1 Kondoh Differentiation Signaling Project (ERATO/SORST), Japan Science and
Technology Corporation, 14 Yoshida-Kawaracho, Sakyouku, Kyoto 606-8305,
Japan
2 Graudate School of Sciences, Kyoto University, Sakyo-ku, Kyoto, 606-8502,
Japan
3 University Freiburg, Institute Biology 1 (Zoology), Department of
Developmental Biology, Hauptstrasse 1, D-79104 Freiburg, Germany
4 Graduate School of Frontier Biosciences, Osaka University, 1-3 Yamadaoka,
Suita, Osaka 565-0871, Japan
* Authors for correspondence (e-mail: hirose{at}dsp.jst.go.jp and kondohh{at}fbs.osaka-u.ac.jp).
Accepted 23 February 2004
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SUMMARY |
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Key words: Single cell labeling, Live recording, Computer graphics, Neural subdivisions, Compartments, Retina, Pax6
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Introduction |
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Precursor cells of the nervous system do not stay in fixed areas in the
early embryo but undergo extensive intermingling, a process that begins even
before the onset of gastrulation (Hatada
and Stern, 1994; Kimmel et
al., 1990
). However, after boundaries of neural subdivisions
become morphologically discernible, cells appear to be confined to the area of
individual neural subdivisions (Fraser et
al., 1990
; Inoue et al.,
2000
). Evidence also indicates that regulation of cell movement
(Myers et al., 2002
) and
interactions between groups of cells that form distinct neural subdivisions
(Varga et al., 1999
) is
required for the establishment of functional central nervous system (CNS)
subdivisions. Thus, the formation of cell groups that contribute to the same
neural subdivision is an essential step in development of the organized
CNS.
Behavior of cells in neurogenesis has been studied employing cell labeling
or cell transplantation techniques in zebrafish and chicken embryos
(Couly and Le Douarin, 1988;
Fernandez-Garre et al., 2002
;
Hatada and Stern, 1994
;
Woo and Fraser, 1995
;
Woo and Fraser, 1998
). These
studies indicate progressive separation of precursors of neural subdivisions.
However, in these early studies only the initial and terminal locations of
cell populations were compared, and data on cell movement between those two
time points was missing. Therefore, the information has stopped short of
understanding how cell groups for individual CNS subdivisions are organized
from individual cells.
We report analysis of movement of a number of randomly chosen blastoderm
cells by fluorescent labeling and live recording of single cells. We used
Medaka fish (Oryzias latipes) embryos for analysis, taking advantage
of embryo transparency, the wealth of knowledge on their development, and, in
particular, the recent massive screening of gene mutations that affect nervous
system development (Wittbrodt et al.,
2002). From a practical view point, the slower development of
Medaka embryo compared with zebrafish facilitates the characterization of the
sequence of events with high temporal accuracy. Time-lapse and long time span
recording of a number of the same individual labeled cells in developing
embryos allows understanding of how cell groups are organized and segregated,
and how they undergo morphogenesis in the nervous system.
The analysis indicates that the cells that contribute later to distinct
neural subdivisions start as overlapping populations in early blastoderm;
however, at stage 16+, they almost synchronously segregate into
non-overlapping cell populations that have the characteristics of
`compartments', with cell migration across compartments being inhibited and
cell divisions not yielding daughter cells fated to distinct compartments.
This synchrony of compartmentalization of neural subdivisions is probably
different from that in zebrafish, where neural subdivisions are established in
sequence from anterior to posterior (Woo
and Fraser, 1995). This analysis also indicates how `convergence
and extension' (Myers et al.,
2002
) of neural cells takes place in Medaka, with `extension'
occurring only in the hindbrain and spinal cord.
Precursors for future retina form a single neural compartment at stage 16+
that overlays telencephalon and diencephalon. This retinal compartment was
bisected into bilateral retina precursors in parallel with anterodorsal
movement of the diencephalic precursor compartment, a movement that was
previously indicated by single cell analysis in zebrafish embryos
(Varga et al., 1999). As an
extension of the analysis, we compared establishment of the retinal precursor
compartment and expression of Pax6, observing a temporal lag between
these events, and found that Pax6 expression in the early phase was
confined to laterally located cells in the retina-specified cell
population.
Thus, dynamic, computer graphics-aided analysis of cell movement composed of continuous live recording of single labeled cells provides a powerful new tool for understanding cellular events in the embryo. The same technique should prove useful in non-neural tissues and in other animal species, but transparent fish embryos are especially advantageous, and Medaka allows high temporal resolution of events during gastrulation to be achieved.
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Materials and methods |
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Fluorescent single cell labeling
At stage 13 when embryos were spherical, embryos of 1000 µm ±10
µm diameter were selected for best fit to the computer graphics embryo
model. Single cells were injected at this stage with 3% rhodamine dextran dye
(Molecular Probes D-3308) using the technique described by Varga et al.
(Varga et al., 1999). The
thickened cell layer forming the embryonic shield at the dorsal margin was
used as a landmark to orient the blastoderm. Dechorionated embryos were placed
on a graded silicone mount and the animal pole was oriented upwards. One to
three cells some distance apart in an embryo were then injected with
fluorescent dye.
Time-lapse recording of the labeled cells and data analysis using computer graphics model of the embryo
At intervals of 1-2 hours, fluorescent images of the embryos were
photo-recorded from multiple angles. Cells labeled with fluorescent dye were
observed by mounting an embryo on a 1x1 mm grid reticules in a silicon
mount and photo-recording from two angles (from stage 13 to stage 16) or three
angles (dorsal, lateral and frontal) (stage 16 and later) using a Zeiss Axio
Skop2 FS microscope equipped with a Pixera Penguin 150CL camera. Cell
positions were transferred to the computer graphics model embryo in the
following way. The stage-matched model embryo was oriented at the same angle
as the photo-image of the live embryo using morphological landmarks, e.g.
blastoderm edge and cell thickening along the dorsal midline. Images of real
and model embryos were superimposed, and the position of a fluorescent cell
observed from an angle was plotted onto the model. The plot was also made from
the second angle, and the gravity center of the plots was taken as the cell
position on the computer graphics model embryo. A cell is presented by a dot
with a diameter of 20 µm that approximates cell diameter in real space
(Fig. 1). Trajectories of
labeled cells were generated using recorded positions and the Bezier spline
curve approximation method in CINEMA 4D software (see Movie at
http://dev.biologists.org/supplemental/).
|
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Results |
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The images of a real embryo and stage-matched computer graphics model were superimposed and the positions of fluorescence-labeled cells in the embryo were plotted on the model (Fig. 1A,B). We plotted cells by using images at different angles of the same embryo, and the middle points of cell plots were taken for cell positioning. In practice, cell positions in the computer graphics embryo model were very accurate, and following plots could be merged with previous ones without exception (Fig. 1). After stage 16, cell positions in an embryo were measured from three directions (dorsal, lateral and frontal), as the embryos gained thickness along the midline. The standard deviation of cell position measurements at stage 15 was 7.0 µm (n=50), and that at stage 17 was 3.0 µm (n=50), while the average cell diameter was 20 µm.
Neural progenitors of the blastoderm
At stage 13 (13 hpf), early in gastrulation, the animal quarter of the yolk
surface is covered by two-layered blastoderm. The outer layer consists of flat
cells, while the inner layer is composed of round cells and has variable
thickness from one to two cell diameters (ventral margin) to four to five cell
diameters (dorsal margin). We determined the cell population that gave rise to
neural tissue in later development by single cell labeling. Of 120 randomly
labeled single cells at stage 13, 13 were in the outer layer, and descendants
of all these cells were found in the enveloping layer at stage 24 (44 hpf, 16
somites). Early separation of blastoderm cells into these germ layers is
common to fish species (Ballard,
1973; Kimmel et al.,
1990
; Oppenheimer,
1936
; Warga and
Nusslein-Volhard, 1999
).
Among the remaining 107 cells in the inner layer that developed into embryonic tissues at stage 24, 75 initially located in the dorsal 140° sector (±70° latitude), referred to as the `neurogenic sector' (Fig. 2C), contributed mainly to the neural and placodal tissues. Six cells of the same sector that were close to the blastoderm margin contributed to somitic mesoderm, and 26 cells outside of the dorsal sector contributed primarily to epidermis (Fig. 2C).
|
Initial cell populations that contribute later to the neural subdivisions
The nervous system of stage 24 embryo is subdivided common to vertebrates:
telencephalon, retina, diencephalon, mesencephalon, rhombencephalon, spinal
cord and peripheral nervous system of placodal or neural crest origin
(Fig. 3C). The
fluorescent-labeled cells at stage 13 were color-coded in the
computer-graphics model according to their contribution to neural subdivisions
at stage 24 (Fig. 3A,C-E)
|
Separation of epidermal and neural precursors (stage 13 to stage 16)
Along with progress of epiboly, initial movement of cells was largely
vertical, as indicated by their trajectories at the beginning of gastrulation
(Fig. 4). However, from stage
15 to stage 16, in contrast to epidermal cells that continued vertical
movement toward the vegetal pole, those contributing to neural subdivisions
changed directions and converged near the midline
(Fig. 4A). This difference of
cell movements distinguishes and promotes separation of cell populations that
later contribute to epidermis and neural tissues
(Fig. 4A). Non-overlapping cell
groups of epidermal and neural populations were established by stage 16,
bordered by the cell population of future neural crest/placode (yellow)
(Fig. 3H).
|
Immediately after stage 16+ (22 hpf), vertical movement of future neural cells became largely arrested, then the cells for telencephalon, retina, diencephalon and mesencephalon turned their directions towards the animal pole (Fig. 4B-E), while the cells for future rhombencephalon and spinal cord showed horizontal drift toward the midline (Fig. 4F,G).
As indicated by the rectangles in Fig. 3G-K and more explicitly by the illustrations of cell group positions in Fig. 5A, after stage 16+ cell groups for future tel-, di- and mesencephalon did not change their AP width significantly (telencephalon actually becomes shorter in the AP dimension), and were displaced anteriorly on the embryo coordinate as coherent cell masses. By contrast, the cell group for rhombencephalon became twice as long along the AP axis during the period from stage 16+ to stage 19. The area occupied by the prospective spinal cord cells was more posteriorly shifted and significantly elongated (Fig. 5A).
|
Establishment of compartments for future neural subdivisions (stage 16+)
Spatial coherency of cells belonging to a future subdivision of the CNS
after stage 16+ suggests that neural subdivisions are already established by
this stage, much earlier than neural subdivisions became morphologically
discernible (stage 24, 44 hpf). However, trajectories of individual cells
(Fig. 4) indicate that cells
change their relative positions after stage 16+. These observations suggest
that cells do not cross the boundaries of neural subdivision once established
at stage 16+, while cells change their relative positions within neural
subdivisions, i.e. between the boundaries. To confirm this, we assessed
relative vertical migration of the cells at various AP levels from
diencephalon to rhombencephalon during development from stage 15+ to 24, by
counting the number of cells crossing horizontal lines transverse to the cell
group (Fig. 5B, part a). During
this period, all neural precursors of the corresponding subdivisions were
displaced as a mass along the AP axis, without significant alteration of the
AP length of individual territories. Lines 3 and 8 were placed on boundaries
of the prospective di- and mesencephalon, and that of mes- and
rhombencephalon, respectively (Fig.
5A). As shown in Fig.
5B (part b), up to stage 16, vertical movement in the cell
population was almost uniform along the various AP levels, but at stage 16+,
the movement across lines 3 and 8 was suddenly arrested, while at other AP
levels vertical cell movement continued. This indicates that the cell groups
that later form di, mes- and rhombencephalon organized separate `compartments'
where cells may move around in a compartment but do not cross boundaries with
other compartments.
Vertical cell movement within the compartment remained extensive up to stage 17 (3 hours later), then after stage 19 the vertical cell movement across the lines were gradually attenuated. This temporal change in cell movement activity was also observed along the horizontal axis (Fig. 5B, part c). Measurement of lateral movement by counting cells crossing the midline (Fig. 5B, part c) indicated that this movement is maximum at stage 17, but is attenuated at stage 19.
Establishment of compartments at stage 16+ was supported by the observation on the fate of mitotic daughter cells (Fig. 5C). Some mitoses occurring in the region of cell group border gave rise to daughter cells with split fates up to stage 16, but after stage 16+ even those mitoses that took place very close to the border with another cell group never produced split fate daughters (Fig. 5C), although cell growth was more pronounced after this stage (Fig. 5D). Thus, establishment of a compartment results in clonal separation by the boundary.
Development of individual compartments for neural subdivisions
After cell groups are organized into compartments, there is a period of
extensive cell proliferation around stage 17
(Fig. 5D), followed by a steady
increase of cell number in all compartments. This cell proliferation was not
accompanied by an increase in AP length of the anterior neural compartments
(Fig. 5A), and resulted in a
gain in their width and height (Fig.
3E,K, Fig. 5A). Thus, in Medaka embryos, overall `convergence' of neural precursors towards
the midline clearly occurs as illustrated by cell trajectories
(Fig. 4), but `extension' of
the central nervous system is ascribed only to moderate AP elongation of the
rhombencephalon and spinal cord.
Development and lateral separation of the single retinal population
We analyzed how two retinas are generated and separated into two eyes from
a single, coherent cell group. Retina-forming cells were in a single
compartment at stage 16+ (22 hpf) superficial to telencephalic and
diencephalic compartments, and covering their posterior and anterior halves
(Fig. 6A-C). This single
retinal compartment was split bilaterally from posterior to anterior,
paralleled with dorsal movement of diencephalic precursors
(Fig. 6D-F). At the same time,
the ventral region of the diencephalic cell group elongated anteriorly and
displaced future telencephalic cells in the anterior and dorsal direction
(Fig. 6C,F,I). By stage 19, the
retinal cell group had been completely separated into two lateral groups
(Fig. 6G-I). Thus, the
bilateral retinas were established from an isolated single compartment by
morphogenetic cell movement.
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Discussion |
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Comparison with earlier analysis in particular on zebrafish
Pioneering studies traced cell fates in a large number of blastodermal
cells using single cell labeling or analogous techniques in embryonic chicken
and zebrafish (Fernandez-Garre et al.,
2002; Hatada and Stern,
1994
; Woo and Fraser,
1995
; Woo and Fraser,
1998
). Specific attention was paid to genesis of the CNS, which
occurs the earliest and occupies the largest cell population in the early
embryos. These earlier works recorded positions of labeled cells at limited
time points without following the behavior of cells in between. In this study,
we performed time-lapse recording of single labeled cells over a relatively
long time span (nearly 30 hours), enabling characterization of the sequence of
events in great detail.
Work carried out by Woo and Fraser (Woo
and Fraser, 1995) on zebrafish was particularly informative, and
comparison of data on Medaka and zebrafish reveals common principal processes
in early development in these fish species, but also highlights interesting
differences. Thus, although neural subdivisions in zebrafish appear to be
progressively formed anterior to posterior, in Medaka all neural subdivisions
are formed and compartmentalized more or less synchronously. In Medaka there
are three stages in the process.
This synchrony of events among cell groups for future neural subdivisions
is in a sharp contrast to zebrafish embryos
(Woo and Fraser, 1995), where
at 10 hpf separation of the cell groups for tel-, di- and mesencephalon is
almost complete, but those of mes- and rhombencephalon still share a region of
overlap, and rhombencephalic precursors have not converged to the midline.
Early characteristics of neural progenitor cells
In stage 13 embryos, all neural progenitor cells were located on the dorsal
140° sector of the deep cell layer
(Fig. 2). Territories occupied
by the progenitors of distinct neural subdivisions overlapped extensively
(Fig. 3B). This observation
argues that the later fate of precursor cells in stage 13 embryos is not
strictly specified but depends on conditions such as cell position at a later
developmental stage. Regional overlap of different cell fates in early
blastoderm was also observed by labeling of cells in early zebrafish and
chicken embryos (Hatada and Stern,
1994; Kimmel et al.,
1990
; Woo and Fraser,
1995
).
Organization of the cell groups for future neural subdivisions into `compartments'
It is remarkable that after stage 16+, cells that later contribute to
different neural subdivisions are organized into spatially non-overlapping
cell groups, and cells stop crossing the boundary, although cell movement
within groups reaches a maximum at stage 17
(Fig. 5B, parts b and c).
Analysis of cell movement in the AP direction
(Fig. 5B, part b) did not
detect other boundaries restricting cell movement. Thus, after stage 16+, cell
groups to form the future neural subdivisions acquire the characteristics of
`compartments' first defined in Drosophila embryos
(Garcia-Bellido et al., 1973).
These compartments then develop into individual neural subdivisions.
After the compartment boundaries are formed, mitoses occurring adjacent to
the boundary with the neighboring compartment never give rise to daughter
cells that contribute to another compartment, although mitoses in earlier
stages of development often resulted in split fate for daughter cells
(Fig. 5C). This clonal
separation is another feature of compartments. Clonal separation of cells
after establishment of the compartment boundary is also demonstrated for later
established subdivisions, e.g. rhombomere boundaries of the chicken
(Fraser et al., 1990).
Convergence and extension of the neural primordium in Medaka embryo
In an overall view of the genesis of the CNS primordium, precursors for the
future CNS first converge towards the midline, and then the entire CNS
undergoes AP elongation (`convergence and extension')
(Myers et. al., 2002).
As discussed earlier, convergence of neural precursor cells towards the midline occurs almost synchronously in Medaka embryos, starting at stage 15 and ending at nearly stage 16+, and this movement largely separates cells to be fated to the neural and epidermal lineages (Figs 3, 4). This convergence is not accompanied by a significant AP elongation of the territory occupied by the precursor cells for tel-, di-. mes- and rhombencephalon, and retina.
After compartments for neural subdivisions were established at stage 16+
(22 hpf), those for tel-, di- and mesencephalon do not significantly alter
their AP length (Fig. 3,
Fig. 5A), although the cells
increase in individual compartments (Fig.
5A,D). The rhombencephalic subdivision elongates in the AP
dimension two folds by stage 24 (22 hours later), but the gain in tissue
thickness was more significant (Fig.
5A). The major part of extension of the CNS primordium is
accounted for by elongation of the spinal cord region, which appears to be
caused at least in part by cell multiplication
(Fig. 5A), and also probably by
tissue addition under influence of the `tail organizer'
(Agathon et al., 2003). In the
report by Kimmel et al. (Kimmel et al.,
1994
) on zebrafish embryos where cell clones contributing to the
rhombencephalic region were analyzed, extension of the neural tube by an
intercalation mechanism was suggested. Analogous mechanisms may also be
involved in Medaka development, but this must be only in rhombencephalon and
spinal cord regions.
Bifurcation of retinal compartment
An important observation in this study was that retinal precursor cells at
first form a single compartment at stage 16+, being separated from the
compartments for future telencephalon and diencephalon, and located dorsal to
tel- and diencephalic compartments (Figs
3,
6). Then the retinal precursors
are split into bilateral retinas, which is associated with dorsal rise and
anterior shift of the diencephalic precursors
(Fig. 6). This two-step
mechanism to produce bilateral retinas was first proposed by cell fate tracing
experiments in zebrafish embryos (Varga et
al., 1999). When prospective retina and diencephalic cells in the
superficial region of zebrafish embryos were labeled at 80% epiboly stage, and
their tissue contribution was analyzed at a later stage (24 hpf), retinal
precursor cells clearly formed a single planar cell group distinct from
diencephalic precursors. Diencephalic precursors, initially located posterior
to the retinal precursors, were later located medial and anterior to the
bilateral retinas. In this study using Medaka embryos, we followed the
position of individual labeled cells at close intervals, and successfully
monitored the cells even those located deep in the embryonic tissue. Thus,
spatial reorganization of the compartmentalized cell groups was clearly
displayed in the three dimensional model
(Fig. 6). This not only
confirms the previous proposal (Varga et
al., 1999
), but also establishes the two-step retinal splitting
mechanism associated with anterior movement of the diencephalic precursors.
This mechanism must be common to teleosts, and may presumably be conserved in
other vertebrate species.
However, it has been a generally accepted view that prechordal plate
mesoderm ventral to the prosencephalons produces signals along the midline
that transform retinal precursors into a non-retinal fate, thus yielding two
bilateral retinal cell populations (e.g.
Li et al., 1997). This view is
based on investigations of early retina development in non-fish vertebrates
involving grafting or ablating a cell sheet. In these experiments, cell
populations located in the medial part of the retina field appeared
bipotential for development into retina and diencephalon (e.g.
Couly and Le Douarin, 1988
;
Fernandez-Garre et al., 2002
;
Li et al., 1997
). Considering
the situation in Medaka, the possibility exists that in avian and amphibian
species, retinal and diencephalic divisions are also separated earlier than
the current model and extensively overlap vertically. This vertical overlap is
demarcated in zebrafish by foxb1.2 (mar) gene expression in
prospective median hypothalamic precursors
(Varga et al., 1999
). Thus,
the bipotential nature of cell populations observed in grafting experiments
could be a consequence of a mixed population of specified retinal and
diencephalic cells within grafted pieces of tissues. It is important to
re-examine the topology of the primordia of neural divisions in these species,
although possible occurrence of species-dependent variation of the mechanism
must also be considered.
Another intriguing observation was on the timing of establishment of the
retinal compartment and expression of Pax6 in retinal precursor
cells. Pax6 is known to be involved in establishing characteristics
of retina (Grindley et al.,
1995) and prosencephalon
(Mastick et al., 1997
).
Shortly after the retinal precursor compartment was established (stage 16+, 22
hpf), expression of Pax6 was initiated in the lateral retina
precursor cells but not in the median retinal precursors
(Fig. 7A,B). At stage 16++ (23
hpf, 95% epiboly), Pax6 expression covered all cells that gave rise
to retina, diencephalon and rhombencephalon
(Fig. 7C,D). Thus,
Pax6 expression and establishment of the retinal precursor
compartment was not synchronous as previously supposed, but initiation of
Pax6 expression in the anterior compartments may depend on cues that
were available only to laterally located cells.
It is also interesting to note that in Medaka eyeless mutant
embryos with low temperature-sensitive Rx3 expression
(Loosli et al., 2001), lateral
retinal cells are not formed. However, cells that would normally have the
potency to form retina express Pax6 and have lens-inducing potential.
These cells are embedded as a cell cluster in the prospective diencephalic
region of the neural plate (Winkler et
al., 2000
). It is tempting to speculate that the eyeless
defect lies in the process of bifurcating the retinal cell group at stage 17.
Results from a temperature shift experiment
(Winkler et al., 2000
), where
the formation of bilateral retinas is blocked by low temperatures at stage 17,
would be consistent with this model.
Thus, analysis of specific gene expression and developmental defects of mutant brains based on the cell tracing technique described here is promising for shedding a new light on principles of early neural development.
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ACKNOWLEDGMENTS |
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Footnotes |
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