Download Modulation of cell surface architecture in gastrulating chick embryo

Indian Journal of Experimental Biology
Vol. 48, April 2010, pp. 346-353
Modulation of cell surface architecture in gastrulating chick embryo in response to
altered fibroblast growth factor (FGF) signaling
Seema Borgave & Surendra Ghaskadbi*
Division of Animal Sciences, Agharkar Research Institute, Pune 411 004, India
Received 15 October 2009
Gastrulation is a fundamental process that results in formation of the three germ layers in an embryo. It involves highly
coordinated cell migration. Cell to cell communication through cell surface and the surrounding molecular environment
governs cell migration. In the present work, cell surface features, which are indicative of the migratory status of a cell, of an
early gastrulating chick embryo were studied using scanning electron microscopy. The distinct ultrastructural features of
cells located in the various regions of the epiblast are described. Differences in the surface features of cells from distinct
embryonic regions indicate differences in their migratory capacities. Further, the dynamic nature of these cell surface
features by their response to altered fibroblast growth factor (FGF) signaling, experimentally created by using either excess
FGF or inhibition of FGF signaling are demonstrated.
Keywords: Altered FGF signaling, Cell surface architecture, Chick embryo
In amniotes the epiblast cells ingress through the
developing primitive streak and give rise to
mesodermal and endodermal cells during gastrulation.
Gastrulation involves coordinated movements of cells
either individually or as cell sheets1 and cell-cell
communication is crucial during such migratory
processes2. Cells directly communicate with one
another through their cell surface; this often decides
their respective path of differentiation3. The threedimensional cellular architecture as well as the
surface features of individual cells can be studied with
scanning electron microscopy (SEM). In spite of
being one of the primary processes, gastrulation is not
completely understood in amniotes. The organization
and surface architecture of cells of early gastrulating
chick embryos has been described4-9. These studies
point towards differences in shape and migration rates
of cells located within and outside the primitive
streak, probably due to the differences in requirement
for moving inside through the streak and lateral
movements between ectoderm and endoderm, postinvagination. In some of these reports mesodermal
and endodermal cells from ventral surface have been
studied using scanning electron microscopy (SEM)6-8.
However, there are only a few studies wherein the
________________
*Correspondent author
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E-mail: [email protected]
dorsal surface i.e. epiblast of the early chick embryos
is described4,5. Study of dorsal surface is crucial for
understanding the migratory potentials of epiblast
cells that eventually ingress through the primitive
streak from dorsal side, undergo epithelial to
mesenchymal transition and move ventrally to form
either mesoderm or endoderm. Earlier, Bancroft and
Bellairs4 have studied the surface features of epiblast
cells of embryos from un-incubated egg up to 30 h of
incubation. On the other hand, Jacob et al.5 have
described the morphological features of epiblast cells
from prospective neural plate, anterior primitive
streak and lateral prismatic region. In the present
study the differences in the ultrastructural features of
the epiblast cells from four developmentally
significant regions of an early chick embryo, viz,
Hensen’s node, prospective neural plate, anterior and
posterior regions of the primitive streak have been
studied. To our knowledge, such a detailed account of
cell surface morphology from discrete embryonic
areas does not exist in literature.
Altered fibroblast growth factor (FGF) signaling
causes abnormal development of nervous system and
mesodermal structures in early chick embryo have
been demonstrated10. The abnormal development was
associated with rapid modulation of expression of
brachyury, noggin10 and goosecoid11, genes which are
essential for normal development of the nervous
system and mesodermal structures. Brachyury12 and
BORGAVE & GHASKADBI: ALTERED FGF SIGNALING & CELL SURFACE CHANGES
goosecoid13 encode transcription factors that are
required for the gastrulation movements and
differentiation of mesoderm. Appropriate levels of
FGFs are thought to be essential for the normal
morphogenesis of vertebrate embryos14. Hence it is
likely that the effects of altered FGF signaling in early
chick embryo10,11 are reflected at the level of cell
surface. In the present work, the effects of altered
FGF signaling have been studied on the surface
features of embryos that reflect the migratory status of
cells. Also the dynamic nature of the cell surface in
four important embryonic regions have been
described.
Materials and Methods
Freshly laid White Leghorn chicken eggs were
obtained from a local hatchery. Human recombinant
bFGF and suramin were procured from Sigma (St.
Louise, USA).
In vitro culture and treatment of chick embryo
explants⎯Eggs were incubated for 18 h at 37.5°C to
obtain Hamburger Hamilton15 (HH) stage 4 chick
embryos. Embryos were dissected in Pannet-Compton
saline16 (PC saline, pH 7.4), cultured in vitro by New's
single ring technique17 and staged on the basis of
standard morphological criteria15. The cultures were
randomly divided into 4 groups. Embryos were
treated as described before10,11,18. Two controls,
treated either with PC saline alone or with PC saline
containing BSA, were maintained. Thus, embryos
were treated with either PC saline, BSA (100
ng/culture), 6 nM bFGF (10 ng/culture) or 2 mM
suramin (200 nmoles/culture) as described earlier10,11.
After addition of the test chemical the embryos were
left at room temperature for 30 min to allow proper
diffusion of the chemical and then incubated at
37.5°C for 2 h. These embryos were fixed for SEM.
All experiments were carried out at least in triplicate.
Scanning electron microscopy⎯At the end of 2 h,
the embryos were processed for SEM as described
before9,19. Embryos were fixed in 2.5%
glutaraldehyde (pH. 7.4) for 24 h at 4°C. Embryos
were washed with PBS to remove traces of
glutaraldehyde, dehydrated in a graded series of
ethanol and transferred to amyl acetate gradually.
Amyl acetate was replaced with liquid CO2 in a E3000
critical point drier (Bio-Rad). Three cycles of critical
point drying were carried out to ensure complete
drying. Embryos were mounted flat with their dorsal
side up on metal stubs, coated with gold in E5200
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automatic sputter coater (Bio-Rad), scanned with a
Stereoscan S120 SEM (Cambridge Instruments) and
photographed. A minimum of 15 representative
embryos were scanned from each group.
Results
Cells from the Hensen’s node, prospective neural
plate, anterior and posterior regions of the primitive
streak of stage 4 chick embryos (Fig. 1) were studied
by SEM. Simultaneously, alterations in the cellular
arrangement and cell surface features after 2 h of
treatment with either exogenous FGF or suramin were
studied.
Hensen’s node⎯Cells of the Hensen’s node of a
control embryo were arranged in a uniform sheet (Fig.
2A-C). Cells exhibited various types of surface
projections. Vesicular projections described earlier as
‘glass-bell-like formations’20, appeared as beads along
the cell boundaries. The cells also exhibited numerous
globular projections and a few small, thin thread-like
connecting cords (Fig. 2B, C). At the end of 2 h, FGFtreated embryos exhibited distorted cell surface
features (Fig. 2D-F). The cells appeared to be
crawling over each other and surface features were
not clearly discernible. The cells exhibited varied
sizes and shapes with more connecting cords as
compared to controls (Fig. 2A-C). In suramin-treated
embryos, the sheet of cells in Hensen’s node appeared
wavy (Fig. 2G). Surface protrusions increased to such
an extent that the cell surfaces were barely visible.
The cells were covered with globular projections (Fig.
2H, I). The surface protrusions were abnormal; they
were shorter, ended in bulb like structures and
appeared hollow which have been described as ‘bowllike projections’5 (Fig. 2I).
Prospective neural plate⎯In a control embryo the
cells of the prospective neural plate were arranged as
a uniform sheet (Fig. 3A-C). These cells possessed
Fig. 1⎯Diagrammatic representation of HH stage 4 embryo
showing the regions that were scanned for cell surface
ultrastructure studies. (A) Hensen's node, (B) prospective neural
plate, anterior (C) and posterior (D) parts of the primitive streak.
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INDIAN J EXP BIOL, APRIL 2010
Fig. 2⎯Scanning electron micrographs of cells of Hensen's node. HH stage 4 chick embryos were incubated in presence of either 6 nM
FGF or 2 mM suramin for 2 h at 37ºC and processed for scanning electron microscopy. (A) A control embryo showing uniform sheet of
cells. (B,C) Magnified views of cells from the same region of (A). (B) Note vesicular projections at cell boundaries (hollow arrow),
globular projections (arrow) and small thread like connecting cords (half-filled arrowhead). (D-F) Comparable area of FGF-treated
embryos showing distorted surface features of cells. (E) The cells appear to crawl over each other and exhibit variation in size and shape
(asterisk). Note increased abundance of connecting cords (half-filled arrowhead in E) and globular protrusions (arrow in F). (G-I) Cells of
the Hensen's node region of suramin-treated embryo. Note wavy nature of the cell sheet (G) and significant increase in the abundance of
small connecting cords (half-filled arrowhead in H) and globular projections (arrow in I). (I) bowl-like surface projections are also seen
(hollow arrow). Scale bar in (G) represents 20 μm (for A,D,G), in (H) 10 μm (for B,E,H) and in (I) 5 μm (for C,F,I).
more number of lengthy thread-like connecting cords
as compared to cells of the Hensen’s node (Fig. 2AC). These cells also exhibited presence of globular
projections on the cell surface. Bowl-like projections
were rarely seen in this region. In FGF-treated
embryos, the cell sheet appeared slightly wavy (Fig.
3D). In comparison to the control, there was a slight
increase in the number of surface protrusions, mainly
globular projections in this region (Fig. 3D-F). In
embryos treated with suramin, the prospective neural
plate cells were not arranged in one plane but
exhibited an undulating pattern (Fig. 3G). Cells were
irregular in shape with uneven surfaces and appeared
to be crowded (Fig. 3G,H). The abundance of bowl-
like projections was more than controls (Fig. 3H,I, for
control Fig. 3B,C).
Anterior region of the primitive streak⎯The cells
of the anterior region of the primitive streak of a
control embryo were arranged in a continuous
uniform sheet and exhibited smooth surfaces (Fig.
4A). Vesicular protrusions were located at cell
boundaries and a few cells showed presence of
globular projections and cilia (Fig. 4A-C). Cell
surfaces appeared flat than those of cells from
Hensen’s node (Fig. 2A-C) and prospective neural
plate (Fig. 3A-C). Also, lengthy connecting cords
were rarely seen in the cells of anterior region of the
primitive streak (Fig. 4B,C). In FGF-treated embryos,
BORGAVE & GHASKADBI: ALTERED FGF SIGNALING & CELL SURFACE CHANGES
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Fig. 3⎯Scanning electron micrographs of cells of prospective neural plate (A-C) A control embryo showing cells as a uniform sheet.
Cellular projections are mainly in the form of connecting cords (half-filled arrowhead in B) and globular projections (arrow in C). (D-F)
Comparable area of FGF-treated embryos showing slightly wavy appearance of the cell sheet (asterisk in D). Note slight increase in
abundance of connecting cords (half-filled arrowhead in E) and small globular projections (arrow in F). (G-I) A suramin-treated embryo.
(G) Note crowding of cells resulting in their irregular arrangement (asterisk). Cells appear with uneven surfaces (half-filled arrowhead in
H) with bowl-like protrusions (arrow in I). Scale bar in (G) represents 20 μm (for A,D,G), in (H) 10 μm (for B,E,H) and in (I) 5 μm (for
C,F,I).
the cells were arranged in a wavy sheet and seemed to
be crawling over each other (Fig. 4D-F). These cells
appeared highly distorted and the number of unevenly
distributed globular protrusions was significantly
increased. FGF treatment led to an increase also in the
proportion of lengthy connecting cords (Fig. 4E,F). In
suramin-treated embryos the cells were crowded and
with irregular surfaces (Fig. 4G-I). The abundance of
all types of cell surface projections was also increased
(Fig. 4G-I).
Posterior region of the primitive streak⎯In a
control embryo, cells located in the posterior one third
region of the primitive streak were arranged as a
continuous sheet. The number of surface projections
(Fig. 5A-C) appeared to be more as compared to the
anterior region of the primitive streak (Fig. 4A-C).
The connecting cords in this region were more
lengthy than any other embryonic region. These cells
also exhibited presence of globular projections and
microvilli. In FGF-treated embryos, globular
projections were slightly more abundant (Fig. 5D-F).
There was a considerable increase in the number of
surface protrusions in suramin-treated embryos that
covered almost the entire cell surfaces (Fig. 5G-I).
The connecting cords were significantly longer (Fig.
5I) as compared to controls (Fig. 5C) and appeared to
be entangled with connecting cords of distant cells
with tuberous protrusions in between (Fig. 5H,I).
Such lengthy connecting cords are thought to be
specialized systems for information transmission4,5.
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INDIAN J EXP BIOL, APRIL 2010
Fig. 4⎯Scanning electron micrographs of anterior region of the primitive streak (A-C) A control embryo. Note cells with smooth
surfaces that are arranged in a uniform sheet. Vesicular projections at cell boundaries (arrow in B) and a few globular protrusions (halffilled arrowhead in C) are seen. Connecting cords are rarely observed. (D-F) Cells from the anterior primitive streak of FGF-treated
embryo arranged in a wavy sheet. (E) Highly distorted shape of the cells and their crawling over each other is seen. (E) Significant
increase in abundance of unevenly distributed globular projections (arrow) and localized increase in connecting cords (hollow arrow) are
observed. (G-I) Comparable area of suramin-treated embryos. (H) Cells appear to be crowded with irregular surfaces (asterisk) and
increase in surface protrusions like globular bodies (arrow) and connecting cords (hollow arrow) is seen. (I) Note presence of bowl-like
projections in these embryos (half-filled arrowhead). Scale bar in (G) represents 20 μm (for A,D,G), in (H) 10 μm (for B,E,H) and in (I) 5
μm (for C,F,I).
Overall, these results show distinct differences in
the arrangement and the distribution of surface
projections of the cells located in different embryonic
regions that are destined to form different tissues.
These cell surface features appear to be dynamic in
nature as evident from their altered appearance after
exposure to exogenous FGF or suramin.
Discussion
The present work describes ultrastructural features
of cells on the dorsal surface of HH stage 4 chick
embryo. Cells of the Hensen's node, prospective
neural plate and anterior and posterior regions of the
primitive streak, which contribute to different tissues
of the embryo, were studied. The cells from different
embryonic regions exhibited distinct surface features
that could mainly be due to their requirement for
migration depending on their fate. These results are in
agreement with previous studies that demonstrate
morphological differences between cells from
different regions of the epiblast4,5. The most
commonly observed surface features in early chick
epiblast were globular projections, glass-bell-like
formations in the form of either bowl-like or vesicular
cell projections and thin thread like connecting cords.
The bowl-like and vesicular projections are thought to
be cytoplasmic connections between adjacent cells
while connecting cords are believed to help in
BORGAVE & GHASKADBI: ALTERED FGF SIGNALING & CELL SURFACE CHANGES
351
Fig. 5⎯Scanning electron micrographs of cells located in the posterior region of the primitive streak. (A-C) A control embryo showing
cells arranged in a continuous sheet. (B) Note presence of globular projections (hollow arrow), microvilli and long thread like connecting
cords (arrow). (D-F) Cells from the comparable region of FGF-treated embryo. Slight enhancement in the abundance of comparatively
short connecting cords (arrow in E) that cover most of the cell surface in F. The globular bodies look swollen in these embryos (arrow in
F). (G-I) Cells of the posterior region of the primitive streak of suramin-treated embryo. (H) Significant increase in the abundance of
microvilli (hollow arrow) and extremely lengthy connecting cords (arrow) is seen. (H,I) Cell surface is barely seen. Note presence of
lengthy connecting cords that appear to be entangled with projections from neighboring cells with tuberous structures in between (arrow
in H,I). Scale bar in (G) represents 20 μm (for A,D,G), in (H) 10 μm (for B,E,H) and in (I) 5 μm (for C,F,I).
communication between cells at longer distances4,5.
All the different types of surface protrusions are
means of cell-cell communication. The number and
appearance of these projections differ depending on
the embryonic region and the developmental stage4,5.
Cells of Hensen's node show a few surface
protrusions at the boundaries indicating active
migration whereas the prospective neural plate cells,
which are with relatively more but evenly distributed
surface protrusions suggest slower migration. The
cells of prospective neural plate appear tightly packed
than Hensen’s node cells. The cells ingressed through
the Hensen’s node move anteriorly to form the
foregut, head process and notochord21. Cells from the
anterior region of the primitive streak possess the
least number of surface protrusions. Cells of the
posterior region of the primitive streak, on the other
hand, exhibit maximum surface projections that are
much longer than those of any other cells. Anterior
primitive streak cells contribute to somites whereas
the cells of posterior primitive streak mainly form
extra-embryonic mesoderm22. In the present study, the
surface features of cells located in the anterior
primitive streak indicate that these cells may possess
extensive migratory activities as compared to cells of
posterior primitive streak. These results are in
agreement with reports4,5,23,24 that in a developing
embryo different groups of cells have different
migration capacities depending on their position and
fate.
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INDIAN J EXP BIOL, APRIL 2010
Endogenous FGF levels were altered with addition
of either exogenous FGF or suramin. Although suramin
affects a variety of growth factors, it predominantly
inhibits FGF action10,11,25-29. FGF is thought to act
through genes like brachyury10 and goosecoid11.
Brachyury is involved in induction and differentiation
of mesoderm12 whereas goosecoid regulates
gastrulation movements and formation of body axis13.
Modulation of expression of both the genes within 2 h
in response to altered FGF signaling was obseved. FGF
signaling is indeed necessary for cell movements
during streak formation in chick embryo30.
Present results indicate that altered FGF signaling
may hamper cellular movements during gastrulation
to varying extent in distinct embryonic areas. FGF is
believed to coordinate gastrulation movements14
through non-canonical Wnt11 signaling pathway31,
Wnt3a signaling pathway and by maintaining Ecadherin levels32. FGF-treated embryos exhibited
down-regulation of brachyury expression in the
primitive streak10. The observations of the present
study suggest that anterior region of the primitive
streak is the active site for ingression of epiblast cells
in early chick embryos. Crowding of cells observed in
this region of FGF-treated embryos may be due to
lack of sufficient amounts of brachyury as Brachyury
is essential for cells to undergo convergent extension
movements12. On the other hand, a few suramintreated embryos showed absence of brachyury
transcripts10 and presence of abnormally lengthy
connecting cord like projections in the posterior onethird region of the primitive streak. These
observations suggest that cells from this region may
have lost their ability to migrate. The surface
architecture of cells of Hensen's node was also
differentially altered due to FGF and suramin.
Differential modulation of goosecoid expression was
observed in the Hensen's node due to FGF and
suramin within 2 h11. The altered cell surface features
together with modulated expression of brachyury and
goosecoid may lead to improper cellular movements
during gastrulation and result in abnormal
morphogenesis of embryos treated with FGF and
suramin.
The present work demonstrates the distinct
differences in the migratory capacities of cells from
various embryonic regions as indicated by cell surface
architecture and dynamic changes in these
ultrastructural features in response to altered FGF
signaling. Earlier it has been shown that embryos with
altered levels of FGF signaling develop abnormal
neural and mesodermal structures10. Collectively, these
studies shows that the abnormal development in FGFand suramin-treated embryos is not only accompanied
by but appears to be an outcome of the rapid
modulation of brachyury10 and goosecoid11 as well as
cell surface architecture.
Acknowledgement
Thanks are due to Drs. Saroj Ghaskadbi, Hemant
Ghate and Vidya Patwardhan for discussions and Mr.
Rajdeep Dongre for technical help with SEM. This
work was supported by Agharkar Research Institute,
Pune.
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