The invertebrate chordate amphioxus gives clues to vertebrate origins.

Amphioxus (cepholochordates) have long been used to infer how the vertebrates evolved from their invertebrate ancestors. However, some of the body part homologies between amphioxus and vertebrates have been controversial. This is not surprising as the amphioxus and vertebrate lineages separated half a billion years ago-plenty of time for independent loss and independent gain of features. The development of new techniques in the late 20th and early 21st centuries including transmission electron microscopy and serial blockface scanning electron microscopy in combination with in situ hybridization and immunocytochemistry to reveal spatio-temporal patterns of gene expression and gene products have greatly strengthened inference of some homologies (like those between regions of the central nervous system), although others (like nephridia) still need further support. These major advances in establishing homologies between amphioxus and vertebrates, together with strong support from comparative genomics, have firmly established amphioxus as a stand-in or model for the ancestral vertebrate.