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We studied the expression of DtOtx, a homeobox gene of the freshwater planarian Dugesia tigrina closely related to the Drosophila orthodenticle (otd) and vertebrate Otx genes, which are known to control head development in both fruit flies and vertebrates. DtOtx was not significantly expressed in adult planarians but it was activated within one hour in regenerating tissues with a clearly asymmetric pattern. Animals sectioned transversally, either between the head and the pharynx, or caudal to the pharynx, give rise to a head-containing fragment regenerating a tail region and to a tail-containing fragment regenerating a head region. DtOtx was found to be activated in both regeneration blastemas but its transcripts were much more abundant in the head-regenerating tissues than in the tail-regenerating tissues. The same asymmetric distribution of DtOtx transcripts was observed in central portions of the body regenerating both head and tail structures and in animals laterally regenerating after a longitudinal cut. These data suggest a role of this gene in patterning the body axis of these primitive bilateria, at least during regeneration.

In recent years the characterization of Hox genes in different phyla has led to the suggestion of a universal role for these genes in animal axis determination. Some phyla, such as Platyhelminthes, have not yielded easily to such analysis, although a range of Hox genes have been shown to be present. In this report we present data concerning the relatively large number of Hox genes with a close similarity to representatives of annelids, supporting a phylogenetic clustering of Platyhelminthes within the spiralian protostomes. We also point out the permanent presence of Hox transcripts in adult planarians, with two classes distinguishable by their different patterns of axial expression: some are expressed uniformly, whilst a second group shows a nested expression with graded anterior expression boundaries. During posterior regeneration the nested Hox genes increase differentially depending on the level of sectioning, and then turn on gradually to recover the differential axial pattern of intact adults. These molecular results and others at the cellular level support the previous hypothesis that Platyhelminthes may have become simplified by progenesis.

Platyhelminthes are widely considered to be the sister group of coelomates (Philippe, H., Chenuil, A. and Adoutte, A. (1994)Development 1994 Supplement, 15-24) and the first organisms to show bilateral symmetry and cephalization. Within this phylum, the freshwater planarians (Turbellaria, Tricladida) have been used as model systems for studying bidirectional regeneration (Slack, J. M. W. (1980) J. Theor. Biol 82, 105-140). We have been attempting to identify potential pattern-control genes involved in the regeneration of planarian heads and tails after amputation. Since Hox cluster genes determine positional identity along the anteroposterior axis in a wide range of animals (Slack, J. M. W., Holland, P. W. H. and Graham, C. F. (1993) Nature 361,490-492), we performed an extensive search for Hox-related genes in the planarian Dugesia(G)tigrina. Sequence analyses of seven planarian Dthox genes (Dthox-A to Dthox-G) reveal high similarities with the homeodomain region of the Hox cluster genes, allowing us to assign planarian Dthox genes to anterior and medial Hox cluster paralogous groups. Whole-mount in situ hybridization studies in regenerating adults showed very early, synchronous and colocalized activation of Dthox-D, Dthox-A, Dthox-C, Dthox-E, Dthox-G and Dthox-F. After one hour of regeneration a clear expression was observed in all Dthox genes studied. In addition, all seemed to be expressed in the same regenerative tissue, although in the last stages of regeneration (9 to 15 days) a differential timing of deactivation was observed. The same Dthox genes were also expressed synchronously and were colocalized during intercalary regeneration, although their expression was delayed. Terminal regeneration showed identical Dthox gene expression in anterior and posterior blastemas, which may prevent these genes from directing the distinction between head and tail. Finally, continuous expression along the whole lateral blastema in sagittal regenerates reflected a ubiquitous Dthox response in all types of regeneration that was not related specifically with the anteroposterior polarity.