On p. 4794, the incorrect plasmid was referenced. The plasmid used was pHyVec16 (GenBank Accession Number KP145000).
Genome sequencing has revealed examples of horizontally transferred genes, but we still know little about how such genes are incorporated into their host genomes. We have previously reported the identification of a gene (flp) that appears to have entered the Hydra genome through horizontal transfer. Here we provide additional evidence in support of our original hypothesis that the transfer was from a unicellular organism, and we show that the transfer occurred in an ancestor of two medusozoan cnidarian species. In addition we show that the gene is part of a bicistronic operon in the Hydra genome. These findings identify a new animal phylum in which trans-spliced leader addition has led to the formation of operons, and define the requirements for evolution of an operon in Hydra. The identification of operons in Hydra also provides a tool that can be exploited in the construction of transgenic Hydra strains.
Developmental processes such as morphogenesis, patterning and differentiation are continuously active in the adult Hydra polyp. We carried out a small molecule screen to identify compounds that affect patterning in Hydra. We identified a novel molecule, DAC-2-25, that causes a homeotic transformation of body column into tentacle zone. This transformation occurs in a progressive and polar fashion, beginning at the oral end of the animal. We have identified several strains that respond to DAC-2-25 and one that does not, and we used chimeras from these strains to identify the ectoderm as the target tissue for DAC-2-25. Using transgenic Hydra that express green fluorescent protein under the control of relevant promoters, we examined how DAC-2-25 affects tentacle patterning. Genes whose expression is associated with the tentacle zone are ectopically expressed upon exposure to DAC-2-25, whereas those associated with body column tissue are turned off as the tentacle zone expands. The expression patterns of the organizer-associated gene HyWnt3 and the hypostome-specific gene HyBra2 are unchanged. Structure-activity relationship studies have identified features of DAC-2-25 that are required for activity and potency. This study shows that small molecule screens in Hydra can be used to dissect patterning processes.
No abstract
TheHydranervous system is the paradigm of a simple nerve net. Nerve cells inHydra, as in many cnidarian polyps, are organized in a nerve net extending throughout the body column. This nerve net is required for control of spontaneous behavior: elimination of nerve cells leads to polyps that do not move and are incapable of capturing and ingesting prey (Campbell, 1976). We have re-examined the structure of theHydranerve net by immunostaining fixed polyps with a novel pan-neuronal antibody that stains all nerve cells. Confocal imaging shows that there are two distinct nerve nets, one in the ectoderm and one in the endoderm, with the unexpected absence of nerve cells in the endoderm of the tentacles. The nerve nets in the ectoderm and endoderm do not contact each other. High-resolution images show that the nerve nets consist of bundles of parallel overlapping neurites. Transmission and serial block face scanning electron microscopy show that nerve bundles in the ectoderm are closely associated with ectodermal muscle processes. Nerve bundles in the endoderm are separate from muscle processes. The occurrence of bundles of neurites supports a model for continuous growth and differentiation of the nerve net by lateral addition of new nerve cells to the existing net. This model was confirmed by tracking newly differentiated nerve cells.
Studies using chemical genetics allow the researcher to perform the equivalent of classical genetics in real time using molecules that can produce a phenotype when applied to either cultured cells or a whole organism. We have initiated a chemical genetic screen using the AEP strain of Hydra vulgaris to identify bioactive molecules that modulate signaling pathways involved in development and regeneration. One compound, DAC‐2–25, induced the growth of ectopic tentacles in regenerating heads and buds. Chronic exposure to DAC‐2–25 induces ectopic tentacles in H. vulgaris AEP, H. vulgaris 950f, and H. viridissima, but not H. vulgaris Zurich. In chronically treated animals, the first ectopic tentacles appear just below the tentacle ring. Subsequent tentacles emerge in a wave down the body column. We are using AEP/Zurich chimeras to identify which cell lineage is targeted by DAC‐2–25. Structure‐activity relationship (SAR) studies have been initiated to determine what features of the molecule are required for activity. Ultimately we will use affinity chromatography coupled with mass spectrometry to identify the protein target of DAC‐2–25. Crosses of AEP and Zurich have been produced with the intention of doing bulk segregant analysis as an alternative approach to identifying the gene encoding the DAC‐2–25 target protein.
The Hydra nervous system is the paradigm of a “simple nerve net”. Nerve cells in Hydra, as in many cnidarian polyps, are organized in a nerve net extending throughout the body column. This nerve net is required for control of spontaneous behavior: elimination of nerve cells leads to polyps that do not move and are incapable of capturing and ingesting prey (Campbell, 1976). We have re-examined the structure of the Hydra nerve net by immunostaining fixed polyps with a novel pan-neuronal antibody that stains all nerve cells. Confocal imaging shows that there are two distinct nerve nets, one in the ectoderm and one in the endoderm, with the unexpected absence of nerve cells in the endoderm of the tentacles. The nerve nets in the ectoderm and endoderm do not contact each other. High-resolution images show that the nerve nets consist of bundles of parallel overlapping neurites. Transmission and serial block face scanning electron microscopy show that nerve bundles in the ectoderm are closely associated with ectodermal muscle processes. Nerve bundles in the endoderm are separate from muscle processes. The occurrence of bundles of neurites supports a model for continuous growth and differentiation of the nerve net by lateral addition of new nerve cells to the existing net. This model was confirmed by tracking newly differentiated nerve cells.
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