Genes of the Hox cluster are restricted to the animal kingdom and play a central role in axial patterning in divergent animal phyla. Despite its evolutionary and developmental significance, the origin of the Hox gene cluster is obscure. The consensus is that a primordial Hox cluster arose by tandem gene duplication close to animal origins. Several homeobox genes with high sequence identity to Hox genes are found outside the Hox cluster and are known as 'dispersed' Hox-like genes; these genes may have been transposed away from an expanding cluster. Here we show that three of these dispersed homeobox genes form a novel gene cluster in the cephalochordate amphioxus. We argue that this 'ParaHox' gene cluster is an ancient paralogue (evolutionary sister) of the Hox gene cluster; the two gene clusters arose by duplication of a ProtoHox gene cluster. Furthermore, we show that amphioxus ParaHox genes have co-linear developmental expression patterns in anterior, middle and posterior tissues. We propose that the origin of distinct Hox and ParaHox genes by gene-cluster duplication facilitated an increase in body complexity during the Cambrian explosion.

The HB9 homeobox gene has been cloned from several vertebrates and is implicated in motor neuron differentiation. In the chick, a related gene, MNR2, acts upstream of HB9 in this process. Here we report an amphioxus homologue of these genes and show that it diverged before the gene duplication yielding HB9 and MNR2. AmphiMnx RNA is detected in two irregular punctate stripes along the developing neural tube, comparable to the distribution of 'dorsal compartment' motor neurons, and also in dorsal endoderm and posterior mesoderm. We propose a new homeobox class, Mnx, to include AmphiMnx, HB9, MNR2 and their Drosophila and echinoderm orthologues; we suggest that vertebrate HB9 is renamed Mnx1 and MNR2 be renamed Mnx2.

Hedgehog proteins are important cell-cell signalling proteins utilized during the development of multicellular animals. Members of the hedgehog gene family have not been detected outside the Metazoa, raising unanswered questions about their evolutionary origin. Here we report a highly unusual hedgehog-related gene from a choanoflagellate, a close unicellular relative of the animals. The deduced C-terminal domain, Hoglet-C, is homologous to the autocatalytic domain of Hedgehog proteins and is predicted to function in autocatalytic cleavage of the precursor peptide. In contrast, the N-terminal Hoglet-N peptide has no similarity to the signalling peptide of Hedgehog (Hh-N). Instead, Hoglet-N is deduced to be a secreted protein with an enormous threonine-rich domain of unprecedented size and purity (over 200 threonine residues) and two polysaccharide-binding domains. Structural modelling reveals that these domains have a novel combination of features found in cellulose-binding domains (CBD) of types IIa and IIb, and are expected to bind cellulose. We propose that the two CBD domains enable Hoglet-N to bind to plant matter, tethering an amorphous nucleophilic anchor, facilitating transient adhesion of the choanoflagellate cell. Since Hh-C and Hoglet-C are homologous, but Hh-N and Hoglet-N are not, we argue that metazoan hedgehog genes evolved by fusion of two distinct genes.

No abstract