Graduate Program in Zoology, Section of Molecular Cell and Developmental Biology and Institute of Cellular and Molecular Biology, The University of Texas at Austin, TX 78712, USA
Author for correspondence (e-mail:
dhkuo{at}berkeley.edu)
Accepted 16 September 2004
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SUMMARY |
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Key words: Leech, Helobdella, O/P equivalence group, Cell-cell interaction, Evolution of a developmental mechanism
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Introduction |
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The O/P lineages in the segmental ectoderm of glossiphoniid leeches also
form an equivalence group (Weisblat and
Blair, 1984; Zackson,
1984
; Huang and Weisblat,
1996
; Keleher and Stent,
1990
). The segmented ectoderm and mesoderm of the leech are
derived from five bilateral pairs of stem cells called teloblasts. There are
four pairs of ectodermal teloblasts (N, O/P, O/P and Q) and one pair of
mesodermal teloblasts (M), each of which gives rise to a distinct set of
differentiated descendants. Through repeated asymmetric cell divisions, each
teloblast produces a bandlet, i.e. a string of primary blast cells that are
segmental founder cells for their respective teloblast lineage. Bandlets
arising from the five ipsilateral teloblasts come together in parallel to form
the germinal band, with the four ectodermal bandlets arranged along the
dorsoventral axis such that the n bandlet is the ventralmost and the q bandlet
is the dorsalmost (Fig. 1). The
m (mesodermal) bandlet underlies the four ectodermal bandlets.
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Three different cell interactions have been proposed to govern cell fate
specification of the O/P lineage. In experiments in which the P lineage is
ablated, the presumptive (i.e. positionally defined) O lineage `transfates'
into the P fate. By contrast, the presumptive P lineage does not transfate
following the ablation of the O lineage
(Weisblat and Blair, 1984;
Shankland and Weisblat, 1984
;
Zackson, 1984
). This finding
was initially interpreted as implying that the P lineage suppresses the P fate
and/or induces the O fate in the O lineage. However, later experiments also
revealed an O fate-inducing signal from the micromere-derived provisional
integument that covers the germinal band
(Ho and Weisblat, 1987
), and a
P fate-inducing signal from the Q lineage
(Huang and Weisblat, 1996
). To
accommodate these results, Huang and Weisblat
(Huang and Weisblat, 1996
) have
proposed a model in which the `interaction' between the O/P pair is purely
steric, i.e. the presence of the p bandlet prevents the o bandlet from
physically contacting, and being induced to the P fate by, the q bandlet.
One potential source of confusion is that these data were obtained from
three different Helobdella species: H. triserialis
(Weisblat and Blair, 1984;
Shankland and Weisblat, 1984
;
Zackson, 1984
;
Ho and Weisblat, 1987
), H.
stagnalis (Zackson, 1984
)
and H. robusta (Huang and
Weisblat, 1996
). The authors assumed that the developmental
mechanisms underlying O/P specification should be very similar in these
species given their nearly identical patterns of embryonic cell lineage and
cell fate. However, these morphological traits are also conserved in some more
distantly related clitellate species in which the O and P lineages behave
differently in response to certain experimental paradigms
(Goto et al., 1999
;
Keleher and Stent, 1990
). In
addition, a variation in developmental cell fates between Helobdella
species has been reported for the cell lineage arising from one of the
embryonic micromeres (Huang et al.,
2002
). It is possible that the O/P specification mechanism also
varies between different Helobdella species, and in the present study
we provide evidence that this is the case.
In this study, the cell-cell interactions involved in O/P specification are
experimentally characterized in three laboratory populations belonging to the
leech genus Helobdella. As noted above, it was previously shown that
the Q lineage plays a central role in O/P specification in a population of
H. robusta originating from Sacramento, CA
(Huang and Weisblat, 1996).
Here, we show that there is a significant difference in the response of the
O/P lineages to the ablation of the Q lineages in a second leech population
from Austin, TX [also described as H. robusta (see below)] in which
another signaling pathway functions as a redundant component in the O/P
specification mechanism. In addition, we find no evidence that the
micromere-derived provisional integument influences O/P fate specification in
H. robusta (Austin), in contrast to a previous report from H.
triserialis (Ho and Weisblat,
1987
). These data suggest that the cell fate specification
mechanism of the O/P equivalence group, similar to that of the nematode VPCs,
has undergone divergence in closely related populations, while retaining a
high degree of evolutionary conservation in cell lineage and terminal
morphology.
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Materials and methods |
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A morphologically similar but distinguishable leech population originating
from Galt, CA, has been previously described as Helobdella sp. (Galt)
(Huang et al., 2002). Molecular
data suggest that these leeches form an outgroup to the two H.
robusta populations (A. Bely, personal communication).
Embryo culture and staging are as described by Kuo and Shankland
(Kuo and Shankland, 2004).
Injection of cell lineage tracer and ablation of teloblasts
Cell lineage tracer labeling was carried out by pressure injecting the OP
proteloblast or an O/P teloblast with a 1:1 mixture of 100 mg/ml
tetramethylrhodamine dextran (lysine fixable) or fluorescein dextran (lysine
fixable) (Molecular Probes, Eugene, OR) and 4% Fast Green (Sigma) in 200 mM
KCl. Teloblast ablation was carried out by pressure injection into the target
teloblast of a 1:3:4 mixture of 0.83 mg/ml ricin A chain (Sigma), 100 mg/ml
fluorescein dextran and 4% Fast Green (Sigma) in 200 mM KCl. Operated embryos
were raised separately in 24-well culture plates and were fixed at stage 7/8
or late stage 9 with a 1:1 mixture of 8% formaldehyde (Pella) and
HEPES-buffered saline (50 mM HEPES, 150 mM NaCl, pH 7.4) and counterstained
with 2.5 µg/ml Hoechst 33258 at 4°C overnight. Stage 7/8 embryos were
used for examination of blast cell cleavage patterns. The size and spatial
pattern of the blast cells were determined from their nuclei as revealed by
Hoechst 33258 staining (Zackson,
1984). To examine the differentiated pattern elements that arose
from labeled O/P lineages, stage 9 embryos were dissected and mounted on
slides in buffered glycerol and viewed by fluorescence microscopy.
Ablation of the micromere-derived provisional integument
The micromere-derived provisional integument was ablated by a
lineage-specific photo-oxidation technique
(Shankland, 1984). This
procedure was modified from Ho and Weisblat
(Ho and Weisblat, 1987
) to
accommodate differences in developmental timing of the different leech
species. First, the target micromeres (opq'' and n') were injected
with fluorescein dextran at stage 6a. At late stage 7/early stage 8, an
intense 485 nm light beam was focused on the labeled cells through the 40
x water immersion objective of a compound microscope to selectively kill
the fluorescein-containing descendants of the injected micromeres. In these
same experiments, a 1:1 mixture of 100 mg/ml biotin dextran (lysine fixable)
(Molecular Probes, Eugene, OR) and 4% Fast Green (Sigma) in 200 mM KCl was
pressure injected into the O/P teloblasts as a lineage tracer. Roughly 24-30
hours after the irradiation procedure, the embryos were fixed, counterstained
and washed as described above. To visualize biotin dextran lineage tracer,
fixed embryos were incubated in a saline buffer containing 0.8% Triton X-100
and 5 µg/ml avidin-rhodamine complex (Vector) at room temperature for 2
hours and then washed extensively.
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Results |
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The blast cell clones in a given bandlet exhibit an anteroposterior
gradient of clonal age that reflects their order of birth. The older
(anterior) blast cell clones exhibit cleavage patterns of more advanced
stages, while younger (posterior) blast cell clones exhibit cleavage patterns
at earlier stages. The first divisions of the primary o and p blast cells are
both asymmetric, but the degree of asymmetry is significantly greater in the o
bandlet (Shankland, 1987b;
Shankland, 1987c
)
(Fig. 2A,B). The subsequent
blast cell divisions also exhibit lineage-specific patterns. These cleavage
patterns are indistinguishable in the populations examined here.
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In a unilateral Q teloblast ablation experiment, transient contact with the
q bandlet of the contralateral germinal band is sufficient to specify o/p
blast cells to the P fate in H. robusta (Sacramento)
(Huang and Weisblat, 1996). As
described below, we obtained the same result from unilateral Q teloblast
ablation experiments in H. robusta (Austin). To eliminate completely
the influence of the Q lineage, the two Q teloblasts were ablated bilaterally
in the following experiments unless otherwise specified.
H. robusta (Austin)
After bilateral ablation of the Q teloblasts, the two ipsilateral O/P
teloblasts were injected with fluorescent dextran lineage tracer. In one set
of experiments, the cleavage pattern of blast cell clones in 52 pairs of o/p
bandlets was scored at stage 8 (Fig.
4A,B; Table 1, row
C). In 51 o/p pairs, the blast cell clones in the presumptive o bandlet
consistently displayed an O-type cleavage pattern. In 32 out of these 51
pairs, blast cell clones in the presumptive p bandlet displayed a P-type
cleavage pattern. But in the other 19 pairs, the majority of individual blast
cell clones in the presumptive p bandlet expressed a P-type cleavage pattern,
but some presumptive p blast cell clones expressed an O-type cleavage pattern.
In one atypical embryo, it appeared that a physical inversion of the o/p
bandlets had taken place at the posterior end of the germinal band, and in
this case each bandlet maintained the cleavage pattern characteristic of its
original position (data not shown). These results are not consistent with the
previous observations from H. robusta (Sacramento)
(Huang and Weisblat, 1996).
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H. robusta (Sacramento)
To ensure that the apparent difference in the response of the O/P lineages
to bilateral ablation of the Q lineages is not an artifact produced by some
deviation in the experimental procedure, we likewise performed bilateral
ablation of the Q teloblasts in embryos of H. robusta (Sacramento).
Following bilateral ablation of the Q lineages, the O/P lineages were labeled
with fluorescent lineage tracer. Under these conditions, we found that the
cleavage pattern of all o/p bandlets was uniformly O-type in the Sacramento
population (n=10) (Fig.
4C; Table 1, row
T). In stage 9 embryos, we found that the labeled O/P lineage produced only O
pattern elements, and that P pattern elements were completely missing
(n=17) (Table 2, row
G). In many cases, we could detect duplicated O pattern elements that were
labeled with the two different fluorophores and hence arose independently from
the two ipsilateral O/P teloblasts (Fig.
6E-J). These findings confirm that both ipsilateral O/P lineages
adopt the O fate when the Q lineage is absent in H. robusta
(Sacramento) (Huang and Weisblat,
1996), and verify that the disparate results obtained with H.
robusta (Austin) are not a procedural artifact.
Helobdella sp. (Galt)
To explore further the diversity of O/P specification mechanism in the
genus Helobdella, bilateral ablation of the Q teloblasts was also
performed in the more distantly related Helobdella sp. (Galt). The
O/P lineages were labeled with fluorescent lineage tracer, and the cleavage
pattern of the o/p blast cell clones examined in stage 8 embryos. It was found
that the o/p blast cell clones in the operated embryos uniformly express an
O-type cleavage pattern (n=24)
(Fig. 4D;
Table 1, row U). The response
of the O/P lineage to bilateral ablation of the Q lineages in
Helobdella sp. (Galt) is therefore largely the same as that of H.
robusta (Sacramento), which suggests that similar mechanisms may be
responsible for O/P specification in these two species.
O/P specification mechanism of H. robusta (Austin): mesoderm is involved in O/P specification
The observation that the dorsal o/p bandlet, but not the ventral o/p
bandlet, of H. robusta (Austin) can adopt a partial or complete P
fate in the absence of the Q lineages implies the existence of a redundant
inductive signal that may emanate from some other cell population. To identify
the source of this redundant signal, we examined the effects of ablating
either the M teloblast or the N teloblast, whose descendant lineages are
adjacent to the o/p bandlets in a normal germinal band and thus are prime
candidates to influence O/P specification.
Ablation of the M or N lineage alone does not produce any consistent
alteration in the O/P lineage of H. robusta (Austin), consistent with
previous observations from H. robusta (Sacramento)
(Huang and Weisblat, 1996). In
experiments in which the M lineage or the N lineage was individually ablated,
we saw no visible effect on the O/P cleavage pattern except in one case
(Table 1, rows D-G). In this
particular embryo, the M lineages of which were bilaterally ablated, both the
presumptive o and p bandlets were in contact with the q bandlet at the
posterior end of the germinal band, and both displayed a P-type cleavage
pattern. The same atypical phenomenon was also reported by Huang and Weisblat
(Huang and Weisblat, 1996
), and
was used to support the notion that an interaction with the Q lineage alone
can induce P fate in the O/P lineage.
In order to detect whether the N or M lineages play any role in the Q
lineage-independent pathway, either the N or the M teloblast was ablated in
combination with the two Q teloblasts. Following ablation of the right N
teloblast and two Q teloblasts, the cleavage patterns of the presumptive O and
P lineage does not appear to be significantly different from the cleavage
pattern seen in embryos whose Q lineages were ablated alone
(2=3.28, P=0.070)
(Table 1, row H). However, when
both N and Q teloblasts were ablated bilaterally, the cleavage pattern of the
O/P lineage became highly variable (Table
1, row I). In these embryos, the o/p bandlets frequently failed to
enter the germinal band, and were often found in the peripheral region of the
micromere cap (data not shown). We believe these variable results are a
non-specific effect due to disruption in the relative position of the o/p
bandlets with respect to the germinal band as a whole.
By contrast, combinatorial ablation of the M and Q teloblasts had a
profound and reproducible effect on the cleavage pattern of the o/p blast cell
clones. When either ipsilateral or bilateral M lineage(s) were ablated in
combination with bilateral ablation of the Q lineages, blast cell clones in
both the presumptive o and presumptive p bandlet exhibited uniformly O-type
cleavage patterns on the mesoderm-deficient side
(Fig. 7;
Table 1, rows J,K). The
frequency at which P-type cleavage was expressed in these experiments was
significantly different from that seen when the Q lineages were ablated alone
(2=24.00, P<0.0001). In H. robusta
(Sacramento), combinatorial ablation of the Q and M lineages produces the same
effect (Huang and Weisblat,
1996
).
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Ablation of the micromere-derived provisional integument does not influence O/P specification of H. robusta (Austin)
In addition to the m, n, and q bandlets, the o/p bandlets also contact an
overlying provisional integument arising from the micromere lineages. An
involvement of this provisional integument in O/P specification was previously
demonstrated in H. triserialis, in which ablation of the n' and
opq'' micromere clones causes the presumptive O lineage to adopt various
aspects of the P fate (Ho and Weisblat,
1987). To determine whether these same micromere lineages are
involved in O/P specification of H. robusta (Austin), we ablated the
provisional integument descending from the n' and opq'' micromeres
at embryonic stage 8 and examined the cleavage pattern of the presumptive o
and p bandlets. Unlike H. triserialis, the o and p bandlets
maintained their normal cleavage pattern following this ablation in H.
robusta (Austin) (Table 1,
row L).
When these two micromere clones were ablated together with the two Q
teloblasts in embryos of H. robusta (Austin), the presumptive p
bandlets exhibited a segmentally mosaic cleavage pattern that did not
significantly differ in the rate of P fate specification from embryos in which
only the two Q teloblasts were ablated (2=2.05,
P=0.1517) (Table 1,
row M). These data suggest that ablation of the provisional integument does
not have any significant effect on O/P specification in H. robusta
(Austin).
Q lineage is involved in specification of the P fate in H. robusta (Austin)
In the mesoderm ablation experiments described above, the presumptive p
bandlet consistently expressed its normal P-type cleavage pattern unless the Q
lineages were also ablated. This result suggests that the Q lineage may be
able to specify P fate in H. robusta (Austin), even though other
experiments show that it is not required for P fate specification because of
the existence of a redundant Q lineage-independent pathway. To further
elucidate the significance of the Q lineage in O/P specification, we performed
experiments in which all but one of the four O/P teloblasts were ablated, and
the development of the remaining `lone' O/P lineage was compared in the
presence or absence of the Q lineage.
In the presence of the Q lineages, a `lone' O/P lineage uniformly expressed
a P-type cleavage pattern at stage 7/8 (n=12) and gave rise
consistently to P pattern elements at stage 9 (n=15)
(Table 1, row N;
Table 2, row D). This result is
similar to that obtained in comparable experiments on H. triserialis
and H. stagnalis (Weisblat and
Blair, 1984; Zackson,
1984
).
But in the absence of the Q lineages, the cleavage pattern of blast cell
clones in a `lone' o/p bandlet was exclusively O-type (n=57)
(Fig. 8A;
Table 1, row O). The pattern of
differentiated descendants produced at stage 9 by these o/p bandlets was more
complicated (Table 2, row E).
In five out of eight embryos, the `lone' o/p bandlet gave rise to O pattern
elements exclusively (Fig. 8B).
In the remaining three embryos, the `lone' o/p bandlet gave rise to O pattern
elements in some segments and P pattern elements in other segments
(Fig. 8C). Here, again, there
was a statistically significant inconsistency between cleavage pattern of the
blast cell clones examined in stage 8 embryos and the differentiated pattern
elements observed in stage 9 embryos (2=34.2,
P<0.0001).
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Transient interaction with the contralateral q bandlet is sufficient for P fate specification in H. robusta (Austin)
Huang and Weisblat (Huang and Weisblat,
1996) have shown that a brief contact with the contralateral q
bandlet is sufficient to specify P fate in the O/P lineages of H.
robusta (Sacramento). To determine whether a brief interaction with the
contralateral Q lineage is also sufficient to specify P fate in H.
robusta (Austin), we ablated one of the right O/P teloblasts and the
right Q teloblast, and labeled the surviving right O/P teloblast with lineage
tracer. Consistent with the results from H. robusta (Sacramento), the
labeled O/P lineage consistently expressed the P-type cleavage pattern in
stage-7/8 embryos (n=15) and gave rise to P pattern elements in stage
9 embryos (n=10) (Table
1, row P; Table 2,
row F).
Given the presence of a second M lineage-dependent pathway for P fate specification in H. robusta (Austin), we considered the possibility that the ipsilateral M lineage might be augmenting the P fate-inducing properties of the contralateral Q lineage in this experiment. To test this possibility, we first performed experiments in which the right M and Q teloblasts were ablated (n=10) and found that the cleavage patterns expressed by the two right O/P lineages were normal (Table 1, row Q). Thus, the ipsilateral M lineage is not required for the contralateral Q lineage to specify P fate in the dorsal O/P lineage. Additionally, we performed experiments in which one of the right O/P teloblasts was ablated in combination with the right M and Q teloblasts (n=10) or the right M, N and Q teloblasts (n=9). In each paradigm, the surviving o/p bandlet consistently expressed the P-type cleavage pattern (Table 1, rows R,S).
Similar to what has been reported for H. robusta (Sacramento), these data suggest that a brief contact with the contralateral Q lineage can specify P fate in an O/P lineage of H. robusta (Austin), and that this specification occurs effectively in the absence of the other ipsilateral teloblast lineages.
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Discussion |
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This variation in the pattern of cell-cell interactions suggests that the
developmental pathway underlying the conserved morphology of the O/P lineages
has undergone evolutionary divergence in closely related leech species, and,
depending on the taxonomic status of the two H. robusta populations,
possibly even within a single species. Taken together with the species
variation in nematode vulval development
(Sommer, 2001), this finding
also suggests that a rapid divergence of developmental pathways underlying
conserved morphological patterns may be a widespread feature in the evolution
of developmental equivalence groups.
A mesoderm-dependent pathway of P fate specification in H. robusta (Austin)
H. robusta (Austin) and the other two Helobdella
populations examined here share the following features: (1) the presumptive O
and P lineages both have the developmental potential to adopt either O or P
fate, and are thus equipotent; (2) the default fate of the O/P lineage in the
absence of cell interactions appears to be the O fate; and (3) the q bandlet
alone is able to specify the P fate in an adjacent o/p bandlet. However,
H. robusta (Austin) differs from the others in that the Q
lineage-dependent pathway and a distinct mesoderm-dependent pathway are
redundantly involved in P fate specification. The molecular basis of these
effects is unknown, but one could imagine that such interspecies variation
might arise from either qualitative or quantitative differences in the signals
that pattern O and P cell fates.
Teloblast ablation experiments suggest that the P fate can be specified in
H. robusta (Austin) by a cooperative interaction involving both the
two adjacent o/p bandlets and the underlying m bandlet. Removal of either the
ipsilateral M lineage or one of the two ipsilateral O/P lineages can, in the
absence of the Q lineages, cause the remaining O/P lineage(s) to forsake the P
pathway and follow the O developmental pathway with high frequency. Assuming
that the two O/P lineages are truly equivalent, it would seem likely that the
m bandlet is able to provide extrinsic cues that are sufficient to break that
equivalence. The dorsoventral polarity of the m bandlet, which manifests
itself in the orientation of the cell division of the primary m blast cell
(Zackson, 1984) (D.-H.K.,
unpublished), may have allowed this structure to assume a novel role in the
Austin population as an extrinsic source of dorsoventral asymmetry for the O/P
specification.
We envision that the m bandlet and the two o/p bandlets could interact to specify P fate in H. robusta (Austin) by either of two distinct mechanisms. In one scenario, the m bandlet may be able to directly induce the P fate in the more dorsal of a pair of overlying o/p bandlets after the Q lineages have been ablated. However, this hypothesis does not readily explain why the m bandlet fails to induce the P fate in a single o/p bandlet (Table 2), although it is formally possible that removal of the q bandlet and one o/p bandlet might prevent the remaining o/p bandlet from adopting a position in which it could receive an inductive signal from the m bandlet.
An alternative scenario is that the m bandlet may potentiate a lateral
interaction between the two overlying o/p bandlets, and that it is this
lateral interaction which specifies the P fate in the absence of the Q
lineages. Huang and Weisblat (Huang and
Weisblat, 1996) have proposed that the interaction of the o and p
bandlets is purely steric in H. robusta (Sacramento), i.e. that the p
bandlet merely shields the o bandlet from the P-inducing influence of the
dorsally situated Q lineage. However, our findings argue against this
interpretation for H. robusta (Austin) as the specification of the
definitive P fate persists following bilateral ablation of the Q lineages, and
is either partially or completely eliminated when one of the two ipsilateral
o/p bandlets is also removed (Table
2).
In H. robusta (Austin) embryos in which the Q lineages have been ablated and the M lineages are still present, the presumptive p bandlet expresses an ectopic O-type cleavage pattern in some segments. This abnormal pattern of cell division seems to be correlated with a morphological anomaly that reduces the contact area between the two ipsilateral o/p bandlets (Fig. 5). The ectopic O-type cleavages would appear to result from a delayed specification of the P fate, as other data suggest that these same blast cells do go on to produce an essentially normal pattern of P-type descendants. We do not know what sequence of causal events leads to this anomalous morphology of the germinal band, but one can speculate that a reduction in the contact of the presumptive o and p blast cells may be indicative of an attenuated interaction between them.
Multiple steps in P fate specification
In two of the experiments described here, we observed statistical
inconsistencies in the frequency of P fate specification as manifested by
O-type blast cell cleavage pattern at stage 7/8 and by the formation of P-type
differentiated pattern elements at later stages. This result suggests that
even though the o/p blast cell expresses an O-type cleavage at its first
division, it can still become committed to the definitive P fate by cell-cell
interaction(s) that take place at a later stage of development. In support of
this idea, it has previously been shown in H. triserialis that
presumptive o blast cells that have experienced one or more of their normal
cleavages can nonetheless undergo a partial or complete commitment to produce
P-type differentiated descendants if the adjoining p bandlet is ablated
(Shankland, 1987a;
Shankland, 1987b
). The reverse
has not been observed despite a variety of experimental manipulations,
suggesting that commitment of a primary o/p blast cell to the P pathway may be
an irreversible event that constrains both cleavage pattern and differentiated
cell fate.
Segmental differences have also been reported in the blast cell cleavage
patterns that generate O and P cell fates. In the so-called OP lineage of the
four most rostral segments in the leech, distinct cleavage programs and
cell-cell interaction patterns are used to generate a set of differentiated
pattern elements that are segmentally homologous to the combined O and P
lineages of the midbody and caudal segments
(Shankland, 1987d;
Kuo and Shankland, 2004
).
These findings imply that cleavage programs can be functionally dissociated
from differentiated cell fates in the lateral ectoderm of the leech
Helobdella, raising the possibility that distinct mechanisms may be
involved in their specification.
Role of the micromere-derived provisional integument in the O/P specification pathway
It has previously been shown that the micromere-derived provisional
integument that covers the o/p bandlets is required for normal specification
of the O fate in H. triserialis
(Ho and Weisblat, 1987). But
the developmental fate of the presumptive O lineage was not altered in similar
experiments performed here on H. robusta (Austin). The most
straightforward explanation of these disparate observations is that the role
of the provisional integument in O/P specification, like that of the M
lineage, varies between species.
However, ablation of micromere clones is inherently transient and variable,
apparently because the provisional integument undergoes a wound-healing
response (Ho and Weisblat,
1987). Additionally, there are subtle but nonetheless pertinent
differences in the positioning of specific micromere clones relative to the
time-line of blast cell maturation between H. triserialis and H.
robusta (Austin) (D.-H.K., unpublished). Given these potentially
confounding factors, we would not discount the possibility that the
provisional integument of H. robusta (Austin) could play some role in
O/P specification that was not revealed by our experimental procedure.
In any case, the O-fate inducing signal from the micromere-derived
provisional integument is unlikely to be a pivotal factor in breaking the
equivalence of the O/P lineages in the leech Helobdella as this
epithelium contacts both the o and p bandlets
(Ho and Weisblat, 1987;
Smith and Weisblat, 1994
). But
in some species it may still be involved in promoting O fate specification
once the initial equivalence is broken.
Evolution of the O/P equivalence group
The specification of O and P fates has been experimentally studied in
several clitellate species. In the sludgeworm Tubifex, it has been
shown that the P lineage specifies a pluripotent O/P lineage to adopt the O
fate, but that the specification of the P lineage does not require any of the
other ectodermal lineages (Arai et al.,
2001). On the basis of those results, the authors proposed that
the O and P lineages of Tubifex differ from those of
Helobdella in that they do not represent an equivalence group
(Arai et al., 2001
). It should
be noted that the role of mesoderm has not yet been experimentally studied in
Tubifex, and it is possible that, similar to H. robusta
(Austin), mesoderm may play a role in the specification of P fate.
Nonetheless, the fact that the P fate is specified normally in
Tubifex in the absence of both Q and ipsilateral O lineages strongly
suggests that there is a significant difference in the O/P specification
mechanisms employed by this distantly related clitellate species.
In the leech Theromyzon, the O/P lineages do behave as an equivalence group and their cell fates are determined by relative location in the germinal band (Keleher and Stent, 1992). However, simple ablation of the Q lineage does not alter the O/P specification of the Theromyzon embryo (F. Z. Huang and D. A. Weisblat, personal communication), as it does in H. robusta (Sacramento). Further studies will be required to ascertain whether Theromyzon employs an O/P specification mechanism similar to that of H. robusta (Austin), or relies on some more divergent mechanism.
As in the leech Theromyzon, the O/P lineages of all three Helobdella laboratory populations studied here behave as an equivalence group. In H. robusta (Sacramento) and Helobdella sp. (Galt), the Q lineage is absolutely required for the specification of the P fate. But in H. robusta (Austin), the P fate appears to be specified redundantly by the Q lineage and by a set of interactions that involve the two ipsilateral O/P lineages and the mesodermal M lineage. Thus, either one of these pathways can specify the P fate in this particular laboratory population.
Fig. 9 portrays these findings in the context of a phylogenetic tree. Given the data at hand, the most parsimonious interpretation is that the mesoderm-dependent pathway of P fate specification is an apomorphic (derived) trait that arose recently in the branch leading to H. robusta (Austin). The role of the Q lineage in O/P specification appears to be evolutionarily conserved among Helobdella, and may be a synapomorphy of this group. However, we cannot at this time exclude the possibility that the P fate-inducing ability of the Q lineage in Helobdella is a symplesiomorphic (shared ancestral) trait that was lost in the branch leading to Tubifex.
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ACKNOWLEDGMENTS |
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Footnotes |
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