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First published online 11 March 2009
doi: 10.1242/dev.026922
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1 Max Planck Institute of Molecular Cell Biology and Genetics, Pfotenhauerstr.
108, 01307 Dresden, Germany.
2 Max-Planck-Institute for the Physics of Complex Systems, Nöthnitzer Str.
38, 01187 Dresden, Germany.
3 European Molecular Biology Laboratory, Cell Biology and Biophysics Department,
Meyerhofstr. 1, 69126 Heidelberg, Germany.
* Author for correspondence (e-mail: heisenberg{at}mpi-cbg.de)
Accepted 11 February 2009
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SUMMARY |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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Key words: Gastrulation, Zebrafish, Nuclear movements, Yolk syncytial layer, Cortical flow
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INTRODUCTION |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Although in
most cases nuclei are transported along microtubules by microtubule motor
proteins (Morris, 2000;
Morris, 2003;
Reinsch and Gonczy, 1998), the
actin cytoskeleton has also been implicated
(Reinsch and Gonczy, 1998;
Starr and Han, 2003). In
addition to these intrinsic transport mechanisms, signals from surrounding
tissues and cells have also been involved in nuclear movements. Notably,
studies in Drosophila have provided evidence that the migration of
the oocyte nucleus, which is required to establish the dorsal-ventral axis of
the embryo, crucially depends on localized signals from the surrounding
follicular epithelium (Gonzalez-Reyes et
al., 1995; Roth et al.,
1995).
Extensive nuclear movements have been observed within syncytial
environments in metazoan development
(D'Amico and Cooper, 2001;
Englander and Rubin, 1987;
Foe and Alberts, 1983;
Trinkaus, 1993;
Xiang and Fischer, 2004). One
of the best-studied examples is the Drosophila syncytial
preblastoderm, where nuclei initially move along the anterior-posterior axis
of the embryo by actin-dependent cytoplasmic streaming and are subsequently
transported towards the cortex along microtubules by associated motor proteins
(Baker et al., 1993;
Robinson et al., 1999;
von Dassow and Schubiger,
1994). Although these studies have provided detailed insights into
the molecular and cellular mechanisms that underlie syncytial nuclear
migrations, much less is known about how such migrations are coordinated with
the movements of surrounding cells and tissues in the developing organism.
To obtain insights into the coordination of syncytial nuclear movements
with surrounding cells and tissues, we have chosen to study the yolk syncytial
layer (YSL) in the zebrafish gastrula. The YSL and its nuclei play crucial
roles in embryo patterning and morphogenesis. Nodal/TGFβ signals
emanating from the YSL are thought to be involved in specifying mesoderm and
endoderm cell fates in marginal blastomeres along the circumference of the
embryo (Chen and Kimelman,
2000; Mizuno et al.,
1999; Mizuno et al.,
1996; Ober and Schulte-Merker,
1999; Rodaway et al.,
1999). Furthermore, interfering with the actin and microtubule
cytoskeleton of the YSL leads to defective epiboly movements of both the YSL
and the cellularized blastoderm (Cheng et
al., 2004; Solnica-Krezel and
Driever, 1994; Zalik et al.,
1999), suggesting that proper YSL morphogenesis is required for
blastoderm epiboly. Finally, the YSL has been proposed to induce fibronectin
expression in the overlying embryonic tissue, which is important for heart
progenitor cell migration during gastrulation and somitogenesis stages
(Sakaguchi et al., 2006).
The YSL is formed by marginal blastomeres that collapse and deposit their
nuclei into the cortical cytoplasm of the yolk cell at blastula stage
(Kimmel and Law, 1985;
Trinkaus, 1993). These yolk
syncytial nuclei (YSN) undergo three to five rounds of divisions before
ceasing mitosis at sphere stage. At this stage, the YSL contains several
hundreds of YSN that can be subdivided into two main groups according to their
position within the YSL (D'Amico and
Cooper, 2001). External YSN (eYSN) form a marginal band of YSN
located in front of the enveloping cell layer (EVL). Some of the eYSN move
towards the vegetal pole (epiboly movements), whereas others recede from the
marginal zone and undergo convergence and extension (CE) movements. By
contrast, internal YSN (iYSN) are located below the blastoderm and EVL and
primarily undergo CE movements. Strikingly, CE movements of iYSN appear very
similar to the CE movements of the overlying blastoderm (mesoderm, endoderm
and ectoderm progenitors) (D'Amico and
Cooper, 2001). It has therefore been hypothesized that iYSN
movements are driven by chemotactic signals emanating from overlying
mesendoderm cells in the hypoblast (Cooper
and Virta, 2007). Alternatively, iYSN may direct mesendoderm
movements, or iYSN and mesendoderm may move independently from each other
(D'Amico and Cooper, 2001).
However, direct experimental evidence for any of these hypotheses is still
missing.
In this study, we show that the movements of mesendoderm progenitors and iYSN are coordinated during zebrafish gastrulation. Our findings suggest that mesendoderm progenitors direct iYSN movement by modulating cortical flow within the YSL responsible for nuclear transport. Furthermore, we show that the ability of mesendoderm progenitors to guide iYSN movements requires E-cadherin expression, suggesting that adhesive contact between mesendoderm progenitors and YSL is involved.
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MATERIALS AND METHODS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). Details
can be provided on request.
Nuclear labeling
To label nuclei in live embryos, 1 mg/ml Histone H1 conjugated to Alexa
Fluor 488 (H13188, Invitrogen) was injected in one marginal blastoderm cell at
16-cell stage (1.5 hours post-fertilization; hpf) to visualize mesendoderm
progenitors, and in the YSL between high and sphere stage (3.3-4.0 hpf) to
mark YSN.
mRNA injections
mRNA was synthesized and injected as described
(Montero et al., 2005;
Westerfield, 2000). Details
can be provided on request.
Morpholino oligonucleotide injection
To knock down E-cadherin function, a previously described morpholino
oligonucleotide designed against the 5' end of e-cadherin cDNA
(Babb and Marrs, 2004;
Montero et al., 2005) (Gene
Tools) was injected at the one-cell stage (0.25-0.5 ng).
Polystyrene microsphere injection
Fluoresbrite White-Green Microspheres (Polysciences Europe, Germany) with a
diameter of 0.5 µm were washed twice in water by brief centrifugation and
removal of the supernatant, then incubated for 1 hour in 0.1 µg/µl
bovine serum albumin to block unspecific binding, briefly centrifuged and
re-suspended in water. To insert microspheres into the YSL, a drop of 0.1 nl
was injected at the margin of the YSL at 30% epiboly.
Transplantation experiments
MZoep mutant embryos were used as host embryos, and wild-type
embryos were used as donor embryos. Between sphere and dome stage (4.0-4.3
hpf), dechorionated embryos were transferred into an agarose chamber. About 20
cells from a donor embryo were transferred into the blastoderm margin of a
host MZoep embryo.
Cytochalasin and phalloidin treatment
For cytochalasin treatment, dechorionated 30-50% epiboly stage (5 hpf)
embryos were incubated in 1-2 µg/ml cytochalasin B (Sigma-Aldrich, Germany)
in 1x Danieau's buffer for 1-2 hours at 31°C. After treatment,
embryos were mounted in low melting point (LMP) agarose containing
cytochalasin. For phalloidin experiments, we injected into the YSL 100 pg
Lifeact-GFP mRNA at the 1000-cell stage (3 hpf), 500 pg Histone-Alexa
488 at sphere stage (4 hpf) and 20 pg Amino-Phalloidin, Hydrochloride (Cat.
No. ALX-350-266-M001, Alexis Biochemicals) at 50% epiboly.
Embryo sectioning and immunostaining
Embryos were sectioned and immunostained as described
(Montero et al., 2005).
Details can be provided on request.
Transmission electron microscopy
For analysis of the YSL ultrastructure, transmission electron microscopy
was performed as described (Montero et
al., 2005). Dechorionated embryos were fixed in 2% gluteraldehyde
and 0.5% paraformaldeheyde in 0.1 M phosphate buffer overnight at 4°C.
Two-photon excitation timelapse microscopy
Two-photon imaging was performed as described
(Ulrich et al., 2003). Details
can be provided on request.
Movement analysis in two dimensions
To analyze YSN and microsphere movements in two dimensions (2D), we used
previously described Motion Tracking software
(Helenius et al., 2006;
Rink et al., 2005). Net dorsal
speed was calculated by dividing the net displacement in the x-axis
by the total time. We used the emerging notochord and neural tube as
anatomical landmarks to determine the embryonic midline.
Movement analysis in three dimensions
To analyze nuclear and cell movements in three dimensions (3D), we
implemented an automated tracking algorithm, allowing the estimation of
nuclear trajectories without user interaction
(Stühmer, 2007).
Similarity quantification
In this section we refer to a cell, nucleus or bead generically as an
`element' for convenience, as the same algorithm is used to quantify movement
of all three. The velocity of element i at time point
tk can be estimated from the displacement between two
consecutive timepoints as
where
). The similarity between the motion of element i and
a neighboring element j at time t can be defined as the
normalized internal product of the corresponding velocity vectors, yielding
the cosine of the smallest angle between these vectors:
Elements moving with parallel velocity give the maximum similarity of 1.0, whereas elements moving in different directions give smaller values. A value of -1.0 indicates movement in opposite directions.
Because the image segmentation algorithm that is used for automated tracking in 3D depends on the image resolution, estimated positions can adopt only discrete values. The resulting quantization error is especially large in the z-direction. To reduce this effect of quantization noise, the positions are filtered along the trajectory with a mean filter kernel that averages over the four previous and future time points.
Distance difference quantification
To analyze the movement of iYSN in the presence of mesendoderm progenitors
in our transplantation experiments, we calculated the normalized distance
difference 
dij(t) between each
nearest-neighbor iYSN/mesendoderm progenitor pair (i,j) at
consecutive time points.
where
Flow field analysis
The flow fields were computed with the FlowJ plugin for ImageJ (NIH, USA)
(Abramoff et al., 2000), using
the Lucas-Kanade method (Lucas and Kanade,
1981). Flow vectors where drawn using a custom-built vector field
visualization plugin for ImageJ that exports the computed vectors into SVG
(Scalable Vector Graphics) format.
Hydrodynamic description of progenitor-induced cortical flow
By investigating the constitutive properties of the cortex and the length
and time scales involved in progenitor migration, we develop a suitable
physical description of YSN dynamics. The cytoskeleton is viscoelastic,
displaying elastic behavior on short time scales and viscous behavior on long
time scales. The time scale for elastic relaxation is system dependent, though
typical values are of the order of seconds to minutes
(Pullarkat et al., 2007), e.g.
40 seconds for chick fibroblasts
(Thoumine and Ott, 1997). The
time scale for cortical flow induced by a transplanted patch of progenitors is
given by a/Up50 minutes, where
a50 µm is the radius of a progenitor patch and
Up1 µm/minute is the speed with which the
progenitor patch moves. Since the time scale for cortical flow is much longer
than that for elastic relaxation, the cortex may be described as an
incompressible viscous fluid for the present purpose. In general, the flow
profile is a function of the Reynolds number (the ratio of inertial to viscous
forces) and the system geometry (Landau
and Lifshitz, 1987). We overestimate the Reynolds number as
Re=
Upa/
10-5, where
is the density, which we take to be that of water, and is the viscosity,
which we underestimate to be 10x that of water, approximately the
measured viscosity of cytoplasm in tissue culture cells
(Luby-Phelps, 2000) (the
viscosity of the cortex is expected to be much higher). To specify the
geometry, we model the cortex as an infinite 2D fluid in the plane immediately
below the progenitor patch. We specify that the fluid in contact with the
patch moves with a velocity U, whereas the rest of fluid is free to
move, satisfying the boundary conditions that the velocity is U at
the edge of the patch and zero infinitely far away. This is equivalent to flow
past a circle, for which approximate solutions for the flow profile are known
for low Re (Lamb,
1932; Van Dyke,
1975). Importantly, in the low Re regime, the velocity
decays from the progenitor patch very slowly as log r, r being the
distance from the patch. Finally, we verify that the YSN move along with the
cortical flow (advectively), as opposed to diffusively, by estimating the
Péclet number, Pe=Ua/D, the ratio of
diffusive to advective time scales, where D is the diffusivity. To estimate
D, we use the Stokes-Einstein-Sutherland relation with an effective
viscosity of 1 Pa-s, interpreted from measurements of microspheres in
fibroblasts (Luby-Phelps,
2000), giving D10-4 µm2/s.
This is an underestimation of D, as the YSN are larger than the 0.16
µm beads described previously
(Luby-Phelps, 2000), resulting
in greater effective viscosity. Nevertheless, we obtain Pe50,
which means that advection dominates diffusion and the YSN move with the
progenitor-induced cortical flow.
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RESULTS |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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). To quantitatively determine the extent to which iYSN and
mesendoderm convergence movements are coordinated, we recorded timelapse
movies of paraxial and lateral regions of gastrulating zebrafish embryos in
which we fluorescently labeled iYSN and nuclei of overlying mesendoderm
progenitors (Fig. 1A-C). To
analyze and compare the trajectories of iYSN and mesendoderm cells, we
developed an image analysis software that allows automated tracking of nuclei
in three dimensions (3D) over time (Fig.
1C-F; Movie 1) (Stühmer,
2007). We quantified movement coordination between iYSN and
mesendoderm progenitors by determining the similarity between the movements of
mesendoderm cells and iYSN at short (0-40 µm), medium (40-80 µm) and
long (80-120 µm) distances. The similarity value corresponds to the cosine
of the smallest angle between two velocity vectors
(Costa Lda et al., 2005).
Thus, cells and iYSN moving in the same direction will have high similarity
values (close to 1), whereas cells and iYSN moving in opposite directions will
have low similarity values (close to -1). We found that for short distances
52% of values were higher than 0.5, indicating high movement similarity
(Fig. 1G). The similarity
values were only slightly reduced for medium and long distances
(Fig. 1H,I), suggesting that
movement coordination of iYSN and mesendoderm slowly decays with increasing
distance. We also determined movement similarity among iYSN and among
mesendoderm progenitors, and found a high degree of movement similarity in
both cases (see Fig. S1 in the supplementary material). These observations
indicate that cellular and nuclear movements are highly coordinated not only
between the mesendoderm and YSL, but also within each layer. In addition, we
observed that mesendoderm progenitors move faster than the underlying iYSN
[v(mesendoderm)=1.46 µm/minute; v(iYSN)= 1.06
µm/minute; P<0.05, n=6 embryos]. Taken together, our
observations extend previous findings
(D'Amico and Cooper, 2001) and
show that iYSN and mesendoderm progenitors exhibit highly coordinated
convergence movements over long length scales during gastrulation.
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) that the YSL contains a dense network of
microtubules where iYSN are embedded. To test whether microtubules might be
involved in iYSN movements, we recorded timelapse movies of the microtubule
network by expressing a GFP fusion of the microtubule-associated protein Tau
in the YSL (Geldmacher-Voss et al.,
2003; Kaltschmidt et al.,
2000) (see Movie 2 in the supplementary material). The movement of
labeled microtubules in relation to iYSN was analyzed by determining the
microtubule flow pattern (for details, see Materials and methods) and
comparing it with the movement of iYSN therein. We found that during later
stages of gastrulation (8-10 hpf), the YSL microtubule network converges
towards the dorsal side, similar to the iYSN
(Fig. 2A,D), suggesting that
iYSN move together with the microtubule network rather than along
microtubules. This view was supported by our observation that the movements of
the plus ends of microtubules, visualized by expressing a GFP fusion of the
microtubule plus end-binding protein 3 (EB3)
(Nakagawa et al., 2000;
Stepanova et al., 2003) in the
YSL, did not exhibit any preferential orientation within the YSL and that iYSN
did not show any obvious plus end-directed movements
(Fig. 2B,E; see Movie 3 in the
supplementary material). To determine whether convergence movements are
restricted to iYSN and microtubules, we recorded timelapse movies of cortical
actin movements, visualized by expressing Lifeact-GFP
(Riedl et al., 2008) (see
Movie 4 in the supplementary material). Similar to the iYSN and the
microtubule network, cortical actin exhibited pronounced convergence movements
(Fig. 2C,F), indicating that
the actin and microtubule cytoskeleton undergo convergence movements
together.
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Mesendoderm progenitors direct iYSN convergence movement
The observation that iYSN and mesendoderm progenitors exhibit highly
coordinated convergence movements during gastrulation suggests interaction
between the two. To test whether mesendoderm progenitors are required for iYSN
convergence movements, we investigated iYSN movements in MZoep mutant
embryos, which lack most of their mesendoderm progenitors
(Gritsman et al., 1999). By
analyzing iYSN movement of MZoep embryos in 2D at late gastrulation
stages (8-11 hpf), we found that mutant iYSN from lateral and ventral regions
of the gastrula exhibited strongly reduced convergence movements compared with
wild-type embryos (Fig.
3A,B,D-F; Movies 6-10; see Table S1 in the supplementary material)
(D'Amico and Cooper, 2001). To
quantify the degree of convergence of iYSN movements, we calculated their net
speed along the dorsoventral axis. We found that, in MZoep, the net
dorsal speed is significantly lower than in wild type
(Fig. 3C; see Table S1 in the
supplementary material). By contrast, longitudinal YSN movements in dorsal
regions appeared largely unchanged in mutant embryos from early to
mid-gastrulation stages (6-8 hpf; data not shown). In late gastrulation stages
(8-11 hpf), longitudinal movements of mutant iYSN in dorsal regions occurred
predominantly in an anterior direction, whereas dorsal iYSN in wild-type
embryos undergo longitudinal movements in both anterior and posterior
directions (Fig. 3A,B)
(D'Amico and Cooper,
2001).
Notably, injection of microspheres into the YSL of mutant embryos revealed
that similar to iYSN movements, the convergent cortical flow was strongly
reduced in MZoep mutants, suggesting that mesendoderm influences iYSN
convergence movements by modulating cortical flow (see Fig. S2 and Movie 11 in
the supplementary material). To exclude the possibility that Oep protein
itself is needed within the YSL for iYSN movements independently of the
presence or absence of mesendoderm progenitors, we injected oep-flag
mRNA (Zhang et al., 1998)
into the YSL of MZoep mutant embryos. We observed no rescue of the
MZoep iYSN mutant phenotype in the YSL-injected embryos (see Fig. S3,
Table S1 and Movie 12 in the supplementary material).
Endoderm progenitors have previously been suggested to move in close
contact to the YSL plasma membrane (Warga
and Nusslein-Volhard, 1999). To test whether endoderm progenitors
are specifically required for iYSN convergence, we analyzed casanova
(cas) mutant embryos, which lack endoderm but not mesoderm
progenitors (Alexander et al.,
1999; Dickmeis et al.,
2001; Kikuchi et al.,
2001). In contrast to the situation in MZoep mutant
embryos, iYSN convergence movements appear largely unaffected in cas
mutants (see Fig. S4 and Movie 13 in the supplementary material), indicating
that endoderm progenitors are not specifically required for iYSN convergence.
Taken together, these data suggest a crucial function of mesoderm progenitors
in iYSN convergence movements.
Although these findings demonstrate a requirement for mesendoderm
progenitors in iYSN convergence, they do not indicate whether mesendoderm
progenitors are also sufficient to direct iYSN convergence movements. To
address a possible instructive function of mesendoderm for iYSN movement, we
transplanted a small number of blastodermal cells into the lateral side of
MZoep mutant embryos and monitored the movements of iYSN relative to
the position of the transplanted cells. The majority of these cells underwent
ingression and expressed mesendodermal markers, as previously reported
(Carmany-Rampey and Schier,
2001) (data not shown), indicating that they adopt a mesendodermal
fate. From mid-gastrulation stages onwards, the transplanted cells converged
toward the dorsal side (Fig. 4;
see Movies 14 and 16 in the supplementary material). Strikingly, iYSN in the
transplanted side of MZoep mutant embryos converged together with the
transplanted cells, whereas iYSN on the opposite, non-transplanted side
continued to show only very reduced convergence movements
(Fig. 4A-E; see Movies 15 and
16 in the supplementary material). This was confirmed when we determined the
net dorsal speed of iYSN movement in the presence and absence of transplanted
cells (Fig. 4C; see Table S1 in
the supplementary material). To analyze the convergence movements of iYSN
relative to the transplanted cells in more detail, we imaged the transplanted
region at higher magnifications and tracked their movements in 3D
(Fig. 4F,G; see Movie 14 in the
supplementary material). We found that, as in wild-type embryos, iYSN and
transplanted cells move with high similarity values (see Materials and
methods) at short as well as at long distances
(Fig. 4H,I). These results
suggest that convergence movements of mesendoderm progenitors are able to
induce convergence movements of underlying iYSN.
|
E-cadherin (cdh1 gene in zebrafish) has previously been shown to
be highly expressed in the YSL and mesendoderm progenitors, and to represent a
key component regulating cell-cell interaction both within and between the
forming germ layers during gastrulation
(Fig. 5B)
(Babb and Marrs, 2004;
Kane et al., 2005;
Krieg et al., 2008;
McFarland et al., 2005;
Montero et al., 2005;
Shimizu et al., 2005). To
determine whether E-cadherin is also required for the coordination of iYSN and
mesendoderm progenitor convergence movements, we interfered with E-cadherin
expression by injecting a previously characterized morpholino oligonucleotide
(MO) targeted against e-cadherin and monitored iYSN and mesendoderm
convergence movements in 3D over time. We analyzed the movements of iYSN and
mesendoderm at mid-gastrulation stages (7-8 hpf)
(Fig. 5C; see Movie 17 in the
supplementary material) and found that the similarity values for movement
coordination between iYSN and mesendoderm were reduced in morphant embryos
compared with wild-type embryos. In the morphants, only 35% of the values were
higher than 0.5, in contrast to the 50% observed in the wild type (compare
Fig. 5E with
Fig. 1J). In addition, we
observed that mesendoderm progenitors moved slightly slower in
e-cadherin morphant embryos than in wild-type embryos (see Table S2
in the supplementary material). To verify that the effects in the morphant
embryos are specifically due to the loss of E-cadherin function, we also
analyzed previously described e-cadherin loss-of-function mutants
(weg) (Kane et al.,
1996; Kane et al.,
2005) (Fig. 5D; see
Movie 18 in the supplementary material). In weg mutant embryos, the
similarity values for movement coordination between iYSN and mesendoderm were
also diminished (35% of the values higher than 0.5)
(Fig. 5F), congruous with our
observations in e-cadherin morphant embryos. Taken together, these
data suggest that E-cadherin expression is needed to coordinate iYSN with
mesendoderm progenitor cell movements.
|
;
Gumbiner, 2005). This,
together with our previous observation that iYSN move together with the actin
and microtubule cytoskeleton by cortical flow, suggests that E-cadherin
coordinates mesendoderm progenitor and iYSN movements by interacting with the
YSL actin cytoskeleton. To determine whether partial disruption of the actin
cytoskeleton can phenocopy loss of E-cadherin function in this process, we
incubated embryos with the actin depolymerizing drug cytochalasin B during
gastrulation, which has previously been shown to effectively interfere with
the actin cytoskeleton in the zebrafish gastrula
(Zalik et al., 1999) (see
Movies 19 and 4 in the supplementary material). When we analyzed iYSN and
mesendoderm movements in cytochalasin B-treated embryos
(Fig. 6A,C; see Movie 20 in the
supplementary material), we found reduced similarity values for movement
coordination between iYSN and mesendoderm when compared with wild-type embryos
(only 34% of the values were higher than 0.5)
(Fig. 6E), similar to the
situation in e-cadherin mutants and morphants. To test whether
polymerized actin is required locally within the YSL, we injected the
actin-polymerizing drug phalloidin (Cooper,
1987; Wehland et al.,
1977) directly into the YSL. We found that phalloidin effectively
interfered with actin distribution within the YSL (compare
Fig. 6B with
Fig. 2C; see Movies 21 and 4 in
the supplementary material) and that the similarity values for movement
coordination between mesendoderm and iYSN were reduced in phalloidin-injected
embryos compared with wild-type embryos (only 41% of the values were higher
than 0.5) (Fig. 6D,F; see Movie
22 in the supplementary material). This suggests that polymerized actin within
the YSL plays a crucial function in this process. Taken together, these
results suggest that mesendoderm control of iYSN convergence movements
requires E-cadherin expression and that the YSL actin cytoskeleton plays an
important function therein.
Mesendoderm progenitors induce convergent cortical flow within the YSL
The observation that mesendoderm progenitors are required and sufficient to
direct iYSN convergence movements suggests a function of mesendoderm
progenitors in iYSN movements. It has previously been hypothesized that YSN
movements might be driven by chemotactic signals emanated by mesendodermal
cells in the hypoblast (Cooper and Virta,
2007). To test this hypothesis, we quantified the distance
difference between each iYSN migrating ahead of the transplanted cells
(considering the direction of movement of the cells) and its nearest
neighboring transplanted mesendoderm progenitor. If the iYSN have the tendency
to move toward the transplanted cells, as a result of a chemotactic signal,
their distance should decrease over time (for details, see Materials and
methods). We found that the normalized distance difference between consecutive
timepoints was equally distributed around 0 (exact mean
value=-0.003±0.027) (Fig.
7A), indicating that iYSN and transplanted cells keep a constant
distance between each other. These results argue against mesendoderm
progenitors directly attracting iYSN, e.g. through the release of a
chemotactic signal.
|
|
). Next,
we analyzed the propagation of forces exerted by a transplanted patch of cells
on the YSL cortex. Modeling the cortex as a 2D viscous fluid and the patch of
cells as a translating solid material, we found (1) that the forces exerted by
the patch are long range, inducing cortical flow even far away from the
progenitors (see Fig. 7B for
the flow profile), and (2) that the YSN are advected with this induced flow
(for details, see Materials and methods). Indeed, the convergence movement of
the YSN in our transplantation experiments displays this behavior, supporting
the hypothesis that transplanted mesendoderm progenitors induce iYSN
convergence movements by modulating cortical flow within the YSL. |
DISCUSSION |
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|
TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES |
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).
Here, we show that mesendoderm progenitors control iYSN convergence movements
by modulating cortical flow within the YSL responsible for iYSN transport.
Furthermore, we provide evidence that the coordination between mesendoderm and
iYSN convergence movements depends on E-cadherin expression, suggesting that
adhesive contact between mesendoderm and YSL is involved.
Previous studies have demonstrated that YSN undergo both epiboly and
convergence and extension movements
(D'Amico and Cooper, 2001;
Solnica-Krezel and Driever,
1994; Trinkaus,
1951). Furthermore, interfering with the actin and microtubule
cytoskeleton of the YSL has been shown to disrupt epiboly movements of the YSL
(Cheng et al., 2004;
Hsu et al., 2006;
Koppen et al., 2006;
Solnica-Krezel and Driever,
1994; Zalik et al.,
1999), suggesting a crucial function of the cytoskeleton for YSN
epiboly. By contrast, the role of the YSL cytoskeleton for iYSN convergence
and extension movements had not yet been addressed. Our observation that iYSN,
together with the actin and microtubule cytoskeleton converge by cortical flow
within the YSL indicates that iYSN are not actively transported along
microtubules, as has been demonstrated for other types of nuclear migrations
(Morris, 2000;
Morris, 2003;
Reinsch and Gonczy, 1998;
Xiang and Fischer, 2004).
Instead, the actin and microtubule cytoskeleton appear to function simply as a
medium in which the iYSN are entrained and advected. This notion is further
supported by our observation that beads injected into the YSL undergo
convergence movements similar to neighboring iYSN. These findings are
reminiscent of previous experiments in the rainbow trout, demonstrating that
chalk particles implanted into the YSL cytoplasm undergo pronounced
convergence movements (Long,
1980).
Observations in Drosophila suggest that cytoplasmic streaming is
involved in nuclear distribution within the syncytial preblastoderm and in
mixing and dispersal of cytoplasmic components within the oocyte
(Dahlgaard et al., 2007;
Serbus et al., 2005;
Theurkauf, 1994;
von Dassow and Schubiger,
1994). The mechanisms underlying cortical flow within the YSL are
currently unknown. However, it is conceivable that, analogous to the situation
in the Drosophila preblastoderm
(von Dassow and Schubiger,
1994), acto-myosin contraction combined with local changes in
actin polymerization within the YSL controls this streaming. Whether iYSN
converge solely by cortical flow or whether other transport mechanisms also
contribute to iYSN convergence is presently unclear. Our preliminary
observations of iYSN being stretched and pulled apart (not shown) suggest that
there are cytoskeletal forces that pull on the nuclear envelope and possibly
contribute to iYSN movements.
During gastrulation, highly organized cell and tissue movements lead to the
formation of the embryonic body axis
(Solnica-Krezel, 2005;
Stern, 1992). Consistent with
this and previous observations (D'Amico
and Cooper, 2001), we found that in gastrulating zebrafish embryos
cellular and nuclear movements of mesendoderm and YSL are highly coordinated.
Furthermore, our observations suggest that iYSN convergence movements depend
on mesendoderm convergence and that mesendoderm directs iYSN convergence
movements. Interestingly, YSN have previously been shown to undergo epiboly
movements independently of the overlying blastoderm in zebrafish and killifish
(Fundulus) (Kane et al.,
1996; Trinkaus,
1951; Trinkaus,
1984). This apparent difference in blastoderm dependence between
epiboly and convergence movements of YSN is probably due to different
processes controlling these movements. Although YSN epiboly has been proposed
to depend on parallel filaments of microtubules polarized towards the vegetal
pole (Solnica-Krezel and Driever,
1994), we show that iYSN convergence movements are directed by
cortical flow through contact with the overlying mesendoderm. Our observation
that microspheres injected into the YSL strongly converge to the dorsal side,
while showing only very reduced epiboly movements (not shown) points at the
interesting possibility that YSN epiboly involves active nuclear migration,
whereas iYSN convergence does not.
Previous studies have hypothesized that iYSN movements might be driven by
chemotactic signals emanating from mesendodermal cells in the hypoblast
(Cooper and Virta, 2007).
However, we found no evidence for mesendoderm attracting neighboring iYSN,
arguing against the assumption that mesendoderm directs iYSN convergence
through chemotactic signaling. By contrast, we provide evidence that iYSN
converge by cortical flow within the YSL and that mesendoderm progenitors
control this cortical flow through E-cadherin-mediated contact with the YSL.
Mesendoderm progenitors most probably establish a physical link to the YSL
plasma membrane via E-cadherin, which then enables mesendoderm progenitors
undergoing convergence movements to drag the YSL plasma membrane and
associated cortical cytoskeleton in the same direction. In such a scenario,
E-cadherin-mediated adhesion between mesendoderm and YSL would be required to
establish sufficient friction between these layers to allow the effective
pulling of the mesendoderm on the YSL. The plausibility of this notion is
supported by our hydrodynamic analysis showing that the flow of a viscous
fluid induced by a solid patch moving over its surface closely resembles the
cortical flow within the YSL induced by mesendoderm progenitors moving over
it.
Questions remain about the specific role of coordinated convergence
movements of the blastoderm and underlying iYSN during embryogenesis. Clearly,
transcription from iYSN is crucial for a variety of different embryonic
processes, ranging from mesendoderm cell fate induction to heart progenitor
cell migration (Chen and Kimelman,
2000; Mizuno et al.,
1999; Mizuno et al.,
1996; Ober and Schulte-Merker,
1999; Rodaway et al.,
1999; Sakaguchi et al.,
2006). In addition, dorsally restricted expression of the homeobox
transcription factor hex within the iYSL has been associated with
dorsoventral patterning of the gastrula, suggesting that the spatially
regulated transcription of this gene within the YSL is important for its
activity (Ho et al.,
1999).
Future experiments will have to determine whether iYSN convergence movements are indeed required for blastoderm patterning and/or morphogenesis by specifically interfering with iYSN convergence movements within the YSL.
Supplementary material
Supplementary material for this article is available at
http://dev.biologists.org/cgi/content/full/136/8/1305/DC1
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Footnotes |
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REFERENCES |
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TOP
SUMMARY
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
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