1 Department of Biology, Rutgers, The State University of New Jersey, Camden, NJ
08102, USA
2 Department of Molecular and Cell Biology, 385 LSA, University of California,
Berkeley, CA 94720-3200, USA
* Author for correspondence (e-mail: dshain{at}camden.rutgers.edu)
Accepted 19 May 2004
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SUMMARY |
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Key words: Theromyzon rude, Leech, Nervous system, Cell lineage, engrailed
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Introduction |
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Essential features of glossiphoniid leech development are shown
schematically in Fig. 1.
Segments arise sequentially from five bilateral pairs of stem cells called M,
N, O, P and Q teloblasts. Each teloblast divides repeatedly, giving rise to
segmental founder cells in a coherent column called a bandlet. During
gastrulation, bandlets coalesce, first bilaterally and then longitudinally
along the ventral midline to form the germinal plate. Cells in each lineage
divide throughout gastrulation, leading eventually to the differentiation of
definitive mesodermal and ectodermal progeny within the germinal plate. Both
mesodermal (M) and ectodermal (N, O, P and Q) lineages contribute neurons to
the 32 bilaterally symmetric, segmentally iterated ventral neuromeres of the
central nervous system (CNS). In addition to a rostral, unsegmented,
supraesophagael ganglion, the CNS comprises four fused neuromeres in the
anterior subesophageal ganglion, seven fused neuromeres in a caudal ganglion
associated with the posterior sucker and 21 neuromeres occurring as distinct
midbody ganglia, separated from adjacent ganglia by interganglionic connective
nerves (Stent et al., 1992).
Each midbody ganglion contains
300-400 neurons, depending on the species
(Macagno, 1980
). Cells arising
from N, O, P and Q lineages differentiate into specific subsets of central and
peripheral neurons and epidermal cells
(Kramer and Weisblat, 1985
;
Braun and Stent, 1989a
). More
than two-thirds of the ganglionic neurons arise from the N teloblasts
(Kramer and Weisblat, 1985
);
consequently, the N lineage has been used to follow morphological aspects of
gangliogenesis (Fig. 1)
(Shain et al., 1998
;
Shain et al., 2000
).
|
In this study, we sought to identify modifications in development that
account for the observed differences in the mature nervous systems of these
leeches. In addition, because Theromyzon embryos are accessible to
experimental manipulation during early development, we were able to ablate
specific cell lineages, or subsets of lineages, to determine the cellular
requirements for forming specific nerves. We were particularly interested in
determining the significance of two transverse stripes of N lineage-derived
cells that transiently connect the posterior margin of the ganglion to ventral
body wall (Shain et al.,
1998), and which contribute three N-derived, peripheral neurons to
the nervous system (nz1-3) (Braun and
Stent, 1989a
). Previous studies indicated that these cells may
play a role in establishing the normal projection of the PP segmental nerve
(Shain et al., 1998
). We show
here that cells within the anterior, transverse stripe of N-derived cells
appear to position an O lineage-derived neuron, PD, that pioneers
the PP segmental nerve tract.
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Materials and methods |
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Lineage tracer injections and cell ablations
Fluorescent lineage tracer [either fluorescein-dextran amine (FDA,
Molecular Probes)] or tetramethylrhodamine-dextran amine (RDA, Molecular
Probes) was injected into M, N, O, P or Q teloblasts as previously described
(Weisblat et al., 1980). Cell
ablations were achieved by irradiating FDA-labeled cells with the focused beam
of a 488 nm argon laser (Hobbs or Lexel, Model 65)
(Braun and Stent, 1989b
) or by
`over-injecting' the cell of interest as described
(Shain et al., 2000
) with
DNase (Blair, 1982
) or ricin A
chain (Nelson and Weisblat,
1992
).
Immunohistochemistry
Fixed, dissected germinal plates were prepared and immunostained as
previously described (Shain et al.,
2000). Monoclonal antibodies specific to either leech muscle (Lan
3-14) (Zipser and McKay, 1981
)
or mouse acetylated
-tubulin (Sigma) were used at a 1:1000 dilution;
Cy3- and Cy5-conjugated secondary antibodies (Jackson Lab) were diluted 1:400.
TOTO-3 nuclear staining was performed according to the specifications of the
manufacturer (Molecular Probes).
Microscopy
Germinal plates were viewed and photographed using either a Zeiss Axiophot
microscope or a confocal microscope (BioRad model MRC-1000/1024). Specimens
were photographed using Ektachrome 400 film (Kodak) and scanned with a
SprintScan 35 Plus (Polaroid) slide scanner. Adjustment of color levels and
merging of images was performed with Adobe Photoshop 6.0. In generating
digitally merged fluorescence images, the shades used to pseudocolor different
signals were selected to optimize clarity and consistency, rather than to
mimic the spectra of the fluorophores employed.
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Results |
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At 60 hours clonal age, axon fibers projected laterally from the
posterior aspect of the ganglion to establish the PP nerve. The appearance of
these processes coincided approximately in time and location with the
formation of two transient, ventrolateral stripes of N lineage-derived cells
near the posterior ganglionic boundary; cells in the anterior stripe express
the leech engrailed-class protein (Wedeen et al., 1991;
Lans et al., 1993
;
Shain et al., 1998
).
Peripheral, longitudinal fibers originated from the pz8/cf3 cluster at
65 hour clonal age. These bilaterally paired nerves projected across
segmental boundaries and also moved laterally with the expanding germinal
plate until
100 hours clonal age, at which time they retracted. The first
fiber(s) corresponding to the UP segmental nerve were observed at
70
hours. Initially, these appeared to project from the N-derived nz3 neuron into
the ipsilateral root of the PP nerve, and later sent processes lateral and
parallel to the PP nerve. Processes of the DP nerve appeared at
80 hours
clonal age as a branch of the PP nerve, distal to the bifurcation of UP and
PP. DP nerve projections of the Retzius neuron in glossiphoniid leeches have
been described previously (Stuart et al.,
1987
; Elsas et al.,
1995
).
By clonal age 120 hours, the neuroarchitecture of the T. rude
nervous system was essentially as described for a mature glossiphoniid leech
(Braun and Stent, 1989a
). In
contrast to Hirudo (Jellies et
al., 1996
), there was no condensation of the AA and MA nerves,
thus the three segmental nerves remained distinct at the edge of the ganglion;
also by this time, more than 10 well-defined commissures were evident across
the midline. The longitudinal connectives and Faivre's nerve were considerably
larger in diameter than at earlier stages, and ganglia had become encapsulated
by connective tissue sheaths.
Normal axon contributions from each teloblast lineage
To establish the contributions of each teloblast lineage (M, N, O, P and Q)
to the development of specific nerves, subsets of neurons were labeled by
unilateral injections of FDA into each teloblast and the distribution of
labeled axons was observed by epifluoresence in the nerves of midbody ganglia
in which the N lineage cells were 100 hours clonal age. A summary of data
obtained from this analysis is presented in
Table 1. The neuroectodermal
(N) lineage displayed the broadest distribution of axon projections,
contributing processes bilaterally to all 11 nerve tracts that were scored.
The other ectodermal lineages (O, P and Q) contributed primarily to
ipsilateral segmental nerves (AA, MA and PP) and longitudinal connectives,
while the mesodermal (M) lineage, contributed processes only to the
longitudinal connectives and Faivre's nerve.
|
|
No dramatic modifications of the normal nerve pattern were observed upon
unilateral O teloblast ablations. However, the PP nerve appeared to be
retarded in its growth and deviated from its normal projection parallel to the
AA and MA nerves (Fig. 3C).
Unilateral P teloblast ablations resulted in deficiencies in the size and
position of the MA nerve tract. Although an MA nerve often formed in the
absence of ipsilateral, P-derived progeny, its diameter was reduced,
suggesting a significant decrease in the number of axons occupying that nerve.
In addition, the MA nerve often projected into the ganglion either anterior or
posterior to its normally central position
(Fig. 3D). [P lineage ablations
are feasible in T. rude because the O-to-P fate change that
invariably results from loss of the P lineage teloblast in Helobdella
(Weisblat and Blair, 1984;
Shankland and Weisblat, 1984
;
Huang and Weisblat, 1996
)
occurs in only some cases in Theromyzon
(Keleher and Stent, 1990
).]
Unilateral Q teloblast ablations induced no gross changes in the normal
pattern of axons (not shown).
In a related study, Braun and Stent
(Braun and Stent, 1989b)
unilaterally photoablated several segments' worth of n, o, p and q progeny
within the germinal plate of Helobdella triserialis, and monitored
axon growth by labeling contralateral, N-derived motoneurons that crossed the
midline. Our experiments were conducted in Theromyzon using teloblast
ablations so that the lineage in question was never present within the
germinal plate. Although the results of the two studies are generally
consistent, several phenotypes observed here (e.g. MA/PP fusion, developmental
delays, abnormal position of some nerves) were not observed in the earlier
study. We attribute these differences to the timing and efficacy of the
ablations, although species-specific effects cannot be ruled out.
These differences notwithstanding, preventing the normal contributions of
the individual O, P and Q lineages had no large effects on nerve formation in
either species (i.e. H. triserialis or T. rude), despite the fact
that these lineages contribute processes to all ipsilateral nerve tracts
except UP. To test for the possibility that these three ectodermal lineages
have redundant effects on nerve patterning, we examined germinal plates from
embryos in which the O, P and Q lineages had all been ablated on one side. In
such embryos [and in embryos with bilateral OPQ ablations (not shown)], all of
the major longitudinal and segmental nerves still formed, but their positions
were abnormal (Fig. 3E). This
suggests that axons from the remaining cells (primarily ipsilateral and
contralateral N-derived neurons) have the capacity to establish all of the
major nerve tracts in leech. Conversely, embryos in which both N teloblasts
were ablated (resulting in the absence of 2/3 of ganglionic neurons) also
formed the major longitudinal and segmental nerves, although the MA and PP
nerves coalesced as described above and there was a marked reduction in the
size of the AA nerve (not shown), suggesting that O-, P- and Q-derived neurons
can also pioneer most nerve tracts in the absence of the N lineages.
Correlation between muscle fiber and segmental nerve placement
The severe disorganization of nerve tracts following unilateral mesodermal
ablations (Fig. 3A) prompted us
to examine the relationship between nerve and muscle fibers during
development. For this purpose, we labeled the neuroectoderm by injecting an N
teloblast with RDA, fixed and dissected the embryos after 100 hours
further development, and then differentially labeled axons (ACT) and muscle
fibers (Lan 3-14) in the dissected germinal plates
(Fig. 4). More than 30
bilateral pairs of longitudinal muscle fibers and between 20-25 circular
muscle fibers per segment were apparent in our preparations. These were
arranged in stereotypical patterns so that homologous muscle fibers could be
recognized in each segment. By this criteria, the MA nerve, which enters each
ganglion approximately at its center (see
Fig. 2), always projected
between the two circular muscle fibers that displayed the greatest inter-fiber
separation (Fig. 4). The AA and
PP nerves consistently ran between sets of circular muscle fibers that were
about five fibers anterior and posterior of the MA nerve, respectively. The UP
nerve appeared less constrained, running between muscle fibers that were
approximately three to seven fibers posterior to PP. Aside from the short,
oblique regions of segmental nerves that exited the ganglion, the segmental
nerves showed a strong propensity to maintain their position between these
specific pairs of muscle fibers. Similar results have been reported previously
(Braun and Stent, 1989a
), who
found that a distance of at least 5 µm (in the smaller embryos of
Helobdella) separates nerve tracts from their nearest circular muscle
fiber. Similar rules may apply for longitudinal nerves that run between
stereotypical sets of longitudinal muscles on either side of the midline
(Fig. 4). In light of the
severe disruptions on nerve patterning induced by mesoderm ablations
(Fig. 3A), these results
suggest that proper patterning of the segmental nerves requires cues provided
by the muscle fibers, or that both segmental nerves and muscle fibers are
patterned in parallel by some other set of mesodermally derived cues.
|
|
The close association of N- and O-derived cells near the nascent PP nerve
suggests that these cells might be required for the normal formation of PP.
Indeed, unilateral ablations of N or O teloblast lineages resulted in
characteristic modifications of the PP nerve (see
Fig. 3B,C). To identify cells
in the N lineage involved in PP nerve patterning, individual primary n blast
cells (nf or ns; see Fig. 1)
were ablated and the resultant embryos were later immunostained with ACT.
Ablation of either nf or ns prevents approximately one-half (75) the
normal complement of N-derived neurons from forming in the experimental
hemiganglion; an ns ablation causes a deficiency in the anterior lobe of the
hemiganglion, while an nf ablation causes a deficiency in the posterior lobe,
including both ventrolateral stripes
(Ramirez et al., 1995
;
Shain et al., 2000
). In all
segments from which the contributions of the ns primary blast cell were
missing, the patterning of the PP nerve appeared normal (n=4;
Fig. 6B). In segments showing
the effects of nf primary blast cell ablations, however, the putative PP nerve
arose as a posterior branch of the MA nerve near the ganglionic margin
(n=9; Fig. 6C), a
phenotype similar to that observed in unilateral N teloblast ablations
described earlier (cf. Fig.
3B). In many injections, blast cells were not killed but rather
went on to produce an abnormally low number of progeny (between 5-10); in
these cases, no ventrolateral stripes were formed (e.g.
Fig. 6C) and the PP nerve was
condensed with the MA nerve.
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Discussion |
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In Hirudo, the first neurites projecting from the ganglion
correspond to the DP nerve, which forms the future posterior nerve root
(Jellies et al., 1996). The MA
nerve, which occupies the position of the future anterior nerve root, appears
a few hours later and is followed by the PP and AA nerves, both of which form
distinct projections from the ganglion
(Jellies et al., 1996
). Thus,
four distinct nerve roots (AA, MA, PP and DP) appear initially in
Hirudo. Later condensation of the AA nerve with the MA nerve, and the
PP nerve with the DP nerve leaves just two segmental nerve roots in the
adult.
The first nerves to appear in T. rude are MA and AA (see
Fig. 2). MA is pioneered by a
peripheral, P-derived cell(s) as in Hirudo
(Jellies et al., 1996). The PP
nerve root forms in T. rude after MA and AA are well established,
which is in reverse order of that described in Hirudo
(Jellies et al., 1996
). And an
additional prominent segmental nerve called the ultraposterior (UP) nerve
(Kramer and Goldman, 1981
;
Braun and Stent, 1989a
), which
is absent in Hirudo, branches from the PP nerve outside of the
ganglionic margin. The DP nerve also branches from PP outside of the ganglion
and is distal to the PP/UP branch point
(Braun and Stent, 1989a
).
Collectively, T. rude projects five segmental nerves from three
segmental nerve roots in each hemiganglion, and no condensation is observed.
[Although the AA and PP nerve roots shift towards the center of the ganglion
as in Hirudo, they remain distinct (see
Fig. 2).] We note that the
initial condensation step in Hirudo joins the DP and PP nerves, while
the AA and MA nerves remain independent. At this stage of Hirudo
development, the axon architecture resembles that of T. rude and
other glossiphoniid leeches (Braun and
Stent, 1989a
; Braun and Stent,
1989b
), and may represent an ancestral condition.
The development of the DP nerve also differs between Hirudo and
Theromyzon. In Hirudo, DP is the first segmental nerve to
emerge from the ganglionic primordia and forms the most prominent projection
(Jellies et al., 1996). By
contrast, the DP nerve in T. rude is not well defined, appearing only
late in the development of the segmental nerves as a relatively minor
bifurcation of the PP nerve (see Fig.
2). The significance of this observation remains unclear, but
there is a correlation between swimming behavior and the prominence of the DP
nerve that transcends taxonomic groupings. The glossiphoniid leeches
Theromyzon and Helobdella, which do not swim, project a
rudimentary DP nerve late in development
(Braun and Stent, 1989a
)
(Fig. 2), while Hirudo
(Family Hirudiniformes) and also the glossiphoniid leech Haementeria
ghilianii, both of which do swim, form a prominent DP nerve early in
embryogenesis (Kuwada, 1985
;
Jellies et al., 1996
).
Cell ablations
In the leech nervous system, identified neurons arise by stereotyped
patterns of cell division and migration from each of the five teloblast
lineages (M, N, O, P and Q) (Zackson,
1984; Kramer and Weisblat,
1985
; Torrence and Stuart,
1986
; Bissen and Weisblat,
1987
; Shankland,
1987
; Bissen and Weisblat,
1989
; Braun and Stent,
1989a
; Shain et al.,
1998
). In other animals, specific cells have been shown to provide
positional cues used by navigating growth cones
(Bentley and Keshishian, 1982
;
Palka et al., 1992
;
Goodman and Shatz, 1993
), and
similar results have been obtained in leech
(Braun and Stent, 1989b
). In
this study, we removed subsets of definitive progeny from one side of the
embryo by ablating specific teloblast lineages or sublineages, so that
developing axons were challenged to find their targets in the absence of
normal positional cues. The effects of mesodermal deficiencies were most
dramatic, including severely disorganized arrays of axons
(Fig. 3A). These results are
consistent with previous studies showing that mesodermal ablation leads to
massive disorganization of ectodermal patterning in Helobdella
(Blair, 1982
;
Torrence et al., 1989
). The
strong correlation between the position of muscle fibers and nerve tracts (see
Fig. 4) further supports the
notion that mesoderm is a crucial component of axon guidance in leech
(Torrence et al., 1989
).
Unilateral ablations of other teloblast lineages displayed less severe, but
more specific effects on nerve patterning. In the absence of N lineage-derived
progeny, the ipsilateral UP nerve failed to form and the ipsilateral PP nerve
appeared to have coalesced with the MA nerve within the hemiganglion and arose
instead as a posterior branch of the MA nerve at the margin of the ganglion.
The absence of UP is consistent with previous studies demonstrating that the
N-derived neuron nz3 is required for UP nerve formation
(Braun and Stent, 1989b).
Ablation of the O lineage also disrupted ipsilateral PP nerve formation
although to a lesser extent. These observations suggest either a cooperative
or epistatic interaction between N and O lineage derivatives is required to
form the normal PP nerve, as discussed below.
Formation of the PP segmental nerve
Several lines of evidence suggest that the O lineage-derived mechanosensory
neuron PD plays a role in establishing the normal trajectory of the
PP segmental nerve. Previous studies have shown that PD homologs in
other glossiphoniid species extend processes medially toward the ipsilateral
connective and also peripherally via the PP nerve root
(Kuwada and Kramer, 1983;
Kramer and Weisblat, 1985
;
Kuwada, 1985
;
Kramer and Goldman, 1981
;
Braun and Stent, 1989a
). In
this study, ACT immunostaining confirmed those results for T. rude
and also revealed an axon process growing laterally from the ipsilateral
connective towards PD (see Fig.
2). When PD was missing (i.e. in unilateral O
ablations), a PP nerve tract eventually formed and exited the ganglion but its
peripheral trajectory was abnormal (see
Fig. 3C). Together, these
observations suggest that the process(es) emanating from the ipsilateral
connective can pioneer the PP nerve in the absence of the PD
neuron, but that they normally grow out along a pre-existing pathway
established by the PD axon.
We have shown previously that a ventrolateral stripe of N lineage-derived
cells forms a transient bridge that connects the ganglion with the ventral
body wall (Shain et al.,
1998). The close association of these cells with early fibers in
the PP nerve tract (see Fig. 5)
suggested that this interaction is required for establishing the PP nerve.
Previous studies indicate that the two distalmost cells in the anterior
ventrolateral stripe (i.e. the future nz1 and nz2 neurons) lie along the PP
nerve pathway (Braun and Stent,
1989a
). But ablating these cells did not disrupt formation of the
PP nerve (Braun and Stent,
1989b
), in apparent contradiction with results presented here (see
Fig. 6C,D). This discrepancy
appears to result from the difference in timing and also in the number of
cells that were ablated in each study. Braun and Stent
(Braun and Stent, 1989b
) did
not ablate nz1 and nz2 until after they had separated from the hemiganglion.
Our results indicate that the PP nerve has already formed by that time (see
Fig. 5A). In our present
experiments, ablations were performed prior to the lateral migration of cells
within the stripe, well before the first fibers of the PP nerve appeared. This
lesion not only prevented formation of nz1 and nz2, but also cells at the
medial end of the N-derived ventrolateral stripe that are closely associated
with the O-derived PD neuron (see
Fig. 5B,
Fig. 7). Although Braun and
Stent (Braun and Stent, 1989b
)
made unilateral ablations in the n bandlet, these appear not to have removed
all N-derived progeny in the hemiganglion and the remaining cells were
presumably sufficient to effect the migration of the PD neuron.
PD arises as part of an O-derived cell cluster within the medial
aspect of the ganglionic primordia, then migrates posteriorly before
initiating axonogenesis (Braun and Stent,
1989a
; Torrence and Stuart,
1986
) (Fig. 7). In
N teloblast ablation experiments, PD fails to migrate posteriorly
(Fig. 7) before differentiating
and therefore projects its peripheral process from the central cluster of
O-derived cells along the MA nerve. Apparently, the axons growing out from the
ipsilateral connective detect the ectopic PD axon and follow it to
the periphery instead of pioneering a posterior tract (as they would if
PD were absent and the ipsilateral N lineage were present)
(Fig. 3C,E); this results in
coalescence of the MA and PP tracts within the ganglion. Interestingly, the
displaced PP axons assume their normal posterior position shortly after
exiting the ganglion, apparently in response to cues from the O-derived oz2,
oz3 neurons, which may act as guidepost cells, similar to those found in the
embryonic appendages of insects (Bate,
1976
; Keshishian and Bentley,
1983a
; Keshishian and Bentley,
1983b
).
In summary, we propose that the posterior migration of PD is required for normal formation of the PP nerve root, and this migration is dependent upon N-derived progeny. In the absence of the N lineage (specifically, one or more cells within the nf.a clone), the O lineage-derived PD neuron fails to migrate posteriorly. Its peripheral process exits the ganglion abnormally via the ipsilateral MA nerve as a result and other neurons that would normally project out the PP nerve also adopt this route, leading to coalescence of the PP and MA nerves. In the absence of the O lineage-derived PD neuron, other axons, possibly from N-derived progeny (Table 1), may pioneer the PP nerve independently, but only after a developmental delay (see Fig. 3C).
This model not only explains the dual requirement for N and O lineage
derivatives in forming the normal PP nerve, but also leads us to predict
segment-specific differences in the development of the PP nerve. The basis for
this prediction lies in the fact that in the O teloblast lineage, only one
blast cell (rather than two as in the N lineage) is required to generate a
segment's worth of definitive progeny
(Zackson, 1984;
Weisblat and Shankland, 1985
).
As the rate of blast cell production is about the same in all lineages, this
means that there is segment-specific discrepancy in the clonal ages of
consegmental N versus O lineage derivatives. In anterior segments, N-derived
and O-derived progeny arise from blast cells born within a few hours of one
another, whereas in posterior segments, O-derived progeny arise from blast
cells born more than a day before the n blast cells whose progeny occupy the
same segment. That is, o blast cell clones for the posterior segments are much
older than the consegmental n blast cell clones. As far as we know, cell
division patterns and the expression of developmental regulators such as the
leech engrailed-class gene appear to proceed autonomously within the
different blast cell clones (Lans et al.,
1993
; Seaver and Shankland,
2001
). If so, we anticipate that, in posterior segments relative
to anterior segments, N-derived signals required to trigger the rearward
migration of the PD neuron arise much later in the terms of the age
of the O clone from which the PD neuron descends, suggesting that
PD migration and subsequent formation of the PP nerve is delayed in
posterior segments.
Finally, we note that cells in the ventrolateral stripe of N-derived
progeny that appear to interact directly with the O-derived, PD
neuron are those cells which express the leech engrailed-class gene
in the early N lineage (Wedeen and
Weisblat, 1991; Lans et al.,
1993
). In Drosophila, engrailed is expressed in stripes
of cells that specify the posterior compartment of each segment
(Kornberg, 1981a
;
Kornberg, 1981b
;
Lawrence et al., 1999a
). By
acting upstream of the hedgehog protein, which is secreted by
engrailed-expressing cells in the posterior compartment
(Lee et al., 1992
),
engrailed influences the affinities and positioning of cells in the
anterior compartment of Drosophila
(Lawrence et al., 1999b
). By
analogy, one could imagine that cells expressing the engrailed-class
gene in the anterior, ventrolateral stripe of leech may influence local
cell-cell affinities that affect the migration of PD, and thus the
position of the posterior nerve root. Whether the expression of the
engrailed-class protein is required for the migration of PD remains
to be determined, but these cells do not seem to express the only
hedgehog-class gene that has been identified in leech
(Kang et al., 2003
).
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ACKNOWLEDGMENTS |
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