- Email: [email protected]
Identification of runt family genes involved in planarian regeneration and tissue homeostasis
Accepted Manuscript Identification of runt family genes involved in planarian regeneration and tissue homeostasis Zimei Dong, Yibo Yang, Guangwen Chen, Dezeng Liu PII:
S1567-133X(17)30234-X
DOI:
10.1016/j.gep.2018.04.006
Reference:
MODGEP 1091
To appear in:
Gene Expression Patterns
Received Date: 29 December 2017 Revised Date:
21 March 2018
Accepted Date: 6 April 2018
Please cite this article as: Dong, Z., Yang, Y., Chen, G., Liu, D., Identification of runt family genes involved in planarian regeneration and tissue homeostasis, Gene Expression Patterns (2018), doi: 10.1016/j.gep.2018.04.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Identification of runt family genes involved in planarian regeneration
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and tissue homeostasis
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Zimei Dong1, Yibo Yang1, Guangwen Chen *1, Dezeng Liu1
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Henan, China
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*Author to whom all Correspondence should be addressed.
Henan, China. Email: [email protected]
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Abstract
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College of Life Science, Henan Normal University, Xinxiang, 453007
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College of Life Science, Henan Normal University, Xinxiang, 453007
The runt family genes play important roles in physiological
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processes in eukaryotic organisms by regulation of protein transcription,
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such as hematopoietic system, proliferation of gastric epithelial cells and
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neural development. However, it remains unclear about the specific
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functions of these genes. In this study, the full-length cDNA sequences of
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two runt genes are first cloned from Dugesia japonica, and their roles are
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investigated by WISH and RNAi. The results show that: (1) the Djrunts
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are conserved during evolution; (2) the Djrunts mRNA are widely
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expressed in intact and regenerative worms, and their expression levels
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are up-regulated significantly on day 1 after amputation; (3) loss of
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Djrunts function lead to lysis or regeneration failure in the intact and
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regenerating worms. Overall, the data suggests that Djrunts play
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important roles in regeneration and homeostatic maintenance in
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planarians.
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Keywords: runt gene; stem cells; planarian; regeneration
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1. Introduction
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Runt-related transcription factors (RUNX) belong to the family of
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related transcriptional regulatory factors, which they trigger expression of
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related genes by binding to different DNA (Vander Zwan et al. 2003).
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The first runx is identified and characterized in Drosophila melanogaster
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and it is involved in embryogenesis (Kania et al. 1990), followed by,
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three runx members are found in mammals, runx1, runx2 and runx3, their
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functions
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development, gastric epithelial cell proliferation and neural development
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(Okuda et al. 1996; Otto et al. 1997; Inoue et al. 2002; Li et al. 2002).
associated
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The amino acid sequences of runx contain two well-conserved
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domains: one is a 128-amino acids RUNT domain, which is usually
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locate at or near the N-terminus of the protein and specifically combine
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with the DNA sequences (Kagoshima et al. 1993; Coffman 2003). In
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mammals, the RUNT domain binds with the protein CBFβ, a kind of
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cytoplasmic protein associated with actin cytoskeleton, to form a
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heterodimer, which maintains the stability of the RUNT domain and
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enhances DNA binding capacity (Bravo et al. 2001; Zhang et al. 2003;
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Tang et al. 2000). RUNX1 and CBFβ are essential for definitive
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hematopoiesis, where they regulate expression of genes associated with
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proliferation, differentiation, and survival of stem and progenitor cells
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(Friedman 2009; de Bruijn & Speck 2004; Wang et al. 2010; Link et al.
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2010). The other conserved domain is the C-terminal pentapeptide
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sequence VWRPY, which is usually rich in proline, serine and threonine
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(Coffman 2003; Javed et al. 2001).
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At present, some runx are also found in many invertebrates, for
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example, there is only one runx member in the sponge, sea anemone, sea
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urchin, spider and nematode species, respectively (Damen et al. 2000;
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Robertson et al. 2002; Sullivan et al. 2008). In contrast, there contains
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four runx members in insects, such as fruit flies, mosquitoes and bees
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(Bao & Friedrich 2008). In Drosophila, runt genes regulate many
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physiological processes, for instance, in the central nervous system, runt
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gene is necessary for the development of neural subunits, in addition, runt
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genes are involved in sex determination, segmentation, hematopoiesis,
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and eye development (Lebestky et al. 2012; Coffman 2003).
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Planarian Dugesia japonica is a free-living member of the
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Platyhelminthes, which is renowned for their regenerative capacity and is
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an attractive model organism for the study of tissue regeneration. The
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pluripotent stem cells existed in planarian, called neoblasts, is the leader
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in planarian reconstruction of tissue and organs. Neoblasts is the only cell
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type with the ability to proliferate and differentiate in vivo with its small
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size, high mass ratio and oval or round shape, and it can differentiate into
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various types of cells including neurons and germinal cells (Saló 2006;
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Handberg et al. 2008), accounting for approximately 20-30% of the total
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cell numbers in planarian (Bravo 1987), and evenly distribute in all of the
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parenchymal tissues except the central nervous system and pharynx.
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Injury induces rhabdite release of inclusions and formation of a protective
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mucosa on the wound surface (Reisinger & Kelbetz 1964), at the same
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time, neoblasts are activated to migrate to the wound, dividing and
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proliferating at the wound to form undifferentiated white regenerative
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blastemata (Eisenhoffer et al. 2008; Wenemoser & Reddien 2010). The
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blastema continues to grow and differentiate into various tissues,
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eventually forming a complete planarian (Saló & Baguà 1989). In spite
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of this, the molecular mechanisms underlying regeneration is only
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starting to be understood. Here, the two runt genes are first cloned from
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D. japonica, and we find that Djrunts are required for planarian
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regeneration and tissue homeostasis. Our work provides basic data to
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elucidate the functions of runt genes and the molecular mechanism of
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planarian regeneration.
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2. Results
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2.1. cDNA cloning and homology analysis of Djrunts
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The full-length cDNA of Djrunt-1 is 1077 bp, including a 5′
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untranslated region (UTR) of 60 bp, a 3′-UTR of 48 bp and an open
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reading frame (ORF) of 969bp encoding a polypeptide of 322 amino
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acids (Fig. S1a). The full-length cDNA of Djrunt-2 is 1644 bp, which
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include a largest ORF of 516 bp, encoding a polypeptide of 172 amino
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acids (Fig. S1b). The deduced amino acid sequences of two genes show
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lower similarity (22.36% on protein level) (Fig. S2). Sequence homology
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search using BLASTn reveals that the Djrunts sequences share high
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similarity with other eukaryotic organisms’ runt sequences. The deduced
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amino acid sequence of Djrunt-1 show identity of 59.3% with Schmidtea
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mediterranea, 32.8% with Ascaris suum, 39.5% with Lottia gigantea,
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33.3% with Papilio polytes, and 38.0% with Pantherophis guttatus,
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respectively. The amino acid sequence of Djrunt-2 display high identity
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with those of runt-2 from S. mediterranea (93.8%), Schistosoma mansoni
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(51.7%), L. gigantea (49.0%), Halyomorpha halys (47.7%), Bombyx mori
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(40.7%) and Homo sapiens (40.7%). These results show that DjRUNT-1
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and DjRUNT-2 are highly conserved proteins among species.
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To shed light on the evolutionary position of DjRUNTs, the
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phylogenetic trees are constructed using the amino acid sequences of
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RUNTS from different Species (Fig. 1a, b). The both phylogenetic trees
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show that the vertebrates clustered together, for the invertebrates, the
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animals from the same phylum clustered together, respectively. The D.
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japonica and S. mediterranea, belonging to same genera, are clustered
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together and locate at the base of the tree with the original evolution
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status. while this evolutionary pattern is in accordance with the concept of
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traditional taxonomy. The high bootstrap values support the precision of
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topology.
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Fig.1 Phylogenic trees constructed by the neighbor-joining method
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according to amino acid sequences of runt-1 (a) and runt-2 (b). Numbers
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at the nodes denote the bootstrap percentages of 1000 pseudoreplicates.
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2.2. Expression patterns of Djrunts in intact and regenerative worms The spatial and temporal expression patterns of Djrunts in intact and
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regenerative worms are highly similar (Fig. 2). In intact worms, the
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mRNA of both genes are mainly distributed in each side of the body and
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the margin of the head, but the signal is not very strong, while in
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regenerative worms, the positive signals of both genes are strongly
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expressed in the regenerative blastemas whether in the fragments
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regenerating tail or head and throughout the regeneration process (Fig. 2).
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The RNAi worms, which are intact and regenerated 1 day with riboprobes
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for the two genes, are used as negative control hybridization. For these
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worms, there are almost no or very weak positive signals.
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Fig.2 Expression analyses of Djrunt-1 and Djrunt-2 in intact and
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regenerating worms by Whole-mount in situ hybridization. Dorsal view
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of intact and regeneration fragments followed by 1, 3, 5 and 7 days after
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decapitation. The arrows indicate the regions where the expressions are
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up-regulation. In all images, anterior is to the top. Scale bar: 0.5 mm.
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Quantitative real-time PCR is performed to examine the temporal
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expression levels of Djrunts during head regeneration. The worms are
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amputated transversely and the RNA is extracted from the entire tail
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fragments of the 0 d, 2 h, 6 h, 1 d, 2 d, 3 d, 5 d and 7 days after
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amputation and the intact worms respectively. Three specimens are
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included in each biological replicate and the intact worms are treated as
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the control. The expression pattern of Djrunt-1 is very similar to that of
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Djrunt-2. Their expression levels are changed dynamically and exhibited
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up-regulation throughout head regeneration compared with that of the
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control, Djrunt-1 and Djrunt-2 expression values are distinctly
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up-regulated at 6 h and reached the peak on the first day after amputation,
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then, its expression levels are gradually down-regulated (Fig. 3a, b).
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Fig.3 The expression patterns of Djrunt-1 (a) and Djrunt-2 (b) during
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head regeneration. Djβ-actin is used as the internal control. Vertical bars
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represent the mean±SD (N=3). Asterisks indicate statistical differences
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(*P<0.05; **P<0.01).
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2.3. Djrunts RNAi affects planarian regeneration and tissue
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homeostasis
To further examine the role of runts in D. japonica, depletion of the
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Djrunt-1, Djrunt-2 and the both Djrunts together are performed by RNAi
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in intact and regenerative worms. The RNA from each experimental
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group is extracted from Djrunts (RNAi) animals on the 10th day of
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regeneration (12 days from the last feeding). and 3 entire tail fragments
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are used for each group. The planarians interfered by L4440-GFP are
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used as the control. The effectiveness of RNAi is confirmed by WISH
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and qRT-PCR, significant reduction in endogenous Djrunts RNA levels
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and no or very weak positive signals are observed in RNAi worms with
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respect to the controls, albeit they are not completely eliminated after
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RNAi. Furthermore, Djrunt-1 RNAi has a little effect on the expression
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of Djrunt-2 and vice versa, while, their expression levels exhibit
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considerable reduction after co-interference of Djrunts (Fig. 4).
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Fig.4 qRT-PCR to measure mRNA levels of Djrunts (on the 12th day of
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the last feeding). Three worms are used for extracted RNA of every group.
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Vertical bars represent the mean±SD (N=3). The planarians interfered by
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L4440-GFP are used as the control. Asterisks indicate statistical differences
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(*P<0.05; **P<0.01).
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The phenotype defects are observed in intact and regenerative
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worms after the two genes RNAi (Fig. 5). Knockdown of Djrunt-1 result
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in 30% of intact worms to lyse (6/20) (Fig. 5c). In general, the
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abnormalities begin to appear on the third day of regeneration, the
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ulceration occurs at the wound and the body surface, and then the worms
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are lysed. 50% of the head regeneration worms following Djrunt-1 RNAi
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appear abnormalities after 18 days of regeneration (10/20), including lysis
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(4/20), no regeneration (5/20), and lesions (1/20) (Fig. 5a); and 45% of
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Djrunt-1 RNAi worms emerge abnormal phenotype during tail
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regeneration (9/20), including lysis (8/20) and no regeneration (1/20) (Fig.
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5b). Djrunt-2 RNAi worms have no obvious morphological changes on
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intact worms compared to the controls, but in the head regeneration, 25%
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of worms show lesions and no regeneration (5/20) (Fig. 5b), while, 35%
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of the head fragments appear lysis or no regeneration(7/20) during tail
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regeneration (Fig. 5a). When the both genes are co-interfered, the RNAi
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animals exhibit morphological abnormality on the second day after last
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feeding. For the regenerative worms, 65% (13/20) of the tail fragments
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and 80% (16/20) of head fragments are unable to heal on the wound
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surface and lose regeneration ability, then they are gradually lysed after
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18 days of regeneration (Fig. 5a, b). In intact worms, 60% of RNAi
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worms appear lesions and lysis (12/20) (Fig. 5c).
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Fig.5 Representative images of phenotypes observed following Djrunts
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RNAi. (a) Phenotypes in head regeneration worms, (b) Phenotypes in tail
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regeneration worms, (c) Phenotypes in intact worms. Scale bar: 0.5 mm.
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To further decipher Djrunts function and explore the reason of
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phenomenon after RNAi, we analyze the expression levels of some
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marker genes following Djrunts RNAi: neoblast markers (Djpiwi-A,
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Djpiwib, Djpiwi-1, and Djpcna ), cell cycle regulator (Djcyclinb), stem
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cell progeny markers (Djp53, DjNB21.11e and DjAGAT-2). The results
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show that the expression levels of Djpiwi-A, Djpiwi-1, Djpcna and
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DjcyclinB are all down-regulated, whereas there are no any significant
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changes on the Djpiwi-B. In addition, the DjNB21.11e, Djp53 and
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DjAGAT-2 are strongly up-regulated (Fig. 6).
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Fig.6 qRT-PCR to measure mRNA levels of marker genes after Djrunts
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RNAi. Vertical bars represent the mean±SD (N=3). Asterisks indicate
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statistical differences (*P<0.05; **P<0.01).
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In this study, the cDNA sequences of the runt family: Djrunt-1 and
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Djrunt-2 are cloned for the first time. Homology analysis show that the
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both genes are conserved during animal evolution. In vertebrates and
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most invertebrates, the C-terminus of the RUNT protein has a more
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conserved "VWRPY" pentapeptide sequence (Kaindl 2014; Ugarte et al.
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2013; Clark et al. 2007). Whereas, the C-terminus motif of the
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DjRUNT-1 is an "IWRPF" pentapeptide sequence. Intriguingly, there is
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no marked "VWRPY" pentapeptide sequence in C-terminal of the
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DjRUNT-2. In general, there is only one Runx member in many
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invertebrates (Damen et al. 2000; Robertson et al. 2002; Sullivan et al.
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2008), while the two members are found in planarians, we speculate that
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the Djrunt-2 might be caused by the mutation of Djrunt-1, which is a
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mutant subtype of the Djrunt-1.
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Two runx members, Djrunt-1 and Djrunt-2, are found in D. japonica.
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Here, the results of qPCR show that the expression levels of the two
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genes increased at 6 h and 24 h after amputation, in which the neoblasts
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cells are proliferating, this is in line with previous reports in S.
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mediterranea: runts are up-regulated in the early stages of regeneration
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(Wurtzel et al. 2015; Wenemoser
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positive signals of both genes are mainly distributed in parenchymal
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tissues and regenerative blastemas, where the neoblasts cells are
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expressed (Wenemoser & Reddien 2010), which is consistent with that of
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in S. mediterranea. For the Djrunts RNAi animals, the wound can not be
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healed normally so that the worms display lysis or lost their regenerative
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capacity. In S. mediterranea, runt-1 (RNAi) animals appear regeneration
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defect of eyes during head regeneration (Wenemoser
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in this study, the runt-1 RNAi worms phenotype are more severe in D.
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japonica than in S. mediterranea, and the RNAi worms are completely
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failure to regenerate or to lyse during head regeneration. Although, the
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planarians S. mediterranea and D. japonica belong to the same genus, but
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there is the obvious difference from protein sequences between the
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RUNTs, especially RUNT-1, the identity of the RUNT-1 between two
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species is only 59.3%.The different phenotype of the runt-1 RNAi worms
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might be caused by the difference of RUNT-1 sequences between two
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et al. 2012), while
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species. The cells in planarian tissues are constantly replaced by newly
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differentiated cells derived from stem cells, when this mechanism is
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damaged, planarians exhibit morphological abnormalities (Reddien et al.
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2005). Djpiwi-A (the ortholog of smedwi-1, which is co-expressed with
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runt-1 in S. Mediterranea. (Wenemoser et al. 2012) ) is specifically
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distribute in neoblasts, while Djpiwi-B (the ortholog of smedwi-2) is
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mainly distribute in the nucleus of neoblasts and also in differentiated
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tissues (Shibata et al. 2016), and Djpiwi-1, defines a subpopulation of
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planarian stem cells (Rossi et al. 2006). In this study, the expression
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levels of Djpiwi-A and Djpiwi-1 are significantly down regulate following
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Djrunts RNAi, interestingly, the expression level of Djpiwi-B has no
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significant change compared with that of the controls. The expression
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levels up-regulation of these neoblast markers indicate that Djrunts might
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promote proliferation of neoblasts. In mitosis, RUNX is found on
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centrosomes, spindles, and intermediates (Chuang et al. 2012), and
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RUNX2 binds to promoters of various cell cycle-related genes and
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regulates histone modifications during mitosis (Chuang et al. 2016). To
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further confirm this, we examine the expression levels of Djpcna, a
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helper protein gene necessary for DNA synthesis, which is expressed at
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the late Gl and S phases of the cell cycle (Takahashi & Caviness 1993),
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and DjcyclinB, the cyclin gene which regulates cell entry into mitosis.
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(Basto et al. 2007). Previous studies have shown that in the mitosis of
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osteoblasts, RUNX1 levels increased during G1 to S and maintain high
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levels during G2 and M phases (Bernardin-Fried et al. 2004), while
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RUNX2 protein levels are higher in early G1 phase and decreased in
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early S phase (Galindo et al. 2005), which shows that changes in RUNX
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activity affect the progression of the cell cycle. As expected, the
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expression levels of Djpcna and DjcyclinB are significantly decreased
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following Djrunts RNAi.
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The study in S. mediterranea shows that the runts, especially runt-1, can promote neoblast differentiation (Wenemoser
et al. 2012) and the
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studies of in other species, runx1 is involved in regulation of
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hematopoietic stem cell differentiation (Okuda et al. 1996), as well as
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RUNX2 is a necessary transcription factor during the early differentiation
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of osteoblast (Li et al. 2016), and it participates in the regulation of gene
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expression on differentiation in osteoblast. To ascertain the roles in
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differentiation of Djrunts in D. japonica, the stem cell progeny markers:
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Djp53, DjNB21.11e and DjAGAT-2 are investigated, which are located in
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early epidermal-committed stem cell progeny (Djp53, DjNB21.11e) and
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late epidermal-committed stem cell progeny (DjAGAT-2) (Nogi & Levin
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2005; Pearson & Sánchez Alvarado 2010). However, it is surprising that
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these genes significantly increased following Djrunts RNAi. In S.
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mediterranea, Smed-p53 is located in the newly made stem cell progeny,
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when it is knocked out, the cells proliferation increases and
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differentiation reduces (Pearson & S á nchez Alvarado 2010). Some
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researches have shown that RUNX is involved in the developmental
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balance between cell proliferation and differentiation in many systems
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(Coffman 2003). In Caenorhabditis elegans, RNT-1, the runx homologue,
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is required for the development of larvae and may interact directly with
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cell cycle regulators, as well as the balance between proliferation and
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differentiation (Kagoshima et al. 2007; Nimmo et al. 2005; Xia et al.
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2007). So, we speculate that the Djrunts may regulate the balance
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between proliferation and differentiation in the intact and regenerating
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worms, In other words, the failure of neoblast maintenance may also
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result from loss of proliferative potential and subsequent premature
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differentiation. While these conclusions need to be further confirmed
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through some more direct methods in the future, such as BrdU labeling,
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double fluorescent in situ hybridization and so on. And further studies
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will be required to elucidate the precise mechanism of action of Djrunts
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genes.
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Djrunts knockdown is more efficient for double knockdowns than
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for individual genes and the RNAi phenotypes are more severe. Some
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researchers have shown that aside from cross repression, RUNX proteins
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cooperate with each other, chondrocyte maturation is highly dependent on
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the regulation of both Runx2 and Runx3 (Yoshida et al. 2004), whereas
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regulation of sternal morphogenesis involves cooperation of Runx2 and
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Runx1 (Kimura et al. 2010). In addition, the expression patterns of
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Djrunt-1 and Djrunt-2 are very similar, but Djrunt-2 RNAi animals do
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not display any abnormal in intact individuals and only a few
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abnormalities in regenerative fragments compared to those of Djrunt-1 or
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both genes RNAi worms. Accordingly, we suspect that Djrunt-1 and
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Djrunt-2 may interact with each other. Djrunt-1 play critical roles and the
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two genes are synergistic in regulating regeneration and tissue
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homeostasis.
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4. Materials and methods
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4.1. Animals and sample preparation
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Planarians used in this work are collected from Shilaogong, Hebi
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City, Henan Province, China. Animals are cultured in autoclaved tap
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water in dark at 20℃ and starved for at least 2 weeks before being used in
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all experiments. Regenerating worms are obtained by transverse
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amputation at the pre-pharyngeal and post-auricle level from intact
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worms. This study does not involve endangered or protected species, and
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the collection of specimen is approved by the Forestry Department of
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wild animal protection, Henan Province, China.
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4.2. Isolation of the Djrunts genes
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Total RNA is extracted using RNAiso plus (Takara, China), and 2µg
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total RNA is used for reverse transcription according to the
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manufacturers’ instructions of M-MLV reverse transcriptase (TaKaRa,
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China). All primers used in this study are shown in Table. S1. The
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Expressed sequence tag (EST) fragments of Djrunt-1 and Djrunt-2 genes
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are first obtained with two pairs of specific primers: Djrunt-1 F, Djrunt-1
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R and Djrunt-2 F, Djrunt-2 R (Table. S1). Based on the EST, the 5′-gene
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specific primers (GSP) (Djrunt-1 5′GSP1 and GSP2; Djrunt-2 5′GSP1
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and GSP2) and 3′-gene specific primers (Djrunt-1 3′GSP1 and GSP2;
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Djrunt-2 3′GSP1 and GSP2) are designed (Table. S1). The corresponding
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full-length transcripts are
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complementary DNA (cDNA) ends (RACE) using both 5′and 3′-Full
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RACE kits (TaKaRa, China) according to the manufacturer’s instructions.
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The PCR products are gel-purified and ligated into the pMD19-T vector,
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and then submit for sequencing. The sequences were verified and
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deposited into the GenBank database (GenBank accession number:
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Djrunt-1, KX885484; Djrunt-2, KX885485).
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4.3. Sequence analysis and phylogenetic tree construction
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amplification of
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Amino acid sequences of DjRUNT-1 and DjRUNT-2 are deduced
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from their cDNA sequences. The identity of the protein sequences is
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calculated using the MegAlign program in DNAStar software package. In
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addition, Phylogenetic trees are carried out from the amino acid sequence
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alignments by the neighbor-joining (NJ) method using Mega 4.0 program
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(http://www.megasoftware.net/). Statistical support is provided by 1000
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bootstrap replications.
363
4.4. Quantitative real-time PCR
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Quantitative real-time PCR is performed as described previously
365
(Dong et al. 2012). The specially designed primers of Djrunt-1, Djrunt-2
366
and some marker genes (Djpiwi-A, Djpiwi-B, Djpiwi-1, Djpcna,
367
DjcyclinB, DjNB21.11e, Djp53, DjAGAT-2, ) are used for real-time PCR;
368
Djβ-actin is used as the reference gene in all experiments. Primers
369
(Table. S1) are designed using Primer Premier 5.0 software, and all
370
primers generate a single PCR band of the expected size. PCR products
371
are verified by DNA sequencing. The levels of relative expression are
372
calculated and quantified with the 2-ΔΔCt method.
373
4.5. Whole-mount in situ hybridization
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Whole-mount in situ hybridization (WISH) is carried out as
375
described previously (Pearson et al. 2005; Dong et al. 2014). The
376
digoxigenin-labelled probes are designed according to Djrunt-1 (from
377
position 211 to 595) and Djrunt-2 (from position 50 to 568) sequences,
378
respectively. The probes are synthesized using the RNA in vitro labeling
379
kit (Roche). Intact and regenerative worms (1, 3, 5, and 7 days after being
380
decapitated) are used for WISH. Control experiments are performed using
381
the sense probe.
382
4.6. RNA interference
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Planarians are fed bacteria to induce to express double stranded
384
RNA (dsRNA) against the Djrunt-1 and Djrunt-2 genes as previously
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described (Dong et al. 2017). The conserved domains of Djrunt-1 (from
386
position 262 to 879) and Djrunt-2 (from position 155 to 665) genes are
387
amplified from cDNA clone using primers (Djrunt-1 RNAi; Djrunt-2
388
RNAi) and then cloned into the L4440 vector using XbaI and KpnI. For
389
the RNAi experiment, worms are fed 3 times over 5 days (1st, 3rd and 5th)
390
and are amputated into 2 fragments pre-pharyngeally at 24 h after the last
391
feeding (Fig.7). The effectiveness of RNAi is confirmed by qRT-PCR and
392
WISH, the L4440-GFP is used as the control. The morphological changes
393
of intact and regenerative worms after RNAi are observed using the
394
stereomicroscope, and the images are captured with a Leica camera
395
(DFC300FX, Germany).
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Fig.7 RNAi feeding schedules. (dpa: days post amputation)
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4.7. Statistical analysis The SPSS 13.0 software is used as statistical analysis. Data is
401
analyzed by one-way ANOVA followed by post-hoc multiple
402
comparisons using the LSD test and Dunnett’s test. Differences are
403
regarded as statistically significant at P < 0.05 and highly significant at P
404
< 0.01.
405
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Conflict of Interest Statement:
407
None.
408
Acknowledgements
409
This work was supported by the National Natural Science Foundation of
410
China (grants numbers Nos.31570376, 31471965 and u1604173).
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Appendix A. Supplementary Materials
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Sequence(5'-3')
Djrunt-1 EST-F
CACGAAGCTACCTCAGCATTGG
Djrunt-1 EST-R
ATTTTCCTCTTCCACTGCGAC
Djrunt-1 3'GSP1
AGTGTGGTTATCGGCCAGTAATC
Djrunt-1 3'GSP 2
GCTACCTCAGCATTGGCGAT
Djrunt-1 5'GSP 1
CAATGTTTGGACTAGCTTCTATATG
Djrunt-1 5'GSP 2
GGTGATCTGGGATAACATAATCCTG
Djrunt-1 RT-F
TTTATTAGTCGCAGTGGAAGAGG
Djrunt-1 RT-R
ATCTGTTGGGCGTATAAACTGAG
Djrunt-2 EST-R
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CACTGGCGCTCCAACAAGAC GGCCCATCGACGGTTACTTTGAT
TGATTGAGCACATTGTCAGGAAG
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Djrunt-2 3'GSP1
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Djrunt-2 EST-F
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Primers
ACAGTGTTCCATTTTGATTGAGC
Djrunt-2 5'GSP1
TGATTCTCCAATGTGAAGGTAACTG
AC C
Djrunt-2 3'GSP2
Djrunt-2 5'GSP2
GGATACGATTCCATAGTTATTGTG
Djrunt-2 RT-F
AACATCACAATAACTATGGAATCGT
Djrunt-2 RT-R
CTGACAATGTGCTCAATCAAAATG
Djβ-actin RT-F
ACACCGTACCAATCTATG
Djβ-actin RT-R
GTGAAACTGTAACCTCGT
Djpiwi-A RT-F
GATGGTGTAGGAGATTCGCAACT
Djpiwi-A RT-R
CGGAGATGCAGTGCCTTTAGTAGT
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Djpiwi-B RT-R
ATTGTCCCCAACACCATCTCG
Djpiwi-1 RT-F
AAACGAAGCACCCGAAGATATG
Djpiwi-1 RT-R
CCCACTTATTTGACATACCCTGAG
Djpcna RT-F
GTAGAGAAATGAGTCAAATGGGTG
Djpcna RT-R
TCAATGGTCACGGCATCACT
DjcyclinB RT-F
AGTCACGATGTTGGGAAAATGC
DjcyclinB RT-R
TTTCTCAATAACCCTGGCGTGT
Djp53 RT-F
GATCAACTAAGCCTTTCAGCCAT
Djp53 RT-R
CAAAGCACAGACAGCAGAGTCAT
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Table S1. Primers used in this study
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635 636 637 638 639 640 641 642 643 644
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CGATTCAGGCATCTGTATTCTCG
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Djpiwi-B RT-F
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Fig. S1. The nucleotide and deduced amino acid sequences of the Djrunt-1 (a) and
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Djrunt-2 (b) cDNA. The grey is “RD” domain, the red is conserved phosphorylation
652
sites, the green "IWRPF" is
653
codon, and underlined sequence is the poly a signal.
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C-terminus conserved sequence, “*” is the termination
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Fig. S2. An alignment of the amino acids deduced by Djrunt-1 and Djrunt-2.
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Highlights
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The full-length cDNA sequences of Djrunt genes were first cloned. The expression levels of Djrunts were up-regulated on day 1 after amputation. The Djrunts play important roles in regeneration and homeostatic maintenance.
S1567-133X(17)30234-X
DOI:
10.1016/j.gep.2018.04.006
Reference:
MODGEP 1091
To appear in:
Gene Expression Patterns
Received Date: 29 December 2017 Revised Date:
21 March 2018
Accepted Date: 6 April 2018
Please cite this article as: Dong, Z., Yang, Y., Chen, G., Liu, D., Identification of runt family genes involved in planarian regeneration and tissue homeostasis, Gene Expression Patterns (2018), doi: 10.1016/j.gep.2018.04.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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Identification of runt family genes involved in planarian regeneration
2
and tissue homeostasis
3
Zimei Dong1, Yibo Yang1, Guangwen Chen *1, Dezeng Liu1
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1
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Henan, China
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*Author to whom all Correspondence should be addressed.
Henan, China. Email: [email protected]
9
Abstract
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College of Life Science, Henan Normal University, Xinxiang, 453007
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College of Life Science, Henan Normal University, Xinxiang, 453007
The runt family genes play important roles in physiological
12
processes in eukaryotic organisms by regulation of protein transcription,
13
such as hematopoietic system, proliferation of gastric epithelial cells and
14
neural development. However, it remains unclear about the specific
15
functions of these genes. In this study, the full-length cDNA sequences of
16
two runt genes are first cloned from Dugesia japonica, and their roles are
17
investigated by WISH and RNAi. The results show that: (1) the Djrunts
18
are conserved during evolution; (2) the Djrunts mRNA are widely
19
expressed in intact and regenerative worms, and their expression levels
20
are up-regulated significantly on day 1 after amputation; (3) loss of
21
Djrunts function lead to lysis or regeneration failure in the intact and
22
regenerating worms. Overall, the data suggests that Djrunts play
23
important roles in regeneration and homeostatic maintenance in
24
planarians.
25
Keywords: runt gene; stem cells; planarian; regeneration
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1. Introduction
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Runt-related transcription factors (RUNX) belong to the family of
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related transcriptional regulatory factors, which they trigger expression of
29
related genes by binding to different DNA (Vander Zwan et al. 2003).
30
The first runx is identified and characterized in Drosophila melanogaster
31
and it is involved in embryogenesis (Kania et al. 1990), followed by,
32
three runx members are found in mammals, runx1, runx2 and runx3, their
33
functions
34
development, gastric epithelial cell proliferation and neural development
35
(Okuda et al. 1996; Otto et al. 1997; Inoue et al. 2002; Li et al. 2002).
associated
with
hematopoietic
systems,
skeletal
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The amino acid sequences of runx contain two well-conserved
37
domains: one is a 128-amino acids RUNT domain, which is usually
38
locate at or near the N-terminus of the protein and specifically combine
39
with the DNA sequences (Kagoshima et al. 1993; Coffman 2003). In
40
mammals, the RUNT domain binds with the protein CBFβ, a kind of
41
cytoplasmic protein associated with actin cytoskeleton, to form a
42
heterodimer, which maintains the stability of the RUNT domain and
43
enhances DNA binding capacity (Bravo et al. 2001; Zhang et al. 2003;
44
Tang et al. 2000). RUNX1 and CBFβ are essential for definitive
45
hematopoiesis, where they regulate expression of genes associated with
46
proliferation, differentiation, and survival of stem and progenitor cells
47
(Friedman 2009; de Bruijn & Speck 2004; Wang et al. 2010; Link et al.
48
2010). The other conserved domain is the C-terminal pentapeptide
49
sequence VWRPY, which is usually rich in proline, serine and threonine
50
(Coffman 2003; Javed et al. 2001).
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At present, some runx are also found in many invertebrates, for
52
example, there is only one runx member in the sponge, sea anemone, sea
53
urchin, spider and nematode species, respectively (Damen et al. 2000;
54
Robertson et al. 2002; Sullivan et al. 2008). In contrast, there contains
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four runx members in insects, such as fruit flies, mosquitoes and bees
56
(Bao & Friedrich 2008). In Drosophila, runt genes regulate many
57
physiological processes, for instance, in the central nervous system, runt
58
gene is necessary for the development of neural subunits, in addition, runt
59
genes are involved in sex determination, segmentation, hematopoiesis,
60
and eye development (Lebestky et al. 2012; Coffman 2003).
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Planarian Dugesia japonica is a free-living member of the
62
Platyhelminthes, which is renowned for their regenerative capacity and is
63
an attractive model organism for the study of tissue regeneration. The
64
pluripotent stem cells existed in planarian, called neoblasts, is the leader
65
in planarian reconstruction of tissue and organs. Neoblasts is the only cell
66
type with the ability to proliferate and differentiate in vivo with its small
67
size, high mass ratio and oval or round shape, and it can differentiate into
68
various types of cells including neurons and germinal cells (Saló 2006;
69
Handberg et al. 2008), accounting for approximately 20-30% of the total
70
cell numbers in planarian (Bravo 1987), and evenly distribute in all of the
71
parenchymal tissues except the central nervous system and pharynx.
72
Injury induces rhabdite release of inclusions and formation of a protective
73
mucosa on the wound surface (Reisinger & Kelbetz 1964), at the same
74
time, neoblasts are activated to migrate to the wound, dividing and
75
proliferating at the wound to form undifferentiated white regenerative
76
blastemata (Eisenhoffer et al. 2008; Wenemoser & Reddien 2010). The
77
blastema continues to grow and differentiate into various tissues,
78
eventually forming a complete planarian (Saló & Baguà 1989). In spite
79
of this, the molecular mechanisms underlying regeneration is only
80
starting to be understood. Here, the two runt genes are first cloned from
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D. japonica, and we find that Djrunts are required for planarian
82
regeneration and tissue homeostasis. Our work provides basic data to
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elucidate the functions of runt genes and the molecular mechanism of
84
planarian regeneration.
85
2. Results
86
2.1. cDNA cloning and homology analysis of Djrunts
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The full-length cDNA of Djrunt-1 is 1077 bp, including a 5′
88
untranslated region (UTR) of 60 bp, a 3′-UTR of 48 bp and an open
89
reading frame (ORF) of 969bp encoding a polypeptide of 322 amino
90
acids (Fig. S1a). The full-length cDNA of Djrunt-2 is 1644 bp, which
91
include a largest ORF of 516 bp, encoding a polypeptide of 172 amino
92
acids (Fig. S1b). The deduced amino acid sequences of two genes show
93
lower similarity (22.36% on protein level) (Fig. S2). Sequence homology
94
search using BLASTn reveals that the Djrunts sequences share high
95
similarity with other eukaryotic organisms’ runt sequences. The deduced
96
amino acid sequence of Djrunt-1 show identity of 59.3% with Schmidtea
97
mediterranea, 32.8% with Ascaris suum, 39.5% with Lottia gigantea,
98
33.3% with Papilio polytes, and 38.0% with Pantherophis guttatus,
99
respectively. The amino acid sequence of Djrunt-2 display high identity
100
with those of runt-2 from S. mediterranea (93.8%), Schistosoma mansoni
101
(51.7%), L. gigantea (49.0%), Halyomorpha halys (47.7%), Bombyx mori
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(40.7%) and Homo sapiens (40.7%). These results show that DjRUNT-1
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and DjRUNT-2 are highly conserved proteins among species.
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To shed light on the evolutionary position of DjRUNTs, the
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phylogenetic trees are constructed using the amino acid sequences of
106
RUNTS from different Species (Fig. 1a, b). The both phylogenetic trees
107
show that the vertebrates clustered together, for the invertebrates, the
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animals from the same phylum clustered together, respectively. The D.
109
japonica and S. mediterranea, belonging to same genera, are clustered
110
together and locate at the base of the tree with the original evolution
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status. while this evolutionary pattern is in accordance with the concept of
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traditional taxonomy. The high bootstrap values support the precision of
113
topology.
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Fig.1 Phylogenic trees constructed by the neighbor-joining method
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according to amino acid sequences of runt-1 (a) and runt-2 (b). Numbers
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at the nodes denote the bootstrap percentages of 1000 pseudoreplicates.
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2.2. Expression patterns of Djrunts in intact and regenerative worms The spatial and temporal expression patterns of Djrunts in intact and
121
regenerative worms are highly similar (Fig. 2). In intact worms, the
122
mRNA of both genes are mainly distributed in each side of the body and
123
the margin of the head, but the signal is not very strong, while in
124
regenerative worms, the positive signals of both genes are strongly
125
expressed in the regenerative blastemas whether in the fragments
126
regenerating tail or head and throughout the regeneration process (Fig. 2).
127
The RNAi worms, which are intact and regenerated 1 day with riboprobes
128
for the two genes, are used as negative control hybridization. For these
129
worms, there are almost no or very weak positive signals.
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Fig.2 Expression analyses of Djrunt-1 and Djrunt-2 in intact and
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regenerating worms by Whole-mount in situ hybridization. Dorsal view
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of intact and regeneration fragments followed by 1, 3, 5 and 7 days after
134
decapitation. The arrows indicate the regions where the expressions are
135
up-regulation. In all images, anterior is to the top. Scale bar: 0.5 mm.
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Quantitative real-time PCR is performed to examine the temporal
138
expression levels of Djrunts during head regeneration. The worms are
139
amputated transversely and the RNA is extracted from the entire tail
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fragments of the 0 d, 2 h, 6 h, 1 d, 2 d, 3 d, 5 d and 7 days after
141
amputation and the intact worms respectively. Three specimens are
142
included in each biological replicate and the intact worms are treated as
143
the control. The expression pattern of Djrunt-1 is very similar to that of
144
Djrunt-2. Their expression levels are changed dynamically and exhibited
145
up-regulation throughout head regeneration compared with that of the
146
control, Djrunt-1 and Djrunt-2 expression values are distinctly
147
up-regulated at 6 h and reached the peak on the first day after amputation,
148
then, its expression levels are gradually down-regulated (Fig. 3a, b).
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Fig.3 The expression patterns of Djrunt-1 (a) and Djrunt-2 (b) during
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head regeneration. Djβ-actin is used as the internal control. Vertical bars
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represent the mean±SD (N=3). Asterisks indicate statistical differences
153
(*P<0.05; **P<0.01).
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2.3. Djrunts RNAi affects planarian regeneration and tissue
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homeostasis
To further examine the role of runts in D. japonica, depletion of the
158
Djrunt-1, Djrunt-2 and the both Djrunts together are performed by RNAi
159
in intact and regenerative worms. The RNA from each experimental
160
group is extracted from Djrunts (RNAi) animals on the 10th day of
161
regeneration (12 days from the last feeding). and 3 entire tail fragments
162
are used for each group. The planarians interfered by L4440-GFP are
163
used as the control. The effectiveness of RNAi is confirmed by WISH
164
and qRT-PCR, significant reduction in endogenous Djrunts RNA levels
165
and no or very weak positive signals are observed in RNAi worms with
166
respect to the controls, albeit they are not completely eliminated after
167
RNAi. Furthermore, Djrunt-1 RNAi has a little effect on the expression
168
of Djrunt-2 and vice versa, while, their expression levels exhibit
169
considerable reduction after co-interference of Djrunts (Fig. 4).
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Fig.4 qRT-PCR to measure mRNA levels of Djrunts (on the 12th day of
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the last feeding). Three worms are used for extracted RNA of every group.
173
Vertical bars represent the mean±SD (N=3). The planarians interfered by
174
L4440-GFP are used as the control. Asterisks indicate statistical differences
175
(*P<0.05; **P<0.01).
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The phenotype defects are observed in intact and regenerative
178
worms after the two genes RNAi (Fig. 5). Knockdown of Djrunt-1 result
179
in 30% of intact worms to lyse (6/20) (Fig. 5c). In general, the
180
abnormalities begin to appear on the third day of regeneration, the
181
ulceration occurs at the wound and the body surface, and then the worms
182
are lysed. 50% of the head regeneration worms following Djrunt-1 RNAi
183
appear abnormalities after 18 days of regeneration (10/20), including lysis
184
(4/20), no regeneration (5/20), and lesions (1/20) (Fig. 5a); and 45% of
185
Djrunt-1 RNAi worms emerge abnormal phenotype during tail
186
regeneration (9/20), including lysis (8/20) and no regeneration (1/20) (Fig.
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5b). Djrunt-2 RNAi worms have no obvious morphological changes on
188
intact worms compared to the controls, but in the head regeneration, 25%
189
of worms show lesions and no regeneration (5/20) (Fig. 5b), while, 35%
190
of the head fragments appear lysis or no regeneration(7/20) during tail
191
regeneration (Fig. 5a). When the both genes are co-interfered, the RNAi
192
animals exhibit morphological abnormality on the second day after last
193
feeding. For the regenerative worms, 65% (13/20) of the tail fragments
194
and 80% (16/20) of head fragments are unable to heal on the wound
195
surface and lose regeneration ability, then they are gradually lysed after
196
18 days of regeneration (Fig. 5a, b). In intact worms, 60% of RNAi
197
worms appear lesions and lysis (12/20) (Fig. 5c).
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Fig.5 Representative images of phenotypes observed following Djrunts
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RNAi. (a) Phenotypes in head regeneration worms, (b) Phenotypes in tail
201
regeneration worms, (c) Phenotypes in intact worms. Scale bar: 0.5 mm.
202 203
To further decipher Djrunts function and explore the reason of
204
phenomenon after RNAi, we analyze the expression levels of some
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marker genes following Djrunts RNAi: neoblast markers (Djpiwi-A,
206
Djpiwib, Djpiwi-1, and Djpcna ), cell cycle regulator (Djcyclinb), stem
207
cell progeny markers (Djp53, DjNB21.11e and DjAGAT-2). The results
208
show that the expression levels of Djpiwi-A, Djpiwi-1, Djpcna and
209
DjcyclinB are all down-regulated, whereas there are no any significant
210
changes on the Djpiwi-B. In addition, the DjNB21.11e, Djp53 and
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DjAGAT-2 are strongly up-regulated (Fig. 6).
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Fig.6 qRT-PCR to measure mRNA levels of marker genes after Djrunts
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RNAi. Vertical bars represent the mean±SD (N=3). Asterisks indicate
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statistical differences (*P<0.05; **P<0.01).
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In this study, the cDNA sequences of the runt family: Djrunt-1 and
219
Djrunt-2 are cloned for the first time. Homology analysis show that the
220
both genes are conserved during animal evolution. In vertebrates and
221
most invertebrates, the C-terminus of the RUNT protein has a more
222
conserved "VWRPY" pentapeptide sequence (Kaindl 2014; Ugarte et al.
223
2013; Clark et al. 2007). Whereas, the C-terminus motif of the
224
DjRUNT-1 is an "IWRPF" pentapeptide sequence. Intriguingly, there is
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no marked "VWRPY" pentapeptide sequence in C-terminal of the
226
DjRUNT-2. In general, there is only one Runx member in many
227
invertebrates (Damen et al. 2000; Robertson et al. 2002; Sullivan et al.
228
2008), while the two members are found in planarians, we speculate that
229
the Djrunt-2 might be caused by the mutation of Djrunt-1, which is a
230
mutant subtype of the Djrunt-1.
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Two runx members, Djrunt-1 and Djrunt-2, are found in D. japonica.
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Here, the results of qPCR show that the expression levels of the two
233
genes increased at 6 h and 24 h after amputation, in which the neoblasts
234
cells are proliferating, this is in line with previous reports in S.
235
mediterranea: runts are up-regulated in the early stages of regeneration
236
(Wurtzel et al. 2015; Wenemoser
237
positive signals of both genes are mainly distributed in parenchymal
238
tissues and regenerative blastemas, where the neoblasts cells are
239
expressed (Wenemoser & Reddien 2010), which is consistent with that of
240
in S. mediterranea. For the Djrunts RNAi animals, the wound can not be
241
healed normally so that the worms display lysis or lost their regenerative
242
capacity. In S. mediterranea, runt-1 (RNAi) animals appear regeneration
243
defect of eyes during head regeneration (Wenemoser
244
in this study, the runt-1 RNAi worms phenotype are more severe in D.
245
japonica than in S. mediterranea, and the RNAi worms are completely
246
failure to regenerate or to lyse during head regeneration. Although, the
247
planarians S. mediterranea and D. japonica belong to the same genus, but
248
there is the obvious difference from protein sequences between the
249
RUNTs, especially RUNT-1, the identity of the RUNT-1 between two
250
species is only 59.3%.The different phenotype of the runt-1 RNAi worms
251
might be caused by the difference of RUNT-1 sequences between two
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et al. 2012), while
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species. The cells in planarian tissues are constantly replaced by newly
254
differentiated cells derived from stem cells, when this mechanism is
255
damaged, planarians exhibit morphological abnormalities (Reddien et al.
256
2005). Djpiwi-A (the ortholog of smedwi-1, which is co-expressed with
257
runt-1 in S. Mediterranea. (Wenemoser et al. 2012) ) is specifically
258
distribute in neoblasts, while Djpiwi-B (the ortholog of smedwi-2) is
259
mainly distribute in the nucleus of neoblasts and also in differentiated
260
tissues (Shibata et al. 2016), and Djpiwi-1, defines a subpopulation of
261
planarian stem cells (Rossi et al. 2006). In this study, the expression
262
levels of Djpiwi-A and Djpiwi-1 are significantly down regulate following
263
Djrunts RNAi, interestingly, the expression level of Djpiwi-B has no
264
significant change compared with that of the controls. The expression
265
levels up-regulation of these neoblast markers indicate that Djrunts might
266
promote proliferation of neoblasts. In mitosis, RUNX is found on
267
centrosomes, spindles, and intermediates (Chuang et al. 2012), and
268
RUNX2 binds to promoters of various cell cycle-related genes and
269
regulates histone modifications during mitosis (Chuang et al. 2016). To
270
further confirm this, we examine the expression levels of Djpcna, a
271
helper protein gene necessary for DNA synthesis, which is expressed at
272
the late Gl and S phases of the cell cycle (Takahashi & Caviness 1993),
273
and DjcyclinB, the cyclin gene which regulates cell entry into mitosis.
274
(Basto et al. 2007). Previous studies have shown that in the mitosis of
275
osteoblasts, RUNX1 levels increased during G1 to S and maintain high
276
levels during G2 and M phases (Bernardin-Fried et al. 2004), while
277
RUNX2 protein levels are higher in early G1 phase and decreased in
278
early S phase (Galindo et al. 2005), which shows that changes in RUNX
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activity affect the progression of the cell cycle. As expected, the
280
expression levels of Djpcna and DjcyclinB are significantly decreased
281
following Djrunts RNAi.
282
The study in S. mediterranea shows that the runts, especially runt-1, can promote neoblast differentiation (Wenemoser
et al. 2012) and the
284
studies of in other species, runx1 is involved in regulation of
285
hematopoietic stem cell differentiation (Okuda et al. 1996), as well as
286
RUNX2 is a necessary transcription factor during the early differentiation
287
of osteoblast (Li et al. 2016), and it participates in the regulation of gene
288
expression on differentiation in osteoblast. To ascertain the roles in
289
differentiation of Djrunts in D. japonica, the stem cell progeny markers:
290
Djp53, DjNB21.11e and DjAGAT-2 are investigated, which are located in
291
early epidermal-committed stem cell progeny (Djp53, DjNB21.11e) and
292
late epidermal-committed stem cell progeny (DjAGAT-2) (Nogi & Levin
293
2005; Pearson & Sánchez Alvarado 2010). However, it is surprising that
294
these genes significantly increased following Djrunts RNAi. In S.
295
mediterranea, Smed-p53 is located in the newly made stem cell progeny,
296
when it is knocked out, the cells proliferation increases and
297
differentiation reduces (Pearson & S á nchez Alvarado 2010). Some
298
researches have shown that RUNX is involved in the developmental
299
balance between cell proliferation and differentiation in many systems
300
(Coffman 2003). In Caenorhabditis elegans, RNT-1, the runx homologue,
301
is required for the development of larvae and may interact directly with
302
cell cycle regulators, as well as the balance between proliferation and
303
differentiation (Kagoshima et al. 2007; Nimmo et al. 2005; Xia et al.
304
2007). So, we speculate that the Djrunts may regulate the balance
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between proliferation and differentiation in the intact and regenerating
306
worms, In other words, the failure of neoblast maintenance may also
307
result from loss of proliferative potential and subsequent premature
308
differentiation. While these conclusions need to be further confirmed
309
through some more direct methods in the future, such as BrdU labeling,
310
double fluorescent in situ hybridization and so on. And further studies
311
will be required to elucidate the precise mechanism of action of Djrunts
312
genes.
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Djrunts knockdown is more efficient for double knockdowns than
314
for individual genes and the RNAi phenotypes are more severe. Some
315
researchers have shown that aside from cross repression, RUNX proteins
316
cooperate with each other, chondrocyte maturation is highly dependent on
317
the regulation of both Runx2 and Runx3 (Yoshida et al. 2004), whereas
318
regulation of sternal morphogenesis involves cooperation of Runx2 and
319
Runx1 (Kimura et al. 2010). In addition, the expression patterns of
320
Djrunt-1 and Djrunt-2 are very similar, but Djrunt-2 RNAi animals do
321
not display any abnormal in intact individuals and only a few
322
abnormalities in regenerative fragments compared to those of Djrunt-1 or
323
both genes RNAi worms. Accordingly, we suspect that Djrunt-1 and
324
Djrunt-2 may interact with each other. Djrunt-1 play critical roles and the
325
two genes are synergistic in regulating regeneration and tissue
326
homeostasis.
327
4. Materials and methods
328
4.1. Animals and sample preparation
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Planarians used in this work are collected from Shilaogong, Hebi
330
City, Henan Province, China. Animals are cultured in autoclaved tap
331
water in dark at 20℃ and starved for at least 2 weeks before being used in
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all experiments. Regenerating worms are obtained by transverse
333
amputation at the pre-pharyngeal and post-auricle level from intact
334
worms. This study does not involve endangered or protected species, and
335
the collection of specimen is approved by the Forestry Department of
336
wild animal protection, Henan Province, China.
337
4.2. Isolation of the Djrunts genes
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Total RNA is extracted using RNAiso plus (Takara, China), and 2µg
339
total RNA is used for reverse transcription according to the
340
manufacturers’ instructions of M-MLV reverse transcriptase (TaKaRa,
341
China). All primers used in this study are shown in Table. S1. The
342
Expressed sequence tag (EST) fragments of Djrunt-1 and Djrunt-2 genes
343
are first obtained with two pairs of specific primers: Djrunt-1 F, Djrunt-1
344
R and Djrunt-2 F, Djrunt-2 R (Table. S1). Based on the EST, the 5′-gene
345
specific primers (GSP) (Djrunt-1 5′GSP1 and GSP2; Djrunt-2 5′GSP1
346
and GSP2) and 3′-gene specific primers (Djrunt-1 3′GSP1 and GSP2;
347
Djrunt-2 3′GSP1 and GSP2) are designed (Table. S1). The corresponding
348
full-length transcripts are
349
complementary DNA (cDNA) ends (RACE) using both 5′and 3′-Full
350
RACE kits (TaKaRa, China) according to the manufacturer’s instructions.
351
The PCR products are gel-purified and ligated into the pMD19-T vector,
352
and then submit for sequencing. The sequences were verified and
353
deposited into the GenBank database (GenBank accession number:
354
Djrunt-1, KX885484; Djrunt-2, KX885485).
355
4.3. Sequence analysis and phylogenetic tree construction
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amplification of
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Amino acid sequences of DjRUNT-1 and DjRUNT-2 are deduced
357
from their cDNA sequences. The identity of the protein sequences is
358
calculated using the MegAlign program in DNAStar software package. In
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addition, Phylogenetic trees are carried out from the amino acid sequence
360
alignments by the neighbor-joining (NJ) method using Mega 4.0 program
361
(http://www.megasoftware.net/). Statistical support is provided by 1000
362
bootstrap replications.
363
4.4. Quantitative real-time PCR
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Quantitative real-time PCR is performed as described previously
365
(Dong et al. 2012). The specially designed primers of Djrunt-1, Djrunt-2
366
and some marker genes (Djpiwi-A, Djpiwi-B, Djpiwi-1, Djpcna,
367
DjcyclinB, DjNB21.11e, Djp53, DjAGAT-2, ) are used for real-time PCR;
368
Djβ-actin is used as the reference gene in all experiments. Primers
369
(Table. S1) are designed using Primer Premier 5.0 software, and all
370
primers generate a single PCR band of the expected size. PCR products
371
are verified by DNA sequencing. The levels of relative expression are
372
calculated and quantified with the 2-ΔΔCt method.
373
4.5. Whole-mount in situ hybridization
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Whole-mount in situ hybridization (WISH) is carried out as
375
described previously (Pearson et al. 2005; Dong et al. 2014). The
376
digoxigenin-labelled probes are designed according to Djrunt-1 (from
377
position 211 to 595) and Djrunt-2 (from position 50 to 568) sequences,
378
respectively. The probes are synthesized using the RNA in vitro labeling
379
kit (Roche). Intact and regenerative worms (1, 3, 5, and 7 days after being
380
decapitated) are used for WISH. Control experiments are performed using
381
the sense probe.
382
4.6. RNA interference
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Planarians are fed bacteria to induce to express double stranded
384
RNA (dsRNA) against the Djrunt-1 and Djrunt-2 genes as previously
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described (Dong et al. 2017). The conserved domains of Djrunt-1 (from
386
position 262 to 879) and Djrunt-2 (from position 155 to 665) genes are
387
amplified from cDNA clone using primers (Djrunt-1 RNAi; Djrunt-2
388
RNAi) and then cloned into the L4440 vector using XbaI and KpnI. For
389
the RNAi experiment, worms are fed 3 times over 5 days (1st, 3rd and 5th)
390
and are amputated into 2 fragments pre-pharyngeally at 24 h after the last
391
feeding (Fig.7). The effectiveness of RNAi is confirmed by qRT-PCR and
392
WISH, the L4440-GFP is used as the control. The morphological changes
393
of intact and regenerative worms after RNAi are observed using the
394
stereomicroscope, and the images are captured with a Leica camera
395
(DFC300FX, Germany).
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Fig.7 RNAi feeding schedules. (dpa: days post amputation)
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4.7. Statistical analysis The SPSS 13.0 software is used as statistical analysis. Data is
401
analyzed by one-way ANOVA followed by post-hoc multiple
402
comparisons using the LSD test and Dunnett’s test. Differences are
403
regarded as statistically significant at P < 0.05 and highly significant at P
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< 0.01.
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Conflict of Interest Statement:
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None.
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Acknowledgements
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This work was supported by the National Natural Science Foundation of
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China (grants numbers Nos.31570376, 31471965 and u1604173).
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Appendix A. Supplementary Materials
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Sequence(5'-3')
Djrunt-1 EST-F
CACGAAGCTACCTCAGCATTGG
Djrunt-1 EST-R
ATTTTCCTCTTCCACTGCGAC
Djrunt-1 3'GSP1
AGTGTGGTTATCGGCCAGTAATC
Djrunt-1 3'GSP 2
GCTACCTCAGCATTGGCGAT
Djrunt-1 5'GSP 1
CAATGTTTGGACTAGCTTCTATATG
Djrunt-1 5'GSP 2
GGTGATCTGGGATAACATAATCCTG
Djrunt-1 RT-F
TTTATTAGTCGCAGTGGAAGAGG
Djrunt-1 RT-R
ATCTGTTGGGCGTATAAACTGAG
Djrunt-2 EST-R
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CACTGGCGCTCCAACAAGAC GGCCCATCGACGGTTACTTTGAT
TGATTGAGCACATTGTCAGGAAG
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Djrunt-2 3'GSP1
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Djrunt-2 EST-F
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Primers
ACAGTGTTCCATTTTGATTGAGC
Djrunt-2 5'GSP1
TGATTCTCCAATGTGAAGGTAACTG
AC C
Djrunt-2 3'GSP2
Djrunt-2 5'GSP2
GGATACGATTCCATAGTTATTGTG
Djrunt-2 RT-F
AACATCACAATAACTATGGAATCGT
Djrunt-2 RT-R
CTGACAATGTGCTCAATCAAAATG
Djβ-actin RT-F
ACACCGTACCAATCTATG
Djβ-actin RT-R
GTGAAACTGTAACCTCGT
Djpiwi-A RT-F
GATGGTGTAGGAGATTCGCAACT
Djpiwi-A RT-R
CGGAGATGCAGTGCCTTTAGTAGT
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Djpiwi-B RT-R
ATTGTCCCCAACACCATCTCG
Djpiwi-1 RT-F
AAACGAAGCACCCGAAGATATG
Djpiwi-1 RT-R
CCCACTTATTTGACATACCCTGAG
Djpcna RT-F
GTAGAGAAATGAGTCAAATGGGTG
Djpcna RT-R
TCAATGGTCACGGCATCACT
DjcyclinB RT-F
AGTCACGATGTTGGGAAAATGC
DjcyclinB RT-R
TTTCTCAATAACCCTGGCGTGT
Djp53 RT-F
GATCAACTAAGCCTTTCAGCCAT
Djp53 RT-R
CAAAGCACAGACAGCAGAGTCAT
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Table S1. Primers used in this study
629 630
635 636 637 638 639 640 641 642 643 644
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CGATTCAGGCATCTGTATTCTCG
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Djpiwi-B RT-F
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Fig. S1. The nucleotide and deduced amino acid sequences of the Djrunt-1 (a) and
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Djrunt-2 (b) cDNA. The grey is “RD” domain, the red is conserved phosphorylation
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sites, the green "IWRPF" is
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codon, and underlined sequence is the poly a signal.
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C-terminus conserved sequence, “*” is the termination
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Fig. S2. An alignment of the amino acids deduced by Djrunt-1 and Djrunt-2.
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Highlights
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The full-length cDNA sequences of Djrunt genes were first cloned. The expression levels of Djrunts were up-regulated on day 1 after amputation. The Djrunts play important roles in regeneration and homeostatic maintenance.