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
Yun-Wei A. Hsu1,
Daniel I. Messinger1,
J. Sook Chung2,3,
Simon G. Webster2,
Horacio O. de la Iglesia1 and
Andrew E. Christie1,4,*
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
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Fig. 1. Schematic representation of the nervous system of a brachyuran crab
illustrating the relative locations of the known neuroendocrine organs of
Cancer species. The central nervous system (CNS) of brachyurans is
generally considered to consist of the supraesophageal (SoG) and fused
thoracic ganglia, which are connected via the circumesophageal
connectives (cocs). The optic nerves (optns) link the SoG
with the ganglia of the eyestalks, the location of the neuroendocrine sinus
gland (SG). Another well-known neuroendocrine site is the pericardial organ
(PO), which is located in the pericardial chamber surrounding the heart. The
POs consist of elaborations of the segmental nerves (sns), which
project from the fused thoracic ganglia. Two additional neuroendocrine sites,
the anterior cardiac plexus (ACP) and the anterior commissural organ (ACO),
are contained within the stomatogastric nervous system (STNS), an offshoot
from the CNS that overlies the foregut. The ACPs are located on the anterior
cardiac nerves (acns) and the ACOs are located within the commissural
ganglia (CoGs). For the sake of future discussion, the stomatogastric ganglion
(STG) is also shown in this schematic. It should be noted that this
illustration is not drawn to scale and that other portions of the nervous
system have been excluded for the sake of simplicity.
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Fig. 2. Schematic diagram of the optic ganglia of Cancer species
highlighting the location and organization of the X-organ-sinus gland (XO-SG)
system. The nervous system contained within the eyestalk consists of several
distinct regions, including the medulla terminalis (MT), the medulla interna
(MI), the medulla externa (ME), the lamina ganglionaris (LG) and the retina.
This system of ganglia is connected to the supraesophageal ganglion (SoG)
via the optic nerve (optn). Located in the MT is a loosely
associated collection of neurosecretory somata that are collectively termed
the X-organ (XO). The release site of hormones produced by these somata is the
sinus gland (SG), which is located at the junction of the MI and ME. The sinus
gland tract (sgt) links the XO and SG.
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Fig. 3. Moult-inhibiting hormone (MIH)-like immunoreactivity in the X-organ-sinus
gland (XO-SG) system of Cancer productus. (A) MIH-like labeling in
the SG. Whole-mount immunolabeling of the nervous system contained within the
eyestalk with antibody to MIH consistently stained nerve terminals in the SG.
This labeling could be unambiguously traced via immunopositive axons
in the sinus gland tract (sgt) to somata in the XO. This micrograph
is a brightest pixel projection of 29 optical sections taken at 1.95 µm
intervals. (B) MIH-like labeling in the XO. Immunoprocessing using anti-MIH
consistently labeled 30 or so somata in the XO. This micrograph is a brightest
pixel projection of 19 optical sections taken at 1.95 µm intervals. A and B
are taken from the same preparation and are shown at the same magnification.
Scale bar in B, 150 µm.
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Fig. 4. The distribution of crustacean hyperglycemic hormone (CHH)-like and CHH
precursor-related peptide (CPRP)-like labels in the sinus gland (SG) differ
from those of moult-inhibiting hormone (MIH) and mandibular organ-inhibiting
hormone (MOIH) in Cancer productus. Within the SG, terminals labeled
by the CHH and CPRP antibodies had the appearance of flocculent tendrils,
whereas those revealed by the MIH and MOIH antibodies appeared more oval and
blob-like. (A) CHH-like immunopositive terminals in the SG. (B) CPRP-like
immunopositive terminals in the SG. (C) MIH-like immunopositive terminals in
the SG. (D) MOIH-like immunopositive terminals in the SG. All micrographs are
single optical sections shown at the same magnification. Scale bar in D, 50
µm.
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Fig. 5. Double-immunolabeling of the X-organ (XO) with antisera to crustacean
hyperglycemic hormone (CHH)-family members and CHH precursor-related peptide
(CPRP) in Cancer productus. (A1-3) Moult-inhibiting hormone
(MIH)/mandibular organ-inhibiting hormone (MOIH) double labeling in the XO.
This series of micrographs consists of pseudo-colored single optical sections
collected from a single focal plane in an XO labeled with both anti-MIH (A1,
pseudo-colored green) and anti-MOIH (A2, pseudo-colored red). When A1 and A2
were merged (A3), complete overlap in the labeled structures was revealed
(i.e. yellow, but not green or red, coloration is seen in all immunopositive
structures in the micrograph), suggesting that the MIH- and MOIH-like labels
are contained in a common set of XO somata. The same result was seen in
preparations pairing anti-CHH/anti-CPRP (data not shown). (B1-3) MIH/CPRP
double labeling in the XO. This series of micrographs consists of
pseudo-colored single optical sections collected from a single focal plane in
an XO labeled with both anti-MIH (B1, pseudo-colored green) and anti-CPRP (B2,
pseudo-colored red). When B1 and B2 were merged (B3), no overlap in the
labeled structures was revealed (i.e. a lack of yellow colored structures in
the micrograph), suggesting that the MIH- and CPRP-like labels are contained
in distinct sets of XO somata. Though not shown, the same result was seen in
preparations pairing anti-MIH/anti-CHH, anti-MOIH/anti-CHH and
anti-MOIH/anti-CPRP. A1,2 and B1,2 are all shown at the same scale. Likewise
A3 and B3 are shown at the same magnification. Scale bars in B2 and B3, 100
µm.
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Fig. 6. Crustacean hyperglycemic hormone (CHH)-like, CHH precursor-related peptide
(CPRP)-like and mandibular organ-inhibiting hormone (MOIH)-like labeling in
the segmental nerves (sn) and pericardial organ (PO) of Cancer
productus. (A) CHH-like labeling in the sn anterior to the PO.
Two bipolar somata were labeled with the CHH antibody in the anterior bar of
the PO or in the sns just anterior to it (one stained soma shown;
arrow). Staining in these somata was uniform in its distribution, with the
label filling the axons emanating from the cell bodies. As this micrograph
shows, many small diameter axons and superficially located nerve terminals
were also labeled by the CHH antibody in the sn. This micrograph is a
brightest pixel projection of 34 optical sections taken at 1.95 µm
intervals. (B) CPRP-like labeling in the sn anterior to the PO. As
with the CHH antibody, two bipolar somata were labeled in the
sn/anterior bar area of the PO by the CPRP antibody (two shown;
arrows). Here, labeling within the somata was granular in appearance, and
little immunoreactivity was present in the axons emanating from the cell
bodies. Similar to the CHH label, many small diameter axons and superficially
located nerve terminals in the sn were stained by the CPRP antibody.
This micrograph is a brightest pixel projection of 21 optical sections taken
at 1.95 µm intervals. (C) CHH-like labeling in the anterior bar of the PO.
In addition to the bipolar neurons seen in the sn, a single
multipolar soma was routinely labeled by the CHH antibody in the anterior bar
of the PO (arrow). As with the other PO somata labeled by this antiserum,
staining in this cell body was uniform and diffuse in appearance. In addition
to the soma, an extensive network of fine fibers, as well as superficial nerve
terminals, were labeled by the CHH antibody within the anterior bar of the PO.
Although not shown, the CPRP antibody produced an identical pattern of
labeling in this portion of the PO, with the exception that labeling in the
multipolar soma was granular in appearance rather than uniformly diffuse. This
micrograph is a brightest pixel projection of 37 optical sections taken at
1.95 µm intervals. (D) MOIH-like labeling in the anterior bar of the PO. As
with the CHH and CPRP labels, the MOIH antibody stained a network of fine
fibers and superficially located nerve terminals in the anterior bar of the
PO, though the immunoreactivity was less extensive and weaker than with the
other sera. However, no somata were labeled by the MOIH antiserum, nor were
any labeled in the sns near the anterior bar. This micrograph is a
brightest pixel projection of 26 optical sections taken at 1.95 µm
intervals. All scale bars, 100 µm.
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Fig. 7. Co-localization of crustacean hyperglycemic hormone (CHH)-like and CHH
precursor-related peptide (CPRP)-like immunoreactivity in the pericardial
organ (PO) of Cancer productus. This series of micrographs consists
of pseudo-colored single optical sections collected from a single focal plane
from a PO labeled with both anti-CPRP (A, pseudo-colored green) and anti-CHH
(B, pseudo-colored red). When A and B are merged (C), complete overlap in the
labeled structures was revealed (i.e. yellow, but not green or red, coloration
is seen in all immunopositive processes in the micrograph), suggesting that
the CPRP- and CHH-like labels are contained in a common set of structures in
the PO. A and B are shown at the same magnification. Scale bars in B and C, 25
µm.
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Fig. 8. Mandibular organ-inhibiting hormone (MOIH)-like labeling in the anterior
cardiac plexus (ACP) of Cancer productus. (A) MOIH-like labeling in
the ACP. Immunoprocessing of the stomatogastric nervous system with the MOIH
antibody consistently produced labeling in the ACP. This micrograph is a
brightest pixel projection of 16 optical sections taken at 1.95 µm
intervals. (B) MOIH-like labeling at the junction of the anterior cardiac
(acn) and stomatogastric (stn) nerves. Labeling in each ACP
arose from four axons that projected into each acn from the
stn (axons in the stn denoted by arrows). This micrograph is
a brightest pixel projection of 14 optical sections taken at 1.95 µm
intervals. (C) MOIH-like labeling at the junction of the stn and the
superior esophageal (son) nerves. The four axons that are the source
of the MOIH-like labeling in the ACP can be traced through the stn to
its junction with the paired sons. Here, two of the four axons enter
the left son and two the right son. Within each
son, MOIH-like labeling in the axons became weak near the junction of
the nerve with the commissural ganglion, and further tracing was not possible
from immunoreactivity alone (not shown). This micrograph is a brightest pixel
projection of 17 optical sections taken at 1.95 µm intervals. Both B and C
are shown at the same magnification. Scale bars in A and C, 75 µm.
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Fig. 9. Mandibular organ-inhibiting hormone (MOIH)-like labeling in the
stomatogastric ganglion (STG) of Cancer productus. (A) MOIH-like
labeling in the STG. Among the regions of the stomatogastric nervous system
labeled by the MOIH antibody was the STG. Here, staining was present in two
intrinsic somata as well as in the neuropil within the ganglion. The
immunopositive neuropil appears to originate from both the arborizations of
the intrinsic immunopositive somata as well as from the arborizations of
projection neurons, which send axons to the STG via the
stomatogastric nerve (stn). (B) Higher magnification image of the
MOIH-immunopositive somata shown in A. Note that the staining in these cell
bodies is cytoplasmic and distinctly punctate. The micrographs shown in both A
and B are single optical sections. Scale bars in A and B, 75 µm.
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Fig. 10. Schematic representation of mandibular organ-inhibiting hormone (MOIH)-like
immunoreactivity in the stomatogastric nervous system (STNS) of
Cancer species. In this diagram, filled circles represent
immunopositive somata, thick lines within nerves represent immunopositive
axons and tangles of thin lines represent regions of immunopositive neuropil
or neuroendocrine release sites. In addition to labeling the anterior cardiac
plexus (ACP) and the axons innervating it, MOIH-like labeling was also evident
in other regions of the STNS. In brief, approximately a dozen somata were
labeled in each commissural ganglion (CoG), as were two in the stomatogastric
ganglion (STG). No immunopositive somata were present in the esophageal
ganglion (OG) nor were any seen in the nerves of the STNS. Immunopositive
neuropil was present in the CoGs and the STG. Extraganglionic neuropil was
present in the superior esophageal nerves (sons; most commonly in the
vicinity of the dorsal posterior esophageal nerve (dpon)], at the
junction of the sons, the esophageal nerve (on) and the
stomatogastric nerve (stn), as well as in the stn proper
[commonly near the insertion point of the anterior cardiac nerves
(acns)]. The immunopositive neuropil in the STG probably originates
from both the arborizations of the intrinsic somata as well as from the
arborizations of approximately six axons projecting from the stn
(approximately three from each son). Immunopositive axons were also
present in the circumesophageal connectives (cocs), which link the
STNS to the supraesophageal and thoracic ganglia, as well as occasionally in
the anterior lateral nerves (alns; one axon in each aln)
that emanate from the STG and innervate muscles of the gastric mill region of
the foregut.
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