1 Department of Organismal Biology and Anatomy, University of Chicago, Chicago,
IL 60637, USA
2 Department of Molecular Genetics and Cell Biology, University of Chicago,
Chicago, IL 60637, USA
3 Howard Hughes Medical Institute, University of Chicago, Chicago, IL 60637,
USA
* Author for correspondence (e-mail: npatel{at}midway.uchicago.edu)
Accepted 11 September 2002
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SUMMARY |
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Key words: Pattern formation, Cell lineage, Fate map, Crustacea, Parhyale
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INTRODUCTION |
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Many crustaceans (and a small number of insects), however, do display total
cleavage during early embryogenesis, but very few studies have been undertaken
to determine the extent to which invariant cell lineages occur in these
animals. In some cases, observations based on tracking cell morphologies in
crustacean embryos have suggested the presence of invariant lineages
(Grobben, 1879;
Bigelow, 1902
;
Fuchs, 1914
; Hertzler et al.,
1992), and a single study making use of the injection of a tracer has
demonstrated the origin of mesendoderm material from a single blastomere at
the four-cell stage in the indirect developing shrimp Sicyonia
(Hertzler et al., 1994
).
In this study, we describe experiments designed to trace the cell lineage
pattern in the amphipod crustacean, Parhyale hawaiensis. Previous
authors have noted the unique arrangement of blastomeres found in this group
of crustaceans, but had not established lineage data
(Langenbeck, 1898;
Weygoldt, 1958
;
Scholtz, 1990
). In amphipods,
the first and second cell divisions are slightly unequal, but the third
division is highly unequal and thus generates a set of four macromeres and
four micromeres. Through the injection of various lineage tracers into the
blastomeres of the eight-cell stage embryo, we demonstrate that the fates of
the macromeres and micromeres in Parhyale are restricted to
individual germ layers. The ectoderm is generated by three macromeres, the
visceral mesoderm by the fourth macromere, the somatic mesoderm is generated
by two micromeres, and the endoderm and the germ cells are generated by the
two other micromeres. With the notable exception of germline formation in some
insects (Kahle, 1908
), this is
the first demonstration of distinct blastomere cell fates in an arthropod.
There is, however, no obvious resemblance between the lineage patterns of
Parhyale and those of the nematodes, spiralians and deuterostomes,
the lineages of which are known. Thus, it would appear that this level of
blastomere fate determination has evolved independently in this group of
crustaceans. Finally, we believe that this crustacean has several properties,
including ease of culturing, ready accessibility to all embryonic stages and
relatively rapid generation time, that make it a useful system for detailed
analyses of many aspects of crustacean development.
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MATERIALS AND METHODS |
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The first three cleavages take place within the first 8 hours of
development and result in an eight-cell embryo composed of four macromeres and
four micromeres (Fig. 1A,B). By
12 hours, there are roughly 100 cells distributed relatively evenly at the
surface of the egg (Fig. 1C)
and all the cells are approaching the same size (as the macromeres have
divided more than the micromeres). At 18 hours many of the cells have
condensed towards specific regions of the egg and the onset of gastrulation
begins shortly after this, as some cells move to more internal position within
the egg. At 3 days (Fig.
1D), a distinct germband with head lobes is visible. At this time,
the ectodermal cells begin to arrange themselves into a precise pattern of
rows and columns, and this organizational process proceeds in an
anterior-to-posterior direction across the germband. The initial rows that are
formed undergo a subsequent precise pattern of divisions to yield individual
parasegments, and again these divisions progress anterior to posterior along
the germband. At 4 days (Fig.
1E), the germband has lengthened considerably and is folded in its
posterior region, and appendages are clearly visible in the anterior regions
of the animal. At 6 days (Fig.
1F), all the appendages are visible and internal organs such as
the gut can be seen forming. At 9 days
(Fig. 1G) organogenesis appears
nearly complete, and the embryo has the same morphology as a hatchling, which
in turn is very similar in morphology to a full grown adult
(Fig. 1H).
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In our study, we injected the blastomeres of two-, four- and eight-cell
stage embryos to track their lineage. TF-4 needles (World Precision
Instruments) are pulled with a horizontal puller P-90 (Sutter) and filled with
the appropriate labeled dextran or mRNA. To keep them in place during the
injection, the embryos are placed on a slide next to small strip of 2% agar in
50% seawater. Once properly oriented, the embryos are injected with an IM 300
Microinjector (Narishige) on a Zeiss Axiovert microscope. The following
tracers were used: rhodamine-conjugated dextran [2.0 µg/µl TRITC dextran
(Mr 500,000), Sigma], fluorescein-conjugated dextran [2.0
µg/µl FITC dextran Mr 150,000) Sigma],
Biotin-conjugated dextran [Biotin dextran, 1 µg/µl (70,000
Mr), Sigma], and capped mRNA (1 µg/µl) encoding
either green fluorescent protein (GFP) or Discosoma Red fluorescent
protein (DsRed. T1) (Bevis and Glick,
2001). For double injections of fluorescent tracers, several
embryos were injected with one tracer, the location of the tracer was
confirmed by fluorescence microscopy, the embryos were placed back on a slide,
oriented appropriately and injected for a second time (in a different cell)
with a different tracer.
Biotin-coupled dextran as single tracer
The embryos injected with Biotin dextran were fixed in one of two ways,
either by formaldehyde fixation or by boiling. Fixation by formaldehyde is
done for 15 minutes in 3.7% formaldehyde in PBS (pH 7.0) at room temperature.
While in the fixation solution, a hole was poked in the egg and the two outer
membranes were removed with tungsten needles. When fixed in this manner (and
stained as described below), it was possible to further dissect the embryos
and flatten them afterwards on a slide; the tissue and the yolk stayed white
and the DAPI staining was bright and clear. However, because a hole must be
made initially, on either the ventral or the dorsal side, the distribution of
clones could be scored accurately only on side or the other side. Fixation by
boiling was achieved by immersing the embryos for 10 seconds in 95°C PBS
(pH 7.0) followed by transfer to ice-cold PBS. This method of fixation makes
dissections easier, the two membranes can be easily removed without damaging
the embryo, and the whole embryo can be scored from all sides. However, the
boiling hardens the embryos so that they cannot be flattened later, it turns
the yolk a yellow color, and the DAPI staining of boiled embryos is weaker and
has more background than that of formaldehyde-fixed embryos. After either
method of fixation, embryos were incubated with HRP-conjugated streptavidin
(Molecular Probes) at a dilution of 1:1000 in PT (PBS + 0.01% Triton), washed
in PBS, developed with 1 mg/ml DAB + 0.6 mg/ml NiCl + 0.01%
H2O2 for 10 minutes, washed in PBS, stained with DAPI at
1 µg/µl in PBS, and cleared in 50% and 70% glycerol in 1xPBS.
Pictures were taken with a Zeiss Axiophot using a Kontron 3012 (Jenoptik)
digital camera. Data were assembled using Adobe Photoshop 6.0.
Fluorochrome-coupled dextrans and mRNAs for DsRed and GFP as single
and double tracers
The embryos injected with fluorochrome-coupled dextrans or mRNA for the
fluorescent proteins EGFP or DsRed.T1 can be scored live over the whole period
of embryogenesis. Pictures were taken at approximately 12 hours, 18 hours
(just before gastrulation), 3 days (germband) and 6 days (organogenesis).
Pictures were taken with a Zeiss Axiophot using a Sony digital camera. Data
were assembled using Adobe Photoshop 6.0.
FITC dextran has a higher background problem because of tissue
autofluorescence than does TRITC dextran. mRNAs for the fluorescent proteins
EGFP and DsRed.T1 were made from expression vectors that were made by cloning
the GFP- and DsRed.T1-coding regions from pEGFP-1 (Clontech) and pDsRed.T1
(Bevis and Glick, 2001) into
the expression vector pSP (gift of Angus MacNicol) and capped transcripts
generated using the SP6 Ambion mMessageMachine kit. Expression was detected by
fluorescence 1.5 hours after injection of DsRed.T1 mRNA and 2-3 hours for EGFP
mRNA. The GFP signal was relatively weak in our hands, although the DsRed.T1
signal was as strong as the signal from TRITC labeled dextrans.
Relative merits of histochemistry versus fluorescence
Biotin dextran is a useful tracer because, after fixation and subsequent
enzymatic development, there is a high signal/noise ratio and the preparations
are permanent. The Biotin dextran method also has a spatial resolution at the
single cell level and allows for simultaneous DAPI staining. The main drawback
of the Biotin dextran method is that embryos must be fixed, and thus each
injection yields data for only a single time point. Fluorescent tracers are
useful as they allow for continuous in vivo observation of the clones.
However, this method does not allow us to collect DAPI data simultaneously,
and there is a loss of fluorescent signal upon fixation. TRITC dextran and
FITC dextran labeling provide excellent spatial resolution until gastrulation;
after that, this technique does not produce as good a spatial resolution as
the Biotin dextran method (although this may be resolved by improved optical
techniques). The fluorescent proteins GFP and DsRed.T1 show the same
advantages and disadvantages as the fluorochromes. In some cases, we have used
a 1:1 mix of Biotin dextran plus one of the fluorescent tracers to take
advantage of the strengths of each technique.
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RESULTS |
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In our initial experiments, we injected individual cells of the eight-cell embryo with either Biotin-dextran or DsRed.T1 mRNA as lineage tracers (see Materials and Methods). We then analyzed the distribution of clones in germband stages (3-4 days of development) and during organogenesis (6-7 days of development). Table 1 summarizes the number of clones analyzed at various stages for each injected blastomere. The fate of `El', `Ep', `Er', `ml' and `mr' is easy to recognize at the germband stage, but the fate of `g', `en' and `Mv' only become clear at about 6 days of development when organ formation has begun. Having established the fate of these clones, we then went back to analyze the distribution, proliferation and migration of the clones during earlier stages (between the time of injection and the establishment of the germband at 2-3 days). Below, we begin with a description of the fate of the clones at the germband and organogenesis stages, and then describe the way in which these clones behave and move during earlier stages of development.
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The ectoderm is a composite of the macromere clones `El', `Er' and
`Ep'
The progeny of `El', `Er' and `Ep' are strictly ectodermal; all their
progeny are restricted to the ectoderm (and ectodermal derivatives such as the
nervous system) and the entire ectoderm can be traced back to these three
macromeres.
At 3 days, when the initial germband is well organized, the cells from
`El' and `Er' make up, respectively, the anterior left and anterior right
ectoderm of the germband, and arrange themselves into a very precise pattern
of rows and columns of cells (Fig.
2A,C). The `Ep' clone forms the posterior ectoderm of the germband
(Fig. 2E), the cells of this
clone will also eventually organize into rows and columns, but do so later
than the more anterior ectoderm (Fig.
2F). In addition, the cells of the `Ep' clone also produce the
midline of the ectoderm extending all the way up to the gnathal region of the
embryo, and thus generate the central midline that separates the `El' and `Er'
clones (Fig. 2E,F) along much
of the length of the embryo. Interestingly, distinct behaviors of midline
cells have also been found at later stages of development in the amphipod
Orchestia (Gerberding and
Scholtz, 1999
; Gerberding and
Scholtz, 2001
).
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The `El', `Ep' and `Er' clones intermix but remain a monolayer. As the germband first begins to form, `El' and `Er' form clones with a relatively small cell size and high cell density positioned at the anterior part of the forming germband. By contrast, the `Ep' clone displays a relatively larger cell size and lower cell density and is spread out over a region of the posterior ventral side and most of the posterior dorsal side of the egg; as the germband continues to condense, the `Ep' clone proliferates and compacts to form the most posterior part of the germband (data not shown). Given that the germband is formed by the condensation of cells from the surface of the egg, it is not surprising that some mixing of the cells between the three clones does occur, but the mixing is restricted in a predictable way. In the head (anterior to the future gnathal region), there is no distinct midline and cells from `El' and `Er' mix extensively across the midline. In the region of the gnathal and thoracic segments, however, the `Ep' clone establishes a well-defined midline, and the `El' and `Er' clones maintain a strictly left-right distinction (Fig. 2A-D). The anteroposterior boundary between `El'+`Er' domain versus the `Ep' clone varies from embryo to embryo, but is generally somewhere within the posterior thorax or anterior abdomen, in a few cases the contribution of `Ep' can be surprisingly small (Fig. 2B,D,F). Possibly, this variability in the composition of the germband ectoderm from the three clones results from the variable degree of inequality in the first and second cleavages that determine the relative sizes of the different macromeres. This boundary is usually quite irregular (i.e. not defined by any specific row of cells), and in addition, there can be scattered cells from `El' and `Er' that end up incorporated in a random manner into the developing abdomen. In summary, `El', `Er' and `Ep' clones can be characterized as occupying anterior left, anterior right and posterior ectoderm, respectively, but with some expected patterns of mixing occurring.
The somatic mesoderm is a composite of the micromere clones `ml' and
`mr'
The `ml' and `mr' clones were analyzed during germband formation and
organogenesis and were found to generate the somatic mesoderm and produce no
other cell type than somatic mesoderm.
The germband mesoderm is assembled out of two clones. At the germband stage, the clones originating from `ml' and `mr' are found immediately underneath the ectoderm (Fig. 3A). The cells originating from `ml' and `mr' are found on the left and right sides of the embryo respectively, and we never observed any violation of this left-right allocation. At day 3, each unilateral clone is subdivided into two populations, one consists of a randomly arranged anterior population of cells (non-teloblastic mesoderm) that will form the mesoderm of the head and heart, and the second is a stereotypically arranged set of posterior cells (teloblastic mesoderm) that will go on to form all the rest of the somatic mesoderm (Fig. 3A,B).
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The anterior, non-teloblastic parts of the `ml' and `mr' clones form the mesoderm of head (and its associated appendages) and two distinct circular structures on either side of the head that are not associated with segments or appendages (Fig. 3B). During organogenesis, these circles disperse and the cells migrate dorsally (Fig. 3D), moving jointly with endoderm and visceral mesoderm (see below). Before hatching, labeled cells can also be found mediodorsally in the putative heart rudiment (data not shown).
The posterior region of the mesoderm is the teloblastic mesoderm. On each side of the embryo, four mesodermal stem cells called mesoteloblasts differentiate at the very posterior end of the clones (Fig. 3A). As these stem cell divide, they move posteriorly one segment at a time in the embryo, leaving behind a row of four progeny in each segment as they do so. This establishes a pattern of four mesodermal precursor cells per segment on each side of the embryo (Fig. 3B). By day 4.5, the mesoteloblasts have finished the generation of segmental mesoderm progeny. These segmental mesodermal progeny then begin to divide (Fig. 3C) and eventually form the muscle cells of the appendages and body wall (Fig. 3D,E).
The germ cells originate from micromere `g'
The lineage of `g' is restricted to the germ line and there is no other
source for the germ cells. During development, the `g' clone settles at the
prospective gonads.
During early germband formation at day 3, the cells in the `g' clone lie in single cluster underneath the ectoderm at the level of the future mandibular segment (Fig. 4A). As development proceeds, the clone splits at the midline into two bilaterally symmetric populations of cells (Fig. 4B) and by day 4 reach a position lateral to the germband at the level of the future gnathal segments (Fig. 4C). The two clusters of cells derived from `g' keep migrating until they reach the dorsal median where the heart rudiment forms. By day 9, the cells of the `g' clone are found within the developing gonads in a dorsal position adjacent to the gut at the level of the fourth thoracic segment (Fig. 4D), and it is at this position that the ovaries and testes are centered in adult animals.
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The endoderm originates from micromere `en' and the visceral mesoderm
originates from macromere `Mv'
The fate of `en' and `Mv' clones is most obvious during organogenesis (6-7
days). At this stage, it is clear that `en' and `Mv' generate the gut, i.e.
endoderm and visceral mesoderm. Endoderm and visceral mesoderm are in
immediate proximity and therefore so close together that overlap can not be
exclude in all cases. However, the variety of methods used all provide
evidence that `en' generates the entire endoderm and `Mv' generates the entire
visceral mesoderm, and the lineages are restricted to these tissues
respectively.
During organogenesis, the endoderm and visceral mesoderm clones form the
gut tube around a central yolk. The central yolk is formed early by a
separation of the outer cytoplasm from the inner yolk. The redistribution of
the cytoplasm can easily be visualized as all the three dextran-coupled
tracers as well as the DsRed protein are found preferentially in the
cytoplasm. From the one-cell to the eight-cell stage, cytoplasmic signal is
found throughout the whole injected cell. Starting at the 16-cell stage, the
cytoplasm becomes successively localized at the surface and excluded from the
inner yolk. This observation, however, does not allow us to conclude whether
the inner yolk is cellular or acellular. If the yolk is cellular, the yolk
cells could either originate from cells that stay central from early on or
from cells that settle within the yolk secondarily. Alternatively, an
acellular yolk could be formed by having the cells divide without a
corresponding nuclear division. Indeed, previous observations of dissociated
cells from early embryos of gammarid amphipods suggested that the cytoplasm
and the yolk become separated by tangential divisions of the cells without
divisions of their nuclei, resulting in small outer cells with cytoplasm and
nuclei, and a bigger inner yolk compartment that is anuclear
(Rappaport, 1960).
At germband formation, the `en' clone comprises no more than eight cells (data not shown). The clone is the most dorsal clone and its cells are flat and spread out over the dorsal yolk. At the same time, the `Mv' clone comprises about two dozens of cells and is located between the ectoderm clones of `Er' and `El' and the dorsal `en' clone (data not shown). During formation of the midgut, the `en' clone extends ventrally and posteriorly from its initial dorsal anterior position (Fig. 5A-D), while the `Mv' clone extends dorsally and posteriorly from its initial anterior lateral position (Fig. 5E-G). During this movement, both clones extend together and the leading edge comprises cell of both clones. In double labels with FITC- and TRITC-labeled dextrans, however, it is clear that `en' cells are situated internal to the `Mv' cells (Fig. 5J-L). At the end of this extension process, `en' and `Mv' form the two layered sheath of midgut around a tube-shaped yolk mass (Fig. 5D,H). We also analyzed sections through the midgut of labeled 7-day-old embryos to confirm that the `en'-derived cells were internal to the `Mv'-derived cells (data not shown).
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The clones show distinct proliferation and migration patterns prior
to germband formation
Having analyzed the cell fate of each of the four macromere and four
micromere lineages at germband and later stages, we decided to investigate
some earlier stages in order to understand more about the behavior of these
lineages in the steps leading up to the formation of the initial germband. We
focused our analyses on two stages. First, at 12 hours of development, at
which time the cells are more or less uniformly distributed around the surface
of the egg (Fig. 1C,
Fig. 6B). Owing to the
appearance of the embryo at this time, we have nicknamed this the `soccerball'
stage. Second, at 18 hours of development, when gastrulation is just about to
begin. At this stage, there is a rosette shaped cluster of cells that is
easily visible in living embryos (Fig.
6C), and we have nicknamed this the `rosette' stage.
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The events prior to germband formation are addressed by double fluorescent labels. Because there are no clear morphological landmarks for orienting the embryo at the soccerball stage, it is difficult to compare the position of individually labeled clones. In addition, the overall shape of the egg at this stage does not provide a reproducible way of orienting the embryo forming within. However, by injecting pairs of blastomeres with different tracers (one with FITC dextran and another with TRITC dextran, see also row four of Table 1) we found that we could understand the relative orientation of all the clones. The `Mv'+`Ep' and `g'+`en' pairs are situated along the AP axis, and the AP axis is the line connecting them. The `El'+`Er' and `ml'+`mr' pairs are situated to the right and the left side of the AP axis, therefore the line connecting them is perpendicular to the AP axis.
Proliferation rates and relative area are different between clones and change over time within clones. During the 4 hours between the eight-cell stage and the 100-cell soccerball stage, the numbers of cells, in each clone, the area on the egg surface that they cover, and their relative locations are all changing simultaneously. At the soccerball stage, `El', `Ep' and `Er' comprise about 12 to 15 cells (Fig. 6E), `Mv' comprises about eight cells (Fig. 6B). The micromeres have undergone two to three divisions, and thus there are four to eight progeny of each micromere at the soccerball stage (Fig. 6H,L). At the eight-cell stage, the macromeres of course cover a greater area than the micromeres. By the soccerball stage, progeny of the three macromeres `El', `Ep' and `Er' have increased their area relative to the progeny of macromere `Mv'. The progeny of micromere `en' increase their area of coverage relative to the other micromere lineages (Fig. 6H).
Migration patterns are different between clones. Relative to each other, the clones move extensively up to the formation of the germband and beyond. The clone that is proliferating and moving the least seems to be the `en' clone; thus, we define the position of the `en' clone to be fixed, and describe the movements of the other cells relative to the `en' clone. After the eight-cell stage, the progeny of the three other micromeres, `mr', `g' and `ml', leave their dorsal and superficial positions next to `en' and migrate to ventral and internal positions. During the movements, they pass through the anterior tip of the egg. The `g' clone takes a medial path and crosses over the `Mv' clone (Fig. 6H,J). The `mr' and `ml' cells take lateral paths (right and left) and do not cross the progeny of `Mv' (Fig. 6L,M). The two macromeres `Er' and `El' expand from their ventromedial position towards the anterior tip of the egg (Fig. 6E,F). The `Ep' cells follow `Er' and `El' anteriorly, but remain posterior to them at all times (Fig. 6B,C). In terms of movements, the behavior of the `Mv' clone is simialr to the `en' clone in that its cells move very little and remain superficial until germband formation (Fig. 6B,C).
During the rosette stage, the somatic mesoderm ingresses laterally, the endoderm remains superficial. At the rosette stage, the cleavage mode changes to superficial cleavage and cells start to condense at two locations. One of these condensations has the shape of a rosette that consists of about 15 cells and is situated at the future dorsal side. The rosette can be further subdivided into a central, deeper ring of cells and an outer, more superficial ring of cells. The other condensation appears a bit later than the rosette and is located at the future ventral side of the embryo. It comprises more cells than does the rosette, but all these cells remain superficial. Double labels elucidate the differential contributions of the clones to these two condensations. The deeper cells of the rosette are the `Mv' progeny (Fig. 6C,J). Double labels of `El'+`Er' show that the ventral condensation initially comprises cells derived from `El' and `Er'. The cells joining it later and more posterior originate from `Ep' (data not shown). Double labels of `ml' and `mr' show that the progenitors for the somatic mesoderm ingress separately at the anterior-right and anterior-left edge of the ventral condensation a few hours after the rosette stage (data not shown). The `en' clone remains superficial throughout the soccerball stage and only ingresses during the germband stage.
The first, second and third cleavage set up the macromeres and
micromeres as well as the AP and DV axes
Through the observation of living embryos and the injection of tracers into
two- and four-cell embryos, we were also able to deduce the division pattern
that leads to the eight-cell stage (data not shown). A summary of the results
is shown in Fig. 7.
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There are two arrangements of the blastomeres at the eight-cell stage that
show mirror symmetry (Fig. 7E).
In one case, the sister cells `Mv' and `g' are located to the left of the
prospective AP axis, in the other case, they are located to the right. The two
arrangements also affect the cell pedigree. If `Mv' and `g' are to the left,
they share a common progenitor at the two-cell stage with `Er' and `mr'
(Fig. 7F). If they are located
to the right, they share a progenitor with `El' and `ml'
(Fig. 7G). Note that this does
not affect the architecture of the pedigree, in both cases, each of the two
cells at the two-cell stage generates paired ectoderm and mesoderm progenitors
and non-paired progenitors for either ectoderm and endoderm or mesoderm and
germ cells. A similar mirror symmetry pattern during early cleavage that still
yields identical embryos has also been reported for other crustaceans and for
spiralians (Baldass, 1941;
Luetjens, 1995).
Some progenitors for the germ layers are paired, some unpaired. The four clones derived from macromeres `Mv' and `Ep' and micromeres `g' and `en' demarcate the AP axis of the embryo and start as bilateral cell populations situated on the median axis. Conversely, the four clones derived from macromeres `El' and `Er' and micromeres `ml' and `mr' begin as unilateral cell populations on either the left or the right side of the embryo. The `ml' and `mr' clones maintain their perfect left/right allocation while the `El' and `Er' clones display some left/right mixing (see above).
The blastomeres at the eight-cell stage can be depicted as a fate map that predicts where the daughters of the blastomeres end up at the germband (Fig. 7A-D). At the eight-cell stage, the material for the germ layers is located along the AP axis in the following orientation: `Mv' most anterior; `g'; `Er'/`mr'/`ml'/`El' in the middle; `en'; `Ep' most posterior (Fig. 7A,C). At the germband stage (i.e. after the initial processes of proliferation, migration and mesoderm ingression), the material is reconfigured along the AP axis. The order then is endoderm `en', visceral mesoderm `Mv', anterior ectoderm `El'+`Er', germ cells `g' and somatic mesoderm `ml'+`mr' (underneath `El' + `Er'), and `Ep' derived ectoderm at the posterior (Fig. 7B,D). During organogenesis, there are further rearrangements so that the endoderm `en' and visceral mesoderm `Mv' have formed the midgut, which runs almost the entire length of the embryo.
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DISCUSSION |
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How do the comprehensive data on Parhyale compare to the
partial fate maps and cell lineages of other crustaceans?
Previous studies of crustacean development had established early fate maps
for several species (reviewed by Shiino,
1957; Anderson,
1973
; Weygoldt,
1994
). With one exception, these fate maps are not the result of
labeled lineage analysis, but instead are based on tracing cells of particular
morphology during the first few division of the embryo (and usually in
sectioned material). For example, in several species, germline cell are picked
out because of the unique appearance of the cytoplasm and their relatively
slow proliferation rate and the endoderm is picked out by its very internal
position in the embryo. Furthermore, these lineage analyses do not follow the
fate of the cells up to the time that the final body plan is established.
While these fates maps are incomplete, and need to be tested by the injection
of lineage tracers, they nevertheless help to illustrate the diversity seen in
early crustacean development (Weygoldt,
1979
). The fate map and cell lineage pattern we have established
here for Parhyale bears similarities to that in other crustaceans,
but surprisingly not to those of closely related malacostracan taxa, but
instead to those of more distantly related non-malacostracan taxa.
Crustaceans are generally divided into five major groups of largely
unresolved evolutionary relation, Remipedia, Cephalocarida, Branchiopoda,
Maxillopoda and Malacostraca, and it is the latter to which Parhyale
belongs. Our results allow us to establish a fate map for Parhyale
and compare it with descriptions of other crustacean fate maps at a similar
stage. By definition, the position where the prospective mesoderm is
internalized is the blastopore. In Parhyale the prospective mesoderm
ingresses underneath the ectoderm in an arc and thus the blastopore is more
like a lip (A. Price and N. Patel, unpublished), while in at least a few other
crustaceans a simpler pore-shaped blastopore exists. In Parhyale, the
prospective endoderm is situated anteriorly of the prospective mesoderm
(Fig. 8A). The inner germ
layers move beneath the ectoderm from an anterior blastopore. In the closest
relatives of the amphipods, the peracaridan malacostracans, the blastopore is
located posterior to the ectoderm and the endoderm is located posterior of the
mesoderm (McMurrich, 1895;
Manton, 1928
). In more
distantly related malacostracans that have total cleavage, the endoderm is
located dorsal, not anterior, to the mesoderm
(Fig. 8E)
(Taube, 1909
;
Hertzler, 2002
). However, in
the case of the branchiopods, the situation is similar to Parhyale.
The blastopore is posterior, but the endoderm material is situated in front of
the mesoderm (Grobben, 1879
;
Kühn, 1913
;
Baldass, 1941
;
Weygoldt, 1994
)
(Fig. 8C,I). In the
maxillopodans (cirripeds and copepods), the situation is like that in
Parhyale and the branchiopods
(Fig. 8C,G)
(Bigelow, 1902
;
Fuchs, 1914
;
Delsman, 1917
;
Shiino, 1957
). The lack of
similarity between the fate map of Parhyale and those of more closely
related malacostracan crustaceans and its similarity to those of distantly
related non-malacostracan crustaceans suggests that this may be an example of
convergent evolution.
|
Heterochrony of germ layer determination
Some cell lineage data, again based usually on tracing cells by their
morphology, is also available for several crustacean taxa with total cleavage.
In some of the taxa with total cleavage, cleavage is equal in the sense that
early blastomeres cannot be distinguished
(Müller-Calé,
1913; Benesch,
1969
). In others, cleavage is more or less unequal and early
blastomeres are distinguishable and are described as progenitors for germ
layers. In general, the putative progenitors for mesoderm, endoderm and germ
cells are derived from few cells at an early stage. A comparison between
Parhyale and these other crustaceans reveals that different numbers
of cell cycles can occur before lineages are restricted to specific germ
layers and different numbers of cells are used to generate the germ layers.
For reasons of space, only comparisons for the endoderm and germ line are
discussed here.
In the malacostracan shrimp, Sicyonia, the endoderm originates
jointly with the mesoderm and the germline from one of the four blastomeres
after the third division, as the only previous lineage tracer injection
experiments carried out in crustaceans shows
(Hertzler et al., 1994). The
fifth and sixth division at the 31- and 62-cell stage each generate an
endoderm progenitor (Fig. 8F)
(Hertzler, 2002
). In the
maxillopodan barnacles, the fourth division at the 16-cell stage generates
single progenitors of endoderm and mesoderm, and the endoderm progenitor is
considerably bigger than all other cells
(Fig. 8C)
(Bigelow, 1902
;
Delsman, 1917
;
Shiino, 1957
;
Anderson, 1969
) In the
branchiopodan waterfleas, the fourth division at the 16-cell stage sets up
single endoderm and germline progenitors of average size
(Fig. 8I) (Grobben, 1878;
Kühn, 1913
). In the
maxillopodan copepod Cyclops, the same process happens at the fifth
division at the 32-cell stage (Fig.
8G) (Fuchs, 1914
).
This five taxa comparison shows that the endoderm progenitor `en' of
Parhyale is generated earlier than in other crustaceans. In addition,
it is a micromere that is the sister of the ectoderm progenitor `Ep'
(Fig. 8B); in other
crustaceans, however, the endoderm progenitors are either average sized or
macromeres and, in lineage terms, are most related to the progenitors of
either the mesoderm or the germ line (Fig.
8D,F,H,J).
Similarly, a comparison between our results for Parhyale and those
for other crustaceans shows that the germ line progenitor `g' in
Parhyale is generated earlier than in other crustaceans. In
Sicyonia, the germ cell lineage separates from the mesoderm at the
seventh division at the 122-cell stage
(Fig. 8F)
(Hertzler, 2002). In the
barnacles, no germline is detected at the 64-cell stage
(Bigelow, 1902
;
Delsman, 1917
;
Shiino, 1957
;
Anderson, 1969
). In the
waterfleas and copepods, the germline is set up as the sister cell of the
endoderm progenitor at the 16- and 32-cell stages, respectively
(Fig. 8H,J) (Grobben, 1878;
Kühn, 1913
;
Fuchs, 1914
).
These comparisons of the relative timing of cell lineage restrictions can
be extended outside the crustaceans as well because the determination of germ
layers is an ancient process that dates back to the common ancestor of
protostomes and deuterostomes. The insect Miastor, a midge, offers an
example where a germline progenitor is separated from the rest of the egg at
the syncytial eight-cell stage by the deployment of a membrane surrounding
only the germline progenitor (Kahle,
1908). Outside of the arthropods, early embryonic patterns of
invariant cell lineage are found in species off nematodes, annelids and
ascidians. C. elegans generates a gut progenitor at the third
division and a germline progenitor at the fourth division
(Fig. 8K)
(Sulston et al., 1981
). In
basal spiralians, the progenitors for the endoderm and mesoderm are generated
at the sixth cell division, in leeches and other clitellates they are
generated much later (Fig. 8L);
the germline in clitellates is found to descend jointly with the muscle
mesoderm from mesoderm stem cells (Goto et
al., 1999
; Kang et al.,
2002
). In ascidians, the various germ layers are composed from the
combination of many cell lineages of the 64-cell stage; a germline has not
been detected by this stage (Nishida,
1987
). In conclusion, germ layer determination usually takes place
between the third and sixth division and the generation of germ cells can
occur much later than this. It is debatable whether there are features of germ
layer determination that are homologous between arthropods, nematodes,
spiralians and ascidians, but it is clear that the lineage patterns found in
Parhyale are particularly noteworthy because they occur much earlier
than in other animals.
Is the link between the cell lineage and the germ layers in
Parhyale incidental or functional?
Cell lineage and cell fate are linked to various degrees in different
developmental systems (Goldstein and
Freeman, 1996; Moody,
1999
). The nematode C. elegans has an invariant cell
lineage, and some aspects of cell fate are linked to cellular asymmetries set
up during the pattern of cell division, but other experiments show that
several cell fate decisions can be uncoupled from the cell division pattern
(Schnabel, 1997
). Within
annelids, comparisons of cell division patterns that at first appear rather
different do reveal the conservation of certain patterns, hinting that a
certain series of divisions may be necessary to determine the different cell
fates and are therefore conserved during evolution
(Schneider et al., 1991
;
Dohle, 1999
). In the frog
Xenopus, the arrangement of blastomeres varies, but if embryos are
selected for a stereotypic arrangement of blastomeres, the resulting lineages
are invariant in the sense that blastomere fate is predictable and restricted
(Moody, 1987a
;
Moody, 1987b
). However, the
fact that there are also non-stereotypical arrangements that give rise to
identical animals demonstrates a primacy of regional determinants over cell
lineage (Moody, 1990
).
Although we find a stereotyped arrangement of micromeres and macromeres in
Parhyale, and an invariant lineage pattern with regards to the
formation of different germ layers, we do not know how yet how tightly cell
fate is tied to cell lineage in Parhyale. The isolation of
blastomeres as done in shrimp (Kajishima, 1951;
Hertzler et al., 1994
;
Wang and Clark, 1996
), and
cell ablation experiments will allow us to investigate this issue and assess
the contributions of cell lineage and positional information during
Parhyale development. In addition to these questions of cell fate
determination during early embryogenesis, we believe that Parhyale
holds promise as a useful crustacean for the investigation of many
developmental problems, particularly comparative questions aimed at
understanding the evolution of pattern formation within the arthropods.
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ACKNOWLEDGMENTS |
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