is expanded by a thickening of the walls and expansion of the central canal of spinal cord into three primary brain vesicles: The prosencephalon (forebrain), mesencephalon (midbrain) and rhombencephalon (hindbrain), further differentiated in the various vertebrate groups. Two laterally placed eyes form around outgrowths from the midbrain, except in hagfish, though this may be a secondary loss. The forebrain is well-developed and subdivided in most tetrapods, while the midbrain dominates in many fish and some salamanders. Vesicles of the forebrain are usually paired, giving rise to hemispheres like the cerebral hemispheres in mammals. The resulting anatomy of the central nervous system, with a single hollow nerve cord topped by a series of (often paired) vesicles, is unique to vertebrates. All invertebrates with well-developed brains, such as insects, spiders and squids, have a ventral rather than dorsal system of ganglions, with a split brain stem running on each side of the mouth or gut. Molecular signatures In addition to the morphological characteristics used to define vertebrates (i.e. the presence of a notochord, the development of a vertebral column from the notochord, a dorsal nerve cord, pharyngeal gills, a post-anal tail, etc.), molecular markers known as conserved signature indels (CSIs) in protein sequences have been identified and provide distinguishing criteria for the subphylum Vertebrata. Specifically, 5 CSIs in the following proteins: protein synthesis elongation factor-2 (EF-2), eukaryotic translation initiation factor 3 (eIF3), adenosine kinase (AdK) and a protein related to ubiquitin carboxyl-terminal hydrolase are exclusively shared by all vertebrates and reliably distinguish them from all other metazoan. The CSIs in these protein sequences are predicted to have important functionality in vertebrates. A specific relationship between Vertebrates and Tunicates is also strongly supported by two CSIs found in the proteins Rrp44 (associated with exosome complex) and serine palmitoyltransferase, that are exclusively shared by species from these two subphyla but not Cephalochordates, indicating Vertebrates are more closely related to Tunicates than Cephalochordates. Evolutionary history External relationships Originally, the "Notochordata hypothesis" suggested that the Cephalochordata is the sister taxon to Craniata (Vertebrata). This group, called the Notochordata, was placed as sister group to the Tunicata (Urochordata). Although this was once the leading hypothesis, studies since 2006 analyzing large sequencing datasets strongly support Olfactores (tunicates + vertebrates) as a monophyletic clade, and the placement of Cephalochordata as sister-group to Olfactores (known as the "Olfactores hypothesis"). As chordates, they all share the presence of a notochord, at