A new case of neuropeptide coexpression (RGamide and LWamides) in Hydra,found by whole-mount,two-color double-labeling in situ hybridization

The freshwater polyp Hydra has a primitive nervous system that expresses at least six different neuropeptide genes: (1) three genes, coding for the preprohormones-A, -B, and -C that each gives rise to a variety of peptides with the C-terminal sequence Arg-Phe-NH(2) (the Hydra-RFamides); (2) one gene, coding for a preprohormone that gives rise to five peptides with the C-terminal sequence Leu-Trp-NH(2) (the Hydra-LWamides); (3) one gene, coding for a preprohormone that produces a peptide with the C-terminal sequence Lys-Val-NH(2) (Hydra-KVamide, also called Hym-176); and (4) one gene, coding for a preprohormone that gives rise to a peptide with the C-terminal sequence Arg-Gly NH(2) (Hydra-RGamide, also called Hym-355). In a previous paper, we described that a population of neurons in the peduncle (a region just above the foot) of Hydra coexpresses the preprohormone-A and KVamide genes, whereas neurons in the other regions only express either the preprohormone-A, -B, -C, LWamide, or the KVamide genes. Here, we investigated the RGamide gene expression, using whole-mount, two-color double-labeling in situ hybridization, and found that neurons in the basal disk (foot), gastric region, hypostome (a region around the mouth), and tentacles coexpress this gene together with the LWamide gene. A small population of neurons in the hypostome and upper gastric region expresses only the LWamide gene. No coexpression of the RGamide gene with any of the other neuropeptide genes was observed. This is the second example of coexpression of two neuropeptide genes in cnidarians. It demonstrates that many neurons in the primitive nervous systems of cnidarians use combinations ("cocktails") of neuropeptides for their signaling. It also shows that Hydra has at least seven neurochemically different populations of neurons.