Regeneration‐dependent conditional gene knockdown (Readyknock) in planarian: Demonstration of requirement for Djsnap‐25 expression in the brain for negative phototactic behavior

Abstract

Freshwater planarians have a simple and evolutionarily primitive brain structure. Here, we identified the Djsnap-25 gene encoding a homolog of the evolutionarily conserved synaptic protein SNAP-25 from the planarian Dugesia japonica and assessed its role in brain function. Djsnap-25 was expressed widely in the nervous system. To investigate the specific role of Djsnap-25 in the brain, we developed a unique technique of RNA interference (RNAi), regeneration-dependent conditional gene knockdown (Readyknock), exploiting the high regenerative capacity of planarians, and succeeded in selectively eliminating the DjSNAP-25 activity in the head region while leaving the DjSNAP-25 activity in the trunk region intact. These knockdown animals showed no effect on brain morphology or on undirected movement of the trunk itself. Light-avoidance behavior or negative phototaxis was used to quantitatively analyze brain function in the knockdown animals. The results suggested that the DjSNAP-25 activity within the head region is required for two independent sensory-processing pathways that regulate locomotive activity and directional movement downstream of distinct primary sensory outputs coming from the head margin and the eyes, respectively, during negative phototaxis. Our approach demonstrates that planarians are a powerful model organism to study the molecular basis of the brain as an information-processing center.

Planarian Hedgehog/Patched establishes anterior–posterior polarity by regulating Wnt signaling

“…38. After whole-mount in situ hybridization, specimens were subjected to immunohistochemistry using anti-DjAADCA or anti-DjSyt antibody essentially as described (15,32). In brief, specimens were incubated with the primary antibody (1:1,000 dilution) overnight at 4°C.…”

“…The primer set for the Djptc gene was not complementary to its dsRNA, rather, primers were designed to correspond to the 3Ј end of the Djptc gene and the dsRNA was designed to correspond to the 5Ј end. Measurements were performed in quadruplicate for technical replicates (coefficient of variation was 0.1-2.8% for all primer sets), and were normalized to the expression level of DjG3PDH (32). In the analyses of trunk regenerants, the expression level in the anterior end of the control fragment was taken as the standard for the relative gene expression (RGE) value, except in the case of Djwnt11-2, for which the level of the posterior end of the control fragment was taken as the standard because its expression was not detectable in the anterior end.…”

DjCBC‐1, a conserved DEAD box RNA helicase of the RCK/p54/Me31B family, is a component of RNA‐protein complexes in planarian stem cells and neurons

“…Whole-mount in situ hybridization using alkaline phosphatase and NBT/BCIP color development was performed as described by Umesono et al (1997) and Takano et al (2007).…”

Brain regeneration from pluripotent stem cells in planarian

“…In addition, a gene-knockdown method using RNA interference (RNAi) in planarians has been developed (Sánchez Alvarado & Newmark 1999;Newmark et al 2003;Orii et al 2003). Recently, we have succeeded in conditional gene knockdown by the combination of RNAi and the regeneration ability of planarians, named 'Readyknock' ( Takano et al 2007). The availability of these methods has greatly facilitated our ability to investigate the genetic programmes underlying brain regeneration.…”