Expression Patterns of the <i>Drosophila</i> Neuropeptide CCHamide-2 and Its Receptor May Suggest Hormonal Signaling from the Gut to the Brain

<div><p>The insect neuropeptides CCHamide-1 and -2 are recently discovered peptides that probably occur in all arthropods. Here, we used immunocytochemistry, <i>in situ</i> hybridization, and quantitative PCR (qPCR), to localize the two peptides in the fruitfly <i>Drosophila melanogaster</i>. We found that CCHamide-1 and -2 were localized in endocrine cells of the midgut of larvae and adult flies. These endocrine cells had the appearance of sensory cells, projecting processes close to or into the gut lumen. In addition, CCHamide-2 was also localized in about forty neurons in the brain hemispheres and ventral nerve cord of larvae. Using qPCR we found high expression of the CCHamide-2 gene in the larval gut and very low expression of its receptor gene, while in the larval brain we found low expression of CCHamide-2 and very high expression of its receptor. These expression patterns suggest the following model: Endocrine CCHamide-2 cells in the gut sense the quality of food components in the gut lumen and transmit this information to the brain by releasing CCHamide-2 into the circulation; subsequently, after binding to its brain receptors, CCHamides-2 induces an altered feeding behavior in the animal and possibly other homeostatic adaptations.</p> </div

The anterior midgut of adult flies containing CCHamide-2 immunoreactive endocrine cells.

<p>(See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g003" target="_blank">figure 3</a> for a schematic drawing.) A. An overview of the anterior midgut showing the gut lumen and CCHamide-2 containing endocrine cells that project small hair-like processes (arrows) into the lumen. Note that these cells are 25-50 µm. Scale bar = 50 µm. B. Close up of the endocrine cells in the anterior midgut, showing that their bases are in contact with the body cavity and that their more slender apical parts project into the lumen. Scale bar = 20 µm.</p

A double-labeling experiment as in Figure 6, but now showing the posterior part of the ventral nerve cord.

<p>A. A dorsal view of the dorsal plane of the ventral nerve cord stained with the synapsin monoclonal antibody, showing the two columns of neuropil of the ventral nerve cord (red). B. The same focal plane as in A, but now showing staining with the CCHamide-2 antiserum (green). C. Merge of A and B. Dorsally located CCHamide-2 immunoreactive processes can be seen projecting in a longitudinal orientation over the whole nerve cord. Scale bar = 20 µm.</p

qPCR data for the expression of the <i>D. melanogaster</i> CCHamide-1 gene (CG14358), the CCHamide-1 receptor gene (CG14593), the CCHamide-2 gene (CG14375), and CCHamide-2 receptor gene (CG30106) in different organs or body parts of larval and adult flies.

<p>Third instar larval brain (a); third instar larval gut (b); third instar larval carcass (= rest of the body without brain and gut) (c); adult male brain (d); adult male gut (e); adult male carcass (= rest of the body without brain and gut) (f); adult female brain (g); adult female gut (h); adult female carcass (= rest of the body without brain and gut) (i). At least 25 organs were pooled for each qPCR measurement. These pools are the same in (A)-(D). The mRNA concentrations in each panel are given relative to column b (b=1). Other conditions are as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g008" target="_blank">Figure 8</a>. A. Expression of the <i>D. melanogaster</i> CCHamide-1 gene. B. Expression of the <i>D. melanogaster</i> CCHamide-1 receptor gene. C. Expression of the CCHamide-2 gene. D. Expression of the CCHamide-2 receptor gene. Statistical analyses showed that there is a significant difference (P < 0.001) between the columns a and b; d and e; and g and h in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9C and Figure 9D</a>.</p

Schematic drawing of the localizations of the CCHamide-2 immunoreactive neurons and neuropil in the CNS of third instar larvae.

<p>The drawing shows the two hemispheres and the ventral nerve cord. The central neuropil of the larval CNS (stained with a synapsin mouse monoclonal antibody, cf. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g006" target="_blank">Figure 6 and Figure 7</a>) is outlined by green lines and shades. Neuronal perikarya and neuropil are drawn in red. Weakly immunoreactive perikarya are drawn as open red symbols. The perikarya indicated by 1 and 2 in the right hemisphere are located dorsally in each hemisphere; the perikarya indicated by 4, and 5, and 6 are located in the ventral parts of the hemispheres. The neuropils indicated by 3 as red dots in the anterior parts of the central neuropil are located partially ventrally and partially medially between the levels of the neurons 1 and 4. All perikarya in the ventral nerve cord are located in the ventral part of this nerve cord. They belong to the three fused thoracic ganglia.</p

Endocrine cells in the body wall of the midgut of third instar <i>D. melanogaster</i> stained for CCHamide-2.

<p>A. An overview, showing numerous endocrine cells in the anterior part of the anterior midgut (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g003" target="_blank">Figure 3</a> for a schematic drawing). Scale bar = 100 µm. B. A closer view of the endocrine cells, showing protrusions (arrow) directed toward the lumen of the gut. Scale bar = 20 µm. C. Close-up of a single endocrine cell. The basal part of the cell is in contact with the body circulation (the border of the gut epithelial cells and body cavity is stippled). Note that these cells are 25-40 µm in length. Scale bar = 20 µm.</p

Cartoon of our proposed mechanism for the hormonal action of CCHamide-2 in third instar larvae.

<p>This hypothetical model is based on the sensory nature of the CCHamide-2 immunoreactive endocrine cells in the gut (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g001" target="_blank">Figure 1</a>) and on our findings that there is abundant expression of the CCHamide-2 receptor gene in the brain (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9D</a>, a), while this expression is virtually absent in the gut (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9D, b</a>), combined with the high CCHamide-2 peptide gene expression in the gut (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g009" target="_blank">Figure 9C, b</a>). This model predicts that the quality of the food in the gut lumen is sensed by the CCHamide-2 immunoreactive endocrine cells in the gut wall. These cells release the CCHamide-2 peptides into the circulation, after which they reach the brain and bind to the CCHamide-2 receptors. This binding starts a cascade that leads to an altered (adapted) feeding behavior of the animal. In addition to this long-distance hormonal CCHamide-2 signaling, there is a local (synaptic or paracrine) CCHamide-2 signaling in the larval CNS, possibly also associated with feeding.</p

Neurons in the larval CNS stained with CCHamide-2 antisera.

<p>These preparations are somewhat compressed, because of mounting without the application of spacer rings. These spacer rings were present in the preparations shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g006" target="_blank">Figure 6 and Figure 7</a>. For a schematic representation of the neurons and neuropil in the two larval brain hemispheres and ventral nerve cord, see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g005" target="_blank">Figure 5</a>. A. Ventral view of one hemisphere of the brain, showing 7-8 immunoreactive neurons located in the central region (long arrows) and processes (neuropil) projecting to other regions of the brain (short arrow). This neuropil corresponds to the neuropil indicated by number 3 in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0076131#pone-0076131-g005" target="_blank">Figure 5</a>. A piece of the ring gland (asterisk) is also visible, as well as a piece of the ventral nerve cord (VNC). Scale bar = 50 µm. B. Ventral view of another brain hemisphere with the ventral nerve cord. At least 12 nerve cells (short arrows) can be seen here symmetrically ordered along the midline of the ventral nerve cord (VNC). Scale bar = 50 µm.</p

qPCR data for the expression of the <i>D. melanogaster</i> CCHamide-1 gene (CG14358), the CCHamide-1 receptor gene (CG14593), the CCHamide-2 gene (CG14375), and the CCHamide-2 receptor gene (CG30106), in different developmental stages.

<p>Embryos, 20-24 h after egg laying (a); first instar larvae, 48 h after egg laying (b); second instar larvae, 72 h after egg laying (c); third instar larvae, 96 h after egg laying (d); pupae, 7 d after egg laying (e); and adult male (f) and female (g) flies both 10 d after egg laying, or 1 d after eclosion. At least 25 animals were pooled from each developmental stage. These pools are the same in (A)-(D). The mRNA concentrations in each panel are given relative to column g (g=1). The qPCR experiments were run as triplicates. The vertical bars in each column (which are sometimes smaller than the lines of the column) represent SEM. A. Expression of the <i>D. melanogaster</i> CCHamide-1 gene in the different developmental stages. B. Expression of the <i>D. melanogaster</i> CCHamide-1 receptor gene. C. Expression of the CCHamide-2 gene. D. Expression of the CCHamide-2 receptor gene.</p

Schematic representations of the guts from third instar larvae and adult flies.

<p>The numbers give total numbers of counted CCHamide immunoreactive endocrine cells per midgut segment. These numbers vary from animal to animal and we have, therefore, given both the lowest and highest number counted in at least 50 animals. The colors indicate the concentrations of CCHamide immunostained cells from blue (low concentration) to green (intermediate) to red (high concentration). A. third instar larvae. Both the CCHamide-1 and -2 endocrine cells occur in the anterior midgut and in the anterior part of the posterior midgut. B. Adult male/female flies. The CCHamide-1 and -2 endocrine cells occur mainly in the anterior midgut and to a somewhat lesser extend in the posterior midgut.</p