Girardia dorotocephala transcriptome sequence, assembly, and validation through characterization of piwi homologs and stem cell progeny markers

Abstract

Planarian flatworms are popular models for the study of regeneration and stem cell biology in vivo. Technical advances and increased availability of genetic information have fueled the discovery of molecules responsible for stem cell pluripotency and regeneration in flatworms. Unfortunately, most of the planarian research performed worldwide utilizes species that are not natural habitants of North America, which limits their availability to newcomer laboratories and impedes their distribution for educational activities. In order to circumvent these limitations and increase the genetic information available for comparative studies, we sequenced the transcriptome of Girardia dorotocephala, a planarian species pandemic and commercially available in North America. A total of 254,802,670 paired sequence reads were obtained from RNA extracted from intact individuals, regenerating fragments, as well as freshly excised auricles of a clonal line of G. dorotocephala (MA-C2), and used for de novo assembly of its transcriptome. The resulting transcriptome draft was validated through functional analysis of genetic markers of stem cells and their progeny in G. dorotocephala. Akin to orthologs in other planarian species, G. dorotocephala Piwi1 (GdPiwi1) was found to be a robust marker of the planarian stem cell population and GdPiwi2 an essential component for stem cell-driven regeneration. Identification of G. dorotocephala homologs of the early stem cell descendent marker PROG-1 revealed a family of lysine-rich proteins expressed during epithelial cell differentiation. Sequences from the MA-C2 transcriptome were found to be 98–99% identical to nucleotide sequences from G. dorotocephala populations with different chromosomal number, demonstrating strong conservation regardless of karyotype evolution. Altogether, this work establishes G. dorotocephala as a viable and accessible option for analysis of gene function in North America.

What is the role of PIWI family proteins in adult pluripotent stem cells? Insights from asexually reproducing animals, planarians

“…In summary, the localization and biological functions of the three ortholog groups of PIWIs seem to be conserved among planarian species; there are two cytoplasmic PIWIs and one nuclear PIWI in the fly ovary (Brennecke et al, 2007). Planarians have both a sexual and an asexual reproductive system (Kobayashi & Hoshi, 2002;Shibata et al, 1999), and piwi family genes are expressed in neoblasts as well as in germline cells (Almazan et al, 2018;Guo et al, 2006;Nakagawa et al, 2012;Palakodeti et al, 2008;Shibata et al, 2016). In asexual reproduction, neoblasts are the source of all cells constituting their offspring.…”

“…Knockdown of genes in the Piwi-2/B group caused a severe regenerative defect in planarians (Almazan et al, 2018;Nakagawa et al, 2012;Reddien et al, 2005;Shibata et al, 2016). Studies on the Piwi-2/B group using D. japonica and S. mediterranea suggest a common requirement of PIWI-2/B for the planarian aPSC system by showing stepwise defects in the RNAi animals (Reddien et al, 2005;Shibata et al, 2016).…”

“…Phylogenic analysis of the PIWIs in the animal kingdom showed that planarian PIWIs can be categorized into three ortholog groups: PIWI-1/A, PIWI-2/B, and PIWI-3/C groups (Figure 3, Table 1). Genes in Piwi-1/A are transcriptionally expressed specifically in neoblasts and code for cytoplasmic PIWIs (Almazan, Lesko, Markey, & Rouhana, 2018;Guo et al, 2006;Nakagawa, Ishizu, Hasegawa, Kobayashi, & Matsumoto, 2012;Yoshida-Kashikawa et al, 2007). Genes in Piwi-2/B are transcriptionally expressed in neoblasts and brain neurons, and code for nuclear PIWIs (Almazan et al, 2018;Kim et al, 2019;Nakagawa et al, 2012;Reddien, Oviedo, Jennings, Jenkin, & Sánchez Alvarado, 2005;Shibata et al, 2016;Zeng et al, 2013).…”

“…Genes in Piwi-1/A are transcriptionally expressed specifically in neoblasts and code for cytoplasmic PIWIs (Almazan, Lesko, Markey, & Rouhana, 2018;Guo et al, 2006;Nakagawa, Ishizu, Hasegawa, Kobayashi, & Matsumoto, 2012;Yoshida-Kashikawa et al, 2007). Genes in Piwi-2/B are transcriptionally expressed in neoblasts and brain neurons, and code for nuclear PIWIs (Almazan et al, 2018;Kim et al, 2019;Nakagawa et al, 2012;Reddien, Oviedo, Jennings, Jenkin, & Sánchez Alvarado, 2005;Shibata et al, 2016;Zeng et al, 2013). Meanwhile, genes in Piwi-3/C are transcriptionally expressed in neoblasts and brain neurons and code for cytoplasmic PIWIs localized in CBs (Kashima, Kumagai, Agata, & Shibata, 2016;Kim et al, 2019;Palakodeti et al, 2008;Rouhana, Vieira, Roberts-Galbraith, & Newmark, 2012).…”

“…Although genes in the Piwi-1/A ortholog group are expressed specifically in neoblasts and have been used as neoblast markers, knockdown of these genes did not cause any altered phenotype in asexual worms of D. japonica, S. mediterranea, D. ryukyuensis, etc. (Table 1) (Almazan et al, 2018;Nakagawa et al, 2012;Reddien et al, 2005;Shibata et al, 2016). Alternatively, Nakagawa et al (2012) showed that Drpiwi-1 is indispensable for germline development during sexualization of D. ryukyuensis (Table 1).…”