Downloaded from rnajournal.cshlp.org on June 4, 2017 - Published by Cold Spring Harbor Laboratory Press Identification of neoblast- and regeneration-specific miRNAs in the planarian Schmidtea mediterranea VIDYANAND SASIDHARAN,1 YI-CHIEN LU,2 DHIRU BANSAL,1 PRANAVI DASARI,1 DEEPAK PODUVAL,1 ASWIN SESHASAYEE,3 ALISSA M. RESCH,4 BRENTON R. GRAVELEY,4,5 and DASARADHI PALAKODETI1,5 1Institute for Stem Cell Biology and Regenerative Medicine, National Center for Biological Sciences, Bangalore 560065, India 2Department of Pathology and Laboratory Medicine, Weill Cornell Medical College, New York, New York 10065, USA 3National Center for Biological Sciences, Bangalore 560065, India 4Department of Genetics and Developmental Biology, Institute for Systems Genomics, University of Connecticut Stem Cell Institute, University of Connecticut Health Center, Farmington, Connecticut 06030, USA ABSTRACT In recent years, the planarian Schmidtea mediterranea has emerged as a tractable model system to study stem cell biology and regeneration. MicroRNAs are small RNA species that control gene expression by modulating translational repression and mRNA stability and have been implicated in the regulation of various cellular processes. Though recent studies have identified several miRNAs in S. mediterranea, their expression in neoblast subpopulations and during regeneration has not been examined. Here, we identify several miRNAs whose expression is enriched in different neoblast subpopulations and in regenerating tissue at different time points in S. mediterranea. Some of these miRNAs were enriched within 3 h post- amputation and may, therefore, play a role in wound healing and/or neoblast migration. Our results also revealed miRNAs, such as sme-miR-2d-3p and the sme-miR-124 family, whose expression is enriched in the cephalic ganglia, are also expressed in the brain primordium during CNS regeneration. These results provide new insight into the potential biological functions of miRNAs in neoblasts and regeneration in planarians. Keywords: microRNA; neoblast; planaria; regeneration INTRODUCTION Wound healing and blastema formation take place within 2–3 d after amputation. The later stages of regeneration in- Planarians are fresh water invertebrates belonging to the phy- volve repatterning of old and new tissues within 2 wk of lum Platyhelminthes. They share common traits such as post-amputation, resulting in complete restoration of normal bilateral symmetry, triploblasticity, and encephalization with metazoans. Additionally, planarians possess well-defined morphology of the animal (Lobo et al. 2012). This complex anterior-posterior and dorsoventral axes. Planarians are phenomenon is controlled by a variety of genes that code well known for their enormous regenerative ability—a tiny for proteins involved in chromatin modifications, various body fragment can fully regenerate within a week. The regen- signaling pathways, and post-transcriptional regulatory pro- erative prowess of planarians has been attributed to special- cesses (Aboobaker et al. 2011; Reddien et al. 2011; King ized pluripotent cells called neoblasts. Advances in cellular and Newmark 2012). Recently Wenemoser et al. (2012) and molecular techniques have helped reveal the mecha- have identified waves of gene expression profiles that peak nisms that govern neoblast function and regeneration. within 30 min following amputation and gradually taper Following amputation or injuries, planarians respond by down by 24 h, indicating tight regulation of gene expression wound healing, which involves wound closure by migration during regeneration. of adjacent epithelial cells, followed by the migration and rap- MicroRNAs are short noncoding RNAs (18–24 nt) that id proliferation of neoblasts to form an undifferentiated tis- play a major role in post-transcriptional gene regulation in ′ sue called blastema. The initial steps of regeneration also metazoans. They usually bind to sequences in the 3 UTRs involve selective loss of old cells near the wound region via of mRNAs and silence gene expression by either mRNA deg- apoptosis, followed by remodeling of new and old tissues. radation or translational repression. miRNAs are involved in controlling diverse biological functions such as embryo- 5Corresponding authors genesis, development, and regeneration by fine tuning pro- E-mail [email protected], [email protected] tein expression levels (Bartel et al. 2004). In a recent study, E-mail [email protected] Article published online ahead of print. Article and publication date are at knockdown of Smed-Ago, a key factor involved in miRNA/ http://www.rnajournal.org/cgi/doi/10.1261/rna.038653.113. siRNA biogenesis in planarians, led to a drastic reduction 1394 RNA 19:1394–1404; © 2013; Published by Cold Spring Harbor Laboratory Press for the RNA Society Downloaded from rnajournal.cshlp.org on June 4, 2017 - Published by Cold Spring Harbor Laboratory Press Neoblast- and regeneration-specific miRNAs in planaria of the neoblast population and defects in regeneration, sug- (posterior regenerating tissue) and separately from tails that gesting a role of miRNAs/siRNAs in neoblast function and re- were regenerating heads (anterior regenerating tissue) (Sup- generation (Rouhana et al. 2010; Li et al. 2011). Previous plemental Fig. 1B). These time points were selected so that studies have identified several miRNAs that are expressed various regenerative processes, such as wound healing, neo- in neoblasts (Palakodeti et al. 2006; Friedländer et al. 2009; blast proliferation, differentiation, and patterning were rep- Lu et al. 2009), but little is known about the differential ex- resented. Small RNA libraries were also prepared from pression of miRNAs in proliferating neoblasts and neoblast unamputated animals, which served as a baseline control progeny. Recently, Tian et al. (2012) and Qin et al. (2011) for miRNA expression levels. Deep sequencing of the small identified several miRNAs that are enriched in 3-d regenerat- RNA libraries was performed on an Illumina HiSeq 1000/ ing tissues. However, a systematic analysis of miRNA expres- Illumina GAIIx. Consistent with previous studies (Palakodeti sion in anterior and posterior regenerating tissues at various et al. 2008; Friedländer et al. 2009), we observed two dis- time points has not been conducted. tinct small RNA populations, one of 18–24 nt representing Here, we studied miRNA expression in several purified miRNAs and siRNAs, and a second of 31–32 nt representing subpopulations of neoblasts and during regeneration in piRNAs. Since our focus here is on miRNA expression, all Schmidtea mediterranea. Small RNA sequencing from various subsequent analysis was restricted to the 18- to 24-nt popu- FACS-sorted neoblast populations identified miRNAs whose lation. We obtained 50–55 million 18- to 24-nt reads from expression is enriched in proliferating neoblasts and neoblast the regenerating time point libraries and 2–3 million 18- to progeny. Sequencing of small RNAs throughout regeneration 24-nt reads from the FACS-purified cell population libraries revealed several miRNAs that are expressed within 3–24 h (Supplemental Fig. 1C). The raw reads were aligned to the S. post-amputation and miRNAs that are asymmetrically ex- mediterranea draft genome using Bowtie (Langmead et al. pressed during regeneration. For example, members of the 2009) without any mismatches. Approximately 80%–90% mir-124 family are up-regulated in anterior regenerating of the total raw reads aligned to the S. mediterranea genome tissue but not in posterior regenerating tissue. We also found (Supplemental Fig. 1C). The reads were also mapped to a da- that the mature miRNAs from the sme-miR71b/miR2d/ tabase of known S. mediterranea miRNAs (miRbase). Inter- miR752/miR13 miRNA cluster are differentially enriched in estingly, only 36%–40% of the reads obtained from the proliferating neoblasts and neoblast progeny. These studies neoblast populations aligned to known miRNAs, whereas suggest that the abundance of mature miRNAs from the 45%–55% of the reads obtained from the regenerating tissue sme-miR71b/miR2d/miR752/miR13 miRNA cluster is post- aligned to known miRNAs (Supplemental Fig. 1C). The transcriptionally regulated. Together, these results highlight unaligned reads could be novel miRNAs, siRNAs, or degrada- miRNA-mediated post-transcriptional regulation as a key tion fragments of larger RNA species. We next used miR- regulator of gene expression involved in stem cell function Deep2 (Friedländer et al. 2012) to identify novel miRNAs. and regeneration in planarians. After filtering the miRDeep2 predicted list using a miRDeep2 score cutoff of +10 and a Randfold P-value cutoff of <0.05, we identified 15 potential new miRNA loci. Of these, two RESULTS are new members of the sme-miR-754 and sme-miR-2182 miRNA families, while the 13 others appear to be novel pla- Deep sequencing of small RNAs from regenerating narian-specific miRNAs (Supplemental Table1; Supplemen- tissue and neoblast populations tal Fig. 4B). Only 0.01% of the aligned small RNA reads map Previous studies of planarian miRNAs have revealed several to the 15 new miRNA loci. The majority of the reads that strain- and neoblast-specific miRNAs. Most of these studies failed to map to the miRNAs aligned to regions of the ge- involved comparing small RNA profiles between irradiated nome to which piRNAs align, suggesting that these reads and nonirradiated animals or isolating a mixture of prolifer- could either be the degradation products