We have characterized the genomic organization of Dth-1 and Dth-2, planarian homeobox-containing genes, previously described at the cDNA level (J. Garcia-Fernandez, J. Baguna and E. Salo (1991), Proc. Natl. Acad. Sci. USA, 88, 7338–7342). Genomic analysis shows that Dth-1 and Dth-2 genes encode proteins of 533 and 363 amino acids respectively. The open reading frame of Dth-1 is interrupted by two large introns of 8 kb and 12 kb Dth-2 also shows two introns, but these are short (42 bp and 44 bp) and the second interrupts helix III at position 44–45, as is the case with other homeobox genes from such divergent animals as Drosophila, honeybee, C. elegans, ascidians, and mouse, which suggests an ancient evolutionary relationship between these genes. The spatial distribution of transcripts in adult tissues, determined by in situ hybridization, demonstrates that Dth-1 is expressed at a high level in the gastrodermal cells, while Dth-2 is expressed in the peripheral parenchyma, at higher levels in the dorsal than the ventral regions. Their specific spatial distribution suggests a possible role for these homeobox genes in determination and/or differentiation of specific cell types. The expression pattern of both genes is more or less continuous, but in Dth-1 clustered discontinuous labelling in areas surrounding the gastrodermis may indicate a specific expression of this gene in groups of undifferentiated cells (neoblasts) already committed or determined to gastrodermal cell fates. In situ hybridization analysis during early regeneration shows expression only in the postblastema (stump) differentiated areas while no expression has been detected in the undifferentiated blastema, indicating that neither gene has a role in pattern formation mechanisms known to occur at the early stages of regeneration (0-3 days). Hence, Dth-1 and Dth-2 are planarian homeobox genes presumably involved in specific cell or tissue determination and/or differentiation.

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