A strategy for mapping bicoid on the phylogenetic tree

In Drosophila, a gradient of Bicoid protein (BCD) originates from prelocalized mRNA at the anterior pole of the egg and establishes developmental programs including those for the larval head and thorax in a concentration-dependent manner [1,2]. Orthologous proteins have been reported only from cyclorrhaphan flies [3,4]. However, a BCD-like determinant has been postulated for a large variety of insects including leafhopper, beetles and midges [5–7]. It therefore remains to be determined whether diverged bicoid (bcd) orthologs exist in other insect orders. bcd encodes a homeodomaincontaining transcription factor, and is located immediately upstream of zerknüllt (zen) in the Hox gene complex (Hox-C) of various drosophilids [8,9]. Based on these findings M. Akam (in [10]) suggested that bcd originated as a result of a gene duplication involving zen. However, it has been difficult to test this hypothesis due to rapid sequence evolution of bcd and zen [3]. Only recently, sequence analysis of a bcd homolog from the basal cyclorrhaphan fly Megaselia (Phoridae) provided direct support for a sister-gene relationship of bcd and zen, and implicated the position of bcd upstream of zen in the Hox-C as ancestral [11]. We tested for linkage of bcd and zen in the blowflies Calliphora erythrocephala and Lucilia sericata (Calliphoridae, Diptera) where bcd homologs have been identified previously [4]. Using degenerate PCR primers we isolated zen homeoboxes from both species (Figure 1a). Specific nested primer pairs, when used in long range PCR, amplify single genomic DNA fragments of 12 kb (Calliphora) and 16 kb (Lucilia), linking the homeoboxes of the bcd and zen homologs (Figure 1b). The identity of each fragment was verified by Southern-blot hybridization and terminal sequencing . Thus, bcd is linked to a Hox class 3 gene in blowflies (Calyptratae) as it is in drosophilids (Acalyptratae), and the 5′ to 3′ orientation of bcd and zen with respect to one another is conserved. This linkage was probably inherited from the common ancestor of the monophyletic Schizophora (Acalyptratae and Calyptratae) [12], which comprise the majority of family-level diversity of Diptera [13]. Our observations strongly support the hypothesis that bcd arose as a tandem duplication of zen within the Hox-C (Figure 2). We conclude that analysis of the relevant Hox-C portion of selected species provides a means to map the origin of bcd on the phylogenetic tree. In the red flour beetle Tribolium castaneum, a holometabolous insect distantly related to flies, orthologs of the eight arthropod homeotic genes [14], as well as ftz [15] and zen [16] are arrayed in the same order as their Drosophila counterparts (Figure 2), an order that has been maintained for over 300 million years. To determine whether a highly diverged bcd ortholog is located in its predicted position in the Tribolium Hox-C we sequenced a BAC clone spanning the region from the 5′ exon of mxp/Hox2 to Tcftz (Genbank accession AF321227). We analyzed this sequence using BLAST (NCBI), and the BCM genefinder program (Baylor College of Medicine Web site) to predict open reading frames and putative transcription units. We found a second zen gene (see the homeodomain comparisons in Figure 1a) immediately downstream of the one previously identified. These genes are most likely the result of an independent duplication in the lineage leading to Tribolium. Although known transcription units were faithfully predicted, no other homeodomain-encoding sequences were found. The Tribolium Hox-C does not, therefore, contain a bcd ortholog in the interval between mxp/Hox2 and TcDfd/Hox4. Conservation of the relative positions of zen and bcd in blowflies Magazine R43