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The vertebrate embryonic hindbrain is segmented into rhombomeres. Gene expression studies suggest that amphioxus, the closest invertebrate relative of vertebrates, has a hindbrain homolog. However, this region is not overtly segmented in amphioxus, raising the question of how hindbrain segmentation arose in chordate evolution. Vertebrate hindbrain segmentation includes the patterning of cranial motor neurons, which can be identified by their expression of the LIM-homeodomain transcription factor islet1. To learn if the amphioxus hindbrain homolog is cryptically segmented, we cloned an amphioxus gene closely related to islet1, which we named simply islet. We report that amphioxus islet expression includes a domain of segmentally arranged cells in the ventral hindbrain homolog. We hypothesize that these cells are developing motor neurons and reveal a form of hindbrain segmentation in amphioxus. Hence, vertebrate rhombomeres may derive from a cryptically segmented brain present in the amphioxus/vertebrate ancestor. Other islet expression domains provide evidence for amphioxus homologs of the pineal gland, adenohypophysis, and endocrine pancreas. Surprisingly, homologs of vertebrate islet1-expressing spinal motor neurons and Rohon-Beard sensory neurons appear to be absent. (C) 2000 Academic Press.