Two-color double-labeling in situ hybridization of whole-mount Hydra usingRNA probes for five different Hydra neuropeptide preprohormones: evidence for colocalization

The freshwater polyp Hydra magnipapillata has a primitive nervous system th at produces at least. three distinct classes of neuropeptides: various pept ides having the C-terminal sequence Arg-Phe-NH2 (the Hydra-RFamide family), Leu-Trp-NH2 (the Hydra-LWamide family), and a single peptide having the C- terminal sequence Lys-Val-NH2 (Hydra-KVamide). The various Hydra-RFamides a re synthesized by three different preprohormones: preprohormone-A, -B, and -C. The various Hydra-LWamides are synthesized by a single preprohormone (p repro-Hydra-LWamide), as is Hydra-KVamide (prepro-Hydra-KVamide). Using a w holemount double-labeling two-color in situ hybridization technique and RNA probes specific for each of these five Hydra preprohormone mRNAs, we found that specific sets of neurons express each of the five preprohormones, exc ept for the peduncle region of Hydra tan area just above the basal disk), w here a population of neurons exists that expresses both preprohormones-A an d prepro-Hydra-KVamide mRNAs. The functional significance of this coexpress ion is unclear. This is the first report on the coexpression of two well-ch aracterized preprohormones (yielding two well-characterized neurohormone fa milies) in cnidarians. This report also shows that there are at least six n eurochemically different populations of neurons in Hydra.