%0 Journal Article %J Dev Biol %D 2022 %T DDX24 is required for muscle fiber organization and the suppression of wound-induced Wnt activity necessary for pole re-establishment during planarian regeneration. %A Sarkar, Souradeep R %A Dubey, Vinay Kumar %A Jahagirdar, Anusha %A Lakshmanan, Vairavan %A Haroon, Mohamed Mohamed %A Sowndarya, Sai %A Sowdhamini, Ramanathan %A Palakodeti, Dasaradhi %X

Planarians have a remarkable ability to undergo whole-body regeneration. Successful regeneration outcome is determined by processes like polarity establishment at the wound site, which is followed by pole (organizer) specification. Interestingly, these determinants are almost exclusively expressed by muscles in these animals. However, the molecular toolkit that enables the functional versatility of planarian muscles remains poorly understood. Here we report that SMED_DDX24, a D-E-A-D Box RNA helicase, is necessary for planarian survival and regeneration. We found that DDX24 is enriched in muscles and its knockdown disrupts muscle fiber organization. This leads to defective pole specification, which in turn results in misregulation of many positional control genes specifically during regeneration. ddx24 RNAi also upregulates wound-induced Wnt signalling. Suppressing this ectopic Wnt activity rescues the knockdown phenotype by enabling better anterior pole regeneration. To summarize, our work highlights the role of an RNA helicase in muscle fiber organization, and modulating amputation-induced wnt levels, both of which seem critical for pole re-organization, thereby regulating whole-body regeneration.

%B Dev Biol %V 488 %P 11-29 %8 2022 May 04 %G eng %R 10.1016/j.ydbio.2022.04.011 %0 Journal Article %J Bio Protoc %D 2022 %T Flow Cytometry Analysis of Planarian Stem Cells Using DNA and Mitochondrial Dyes. %A Haroon, Mohamed Mohamed %A Vemula, Praveen Kumar %A Palakodeti, Dasaradhi %X

Planarians are free-living flatworms that emerged as a crucial model system to understand regeneration and stem cell biology. The ability to purify neoblasts, the adult stem cell population of planaria, through fluorescence-activated cell sorting (FACS) has tremendously increased our understanding of pluripotency, specialization, and heterogeneity. To date, the FACS-based purification methods for neoblasts relied on nuclear dyes that discriminate proliferating cells (>2N), as neoblasts are the only dividing somatic cells. However, this method does not distinguish the functional states within the neoblast population. Our work has shown that among the neoblasts, the pluripotent stem cells (PSCs) are associated with low mitochondrial content and this property could be leveraged for purification of the PSC-enriched population. Using the mitochondrial dye MitoTracker Green (MTG) and the nuclear dye SiR-DNA, we have described a method for isolation of PSCs that are viable and compatible with downstream experiments, such as transplantation and cell culture. In this protocol, we provide a detailed description for sample preparation and FACS gating for neoblast isolation in planaria.

%B Bio Protoc %V 12 %P e4299 %8 2022 Jan 20 %G eng %N 2 %R 10.21769/BioProtoc.4299