
doi:10.1016/j.ydbio.2011.05.296
Program/Abstract # 340
Transforming growth factor-beta-related signaling in
blastocyst morphogenesis
Yijing Chena, Jiami Guob, Jibiao Lib
aKent State University Biological Sciences, Kent, OH, USA
bKent State University, Kent, OH, USA
Studies of mouse preimplantation development using embryos
cultured in vitro establish the importance of autocrine/paracrine growth
regulatory pathways in controlling preimplantation development.
Signaling molecules of the Transforming Growth Factor beta (TGF-β)
superfamily are potent regulators of development and tissue home-
ostasis. Smad4 is a central mediator of the TGF-beta-related signaling
and is important for numerous processes including cellular growth,
differentiation, migration, and extracellular matrix production. Mice
lacking Smad4 die around peri-gastrulation period due to defects in
epiblast proliferation, mesoderm formation and extraembryonic devel-
opment. Maternal gene products play key roles in shaping the earliest
developmental programs in awide variety of organisms. Relatively little
is known about the functions of mammalian maternal gene products.
Maternal Smad4 gene products are abundant in unfertilized eggs aswell
as cleaving blastomeres of preimplantation mouse embryos. We have
conditionally inactivated Smad4 in the female germline. Eggs depleted
of maternal Smad4 gene products complete meiosis and are fertilized
normally. However, preimplantation development is severely compro-
mised in embryos derived from such eggs evenwhen they are fertilized
by wild type sperms, demonstrating the importance of maternally
derived Smad4 gene products in early mouse development. The
consequences of disrupting maternal TGF-β related signaling on
blastocyst morphogenesis, including blastomere cleavage, cell polarity
establishment and lineage segregation, will be presented.
doi:10.1016/j.ydbio.2011.05.297
Program/Abstract # 341
Wnt8a is a target of miR430 post-transcriptional regulation
Arne C. Lekven, Annika Butler, Kevin Baker,
Amy Whitener, Anand Narayanan
Texas A&M University, College Station, TX, USA
Wnt/β-catenin signaling in early vertebrate embryos patterns the
D/V and A/P axes, and Wnt8a is one of the earliest acting genes that
interconnects both D/V and A/P patterning. In the Zebrafish, Wnt8a, the
earliest zygotically expressed Wnt gene, acts over broad domains to
regulate D/V and A/P patterning of themesoderm and ectoderm.Wnt8a
acts in two phases to regulate different developmental choices. In early
gastrula stages, Wnt8a signaling prevents expansion of the Dorsal
Organizer and induces posterior neural plate fates. During mid to late
gastrulation, Wnt signaling promotes growth of the posterior embryo.
Because of its critical role during axis patterning, Wnt8a regulation is
likely to occur at several levels, including post-transcriptionally. We
have used a combination of transgenic and transient sensor assays and
target protector morpholinos in the Zebrafish to test the hypothesis
that post-transcriptional regulation of Wnt8a occurs through the
action of microRNAs. Our results show that Wnt8a is a target of
several microRNAs during early development, a major regulator
beingmiR430. We identified twomiR430 binding sites inWnt8a UTR
elements, and these sites are necessary and sufficient forWnt8a post-
transcriptional regulation. Protecting Wnt8a transcripts from
miR430 regulation results in elevatedWnt8a expression and embryo
patterning defects. Thus, microRNA-dependent regulation of Wnt8a
is crucial to normal embryonic axis patterning.
doi:10.1016/j.ydbio.2011.05.298
Program/Abstract # 342
A dorsalized and cell migration maternal effect mutant
in Zebrafish
Yvette Langdon, Mary Mullins
Philadelphia, PA, USA
Abstracts 205


Understanding inter-strain differences in pre-implantation mouse development 

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Understanding inter-strain differences in pre-implantation mouse development

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