Hox genes disobey colinearity and do not distinguish head from tail during planarian regeneration.

The Hox cluster genes encode a family of transcriptions factors that contain the homeodomain and determine the anteroposterior positional values in a wide range of metazoan, from nematodes to vertebrates. The genomic organisation of these genes is colinear with their expression domains in development (Duboule and Dolle, 1989). In contrast, during axolotl limb regeneration (Gardiner et a1.1995) Hox genes are re-expressed non-colinearly, and seem to play an active function in proximodistal determination. Planarians can regenerate along any body axis: anteriorly (head regeneralion), posteriorly (Iail regeneration), bilaterally (left to right and right to left) and intercalary (between head and tail). Since planarians show clear anteroposterior (headto-tail) polarity, we reasoned that Dthox genes may be instrumental in defining this polarity in novel patterning events that do not occur in development or in amphibian regeneration. We used two sets of degenerate oligonucleotides complementary to the conserved first and third helix of the homeobox to amplify, by PCR, genomic DNA or cDNA from regenerative blastemas or libraries. Subsequent screening of genomic and cDNA libraries with the PCR-amplified sequences allowedus to isolateseven differentDugesia(G)tigrinaHox genes, whichwe catl DthoxA through Dthox-G. Whole-mount in situ hybridisation studies were performed to study the patterns of Dlhox expression from the beginning (1 hour) to the end (15 days) of different types of regeneration. Negative controls were performed with sense probes (Figure 2A). Sequence analysis and comparison with the homeodomain and flanking regions of Hox cluster genes allowed us to order the planarian Dthox genes in three main groups. Dthox-B and -G show the highest similarity at the amino acid level (70%) with representatives of the paralogous groups(PG) 2 and 3. Some specific residues scattered through the homeodomain and in the flanking regions related to PG's 2 and 3 were conserved. The low percentage of similarity between both genes (56%) allowed us to consider them as independent. The global similarity of Dthox-G with the PG2/3 representatives is low, suggesting that the orthology of Dthox-G with PG2/3 is extremely speculative, so this gene could be considered as unclassified. Similar results have been obtained in the Po/yeelis nigra homologous gene Pnox-4 (Balavoine and Telford. 1995). Dthox-A and -0 are most similar to PG 4 and 5 respectively. with 82% in the homeodomain and a clear conservation in the flanking region immediately downstream. The percentage of similarity (70%) and different intron positions between both genes suggest an independent or old common origin.Partial homeobox sequence of Pnox-8, which is classified as orthologous to Dfd/PG-4 (Balavoine and Tellord., 1995) detines this homology with Dthox-D.The last group (Dthox -C -E and -F) is similar to different medial PGs (6 to 8): Dthox C and E share a high similarityin their homeodomain (93%) and two new intron positions in their homeoboxes; this is one of the firsts indications that Dugesia tigrina could be a tetraploid diploidizated organism with two putative clusters. Moreover, they are most similar to amphioxus PG7 (93-92%) and to Drosophila Antp gene (90%); two downstream flanking positions are conserved with the Antp sequence and some other positions show conservative changes. On this basis Dthox.C and .E are considered as putative orthologues of Antp as well as Pnox-7 from Po/ycelis nigra (Balavoine and Telford, 1995), and Smox-l from Schistosoma mansoni (Webster and Mansour, 1993). Finally, Dthox-F presented the highest similarity in the homeodomain and flanking sequences with Ubx. Abd-A and the Annelida genes CTsx-2, lox-2 and Lox-4 (Dick and Buss. 1994; Wong et al.. 1995). Partial homeodomain sequences of Polycelis nigra genes Pnox-1a,b define a clear homology to Dthox-F. In the homeodomain the identity is between 85-88%, sharing three specific positions (R:2, H:24, l:56) with Ubx and Abd-A. The downstream two-thirds of the homeodomain share near 100% homology with only one conservative substitution and four specific residues with CTsx-2. A high conservation in the downstream region was also observed with Ubx, allowing us to define a charged motif defined by QI(R/K)(EID)lNE. The high degree of similarity allowed us to consider Dthox-F as a putative orthologue of Ubx/Abd-A.