DjSlk is required for the survival of planarian Dugesia japonica 1 ISJ 14: 1-8, 2017 ISSN 1824-307X RESEARCH REPORT RNAi of CNS-expressed gene DjSlk induces morphogenetic malformation and death in planarian Dugesia japonica X Chen1,2, H Zhen1, S Wu1, Q Lu1, Q Pang1, B Zhao1 1Laboratory of Developmental and Evolutionary Biology, School of Life Sciences, Shandong University of Technology, Zibo 255049, P.R. China 2Translational Medicine Center, The Sixth People′s Hospital of Zhengzhou, Zhengzhou 450000, P. R. China Accepted December 13, 2016 Abstract Ste20-like kinases are critically multifuctional proteins which play important roles in varieties of celluar processes and physiological events. Here, We characterized a Ste20-like kinase gene (DjSlk) in planarian Dugesia japonica. Whole-mount in situ hybridizations revealed that DjSlk was expressed in the central nervous system (CNS) including cephalic ganglia and ventral nerve cords (VNCs) in intact and regenerating animals. After RNA interference (RNAi) of DjSlk, adult planarians became immobilized and wrinkled, then swelled and lysed eventually. DjSlk RNAi treated regenerating planarians could form the entire animals, and then displayed the similar phenotype transformation. These results suggest that loss of function of DjSlk leads to morphogenetic malformation of planarian D. japonica probably via regulating cell volume instead of disrupting the balance between cell proliferation and apoptosis. Key Words: CNS; DjSlk; expression; morphogenetic malformation; cell volume Introduction Endowed with abundant pluripotent stem cells named neoblasts which proliferate and differentiate to all cell types in response to amputation and/or injury ， planarians can regenerate the complete worms with all organs from almost any tiny fragments (Newmark and Sánchez Alvarado, 2002; Reddien and Sánchez Alvarado, 2004). Neoblast is the only source to provide new cells during turnover and regeneration (Wagner DE et al., 2011). Elimination of neoblasts by irradiation, planarians demonstrate the typical phenotype defects such as head regression, dorsal curling and lesions during homeostasis and fail to shape the whole animals for amputated pieces (Newmark and Sánchez Alvarado, 2002; Reddien and Sánchez Alvarado, 2004; Saló et al., 2009; Scimone et al., 2010). Planarians can acquire similar phenotypes by silencing some genes related to neoblast maintenance, proliferation, differentiation, and apoptosis (Reddien et al., 2005; Guo et al., 2006; Pearson and Sánchez Alvarado, 2010; Li et al., 2011). ___________________________________________________________________________ Corresponding author: Bosheng Zhao School of Life Sciences Shandong University of Technology 266 Xincun W estern Road, Zibo 255049, P.R. China E-mail: zhaobosheng@sdut.edu.cn Sterile 20 (ste20) is originally found as mitogen-activated protein kinase kinase kinase kinase (MAP4K) involved in the mating response of haploid yeast Saccharomyces cerevisiae (Wu et al., 1995). Its homologs in other organisms form the ste20-like kinase (SLK) superfamily and are mainly regarded as upstream regulators of the MAPK pathways (Dan et al., 2001; Ling et al., 2008). Ste20-like kinases play crucial roles in various cellular processes including cell growth, cell migration, apoptosis, cell-cycle control, cell shape change and stress responses (Dan et al., 2001; Strange et al., 2005; Ling et al., 2008). In this paper, we identified a Slk gene in planarian Dugesia japonica and studied its tempospacial expression pattern and loss-of-function phenotype in intact and regenerating animals. Materials and Methods Animals The planarians Dugesia japonica collected in Boshan, Shandong, China, are maintained in autoclaved tap water at 20 ℃ and 6 - 10 mm long animals were starved for at least one week before use in experiments. Cloning and Sequence Analysis of cDNA The DjSlk cDNA was derived from random mailto:zhaobosheng@sdut.edu.cn 2 sequencing of a planarian cDNA library. Comparison against the GenBank protein database was performed using the BLAST network server at the National Center for Biotechnology Information (NCBI). Multiple protein sequences were aligned using the MegAlign program by the CLUSTAL method in DNASTAR software package (Burland, 2000). Whole-mount in situ hybridization Whole-mount in situ hybridizations were performed as described previously (Pearson et al., 2009). The digoxigenin (DIG)-labelled antisense RNA probes were synthesized in vitro. Hybridizations were carried out at 56 ℃ for 17 h, the BCIP/NBT mixture solution (Roche) was used for color development. For regeneration experiments, animals were amputated pre- and post-pharyngeally and left to regenerate, the head-, trunk-, and tail-pieces were collected at the times indicated. Quantitative real-time PCR Quantitative real-time PCR was used to monitor the quantitative expression of the DjSlk as described previously (Yu et al., 2015) in intact planarians, regenerating trunk fragments， and regenerating trunk fragments of RNAi-treated planarians at different times after amputation. The cDNA was synthesized using a First-Strand System kit from Invitrogen after total RNAs were extracted using RNAiso Reagent (TaKaRa). qPCR reactions were performed using Fast Start Universal SYBR Green Master (Rox) (Roche, Switzerland) according to the manufacturer’s protocol. Three samples for each condition were run in parallel by a 7,500 Real Time PCR System (Applied Biosystems). Data were normalized to the expression of the internal control DjEF2. The following sets of specific primers were used: DjSlk mRNA, 5′-CGAAGGACAAAGGCACAT-3′, 5′-GAGCGAACACCAGGAACT-3′. DjEF2 mRNA, 5 ′ - T T A A T G A T G G G A A G A T A TG T T G - 3 ′; 5 ′ - G T A C C A T A G G A T C T G A T T T T G C - 3 ′. The data were analyzed using SPSS 16.0 software. The significance of differences was analyzed by one-way analysis of variance (ANOVA) followed by a Tukey's post-hoc analysis to identify differences between the experimental and intact planarians. Data presented are means ± SD. Values of p < 0.05 were considered to be significant. RNA interference DjSlk was cloned into the L1440 plasmid with two T7 primers, and then dsRNA was synthesized according to the manufactrue’s instructions (MEGAscript® RNAi Kit). Animals were injected at day 0, day 2, 4, and 6. For regeneration studies, animals were cut at day 10. Control animals were injected with deionized sterile water. Immunostaining Animals were killed in 2 % HCl for 5 min at RT and fixed in 4 % paraformaldehyde solution for 3 h at 4 °C, then dehydrated in 100 % methanol solution for 1 h at -20 ℃. The following procedures were processed as described elsewhere (Robb and Sánchez Alvarado, 2002; Inoue et al., 2007; Cebria, 2007, 2008). The primary antibody anti-SYNORF1, a mouse monoclonal antibody specific for synapsin (Developmental Studies Hybridoma Bank) was used at a dilution of 1:25. The secondary antibody Dylight 594 AffiniPure Goat anti-mouse IgG(H+L) (EarthOX) was used at a dilution of 1:200. Results Sequence analysis of DjSlk The cDNA clone obtained from planarian D. japonica cDNA library is about 1,100 bp with the longest open reading frame of 879 bp. It encodes for a deduced protein of 292 amino acids with predicted molecular mass of approximately 32.4 kDa (Fig. 1) Fig. 1 The nucleotide and deduced amino acid sequence of DjSlk. 3 Fig. 2 Alignment of subdomain XI of Ste20-like kinases, including DjSLK using the MegAlign program (DNASTAR) by the CLUSTAL W method. Shaded (with solid black) residues are the amino acids that match the consensus. GenBank accession numbers: Aplysia californica (XP_005111485.1), Ailuropoda melanoleuca (XP_002914907.1), Crassostrea gigas (EKC21462.1), Canis lupus (XP_003433112.1), Clonorchis sinensis (GAA52112.1), Drosophila melanogaster (NM_142339.2), Danio rerio (XP_005165982.1), Felis catus (XP_003980538.1), Latimeria chalumnae (XP_005992350.1), Loa loa (EJD76726.1), Schistosoma japonicum (CAX753595.1), Salmo salar (ACI33699.1). Initial BLASTP search at NCBI revealed that this gene belongs to Ste20-like kinase. However, its 5′ end is missed and only subdomain XI is entire (Hanks and Hunter, 1995). The deduced amino acids aligned with other subdomain XI of Ste20-like kinases showed that DjSLK shares 39.2 % - 72.2 % similarity with its homlogs in other organisms (Fig. 2). Ste20 kinases consist of the P21-activated kinase (PAK) and germinal center kinase (GCK) families according to the relative location of kinase domain, these two families can be further subdivided into PAK I and PAK II and GCK I to VIII subfamilies, respectively (Dan et al., 2001; Strange et al., 2005; Ling et al., 2008). Due to loss of most subdomains, the closest homlogs can not be ascertained and the gene is termed ste20 like kinase (DjSlk) in this study. DjSlk expression pattern in adult and regenerating planarians In order to analyse the expression pattern of the planarian DjSlk gene we performed whole mount in situ hybridization on intact and regenerating animals. In intact planarians, DjSlk was expressed in central nervous system which possesses an inverted U-shaped pair of cephalic ganglia and two longitudinal ventral nerve cords that project posteriorly along the worm (Cebrià et al., 2002; Cebrià, 2007; Agata and Umesono, 2008) (Fig. 3B). And DjSlk localized in both the central spongy region and the lateral branches in the cephalic ganglia (Fig. 3B). In regenerating animals, DjSlk transcripts could always be detected in CNS in the head-, trunk-, and tail-pieces. During the initial regeneration stages after amputation, DjSlk expression was not detected Fig. 3 Expression of DjSlk in intact planarian (A) An intact planarian processed and hybridized using the DjSlk sense probe. No signal was seen in the control. (B) Ventral view of intact planarian, expression of DjSlk is mainly present in the CNS. Anterior is to the left. Scale bar: 500 μm. 4 Fig. 4 Expression of DjSlk during regeneration. Expression of DjSlk in regeneration of day 1 (A - C), day 3 (D - F), day 5 (G - I), day 7 (J - L), and day 9 (M - O) after amputation. DjSlk is detected in the pre-exiting and newly regenerated CNS. Anterior is to the left. Scale bar: 300 μm. within the head and tail blastema, but it was detected in the preexisting CNS (Figs 4A - C). At 3 and 5 days of regeneration, new neural cells in front of the commissure differentiated, and CNS recovered most of its function (Cebria, 2007). DjSlk expression was detected in the preexisting and newly regenerated CNS (Figs 4D - I). With the development of regeneration, the original expression was gradually reestablished (Figs 4J - O). The Relative quantitative real-time PCR analysis was performed to investigate the change of expression of DjSlk mRNA during planarian regeneration. We examined RNA samples from normal intact planarians and trunk fragments regenerated for 1 day, 5 Fig. 5 qRT-PCR analysis of DjSlk expression in intact and regenerating truck fragments at 1, 3, 5, 7, 9 days after amputation. Data was expressed as the ratio of DjSlk to DjEF2α mRNA. Error bars represent the  SD for three independent PCR amplifications and quantifications. *p < 0.05 or **p < 0.01 compared to control intact planarians. Fig. 6 qRT-PCR analysis of DjSlk RNAi efficiency in adult intact planarians. Error bars represent the  SD for three independent PCR amplifications and quantifications. **p < 0.01 is the comparison between control intact animals and RNAi intact animals. Template 6 Fig. 7 Abnormal appearance change in intact (B-E) and regenerating planarians (B′-E′) after DjSlk RNAi. (A) Control animal, microinjection of water in adult animal after 2 months. (B-E) After RNAi, planarians became immobilized and wrinkled (B), swelled (C), lysed (D), and died (E). (A′) Contral animal, day 30 of regeneration after amputation. (B′-E′) The appearance transformation in regenerating trunk fragments after RNAi-treated planarians. Anterior is to the top. Scale bar: A - E = 800 μm, A′- E′ = 200 μm. 3 days, 5 days, 7 days, and 9 days, respectively. The resluts indicated that DjSlk mRNA was gradually increased during regeneration compared to normal intact planarians and achieved to the maximal leval at 7 days (p < 0.01) after amputation. Then it declined to almost normal level at regeneration day 9 (Fig. 5). DjSlk RNAi induces morphogenetic defects and death of planarians To study the role of DjSlk gene in the homeostasis and regeneration of the planarian, we knocked down the endogenous expression of DjSlk using RNAi. Real-time PCR analysis of DjSlk mRNA showed that the RNAi-treatment efficiently down-regulated the expression of DjSlk in intact planarians ( p< 0.01) (Fig. 6). After 5 days of injecting DjSlk dsRNA, the intact animals became immobilized and wrinkled (Fig. 7B, n = 6/13). Then the planarians swelled and started to lyse at day 9 and day 15, respectively (Figs 7C and D, n = 6/13). And the animals completely lysed at about 25 days after the treatment (Fig. 7E, n = 6/13). In contrast, control animals lived without any abnormal change even for 2 months (Fig. 7A, n = 10/10). DjSlk RNAi treated trunk fragments could regenerate the whole bodies, and then showed the same appearance change starting at 14 days after amputation (Figs 7B’ - E’). Immunostaining with an anti-SYNORF1 antibody against synapsin revealed that DjSlk RNAi didn′t interfere with CNS intactness and regeneration (Fig. 8). Discussion Ste20 kinases function in morphological events in different organisms (Strange et al., 2005). In yeast mating pathway, cell shrinkage activates ste20 kinase and suppresses mating defects (Strange et al., 2005). One ste20 kinase named proline-alanine-rich ste20-related kinase (PASK) is strongly expressed in neurons and transporting epithelia in rats (Ushiro et al., 1998). PASK can interact with and phosphorylate Na-K-2CL (NKCC) and K-CL (KCC) cotransporters to regulate cell volume (Piechotta et al., 2002; Dowd and Forbush, 2003; Strange et al., 2005). During cell swelling, loss of kinase activity of PASK results in dephosphorylation of both cotransporters which lead to inhibit NKCC and activate KCC (Strange et al., 2005). In this study, DjSlk was expressed in CNS and RNAi enventully induced swelling and lysing of planarians. And the defects probably resulted from cell swelling and osmotic lysis after loss of kinase 7 Fig. 8 Immunostaining with anti-SYNORF1 in DjSlk-RNAi-treated planarians during homeostasis (A - B and A' - B') and regeneration (C - D and C' - D'), the defects of CNS including brain and VNC were not detected. (A) Intact planarian was injected with water as control. (B) DjSlk-RNAi-treated intact planarian at 9 days. (C) Immunostaining of normally regeneration of the cutting head sample was detected at 15 days after amputation. (D) DjSlk-RNAi-treated intact planarian was cut head to regenerate at 15 days. A ′- D′ the magnification of head in A - D, respectively. Anterior is to the up. Scale bars: A - D = 500 μm; A′ - D′ = 100 μm. activity of DjSlk. Meanwhile, the irradiation-treated typical phenotype change did not occur in intact planarians, and amputated fragments could regenerate the whole bodies after silencing DjSlk. All these results suggest that, just like PASK, DjSlk regulates cell volume instead of involving in neoblast maintenance, proliferation, differentiation, and apoptosis like other neoblast-related genes. Acknowledgments We thank Dr. Yuqi Huo from Translational Medicine Center, The Sixth People′s Hospital of Zhengzhou, for reading and discussing the manuscript. 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