Expression of a retinal homeobox (Rx) gene during planarian regeneration

Abstract

Retinal homeobox (Rx) genes, with representatives in vertebrates and invertebrates, encode fundamental regulators of early eye and brain formation. Here we describe the spatiotemporal expression profile of a candidate planarian orthologue of Rx during regeneration in Dugesia japonica and Schmidtea mediterranea. Although low levels of Rx transcripts were found throughout the body of intact planarians, high levels of Rx expression were specific to regenerating tissue in both head and tail fragments. We also observed that Rx was never expressed in the simple rhabdomeric planarian eyes, supporting the notion that only formation of eyes that use the ciliary type of photoreceptors requires Rx function. KEY WORDS: paired-class homeobox gene, Rx, planarian, regeneration, eyeRx paired-type homeobox transcription factors have been isolated in several vertebrate and invertebrate species (Arendt et al., 2004; Bailey et al., 2004 and references therein;D'Aniello et al., 2006). A growing body of evidence suggests that Rx genes, early expressed in the anterior neural plate, are essential for the proliferation and specification of retina progenitor cells in chordates. In invertebrates, such as Drosophila, Rx does not seem to play a similar role. In fact, Drosophila Rx is not required for the formation of the visual system, but only for brain and clypeus development (Davis et al., 2003). In addition, an Rx ortholog of the annelid Platynereis has been found expressed in the ciliary photoreceptor cells of the brain, but not in the rhabdomeric photoreceptors of the larval and differentiating adult eyes (Arendt et al., 2004). Based on these observations, it has been proposed that an Rx ancestral function was related to the regulation of genes involved in brain development. During evolution, Rx genes became then essential for development of photoreceptors of ciliary type. No data are available on the function of Rx genes in a context different from the embryonic development, such as regeneration. Planarians (Platyhelminthes, Lophotrochozoa) offer a suitable model to investigate the role of Rx during regeneration. These organisms indeed can regenerate heads, including a complete brain, as well as other body parts in a short time, by virtue of the activity of adult stem cells, called neoblasts Saló 2006;Sánchez Alvarado, 2006). Planarian brain consists of two anterior cephalic ganglia connected to two ventral Int. J. Dev. Biol. 52: 1113-1117 (2008) nerve cords that run along the body (Cebrià, 2007). A pair of simple rhabdomeric eyes is generally located on its dorsal side. Despite the planarian central nervous system (CNS) seems quite simple at the morphological level, it possesses a functionally and molecularly complex structure (Cebrià, 2007 and references therein).We have cloned and sequenced the cDNA of a planarian Rx homolog in Dugesia japonica (GI strain) and Schmidtea mediterranea (asexual strain). Animals were maintained in autoclaved stream water at 18°C and starved for two weeks before being used in the experimen...

Transcriptome Analysis of the Planarian Eye Identifies ovo as a Specific Regulator of Eye Regeneration

“…In contrast to Otx, Eya, Six-1/2, Meis , and SoxB , several transcription factors that have prominent roles in Drosophila and/or vertebrate eye formation – Pax6, Dachshund, Rax , and Six3 - have no apparent role in planarian eye formation (Figure 7E) (Lapan and Reddien, 2011; Mannini et al, 2008; Martin-Duran et al, 2012; Pineda et al, 2002), a conclusion extended by our eye transcriptome and embryonic eye expression data. Therefore, planarian data does not support the argument that these genes are ancestral regulators of eyes.…”

“…Genes previously described to be expressed in planarian eyes all displayed at least 4-fold enrichment in our eye reads, compared to reads from control tissue (ventral, anterior tissue enriched for neurons), in terms of fragments per kilobase per million fragments mapped (FPKM) values (Figure 1D) (See Experimental Procedures for details). pax6A , pax6B , rax , six3 , and dachshund are orthologs of genes with roles in eye development in other organisms, but have been described to lack detectable expression and function in the planarian eye (Lapan and Reddien, 2011; Mannini et al, 2008; Pineda et al, 2002; Pineda and Salo, 2002); all of these genes displayed FPKM values of zero (Figure 1D), demonstrating specificity of the data and further suggesting that these genes have no role in planarian eye regeneration. We conclude that transcript levels in the RNA-seq dataset can be highly predictive of gene expression in vivo .…”

Berberine exposure triggers developmental effects on planarian regeneration

“…To acquire a more thorough understanding about the effect of 600 μM berberine treatment on other neuronal cell types, we also compared gene transcription of other markers between berberine-treated planarians and controls. We selected Dj-gad , a marker of GABAergic neurons25 and Dj-rax , a gene expressed in a subset of nerve cells26. While no difference in Dj-gad expression level was observed, a significant reduction of Dj-rax transcripts was detected in berberine-treated planarians.…”

Onecut is a direct neural-specific transcriptional activator of Rx in Ciona intestinalis

“…In invertebrate species, although Rx genes are expressed in the nervous system, they do not share any expression in the eye structures (Arendt et al, 2004;Eggert et al, 1998;Mannini et al, 2008). Therefore, it seems that Rx gene function was likely restricted to the brain in the common ancestor of vertebrate and invertebrate, while during Deuterostome evolution Rx genes acquired a new role in the development of the eye structures.…”