THE JOURNAL OF COMPARATIVE NEUROLOGY 397:394–402 (1998)
Development and Pathway Formation of
Peripheral Neurons During Leech
Embryogenesis
YUEQIAO HUANG,1 JOHN JELLIES,2 KRISTEN M. JOHANSEN,1
AND JØRGEN JOHANSEN1*
1Department of Zoology and Genetics, Iowa State University, Ames, Iowa 50011
2Department of Biological Sciences, Western Michigan University,
Kalamazoo, Michigan 49008
ABSTRACT
By labeling the germinal plates of staged leech embryos with monoclonal antibodies to the
immunoglobulin superfamily member Tractin, we have documented the distribution and
initial development of peripheral neurons in a hirudinid leech. We find, in addition to sensillar
and extrasensillar sensory neurons, that there are 21 identifiable peripheral neurons in each
hemisegment. These neurons are found in highly stereotyped positions, and all but two of
them are associated with the segmental nerves. We show that eight of the peripheral neurons
have the characteristic morphology of stretch receptor neurons and that they form a
circumferentially distributed grid aligned in such a way that each of five specialized
longitudinal muscle fascicles are monitored by at least two stretch receptor cells covering
ventral, lateral, and dorsal regions of the body wall. Furthermore, we show that, in contrast to
the dorsal posterior nerve, which is pioneered by central projections, the pathways of the three
remaining segmental nerves are likely to be pioneered or guided by peripheral neurons.
J. Comp. Neurol. 397:394–402, 1998. r 1998 Wiley-Liss, Inc.
Indexing terms: Hirudo; axon; monoclonal antibody; Tractin; navigation
The leech central nervous system (CNS) consists of a
fixed number of 32 ganglia: 21 similar midbody ganglia, 4
ganglia fused into a head brain, and 7 ganglia fused into a
tail brain, and a supraesophageal ganglion (Muller et al.,
1981). Each midbody ganglion contains about 400 neurons, which are relatively large, some being up to 100 µm
in diameter (Macagno, 1980). For these reasons leech CNS
neurons have been well mapped and characterized (Muller
et al., 1981). In contrast, little is known about the number
and distribution of neurons constituting the peripheral
nervous system (PNS). Using antibodies to Tractin, a novel
immunoglobulin superfamily member recently cloned in
leech (Huang et al., 1997), we provide here a detailed map
of the localization and initial development of peripheral
neurons in a hirudinid leech. Tractin is expressed on the
soma and processes of both peripheral and central neurons
as they differentiate and therefore serves as an ideal
marker for neuron development and process outgrowth.
By labeling the germinal plate of staged leech embryos
with this antibody, we show that early in development, in
addition to the seven groups of sensillar sensory neurons
that have been previously characterized (Johansen et al.,
1992; Jellies et al., 1994; Jellies and Johansen, 1995), 21
r 1998 WILEY-LISS, INC.
identifiable peripheral neurons are present in each
hemisegment. All but two of these neurons are aligned
with the major nerves, and since their differentiation
precedes extension of axons from the CNS, they are
candidates to serve as guides for the formation of common
nerve pathways.
MATERIALS AND METHODS
Experimental preparations
For these experiments embryos of the hirudinid leech
species Hirudo medicinalis were used. Breeding, maintenance, and staging of Hirudo embryos at 22–25°C were as
previously described (Fernández and Stent, 1982; Jellies
Grant sponsor: National Institutes of Health; Grant number: NS 28857;
Grant sponsor: National Science Foundation; Grant number: 9724064;
Grant sponsor: Hatch Act; Grant sponsor: State of Iowa.
*Correspondence to: Jørgen Johansen, Department of Zoology and Genetics, 3156 Molecular Biology Building, Iowa State University, Ames, IA
50011. E-mail: jorgen@iastate.edu
Received 6 February 1998; Revised 27 April 1998; Accepted 28 April 1998
PERIPHERAL NEURON DEVELOPMENT IN LEECH
et al., 1987), except that embryos were maintained in
embryo water that was made as sterile-filtered solutions of
0.0005% commercial sea salt (Instant Ocean). Embryonic
day 10 (E10) was characterized by the first sign of a tail
sucker; E30 is the termination of embryogenesis. There
are about 10–20 embryos in each cocoon, and these sibling
embryos develop synchronously. Dissections of the embryos were performed in leech saline solutions with the
following composition (in mM): 110 NaCl, 4 KCl, 2 CaCl2,
10 glucose, 10 HEPES, pH. 7.4. In some cases 8% ethanol
was added and the saline solution cooled to 4°C to inhibit
muscle contractions.
Antibodies
Two previously reported monoclonal antibodies (mAbs),
Tractin-4G5 (IgG1) (Huang et al., 1997) and an antibody to
acetylated tubulin (IgG2B) (Sigma) (Jellies et al., 1996),
were used in these studies. In addition, a new mAb,
Tractin-4F1 (IgG2A), was made to a synthetic peptide
based on sequence from the second immunoglobulin domain of Tractin (Huang et al., 1997). The peptide synthesized had the following sequence: CYNLDYEGNFHFANVMEEDHR-NH2 (QCB, Hopkinton, MA). A cysteine (in
bold) was added to the end of the peptide for coupling
purposes. The peptide was covalently coupled to keyhole
limpet hemocyanin (Pierce, Rockford, IL) carrier protein
with sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate (Pierce) per the manufacturer’s instructions. For mAb production, Balb C mice were injected with
50 µg of the keyhole limpet hemocyanin-coupled peptide at
21-day intervals. After the third boost, spleen cells of the
mice were fused with Sp2 myeloma cells, and a monospecific hybridoma line was established using standard procedures (Harlow and Lane, 1988). Tractin-4F1 ascites was
obtained by injecting four mice intraperitoneally with
antibody-producing hybridoma cells. All procedures for
mAb production were performed by the Iowa State University Hybridoma Facility and were approved by the University’s animal care committee in accordance with National
Institutes of Health (NIH) guidelines.
Immunohistochemistry
The results of this paper are based on the immunocytochemical labeling of more than 100 individual embryos,
each of which has multiple segments in different sequential stages of development. Dissected Hirudo embryos
were fixed overnight at 4°C in 4% paraformaldehyde in 0.1
M phosphate buffer, pH 7.4. The fixed embryos were
incubated overnight at room temperature with diluted
antibody (acetylated tubulin mAb, 1:1,000; Tractin-4G5 or
4F1 mAb ascites 1:1,000) in phosphate-buffered saline
(PBS) containing 1% Triton X-100, 10% normal goat
serum, 0.001% sodium azide, washed in PBS with 0.4%
Triton X-100, and incubated with horseradish peroxidase
(HRP)-conjugated goat anti-mouse antibody (Bio-Rad, Hercules, CA; 1:200 dilution). After washing in PBS, the
HRP-conjugated antibody complex was visualized by reaction in 3,38 diaminobenzidine tetrahydrochloride (DAB;
0.03%) and H2O2 (0.01%) for 10 minutes. The final preparations were dehydrated in alcohol, cleared in xylene, and
embedded as whole-mounts in Depex mountant. Doublelabeled preparations were obtained by a subsequent incubation in the other primary antibody and by using fluorescently conjugated subtype-specific secondary antibodies. A
395
rabbit anti-mouse IgG Texas Red-conjugated secondary
antibody (Cappel, Malvern, PA) was used for Tractin-4G5
or 4F1 and a rabbit anti-mouse IgG2B fluorescein isothiocyanate (FITC)-conjugated secondary antibody (Cappel) for
the acetylated tubulin antibody. Fluorescently labeled
preparations were mounted in glycerol with 5% n-propyl
gallate. The labeled preparations were photographed on a
Zeiss Axioskop under brightfield illumination except for
the panels in Figure 3, for which Nomarski optics were
used. The color positives were digitized using Adobe Photoshop and a Nikon Coolscan slide scanner. In Photoshop the
images were converted to black and white and image
processed before being imported into Freehand (Macromedia) for composition and labeling.
RESULTS
Figure 1 shows a hemisegment of an E11 Hirudo embryo
labeled with the Tractin-4G5 mAb. The labeling demonstrated the entire distribution of peripheral neurons,
many of which have not been previously described, that
were associated with the four major nerves (AA, anterioranterior; MA, medial-anterior; DP, dorsal-posterior; PP,
posterior-posterior) and the seven groups of sensillar
sensory neurons, as well as the two peripheral neurons,
the nephridial nerve cell (NNC), and the third stretch
receptor neuron (HO3), that were not directly aligned with
the segmental nerves. The largest group of peripheral
neurons was located within the sensilla, which consists of
clusters of mixed sensory cells composed of chemoreceptors, photoreceptors, and mechanoreceptors found on the
central annulus of each segment (Muller et al., 1981;
Phillips and Friesen, 1982; Johansen et al., 1994). The
sensilla are termed S1–S7, with the most ventral sensillum designated S1. S1–S5 sensillar neurons send their
axons toward the CNS through the MA nerve, whereas the
S6 and S7 neurons extend their axons through the DP
nerve (Fig. 1).
The second most prominent group of peripheral neurons
labeled were the HO cells (also affectionately known as
‘‘Hoover’’ cells because of their resemblance to the vacuum
cleaner) (Fig. 2), which have been shown to function as
stretch receptors in the body wall (Blackshaw et al., 1982;
Blackshaw and Thompson, 1988; Blackshaw, 1993). In
adult leeches three HO cells have been described per
hemisegment that are situated within the sheath of the
segmental nerves in the region of the ventral body wall
(Blackshaw and Thompson, 1988). They have a characteristic morphology with two flat, fan-shaped dendrites proximal and distal to the cell body, and they extend the largest
diameter axons found in the leech along defined pathways
into the CNS. The fan-shaped dendrites are in close
association with longitudinal muscle fibers, which insert
through the nerve sheath and contact the surfaces of the
flattened dendrites (Blackshaw, 1993). However, from our
developmental studies, it became clear that in addition to
the three ventrally situated HO cells five more peripheral
neurons per hemisegment exhibited the distinctive morphology of HO cells for a total of eight, which we have
designated HO1–8 (Figs. 1, 2). Labeling with Tractin-4G5
antibody also revealed that there are two types of HO cells.
One type displays both a proximal and a distal fan-shaped
dendrite (HO1, HO3, and HO6), and a second type appeared to possess only the proximal dendritic fan (HO2,
396
Fig. 1. Localization of peripheral nerve cells in the 5th segment of
an E13 Hirudo embryo. The figure shows one hemisegment of the
germinal plate labeled with Tractin-4G5 antibody. There are four
major segmental nerves (AA, anterior-anterior; MA, medial-anterior;
DP, dorsal-posterior; PP, posterior-posterior) emanating from the
central ganglion (G). The seven groups of sensory neurons constituting
the sensilla are labeled S1–S7. A group of seven cells form the anterior
root ganglion (ARG), which is located at the future junction between
the AA and MA nerves. Among functionally identifiable neurons are
the eight stretch receptor neurons (HO1–8) and the nephridial nerve
cell (NNC). In addition to these cells the labeling reveals the location of
a number of neurons associated with the segmental nerves: the
anterior nerve cell (ANC), the lollipop cell (LPC), the medial anterior
nerve cell (MANC), and the two posterior nerve cells (PNC1 and 2).
Anterior is to the left and dorsal is up. Scale bar 5 35 µm.
HO4–5, and HO7–8) (Fig. 2B). All the HO cells, except
HO3, were associated with either the AA nerve (HO1,
HO4, and HO6) or the PP nerve (HO2, HO5, HO7, and
HO8) and were arranged in vertical parallel rows. This is
illustrated in Figure 2, in which an E14 embryo has been
labeled with a mAb to acetylated tubulin (ACT). At late
embryonic stages the ACT mAb labels the cell body of some
Y. HUANG ET AL.
peripheral neurons including the HO cells in addition to
central nerve projections and a specific subset of five
longitudinal muscle fascicles (Jellies et al., 1996). By
observing the preparations at high magnification using
Nomarski optics, we ascertained that the latter corresponded to the longitudinal muscle fascicles (LMF1–5),
which penetrated the nerve sheaths and interacted with
the dendritic fans of the HO cells (Fig. 2). In this arrangement each longitudinal muscle fascicle was monitored by
at least two spatially separated HO cells per hemisegment
covering ventral, lateral, and dorsal regions of the body
wall. Since there were only three HO cells along the AA
nerve and the HO6 cell body was located at the level of
LMF4, HO6 extended a long distal dendrite, which ended
in a club-like fan into which LMF5 inserted (Fig. 3A). In
the PP nerve, LMF5 was contacted by the dendritic fan of
HO8, which, although localized in the PP nerve, sends its
axon to the CNS through the DP nerve (Fig. 2B).
In addition to the sensillar neurons and the putative
stretch receptors, labeling with the Tractin-4G5 mAb
revealed a number of neurons associated with the segmental nerves, which are of unknown function. Along the AA
nerve there were two such cells, one, the anterior nerve
cell (ANC) situated close to the HO4 stretch receptor
neuron, and the other, which we have named the lollipop
cell (LPC) based on its appearance in Lucifer yellow fills
(Fig. 4B in Jellies et al., 1996), located close to the HO1 cell
(Fig. 1). A small ganglion named the anterior root ganglion
(ARG; Rude, 1969; Lent et al., 1993; Johansen and Johansen, 1995) was located just distal to the future junction
between the AA and MA nerve (Jellies et al., 1996).
Nomarski images of Tractin-4G5 antibody labeling showed
that this ganglion was made up of a cluster of seven cells
(Fig. 3B), one of which has been demonstrated to be
dopaminergic (Lent et al., 1984). Furthermore, along the
MA nerve between the position of the S2 and S3 sensilla
was located a hitherto undocumented cell that we have
designated the medial anterior nerve cell (MANC; Figs. 1,
2B). No peripheral neurons appeared to be situated along
the DP nerve; however, two such neurons, the posterior
nerve cells 1 and 2 (PNC1 and 2) were clustered together
with the HO2 stretch receptor at the ventral root of the PP
nerve. Only two peripheral neurons (Fig. 1) were not
associated with the segmental nerves or the sensilla at this
embryonic stage: one was the third ventrally situated
stretch receptor neuron, HO3, and the other was the NNC
cell, which innervates the nephridium and bladder (Wenning, 1983). An interesting aspect of leech development is
the finding that numerous extrasensillar sensory neurons
(xsn), as defined by the Lan3-2 antibody, start to differentiate relatively late in development at E16 and then continue to increase in number throughout the life span of the
leech (Peinado et al., 1990; Johansen et al., 1992; Gascoigne and McVean, 1993). These neurons were also labeled by the Tractin-4G5 mAb and could be observed to
form small clusters of sensory neurons aligned in vertical
rows at the middle of each of the five segmental annuli
(Fig. 3C). These late differentiating sensory neurons use
CNS efferents as guides for the extension of their axons to
the CNS (Jellies et al., 1995, 1996).
In the leech the formation of both the central and
peripheral nervous systems proceeds in a rostrocaudal
sequence with each posterior segment approximately 2.5
PERIPHERAL NEURON DEVELOPMENT IN LEECH
397
Fig. 2. Distribution of stretch receptor neurons associated with
specific longitudinal muscle fascicles. A: Hemisegment of an E14
embryo labeled with an antibody to acetylated tubulin. The antibody
labels an epitope expressed by all centrally located neurons, by five
discrete longitudinal muscle fascicles (double-headed arrows), as well
as by a subset of peripheral neurons that include the HO cells. Single
arrows indicate the position of the soma of the eight stretch receptor
neurons. B: Schematized diagram of the labeling in A, showing the
spatial relationship between the stretch receptor neurons (HO1–8),
the segmental nerves (AA, anterior-anterior; MA, medial-anterior; DP,
dorsal-posterior; PP, posterior-posterior) and the longitudinal muscle
fascicles (LMF1–5) associated with the HO cells. There are two types
of stretch receptor neurons: one type with both a proximal and a distal
fan-shaped dendrite (HO1, HO3, and HO6) and a type with only the
proximal dendritic fan (HO2, HO4–5, and HO7–8). Anterior is to the
left and dorsal is up. Scale bar 5 75 µm.
hours later in development than the more anterior one
(Jellies and Kristan, 1991). Consequently, since there are
32 segments, an embryo exhibits segments in different
stages of development spanning a period of about 2–3
days, which greatly facilitates analysis of neuronal differentiation (Johansen et al., 1994). Figure 4 shows the entire
germinal plate of an E8 embryo labeled with Tractin-4F1
antibody allowing the relative temporal developmental
sequence of the CNS and PNS to be followed.
The first neurons to differentiate as revealed by the
Tractin-4F1 antibody were within the CNS, as indicated
by the arrows in the most posterior segments of the
embryo in Figure 4. However, within the next 10–15 hours
of development, groups of antibody-positive neurons were
also detectable in the periphery. The progression of their
development from three different hemisegments spaced
9–15 hours apart and indicated by A, B, and C (Fig. 4) is
shown at higher magnification in Figure 5. What appears
to be the primordia for the three most dorsal peripheral
neurons (HO4, HO6, ANC) associated with the AA nerve,
the S3 sensillum, and the three ventrally situated cells
(PNC1 and 2, HO2) were the first to be antibody positive in
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Y. HUANG ET AL.
Fig. 3. Tractin-4G5 antibody labeling of peripheral neurons. A: The
HO6 stretch receptor neuron extends a long distal dendrite (arrowheads), which ends in a club-like fan into which LMF5 inserts (top
arrow). The proximal dendrite interacts with LMF4 (bottom arrow).
xsn indicates a group of the late differentiating extrasensillar sensory
neurons. B: The seven neurons constituting the anterior root ganglion
(ARG) and the medial anterior nerve cell (MANC). C: Extrasensillar
sensory neurons (arrowheads) form small clusters aligned in vertical
rows at the middle of each of the five segmental annuli. The images in
all three panels were obtained using Nomarski optics. Scale bar 5 50
µm in A,C, 20 µm in B.
the periphery (Fig. 5A). They appeared as irregularly
shaped cells from which numerous fine filopodia extended.
Our stainings do not have a resolution that allowed us to
identify or follow the differentiation of the individual
neurons within these groups. However, by following the
progression of the labeling segment by segment, it was
possible to determine which progeny was derived from
which group of primordia.
The second group of peripheral neurons to differentiate,
as illustrated in Figure 5B, comprised the ones giving rise
to the S6 and S5 sensilla, the HO1 and LPC cells, and the
HO7 and HO8 cells. At this time axons from the peripheral
neurons destined to be situated along the future AA, MA,
and PP nerves have reached the CNS (arrowheads, Fig.
5B) whereas the first axons projecting from the CNS are
not extended until several hours later. We have previously
demonstrated by labeling with the ACT antibody and by
Lucifer Yellow dye fills that in Hirudo the first axons to
emerge from the CNS are those of the dorsal P-cell that
pioneer the DP nerve (Jellies et al., 1996). In the E8
embryo of Figure 4 this axon was only detectable in the
most anterior ganglia, as shown in Figure 5C. Additionally,
these findings were confirmed by fluorescent double labeling with Tractin and ACT antibody of the same embryos.
Thus, in contrast to the DP nerve, formation of the three
other segmental nerves (AA, MA, and PP) is pioneered or
guided by peripheral neurons. When the segments reach
the developmental stage illustrated in Figure 5C, the
primordia for all the peripheral neurons depicted in Figure
1 have become Tractin antibody positive, including the
NNC, ARG, and HO3 cells. As the germinal plate expands,
the individual neurons separate and migrate to their final
positions. For example, the NNC neuron differentiated
among the group forming the ARG (Fig. 5C) before migrating away from these neurons to its eventual position just
anterior to the AA nerve at the base of the nephridium, and
the MANC cell emerged among the S3 sensillar neurons
before migrating to its final position on the MA nerve
midway between the S3 and S2 sensilla. At the same time
projections from the CNS grow out along the peripheral
neurons and their axons, with the latter determining the
course of the common segmental nerve pathways (Fig. 6),
and the mature pattern of the PNS is gradually established (Fig. 1).
PERIPHERAL NEURON DEVELOPMENT IN LEECH
399
DISCUSSION
In this study we have used antibodies to the immunoglobulin superfamily member Tractin to document the
distribution and development of peripheral neurons in a
hirudinid leech. We find that in addition to the sensillar
and extrasensillar sensory neurons, there are 21 identifiable peripheral neurons present in each hemisegment in
highly stereotyped positions and that all but 2 of these
neurons are associated with the segmental nerves.
The most prominent of these cells are the eight putative
stretch receptor neurons. That some of them function as
stretch receptor neurons has been demonstrated by recording intracellularly from the axon while stretching the
region of body wall muscle associated with the flattened
dendrites (Blackshaw, 1993). Interestingly, the soma is
spiking whereas the axon is not. Consequently action
potentials are not propagated actively but rather electronically along the large-diameter axons, which have very
large length constants providing for high-speed conduction
to synapses in the CNS (Blackshaw, 1993). The receptors
hyperpolarize during stretch but have a marked excitatory
response to release from stretch (Blackshaw and Thompson, 1988). In skeletal muscles of vertebrates and some
invertebrates, stretch receptor neurons are associated
with discrete receptor muscles (intrafusal fibers) involved
in mediating the stretch response. Our identification of
five longitudinal muscle fibers that are in contact with HO
neurons and that, in contrast to any other muscle type,
express acetylated tubulin suggests that these muscle
fibers may serve a similar specialized function associated
with the stretch response in leech. Moreover, the eight HO
cells form a circumferentially distributed grid in such a
way that each of the five specialized longitudinal muscle
fibers are monitored by at least two different HO cells. This
arrangement is ideally suited to provide rapid spatial
information regarding stretch from the entire body wall.
We have previously shown that in hirudinid leeches the
MA nerve is pioneered by peripheral neurons from the S3
sensillum, whereas the DP nerve is pioneered by the
centrally situated PD-neuron (Jellies et al., 1994, 1996). In
this study we have extended these observations and show
that like the MA nerve, the course of the AA and PP nerves
is also pioneered by peripheral neurons, leaving only the
DP nerve to be established by the CNS. Similar conclusions that peripheral neurons play a major role in segmental nerve formation have been reached in the glossiphoniid
leech Helobdella, in which the contributions of different
parts of the nervous system were followed using fluorescent cell lineage tracers injected into their precursor
teloblasts (Braun and Stent, 1989a,b). These studies
showed that several peripheral neurons in Helobdella
differentiate in a position to guide outgrowth of ganglionic
neurons into the segmental nerves and that ablation of
Fig. 4. Tractin-4F1 antibody labeling of the entire germinal plate
from an E8 embryo showing the formation of the central nervous
system (CNS) and peripheral nervous system (PNS). The development
proceeds in a clear rostrocaudal gradient exhibiting segments in
different stages of development. The arrows point to the first group of
CNS neurons to differentiate. The boxed areas A, B, and C indicate
three hemisegments in different stages of development shown at
higher magnification in Figure 5. The supraesophageal ganglion and
the head ganglia are at the top and the tail ganglia at the bottom of the
figure. Scale bar 5 125 µm.
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Y. HUANG ET AL.
Fig. 5. Developmental progression of differentiation of peripheral
neurons in three different segments of an E8 embryo. Each panel
corresponds to the areas indicated as A, B, and C in Figure 4. The
approximate boundary of differentiated neurons in the central ganglia
(G) are indicated by the stippled line. Anterior is to the left and dorsal
is up in these panels. A: At this developmental stage the first three
groups of peripheral neurons have become Tractin-4F1 antibody
positive. These groups give rise to the HO4, HO6, and ANC cells; the
S3 sensillar neurons; and the HO2 and PNC1 and 2 cells, respectively.
B: This panel shows a more anterior hemisegment approximately 15
hours further in development than the one in A. At this stage
additional groups of peripheral sensory neurons have become Tractin4F1 antibody positive, including the HO1 and LPC cells; the S5 and S6
sensilla; and the HO7 and HO8 stretch receptors. Projections from
these groups of peripheral neurons have entered the ganglion (arrows), defining the future course of the anterior-anterior (AA), medialanterior (MA), and posterior-posterior (PP) segmental nerves. C: In
this composite micrograph of a hemisegment 9 hours further in
development than the one in B, groups of neurons giving rise to all the
peripheral neurons associated with the segmental nerves as well as
the nephridal nerve cell (NNC) and HO3 cells are labeled by the
Tractin-4F1 antibody. In addition, the first peripheral projection of the
central neuron pioneering the dorsal-posterior (DP) nerve is clearly
identifiable. The panels are from actual labelings but do not represent
a true photographic record since the relative contrast of some parts of
the images has been artificially enhanced for increased clarity during
image processing. Scale bar 5 20 µm.
some of these peripheral neurons perturb segmental nerve
formation (Braun and Stent, 1989a,b). In glossiphoniid
leeches, peripheral neurons are derived from four different
kinship groups formed by the n, o, p, and q bandlets of
precursor cells (Braun and Stent, 1989a). During formation of the germinal plate, the precursors often migrate
over considerable distances before reaching the destinations, where they differentiate into neurons. Although
lineage studies of this kind have not been performed in
hirudinid leeches, similar morphogenetic processes are
likely to be involved in generating the PNS. Thus, when
the peripheral neurons in Hirudo differentiate and become
Tractin antibody positive, their position may reflect the
previous migration of undifferentiated precursor cells.
After the differentiation of precursor cells into neurons,
morphogenetic movement continues to play a major role in
the development of the PNS. For example, as the germinal
plate expands, what had first differentiated as closely
apposed groups of neurons separate and migrate to their
final destinations, whereas parts of the segmental nerve
pathways that initially formed as discrete tracts merge by
a secondary condensation (Jellies et al., 1996). However, at
the time these morphogenetic processes occur, the main
features of the trajectories of the major common nerve
pathways between the CNS and PNS have already been
established.
Of the 21 peripheral neurons described here, only 9 can
so far be functionally identified (HO1–8 and the NNC).
Thus, the nature of the remaining cells is not known.
However, since the peripheral neurons in leech are amenable to physiological (Blackshaw and Thompson, 1988;
Wenning et al., 1993) as well as morphological studies
PERIPHERAL NEURON DEVELOPMENT IN LEECH
401
Fig. 6. The anterior germinal plate from an E9 embryo labeled with Tractin-4G5 antibody. The
location of the ganglion (G) and the four common segmental nerve pathways (AA, anterior-anterior; MA,
medial-anterior; DP, dorsal-posterior; PP, posterior-posterior), which at this stage are made up of both
central and peripheral axons are indicated for one of the hemisegments. Scale bar 5 135 µm.
using dye injections (Jellies et al., 1996), the complete map
of their distribution and localization provided here should
greatly facilitate their further functional analysis.
sponses, receptive fields and terminal arborizations of nociceptive cells
in the leech. J. Physiol. 326:251–260.
Braun, J. and G.S. Stent (1989a) Axon outgrowth along segmental nerves in
the leech. I. Identification of candidate guidance cells. Dev. Biol.
132:471–485.
ACKNOWLEDGMENTS
Braun, J. and G.S. Stent (1989b) Axon outgrowth along segmental nerves in
the leech. II. Identification of actual guidance cells. Dev. Biol. 132:486–
501.
We thank Dr. Paul Kapke at the Iowa State University
Hybridoma Facility for expert technical assistance and
help with generating and maintaining the monoclonal
antibody lines. This work was supported by NIH grant NS
28857 (JJo) and by NSF grant 9724064 (JJe). This is
Journal Paper No. J-17793 of the Iowa Agriculture and
Home Economics Experiment Station, Ames, Iowa, Project
No. 3371, supported by the Hatch Act and State of Iowa
funds.
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