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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

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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 Chung^2,3, Simon G. Webster², Horacio O. de la Iglesia¹ and Andrew E. Christie^1,4,*

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

View larger version (24K): [in a new window]   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.

View larger version (12K): [in a new window]   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.

View larger version (66K): [in a new window]   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.

View larger version (141K): [in a new window]   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.

View larger version (88K): [in a new window]   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.

View larger version (129K): [in a new window]   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.

View larger version (68K): [in a new window]   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.

View larger version (56K): [in a new window]   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.

View larger version (89K): [in a new window]   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.

View larger version (26K): [in a new window]   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.