Djeyes absent ( Djeya ) controls prototypic planarian eye regeneration by cooperating with the transcription factor Djsix-1

Abstract

A conserved network of nuclear proteins is crucial to eye formation in both vertebrates and invertebrates. The finding that freshwater planarians can regenerate eyes without the contribution of Pax6 suggests that alternative combinations of regulatory elements may control the morphogenesis of the prototypic planarian eye. To further dissect the molecular events controlling eye regeneration in planarians, we investigated the role of eyes absent (Djeya) and six-1 (Djsix-1) genes in Dugesia japonica. These genes are expressed in both regenerating eyes and in differentiated photoreceptors of intact adults. Through RNAi studies, we show that Djsix-1 and Djeya are both critical for the regeneration of normal eyes in planarians and genetically cooperate in vivo to establish correct eye cell differentiation. We further demonstrate that the genetic interaction is mediated by physical interaction between the evolutionarily conserved domains of these two proteins. These data indicate that planarians use cooperatively Djsix-1 and Djeya for the proper specification of photoreceptors, implicating that the mechanism involving their evolutionarily conserved domains can be very ancient. Finally, both Djsix-1 and Djeya double-stranded RNA are substantially more effective at producing no-eye phenotypes in the second round of regeneration. This is probably due to the significant plasticity of the planarian model system, based on the presence of a stable population of totipotent stem cells, which ensure the rapid cell turnover of all differentiated cell types.

Planarian regeneration: achievements and future directions after 20 years of research

“…Since the initial characterization of the Pax6 gene, we have continued the search for planarian orthologs of genes involved in eye determination (Table 1). We confirmed that the initial eye genetic network is highly conserved in planarians compared to other metazoans, with the characterization of orthologs of sine oculis, eyes absent and opsin genes in different planarian species (Mannini et al, 2004. Loss of function of sine oculis and eyes absent confirms their function in eye determination during regeneration and maintenance in adult planarians (Fig.…”

“…Loss of function of sine oculis and eyes absent confirms their function in eye determination during regeneration and maintenance in adult planarians (Fig. 5A, B) (Mannini et al, 2004, Pineda et al, 2000. Also, the production of transgenic planarians carrying several dimeric Pax6 binding sites plus an enhanced green fluorescent protein reporter gene produces a new phenotype in which the planarians show green eyes (Fig.…”

“…Eye cells (Callaerts et al, 1999) (González-Estévez et al, 2003, Pineda et al, 2001 Sine oculis Gt Gtso Eye cell determination and maintenance (Pineda et al, 2000, Pineda et al, 2001 Eyes absent Dj Djeya Eye cell determination and maintenance (Mannini et al, 2004) Opsin Gt Gtops Photoreceptors (Pineda et al, 2000) Cell-Specific Markers Gastrodermal cells Parenchyma cells (García-Fernàndez et al, 1991, García-Fernàndez et al, 1993a Myosin heavy chain Gt Gtmhc Muscle cells (Cebrià et al, 1999, Cebrià et al, 1997 Regional Markers C-type multidomain free lectins Scarf1,2,3 Gtlec1,2a,2b,2c Parenchyma pre-pharyngeal region (Shagin et al, 2002) …”

DjPum, a homologue ofDrosophila Pumilio,is essential to planarian stem cell maintenance

“…Negative controls were carried out by injection of water, or β-Gal dsRNA or Djeya dsRNA. Djeya dsRNA was obtained as described by Mannini et al (Mannini et al, 2004). Intact specimens were injected three times, one injection every 2 days, for a week, then transected and allowed to regenerate.…”

Expression of hsp90 mediates cytoprotective effects in the gastrodermis of planarians

“…About 10 10 -10 11 molecules of dsRNA were injected in each specimen with a Nanoject microinjector apparatus (Drummond). Injections schedule was as reported in Mannini et al (2004). Amputated fragments were analyzed during the second round of regeneration.…”