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First published online 17 July 2008
doi: 10.1242/dev.022228
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1 Laboratory of Experimental Ontogeny, Program of Anatomy and Developmental
Biology, Institute of Biomedical Sciences, University of Chile, Clasificador 7
- Correo 7, Santiago, Chile.
2 Max-Planck-Institute of Molecular Cell Biology and Genetics,
Pfotenhauerstr.108, 01307 Dresden, Germany.
Author for correspondence (e-mail:
mconcha{at}med.uchile.cl)
Accepted 19 June 2008
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Enveloping layer, Epiboly, Organizer, Organogenesis, Kupffer's vesicle, Left-right asymmetry, Zebrafish
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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;
Nonaka et al., 1998;
Tanaka et al., 2005).
Although the conservation and function of laterality organs are well
established in vertebrates (Okada et al.,
2005; Schweickert et al.,
2007), comparatively little is known about their embryonic origin
and the morphogenetic mechanisms leading to their formation. To obtain
insights into these events, we analysed the formation of Kupffer's vesicle
(KV), the zebrafish organ of laterality
(Essner et al., 2005;
Kramer-Zucker et al., 2005).
KV is a transient organ proposed to arise from non-involuting, highly
endocytic marginal blastomeres (deep NEM cells) that are positioned below
surface-enveloping layer cells (EVL-NEM cells) at late blastula stage. Fate
map studies have suggested that at the onset of gastrulation, deep NEM cells
become displaced from the blastoderm margin to form a distinct group of dorsal
forerunner cells (DFCs) (Cooper and
D'Amico, 1996; D'Amico and
Cooper, 1997; Melby et al.,
1996). DFCs then move to the vegetal pole in close contact with
the overlying EVL margin and become transformed into an epithelial vesicle
(KV) that later produces a cilia-based flow within its interior lumen
(Amack et al., 2007;
Essner et al., 2005;
Kramer-Zucker et al., 2005).
During late somitogenesis, the vesicle collapses and DFCs become incorporated
into notochord, somites and tail mesenchyme
(Cooper and D'Amico, 1996).
Despite these remarkably detailed studies, crucial questions remain concerning
the cellular and molecular mechanisms that lead to DFC formation and the
subsequent transformation of this cell group into a functional organ.
In this study, we demonstrate that DFCs are formed by ingression of surface epithelial cells at the dorsal margin of the zebrafish gastrula, and that Nodal/TGFβ signals are both required and sufficient to induce this process. We further show that, after ingression, a subset of cells within the DFC cluster retains its original polarisation through persistent apical contact with the overlying EVL. These pre-polarised cells serve as `seeds' around which the remainder of DFCs assemble and epithelialise to form the mature KV. These findings point to a conserved mechanism of Nodal/TGFb-induced outer epithelium internalisation during laterality organ formation and demonstrate a novel mechanism by which external apicobasal polarity is propagated from the surface epithelium to an interior organ during vertebrate development.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) and
Tg(β-actin:HRAS-EGFP)
(Cooper et al., 2005). Embryos
were grown at 31°C unless otherwise indicated, manipulated in E3 zebrafish
embryo medium or Danieau buffer, and staged according to morphology
(Kimmel et al., 1995) and age
[hours post-fertilisation (hpf)].
mRNA injections
For mRNA synthesis, pCS2+ expression vectors containing the cDNAs for
different constructs were linearised with NotI and transcribed by SP6
mRNA polymerase as described (Montero et
al., 2003). For mosaic expression, one marginal blastomere was
injected at the 64-cell stage. mRNA amounts used for each experiment are given
in the figure legends.
Immunohistochemistry
Embryos were fixed in 2% or 4% paraformaldehyde (PFA) and stained as
described (Koppen et al.,
2006). Stained embryos were mounted on agarose-coated dishes in
phosphate buffered saline/0.5% Triton X-100 medium or embedded in 1%
low-melting-point agarose. The following primary antibodies and dilutions were
used: mouse anti-ZO-1 (Invitrogen, 1:200), rabbit anti-aPKC-
(Santa Cruz
Biotechnology, 1:200) and mouse anti-acetylated 
-tubulin (Sigma,
1:400). Samples were imaged on a Zeiss LSM META confocal microscope using an
Achroplan 40x/0.8 W dipping objective or a Plan-Apochromat 40x/1.2
W objective.
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Image quantification
Lumen volume analysis was performed by generating 3D renderings of
anti-aPKC- staining using Volocity software. KV cilia length
measurements were performed on z-projections of dorsal confocal
stacks of Tg(sox17:GFP) embryos stained with anti-acetylated
-tubulin using ImageJ software. The number of DFCs during epiboly was
determined using Imaris software (Bitplane), employing the spot recognition
function to count cell nuclei of sox17:GFP-expressing DFC
clusters.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) (see Fig.
1M for illustration).
As expected, we found that at the sphere stage, DFCs were not yet
discernible at the future dorsal side of the embryo. Instead, we observed
dorsal surface epithelial (DSE) cells that were in direct contact with the
yolk syncytial layer and exhibited filopodia-like protrusions at their leading
edge (Fig. 1A,G,J,M).
Strikingly, we found that during subsequent development, a subset of marginal
and submarginal DSE cells internalise through a process of ingression beneath
the surface epithelium (Fig. 1;
see Movies 1 and 2 in the supplementary material; data not shown). Ingression
occurred as trailing cells of the EVL moved over the DSE cells, displacing
them beneath the surface. Once ingressed, DSE cells were clearly recognisable
as DFCs by their position below the EVL margin and ahead of the deep cell
margin. We will therefore refer to these newly ingressed cells as DFCs. These
cells most likely correspond to the previously described deep NEM cells
(Cooper and D'Amico, 1996;
D'Amico and Cooper, 1997).
However, whereas deep NEM cells were observed to lie within the dorsal
blastoderm margin, we found newly ingressed DSE cells to be positioned ahead
of the deep cell margin. This discrepancy is most likely due to the different
labelling and imaging methods used to identify DFCs. Together, we conclude
that DFCs form via the ingression of marginal and submarginal DSE cells
beneath the surface epithelium.
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DSE cell ingression was often immediately preceded by a cell division
(Fig. 1G-I,M; see Movie 2 in
the supplementary material). Typically, the division occurred in the plane of
the epithelium, giving rise to two ingressing cells. However, in younger
embryos (around the `high' stage, 3.3 hpf), we frequently observed
delaminating divisions of DSE cells, which gave rise to a DSE cell and a deep
cell. We observed that in a few instances, the newly formed DSE cell became a
DFC, while the second daughter cell exclusively became a deep cell (see Fig.
S1 and Movie 4 in the supplementary material). This finding is surprising, as
previous analysis of cell division patterns of the zebrafish blastula had
suggested that a DSE cell resulting from a delaminating division is committed
to the EVL fate (Kane et al.,
1992). Together, we conclude that the type of DSE cell division
does not strictly correlate with EVL versus DFC fate.
DFC formation took place concurrently with the onset of embryonic epiboly,
during which marginal deep cells, marginal EVL cells and DFCs all advance
towards the vegetal pole (Cooper and
D'Amico, 1996; Melby et al.,
1996). We found that the conversion of DSE cells into DFCs
consistently completed by 50% epiboly, immediately prior to the formation of
the embryonic organizer (shield). As epiboly progressed, the DFC group became
increasingly separated from the dorsal marginal deep cells and remained in
direct contact with the advancing dorsal EVL margin
(Fig. 1G-I,M)
(D'Amico and Cooper, 1997).
Furthermore, the number of DFCs increased between shield stage and 80% epiboly
from 31±9 (n=16 embryos, mean±s.d.) to 50±11
(n=9), as determined using a Tg(sox17:GFP) transgenic line
(see below). This increase is likely to be due primarily to cell division
within the cell cluster, based on our analysis of time-lapse movies (data not
shown). These numbers are higher than those determined previously
(Cooper and D'Amico, 1996),
probably because of the different methodologies applied to identify DFCs (see
Materials and methods).
Ongoing with vegetal DFC progression, DFCs rapidly converged to the dorsal
side by intercalating between each other, thereby transforming the initially
wide array of cells into a more compact and oval-shaped cluster by about 80%
epiboly (Fig. 1D-I; see Movie 5
and Figs S2 and S3 in the supplementary material). This convergence was
notably mirrored by dorsal convergence of the overlying dorsal marginal EVL
cells (Fig. 1A-C,G-I).
Strikingly, a subset of DFCs became increasingly bottle-shaped during epiboly
and remained persistently linked to the dorsal EVL margin via attachment
points that were enriched for the tight-junction components ZO-1 (Tjp1 - ZFIN)
and protein kinase C zeta (aPKC-; Prkcz - ZFIN)
(Fig. 2A-B' and data not
shown). By contrast, the remainder of cells in the cluster appeared
unpolarised, forming dynamic cell protrusions without a preferential
orientation (see Movie 5 and Fig. S3 in the supplementary material). This
suggests that tight-junction based DFC-EVL attachment couples the vegetal
movement and convergence of both tissues. This finding is consistent with
previous analyses suggesting that the movement of DFCs and of the EVL are
coupled via physical tissue linkage
(D'Amico and Cooper, 1997;
Solnica-Krezel et al.,
1996).
|
).
Consistent with the above results, we found that multiple subgroups of
bottle-shaped DFCs were attached to overlying dorsal marginal EVL cells via
ZO-1-rich subcellular structures at 80% epiboly
(Fig. 2C,C'). After
reaching the vegetal pole during the final phase of epiboly, the cluster
became separated from the overlying dorsal marginal EVL cells, most likely
owing to dorsal marginal deep cells moving between the two tissues during the
vegetal sealing of the embryo (Fig.
2D,D'; see Fig. S2 in the supplementary material). Using
both live imaging and immunolabelling, we found that during this separation
the apices of bottle-shaped DFCs turned towards the inside of the cluster and
generated multiple three-dimensional rosette structures around ZO-1-rich
apical focal points (Fig.
2D,D',F,F'; see Movie 6 in the supplementary
material). At the same time, the DFC cluster became increasingly compact and
acquired the shape of a flattened sphere
(Fig. 2C-D'; see Figs S2
and S3 in the supplementary material). The separation of the DFC cluster from dorsal marginal EVL cells and its rearrangement into rosettes was quickly followed by the formation of an interior vesicle lumen (Fig. 3A-C; see Movie 7 in the supplementary material). This occurred as the initially widely spaced apical focal points within the cluster coalesced into a dense network around which cells arranged into a single large rosette structure, incorporating previously unpolarised DFCs. During these rearrangements, small extracellular lumina typically formed at multiple apical focal points (Fig. 3A,B,D,E,G,H) and rapidly fused into a single ZO-1-lined lumen (Fig. 3C,F,I). Lumen volume then increased approximately 12-fold between the 1-somite and 4-somite stages (see Fig. S4 in the supplementary material). Live imaging of DFCs expressing GFP revealed that cytoplasmic, vacuole-like structures fused with the apical membrane domain as the lumen formed, suggesting that apical vacuole fusion contributes to apical membrane expansion and lumen growth (see Movie 8 in the supplementary material). Interestingly, we found that lumen formation was directly coupled to ciliogenesis. Small, tubulin-rich cilia were first detected at the onset of lumen initiation, when the lumen-facing membrane domain of polarised DFCs started developing cilia (Fig. 3J-L). Lumen volume and cilia length continuously increased during KV maturation (between bud and 4-somite stage), while cilia number remained essentially constant (see Fig. S4 in the supplementary material). This suggests that no additional cilia-forming cells are recruited to the cluster during lumen formation and expansion. Together, these observations suggest that initial DFC polarisation is established during epiboly and contributes to the subsequent formation of rosette structures by a subset of polarised DFCs. Later epithelial differentiation to form KV involves concurrent lumen formation and ciliogenesis.
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). Importantly, recent reports have shown that DFC number
increases or decreases in response to enhanced or reduced Nodal signalling,
respectively (Choi et al.,
2007). In agreement with this, we observed that overexpression of
the Nodal antagonist Lefty strongly decreased the DFC number
(Fig. 4B,E,H). In addition,
maternal-zygotic one-eyed pinhead mutants, which are defective for
Nodal signalling, completely lacked recognisable DFCs at 80% epiboly (data not
shown). Together, this strongly suggests that Nodal signalling is required for
surface cell internalisation to generate DFCs. To test whether Nodal signalling acts instructively during DFC formation, we performed gain-of-function experiments by overexpressing the Nodal signalling ligand Cyclops (Cyc; Ndr2 - ZFIN). Strikingly, we observed that numerous DFC clusters were generated from DSE cells along the whole circumference of the embryo (Fig. 4C). A major dorsal cluster was typically observed that showed increased DFC number at 80% epiboly (104±50, n=5 embryos, mean±s.d.) compared with uninjected controls (50±11, n=9). The enlarged DFC clusters were attached to the EVL margin via apical cell domains, similar to in controls (Fig. 4A,C). Multiple DFC clusters were also observed at the bud stage and early somite stages. These clusters typically contained the characteristic interior apical focal points of controls and underwent partial lumen formation (Fig. 4F,I, and data not shown). These results indicate that enhanced Nodal signalling is sufficient to induce ectopic DFC clusters that undergo partially normal morphogenetic transformation.
To gain insight into the dynamics of ectopic DFC formation in Nodal-overexpressing embryos, we performed time-lapse confocal imaging of Tg(β-actin:HRAS-EGFP) embryos co-injected with cyc mRNA and fluorescently labelled dextran (to identify cyc-expressing cells). Importantly, we found that in these embryos, DFCs formed via the same principal mechanism observed in controls (see Fig. 1). Namely, surface epithelial cells located at the Cyc-overexpressing side of the embryo became transformed into DFCs-like cells through their displacement beneath the EVL sheet after sphere stage (Fig. 4J-L'; see Movie 9 in the supplementary material). In addition, we found this behaviour predominantly in the dextran-negative cells (n=3 movies), suggesting that Cyc induces cell ingression in a non-cell-autonomous manner. In summary, our findings indicate that DFC formation through ingression of surface epithelial cells is controlled by Nodal signalling, which plays both instructive and permissive roles in this process.
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DISCUSSION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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) for
embryonic stages]. Notably, this cell behaviour predominantly occurs in a
region of the dorsal blastoderm margin that is homologous to the organizer
epithelium of Xenopus (Cooper and
Virta, 2007; Shih and Keller,
1992). This suggests that DFCs are formed through a previously
unrecognised process of organizer epithelium ingression in zebrafish.
Interestingly, this organizer epithelial cell ingression represents the
earliest morphogenetic cell behaviour specifically linked to the ontogeny of
the organizer region in zebrafish. Furthermore, it adds to the notion that the
organizer region can be subdivided into domains of distinct morphogenetic
behaviours that generate distinct organ rudiments such as KV, prechordal plate
and notochord (Cooper, M. S., Virta, V. C.
(2007);
Montero et al., 2003).
Together, these findings provide novel insights into cell lineage decisions
and morphogenesis of the organizer region at the onset of zebrafish
gastrulation.
Previous analysis has suggested that KV formation involves a
mesenchymal-to-epithelial transition of the DFC cluster
(Amack et al., 2007). Here we
show that vesicle formation involves an early phase in which epithelial
surface cells convert into deep DFCs with mesenchymal behaviour, followed by a
phase of re-epithelialisation that transforms the DFC cluster into a ciliated
epithelial vesicle. Intriguingly, a subset of polarised DFCs persistently
retains epithelial properties throughout the organogenetic process. These DFCs
initially form apical contacts with the EVL, which is likely to facilitate
their vegetal movement. Subsequently, the polarity of these cells is
maintained as they become internalised and form the `seeds' around which the
remaining mesenchymal DFCs assemble into polarised rosettes. This observation
suggests that epithelial polarity is not initiated de novo within the DFC
cluster, but is instead derived from the apicobasal polarity of the surface
epithelium. Thus, the initial apicobasal polarity of an embryonic surface
tissue provides the polarising cue that underlies the formation of a polarised
interior organ. These findings provide important new insights into
embryological mechanisms underlying the initiation and propagation of
epithelial polarity.
Our finding that KV derives from an embryonic surface epithelium offers new
perspectives concerning the evolutionary conservation of laterality organ
formation in vertebrates. Similar to KV in zebrafish, the amphibian laterality
organ (the gastrocoel roof plate) is a transient ciliated epithelium that
forms through internalisation of surface epithelium of the dorsal organizer
region (Schweickert et al.,
2007; Shook et al.,
2004). Moreover, the precursors of the mammalian organ of
laterality (the posterior notochordal plate) also appear to be derived from
the epithelial epiblast (Blum et al.,
2007; Hirokawa et al.,
2006; Kinder et al.,
2001). However, whereas in Xenopus, where organizer
surface epithelial cells internalise through involution, in zebrafish they
undergo ingression, suggesting that the cytomechanics of organizer surface
epithelium internalisation has evolved differently along phylogenetic lineages
(reviewed by Cooper and Virta,
2007).
Our evidence for a role of Nodal signalling during organizer epithelium
internalisation and DFC specification additionally suggests that the
conservation of vertebrate laterality organ formation also extends to the
molecular level. In Xenopus, specification of the dorsal organizer
epithelium is dependent on Nodal signals produced by the vegetal endoderm
(reviewed by Shook et al.,
2004). Moreover, specification of the mammalian anterior head
process precursors, a group of cells that contributes to the notochordal
plate, also requires high levels of Nodal signalling
(Vincent et al., 2003;
Yamanaka et al., 2007).
Together, this strongly points to a conserved role for Nodal signalling during
internalisation of outer epithelia, which is likely to be the initial step in
vertebrate laterality organ formation. Further studies analysing vertebrate
species other than zebrafish will be needed to fully elucidate the conserved
cellular and molecular mechanisms by which vertebrate embryos acquired a
cilia-dependent, Nodal flow-based strategy to control the handedness of their
body plan.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/135/16/2807/DC1
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ACKNOWLEDGMENTS |
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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4.2 hpf) and subsequent time
points show the progression of dorsal surface epithelial (DSE) cells towards
the vegetal pole. Some marginal DSE cells form filopodia-like protrusions at
their leading edge (arrows and inset in A). (D-F) Images of the deep
cell layer at equivalent time points to A-C, with the deep cell margin marked
with arrowheads. DFCs (brackets) become visible directly in front of the deep
cell margin shortly after the dome stage (E) and then move progressively away
from the margin (F). (G-I) Fate mapping of DSE cells shows that nine of
these cells at or close to the margin (coloured), but no deep cells, become
converted into DFCs. Cells remaining within the enveloping layer (EVL) are
outlined in white and traced by number. The same principal finding was made in
all five embryos analysed by live imaging. The DSE-to-DFC conversion occurs as
the DSE cells round up and become covered by the trailing EVL cells that form
the new EVL margin. During this process, two of these cells (light green and
dark blue) can be seen to divide once (H,I). During vegetal movement, both EVL
cells and DFCs converge to the dorsal side. (J-L) Three-dimensional
rendering and cutting of the image stacks along the dotted lines shown in G-I
allows simultaneous display of the xy and yz axes, and
reveals the repositioning of red and dark-blue DSE cells below the tissue.
(M) Schematic representation of DSE-to-DFC conversion and deep cell
internalisation as observed in the live recording. Two DSE cells, depicting
the red and dark-blue DSE cells in the movie, become positioned beneath the
EVL to become DFCs. The blue cell had divided immediately prior to
internalisation. The attachment point linking DFCs to the EVL is shown as a
yellow spot. Two deep cells (asterisks) are shown to undergo internalisation
to form hypoblast. Scale bar: 30 µm.
