Allatotropin: an ancestral myotropic neuropeptide involved in feeding

Abstract

Background: Cell-cell interactions are a basic principle for the organization of tissues and organs allowing them to perform integrated functions and to organize themselves spatially and temporally. Peptidic molecules secreted by neurons and epithelial cells play fundamental roles in cell-cell interactions, acting as local neuromodulators, neurohormones, as well as endocrine and paracrine messengers. Allatotropin (AT) is a neuropeptide originally described as a regulator of Juvenile Hormone synthesis, which plays multiple neural, endocrine and myoactive roles in insects and other organisms.

Methods: A combination of immunohistochemistry using AT-antibodies and AT-Qdot nanocrystal conjugates was used to identify immunoreactive nerve cells containing the peptide and epithelial-muscular cells targeted by AT in Hydra plagiodesmica. Physiological assays using AT and AT- antibodies revealed that while AT stimulated the extrusion of the hypostome in a dose-response fashion in starved hydroids, the activity of hypostome in hydroids challenged with food was blocked by treatments with different doses of AT-antibodies.

Conclusions: AT antibodies immunolabeled nerve cells in the stalk, pedal disc, tentacles and hypostome. AT-Qdot conjugates recognized epithelial-muscular cell in the same tissues, suggesting the existence of anatomical and functional relationships between these two cell populations. Physiological assays indicated that the AT-like peptide is facilitating food ingestion.

Significance: Immunochemical, physiological and bioinformatics evidence advocates that AT is an ancestral neuropeptide involved in myoregulatory activities associated with meal ingestion and digestion.

Figure 1. Schematic representation of Hydra sp.

Cross sections of two regions of the hydroid body. A: hypostome. B: pedal disk. C: detail of the nerve cells forming a net on the base of the epidermis and gastrodermis. cm: circular muscular layer formed by the contractile extensions of the nutritive muscle cells; cn: cnidocito; eg: epithelial gland cell; ep: epithelial-muscular cell; g: mucous and enzymatic gland cell; i: interstitial cell; lm: longitudinal muscle layer formed by the contractile extensions of the epithelial-muscular cells; n: nerve cell and ns: neurosensory cell.

Figure 2. AT-like immunoreactivity.

A: Panoramic view of a specimen labeled with anti-AT antiserum showing the presence of immunoreactive material in different parts of the body. B: Similar view in which the primary antibody incubation was replaced for saline solution. C: Similar view as in A showing the co-existence of f-actin filaments and immunoreactive material D: A magnified view showing the spatial relationship between AT-like nerve cells and epithelial-muscular cells. Motor nerve cells (green) and epithelial-muscular cells labeled with phalloidin (red).

Figure 3. AT-like immunoreactivity.

A: confocal 3D reconstruction showing AT-like nerve cells in a net-like arrangement and f-actin filaments. B: confocal 3D reconstruction of a cross-section of the hydroid body wall showing the anatomical relation between AT-like motor nerve cells and epithelial-muscular cells, as well as the colocalization of the cells producing the peptide, with contractile cells. C: Schematic representation of B including sensory nerve cells (blue) (modified from [69]). D and E: Panoramic (D) and detailed view (E) of an hydroid showing the presence of allatotropic-like cells in the pedal disc. Motor nerve cells (green) and epithelial-muscular cells labeled with phalloidin (red).

Figure 4. Qdot-AT and AST-C labeling and AT-immunoreactivity.

A: Pedal disc view showing the distribution of cells labeled with Qdot-AT (_*) B: Magnified view showing the cells in clusters C: View of the body wall of a hydroid showing AT recognizing epithelial-muscular cells at the stalk, as well as the distribution of the cells in a circular arrangement D: Magnified view of the epithelial-muscular circular cells at the stalk. Note the cells in two circular rows, as well as the presence of endocytic vesicles (arrow). E: View of the body wall of a hydroid showing AST-C recognizing epithelial-muscular cells at the stalk. Note that the distribution of the labeled cells is different to the labeling corresponding to the AT peptide. F: Magnified view of the same epithelial-muscular circular cells, also showing endocytic vesicles.

Figure 5. Qdot-AT labeling and AT-immunoreactivity.

A: Panoramic view at the bottom of the stalk showing the pedal disc of a hydroid. B: Magnified view of the stalk suggesting. a morpho-functional relationship between both cell populations. Inset: detailed view of a nerve cell. Note that the morphology and spatial orientation of the cell and its projection clearly resembles the motor nature of the nerve cells. C and D: show both channels independently. Note that the position of nerve cells (C), clearly corresponding with non-labeled spaces in (D) (open circles), showing that nerve cells are intercalated between epithelial-muscular cells recognizing AT peptide and reinforcing the morpho functional relationship between these two populations. Epithelial-muscular cells recognizing AT-Qdots conjugates (red) and AT-like motornerve cells (green). Colocalization of markers (yellowish green).

Figure 6. Myotropic activity of AT-like peptide and feeding.

A: Specimen of H. plagiodesmica after the ingestion of an A. salina egg. B: confocal 3D reconstruction of an hydroid after double labeling with AT-antiserum (green) and nanocristals (red). Note the colocalization of both markers at the level of the hypostome (arrows) and gastroenteron (arrow heads). C: Physiological assay demonstrating the AT involvement in feeding movements. Black columns: Hypostome response of 72 h starved hydroids to treatment with different doses of AT. Grey columns: Hypostome response of 72 h starved hydroids challenged with food and exposed to different doses of AT-antiserum. The results are expressed as percentage of individuals that completely extruded the hypostome. (*): Significant differences between hydroids treated with AT or AT-antiserum and controls.

Figure 7. Phylogram of AT-receptor.

Species from the same phylum are labeled by a similar gray tone blocks. The tree is enrooted using the D. melanogaster FMFRamide receptor [50].