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First published online January 31, 2007
Journal of Experimental Biology 210, 699-714 (2007)
Published by The Company of Biologists 2007
doi: 10.1242/jeb.02696

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Midgut epithelial endocrine cells are a rich source of the neuropeptides APSGFLGMRamide (Cancer borealis tachykinin-related peptide Ia) and GYRKPPFNGSIFamide (Gly¹-SIFamide) in the crabs Cancer borealis, Cancer magister and Cancer productus

Andrew E. Christie^1,2,*, Kimberly K. Kutz-Naber³, Elizabeth A. Stemmler⁴, Alexandra Klein¹, Daniel I. Messinger¹, Christopher C. Goiney¹, Anna J. Conterato⁴, Emily A. Bruns⁴, Yun-Wei A. Hsu¹, Lingjun Li^3,5 and Patsy S. Dickinson⁶

¹ Department of Biology, University of Washington, Box 351800, Seattle, WA 98195-1800, USA
² Mount Desert Island Biological Laboratory, PO Box 35, Old Bar Harbor Road, Salisbury Cove, ME 04672, USA
³ Department of Chemistry, University of Wisconsin-Madison, 1101 University Avenue, Madison, WI 53706-1369, USA
⁴ Department of Chemistry, Bowdoin College, 6600 College Station, Brunswick, ME 04011, USA
⁵ School of Pharmacy, University of Wisconsin-Madison, 777 Highland Avenue, Madison, WI 53705-2222, USA
⁶ Department of Biology, Bowdoin College, 6500 College Station, Brunswick, ME 04011, USA

View larger version (32K): [in this window] [in a new window]   Fig. 1. Schematic representation of the digestive tract, including the midgut, of Cancer crabs. The digestive tract of the investigated Cancer species can be divided into three distinct regions: the foregut [comprised of the oesophagous (OE), the cardiac sac (CS), the gastric mill (GM) and the pylorus (PY)], the midgut (colored) and the hindgut. The midgut region is comprised of midgut proper, the highly branched hepatopancreas (not included in this schematic) and three associated caeca: the paired anterior midgut caeca (AMC), which arise laterally, one on either side of the midgut just posterior to the pylorus, and the single posterior midgut caecum (PMC), which arises dorsally, at or just anterior to the midgut/hindgut transition (MHT). Regions of the midgut where SIFamide-like immunoreactivity has been localized are shown in green; those in which tachykinin-like labeling was seen are colored red.

View larger version (105K): [in this window] [in a new window]   Fig. 2. General organization and morphology of peptidergic endocrine cells in the Cancer anterior and posterior midgut caeca epithelia. Regardless of species (Cancer productus shown), immunolabel (anti-substance P in A and anti-SIFamide in B and C) or location within the midgut caeca (posterior midgut caecum in A and anterior midgut caecum in B and C), the gross organization and morphology of the intrinsic peptidergic endocrine cells was similar. Specifically, all cells possessed an enlarged basal region and extended a thin, beaded projection apically toward the midgut lumen. This organization is shown in longitudinal-section in A and in cross-section in B. The morphology of one peptidergic cell from B (arrow) is shown at higher magnification in C. (A) A single optical section. (B) A brightest pixel projection of 42 optical sections collected at 1.05-µm intervals. (C) A brightest pixel projection of 22 optical sections collected at 0.75-µm intervals. Scale bars, 200 µm (A,B) and 25 µm (C).

View larger version (67K): [in this window] [in a new window]   Fig. 3. General organization and morphology of peptidergic epithelial endocrine cells in the midgut proper (MG) of Cancer crabs. Regardless of species (Cancer productus shown), immunolabel (anti-substance P shown) or location within the midgut proper (posteriormost portion of the midgut shown), the gross organization and morphology of the intrinsic peptidergic endocrine cells was similar. All cells possessed an enlarged basal region and extended a short beaded projection apically toward the midgut lumen. This organization is shown in a low-magnification view of the flattened midgut in A and in a high-magnification view of two immunolabeled cells in B. (A) A brightest pixel projection of 61 optical sections collected at 2.1-µm intervals taken at the midgut/hindgut transition (MHT); the boundary of each region is delineated. Note that no immunolabeling is present in the hindgut (HG). In this image, faint autofluorescence can be seen in the muscle fibers overlying both the MG and HG. (B) A brightest pixel projection of nine optical sections collected at 1.95-µm intervals showing two labeled endocrine cells at high magnification. Note that both immunopositive cells appear to span the entire epithelium, abutting both the midgut lumen and the hemocoel. Scale bars, 200 µm (A) and 25 µm (B).

View larger version (99K): [in this window] [in a new window]   Fig. 4. The nuclei of Cancer midgut epithelial endocrine cells are located within their enlarged basal region [Cancer magister posterior midgut caecum (PMC) is shown as an example]. (A) Cross-section of the posterior midgut caecum showing the overall distribution of (A1) substance P-like immunoreactivity (pseudocolored red) and (A2) DAPI labeling (pseudocolored blue) in this structure. A3 is a pseudocolored merger of images A1 and B2. All images in this set are brightest pixel projections of 40 optical sections collected at 1.95-µm intervals, with the substance P and DAPI images collected sequentially. A1 and A2 are shown at the same scale, with the scale bar in A2 equal to 200 µm. The scale bar in A3 is also equal to 200 µm. (B) One immunopositive endocrine cell from A shown at higher magnification. When the images of the substance P immunoreactivity (B1) and DAPI label (B2) are merged (B3), the nucleus of the epithelial endocrine cell (arrow in B2 and B3) can clearly be seen to reside in the enlarged basal region. It should be noted that many other nuclei are present in this micrograph. The large elongate nuclei in the lower portion of B2 and B3 are probably those of epithelial cells, whereas the small, round nuclei in the upper portion of the image may be those of hemocytes. As in A, the substance P and DAPI images shown in B were collected sequentially. All images in this set are brightest pixel projections of 28 optical sections collected at 1.05-µm intervals. B1 and B2 are shown at the same scale, with the scale bar in B2 equal to 25 µm. The scale bar in B3 is also equal to 25 µm.

View larger version (93K): [in this window] [in a new window]   Fig. 5. SIFamide- and tachykinin-related peptide-like immunopositive endocrine cells are differentially distributed within the Cancer midgut epithelium. In each of the species investigated (Cancer borealis shown), the cells labeled by the SIFamide and substance P antibodies were differentially distributed. Specifically, the SIFamide (SIFa)-stained cells (pseudocolored green) were restricted to the epithelium of the anterior portion of the midgut proper and the anterior midgut caeca (shown in A), whereas substance P (Sub P) immunopositive cells (pseudocolored red) were concentrated in the posterior portion of the midgut proper and the posterior midgut caecum (shown in B). As the few substance P-like immunopositive cells seen in the anterior midgut (arrows) were not among those labeled by the SIFamide antibody, and vice versa (e.g. the red and green, but not yellow, cells present in A), no colocalization of the two peptides is apparent in the midgut. It should be noted that some epithelial endocrine cells possess a short, thin, basal process that projects along the outer surface of the midgut (arrows in B). This type of projection was seen in a subset of both the SIFamide- and TRP-like immunopositive cells (TRP cell shown). A1 (anti-SIFamide) and A2 (anti-substance P) are brightest pixel projections of 28 optical sections collected at 1.05-µm intervals. Both labels were imaged simultaneously. A3 is a merge of A1 and A2. B1 (anti-SIFamide) and B2 (anti-substance P) are brightest pixel projections of 26 optical sections collected at 1.05-µm intervals. Both labels were imaged simultaneously. B3 is a merge of B1 and B2. A1, A2, B1 and B2 are all shown at the same scale, with the scale bar in B2 equal to 200 µm. A3 and B3 are shown at the same scale, with the scale bar in B3 also representing 200 µm.

View larger version (8K): [in this window] [in a new window]   Fig. 6. Direct MALDI-FTMS analysis of midgut tissues. Data presented in this figure are from Cancer borealis, although identical peptide identifications were achieved from both Cancer magister and Cancer productus. Regardless of species or tissue, spectra were measured using DHB as the matrix, with conditions optimized for accumulation of m/z 1500. (A) A representative spectrum from a small piece of posterior midgut caecum (PMC). In PMC samples, an intense peak appearing at m/z 934.49 was consistently detected at a high relative abundance. This peak was identified as APSGFLGMRamide (CabTRP Ia), based upon the m/z value measured using internal calibration with poly(propylene glycol). Spectra of the PMC samples showed no indication of a peak corresponding to GYRKPPFNGSIFamide (Gly1-SIFamide), i.e. m/z 1381.74, or any other known SIFamide isoform. (B) A representative spectrum from a small piece of anterior midgut caecum (AMC). In AMC samples, a peak at m/z 1381.74 (corresponding to the [M+H]+ ion for Gly1-SIFamide) was detected in approximately 95% of the spectra measured; a peak at m/z 934.49 (corresponding to the [M+H]+ ion for CabTRP Ia) was detected in approximately 40% of the spectra. Because of the low intensities of these peptide peaks, only accurate mass measurements were used for peptide identification in this tissue.

View larger version (15K): [in this window] [in a new window]   Fig. 7. Detection of APSGFLGMRamide (CabTRP Ia) in the releasate from posterior midgut caecum (PMC) samples. (A) MALDI-FTMS spectrum of 0.5 µl of sample taken after a single PMC sample was immersed in high-K+ saline containing an inhibitor cocktail for 1 h at 4°C. (B) MALDI-FTMS spectrum of 0.5 µl of sample taken after a single PMC sample was immersed in physiological saline containing an inhibitor cocktail for 1 h at 4°C prior to tissue transfer to high-K+ saline. The arrow indicates the m/z position where CabTRP Ia would be found, if present in the sample. Spectra were measured using DHB as the matrix, and conditions were optimized for ions of m/z 1000, using the accumulation of 30 laser shots. Both A and B are shown at the same (m/z) scale.

View larger version (22K): [in this window] [in a new window]   Fig. 8. Detection of APSGFLGMRamide (CabTRP Ia) in the hemolymph of unfed and fed Cancer productus using MALDI-FTMS. To assess whether or not circulating levels of Gly1-SIFamide and/or CabTRP Ia are influenced by the feeding status of an individual animal, hemolymph samples were collected and analyzed from animals held without food for approximately seven days (A), as well as from those held without food for approximately one week but allowed to feed at will for 2 h prior to sampling (B). As can be seen in the representative spectra, a peak corresponding to the [M+H]+ ion for CabTRP Ia, i.e. m/z 934.49, was detectable only in the hemolymph of unfed animals. Gly1-SIFamide was not detected in spectra from either unfed or fed individuals. With the exception of the inset, both A and B are shown at the same (m/z) scale.

View larger version (50K): [in this window] [in a new window]   Fig. 9. Schematic representations of possible triggers for peptide release, including that of CabTRP Ia, from Cancer midgut epithelial endocrine cells. In these schematics, intrinsic endocrine cells are colored red and epithelial cells are colored grey. At present, the cues triggering secretion of paracrines/hormones from the intrinsic endocrine cells of the crab midgut epithelium are unknown. However, two classes of epithelial endocrine cells (`open-type' and `closed-type') have been proposed, based on ultrastructural morphology (Endo and Nishiitsutsiji-Uwo, 1981; Fujita et al., 1988). (A) In open-type endocrine cells, the apical projections span the entirety of the epithelium, projecting into the gut lumen. It is proposed that these cells monitor the extracellular environment of the lumen, initiating (red arrows) or stopping secretion of hormones/paracrines when a threshold level of some chemical/ionic cue is achieved (shown here as a color gradient in the lumen). (B) In closed-type cells, the apical projections do not extend into the lumen. These cells are believed to be mechanosensory, monitoring changes in distension, which trigger (red arrows) or stop the release of hormonal/paracrine signaling agents. It should be noted that regardless of cell type, it is unclear how large the sphere of influence (pink oval in B) might be for a peptide released from gut epithelial endocrine cells. Likewise, it is not clear whether there is a directionality to release from these cells. Given that our study shows that circulating levels of CabTRP Ia are elevated in starved animals, and previous work has demonstrated a myotropic action for it on the musculature of the foregut (Messinger et al., 2005), we hypothesize that the TRP released from the midgut endocrine cells may play a crucial role in ensuring foregut muscle contraction in times of limited food availability. It should be noted that the endocrine cells in both panels of this schematic are highly stylized and should not be interpreted as representative of the morphology of epithelial endocrine cells in a general sense.