Cell Tissue Res (1996) 286:357–364
© Springer-Verlag 1996
030112.954 030112.391 091313.625 140521.416 140518.836 140518.912 040522.666
080105*500 130103*172
Differential expression of the EF-hand calcium-binding protein calsensin
in the central nervous system of hirudinid leeches
Mark Veldman1, Yueqiao Huang1, John Jellies2, Kristen M. Johansen1, Jørgen Johansen1
1
2
Department of Zoology and Genetics, 3156 Molecular Biology Building, Iowa State University, Ames, IA 50011, USA
Department of Biological Sciences, Western Michigan University, Kalamazoo, MI 49008, USA
&misc:Received: 19 January 1996 / Accepted: 15 May 1996
&p.1:Abstract. By immunocytochemistry the distribution and
developmental expression of the small EF-hand calciumbinding protein calsensin in the peripheral (PNS) and
central nervous system (CNS) of the three hirudinid leech
species Haemopis, Hirudo, and Macrobdella was compared. Labeling with calsensin-specific antibodies demonstrated that there was a pronounced difference in the
distribution of calsensin immunoreactivity in the CNS of
these leeches. In Haemopis more than 70 neurons were
labeled, whereas the number in Hirudo was 51 and in
Macrobdella only 8. Furthermore, the expression of calsensin in identified cells common to all three leech species also differed. Immunoblot analysis indicated that this
variability was not likely to be due to multiple proteins or
isoforms being recognized by the calsensin antibody. Labeling of embryos in various stages of development
shows that the ontogeny of calsensin expression in the
CNS is a gradual process with some neurons expressing
calsensin immediately after completion of neurogenesis,
about one-third of the way through embryogenesis, and
others expressing calsensin only postembryonically. In
contrast to the variability in the pattern and temporal expression by CNS neurons, the early embryonic calsensin
expression in a small subgroup of sensillar PNS neurons
was a shared feature by all three leech species. These
findings suggest that calsensin may have different functional properties in CNS and PNS neurons.
&kwd:Key words: Calsensin – Calcium-binding protein – Immunocytochemistry – Neurons – Leeches, Hirudo medicinalis, Haemopis marmorata, Macrobdella decora
(Annelida)
This work was supported by NIH grant NS 28857 (J.Jo.), by NSF
grant 9696018 (J.Je.), and an NSF training grant DIR 9113595 undergraduate fellowship (M.V.). J. Jellies is a Fellow of the Alfred
P. Sloan Foundation. This is journal paper no. J-16747 of Iowa
Agriculture and Home Economics Experiment Station, Ames, Iowa, project no. 3371, and supported by Hatch Act and State of Iowa funds.
Correspondence to: J. Johansen (Tel.: (515) 294–2358; Fax: (515)
294–0345; E-mail: jorgen@iastate.edu)&/fn-block:
Introduction
Regulation of intracellular calcium plays a key role in
signal transduction events governing many biological
processes. Thus, proteins which either modulate or mediate the actions of calcium ions are important for cell
function. A group of proteins with these properties are
the family of calcium-binding proteins that contain EFhand domains (Kretsinger 1980; Persechini et al. 1989).
The members of this family, which includes calmodulin
and troponin C, are characterized by containing a common calcium-binding motif composed of a loop of 12
contiguous residues with oxygen atoms involved in calcium-binding and two flanking α-helices that stabilize
the complex (Kretsinger 1980; Persechini et al. 1989).
Many EF-hand calcium-binding proteins are found exclusively in the nervous system where they often have a
restricted expression to certain tissues and types of neurons (Baimbridge et al. 1992). The function of many of
the nervous system-specific calcium-binding proteins is
unknown; however, they have proven to be valuable
markers of neuronal subpopulations for anatomical and
developmental studies (Baimbridge et al. 1992).
The leech nervous system is an accessible preparation
for such anatomical studies. Each of the 21 iterated segmental ganglia contains only approximately 400 neurons
(Macagno 1980), many of which are identified and have
known physiological roles (Muller et al. 1981). In addition, the peripheral nervous system (PNS) is well characterized, and both PNS and central nervous system
(CNS) development can be analyzed embryologically by
a variety of techniques (Jellies and Johansen 1995). The
formation of the nervous system proceeds in a rostrocaudal sequence, generally, with each posterior segment being 2.5 h later in development than its immediate anterior segment (Stent et al. 1982). Consequently, since there
are 32 segments, each embryo exhibits ganglia in different stages of development spanning a period of about
2–3 days (Johansen et al. 1992). Neurogenesis appears
to be completed at about embryonic day 10, when the
number of neurons per ganglion is at a peak, and is fol-
358
Fig. 1A–C. Labeling of peripheral sensillar sensory neurons by
lan3-6 antibody in three species of leeches. A The labeling of
lan3-6 in two segments of the germinal plate of an E11 Haemopis
embryo. B The labeling by lan3-6 in three segments of an E10 Hirudo embryo. C The labeling of lan3-6 in three segments of an
E12 Macrobdella embryo. In all three panels some of the seven
groups of sensillar neurons S1-S7 have been indicated by arrows.
CNS, The location of the ganglionic chain. Bars: 60 µm (A),
150 µm (B), 200 µm (C)&ig.c:/f
lowed by a period of cell death, reducing the number of
neurons to the adult pattern and distribution (Stewart et
al. 1986).
Calsensin, a novel 9-kDa calcium-binding protein,
which is nervous system-specific and which contains
two helix-loop-helix calcium-binding domains, was
cloned and characterized in the leech by Briggs et al.
(1995). Although calsensin is also found in the CNS
(Zipser and McKay 1981; Zipser 1982; Macagno et al.
1983), recent studies have mostly focused on the expression of calsensin in the growth cones and axons of a
small subset of peripheral sensory neurons that fasciculate in a single axon tract (Briggs et al. 1993, 1995). In
the present paper using mono- and polyclonal antibodies, we compare the distribution and developmental expression of calsensin in the PNS and CNS in three species of hirudinid leeches. We show that while the expression of calsensin in a subset of peripheral sensory
neurons is a feature shared by all three leech species,
the pattern and temporal expression of calsensin by
CNS neurons is highly variable among the different species.
morata, and Macrobdella decora. The leeches were either captured in the wild or purchased from commercial sources.
Materials and methods
Leech species
For the present experiments we used three different leech species,
namely the hirudinid leeches Hirudo medicinalis, Haemopis mar-
Dissections
Dissections of nervous tissue and embryos were performed in
leech saline solutions with the following composition: 110 mM
NaCl, 4 mM KCl, 2 mM CaCl2, 10 mM glucose, 10 mM HEPES,
pH 7.4. In some cases 8% ethanol was added and the saline solution cooled to 4°C to inhibit muscle contractions. Although the
ganglia of the leech nerve cord are very similar, there are some
segmental variations and specializations. For example, the sex
ganglia (M5 and M6) have many more neurons than other ganglia
(Macagno 1980), and some of the iterated neurons have specialized properties in these ganglia (Johansen et al. 1984; French and
Kristan 1992). For this reason the present analysis of CNS calsensin expression was limited to the largely similar midbody ganglia
in the region from M8–M18. The results of this study are based on
the labeling and examination of several hundred adult ganglia as
well as embryos.
Embryos
Macrobdella and Haemopis embryos were obtained from leeches
captured gravid in the wild, whereas Hirudo embryos were obtained from a laboratory breeding colony (Jellies et al. 1987). The
gravid leeches were placed in boxes with moist peat moss in which
they lay their cocoons. Cocoons were maintained at 24°C and embryos were staged according to the criteria described by Fernandez
and Stent (1982). There were about 10–20 embryos in each cocoon
and these sibling embryos developed almost synchronously.
359
Fig. 2A–F. Differential calsensin expression in the CNS on the
ventral and dorsal surface, respectively, of Haemopis (A, D), Hirudo (B, E), and Macrobdella (C, F) ganglia. The preparations
were labeled by lan3-6 antibody. R, Retzius cell; N, lateral nociceptive cell; PV, ventral pressure cell; PD, dorsal pressure cell; AP,
anterior pagoda cell. Arrows in E point to lighter-labeled neurons
situated on the edge of the ventral surface that are not part of the
dorsal pattern. The panels are from actual labelings but do not represent a true photographic record since the relative contrast of
some of the labeled neurons in A, B, D, and E have been artificially enhanced for increased clarity during image processing.
Bar: 100 µm&ig.c:/f
Immunocytochemistry
peroxidase (HRP)-conjugated goat anti-mouse or anti-rabbit antibody (Bio-Rad; 1:200 dilution). After a wash in PBS, the HRPconjugated antibody complex was visualized by reaction in 3,3′
diaminobenzidine (DAB; 0.03%) and H2O2 (0.01%) for 10 min.
The final preparations were dehydrated in alcohol, cleared in xylene, and embedded as whole-mounts in Depex mountant. Adult
ganglia were fixed in 4% paraformaldehyde, the connective capsules on either the dorsal or ventral side opened with fine forceps,
and the ganglia xylene extracted for better antibody penetration
(Zipser and McKay 1981) before they were processed for antibody labeling in a similar way to the embryos. The labeled preparations were photographed on a Zeiss Axioskop using Ektachrome 64T film. 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.
Two calsensin-specific antibodies, the mAb lan3-6, which is of
the IgG2a subtype (Zipser and McKay 1981; Briggs et al. 1993),
and a polyclonal rabbit antiserum (Frigg) raised against a calsensin fusion protein (Briggs et al. 1995), were used in these studies.
Both of these probes showed identical staining patterns in the
preparations where they were compared (Briggs et al. 1995);
however, the mAb lan3-6 has lower background staining and consequently was the antibody of choice for the immunocytochemical analysis. Dissected Hirudo, Macrobdella, and Haemopis embryos were fixed overnight at 4°C in 4% paraformaldehyde in
0.1 M phosphate buffer, pH 7.4, incubated overnight at room
temperature directly in hybridoma supernatant containing 0.4%
Triton X-100 or in polyclonal antisera diluted 1:800 in phosphate-buffered saline (PBS) with 0.4% Triton X-100, washed in
PBS with 0.4% Triton X-100, and incubated with horseradish
360
SDS-PAGE and Western blotting
Since lan3-6 does not recognize the denatured calsensin protein,
the Frigg antiserum was used for immunoblot analysis (Briggs et
al. 1993, 1995). Protein from dissected nerve cords were homogenized in extraction buffer (20 mM TRIS-HCl, 200 mM NaCl,
1 mM CaCl2, 1 mM MgCl2, 0.2% NP-40, 0.2% Triton X-100,
pH 7.4) containing protease inhibitors, and the resulting homogenate was cleared by brief centrifugation. SDS-PAGE of the nerve
cord homogenates was performed according to standard procedures (Laemmli 1970) and electroblot transfer was performed as
in Towbin et al. (1979). For these experiments we used the BioRad mini-gel system, electroblotting to 0.2 µm nitrocellulose, and
using HRP-conjugated secondary antibody (1:3000) for visualization of primary rabbit calsensin antisera diluted 1:2000 in Blotto.
The signal was developed with DAB (0.1 mg/ml) and H2O2
(0.03%) and enhanced with 0.008% NiCl 2. The Western blots
were digitized using the NIH-image software, a cooled high-resolution CCD camera (Paultek), and a PixelBuffer framegrabber
(Perceptics).
Results
Calsensin expression in peripheral neurons
Among the first peripheral neurons to differentiate are
the sensillar neurons, which are clusters of sensory neurons found on the central annulus of each segment (Phillips and Friesen 1982; Johansen et al. 1992). The sensilla are termed S1-S7, with the most ventral sensillum
closest to the CNS in each hemisegment designated as
S1 (Fig. 1). Figure 1 shows a comparison of calsensin
expression in the segments of embryos of the three leech
species labeled with lan3-6. A common feature of this
labeling in Haemopis, Hirudo, and Macrobdella is that
calsensin is expressed in a subset of peripheral sensillar
sensory neurons (Fig. 1, arrows) that extend axons into
the CNS where they selectively fasciculate with each
other forming a single axon tract (Briggs et al. 1993).
However, within the resolution of the present experiments we cannot determine whether it is an identical
subset in each species. In all three species these neurons
are lan3-6 antibody-positive from their earliest differentiation and extension of axon growth cones (this study,
unpublished results; Stewart et al. 1985; Briggs et al.
1993).
Calsensin expression in CNS neurons
In contrast to the expression of calsensin in the periphery, which appears highly conserved in the hirudinid
leeches examined, the differences in the expression pattern of calsensin in the CNS between species is pronounced. This is illustrated in Fig. 2, which shows lan36 labeling of CNS neurons on the ventral and dorsal surface of representative ganglia from Haemopis, Hirudo,
and Macrobdella. Due to their size and position many of
the labeled neurons can readily be identified (Muller et
al. 1981) as indicated on the figure.
In Haemopis there are approximately 40–45 labeled
neurons on the ventral surface of the ganglia (Fig. 2A)
Fig. 3A, B. Development of calsensin expression in the CNS of
Haemopis embryos. A Two E9 segmental ganglia were labeled
with lan3-6. Very few neurons are labeled by the antibody at this
stage. B E25 ganglion labeled with lan3-6 demonstrates increasing numbers of calsensin-expressing neurons at this stage of development. Both panels were photographed using Nomarski optics. Bars: 25 µm&ig.c:/f
and 30–35 on the dorsal surface (Fig. 2D), which are arranged in a generally bilaterally symmetrical pattern.
However, although highly stereotyped, some variation in
the pattern and staining intensity of the neurons is found
from preparation to preparation preventing the determination of a precise number of labeled neurons in this
species. The variation is in part due to differences in the
quality of the dissections, the variability in location especially of smaller neurons, and the relatively large
number of neurons expressing calsensin in this species.
Furthermore, in either a ventral or dorsal dissection of an
adult ganglion the antibody does not penetrate to the
other surface except around the edges of the ganglion.
Consequently, small variations in the position of neurons
that are close to the edge of the ganglia can result in the
appearance of a variable number of neurons being labeled. This variation notwithstanding, the actual lan3-6
labelings shown in Fig. 2A,D represent a very close approximation to a consensus staining pattern of the ventral and dorsal surface of an Haemopis ganglion based
on the visual examination of several dozen well-stained
ganglia. Among the identifiable neurons prominently labeled are the dorsal (PD) and ventral (PV) pressure cells,
361
Fig. 4A–C. Labeling of Hirudo embryonic ganglia
with lan3-6 antibody. A In this segmental ganglion
from an E8 embryo only the transient bipolar cells
(arrows) are prominently labeled. B In a ganglion from
an E10 embryo the bipolar cells have degenerated and
a few dorsal-posterior neurons have begun to express
calsensin. C At E25 numerous neurons express calsensin. The panels were photographed using Nomarski optics. Bars: 30 µm (A, B), 20 µm (C)&ig.c:/f
the Retzius cells (R), and the lateral nociceptive cells (N;
Muller et al. 1981).
In Hirudo the consensus staining pattern of lan3-6 labeling of the ventral ganglionic surface consists of 14
pairs of bilaterally symmetrically situated neurons and
an unpaired neuron for a total of 29 (Fig. 2B). On the
dorsal surface, 11 cells in each hemisegment are labeled
by the calsensin antibody (Fig. 2E). Likewise, in this
species some variation in the calsensin staining pattern is
encountered due to the presence of neurons located close
to the edge of the ganglion. Nonetheless, the variability
is much less than in Haemopis since comparatively fewer neurons are labeled making their relative positions
easier to determine. Among the identifiable cells, the PV
and PD cells are prominently labeled as in Haemopis;
however, the Retzius cells are not (Fig. 2B). In this species the anterior pagoda cell (AP) and the Nut cell can
also be clearly identified as labeled by the antibody
(Fig. 2B).
In Macrobdella the full extent of calsensin CNS expression is limited to four bilaterally symmetrical, ventral cell pairs for a total of 8 cells (Fig. 2C). No dorsally
situated cells are labeled in this species (Fig. 2F). In the
adult ganglia the calsensin expression is reliably detected by the antibody in the PV, PD, and AP cells, whereas
the labeling of the fourth, unidentified cell type is variable.
Ontogeny of calsensin expression in CNS neurons
We examined the ontogeny of calsensin expression by
labeling dissected leech embryos in various stages of development. Embryonic development of hirudinid leeches
takes about 30 days from the laying of a cocoon until
hatching (Fernandez and Stent 1982; Macagno et al.
1983). Embryonic days are termed E1–E30. The first
neurons in the segmental ganglia of Haemopis embryos
become antibody-positive for calsensin at about E9–E10
(Fig. 3A). However, only a few neurons are initially labeled by the antibody. The rest of the adult pattern only
gradually emerges during the remainder of embryogenesis. Even close to hatching, at E25 (Fig. 3B), several
prominently stained cells in the adult ganglia, including
the Retzius and P cells, remain antibody negative, suggesting that they start expressing calsensin only postembryonically. This gradual temporal pattern of expression
is also observed in Hirudo embryos (Fig. 4). However, in
this species the first cells to label with the antibody at
about E8 is a pair of bipolar cells (Stewart et al. 1987).
These cells are temporary and rapidly degenerate within
days after their differentiation (Stewart et al. 1987) and
are not labeled by the antibody in either Haemopis or
Macrobdella. In Macrobdella the first cells to express
calsensin are the PV cells followed closely in time by the
PD cells (Fig. 5). However, the appearance of immunore-
362
Fig. 6. Calsensin is detected as a single 9-kDa protein band on immunoblots of CNS extracts from Haemopis, Hirudo, and Macrobdella. The CNS extracts were separated by 20% SDS-PAGE, immunoblotted, and labeled with a calsensin-specific polyclonal antiserum (Frigg). The faint high-molecular-weight bands are nonspecific and are also obtained by labeling with preimmune serum
(data not shown)&ig.c:/f
Fig. 5A, B. Labeling of pressure cells in E25 embryonic ganglia
from Macrobdella. Panels are from different segments of the same
embryo. The first neurons to express calsensin in this species are
the ventral pressure cells PV (A) followed shortly by the dorsal
pressure cells PD as indicated in a slightly anterior segment (B),
which is further along in development than the more posterior
segment (A). No other cells express calsensin during the embryonic stages of this species. The preparation was labeled with lan36 antibody and photographed using Nomarski optics. S1, The location of the first sensillum. Bar: 50 µm&ig.c:/f
activity in these cells does not occur before E25 or shortly before hatching, whereas the expression of calsensin
in the two anterolaterally situated cells only appears
postembryonically.
Immunoblot analysis of calsensin in the CNS
The variability in the calsensin staining patterns observed in the CNS of the various leech species could
conceivably be due to multiple proteins or isoforms being recognized by the calsensin antibody. To explore this
possibility we immunoblotted Haemopis, Hirudo, and
Macrobdella CNS protein extracts separated by SDSPAGE and labeled them with a calsensin-specific polyclonal antiserum (Briggs et al. 1995). The results from
such an experiment after 20% SDS-PAGE are shown in
Fig. 6. A single protein band of approximately 9 kDa is
recognized by the calsensin antiserum in each species.
No high molecular weight antibody-positive bands were
observed after 10% SDS-PAGE either (data not shown).
Since the peripheral sensory neurons have extensive axonal projections in the CNS (Briggs et al. 1993), these
results indicate that the calsensin antibodies recognize a
single protein species of identical relative molecular
weight that is present in both PNS and CNS neurons in
all three leech species.
Discussion
The small EF-hand calcium-binding protein calsensin is
expressed both in the PNS and CNS of hirudinid leeches. By immunocytochemistry with calsensin-specific
antibodies we have determined the complete distribution pattern in the CNS of Haemopis, Hirudo, and Macrobdella. We show that there is a pronounced difference
in the distribution of calsensin immunoreactivity in the
CNS of the three leech species and that this variability
is not likely to be due to multiple proteins or isoforms
being recognized by the calsensin antibody. In Haemopis more than 70 neurons are labeled, whereas in Hirudo 51 neurons and in Macrobdella only 8 neurons are
labeled. Furthermore, the expression of calsensin in
identified cells common to all three leech species also
differs. For example the R cells, which have been
shown to have essentially similar physiological properties in all hirudinid leeches examined (Lent 1977), express calsensin in Haemopis but not in Hirudo or Macrobdella. This complex pattern of distribution between
363
species makes it difficult to infer a functional role of
calsensin in CNS neurons, a situation that is compounded by the observation that the ontogeny of calsensin expression is a gradual process, with some neurons expressing calsensin just after neurogenesis is completed
and others obtaining the calsensin phenotype only postembryonically. By counting lan3–6-labeled neurons in
ganglia at different stages of embryonic development,
Macagno et al. (1983) showed that more than half of the
eventually lan3–6-positive neurons did not express calsensin before E25 in Haemopis. This makes it highly
unlikely that calsensin expression in the CNS is correlated with developmental processes, such as synaptogenesis. However, this difference in distribution between species is not unique to calsensin. It has become
quite clear from anatomical studies of many calciumbinding proteins, including parvalbumin and calbindin
in various vertebrates, that there are striking differences
in protein distribution between species. Therefore, caution should be taken in making generalizations about
the occurrence and possible function of calcium-binding
proteins (Baimbridge et al. 1992).
In contrast to the differences in the CNS distribution,
we found that calsensin is expressed in a similar subset
of sensillar neurons in embryos of all three leech species. Briggs et al. (1993) showed that a defining feature
of these neurons is that they fasciculate in a single axon
tract. This correlation has led to the hypothesis that calsensin may play a functional role in growth cone guidance or the selective fasciculation of these neurons
(Briggs et al. 1995). Furthermore, immunoaffinity purification experiments with the lan3-6 antibody show that a
large 200 000 Mr protein selectively copurifies with calsensin from CNS extracts of both Haemopis and Macrobdella leeches (Briggs et al. 1995). These results provide evidence that calsensin may be functioning as a
trigger protein that interacts with or modulates the larger
protein. Calcium-binding proteins are generally classified into two separate functional groups, the trigger and
the buffer proteins (Levine and Dalgarno 1983; Baimbridge et al. 1992). The trigger proteins, such as calmodulin, change their conformation in response to calcium
binding and in turn can then interact with and modulate
other proteins. The buffer proteins, such as parvalbumin
and ICaBP, on the other hand, are believed to passively
regulate the level of intracellular free calcium concentration. These observations raise the possibility that calsensin may have different functions in different classes of
leech neurons. The molecular features of calsensin, its
association with the 200 000 Mr protein, and its restricted expression common to all hirudinid leeches in a small
subset of peripheral neurons that fasciculate together in a
single tract are consistent with the hypothesis that it may
participate in a protein complex mediating calcium-dependent signal transduction events in growth cones and
axons. On the other hand, the large variation from species to species of the distribution and onset of expression
of calsensin in the CNS suggest that calsensin may play
a different role in these neurons, serving as a nonessential buffer protein participating in regulation of calcium
homeostasis possibly in conjunction with other calcium-
binding proteins. If this were the case, it could explain
the variable ontogeny of calsensin expression in different neurons and how such profound differences can arise
between functionally homologous neurons in different
species.
&p.2:Acknowledgements. We wish to thank Dr. Ron McKay for his generous gift of the lan3-6 antibody.
References
Baimbridge KG, Celio MR, Rogers JH (1992) Calcium-binding
proteins in the nervous system. Trends Neurosci15:303–
308
Briggs KK, Johansen KM, Johansen J (1993) Selective pathway
choice of a single central axonal fascicle by a subset of peripheral neurons during leech development. Dev Biol 158:
380–389
Briggs KK, Silvers AJ, Johansen KM, Johansen J (1995) Calsensin: a novel calcium-binding protein expressed in a subset of
peripheral leech neurons fasciculating in a single axon tract. J
Cell Biol 129:1355–1362
Fernandez J, Stent GS (1982) Embryonic development of the hirudinid leech Hirudo medicinalis: structure, development and
segmentation of the germinal plate. J Embryol Exp Morphol
72:71–96
French KA, Kristan WB (1992) Target influences on the development of leech neurons. Trends Neurosci 15:169–174
Jellies J, Johansen J (1995) Multiple strategies for directed growth
cone extension and navigation of peripheral neurons. J Neurobiol 27:310–325
Jellies J, Loer CM, Kristan WB (1987) Morphological changes in
leech neurons after target contact during embryogenesis. J
Neurosci 7:2618–2629
Johansen J, Hockfield S, McKay RDG (1984) Distribution and
morphology of nociceptive cells in three species of leeches. J
Comp Neurol 226:263–273
Johansen J, Johansen KM, Briggs KK, Kopp DM, Jellies J (1994)
Hierarchical guidance cues and selective axon pathway formation. In: Seil FJ (ed) Progress in brain research, vol 103. Elsevier, Amsterdam, pp 109–120
Johansen KM, Kopp DM, Jellies J, Johansen J (1992) Tract formation and axon fasciculation of molecularly distinct peripheral neuron subpopulations during leech embryogenesis. Neuron 8:559–572
Kretsinger RH (1980) Structure and evolution of calcium-modulated proteins. CRC Crit Rev Biochem 8:119–174
Laemmli UK (1970) Cleavage of structural proteins during assembly of the head of bacteriophage T4. Nature 227:680–685
Lent CM (1977) The Retzius cells within the central nervous
system of leeches. Prog Neurobiol 8:81–117
Levine BA, Dalgarno DC (1983) The dynamics and function of
calcium-binding proteins. Biochim Biophys Acta 726:187–
204
Macagno ER (1980) Number and distribution of neurons in leech
segmental ganglia. J Comp Neurol 190:283–302
Macagno ER, Stewart RR, Zipser B (1983) The expression of
antigens by embryonic neurons and glia in segmental ganglia
of the leech Haemopis marmorata. J Neurosci 3:1746–
1759
Muller KJ, Nicholls JG, Stent GS (1981) Neurobiology of the
leech. Cold Spring Harbor Press, Cold Spring Harbor, NY
Persechini A, Moncreif ND, Kretsinger RH (1989) The EF-hand
family of calcium-modulated proteins. Trends Neurosci
12:462–467
Phillips CE, Friesen WO (1982) Ultrastructure of the water-movement-sensitive sensilla in the medicinal leech. J Neurobiol
13:473–486
364
Stent GS, Weisblat DA, Blair SS, Zackson SL (1982) Cell lineage in the development of the leech nervous system. In:
Spitzer NC (ed) Neuronal development. Plenum, New York,
pp 1–44
Stewart RR, Macagno ER, Zipser B (1985) The embryonic development of peripheral neurons in the body wall of the leech
Haemopis marmorata. Brain Res 332:150–157
Stewart RR, Spergel D, Macagno ER (1986) Segmental differentiation in the leech nervous system: the genesis of cell number
in the segmental ganglia of Haemopis marmorata. J Comp
Neurol 253:253–259
Stewart RR, Gao W-Q, Peinado A, Zipser B, Macagno ER (1987)
Cell death during gangliogenesis in the leech: bipolar cells appear and then degenerate in all ganglia. J Neurosci 7:1919–
1927
Towbin H, Staehelin T, Gordon J (1979) Electrophoretic transfer
of proteins from polyacrylamide gels to nitrocellulose sheets.
Proc Natl Acad Sci USA 76:4350–4354
Zipser B (1982) Complete distribution patterns of neurons with
characteristic antigens in the leech central nervous system. J
Neurosci 2:1453–1464
Zipser B, McKay R (1981) Monoclonal antibodies distinguish
identifiable neurones in the leech. Nature 289:549–554