S318
MECHANISMS OF DEVELOPMENT
1 2 6 (2 0 0 9) S3 1 4–S 32 8
a late (postnatal) increase in ossification. To mimic GSK-3b or
embryonic development and tissue homeostasis. SnoN is a nega-
Axin2 loss, we are over-expressing a drug-stabilised b-catenin
tive regulator of the pathway as it acts as a transcriptional repres-
construct in wild-type skull osteoblast cultures, and in skull
sor of target genes. Arkadia is an E3 ubiquitin ligase that was
explant (calvarial) cultures. Using a drug-dependent b-catenin
shown to enhance Nodal signalling during mouse and Xenopus
will allow us to investigate the threshold levels of Wnt signalling
development. Our laboratory has recently shown, in cell culture
required for ossification and the stages at which the b-catenin
experiments, that Arkadia promotes Smad3-dependent TGF-b/
signal is critical for osteoblast differentiation. Our findings from
Activin/Nodal signalling through its degradation of the negative
these investigations will be reported here.
regulator SnoN. We are using the zebrafish to study the functional relevance of this interaction in the context of early embryonic
doi:10.1016/j.mod.2009.06.879
development. We have cloned the two snoN genes and the unique Arkadia gene in zebrafish. Sequence analysis shows highly conserved functional domains compared with mammalian proteins.
21-P015
Overexpression and knockdown analysis of snoN1, snoN2, and
Expression of Jagged2 in the ventral spinal cord depends on Shh
Arkadia are being performed to determine their role during zebra-
activity
fish development. For instance, we found that overexpression of
and
controls
the
switch
from
neurogenesis
to
oligodendrogenesis M. Angeles
SnoN at high levels can result in some Nodal-like phenotypes
Rabadn1, Jordi Cayuso1, Catarina Cruz2, James
but we demonstrate that most overexpressed and endogenous
Briscoe2, Elisa Mart1
SnoN proteins get degraded during gastrulation, at a time that
1
coincides with elevated P-Smad2/3 levels. Experiments are being
2
performed in order to determine what signalling events result
Instituto de Biologa Molecular de Barcelona, Barcelona, Spain National Institute for Medical Research, Mill Hill, London, United
Kingdom
in SnoN degradation and whether Arkadia is involved in this process. In addition, we are generating a Smad3-dependent reporter
Classical studies indicate that in any given region of the
transgenic zebrafish line in order to study transcriptional
CNS, the first cell type to arise are neurons, followed by the
responses dependent on SnoN and Arkadia. In transient assays,
generation of glial cells including oligodendrocytes. A paradig-
we showed that this reporter is sensitive to Nodal signalling
matic case is the motor neuron progenitor domain (pMN),
and SnoN in zebrafish embryos.
from which motor neurons (MN) and oligodendrocytes (OLP) sequentially arise. Studies focused on pMN have indicated
doi:10.1016/j.mod.2009.06.881
complex regulation of the MN–OLP switch that requires: ongoing activity of Shh and Olig2, Notch signalling to preserve progenitors
for
the
late
wave
of
domain-specific
progeny
21-P017
production, and activation of a gliogenic phase-specific tran-
Notch signaling in pulmonary epithelial cell differentiation
scriptional programme.
Michaela Mai1, Oliver Dittrich-Breiholz2, Michael Kracht3, Achim
Following a transcriptome screening for Shh regulated gene expression, we show the Notch-ligand Jagged2 to be transiently expressed in pMN at the time of motor neuron generation, and this expression to be dependent on Shh activity. By loss-of-function experiments we show that reduction of Jagged2 activity, at the time of MN generation, results in the premature differentiation of oligodendrocytes, at the expenses of motor neuron generation. Conversely, by invivo gain-of-function experiments we
Gossler1 1
Institute of Molecular Biology, Hannover Medical School, Hannover,
Germany 2
Institute of Biochemistry, Hannover Medical School, Hannover,
Germany 3
Rudolf-Buchheim-Institute of Pharmacology, Justus-Liebig-Universitt
Giessen, Giessen, Germany
show that Jagged2 prevents Olig2 expression to be extinguished
Lung development starts at E9,5 as an outgrowth from the
from the pMN, thus inhibiting oligodendrocyte differentiation
ventral foregut and continues until 30 days postnatally. At late
by activating Hes5. Together these results show that Jagged2 func-
gestation multipotent pulmonary epithelial cell precursors differ-
tion is required to preserve precursors at the pMN, until the sec-
entiate into six cell types with a specific proximodistal
ond gliogenic phase takes place and oligodendrocytes are
distribution.
generated, and that Shh activity is required to control Jagged2 expression at pMN. doi:10.1016/j.mod.2009.06.880
The appearance of the first differentiated cell type – pulmonary neuroendocrine cells (PNEC) – is thought to be Notch dependent [Ito etal., 2000. Development]. Additionally to the PNEC further invivo investigations ascribe Notch a critical role in terminal cell differentiation of other pulmonary epithelial cell types
21-P016 Functional study of SnoN and Arkadia in the zebrafish Marie-Christine Ramel, Caroline Hill Cancer Research UK London Research Institute, London, United Kingdom
[Dang etal., 2003. Oncogene]; further more a Notch dependent control of the proximodistal patterning of the pulmonary epithelium has been suggested by invitro studies [Tsao etal., 2008. JBC]. However the processes in which Notch acts and which particular components are involved is not well understood. To investigate this invivo we began to analyse few known cell markers on sec-
Regulation of the signalling pathway downstream of Trans-
tions of Dll1 mutant lungs and in lungs of embryos with pharma-
forming Growth Factor-b/Activin/Nodal is critical for proper
cologically inhibited Notch activation. All cell types appear to be
S319
1 2 6 ( 2 0 0 9 ) S 3 1 4 –S 3 2 8
MECHANISMS OF DEVELOPMENT
present but preliminary results suggest an altered distribution of
TrkB, suggesting that it might modulate this pathway. I have
pulmonary epithelial cell types.
found that Sprouty3 strongly inhibits the Ca2+ pathway down-
Additionally, by RNA microarray-analysis of laser-microdis-
stream of BDNF and less efficiently inhibits the MAPK pathway.
sected intrapulmonary epithelium we identified 218 potential tar-
In addition, Sprouty3 expression is reduced when BDNF is
get genes which were affected either by loss of-Dll1, or after
knocked down in the embryo, suggesting that Sprouty3 expres-
Notch-inhibition, or under both conditions. These genes are cur-
sion is regulated by BDNF signalling. Sprouty3 knockdown causes
rently being evaluated in silico as well as by expression analysis
excessive branching in neurons of the spinal cord in Xenopus tad-
in wildtype and Notch signaling mutant embryos.
poles. Conversely, overexpression of Sprouty3 reduces branching. In summary, my data suggest that Sprouty3 regulates axon
doi:10.1016/j.mod.2009.06.882
branching downstream of BDNF signalling, possibly via regulating the Ca2+ pathway.
21-P018 Control of retinoic acid and Shh signalling by Pax6 dosage in the
doi:10.1016/j.mod.2009.06.884
cornea Natalie Dor, Romana Kucerova, Martin Collinson
21-P020
University of Aberdeen, Aberdeen, United Kingdom
The apical–basal polarity kinase aPKC functions as a nuclear
Both Pax6+/ mice and ‘PAX77’ mice, which over-express 5–7 copies of a human PAX6 transgene, exhibit eye defects. We compared the Pax6+/ and PAX77 models to investigate how Pax6 dosage controls development and cell-cycle progression in the PAX77+ mice expressed about 150% wild-type levels of corneal Pax6 and Pax6
cell proliferation and fate during
Xenopus neurogenesis Nitin Sabherwal1, Andrew D. Chalmers2, Nancy Papalopulu1 1
Faculty of Life Sciences, Michael Smith
Building, University of
Manchester, Manchester, United Kingdom
anterior eye segment. +/
determinant and regulates
about 70%. The corneas of both were found to
display increased fragility in comparison to wild-types. PAX77+ corneas display severe wound healing abnormalities which in +/
contrast to Pax6 cannot be rescued by EGF. In contrast to wildtype, neither PAX77+ nor Pax6+/ corneas showed improved healing with Shh peptide or retinoic acid addition. We performed a transgenic reporter analysis of RA signalling and showed that +
+/
increased RA signalling is seen in both PAX77 and Pax6 , in comparison to wild-types. Wound healing abnormalities in the +
+/
PAX77 do not lead to corneal opacity which is seen in Pax6 . Over-expression of Pax6 therefore results in microcornea with a failure in corneal differentiation and wound healing. The phenotype shows both similarities and differences with Pax6
+/
mice.
doi:10.1016/j.mod.2009.06.883
2
Department of Biology and Biochemistry, University of Bath, Bath,
United Kingdom During neurogenesis in Xenopus, apico-basally polarized superficial and non-polar deep cells take-up differential fates – only deep cells become primary neurons while superficial cells stay as progenitors for later wave(s) of neurogenesis. The polarity of the superficial cells is defined by the antagonistic interactions between apically localized atypical protein kinase C (aPKC) and basolateral Lethal Giant Larvae (Lgl). It is unknown whether the proteins affecting polarity of cells also affect their fate, and if so, then how membrane polarity information is transmitted to the nucleus is also not known. We show that over-expression of a constitutively active, membrane-tethered form of aPKC (aPKCCAAX) suppresses primary neurogenesis, and promotes cell proliferation and superficial fate. Unexpectedly, these effects can be accounted for by a nuclear fraction of aPKC-CAAX, pointing towards shuttling of aPKC from membrane to the nucleus. Con-
21-P019
versely, antagonising apical aPKC by Lgl2 over-expression causes
The role of Sprouty3, a new signalling regulator, in Xenopus
depolarization and internalization of superficial cells; these inter-
development
nalized superficial cells form ectopic neurons when supple-
Niki Panagiotaki, Karel Dorey, Nancy Papalopulu, Enrique Amaya
mented with a proneural factor like X-ngnr-1. These findings suggest that aPKC itself is a nuclear determinant, affecting
University of Manchester, Manchester, United Kingdom The Sprouty family consists of four members (Sprouty1, 2, 3
mainly cell proliferation; and in the right environment, loss of aPKC is also permissive for a change in cell fate.
and 4) which are conserved among species. In Xenopus, Sprouty1 and Sprouty2 have been shown to repress the Ca2+ pathway
doi:10.1016/j.mod.2009.06.885
downstream of Fibroblast Growth Factor (FGF), while in mammals, Sprouty1, 2 and 4 inhibit the MAPK pathway downstream of FGF. In both systems Sprouty expression is regulated by the same RTK it regulates, creating a feedback loop. Here, I investigate the function of Sprouty3 during development, which has not been addressed yet. We have found that Sprouty3 expression at the tadpole stage is restricted to the spinal cord and the trigeminal
21-P021 Efficient gene transfer to developing chick astrocytes by Tol2 transposition Masahiro Shibata, Ryosuke Inoue, Noboru Sato
nerve. This expression pattern overlaps with that of the neurotro-
Division of Gross Anatomy and Morphogenesis, Niigata University
phin BDNF (Brain Derived Neurotrophic Factor) and its receptor
Graduate School of Medical and Dental Sciences, Niigata-City, Japan