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ABSTRACTS / Developmental Biology 295 (2006) 393 – 402
The dorsal midline of the vertebrate neural tube has been
appreciated as a signaling center directing cell fate specification,
studied extensively in the spinal cord. Using genetic fate mapping
of the Wnt3a lineage, we demonstrate that the dorsal midline is
also an important source of migratory neuronal precursors, which
participate in a variety of brain structures. Dorsal midline-derived
cells in the midbrain form the mesencephalic nucleus of the
trigeminal sensory system and participate in the periaqueductal
gray and the inferior and superior colliculi. In addition, many
nuclei in the pons and the medulla form as a consequence of
tangential migrations from the dorsal midline. Wnt3a-expressing
precursors in the rhombic lip, a transient dorsal midline structure,
give rise to all granule cells, deep cerebellar nuclei, mossy fiber
precerebellar nuclei and also generate some Purkinje cells and
cerebellar interneurons. Finally, Wnt3a lineage derivatives,
arising from early expression domains along the anterior/
posterior axis of the dorsal neural tube, participate in the
formation of the central auditory system. Previous reports have
indicated that diverse migratory streams originating at the
rhombic lip give rise to cells in the isthmus, mesopontine
tegmental system and cerebellum; our observations, however,
demonstrate that this is not a unique feature of the rhombic lip and
implicate the dorsal midline as a site of widespread neuronal
migration. Our results further suggest that structures eventually
becoming integrated into a functional pathway share a molecular
identity early in their developmental history.
doi:10.1016/j.ydbio.2006.04.216
194
Effects of Shh on Pax6 and proliferation in
transdifferentiation
Christian Gutierrez, Jason R. Spence, Mayur Madhavan,
Katia Del Rio-Tsonis
Miami University, Oxford, OH, USA
Only a handful of organisms are able to regenerate the retina
after injury. The embryonic chick has the ability to regenerate the
retina during a limited window in embryonic development via
stem/progenitor cells located in the ciliary margin of the eye or
through the process of transdifferentiation, where the retina
pigmented epithelium (RPE) dedifferentiates, proliferates and
re-differentiates into laminated retina complete with all of the
cell types. Transdifferentiation only occurs until E4.5 and when
stimulated by a source of fibroblast growth factor (FGF). We
have previously shown that overexpression of Sonic Hedgehog
(Shh) is able to inhibit FGF stimulated transdifferentiation. Here
we examine that Pax6 is regulated by Shh during transdifferentiation. At E3 – E3.5, Pax6 is expressed in the RPE, but by E4
it is present at low levels, and is absent in the RPE by E5. It has
previously been shown that Pax6 can induce transdifferentiation
of the RPE. We demonstrate that FGF is able to induce Pax6
expression in the RPE after the retina has been removed.
Furthermore, we show that Shh is able to dramatically decrease
the number of Pax6 positive RPE cells, indicating that Shh plays
a role in maintaining RPE identity by inhibiting Pax6
397
expression. We have also shown that when FGF is added to
the eye after retinectomy, the increase in Pax6 expression is
correlated with an increase in proliferation as assayed by BrdU
incorporation. Since it has been shown that Shh inhibits Pax6
expression and transdifferentiation, it is expected that Shh will
also limit the amount of proliferation in the RPE.
doi:10.1016/j.ydbio.2006.04.217
195
Adherens junctions in chick optic development
Ricardo M. Borges, C.Y. Irene Yan*
Department of Cell and Developmental Biology,
University of Sao Paulo, Sao Paulo, Brazil
Early optic development begins when the neuroepithelium of
neural tube outpockets bilaterally, forming the optic vesicles.
The optic vesicles grow as a continuous epithelial sheet, contact
the surface ectoderm and invaginate, forming bilayered optic
cups. The inner portion of the optic cup forms the neuroretina
(NR), and the outer portion forms the retinal pigmented
epithelium (RPE). At the time of its contact with the
neuroepithelium, the surface ectoderm thickens into the lens
placode and invaginates to form the lens vesicle. Both optic
vesicle and lens placode invagination requires maintenance of
tissue cohesion. Here, we investigate the dynamics of adherent
junctions localization in the eye during optic invagination in
chick embryos. Imunohistochemistry for components of Adherens Junctions (AJ) showed that, at optic vesicle stages,
neuroepithelial AJ were concentrated in cell membranes lining
the ventricular surface, while the surface ectoderm expressed AJ
homogeneously between cell boundaries. In invaginating optic
vesicles, AJ remained polarized at neuroepithelium ventricular
surface, while surface ectoderm labeling became concentrated at
the distal portion of the lens placode. In optic cups, labeling was
concentrated at the cell membranes that constitute the NR/RPE
border, and around the lumen of the lens vesicle. We also
detected the presence of the GTPases Rac1 and Cdc42 in optic
tissues during these stages, suggesting that localization of AJ
during eye morphogenesis is coordinated with differentiation of
optic tissues and could be regulated by GTPases. Supported by
FAPESP.
doi:10.1016/j.ydbio.2006.04.218
196
Wnt5A, a marker for dorsal retinal pigmented epithelium
C.Y. Irene Yan*, Eliane Rossi, Fernanda Siwiec
Biomedical Sciences Inst., Univ. de São Paulo, São Paulo,
SP, Brazil
The optic vesicle forms an optic cup with distinct threedimensional axial identity after intense morphogenesis and
patterning. These axes can be identified through both anatomical
and molecular landmarks. The proximal –distal axis in the
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ABSTRACTS / Developmental Biology 295 (2006) 393 – 402
bilayered optic cup is defined anatomically by the presence of
the prospective neural retina (NR) in the distal layer and the
retinal pigmented epithelium (RPE) proximally. Accordingly,
molecular markers accompany morphogenesis by restricting
their expression to definite compartments. For instance, in the
optic cup, the prospective neural retina expresses Chx10, and the
RPE, Mitf. However, to facilitate identification of definite events
during oculogenesis, there remains a need to identify additional
markers of optical development. Thus, we performed here a
screen for Wnt ligands that are expressed during eye development. Specifically, we examined the expression of Wnt1, Wnt3,
Wnt4-1 and Wnt5A during chick optic vesicles stages up to optic
cup formation. Of these four genes, only Wnt5A was consistently expressed in the dorsal optic cup. Although Wnt1, Wnt3
and Wnt4-1 were present in the developing nervous system,
neither was found in the optic vesicle or cup. Wnt5A was first
detected at the dorsal region of the RPE at stage HH14. In stages
HH15 and HH16, its expression domain increased ventrally in
the both nasal and temporal domains. Throughout all these
stages, the labeling was restricted to the RPE only, and was
completely absent in the neural retina. Based on these data, we
would like to propose Wnt5A as a marker for early dorsal retinal
pigmented epithelium.
Corresponding author: reneyan@icb.usp.br.
doi:10.1016/j.ydbio.2006.04.219
197
Transdifferentiation in Xenopus laevis eye
Alexander S. Jerome, Maria N. Vergara, Katia Del Rio-Tsonis
Miami University, Oxford, OH, USA
The process of transdifferentiation involves the transformation of a differentiated cell type into another. During this
process, a cell must lose its phenotype, the characteristics that
make it a unique cell type that performs specific functions, and
become ‘‘stem-like’’, being able to proliferate and give rise to
different kinds of cells. This outstanding phenomenon occurs
under very specific circumstances in vertebrates, and its
understanding could have profound implications in the field
of regenerative and developmental biology. The eye of the
Xenopus laevis tadpole provides a good model to analyze this
process. In the present study, we have characterized the process
of transdifferentiation of pigmented eye tissues in Xenopus
tadpoles, at a stage in which the eye is already fully developed.
Our aim is to establish a model that will allow for the study of
the molecular mechanisms that drive the process of transdifferentiation. We have been able to induce the transdifferentiation
of pigmented epithelium explants into lens when transplanted
into host Xenopus eyes. In addition, we have induced retina
regeneration from pigmented tissues after complete removal of
the retina in vivo. We have also assessed the influence of
several growth factors and morphogens on this process.
doi:10.1016/j.ydbio.2006.04.220
198
Characterization of silica spicule formation during the
resuscitation and in vitro cell culture of
Hymeniacidon perleve
Wei Zhang 1, Xupeng Cao 2, Xingju Yu 1
1
Marine Bioproducts Engineering Group, Dalian Institute of
Chemical Physics, Chinese Academy of Sciences,
Dalian, China
2
Graduate School of the Chinese Academy of Sciences,
Beijing, China
The biogenic silica mineralization in an intertidal marine
sponge Hymeniacidon perleve (Porifera: Demospongiae) has
been investigated during the developmental process over one
year period and in an in vitro sponge cell culture. Tissue
samples of different developmental stages from dormancy to
bloom and decline were collected. The structural dimensions
and development characteristics of silica spicules were
measured. It was found that the dimensional characteristics of
spicules were restricted by their material properties. The
spicule development that was closely linked with the sponge
development can be classified as four distinct stages: newly
born, growing, maturing and over-matured. In in vitro cell
culture of archaeocyte-dominant cell populations (ADCP), a
time-lapse microscopic observation was set up for studying the
spicule formation and cell – spicule interaction over 1 month
period. The first spicule appeared on day 10 during the ADCP
culture, and the dynamics of spicule formation mimics the
spicule development in the field. To understand the molecular
regulation of spicule developments, the silicatein gene, which
is responsible for the silicification of sponge spicules has been
cloned. In both tissues development and cell cultures, the
expressions of silicatein are correlated well with the onset and
growth of spicules; however, the changes in the number of
spicules formed lag behind the silicatein gene expression.
doi:10.1016/j.ydbio.2006.04.221
199
ram-6 is required for Caenorhabditis elegans male sensory
rays morphogenesis
H.Y. Lee, Y.M. Lam, S.W. Tsang, K.L. Chow
Hong Kong University of Science and Technology, Hong Kong
Caenorhabditis elegans males develop nine pairs of
bilaterally symmetrical peripheral sensory organs known as
rays. Although they are all morphologically and positionally
distinguishable, each of them develop smooth boundary against
the cuticular fan structure. We are interested in a class of
‘‘ram’’ genes essential for the morphogenesis of these sensory
rays. A new component, designated as ram-6, was identified in
an EMS mutagenesis screen. ram-6 mutants display severe
swollen rays phenotype in all of the rays and was shown to
complement all other known ram genes. Temperature shift
experiments suggested that ram-6 participates not just in ray
development but also in early embryonic development. Mutant