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First published online August 3, 2006
Journal of Experimental Biology 209, 3241-3256 (2006)
Published by The Company of Biologists 2006
doi: 10.1242/jeb.02372
Members of the crustacean hyperglycemic hormone (CHH) peptide family are differentially distributed both between and within the neuroendocrine organs of Cancer crabs: implications for differential release and pleiotropic function
1 Department of Biology, University of Washington, Box 351800, Seattle, WA
98195-1800, USA
2 School of Biological Sciences, University of Wales Bangor, Bangor, Gwynedd
LL57 2UW, UK
3 Center for Marine Biotechnology, University of Maryland Biotechnology
Institute, Baltimore, MD 21202, USA
4 Friday Harbor Laboratories, University of Washington, 620 University Road,
Friday Harbor, WA 98250, USA
* Author for correspondence (e-mail: crabman{at}u.washington.edu)
Accepted 6 June 2006
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Summary |
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 TOP Summary IntroductionMaterials and methodsResultsDiscussionReferences |
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Key words: Cancer crabs, sinus gland, (SG), pericardial organ (PO), anterior cardiac plexus (ACP), anterior commissural organ (ACO), stomatogastric nervous system (STNS), stomatogastric ganglion (STG), neurohormone, crustacean hyperglycemic hormone (CHH), crustacean hyperglycemic hormone precursor-related peptide (CPRP), mandibular organ-inhibiting hormone (MOIH), moult-inhibiting hormone (MIH), immunohistochemistry
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Introduction |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Koller, 1927) demonstrated
that hemolymph-borne agents were responsible for color change in shrimp. In
the decade that followed, Hanström
(Hanström, 1931;
Hanström, 1933;
Hanström, 1935;
Hanström, 1937)
identified a structure located in the eyestalk, the sinus gland (SG) as the
source of these substances. Later, Bliss
(Bliss, 1951) and Passano
(Passano, 1951) independently
demonstrated the neurohemal nature of the SG, a first for any neural structure
in the animal kingdom. In recent years, work from many laboratories has shown
that the SG is one of the major neuroendocrine organs in crustaceans and a
number of hormones contained within it have been identified, including a group
of structurally related peptides that have collectively been termed the
crustacean hyperglycemic hormone (CHH) family.
In crustaceans, the CHH family includes CHHs, moult-inhibiting hormones
(MIHs), gonad-inhibiting hormones (GIHs), vitellogenesis-inhibiting hormones
(VIHs) and mandibular organ-inhibiting hormones (MOIHs), all of which are
large peptides (72-78 amino acids) that share considerable amino acid sequence
and structural homology, including six conserved cysteine residues and three
disulfide bridges (Keller,
1992; de Kleijn and van Herp,
1995; Soyez, 1997;
van Herp, 1998;
Webster, 1998;
Chan et al., 2003;
Chen et al., 2005;
Fanjul-Moles, 2006). As their
diverse names imply, members of the CHH family have been shown to influence a
broad array of physiological processes including, but not limited to, the
regulation of metabolism, somatic growth and reproductive maturation
(Sedlmeier and Keller, 1981;
Sedlmeier, 1982;
Sedlmeier, 1988;
Keller, 1992;
Charmantier-Daures et al.,
1994; Wainwright et al.,
1996; Santos et al.,
1997; Khayat et al.,
1998; van Herp,
1998; Webster,
1998; Chung et al.,
1999; Spanings-Pierrot et al.,
2000; Sonobe et al.,
2001; Chan et al.,
2003; Serrano et al.,
2003).
Based on preprohormone organization, the CHH family can be divided into two
sub-groups: (1) CHH and (2) MIH/GIH/VIH/MOIH
(de Kleijn and van Herp, 1995;
Lacombe et al., 1999;
Chan et al., 2003;
Chen et al., 2005;
Fanjul-Moles, 2006). The
preprohormones of the CHH subgroup always contain a second peptide, commonly
referred to as CHH precursor-related peptide or CPRP, which is C-terminally
flanked by the CHH (Weidemann et al.,
1989; Tensen et al.,
1991; de Kleijn et al.,
1994a; de Kleijn et al.,
1995; Ohira et al.,
1997a; Davey et al.,
2000; Toullec et al.,
2002; Marco et al.,
2003; Chen et al.,
2004; Mettulio et al.,
2004; de la Iglesia et al.,
2005; Hsu et al.,
2005a; Toullec et al.,
2006). The preprohormones of the MIH/GIH/VIH/MOIH sub-group lack
precursor-related peptides (Klein et al.,
1993; de Kleijn et al.,
1994b; Sun, 1994;
Lee et al., 1995;
Ohira et al., 1997b;
Chan et al., 1998;
Gu and Chan, 1998;
Umphrey et al., 1998;
Tang et al., 1999;
Lu et al., 2001;
Yang and Rao, 2001;
Edomi et al., 2002;
Krungkasem et al., 2002;
Ohira et al., 2005). Although
CPRP has been shown to circulate in the hemolymph, the physiological role(s)
it plays in crustaceans remains unknown
(Wilcockson et al., 2002).
In brachyuran crabs, particularly those of the genus Cancer, much
work has been done in an effort to identify and characterize the actions of
the native CHH-family members and CPRPs in the X-organ (XO)-SG system. In this
genus, four distinct CHH isoforms have been identified [two from Cancer
pagurus and two from Cancer productus
(Chung et al., 1998;
de la Iglesia et al., 2005;
Hsu et al., 2005a);
Table 1] as have two isoforms
of MIH [one from Cancer magister and the other from C.
pagurus (Chung et al.,
1996; Umphrey et al.,
1998); Table 1] and
two isoforms of MOIH [both from C. pagurus
(Wainwright et al., 1996);
Table 1]. Similarly, nine
isoforms of CPRP have been characterized from the SG of Cancer
species [one from C. pagurus and four each from C. productus
and Cancer borealis (Chung et
al., 1998; de la Iglesia et
al., 2005; Fu et al.,
2005a; Fu et al.,
2005b; Hsu et al.,
2005a); Table 1].
As Table 1 shows, the isoforms
of any given group are nearly identical in amino acid sequence, even when
comparing isoforms from different species.
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Although most studies concerning CHH-family members and CPRPs have focused
on the SG, there is evidence that some of these peptides are also present in
other regions of the crustacean nervous system
(de Kleijn et al., 1995;
Sun, 1995;
Chang et al., 1999;
Dircksen et al., 2001;
Lu et al., 2001;
Gu et al., 2002;
Wilcockson et al., 2002;
Basu and Kravitz, 2003;
Chen et al., 2004;
Fu et al., 2005b;
Ohira et al., 2005;
Toullec et al., 2006). CHH-
and CPRP-like immunoreactivities have been found in the neuroendocrine
pericardial organ (PO) of C. pagurus
(Wilcockson et al., 2002) and
putative CPRP fragments have been sequenced from this structure in C.
productus (Fu et al.,
2005b). The functional relevance of this dual XO-SG/PO
distribution of CHH in these crabs is currently unknown. However, it has been
postulated that the signals governing its release from each site are distinct.
Moreover, the isoforms present in one versus the other structure may
also be distinct (Dircksen et al.,
2001; Toullec et al.,
2006) and subserving different physiological roles, as has been
shown for the CHHs isolated from the SG and PO of the crab Carcinus
maenas (Dircksen et al.,
2001).
Recently, we began a study to identify and compare the hormone complements
present in the neuroendocrine organs of several Cancer species
(Christie et al., 2004;
Christie and Messinger, 2005;
de la Iglesia et al., 2005;
Fu et al., 2005a;
Fu et al., 2005c;
Hsu et al., 2005a;
Hsu et al., 2005b;
Messinger et al., 2005a;
Messinger et al., 2005b;
Cruz-Bermúdez et al.,
2006). As a first step toward determining the identity, tissue
distributions and physiological roles played by each CHH-family member and
CPRP in the neuroendocrine systems of these species, we undertook the
immunohistochemical mapping studies presented in this report. Specifically,
antisera previously generated against native C. pagurus peptides
(Webster, 1996;
Wilcockson et al., 2002) were
used to map the distributions and co-localization patterns of the CHH family
and CPRP in the neuroendocrine organs of Cancer antennarius, Cancer
anthonyi, C. borealis, Cancer gracilis, Cancer irroratus, C. magister and
C. productus. As the data that follow show, most members of the CHH
family and CPRP were immunologically detected in multiple neuroendocrine
organs, with the complement of peptides present in each site conserved across
species. In at least C. productus, the peptides detected in the XO-SG
and PO were also differentially distributed among the neurons comprising each
site. Collectively, the data presented here represent the most extensive and
complete immunohistochemical survey of CHH family members and CPRP in
crustacean neuroendocrine organs to date. Moreover, our findings support the
hypotheses that: (1) members of CHH family and CPRP are released in response
to varied, tissue-specific cues and (2) the isoforms released from non-XO-SG
sites probably possess functions distinct from those ascribed to them when
secreted from the SG. Some of these data have appeared previously in abstract
form (Hsu et al., 2004).
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Materials and methods |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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Tissue collection
For the collection of tissue, crabs were anesthetized by packing in ice for
30-60 min. After anesthesia, the eyestalks were isolated, then the dorsal
carapace was removed and the foregut and the lateral walls of the pericardial
chamber were dissected free in chilled (approximately 10°C) physiological
saline [composition: 440 mmol l-1 NaCl; 11 mmol l-1 KCl;
13 mmol l-1 CaCl2; 26 mmol l-1
MgCl2; 10 mmol l-1 Hepes; pH 7.4 (adjusted with NaOH)].
To obtain the XO-SG system, the carapace encasing an eyestalk was split both
dorsally and ventrally and one half of the split shell was gently teased away
from the other half. The remaining half of the eyestalk was then pinned in a
wax-lined Pyrex dish filled with chilled physiological saline and the optic
ganglia containing the XO-SG system were subsequently isolated. POs were
obtained by pinning the isolated walls of the pericardial chamber in a
wax-lined Pyrex dish containing chilled physiological saline. The nerve roots
forming each PO were then dissected free from the muscles and connective
tissues of the pericardial wall. For isolating the stomatogastric nervous
system (STNS), which contains two recently identified neuroendocrine organs
(Christie et al., 2004;
Messinger et al., 2005a), the
anterior cardiac plexus (ACP) and the anterior commissural organ (ACO), the
foregut was flattened by making a longitudinal cut from the esophagus to the
pylorus on its ventral side followed by a pair of medial cuts directed along
the ossicles of the cardiac sac/gastric mill. After its opening, the teeth of
the gastric mill were removed and the flattened foregut was pinned, inside
down, in a wax-lined Pyrex dish containing chilled physiological saline. The
STNS was then dissected free from the muscles and connective tissues of the
foregut.
Antibodies and antibody production
Primary antibodies
All of the primary antibodies used in our study were rabbit polyclonal
antibodies generated against native C. pagurus peptides
(Table 1). These peptides were
purified from a common pool of approximately 2000 SGs as described in several
previous publications (Chung et al.,
1996; Wainwright et al.,
1996; Webster,
1996; Chung et al.,
1998). For production of the CHH antibody, native C.
pagurus (Capa)-CHH II was used. The development of this antibody
is described elsewhere (Webster,
1996). For the production of the CPRP antibody, the antigen was
C. pagurus (Capa)-CPRP conjugated to bovine thyroglobulin using
either glutaraldehyde or 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide. The
production of this antibody is also described elsewhere
(Wilcockson et al., 2002). For
the production of the MIH and MOIH antibodies, methods similar to those
employed for the production of the CHH antibody were used. Specifically, New
Zealand white rabbits (one for each peptide) were injected subcutaneously at
multiple sites with 5 nmol of native C. pagurus (Capa)-MIH or native
C. pagurus (Capa)-MOIH I, each dissolved in 0.3 ml of
phosphate-buffered saline and emulsified with an equal volume of complete
Freund's adjuvant. Four weeks after the initial immunization, a booster of 5
nmol of peptide (Capa-MIH or Capa-MOIH I) in Freund's incomplete adjuvant was
injected into each rabbit. Similarly, 8 weeks after the initial immunization,
rabbits were boosted again, this time with 3 nmol of peptide (Capa-MIH or
Capa-MOIH I) in Freund's incomplete adjuvant. Twelve weeks after the initial
immunization, rabbits were terminally exsanguinated under anesthesia and serum
was collected from the retracted clots. Production of all antisera used in
this study was done at the University of Wales Bangor (Bangor, UK).
Secondary and tertiary antibodies
The secondary and tertiary antibodies used in our study were kind gifts
from either Jackson ImmunoResearch Laboratories, Inc. (West Grove,
Pennsylvania, USA) or Molecular Probes (Eugene, Oregon, USA). These included
donkey anti-rabbit IgG labeled with Alexa Fluor 488 (Molecular Probes; catalog
#A-21206), Alexa Fluor 594 (Molecular Probes; catalog #A-21207), FITC (Jackson
ImmunoResearch; catalog # 711-095-152) or Rhodamine Red X (Jackson
ImmunoResearch; catalog # 711-295-152); goat anti-rabbit IgG labeled with
Alexa Fluor 488 (Molecular Probes; catalog # A11008) or Texas Red (Jackson
ImmunoResearch; catalog #111-075-144); unlabeled monovalent goat anti-rabbit
IgG antigen-binding (Fab) fragments (Jackson ImmunoResearch; catalog #
111-007-003) or donkey anti-goat IgG labeled with FITC (Jackson
ImmunoResearch, catalog # 705-095-147).
Whole-mount immunohistochemistry
Single labeling
For single labeling, tissue was fixed overnight in a freshly made solution
of 4% paraformaldehyde (EM grade; Electron Microscopy Sciences, Hatfield,
Pennsylvania, USA; catalog #15710) in 0.1 mol l-1 sodium phosphate
buffer (pH 7.4) followed by five rinses (at 1 h intervals) in a solution of
sodium phosphate buffer containing 0.3% Triton X-100 (P-Triton). After
rinsing, tissue was incubated with gentle agitation in a 1:5000 dilution of
primary antibody for approximately 72 h. Primary antiserum was diluted in
P-Triton, with 10% normal donkey serum (NDS; Jackson ImmunoResearch; catalog
#017-000-121) or normal goat serum (NGS; Jackson ImmunoResearch; catalog
#005-000-121) added to diminish nonspecific binding. Following incubation in
primary antibody, tissue was again rinsed five times at 1 h intervals in
P-Triton and then incubated overnight in a 1:300 dilution of secondary
antibody. As with the primary antibody, secondary antibody incubation was done
in P-Triton with 10% NDS or NGS, using gentle agitation. After secondary
antibody incubation, each preparation was rinsed five times over approximately
5 h in sodium phosphate buffer and then mounted between a glass microscope
slide and coverslip using Vectashield mounting medium (Vector Laboratories,
Inc., Burlingame, California, USA; catalog # H-1000). Fixation and incubation
in both primary and secondary antibody was done at 4°C. All rinses were
done at room temperature (approximately 20°C) without agitation. Secondary
antibody incubation, and all subsequent processing, was conducted in the dark.
Likewise, slides were stored in the dark at 4°C until examined.
To increase our confidence that the immunoreactivities reported here were specific for peptides related to the antigen used for the production of each antiserum, we conducted a series of adsorption controls. In these experiments, native Capa-CHH II, Capa-CPRP, Capa-MIH and Capa-MOIH I isolated from the SG of C. pagurus (Table 1) were used as blocking agents. It should be noted that due to a limited supply of these peptides, adsorption controls were only conducted on C. productus tissues and that the adsorptions used for labeling one preparation were sometimes recycled for use in blocking studies on other samples. In each adsorption experiment, the antibody was incubated with a blocking peptide (approximately 10-5 mol l-1) for 2 h at room temperature prior to applying the solution to the tissue. For each tissue in which an antibody produced labeling, a complete block of staining was achieved only when the antiserum was adsorbed with the peptide used for its production (three preparations for each peptide and antibody in each tissue; data not shown). Adsorption of an antibody with the other peptides had no appreciable effect on immunolabeling (three preparations for each peptide and antibody in each tissue; data not shown).
Double labeling
For preparations that were double-labeled (C. productus tissues
only), the staining protocol for single labeling was modified in order to
permit the use of primary antibody pairs that were both developed in the same
host species (rabbit in this case). In brief, POs and SGs were dissected,
fixed and incubated in the first primary antibody as described above for 1 or
2 days, respectively. Following incubation in the first primary antibody,
tissues were rinsed with P-Triton as described for single labeling, then the
SGs were incubated overnight in a 1:25 dilution of monovalent goat anti-rabbit
IgG Fab fragments that also contained 10% NDS whereas the POs were incubated
for 2 days in a 1:10 dilution of goat anti-rabbit IgG Fab fragments containing
10% NDS. For both tissues, this incubation with Fab fragments was done to
sterically cover and immunologically convert the first primary antibody from
rabbit to goat. After incubation with the Fab fragments, tissues were rinsed
every hour for 5 h and then incubated overnight in a 1:300 dilution of
FITC-conjugated donkey anti-goat IgG containing 10% NDS. Following this
overnight incubation, tissues were rinsed as before then incubated in the
second primary antibody for 1 (PO) or 2 (SG) days. After incubation in the
second primary antibody, tissues were rinsed and then incubated overnight in a
1:300 dilution of Rhodamine Red X-conjugated donkey anti-rabbit IgG containing
10% NDS. All subsequent processing was the same as for the single labeling
described above.
To assess the veracity of our double-labeling protocol, we conducted controls to determine the extent to which the Fab fragments were capable of sterically covering, and hence masking, the first primary antibody from detection via the second secondary antibody. Specifically, a single primary was applied to a tissue, sterically converted to goat via incubation in Fab fragments and then incubated in the Rhodamine Red X-conjugated donkey anti-rabbit IgG. For all antibodies and all tissues in which double labeling was undertaken (3 preparations for each antibody in each tissue), a complete masking of the primary antibody was achieved, and hence little or no cross-talk between the first and second primary antibody sets should have occurred in our double-labeled preparations (data not shown).
Confocal and epifluorescence microscopy
After immunoprocessing, preparations were viewed and data were collected
using one of two Bio-Rad MRC 600 laser scanning confocal microscopes (Bio-Rad
Microscience Ltd., Hemel Hempstead, UK), a Bio-Rad Radiance 2000 laser
scanning confocal microscope or a Nikon Eclipse E600 epifluorescence
microscope. Descriptions of the hardware and software used for imaging on
these systems are extensively documented in previous publications
(Christie et al., 1997;
Christie et al., 2003;
Christie et al., 2004;
Messinger et al., 2005a).
Figure production
For the production of figures, Bio-Rad.pic files collected using
the MRC 600 or Radiance 2000 systems were converted to tif files
using ImageJ (available free of charge from the National Institutes of Health
at
http://rsb.info.nih.gov/ij/).
Individual micrographs and composite figures were produced from the
tif files using a combination of ImageJ and Photoshop (version 7.0;
Adobe Systems Inc., San Jose, California, USA). Schematic diagrams were
produced using Canvas (version 8.0; Deneba Systems Inc., Miami, Florida, USA).
It should be noted that the contrast and brightness of final figures were
adjusted as needed to optimize the clarity of printed images.
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Results |
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TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
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;
Beltz, 1988;
Fingerman, 1992). As stated
earlier, isoforms of all members of the CHH family (i.e. CHH, MIH and MOIH)
have been isolated from the XO-SG system of C. pagurus, as has an
isoform of CPRP (Chung et al.,
1996; Wainwright et al.,
1996; Chung et al.,
1998). Using antibodies generated against C. pagurus
peptides (Webster et al., 1996; Wilcockson
et al., 2002; this study), we immunolabeled eyestalks from C.
antennarius, C. anthonyi, C. borealis, C. gracilis, C. irroratus, C.
magister and C. productus to determine if the XO-SG system of
these species also contains members of these peptide groups.
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4
preparations for each antibody in each species). Within the SG, the nerve
terminals labeled by the CHH and CPRP antisera
(Fig. 4A,B) had a tendril-like
appearance, whereas the terminals labeled by the MIH and MOIH antisera
(Fig. 4C,D) appeared more oval
and blob-like. Regardless of antibody or species, all labeling in the SG could
be traced back to the immunopositive XO somata via labeled axons in
the sinus gland tract (sgt), suggesting that these somata are the sole source
of the SG staining.
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Patterns of co-localization in the XO-SG system of C. productus
As just described, the SG terminals labeled by the CHH and CPRP antibodies
were tendril-like whereas those labeled by the MIH and MOIH antibodies were
distinctly blob-like in appearance in all species
(Fig. 4). For C.
productus, we quantified and compared the number of somata labeled by
each antibody. In this species, the numbers of XO somata labeled by the CHH
and CPRP antibodies were essentially identical [anti-CHH: mean=40±1.90
somata/XO, range=36-49, 6 preparations; anti-CPRP: mean=41±2.12
somata/XO, range= 37-47, 4 preparations (Student's t-test,
P>0.05)] as were the number of somata labeled by the MIH and MOIH
sera [anti-MIH: mean=32±1.38 somata/XO, range=25-36, 8 preparations;
anti-MOIH: mean=29±1.92 somata/XO, range= 22-35, 6 preparations
(Student's t-test, P>0.05)]. By contrast, the number of
somata stained by either the CHH or CPRP antibody was statistically different
from those labeled by either the MIH or MOIH antibody (ANOVA,
P<0.0005; Tukey's test, P<0.05). Collectively, these
findings suggested the potential for CHH/CPRP and MIH/MOIH co-localization in
the XO-SG system, and simultaneously implied that the former two peptides were
contained in subpopulations of neurons distinct from those producing the
latter two peptides. To directly assess the patterns of co-localization
present among the CHH family members and CPRP in C. productus, we
developed a double-immunohistochemistry method utilizing primary antisera
generated in a common host species and conducted double-immunolabeling for all
possible combinations of the four antisera using this method.
As Fig. 5 shows,
co-localization of CHH and CPRP as well as of MIH and MOIH (shown in
Fig. 5A) was seen in XO somata
in each of the C. productus eyestalks labeled using these antibody
combinations (6 preparations for each combination). In none of the eyestalks
labeled with any other combination of primary antisera was any co-localization
evident in XO somata (Fig. 5B;
6 preparations for each antibody combination). All somata that expressed
CHH-like immunoreactivity were co-labeled by the CPRP antibody, a finding that
is not surprising given their production from the same preprohormone in C.
productus (de la Iglesia et al.,
2005; Hsu et al.,
2005a). Interestingly, complete co-localization was also seen in
the double-labels pairing of the MIH and MOIH antibodies
(Fig. 5A), suggesting that all
XO somata that produce MIH also produce MOIH.
|
; Wilcockson et al.,
2002; Fu et al.,
2005b). In brachyuran species, including C. productus,
the PO consists of two or more longitudinal nerve trunks that are connected by
vertical nerve bars (Fig. 1).
The trunks and bars that form each PO are elaborations of the segmental nerves
(sns) that originate from the fused thoracic ganglia
(Fig. 1). Both intrinsically
and extrinsically located cell bodies contribute to the release terminals that
lie under the epineurium that covers the PO
(Cooke and Sullivan, 1982;
Beltz, 1988;
Fingerman, 1992). As stated
earlier, both CHH and CPRP have been detected in the PO of C. pagurus
using immunohistochemistry (Wilcockson et
al., 2002). In this species, all labeling for both peptides
originates from three intrinsic somata located in or near the anterior bar of
the PO (Wilcockson et al.,
2002). To assess if CHH-, CPRP-, MIH- or MOIH-like peptides were
present in the POs of other species of Cancer and to determine their
distributions and patterns of co-localization in C. productus, we
immunoprocessed this tissue with the same set of antisera used in our mapping
of these substances in the SG.
Immunohistochemical detection of CHH, CPRP and MOIH, but not MIH in the PO
Immunoprocessing of POs with the CHH, CPRP and MOIH antisera consistently
produced labeling within this tissue in each of the Cancer species
investigated (Fig. 6;
N3 preparations for each antibody in each species). By contrast,
no staining was seen when POs of any crab were processed with the MIH
antiserum (N3 preparations for each species). For both the CHH
and CPRP antibodies, labeling in the POs of each species consisted of up to
three somata (usually two bipolar and one multipolar) in the anterior bar or
in the sns just anterior to it, as well as fine processes and
peripherally located nerve terminals covering extensive regions of the
anterior and posterior bars and portions of all three nerve trunks
(Fig. 6A,B). For both
antibodies, the vast majority of the immunopositive fine fibers and nerve
terminals appeared to originate from the arborizations of the intrinsic
somata. In each of the CHH-immunopositive somata, labeling was cytoplasmic,
uniformly diffuse and often extended for a considerable distance into the
axons emanating from these cell bodies. By contrast, labeling in the
CPRP-immunopositive somata in all species was cytoplasmic and granular, with
little immunoreactivity evident in their projecting axons.
|
In all animals, MOIH-like staining was weak in the axons and in patches of
superficially located nerve terminals in both the anterior and posterior bars,
as well as in the nerve trunks (Fig.
6D). Whereas no MOIH-like staining was seen in any intrinsic PO
somata in any of the species, approximately four MOIH-immunopositive cell
bodies were present in the fused thoracic ganglia of C. productus (3
preparations; data not shown). As the thoracic ganglia are known to be the
source of many of the inputs to the PO
(Cooke and Sullivan, 1982;
Beltz, 1988;
Fingerman, 1992), these somata
are one potential source of the MOIH staining seen in the PO of this species,
and possibly the others as well.
Patterns of co-localization in the PO of C. productus
In all of the species investigated, the distribution of CHH- and CPRP-like
immunoreactivity in the POs was essentially identical with the exception of
the appearance of labeling in the three intrinsic somata. To determine the
extent of colocalization of the two peptides in C. productus,
double-immunolabeling, similar to that in the SG, was undertaken. As
Fig. 7 shows, a complete
overlap of the two immunoreactivities was found in this tissue (6
preparations). CHH/MOIH (6 preparations) and CPRP/MOIH (6 preparations)
double-labelings were also conducted. However, the relative weakness of the
MOIH label with respect to either the CHH or CPRP labels made assessment of
co-localization difficult. In most POs, the immunoreactivities appeared
distinct, whereas in others, weak co-localization in a small population of
terminals was evident (data not shown). Given the complete overlap of the CHH
and CPRP labels and the fact that they appeared to be derived from a set of
somata not labeled by the MOIH antiserum, we believe that the profiles labeled
by the MOIH antibody are distinct from those containing CHH/CPRP.
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The stomatogastric nervous system
The STNS (Fig. 1) of decapod
species is situated over the foregut and is responsible for controlling the
movement of food items through this portion of the digestive tract
(Selverston and Moulins, 1987;
Harris-Warrick et al., 1992).
Recently, we identified two neuroendocrine organs within the STNS of C.
productus (Christie et al.,
2004; Christie and Messinger,
2005; Messinger et al.,
2005a; Messinger et al.,
2005b). One of these, the ACP, is located on the anterior cardiac
nerves (acns) overlying the cardiac sac region of the foregut
(Christie et al., 2004)
(Fig. 1). This site consists of
a dense collection of nerve terminals located just below the epineurium
covering the acns (Christie et
al., 2004), with all innervation to the site originating from four
somata, one pair in each commissural ganglion (CoG)
(Christie and Messinger, 2005;
Messinger et al., 2005b).
Based on their innervation of the ACP, these neuron pairs were named anterior
commissural neurons 1 and 2 (ACN1/2)
(Christie and Messinger, 2005;
Messinger et al., 2005b). The
other site, the ACO, is located within the anterior medial quadrant of the CoG
(Fig. 1) and consists of a
dense collection of nerve terminals that envelop an extensive network of
hemolymph lacunae that invaginate deep into the ganglion
(Messinger et al., 2005a). The
origin of the fibers that produce the ACO remain unknown, though somata in the
thoracic ganglia are likely candidates
(Messinger et al., 2005a).
Both the ACP and ACO are also present in the STNSs of C. antennarius,
C. anthonyi, C. borealis, C. gracilis, C. irroratus and C.
magister (A.E.C., D.I.M. and E. Savage, unpublished observations). To
determine if the ACP and/or ACO contain any of the CHH-related or CPRP-like
peptides, we immunoprocessed the STNSs from each Cancer species with
the same complement of antisera used to map the SG and PO.
Immunohistochemical detection of MOIH, but not CHH, MIH or CPRP in the ACP
In each of the investigated species, no immunoreactivity was seen anywhere
in the STNSs processed with the CHH, CPRP or MIH antisera (N3
preparations for each antibody in each species). By contrast, in all crabs,
extensive labeling was produced throughout using the MOIH antibody
(N3; Figs 8,
9,
10), including staining of the
ACP (Fig. 8) but not the ACO
(data not shown).
|
|
|
; Christie and Messinger,
2005; Messinger et al.,
2005b). In addition to the staining just described, in all species the MOIH antiserum also labeled an extensive neuropil in the stomatogastric ganglion that appeared to originate from both the arborizations of two intrinsic immunopositive somata and from the arborizations of projection neurons traveling to the ganglion via the stn (Fig. 9). This, and all other MOIH-like labeling in the STNS, is summarized schematically in Fig. 10.
|
Discussion |
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|
TOPSummaryIntroductionMaterials and methodsResultsDiscussionReferences |
|---|
As are all antibodies, the sera used in our study are biological and not
chemical reagents and thus are subject to potential cross-reactions with
antigens other than that to which they were raised
(Saper and Sawchenko, 2003).
As Table 1 shows, significant
sequence homology is present between the C. pagurus CHH-family
members used for antibody production. Despite the sequence similarity of the
antigens, the distinct patterns of labeling seen with each antibody strongly
suggest that they are detecting different complements of peptides.
Furthermore, in all tissues, only adsorption of an antibody by the peptide
used for its production completely eliminated labeling. Thus, we feel
confident that the staining presented here is an accurate reflection of the
distributions of CHH-family members and CPRPs in the neuroendocrine organs of
Cancer species.
Members of the CHH family and CPRP are differentially distributed within the XO-SG system as well as within the PO
In addition to observing a differential distribution of CHH-family peptides
and CPRP among the neuroendocrine organs of various Cancer species,
we also found that these peptides are differentially distributed within both
the XO-SG and PO of at least C. productus. Using a protocol that
allowed double labeling of whole-mount tissue with primary antibodies
generated in the same host species, we found complete overlap of labeling for
CHH and CPRP in both the XO-SG and the PO. Similarly, a complete overlap of
labeling was seen for MIH and MOIH in the XO-SG, but in a different set of
neurons than those containing CHH and CPRP. MOIH was also present in the PO,
again in a set of structures distinct from those possessing CHH and CPRP.
Ours is the first study to show co-localization of CHH- and CPRP-like
immunoreactivities in either the XO-SG or the PO, but the association of these
two peptides is not surprising given that both peptides are produced from a
common preprohormone in C. productus
(de la Iglesia et al., 2005;
Hsu et al., 2005a). More
interesting is the complete overlap of the MIH and MOIH labels in the XO-SG,
which are encoded by different genes (Lu
et al., 2000). As stated earlier, the peptides used for the
production of the MIH and MOIH antibodies exhibit significant amino acid
sequence identity (approximately 61%; Table
1), and thus it is possible that each serum was cross-reacting
with a common peptide. However, we were unable to completely block the MIH
staining in the XO-SG with any peptide other than Capa-MIH, including
Capa-MOIH I. Similarly, the XO-SG label produced by the MOIH antibody was
suppressed only after adsorption by Capa-MOIH and not by Capa-MIH. These
results suggest that the MIH- and MOIH-like labelings were specific for their
respective antigens. Clearly, molecular and/or biochemical characterization of
the complement of native C. productus CHH-family peptides will be
necessary to resolve this issue unambiguously.
Biologically, it is interesting to postulate why MIH and MOIH are present in the same set of XO somata. If MIH and MOIH are contained in the same secretory vesicles, then all signals triggering their release would probably result in the coordinated inhibition of both somatic growth (via the inhibition of steroid production in the Y-organ by MIH) and gonadal growth (via the inhibition of methyl farnesoate production in the mandibular organ by MOIH). If, however, the two peptides are contained in distinct sets of vesicles, then the two peptides might well be released in response to distinct physiological cues. Clearly, the former versus the latter situation would have profound physiological consequences for an animal, and with future studies it will be interesting to determine the physiological significance of the co-localized peptides.
Do multiple tissue localizations imply pleiotropic functions for the CHH family members and CPRP in Cancer species?
As just discussed, we have found that the CHH, MOIH and CPRP are each
present in at least two neuroendocrine sites. With such multiple tissue
localizations, the potential clearly exists for distinct cues triggering the
release of a given hormone from each site. In addition, distinct isoforms of
the CHH, MOIH and CPRP may be present in each tissue, as has been shown for
CHH in several crab species (Dircksen et
al., 2001; Toullec et al.,
2006). In Carcinus maenas, the CHH of the PO, unlike its
SG counterpart, does not possess hyperglycemic activity nor does it appear to
inhibit ecdysteroid synthesis (Dircksen et
al., 2001). Instead, this CHH isoform is proposed to serve either
a sensory or local modulatory role in this species
(Dircksen et al., 2001).
Although unproven, the differential distributions of CHH, CPRP and MOIH
revealed in our study suggest the possibility of pleiotropic functions for
these peptides in Cancer species (e.g. their classically ascribed
functions versus possible roles as locally released neuromodulators). As
additional studies are undertaken, it will be interesting to see if this
hypothesis is borne out.
Conclusions
In conclusion, the data we present show that members of the CHH family and
CPRPs are differentially distributed both between and within the
neuroendocrine organs of crabs of the genus Cancer. These results
represent the most complete immunohistochemical survey of CHH-family peptides
and CPRP in the neuroendocrine organs of crustaceans and provide a foundation
for future studies directed at the identification of the native isoforms
present in each species and tissue, as well as the elucidation of their
physiological roles in members of this genus. The finding of CHH, CPRP and
MOIH-like labeling in multiple neuroendocrine tissues suggest that these
peptides may be released into the circulatory system in response to varied
cues and that some of their isoforms may possess distinct tissue-specific
functions.
|
Acknowledgments |
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