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Arid3a regulates nephric tubule regeneration via evolutionarily conserved regeneration signal-response enhancers

  1. Nanoka Suzuki
  2. Kodai Hirano
  3. Hajime Ogino
  4. Haruki Ochi  Is a corresponding author
  1. Yamagata University, Faculty of Medicine, Japan
  2. Hiroshima University, Japan
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Cite this article as: eLife 2019;8:e43186 doi: 10.7554/eLife.43186

Abstract

Amphibians and fish have the ability to regenerate numerous tissues, whereas mammals have a limited regenerative capacity. Despite numerous developmental genes becoming reactivated during regeneration, an extensive analysis is yet to be performed on whether highly regenerative animals utilize unique cis-regulatory elements for the reactivation of genes during regeneration and how such cis-regulatory elements become activated. Here, we screened regeneration signal-response enhancers at the lhx1 locus using Xenopus and found that the noncoding elements conserved from fish to human function as enhancers in the regenerating nephric tubules. A DNA-binding motif of Arid3a, a component of H3K9me3 demethylases, was commonly found in RSREs. Arid3a binds to RSREs and reduces the H3K9me3 levels. It promotes cell cycle progression and causes the outgrowth of nephric tubules, whereas the conditional knockdown of arid3a using photo-morpholino inhibits regeneration. These results suggest that Arid3a contributes to the regeneration of nephric tubules by decreasing H3K9me3 on RSREs.

https://doi.org/10.7554/eLife.43186.001

Introduction

Kidneys are necessary for the maintenance of homeostasis in vertebrates. The functional loss of this organ leads to severe defects in systems vital to all vertebrates, including human. During evolution, vertebrates evolved the following three complex kidney structures: pronephros, mesonephros, and metanephros. Metanephros is the adult kidney in higher vertebrates, such as humans and mice, whereas mesonephros is the adult kidney in fish and amphibians. Pronephros is the simplest and earliest kidney form (Jones, 2005; Desgrange and Cereghini, 2015). Despite the different levels of complexity of these three kidney types, their functional unit, the nephron, does not differ much (Brändli, 1999; Lienkamp, 2016). In human, there are approximately one million nephrons per kidney (Saxén and Sariola, 1987). Each nephron is composed of a glomerulus, a filtering component, and a nephric tubule, which is divided into the following four basic domains: proximal tubule, loop of Henle, distal tubule, and connecting tubule (Saxén and Sariola, 1987). According to molecular studies, the nephrons of the pronephros in Xenopus laevis can be subdivided into four distinct segments that are homologous to the segments of the metanephric nephrons of mammals (Raciti et al., 2008). Owing to these similarities, X. laevis is considered a suitable model for studying renal regeneration for application to human.

Gentamicin-induced nephropathy and partial nephrectomy showed that numerous vertebrates have the ability to repair damage to nephric structures, although this regenerative ability diverges between species (Nonclercq et al., 1992; Diep et al., 2011; Zhou et al., 2010; Caine and Mclaughlin, 2013). In mammals, nephrons contain mature tubular epithelial cells that have the capacity to regenerate following acute kidney injury (Maeshima et al., 2014). These epithelial cells rapidly lose their brush border, dedifferentiate into mesenchymal-like cells, and then migrate into regions where cells are damaged (Maeshima et al., 2014). Recent cell lineage studies have also suggested that renal stem cells and progenitor cells were found in the metanephric mesenchyme, limb of Henle’s loop, and distal convoluted tubule in mice (Kobayashi et al., 2008; Barker et al., 2012). Although mammalian nephrons possess cells that contribute to repair after injury, the regenerative capacity is restricted to the reconstruction of nephric epithelial cells in damaged regions (Maeshima et al., 2015). In contrast to mammals, which merely reconstitute tubular epithelial cells after injury, previous studies showed that X. laevis and zebrafish regenerate fully coiled and functional nephric tubule architecture after severe damage (Diep et al., 2011; Caine and Mclaughlin, 2013). In zebrafish, the transplantation of lhx1a-positive or six2 mesenchymal cells, which are considered stem cells, into adult fish in which the kidney has been injured by gentamicin reconstructs functional nephrons (Diep et al., 2011). X. laevis also regenerates the functional pronephros that can uptake albumin again after the mechanical loss of proximal tubules (Caine and Mclaughlin, 2013). Both amphibians and fish have a high regenerative capacity, but their regenerative mechanisms appear to differ. Zebrafish use kidney stem cells to repair the functional nephrons, whereas X. laevis appear to use remaining tubule cells to regenerate the functional pronephros (Diep et al., 2011; Caine and Mclaughlin, 2013). Thus, despite the different regenerative mechanisms, numerous vertebrates have the ability to repair injured nephrons.

Recently, studies have established protocols for generating the nephric structure derived from induced pluripotent cells (iPSCs) and embryonic stem (ES) cells by the combined application of the Wnt activator and other signaling factors such as BMP4 (Xia et al., 2013; Takasato et al., 2014; Takasato et al., 2015). In addition, direct reprogramming from fibroblasts into renal tubular epithelial cells, called induced renal tubular epithelial cells (iRECs), has been successfully achieved using a combination of transcription factors whose expression in the kidney is evolutionarily conserved between mice and Xenopus (Kaminski et al., 2016; Lienkamp, 2016). However, although the molecular factors for the construction of nephrons in vivo and in vitro have been identified, the molecular mechanisms that allow the reactivation of developmental genes during regeneration in highly regenerative animals remain unclear.

Genetic studies have shown that BMP and canonical Wnt and Fgf signals first specify intermediate mesoderm to form the pronephric primordium, followed by epithelialization, during kidney development (Krause et al., 2015). In addition to signaling factors, transcription factors such as Osr1, Osr2, Pax8, Hnf1b, Lhx1, and WT1 also coordinately regulate kidney formation (Bouchard, 2004; Lienkamp, 2016). These developmental genes for the kidney are evolutionarily conserved between pronephros formation in Xenopus and the more complex nephrons in mammalian meso- and metanephros (Lienkamp, 2016). During the regeneration of nephrons in zebrafish, transplanted lhx1a-positive mesenchymal cells begin to express developmental genes such as pax2a, wt1a, and fgf8a (Diep et al., 2011). In order to use developmental genes during regeneration, the genes have to be expressed again in regenerating tissues, which requires cis-regulatory elements called enhancers. Enhancers are known to determine the tissue specificity and timing of gene expression, and over 43,000 active enhancers have been identified in the human genome alone (Andersson et al., 2014). To date, numerous enhancers for tissue and organ development have been identified by the deletion mapping of upstream genomic regions and/or candidate screening using the epigenetic landscape (Kleftogiannis et al., 2016), whereas only a few enhancers for tissue regeneration have been reported, such as tissue regeneration enhancer elements for zebrafish heart reconstruction (Kang et al., 2016). Although it is generally assumed that the genome of vertebrates contains more enhancers that promote gene expression in regenerating tissues, the difficulty in identifying the in vivo function of enhancers prevents us from revealing the cis-regulatory architecture for regeneration. We have previously established an in vivo enhancer mapping system using X. laevis transgenesis (Ogino et al., 2008; Ochi et al., 2012; Suzuki et al., 2015). Here, we extended this strategy for identifying enhancers for the regeneration of nephric tubules and for examining the transcriptional mechanisms that regulate the enhancer activities during regeneration. We first found that lhx1 expression was induced within 24 hr after the surgical removal of nephric tubules. In order to explore the induction mechanisms of lhx1 in regenerating nephric tubules, we screened enhancers in genomic loci using a transgenic mapping system and found that genomic elements that are conserved from fish to human, called regeneration signal-response enhancers (RSREs), were activated in regenerating nephric tubules. A putative binding motif of Arid3a, a member of the AT-rich interaction domain family, is commonly found in RSREs for lhx1. We found that Arid3a directly binds to RSREs and Arid3a with H3K9me3 demethylases Kdm4/Jmjd2 reducing H3K9me3 levels on RSREs. We further found that the conditional knockdown of arid3a during regeneration suppressed the regeneration of nephric tubules. We now demonstrate that Arid3a is required for the regeneration of nephric tubules through the RSREs.

Results

Proximal tubules and intermediate tubules have different regenerative capacities

The pronephric kidneys of X. laevis become functional from Nieuwkoop and Faber stages 37–38. A previous study showed that proximal tubules that have been partially nephrectomized at stages 37–38 can regenerate functional nephrons (Raciti et al., 2008; Caine and Mclaughlin, 2013). Since we have previously established the Xla.Tg(Xtr.pax8:EGFP) transgenic line that can be used to trace nephric tubule formation, we first confirmed the regenerative process of nephric tubules using live imaging of stage 37 transgenic embryos (Ochi et al., 2012). Our careful nephric tubule surgery did not damage the glomus, which is situated medially to the tubules displaying podocin expression (Figure 1—figure supplement 1). When the proximal tubules were partially nephrectomized, a coiled structure appeared at around 24 hr (Figures 1A, B and E). This coiled tubule continued to extend from 24 to 48 hr, and then the regenerated tubules reconstructed the nephron between 72 and 120 hr (Figure 1B and E). In contrast, completely nephrectomized proximal tubules failed to form a coiled structure, whereas the remaining tubules, the intermediate tubules, continued to extend from the back to the front (Figure 1C and E). Conversely, when the intermediate tubules were completely removed, no tubule extension was observed (Figure 1D and E). Thus, although Xla.Tg(Xtr.pax8:EGFP) cannot capture all of the cells that contribute to the reconstruction of nephric tubules, our results indicate that the proximal tubules are essential for the regeneration of the coiled structure of the nephric duct. In addition, the intermediate tubules also have the capacity to regenerate nephric tubules, although this capacity is limited compared with that of the proximal tubules. It has been shown in a previous study that apoptotic cells and the expression of matrix metalloproteinase nine dramatically increase within 12 hr after nephrectomy, and these events continue until 24–48 hr (Caine and Mclaughlin, 2013). Our observation that regenerative tubules exhibit a coiled structure at around 24–48 hr together with previous findings suggests that the genetic mechanisms that occur within 48 hr after nephrectomy are crucial for nephron regeneration.

Figure 1 with 1 supplement see all
Live imaging of nephric tubules using transgenic X. laevis.

(A) A schematic image of X. laevis nephron. PT: proximal tubule; IT: intermediate tubule; DT: distal tubule; CT: connecting tubule. Magenta arrows: PT; green arrows: IT; orange arrows: DT. (B) Representative regeneration pattern of partially resected proximal tubules. Proximal tubules regenerate a coiled structure. (C) Regeneration pattern of completely resected proximal tubules. The remaining intermediate tubules extend, but no coiled structure is regenerated. (D) Regeneration pattern of completely resected intermediate tubules. No extension of tubules is observed. (E) Statistics of the regeneration pattern. The statistics of three independent experiments are summarized.

https://doi.org/10.7554/eLife.43186.002

lhx1 expression appears in regenerating nephrons immediately after nephrectomy

In order to explore the molecular mechanisms behind the regeneration of nephric tubules, we examined the expression of the transcription factors osr1, osr2, lhx1, six2, hnf1b, hnf4a, pax2, pax8, and wt1 in regenerating nephric tubules. Among them, osr1, osr2, and lhx1, together with pax8, are known to induce ectopic nephrons when overexpressed in Xenopus embryos (Seufert et al., 1999; Carroll and Vize, 1999; Tételin and Jones, 2010), and lhx1- or six2-expressing mesenchymal cells can reconstruct functional nephrons in kidney-injured zebrafish (Diep et al., 2011). The in situ hybridization analysis showed that the expression of lhx1, pax8, hnf4a, hnf1b, and osr2 appeared within 24 hr after the nephrectomy (Figures 2A, D, G and J, Figure 2—figure supplement 1). Such expression became stronger around 48 hr (Figures 2B, E, H and K, Figure 2—figure supplement 1). Then, the expression of lhx1 disappeared at 96 hr, while the expression of pax8 was still detected in the nephric tubules (Figures 2C, F, I and L). In contrast, the expression of pax2 and osr1 appeared around 48 hr, and that of six2 was not detected in regenerating nephric tubules (Figure 2M–2R, Figure 2—figure supplement 1, data not shown). Although it is possible that primary induced lhx1-, pax8-, hnf4a-, hnf1b-, and osr2-expressing cells are not completely consistent with cells observed in Xla.Tg(Xtr.pax8:EGFP) regenerating nephric tubules, the immediate induction of lhx1, pax8, hnf4a, hnf1b, and osr2 after nephrectomy suggests that the loci of these genes contain enhancers of injury response.

Figure 2 with 1 supplement see all
lhx1 expression appears immediately after nephrectomy.

(A–F) Expression of lhx1 on the control side and nephrectomized side at 24, 48, and 96 hr after nephrectomy. N indicates the number of examined embryos. (D) lhx1 expression appears in regenerating nephric tubules within 24 hr (arrows). (E, F) This expression becomes stronger around 48 hr but disappears at 96 hr. (G–L) Expression of pax8. pax8 expression appears in regenerating nephric tubules and is still detected at 96 hr after nephrectomy. (M–R) Expression of pax2 is not observed at 24 hr after nephrectomy (P, arrow). (S) Quantification of expression signals for lhx1, pax8, and pax2. The significance of differences between the control side and the nephrectomized side at 48 hr is calculated by two-tailed paired t-test (lhx1: p=0.0414; pax8: p=0.0102; pax2: p=0.0453). Lines in boxes indicate the median.

https://doi.org/10.7554/eLife.43186.004

Mapping of RSREs

The expression of lhx1, pax8, and hnf1b was immediately induced after injury. It has been shown in a previous study involving simple mRNA injection using Xenopus embryos that Lhx1 with Pax8 can induce ectopic pronephron structures (Carroll and Vize, 1999). In addition, tissue-specific lhx1 knockout using CRISPR/Cas9 genome engineering clearly showed that Lhx1 is required for the development of nephric tubules in Xenopus (DeLay et al., 2018). These previous findings prompted us to focus on the mechanisms of lhx1 expression in regenerating nephric tubules. In order to explore the enhancers that activate lhx1 gene expression in regenerating nephric tubules, we first searched for evolutionarily conserved noncoding sequences (CNSs) as candidates for the enhancers (Woolfe et al., 2005; Ochi et al., 2012). In this study, we focused on two categories of CNSs. The first one is evolutionarily conserved between frog and zebrafish, which have a high regenerative capacity, and the other is conserved among human, mice, frog, and zebrafish (Figure 3). In order to identify CNSs, we compared the genomic sequence of a 365 kb segment encompassing the human LHX1 gene with the orthologous intervals in mice, opossums, frog (X. tropicalis), and zebrafish genomes using the MultiPipMaker alignment tool (Schwartz et al., 2000). This analysis identified 20 CNSs conserved between human and fish and 17 CNSs conserved between frog and zebrafish (Figure 3A). In order to find the enhancers that genuinely activate gene expression, we used an efficient transgenesis technique for a nonmosaic founder assay of X. laevis (Kroll and Amaya, 1996). Each X. tropicalis lhx1-CNS was cloned into a green fluorescent protein (GFP) reporter plasmid carrying a β-actin basal promoter (Ogino et al., 2008). Each construct was used to generate transgenic embryos, and the left side of the nephrons was then resected, after which the embryos were allowed to proceed to stage 37 (Figure 3B). Their GFP expression was examined 48 hr after nephrectomy (Figure 3B). Reporter constructs without a CNS showed no significant GFP expression (data not shown) (Ochi et al., 2012). When GFP expression was detected in the regenerating nephric duct, we counted this as a positive result (Figure 3C). CNSs for lhx1 conserved between frog and zebrafish showed enhancer activities, whereas CNSs conserved among vertebrates showed much stronger enhancer activities (Figure 3C). It is known that lhx1 is expressed in developing nephrons at the early tailbud stage and specifies the renal progenitor cell field (Taira et al., 1994; Carroll et al., 1999; Cirio et al., 2011). Therefore, we examined whether CNSs that have enhancer activities in regenerating nephric tubules are also activated in developing pronephros. The transgenic reporter analysis using early tailbud embryos showed that CNS17, CNS20, and CNS35 have enhancer activities in the eyes and somites at this stage, while lacking (CNS20 and CNS35) or showing (CNS17) very weak enhancer activities in developing pronephros, suggesting that these CNSs primarily function after nephrectomy (Figure 3—figure supplement 1). These results suggest that enhancers conserved between human and zebrafish regulate the reactivation of developmental genes in regenerating nephric tubules.

Figure 3 with 1 supplement see all
The RSRE for lhx1 is conserved between human and fish.

(A) A diagram of vertebrate lhx1 loci showing the position of CNSs. The magenta boxes indicate CNSs conserved between frog and fish, and the blue boxes indicate CNSs conserved between human and fish. The black boxes indicate the exons. (B) A diagram of the experimental design for mapping RSREs. GFP reporter constructs carrying lhx1-CNSs with the β-actin proximal promoter were subjected to transgenesis. All reporter-injected embryos underwent nephrectomy on the left side at stage 37. Nephrectomized embryos were incubated at 18°C for 48 hr and fixed at stages 42/43. Normally developed embryos were subjected to in situ hybridization in order to examine their GFP expression with maximum sensitivity. (C) A summary of RSRE screening. The green bar indicates % of GFP-positive tadpoles in regenerating nephric tubules. N indicates the number of scored tadpoles. The image shows a representative expression pattern of GFP in regenerating nephric tubules. The green arrow indicates the regenerating nephric tubule.

https://doi.org/10.7554/eLife.43186.006

Arid3a functions as an input transcription factor for lhx1-RSREs

The transgenic reporter system of X. laevis combined with the surgical removal of nephric tubules revealed that the lhx1 loci contain the genomic elements that can activate gene expression in regenerating nephrons. We named these elements RSREs. Generally, enhancers activate gene expression by binding transcription factors (Bulger and Groudine, 2010). Therefore, the RSREs that we identified must also be regulated by transcription factors. In order to examine the transcription mechanisms for RSREs during nephron regeneration, we first searched for the binding motifs of transcription factors on RSREs. To dissect the transcriptional inputs for the RSREs, the open-access database JASPAR ver. 5 was used to define potential transcription factor binding sites (Mathelier et al., 2014). The candidate transcription factors were narrowed down according to their nephric expression using the Expression Atlas (the baseline atlas) (Petryszak et al., 2014), and then they were narrowed down further by phylogenetic footprinting (Figure 4—figure supplement 1). Although many lhx1-CNSs showed enhancer activities in regenerating nephric tubules, we focused on the top three lhx1 RSREs—CNS17, CNS20, and CNS35—which showed strong enhancer activities. The motif analysis showed that transcription motifs for Arid3a and Spib were commonly found in RSREs (Figure 4A). arid3a is known to be expressed in the ectoderm of the early neurula and in the epidermis at the late tailbud stage, whereas spib is expressed in the anterior ventral blood islands at the neurula stage (Callery et al., 2005; Costa et al., 2008). We performed an in situ hybridization analysis in order to determine whether these genes are also expressed in the nephric tubules and found that arid3a is expressed there, whereas spib is not (Figure 4B, Figure 4—figure supplement 2). In addition, we performed immunostaining of Arid3a using Xla.Tg(Xtr.pax8:EGFP), which showed that Arid3a protein was detected in proximal tubules and also in the glomus and/or nephrocoelom (Figure 4B, orange arrows and orange arrowheads, respectively). These results suggest that Arid3a is a good candidate for the input transcription factor for RSREs. Therefore, we next examined whether Arid3a regulates the expression of lhx1 in Xenopus and found that lhx1 was induced by conditionally induced arid3a in heat-shock-inducible transgenic X. laevis (Figure 4C) (Wheeler et al., 2000). We then examined whether Arid3a interacts with RSREs in X. laevis. The Myc-tagged X. tropicalis arid3a mRNA was injected into one-cell-stage X. laevis embryos, and then chromatin immunoprecipitation-qPCR (ChIP-qPCR) was performed using anti-Myc antibody. CNS35 was divided into two segments for qPCR, since it is 580 bp long, which is too long for qPCR. ChIP-qPCR showed that Arid3a directly binds to RSREs, but not to exon 5 and CNS32, which do not include putative Arid3a binding motifs (Figure 4D). Furthermore, a luciferase reporter analysis using 293 T cells derived from human embryonic kidneys was performed to examine whether Arid3a functions as a transcriptional activator for RSREs. We found that Arid3a activates CNS17 and CNS20 enhancer activities, whereas no activation was observed in the CNS35 reporter (Figure 4E). Since CNS35 is located in the intron of aatf, it is possible that the activation mechanism of this CNS is different from those of CNS17 and CNS20.

Figure 4 with 2 supplements see all
Arid3a is an input transcription factor for RSREs.

(A) A summary of transcription factor binding motifs on RSREs. (B) arid3a is expressed in nephric tubules. A lateral view of embryos at stage 31 and its transverse section. The upper panels show the in situ hybridization of arid3a.L. The blue arrows indicate the nephric tubules and the white arrows indicate the epidermis. The lower panels show immunostaining using anti-Arid3a using Xla.Tg(Xtr.pax8:EGFP) transgenic tadpole. White arrows: epidermis; orange arrows: proximal tubules; orange arrowheads: glomus and/or nephrocoelom. (C) Arid3a induces lhx1 expression. Xla.Tg(Xla.hsp70:Xtr.arid3a-2A-EGFP) transgenic X. laevis at stage 23 were treated at 34°C for 15 min, followed by 15 min at 14°C. These steps were repeated three times, and tadpoles were incubated at 18°C. lhx1 expression was observed 48 hr after the heat shock at stages 35–36. The signal intensity of in situ hybridization was measured and subjected to statistical analysis. The significance of differences between the control side and the nephrectomized side was calculated using two-tailed unpaired t-test (p=0.0131). The magenta arrows indicate lhx1 expression in proximal and intermediate tubules. N indicates the number of examined embryos. (D) Arid3a directly binds to CNS17, CNS20, and CNS35. Myc-tagged Xtr.arid3a mRNA-injected tadpoles were used for ChIP-qPCR. CNS32 and exon 5 were used as negative elements. The significance of differences between the control IgG and the anti-Myc for Arid3a was calculated using two-tailed unpaired Mann–Whitney t-test: CNS17, p=0.0286; CNS20, p=0.0022; CNS32, p=0.3143 (not significant); exon5, p=0.7000 (not significant); CNS35 (56-334), p=0.0079; CNS35 (314-580), p=0.0022. The error bars indicate SEM. (E) Arid3a activates CNS17 and CNS20. The luciferase reporter assay was performed using HEK293T cells. The significance of differences between the control vector and the CNS-containing reporter was calculated using two-tailed unpaired t-test (CNS17, p=0.0033; CNS20, p=0.0256). The error bars indicate SEM.

https://doi.org/10.7554/eLife.43186.008

Arid3a promotes cell cycle progression in regenerating nephrons

During tail regeneration in X. laevis, the inhibition of apoptosis results in a failure to induce subsequent cell proliferation (Tseng et al., 2007). During proximal tubule regeneration, apoptosis occurs within 3 hr after nephrectomy, and the number of apoptotic cells is decreased at one day (Caine and Mclaughlin, 2013). Meanwhile, the nephric tubules begin to extend from the remaining tubules (Figure 1). During this regeneration step, cell proliferation must occur, as observed in other types of tissue regeneration (Poleo et al., 2001; Passamaneck and Martindale, 2012). Therefore, we first examined the time course of cell proliferation after nephrectomy (Figure 5—figure supplement 1). Immunofluorescence staining with anti-phosphorylated histone H3 antibody, a marker for mitotic cells, showed that there was no significant difference between the control and the nephrectomized sides until 6 hr after nephrectomy, whereas the number of proliferating cells increased on the nephrectomized side at 24 hr (Figure 5—figure supplement 1). It has been shown in a previous study that Arid3a stimulates cyclin E1/E2F-dependent cell cycle progression (Peeper et al., 2002). Therefore, we next investigated whether Arid3a contributes to the cell cycle progression. Xla.Tg(Xla.hsp70:Xtr.arid3a-2A-mcherry, Xtr.pax8:EGFP) and Xla.Tg(Xtr.pax8:EGFP) transgenic Xenopus embryos at tailbud stage 23 were treated with or without heat shock and then incubated for 48 hr at 18°C. Heat-shock-treated embryos were then sorted by mCherry positivity or negativity (Figure 5—figure supplement 2). These tadpoles were examined for the number of phosphorylated histones H3. No significant difference of phosphorylated histones H3 between the heat-shock-untreated embryos and the heat-shock-treated mCherry-negative embryos was observed (Figure 5—figure supplement 3). In contrast, the number of phosphorylated histones H3 in the mCherry-positive Xla.Tg(Xla.hsp70:Xtr.arid3a-2A-mcherry, Xtr.pax8:EGFP) was significantly increased compared with that in the heat-shock-treated Xla.Tg(Xtr.pax8:EGFP) and heat-shock-untreated Xla.Tg(Xla.hsp70:Xtr.arid3a-2A-mcherry, Xtr.pax8:EGFP) (Figure 5A). Since we showed that Arid3a can induce lhx1 expression, we then wondered whether Lhx1 can also promote the cell cycle. Transgenic X. laevis in which lhx1 was conditionally induced showed an increased number of phosphorylated histones H3, as observed in Arid3a-induced X. laevis (Figure 5B). These results suggest that Arid3a and also Lhx1 promote cell cycle progression after nephrectomy.

Figure 5 with 3 supplements see all
Arid3a promotes cell cycle progression.

(A) The number of phosphorylated histones H3 in the nephrectomized area was increased by the conditionally induced Arid3a. Heat-shocked Xla.Tg(Xla.hsp70:Xtr.arid3a-2A-mcherry, Xtr.pax8:EGFP) was nephrectomized at stage 37, incubated for 72 hr, and then fixed at stages 45/46. The white dotted lines indicate the pax8-expressing cells, and the magenta indicates the phosphorylated histone H3-positive cells. The left graph shows the statistical analysis; one-way analysis of variance (ANOVA) and Tukey’s post hoc test were used. ANOVA p=0.0001; *p = 0.5155 (not significant), **p = 0.001, ***p = 0.0019. The error bars indicate SEM. (B) Lhx1 promotes cell cycle progression in the regenerating area. Heat-shocked Xla.Tg(Xla.hsp70:Xtr.lhx1-2A-mcherry) was nephrectomized at stage 37 and then incubated for 72 hr. One-way ANOVA and Tukey’s post hoc test were used. ANOVA: p=0.0005, *p = 0.0015, and **p = 0.0011. (C) Arid3a with Kdm4a and Arid3b reduced the H3K9me3 levels on RSREs. ChIP analysis was performed using X. laevis. Significant differences were calculated by two-tailed unpaired t-test. The p-values from comparisons between the control and arid3a-, arid3b-, and kdm4a-injected embryos were as follows: *p = 0.0389, **p = 0.0313, and ***p = 0.0456. The error bars indicate SEM.

https://doi.org/10.7554/eLife.43186.011

Arid3a with Arid3b and Kdm4a regulate the chromatin configuration of RSREs

The constitutive heterochromatin marker histone 3 lysine nine trimethylation (H3K9me3) is rapidly remodeled to create a chromatin environment, and the lack of H3K9me2/3 could allow global DNA demethylation (Burton and Torres-Padilla, 2010). Arid3a is known to stimulate the expression of immunoglobulin heavy chain (IgH) and is also known to change the accessibility of the IgH enhancer (Kim and Tucker, 2006; Lin et al., 2007). In addition, it has been shown in a previous study that the complex of Arid3a, Arid3b, and Kdm4c modulates the chromatin configuration of stemness genes for breast cancer by decreasing H3K9me3 (Liao et al., 2016). Therefore, we next investigated whether Arid3a modulates the H3K9me3 level on RSREs. ChIP-qPCR analysis using anti-H3K9me3 showed that H3K9me3 was enriched not only on the RSREs CNS17 and CNS20, but also on CNS32 and exon 5 of lhx1 (Figure 5C). However, no enrichment of H3K9me3 on CNS35 was observed (Figure 5C). Next, we found that the coinjection of arid3a, arid3b, and kdm4a decreased H3K9me3 on the RSREs CNS17 and CNS20, but not on CNS32 (Figure 5C). In addition, the enrichment of H3K9me3 on exon 5 was also decreased (Figure 5C). Thus, we found that Arid3a directly regulates lhx1 expression through changing the chromatin configuration on CNS17 and CNS20. In contrast, although CNS35 shows an enhancer activity in regenerating nephric tubules, both the chromatin modification and the activation mechanism differ from those of CNS17 and CNS20.

Arid3a is required for the regeneration of proximal tubules in Xenopus

Our results suggest that Arid3a is involved in the regeneration of nephric tubules in X. laevis. In order to investigate whether Arid3a is required for the regeneration of nephric tubules, we applied photo-morpholino oligonucleotide (Photo-MO) that was previously established in zebrafish in order to control the timing of gene knockdown (Tallafuss et al., 2012; Cabochette et al., 2015). The effectiveness of antisense-MO for gene knockdown is blocked by annealing with sense Photo-MO. Since Photo-MO is cleaved by ultraviolet (UV) treatment, we were able to control the timing of gene knockdown. We first designed the splicing-blocking morpholino for arid3a.L, since the expression arid3a.L in nephrons is stronger than that of arid3a.S (Figure 4B, Figure 4—figure supplement 1A). The arid3a.L-MO-injected embryos became abnormal during gastrulation, consistent with the phenotype that was previously reported using translational blocking morpholino (Figure 6—figure supplement 1B) (Callery et al., 2005). RT-PCR analysis using control and arid3a.L-MO-injected embryos showed that the level of transcripts that included exon three was dramatically reduced in arid3a.L-MO-injected embryos (Figure 6—figure supplement 1C). In contrast, embryos injected with arid3a.L-MO annealed with arid3a.L-Photo-MO developed normally (Figure 6—figure supplement 1D). Thus, arid3a.L-MO is rendered inactive by binding to arid3a.L-Photo-MO. Next, in order to investigate whether the photosensitive subunit is cleaved by 365 nm light, arid3a.L-photo-MO/arid3a.L-MO-injected embryos were treated with UV for 30 min at stages 29–30 (Figure 6A). RT-PCR analysis showed that the arid3a.L transcript, which included exon 3, was significantly decreased by treatment with UV light, same as upon injection of arid3a.L-MO alone (Figure 6A, Figure 6—figure supplement 1C). Since conditionally induced Arid3a promotes the expression of lhx1, we wondered whether the expression of lhx1 in regenerating nephric ducts is affected. In situ hybridization analyses showed that there was no significant increase of lhx1 in UV-treated embryos, suggesting that Arid3a is essential for the expression of lhx1 after nephrectomy (Figure 6B). Cell cycle progression occurred after nephrectomy (Figure 5—figure supplement 1). Therefore, we also examined whether arid3a knockdown induced by UV affects the cell cycle and found that the number of phosphorylated histone H3-positive cells after nephrectomy in UV-treated embryos was reduced (Figure 6—figure supplement 2). We then examined whether Arid3a contributes to the regeneration of proximal tubules. The proximal tubules of arid3a.L-photo-MO/arid3a.L-MO-injected embryos that were treated with UV were partially removed and subsequently incubated for 72 hr. The regenerated proximal tubules showed a coiled structure in UV-untreated controls (Figure 6C). In contrast, although regenerating proximal tubules extended to the front most of the resected X. laevis failed to reconstruct the coiled structure, indicating that Arid3a is required for the proper regeneration of nephric ducts in X. laevis (Figure 6C).

Figure 6 with 2 supplements see all
Arid3a is required for the regeneration of proximal tubules in X.laevis.

(A) Conditional knockdown of Arid3a using arid3a.L-photo-morpholinos (arid3a.L-Photo-MO). The upper panel shows the experimental design of conditional gene knockdown experiment using Photo-MO. arid3a.L-antisense-splicing-blocking MO (arid3a.L-MO) inactivated by Photo-MO is injected at the one-cell stage, subjected to UV exposure at stages 29–30, and then sacrificed for RT-PCR analyses. The lower panel shows the statistical analysis. The significance of differences between the UV-untreated and UV-treated embryos was calculated by two-tailed unpaired t-test (p=0.0217). The error bars indicate SEM. (B) Conditional knockdown of Arid3a during nephric regeneration causes the reduction of lhx1 expression. The upper panel shows the experimental design. arid3a.L-MO inactivated by Photo-MO is injected at the one-cell stage, followed by UV exposure at stages 29–30, nephrectomy at stages 36–37, and subsequently incubation for 48 hr. The lower panel shows the quantification of lhx1 expression signals. The analysis indicates that there was no significant difference between the control side and the nephrectomized side (two-tailed paired t-test, p=0.0748). N indicates the number of examined embryos. The lines in boxes indicate the median. (C) Arid3a is required for the regeneration of nephric tubules. The upper panel shows the experimental design. arid3a.L-MO inactivated by Photo-MO is injected at the one-cell stage, and UV exposure is performed at stages 29–30, followed by nephrectomy at stages 36–37 and subsequently incubation for 72 hr. The left panel shows a summary of the statistics of three independent experiments.

https://doi.org/10.7554/eLife.43186.015

Discussion

In many cases of tissue regeneration, numerous developmental genes that are evolutionarily conserved among vertebrates become reactivated during regeneration (Poss, 2010; Witman et al., 2011; Halasi et al., 2012; Shibata et al., 2016). Therefore, it has been widely discussed that regeneration recapitulates developmental guidance (Witman et al., 2011; Halasi et al., 2012). Every gene in the genome requires cis-regulatory sequences such as a promoter and an enhancer to activate its expression, indicating that genes expressed in regenerating tissues also utilize enhancers and promoters for their expression (Bulger and Groudine, 2010). However, no extensive analysis has been performed on whether highly regenerative animals utilize unique cis-regulatory elements for regeneration and how such cis-regulatory elements become activated during regeneration. Previously, tissue regeneration enhancer elements were identified on the basis of epigenetic profiling (Kang et al., 2016). The screening of epigenetic profiles on the basis of open chromatin marks is useful to capture the regulatory blueprint in a genome-wide manner, while our one-by-one transgenic screening based on the actual activities in regenerating tissues provides a novel and powerful approach to directly identify tissue specificity and in vivo function. Once we identified the functional enhancers in the regenerating tissues, we were able to search for input transcription factors. Here, our transgenic analysis of functional cis-regulatory elements showed that RSREs for lhx1 are evolutionarily conserved between human and fish, and Arid3a with Arid3b and Kdm4a induces lhx1 by changing the chromatin configuration of RSREs (Figure 7A). In addition, we showed that knockdown of arid3a causes the failure of regeneration, whereas the conditionally induced Arid3a promotes cell cycle progression and causes the outgrowth of nephric tubules (Figure 7B and C, Figure 7—figure supplement 1).

Figure 7 with 1 supplement see all
Model illustrating the Arid3a function in the regeneration of proximal nephric tubules.

(A) Arid3a binds to RSREs on lhx1 and changes the H3K9me3 levels. This chromatin modification allows the induction of lhx1 expression. (B) In the absence of Arid3a, proximal tubules fail to regenerate a complete nephron structure. (C) Excess amounts of Arid3a cause the outgrowth of nephric tubules from the distal nephric duct.

https://doi.org/10.7554/eLife.43186.018

lhx1 is one of the earliest genes to be expressed in the pronephric anlagen, and its expression disappears during proximal tubule development (Carroll et al., 1999; Carroll and Vize, 1999) (Figure 2). As it recapitulates the developmental program, lhx1 expression appeared at the early stage in the construction of kidneys in vivo (Takasato et al., 2015). During in vivo nephric regeneration, lhx1 is one of the earliest genes to be expressed in regenerating nephrons (Figure 2). This lhx1 expression pattern prompted us to examine its noncoding DNA sequences. The Xenopus lhx1/xlim-1 enhancer responding to activin has been reported to encompass approximately 14.2 kb, including 5 kb upstream and the gene body (Rebbert and Dawid, 1997; Watanabe et al., 2002). Since recent genome-wide analyses have shown that enhancers are located not only near the gene body but also far away from it, we compared the genomic sequence of a 365 kb segment encompassing the human LHX1 gene with the orthologous intervals in mice, opossums, X. tropicalis, and zebrafish (Woolfe et al., 2005; Ochi et al., 2012). While the activin-responding lhx1 enhancer is located in intron 1, enhancers that showed high activities in regenerating nephrons are located 177 kbp (CNS17) and 144 kbp (CNS20) upstream of lhx1 and are also found in the intron of the next gene aatf (CNS35) (Figure 3). It is known that many genes have multiple enhancers (Bulger and Groudine, 2010). Such multiple enhancers often play different functional roles, while it is also known that they sometimes show overlapping functions (Osterwalder et al., 2018). CNS17, CNS20, and CNS35 are activated in regenerating nephric tubules, indicating that these enhancers have overlapping functions. On the other hand, we also found that CNS17 and CNS20 are modified by H3K9me3 but not CNS35 (Figures 3C and 5C). It has been shown in recent studies that functionally redundant enhancers provide phenotypic robustness (Osterwalder et al., 2018). Our observation that there are overlapping functions of CNS17, CNS20, and CNS35 in the regenerating nephric tubules, albeit with differential activation mechanisms, suggests that there is a redundancy of cis-regulatory elements for regeneration. In addition, we also found that many CNSs that are only conserved between frog and fish showed weak enhancer activities in regenerating nephric tubules (Figure 3C). Since these animals have a high regenerative capacity, it is possible that such weak multiple enhancers possessing overlapping activities may also be key to boosting the gene expression in regenerating tissues. Further studies of transcriptional mechanisms for frog/fish CNSs may provide novel insights into the unique trait of high regenerative capacity in fish and frog.

In zebrafish, lhx1a and six2 mesenchymal cells reconstruct the functional nephrons (Diep et al., 2011). In contrast, our live imaging using Xla.Tg(Xtr.pax8:EGFP) together with McLaughlin’s previous findings suggests that the wound healing and regrowth of existing tubules occur during the regeneration of the nephric tubules of X. laevis, since the remaining nephric tubule cells extend toward the glomus (Caine and Mclaughlin, 2013). In mammals, mature tubular epithelial cells rapidly lose their brush border and dedifferentiate into mesenchymal-like cells following acute kidney injury, and these cells proliferate to regenerating nephric tubules (Maeshima et al., 2014). Therefore, the regenerative mechanisms of the nephric tubules of X. laevis appear to be much closer to the regeneration of nephric tubules in mammals rather than the reconstruction of nephrons from stem cells. Further live imaging studies using transgenic animals with markers of dedifferentiating cells and regrowing cells are required in order to reveal the detailed regenerative mechanisms of nephric tubules in vertebrates.

Recently developed in vivo organ construction technologies enabled us to make nephric structures derived from iPSCs and ES cells by the combined application of the Wnt activator and other signaling factors such as BMP4 (Xia et al., 2013; Takasato et al., 2014; Takasato et al., 2015). Moreover, direct reprogramming from fibroblasts into renal tubular epithelial cells, called iRECs, has successfully converted mouse and human fibroblasts into renal tubular epithelial cells using a combination of transcription factors: Emx2, Hnf1b, Hnf4a, and Pax8 (Kaminski et al., 2016; Lienkamp, 2016). In Xenopus, it is well known that mRNA injection of Wnt11b, Osr1, Osr2, and Lhx1 with Pax8 can induce ectopic pronephron structures (Seufert et al., 1999; Carroll and Vize, 1999; Tételin and Jones, 2010). Although Arid3a is not strong enough to induce complete ectopic nephrons as with Osr1, Osr2, Lhx1, and Pax8 mRNA injection, our findings suggest that Arid3a may contribute to the improvement of the efficiency of in vitro reconstruction and in vivo direct reprogramming or regeneration of nephrons.

For the expression of stem cell factors, Arid3a changes its localization from cytoplasmic to nuclear, which is regulated by importin-9 (Liao et al., 2016). It has been shown in a recent study that A disintegrin and metalloproteinase 13 (ADAM13) increases the nuclear localization of a cleavage product of Arid3a and promotes the expression of the target gene tfap2 alpha (Khedgikar et al., 2017). ADAMs are known to control a variety of cell functions by modulating the ectodomain shedding of several membrane-anchored signaling molecules (Blobel, 2005). In addition, emerging evidence indicates that ADAMs are required for wound healing and the regeneration of oligodendrocytes (Schönefuß et al., 2012; Palazuelos et al., 2015). Therefore, it is possible that ADAMs change the localization of Arid3a after nephrectomy; nuclear-translocated Arid3a regulates H3K9me3 on RSREs, and this epigenetic alteration triggers lhx1 expression in regenerating nephric tubules. Further studies are necessary to reveal the molecular basis behind the signal transduction from the extracellular region to the Arid3a complex on lhx1 RSREs.

Materials and methods

Key resources table
Reagent type
(species) or
resource
DesignationSource or referenceIdentifiersAdditional

information
Gene
(Xenopus tropicalis)
arid3aThis paperRefSeq: NM_001011106.1
Gene
(Xenopus tropicalis)
arid3bThis paperRefSeq: XM_002938881.4
Gene
(Xenopus tropicalis)
lhx1This paperRefSeq:
NM_001100228.1
Genetic reagent
(Xenopus laevis)
Xla.Tg(Xtr.pax8:EGFP)Ochi, H., et al.,
2012; doi: 10.1038/ncomms1851.
Cell line
(Homo sapiens)
293TRIKEN BRC
CELL BANK
RCB2202, RRID:SCR_003163
Transfected
construct
pGL4.23PromegaE8411
Transfected
construct
pGL-lhx1-CNS17-LucThis paperNew regent. The CNS17
fragments from
IS-lhx1-CNS17-β-GFP
vector introduced
into the
SacI and EcoRV sites
of pGL4.23 vector.
Transfected
construct
pGL-lhx1-CNS20-LucThis paperNew regent.
The CNS20 fragments
from IS-lhx1-CNS17-
β-GFP wereintroduced
into the SacI and EcoRV
sites of pGL4.23 vector.
Transfected
construct
pGL-lhx1-CNS35-LucThis paperNew regent.
The CNS35 fragments
from IS-lhx1-CNS17-
β-GFP vector were
introduced into the
SacI and EcoRV sites
of pGL4.23 vector.
AntibodyAnti-phospho
histone H3 (Ser10)
antibody
Milipore06–570(1:1000)
AntibodyAnti-Arid3a antibodyDSHBPCRP-ARID3A-1E9, RRID:AB_2618410(1:10)
AntibodyAlexa 488
-conjugated
goat anti-rabbit IgG
InvitrogenA11001(1:1000)
AntibodyAlexa 568-conjugated
goat anti-mouse IgG
Invitrogen,A11011(1:1000)
AntibodyAnti-H3K9
(tri-methyl K9)
antibody
Abcam,ab8898(1:750)
AntibodyMouse monoclonal
c-Myc (9E10) antibody
Santa Cruz
Biotechnology Inc.
sc-40, RRID:AB_291323(1:750)
Recombinant
DNA reagent
Xenopus laevis
hsp70 promoter
Wheeler, G. N., et al. 2000; doi.org/10.1016/S0960-9822 (00)00596–0
Recombinant DNA reagentIS-β-GFP reporterOgino, H., et al., 2008; doi: 10.1242/dev.009548
Recombinant DNA reagentpCS-myc-arid3aNew regent. The PCR product was introduced into the XhoI and XbaI sites of the pCS2 + MT plasmid.
Recombinant DNA reagentpCS-his-arid3bNew regent. The PCR product was introduced into the ClaI and XbaI site of the pCS vector.
Recombinant DNA reagenthsp70-myc-arid3a-2A-mcherryThis paperNew regent. The PCR amplified myc-arid3a and 2A-mcherry were introduced into the ClaI and XbaI sites of the IS-hsp70-cloning vector.
Recombinant DNA reagenthsp70-myc-arid3a-2A-EGFPThis paperNew regent. The PCR amplified myc-arid3a and 2A-EGFP were introduced into the ClaI and XbaI sites of the IS-hsp70-cloning vector.
Recombinant DNA reagenthsp70-lhx1-2A-EGFPThis paperNew regent. The PCR amplifiedlhx1 was introduced into the EcoRI sites of the pCS2 + MT plasmid. The PCR amplified myc-lhx1 and 2A-EGFP were introduced into the EcoRI and XbaI sites of the IS-hsp70-cloning vector.
Recombinant DNA reagent (Xenopus laevis)arid3a.LThis paperXelaev18006256mNew regent.
The PCR product was introduced into the EcoRI and XhoI sites of the pBluescript II SK plasmid.
Recombinant DNA reagent (Xenopus laevis)arid3a.SThis paperXelaev18009788mNew regent. The PCR product was introduced into the EcoRI and XhoI sites of the pBluescript II SK plasmid.
Recombinant DNA reagent (Xenopus laevis)spib. LThis paperXelaev18036193m.gNew regent. The PCR product was introduced into the EcoRI and XhoI sites of the pBluescript II SK plasmid.
Recombinant DNA reagent (Xenopus laevis)spib.SThis paperXelaev18037903m.gNew regent. The PCR product was introduced into the EcoRI and XhoI sites of the pBluescript II SK plasmid.
Recombinant DNA reagent (Xenopus laevis)hnf4aThis paperXelaev17043619m, Xelaev17043619mNew regent. The PCR product was introduced into the BamHI and HindIII sites of the pBluescript II SK plasmid.
Recombinant DNA reagent (Xenopus laevis)hnf1bThis paperXelaev18012186m.g, Xelaev18014991m.gNew regent. The PCR product was introduced into the BamHI and HindIII sites of the pBluescript II SK plasmid.
Recombinant DNA reagent (Xenopus laevis)osr1This paperXelaev14054577m.g, Xelaev14010174m.gNew regent.
The PCR product was introduced into the XhoI and BamHI sites of the pBluescript II SK plasmid.
Recombinant DNA reagent
(Xenopus laevis)
osr2This paperXelaev14045820m.g, Xelaev14031017m.gNew regent. The PCR product was introduced into the XhoI and BamHI sites of the pBluescript II SK plasmid.
Recombinant DNA reagent (Xenopus laevis)six2This paperXelaev16000858m, Xelaev16036496mNew regent.
The PCR product
was introduced
into the HindIII and XhoI sites of
the pBluescript II SK plasmid.
Recombinant DNA reagent (Xenopus laevis)lhx1This paperXelaev16044871m.gNew regent. The PCR product was introduced into the SmaI and HindIII sites of the pBluescript II SK plasmid.
Recombinant DNA reagent (Xenopus laevis)pax2Heller and Brändli, 1997:
doi.org/10.1016/S0925-
4773(97)00158-5
Recombinant DNA reagent (Xenopus laevis)pax8Heller and Brändli, 1999:
doi.org/10.1002/(SICI)1520-
6408(1999)24:3/4 < 208::AID-
DVG4 > 3.0.CO;2 J
Recombinant DNA reagent (Mus musculus)kdm4aMammalian Gene Collection (MGC) Clones4207552BC028866
Sequence-based reagent (Xenopus tropicalis)PCR primers for CNSThis paper
Sequence-based reagent (Xenopus laevis)ChIP-qPCR primersThis paper
Sequence-based reagent
(Xenopus laevis)
Photo-Morpholino
oligonucleotide for arid3a,L
This paperGene Tools, LLCAGAGGGAAGCCAGCAGGTACTCACC
Sequence-based reagent (Xenopus laevis)Morpholino
oligonucleotide for arid3a,L
This paperGene Tools, LLCAGTACCTGpTGGCTTCCCT
Sequence-based reagent (Xenopus laevis)PT-PCR primers for arid3a.LThis paper
Sequence-based reagent (Homo sapiens)hg19 chr17-34994909–35360679hg19UCSC Genome Browser, RRID:SCR_005780
Sequence-based reagent (Mus musculus)mm10 chr11-83838963–85151744mm10UCSC Genome Browser, RRID:SCR_005780
Sequence-based reagent (Monodelphis domestica)monDom5 chr2-185210169–185976291monDom5UCSC Genome Browser, RRID:SCR_005780
Sequence-based reagent (Xenopus tropicalis)xenTro3 GL173152-472286-845619xenTro3Xenbase, RRID:SCR_003280
Sequence-based reagent (Danio rerio)danRer10-chr15_27468859–28180541danRer10UCSC Genome Browser, RRID:SCR_005780
Sequence-based
reagent (Danio rerio)
danRer10-
chr5_55422952–55633560
danRer10UCSC Genome Browser, RRID:SCR_005780
Software, algorithmGraphPad Prism 7.0GraphPad SoftwareRRID:SCR_002798
Software, algorithmAdobe PhotoshopAdobeRRID:SCR_014199
Software, algorithmMultiPipMakerSchwartz, S., et al., 2000: doi:
10.1101/gr.10.4.577
RRID:SCR_011806
Software, algorithmJASPAR ver. 5Mathelier, A., et al., 2014: doi: 10.1093/nar/gkt997.RRID:SCR_003030
Commercial assay or kitISOGENNIPPON GENECode No. 317–02503
Commercial assay or kitDual-Luciferase Reporter Assay SystemPromegaE1910
 Commercial assay or kitDynabeads Protein ADynabeads10001D
Chemical compound, drugjetPEI (transfection)Polyplus-transfection SA101–10N

Construction of reporter plasmids

The GFP reporter plasmid carrying a chicken β-actin basal promoter (−55 to +53) was previously described as β-GFP (Ogino and Ochi, 2009). The CNSs were amplified from X. tropicalis genomic DNA by PCR and cloned into β-GFP reporter vectors. The primer sequences used in this study are summarized in Supplementary file 1.

Identification of CNSs

The 365 kb genomic sequence of the human LHX1 locus [hg19 chr17-34994909–35360679 (365,771 bp)] and its orthologous sequences in mice [mm10 chr11-83838963–85151744 (1,312,782 bp)], opossums [monDom5 chr2-185210169–185976291 (766,123 bp)], X. tropicalis [xenTro3 GL173152-472286-845619 (373,334 bp)], and zebrafish [danRer10-chr15_27468859–28180541 (711,683 bp), danRer10-chr5_55422952–55633560 (210,609 bp)] were downloaded from the UCSC Genome Browser and Xenbase Genome Browser. These sequences were aligned using MultiPipMaker (Schwartz et al., 2000).

Cloning of X. tropicalis Arid3a and Arid3b

Full-length cDNA fragments of arid3a and arid3b were amplified from a cDNA pool of X. tropicalis tailbud embryos (stage 26). The product was introduced into the XhoI and XbaI sites of the pCS2 +MT plasmid and the ClaI and XbaI site of the pCS2 vector. The PCR-amplified X. laevis heat-shock promoter, Xtr.arid3a, and 2A-mcherry DNA fragments were constructed using the InFusion cloning method (Wheeler et al., 2000) (Clontech, Palo Alto, CA, USA). For the in situ hybridization probes of X. laevis, arid3a.L and arid3a.S were amplified from a cDNA pool of X. laevis tailbud embryos (stages 35/36). The primers used for the vector construction are listed in Table 1.

Table 1
Primer sequences for the RT-PCR and quantitative RT-PCR.
https://doi.org/10.7554/eLife.43186.020
Xtr.arid3a_full-length-FATGAAGCTGCAAGCGGTG
Xtr.arid3a_full-length-RTCAGGGAGAAGGATTGTTAG
Xtr.arid3b_full-length-FCGATGCCGCCACCATGCACCATCACCACCATCATCACCACCATCACT
Xtr.arid3b_full-length-RCTAGAGTGATGGTGGTGATGATGGTGGTGATGGTGCATGGTGGCGGCAT
Xla.CNS17-qPCR-FCTGAGTGAGTTTCAAATAAAAGGATTAAG
Xla.CNS17-qPCR-RGCTATGTAGAGTGGAATAGAGTTAGAATGA
Xla.CNS20-qPCR-FAATACTCACACAGGGAAGACAGC
Xla.CNS20-qPCR-RAAGGCCAAAATTACTTTTCATTTATCTTA
Xla.CNS32-qPCR-FGGGAATTAACCCCCATGGGAA
Xla.CNS32-qPCR-FTTTGCCTCCCTCCTGATCTATAGG
Xla.exon5-qPCR-FCCAGGTTCCATGCACTCTATG
Xla.exon5-qPCR-RTTTCTGGTGGGTGTGACAAA
Xla.CNS35-qPCR-56–334 FAGTTTATAATCTCTGCCGTGCT
Xla.CNS35-qPCR-56–334 RTGTGCTGCTTGGAATTCAAG
Xla.CNS35-qPCR-314–580 FCTTGAATTCCAAGCAGCACAT
Xla.CNS35-qPCR-314–580 RCCTCAAGAACAATTCTCATTTAAATCCAC
Xla.arid3a-L-exon2-RT-PCR-FCCCAAGCAATCTAGTCAACAGACATTTCC
Xla.arid3a-L-exon4-RT-PCR-RGCTGCACTGGTGATTGAAGTTGGTAG
Xla.lhx1-FTCTACTGTAAAAACGACTTCTTCAGG
Xla.lhx1-RCCATTGACTGATAGAGAAGAAAAGG
Xla.six2.L-FCGAAGCCAAAGAGAGGTACG
Xla.six2.L-RTTGGGATCCTTCAACTCTGG
Xla.six2.S-FACCCGTTGTCCTCTTCAATG
Xla.six2.S-RTGACCTGCTGAATGCAAGT
Xla.osr1-FTCCTTCCTACAAGCCTTCAATGGAC
Xla.osr1-RCTGAACAGAACACAATCATGTACAAGGAATTC
Xla.osr2-FGGGAAGATGGGCAGCAAAGCT
Xla.osr2-RTAGAAGTCCTGTCTGGGGCTGTG
Xla.hnf1b-FTGGCTATGGATGCCTATAGTACTGGCC
Xla.hnf1b-RTGCTGATGCTGCTAGTATCTGTGACAAC
Xla.hnf4a-FCGGCTTTCTGTGAACTTCCACTGG
Xla.hnf4a-RCTACATAGCTTCCTGTTTGGTGATGGTC

Transgenic reporter assay

X. laevis embryos were generated by the sperm nuclear transplantation method with oocyte extracts (Kroll and Amaya, 1996). The manipulated embryos were cultured until stage 37, and all normally developed embryos were subjected to in situ hybridization in order to examine their GFP expression with maximum sensitivity. All CNS-carrying reporters were tested at least three times. The frequency of GFP expression varied depending on the constructs, but all constructs exhibited a reproducible expression pattern.

Nephrectomy

We first undertook training in the surgical removal of nephric tubules using stage 37 Xla.Tg(Xtr.pax8:EGFP) embryos in accordance with McLaughlin’s method (Caine and Mclaughlin, 2013). Then, we removed the nephric tubules of Xla.Tg(Xtr.lhx1-CNSs:EGFP). All nephrectomized X. laevis were cultured at 18°C until 24, 48, 72, and 120 hr after nephrectomy.

Motif analysis for transcription factor binding sites

JASPAR ver. 5, an open-access database, was used to search for potential transcription factor binding sites in nephric enhancers (Mathelier et al., 2014). The candidate transcription factors were narrowed down according to their expression using the Expression Atlas (Petryszak et al., 2014). CNSs were aligned using ClustalW, and conserved sequences for the candidate transcription factor binding sites were further analyzed by phylogenetic footprinting (Blanchette and Tompa, 2002).

In situ Hybridization and Immunostaining

The cDNA clones for X. laevis lhx1, pax8, hnf4a, hnf1b, osr1, osr2, and six2 probes were synthesized from stages 35/36. The primers used for cDNA cloning are listed in Table 1pax2 and pax8 cDNA clones were kind gifts from Dr. Brändli (Heller and Brändli, 1997; Heller and Brändli, 1999). The nephrectomized transgenic X. laevis were subjected to in situ hybridization in order to examine their GFP expression with maximum sensitivity. All CNS-carrying reporters were tested at least three times. The frequency of GFP expression varied depending on the constructs, but all constructs exhibited a reproducible expression pattern. The CNSs that drove nephric expression in more than 10% of the examined embryos were defined as RSREs. For whole-mount immunostaining, nephrectomized X. laevis were fixed in 3.7% formaldehyde/MEM at 4°C overnight. After fixation, embryos were washed with 100% ethanol and then incubated in a 2% BSA/PBS-t blocking solution. The embryos were then transferred to a primary antibody solution. A 1 : 1,000-diluted rabbit anti-phospho-histone H3 (Ser10) antibody (Millipore, 06–570) or 1 : 10-diluted anti-Arid3a antibody (DSHB, PCRP-ARID3A-1E9) was used as the primary antibody in conjunction with 1 : 1,000-diluted Alexa 488-conjugated goat anti-rabbit IgG (A11001; Invitrogen) and 1 : 1,000-diluted Alexa 568-conjugated goat anti-mouse IgG (A11011; Invitrogen) secondary antibodies. Images were acquired using ApoTome.2 (Axio Zoom.V16; Carl Zeiss).

Quantification of in situ Hybridization Signals

In situ hybridization signals were photographed using an Axio Zoom.V16 with AxioCam MRc cameras (Carl Zeiss). The images were then subjected to further analysis using Adobe Photoshop CS (Adobe, San Jose, CA, USA) as described in the Results. The in situ hybridization signals were first inverted, and then the relative luminosity, determined as the signal-positive nephric duct area minus an equal area of background, was measured. Then, the average luminosity was subjected to statistical analysis. The significance of differences between the control side and the nephrectomized side was calculated in Prism7 using two-tailed paired t-test (GraphPad Software).

Quantification of proliferation

The nephrectomized X. laevis processed for immunohistochemistry with phosphor-histone H3 (H3P) were photographed at the same magnification and exposure time. H3P-positive cells were counted in a square region (160 mm2) of regenerating tubules indicated by GFP expression of Xla.Tg(Xtr.pax8:EGFP).

Luciferase reporter assay

For the luciferase reporter assay, lhx1-CNSs were cloned into the pGL4.23 vector (Promega, Madison, WI, USA). HEK293T cells seeded in 24-well plates were transfected with 100 ng of lhx1-CNS luciferase plasmids and 10 ng of Renilla luciferase plasmids using jetPEI (Polyplus Transfection SA, Illkirch, France). The total amount of DNA per well was adjusted to 1 μg with pBKS plasmids. Transfected cells were incubated for 48 hr, and then the luminescence signals were measured following the manufacturer’s protocol (Promega).

ChIP analysis for lhx1-CNSs

ChIP was performed as described previously (Gazdag et al., 2016). Myc-Xtr.arid3a-, Xtr.Arid3b-, and mouse kdm4a mRNA-injected embryos were collected at stages 35/36 and subjected to ChIP using Dynabeads Protein A (Dynabeads, Great Neck, NY, USA). The following antibodies were used: rabbit polyclonal anti-H3K9 (tri-methyl K9) antibody (ab8898; Abcam, Cambridge, MA, USA). and mouse monoclonal c-Myc (9E10) antibody (sc-40; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA). Decrosslinking and elution of DNA were performed for real-time quantitative PCR using StepOne Plus (Thermo Fisher Scientific, Waltham, MA, USA). Injection and ChIP-qPCR were performed at least three times independently.

Conditional knockdown using Photo-Morpholino

The splicing-blocking antisense-MOs for Arid3a.L and photo-morpholinos (Gene Tools, Philomath, OR, USA) used in this study were as follows:

  • arid3a.L-exon2-intron2-MO: AGAGGGAAGCCAGCAGGTACTCACC.

  • arid3a.L-exon2-intron2-photo-MO: AGTACCTGpTGGCTTCCCT.

A ratio of 0.9 : 1 of photo-MO to MO was annealed at room temperature for 30 min, and then 2 nM photo-MO/MO was injected with a GFP tracer into one-cell-stage embryos. Cleavage of the antisense of photo-MO was performed by exposing embryos to UV light (365 nm) for 30 min.

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Decision letter

  1. Marianne E Bronner
    Senior and Reviewing Editor; California Institute of Technology, United States
  2. Iain Drummond
    Reviewer

In the interests of transparency, eLife includes the editorial decision letter and accompanying author responses. A lightly edited version of the letter sent to the authors after peer review is shown, indicating the most substantive concerns; minor comments are not usually included.

[Editors’ note: a previous version of this study was rejected after peer review, but the authors submitted for reconsideration. The first decision letter after peer review is shown below.]

Thank you for submitting your work entitled "Arid3a regulates the nephric tubule regeneration via the evolutionarily conserved regeneration signal-response enhancer" for consideration by eLife. Your article has been reviewed by three peer reviewers, and the evaluation has been overseen by a Senior/Reviewing Editor. The reviewers have opted to remain anonymous.

Our decision has been reached after consultation between the reviewers. Based on these discussions and the individual reviews below, we regret to inform you that your work will not be considered further for publication in eLife.

As you will see from the reviews below, the reviewers have raised several major issues that would need to be addressed before the manuscript could be considered further:

1) You would need to provide evidence that the regeneration specific enhancers indeed are regeneration specific.

2) You would need to provide evidence that Arid3a is expressed in the pronephros.

3) You would need to provide a negative control for the chromatin immunopprecipitation experiments.

I refer you to the full reviews for further details. If some time in the future, you feel you can provide the necessary data, we would be willing to consider new version of the manuscript that contains these new data and every effort would be made to return the manuscript to the same reviewers.

Reviewer #1:

The manuscript by Suzuki et al. describes how Arid3a regulates enhancers of lhx1 gene expression during Xenopusnephron regeneration.

Previous studies have demonstrated that the Xenopusnephron regenerates after partial nephrectomy and that lhx1 gene activation occurs during nephron regeneration in zebrafish. This study indicates that lhx1 enhancer elements enable binding of Arid3a to activate of lhx1 gene expression during nephron regeneration. It also shows that Arid3a recruits chromatin modifiers to these enhancer elements to facilitate lhx1 gene expression and resultant cell proliferation. The topic of the paper is significant in that it is the first paper to indicate a mechanism by which regeneration of nephrons occurs in Xenopus. More broadly, this paper is the first to examine how enhancers facilitate regeneration of the nephron. This paper is appropriate for publication in eLife if the following concerns are addressed in a revised manuscript.

1) The manuscript focuses on Arid3a association with a regeneration response enhancer of lhx1 (and to a certain extent pax8), but the Introduction fails to describe the role of lhx1 (and pax8) in Xenopus development or cite manuscripts related to it known roles in kidney development (Taira et al., 1994; Carroll and Vizeet al., 1999; Carroll, Wallingford and Vize, et al.1999; DeLay et al., 2018).

2) Results related to Figure 1 do not describe when (what stage) the nephrectomy is performed in this study.

3) Because pax8 is one of the first genes expressed during kidney development, the pax8 transgenic line is an excellent choice to examine regeneration. However, since in Figure 2 there is some regenerating tissue that is labeled by lhx1 probes but not pax8 probes, is it possible that the transgenic pax8 line does not label all of the regenerating tissue? If so, this is useful information in the context of Figure 1.

4) Throughout the manuscript it is unclear whether these enhancer elements are important for lhx1 expression during normal development or just during regeneration.

5) In Figure 3C, the image of the control side is not clear as to whether the conserved noncoding sequence drives GFP expression in the nephron in the absence of nephrectomy.

6) In Figure 4B, kidney expression of Arid3a.L is difficult to see in the images.

7) The explanation of Figure 5A is unclear. How does inset panel relate to position of larger image? What does the purple indicate?

Reviewer #2:

This paper identifies enhancers that are used in regeneration of the kidney to drive lhx1 expression. As an important part of regeneration, this is potentially very interesting. However, I don't see experiments addressing whether the regeneration induced enhancers are also normally used in development? What is the implication if they are also active in Development – does it mean that the enhancer is not specific to regeneration? The authors call the enhancers regeneration specific, so this is a crucial point for the paper as presented. While this may be the main point that needs to be addressed in a revision, there are a number of other points that need to be clarified in the paper.

Arid3a binding and function:

Figure 4B: the expression of Arid 3aL in tubules is very hard to see. Perhaps a higher magnification would help.

Figure 4C: are some of these tadpoles nephrectomized as suggested in the legend? I don't see panels that address this.

Figure 4C: is CNE35 a negative control? It also binds Arid3a, but this putative enhancer is not activated in expression, so what is the implication here? The possibility that it only activates the gene in which it is resident is attractive but not tested explicitly, though such a specific context effect would be interesting. In any case, such ChIP experiments should address binding to a negative control element or two. As presented, the data are consistent with general binding of the tagged Arid3a to chromatin in a nonspecific way.

I am confused about the Arid3a expression, according to Xenbase the levels of expression in kidney are similar for Arid3aL and 3aS, and in the figures presented, neither appears to be expressed in the pronephric region, though Arid3aL has an overall higher staining in the ISH than does Arid3aS. However, this is not as well controlled as the RNA seq in Xenbase, though that is for adult kidney.

The logic of the experiments in Figure 6 would be clearer if it were made clear that the photoMO is the sense strand which binds and inhibits the antisense.

The results with the photoactivable MO appear quite convincing, though I am still puzzled by how it targets the pronephros, when the evidence for arid3aL expression is not so clear.

Reviewer #3:

This paper presents a thorough and interesting analysis of conserved non-coding elements in the lhx1 locus. This is significant since lhx1 expression and function is important in kidney development and regeneration. The authors show that CNEs have sites for Arid3a and go on to show that Arid3a can bind and activate lhx1CNEs. They also show that a consequence of Arid3a regulation of lhx1 is increased proliferation. This work will be of interest to kidney development investigators and to labs studying tubule regeneration. The approach is rigorous in that many replicates were performed for each experiment and statistical methods were applied.

1) What distinguishes the Regeneration Signal Responsive Enhancers described in this work from developmental regulatory elements in the lhx1 locus? i.e. are these elements the same as cis-elements active during development? Since the fish to human enhancer conservation was stronger than the fish to frog enhancer conservation, this seems to suggest the RSREs might simply be developmental enhancers reactivated by injury, raising the question of whether they warrant the name "regeneration signal response enhancers". Also, wouldn't it have been more interesting to look at the CNEs that were specific to fish and frog (which can regenerate), even if they were not as strong, and look for binding sites in those that were not present in mouse/human (which don't regenerate as well)?

2) As described by the authors, the rationale for choosing to focus on lhx1a as a regeneration gene conflates two different repair mechanisms in frog and fish when the authors say "Among these, the previous finding that lhx1a-expressing cells regenerate functional nephrons in zebrafish prompted us to focus on the mechanisms of Lhx1 expression in regenerating [frog] nephric tubules (Diep et al., 2011)." The zebrafish repair mechanism involves Six2-expressing adult kidney stem cells while the frog nephrectomy model appears to be a regrowth of an existing tubule, much like mammalian kidney regeneration/wound healing, and this tubule does not express Six2. It is important to be clear about what each model represents in terms of distinct regenerative mechanisms.

3) Have the authors tried other unbiased approaches to identifying proteins bound to RSREs?

4) In Figure 4D, there is no explanation of why CNE35 was split into 56-334 and 314-580.

5) In Figure 5C why would CNE35 act differently from CNE17 and CNE20 in terms of altered chromatin structure?

6) From the data shown I am not convinced Arid3a is expressed in the pronephros. The magenta arrows in Figure 4B seem to be pointing at yolk with no blue in situ reaction product where the pronephros would be. The cross section is too low resolution to see tubule morphology in the stained cross section. Clearer data should be presented.

7) In Figure 4C, the experiment is a little difficult to understand. How many hours after heat shock was lhx1 expression assayed?

8) Figure 6: What accounts for the loss of message in UV treated photo MO condition? Splice blocking MOs usually alter splicing; is there an altered Arid3a mRNA after UV activation of the MO? Is there a control for UV exposure alone on mRNA abundance? 30 minutes of continuous exposure to 365 nm light could have effects of its own. Also the experimental design at the top of 6A should indicate that both photo and regular MO's were injected together.

https://doi.org/10.7554/eLife.43186.024

Author response

[Editors’ note: the author responses to the first round of peer review follow.]

We performed transgenic experiments on the regeneration signal-response enhancer (RSRE) in embryos at the early tailbud stage in order to examine whether RSREs function in developing nephrons. We observed no enhancer activities in developing nephrons at stage 26. The results suggest that the enhancers that we identified on the basis of the activities in regenerating nephric tubules exert their primary function after nephrectomy to induce lhx1, and they are now shown in Fig. 3—figure supplement 1. In order to improve the images of arid3a expression, we again performed in situ hybridization and also performed immunostaining using anti-Arid3a antibody. To provide a negative-control ChIP-qPCR for anti-Myc antibody, we added two additional genomic elements, exon5 and CNS32, which do not contain any putative Arid3a binding motifs. We also performed ChIP-qPCR targeting exon5 and CNS32 using antiH3K9me3 antibody.

As you will see from the reviews below, the reviewers have raised several major issues that would need to be addressed before the manuscript could be considered further:

1) You would need to provide evidence that the regeneration specific enhancers indeed are regeneration specific.

We performed transgenic experiments of RSREs in embryos at the early tailbud stage in order to examine whether RSREs function in developing nephrons. This transgenic reporter analysis showed that RSREs exhibited no enhancer activities in developing nephrons at stage 26, suggesting that the enhancers that we identified on the basis of the activities in regenerating nephric tubules exert their primary function after nephrectomy to induce lhx1.

2) You would need to provide evidence that Arid3a is expressed in the pronephros.

We again performed in situ hybridization and provided better images of this. We also performed immunostaining using anti-Arid3a antibody and found Arid3a protein in the proximal tubules.

3) You would need to provide a negative control for the chromatin immunopprecipitation experiments.

We performed ChIP-qPCR experiments using two additional genomic elements, exon 5 and CNS32, which do not contain any putative Arid3a binding motifs, as a negative control for anti-Myc antibody. We also performed ChIP-qPCR targeting exon 5 and CNS32 using antiH3K9me3 antibody.

I refer you to the full reviews for further details. If some time in the future, you feel you can provide the necessary data, we would be willing to consider new version of the manuscript that contains these new data and every effort would be made to return the manuscript to the same reviewers.

Reviewer #1:

[…] 1) The manuscript focuses on Arid3a association with a regeneration response enhancer of lhx1 (and to a certain extent pax8), but the Introduction fails to describe the role of lhx1 (and pax8) in Xenopus development or cite manuscripts related to it known roles in kidney development (Taira et al., 1994; Carroll and Vize, 1999; Carroll, Wallingford and Vize, 1999; DeLay et al., 2018).

Thank you for this comment. We have included the following text in the “Results” section: “It is known that lhx1 is expressed in developing nephrons at the early tailbud stage and specifies the renal progenitor cell field (Taira et al., 1994; Carroll et al., 1999; Cirio et al., 2011)”. We have also included the following text in the “Results” section: “It has been shown in a previous study involving simple mRNA injection using Xenopus embryos that Lhx1 with Pax8 can induce ectopic pronephron structures (Carroll and Vize, 1999). […] These previous findings prompted us to focus on the mechanisms of lhx1 expression in regenerating nephric tubules”.

2) Results related to Figure 1 do not describe when (what stage) the nephrectomy is performed in this study.

Thank you for this comment. We have included the stage when nephrectomy was performed in the “Results” section and also in the figures.

3) Because pax8 is one of the first genes expressed during kidney development, the pax8 transgenic line is an excellent choice to examine regeneration. However, since in Figure 2 there is some regenerating tissue that is labeled by lhx1 probes but not pax8 probes, is it possible that the transgenic pax8 line does not label all of the regenerating tissue? If so, this is useful information in the context of Figure 1.

We agree that Xla.Tg(Xtr.pax:8:EGFP) does not label all of the regenerating nephric cells. Ideally, a double transgenic line such as pax8:EGFP and lhx1:mechrry would be required to trace all of the regenerating cells. In order to address this point, we have included the following changes: “Although it is possible that primary induced lhx1-, pax8-, hnf4a-, hnf1b-, and osr2expressing cells are not completely consistent with cells observed in Xla.Tg(Xtr.pax8:EGFP) regenerating nephric tubules, the immediate induction of lhx1, pax8, hnf4a, hnf1b, and osr2 after nephrectomy suggests that the loci of these genes contain enhancers of injury response”.

4) Throughout the manuscript it is unclear whether these enhancer elements are important for lhx1 expression during normal development or just during regeneration.

We agree with this comment. Hence, in response to this point, we have examined the enhancer activities of RSREs in embryos at the early tailbud stage. This transgenic reporter analysis showed that CNS20 and CNS35 do not exhibit enhancer activities in developing nephrons at stage 26, at which endogenous lhx1 expression has already occurred. Although some of the CNS17 transgenic reporter embryos showed GFP expression in developing pronephros, most of the TG embryos did not. These results suggest that the primary function of RSREs is to induce lhx1 after nephrectomy (—figure supplement 1Figure 3).

5) In Figure 3C, the image of the control side is not clear as to whether the conserved noncoding sequence drives GFP expression in the nephron in the absence of nephrectomy.

Thank you for pointing that out. We have provided a better image of GFP expression.

6) In Figure 4B, kidney expression of Arid3a.L is difficult to see in the images.

Thank you for pointing that out. We performed the in situ hybridization again and provided better images. We also performed immunostaining using anti-Arid3a antibody and found that Arid3a protein was present in the proximal tubule. We have amended the text describing this as follows: “arid3a is known to be expressed in the ectoderm of the early neurula and in the epidermis at the late tailbud stage, whereas spib is expressed in the anterior ventral blood islands at the neurula stage (Callery et al., 2005; Costa et al., 2008). […] In addition, we performed immunostaining of Arid3a using Xla.Tg(Xtr.pax8:EGFP), whichshowed that Arid3a protein was detected in proximal tubules and also in the glomus and/or nephrocoelom (Figure 4B, orange arrows and orange arrowheads, respectively)”.

7) The explanation of Figure 5A is unclear. How does inset panel relate to position of larger image? What does the purple indicate?

Thank you for this comment. We have amended the figure’s legend as follows: “Heat-shocked Xla.Tg(Xla.hsp70:Xtr.arid3a-2A-mcherry, Xtr.pax8:EGFP) was nephrectomized at stage 37, incubated for 72 h, and then fixed at stages 45/46. The white dotted lines indicate the pax8expressing cells, and the magenta indicates the phosphorylated histone H3-positive cells”.

Reviewer #2:

This paper identifies enhancers that are used in regeneration of the kidney to drive lhx1 expression. As an important part of regeneration, this is potentially very interesting. However, I don't see experiments addressing whether the regeneration induced enhancers are also normally used in development? What is the implication if they are also active in Development – does it mean that the enhancer is not specific to regeneration? The authors call the enhancers regeneration specific, so this is a crucial point for the paper as presented. While this may be the main point that needs to be addressed in a revision, there are a number of other points that need to be clarified in the paper.

Thank you very much for the thoughtful comments. In order to address this main comment, we examined the enhancer activities of RSREs in embryos at the early tailbud stage. This transgenic reporter analysis showed that the RSREs CNS20 and CNS35 have no enhancer activities in developing pronephros. Although some of the CNS17 transgenic reporter embryos showed GFP expression in developing pronephros, most did not. These results suggest that the enhancers that we identified on the basis of the activities in regenerating nephric tubules exert their primary function after nephrectomy to induce lhx1, although it is still difficult to determine definitively whether RSREs are specific for regeneration (Figure 3—figure supplement 1). Therefore, we used the term “regeneration signal-response enhancer” rather than “regeneration-specific enhancer” in our manuscript.

Arid3a binding and function:

Figure 4B: the expression of Arid 3aL in tubules is very hard to see. Perhaps a higher magnification would help.

Thank you for this comment. We performed in situ hybridization again and provided better images. We also performed immunostaining using anti-Arid3a antibody and found that Arid3a protein was present in the proximal tubules. We have amended the part of the manuscript describing this as follows: “arid3a is known to be expressed in the ectoderm of the early neurula and in the epidermis at the late tailbud stage, whereas spib is expressed in the anterior ventral blood islands at the neurula stage (Callery et al., 2005; Costa et al., 2008). […] In addition, we performed immunostaining of Arid3a using Xla.Tg(Xtr.pax8:EGFP), whichshowed that Arid3a protein was detected in proximal tubules and also in the glomus and/or nephrocoelom (Figure 4B, orange arrows and orange arrowheads, respectively)”.

Figure 4C: are some of these tadpoles nephrectomized as suggested in the legend? I don't see panels that address this.

Thank you for this comment. We have amended this figure’s legend as follows:

Xla.Tg(Xla.hsp70:Xtr.arid3a-2A-EGFP) transgenic X. laevis at stage 23were treated at 34°C for 15 min, followed by 15 min at 14°C. These steps were repeated three times, and tadpoles were incubated at 18°C. lhx1 expression was observed 48 h after the heat shock at stages 35-36”. We have also added a panel to explain the experimental design.

Figure 4C: is CNE35 a negative control? It also binds Arid3a, but this putative enhancer is not activated in expression, so what is the implication here? The possibility that it only activates the gene in which it is resident is attractive but not tested explicitly, though such a specific context effect would be interesting. In any case, such ChIP experiments should address binding to a negative control element or two. As presented, the data are consistent with general binding of the tagged Arid3a to chromatin in a nonspecific way.

Thank you for this comment. We have provided negative-control elements, exon 5 and CNS32, which do not contain putative Arid3a binding motifs. ChIP-qPCR analysis showed that no enrichment of Myc-tagged Arid3a was observed on exon 5 and CNS32.

As the reviewer mentioned, CNS35 was identified as an enhancer that is activated in regenerating nephric tubules, and ChIP analysis showed that Arid3a binds to this CNS, whereas Arid3a failed to activate this enhancer when Arid3a and a luciferase reporter were cotransfected into 293T cells. In addition, we also found that the enrichment of H3K9me3 on CNS35 was not detected. As the reviewer pointed out, this lack of a repression mark suggests that Arid3a stays on CNS35 in the naïve genome and that this state allows the immediate induction of lhx1. However, we could not test this attractive possibility in this study. Instead, we included the following sentences in the “Discussion” section: “Such multiple enhancers often play different functional roles, while it is also known that they sometimes show overlapping functions (Osterwalder et al., 2018). […] Our observation that there are overlapping functions of CNS17, CNS20, and CNS35 in the regenerating nephric tubules, albeit with differential activation mechanisms, suggests that there is a redundancy of cis-regulatory elements for regeneration”.

I am confused about the Arid3a expression, according to Xenbase the levels of expression in kidney are similar for Arid3aL and 3aS, and in the figures presented, neither appears to be expressed in the pronephric region, though Arid3aL has an overall higher staining in the ISH than does Arid3aS. However, this is not as well controlled as the RNA seq in Xenbase, though that is for adult kidney.

Thank you for this comment. As you have pointed out, RNA-seq data from Xenbase were used for adult tissues. We apologize for this confusion. Therefore, we removed such data from this manuscript. Instead, we again performed in situ hybridization and provided better images. We also performed immunostaining using anti-Arid3a antibody and found that Arid3a protein was present in the proximal tubules.

The logic of the experiments in Figure 6 would be clearer if it were made clear that the photoMO is the sense strand which binds and inhibits the antisense.

The results with the photoactivable MO appear quite convincing, though I am still puzzled by how it targets the pronephros, when the evidence for arid3aL expression is not so clear.

Thank you for this comment. We apologize for this confusion. Due to the fact that we do not have any method of targeting UV irradiation to the pronephric duct, we treated whole embryos with UV. This indicates that most of the cells in the tadpoles were irradiated. However, we were able to control the timing of UV irradiation. We have thus amended the main text as follows: “Since Photo-MO is cleaved by ultraviolet (UV) treatment, we were able to control the timing of gene knockdown.”

Reviewer #3:

[…] 1) What distinguishes the Regeneration Signal Responsive Enhancers described in this work from developmental regulatory elements in the lhx1 locus? i.e. are these elements the same as cis-elements active during development? Since the fish to human enhancer conservation was stronger than the fish to frog enhancer conservation, this seems to suggest the RSREs might simply be developmental enhancers reactivated by injury, raising the question of whether they warrant the name "regeneration signal response enhancers". Also, wouldn't it have been more interesting to look at the CNEs that were specific to fish and frog (which can regenerate), even if they were not as strong, and look for binding sites in those that were not present in mouse/human (which don't regenerate as well)?

Thank you for this comment. In order to address this point, we performed transgenic reporter experiments of RSREs in embryos at the early tailbud stage. This transgenic reporter analysis showed that the RSREs CNS20 and CNS35 have no enhancer activities in developing pronephros. Although some of the CNS17 transgenic reporter embryos showed GFP expression in developing pronephros, most did not. These results suggest that the enhancers that we identified on the basis of the activities in regenerating nephric tubules primarily function after nephrectomy to induce lhx1, although it is still difficult to determine definitively whether RSREs are specific for regeneration (Supplementary Figure 3—figure supplement 1).

As you have pointed out, it is interesting to investigate unique binding motifs that are only commonly found in fish/frog CNSs. These unique transcription binding motifs may provide the unique trait of high regenerative capacity in fish and frog. Although this possibility is very interesting, we focused here on strongly activated enhancers. As such, instead, we included the following text in the “Discussion” section: “In addition, we also found that many CNSs that are only conserved between frog and fish showed weak enhancer activities in regenerating nephric tubules (Figure 3C). […] Further studies of transcriptional mechanisms for frog/fish CNSs may provide novel insights into the unique trait of high regenerative capacity in fish and frog”.

2) As described by the authors, the rationale for choosing to focus on lhx1a as a regeneration gene conflates two different repair mechanisms in frog and fish when the authors say "Among these, the previous finding that lhx1a-expressing cells regenerate functional nephrons in zebrafish prompted us to focus on the mechanisms of Lhx1 expression in regenerating [frog] nephric tubules (Diep et al., 2011)." The zebrafish repair mechanism involves Six2-expressing adult kidney stem cells while the frog nephrectomy model appears to be a regrowth of an existing tubule, much like mammalian kidney regeneration/wound healing, and this tubule does not express Six2. It is important to be clear about what each model represents in terms of distinct regenerative mechanisms.

Thank you for this comment. As you have pointed out, the regeneration of nephric tubules in frog appears to involve the regrowth of existing tubules. This regenerative mechanism is much closer to the regeneration of nephric tubules in mammals, rather than the reconstruction of nephrons in zebrafish from stem cells. We have thus amended the “Results” section as follows: “It has been shown in a previous study involving simple mRNA injection using Xenopus embryos that Lhx1 with Pax8 can induce ectopic pronephron structures (Carroll and Vize, 1999). […] These previous findings prompted us to focus on the mechanisms of lhx1 expression in regenerating nephric tubules”. We have also included the following text in the “Discussion” section: “In zebrafish, lhx1a and six2mesenchymal cells reconstruct the functional nephrons (Diep et al., 2011). […] Further live imaging studies using transgenicanimals with markers of dedifferentiating cells and regrowing cells are required in order to reveal the detailed regenerative mechanisms of nephric tubules in vertebrates”.

3) Have the authors tried other unbiased approaches to identifying proteins bound to RSREs?

We agree that unbiased approaches to identify the enhancers are crucially important. Since we have established the identification of enhancers that are activated in regenerating nephric tubules in this research, we would like to apply unbiased approaches combining ATAC-seq and transgenic reporter analysis in the future.

4) In Figure 4D, there is no explanation of why CNE35 was split into 56-334 and 314-580.

Thank you for this comment. The sequence length of CNS35 is 580 bp. Since this is too long for qPCR, CNE35 was divided into two segments. We have included the following text to explain this: “CNS35 was divided into two segments for qPCR, since it is 580 bp long, which is too long for qPCR”.

5) In Figure 5C why would CNE35 act differently from CNE17 and CNE20 in terms of altered chromatin structure?

As you have pointed out, the chromatin configuration of CNS35 differs from that of CNS17 and CNS20, and the luciferase reporter analysis also showed that the mechanism of activation of CNS35 also differs from those of CNS17 and CNS20 (Figure 4D). At present, we do not have any explicit data to explain the difference between CNS35 and other RSREs. Therefore, we have included the following text in the “Discussion” section: “CNS17, CNS20, and CNS35 are activated in regenerating nephric tubules, indicating that these enhancers have overlapping functions. […] Our observation that there are overlapping functions of CNS17, CNS20, and CNS35 in the regenerating nephric tubules, albeit with differential activation mechanisms, suggests that there is a redundancy of cis-regulatory elements for regeneration”.

6) From the data shown I am not convinced Arid3a is expressed in the pronephros. The magenta arrows in Figure 4B seem to be pointing at yolk with no blue in situ reaction product where the pronephros would be. The cross section is too low resolution to see tubule morphology in the stained cross section. Clearer data should be presented.

We performed in situ hybridization again and provided better images. We also performed immunostaining using anti-Arid3a antibody and found that Arid3a protein was present in the proximal tubules. We have thus amended the text as follows: “arid3a is known to be expressed in the ectoderm of the early neurula and in the epidermis at the late tailbud stage, whereas spib is expressed in the anterior ventral blood islands at the neurula stage (Callery et al., 2005; Costa et al., 2008). […] In addition, we performed immunostaining of Arid3a using Xla.Tg(Xtr.pax8:EGFP), whichshowed that Arid3a protein was detected in proximal tubules and also in the glomus and/or nephrocoelom (Figure 4B, orange arrows and orange arrowheads, respectively)”.

7) In Figure 4C, the experiment is a little difficult to understand. How many hours after heat shock was lhx1 expression assayed?

Thank you for this comment. We have added the experimental design in Figure 4C.

8) Figure 6: What accounts for the loss of message in UV treated photo MO condition? Splice blocking MOs usually alter splicing; is there an altered Arid3a mRNA after UV activation of the MO? Is there a control for UV exposure alone on mRNA abundance? 30 minutes of continuous exposure to 365 nm light could have effects of its own. Also the experimental design at the top of 6A should indicate that both photo and regular MO's were injected together.

Thank you for this comment. We designed the splicing-blocking morpholino to the arid3a.L exon2-intron2 donor site and also designed RT-PCR primers to exon 2 and exon 4. Therefore, exon 3 of arid3a.L is skipped by arid3a.L-MO, and the level of exon 3-containing transcripts is decreased. In order to confirm whether UV exposure affects mRNA abundance, we performed RT-PCR using UV-untreated and UV-treated wild-type tadpoles. This analysis showed that UV does not affect the abundance of spliced mRNA. We added the result on this in Author response image 1.

Author response image 1
A) A diagram of the experimental design.

B) RT-PCR analysis was performed. Lanes 1: Marker, Lane2: UV untreated embryos, Lane3: UV treated embryos. UV treatment and RT-PCR were performed at three times independently. C) The significance of differences between the UVuntreated and UV-treated wiled type embryos was calculated by two-tailed unpaired t-test (p = 0.4857, not significant). The error bars indicate SEM.

We have also changed Figure 6A as suggested and included the following text: “We first designed the splicing-blocking morpholino for arid3a.L, since the expression arid3a.L in nephrons is stronger than that of arid3a.S (Figure 4B, Figure 4—figure supplement 1A). […] Thus, arid3a.L-MO is rendered inactive by binding to arid3a.L-Photo-MO”.

https://doi.org/10.7554/eLife.43186.025

Article and author information

Author details

  1. Nanoka Suzuki

    Institute for Promotion of Medical Science Research, Yamagata University, Faculty of Medicine, Yamagata, Japan
    Contribution
    Data curation, Formal analysis, Investigation
    Competing interests
    No competing interests declared
  2. Kodai Hirano

    Institute for Promotion of Medical Science Research, Yamagata University, Faculty of Medicine, Yamagata, Japan
    Contribution
    Investigation, Construction of plasmids
    Competing interests
    No competing interests declared
  3. Hajime Ogino

    Amphibian Research Center, Hiroshima University, Higashi-hiroshima, Japan
    Contribution
    Resources, Funding acquisition, Methodology, Construction of plasmids
    Competing interests
    No competing interests declared
  4. Haruki Ochi

    Institute for Promotion of Medical Science Research, Yamagata University, Faculty of Medicine, Yamagata, Japan
    Contribution
    Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Investigation, Writing—original draft, Project administration, Writing—review and editing
    For correspondence
    harukiochi@med.id.yamagata-u.ac.jp
    Competing interests
    No competing interests declared
    ORCID icon "This ORCID iD identifies the author of this article:" 0000-0002-0088-581X

Funding

Japan Society for the Promotion of Science (16K07362)

  • Haruki Ochi

Takeda Science Foundation

  • Haruki Ochi
  • Hajime Ogino

Suzuken Memorial Foundation

  • Haruki Ochi

Japan Society for the Promotion of Science (15K07082)

  • Hajime Ogino

Japan Society for the Promotion of Science (16H04794)

  • Hajime Ogino

Japan Society for the Promotion of Science (25124704)

  • Haruki Ochi

Japan Society for the Promotion of Science (25125716)

  • Haruki Ochi

Japan Society for the Promotion of Science (16H04828)

  • Haruki Ochi

Japan Society for the Promotion of Science (17KT0049)

  • Haruki Ochi

The funders had no role in study design, data collection and interpretation, or the decision to submit the work for publication.

Acknowledgements

This work was supported by Grants-in-Aid for Scientific Research from the Japan Society for the Promotion of Science (JSPS) (Grants nos. 16K07362, 25124704, 25125716, 16H04828, and 17KT0049 to H Ochi and 15K07082 and 16H04794 to H Ogino), as well as grants from CREST (JST to H Ogino), the Takeda Science Foundation (to H Ochi and H Ogino), Suzuken Memorial Foundation, and YU-COE (C) (H Ochi). The genomic DNA of X. tropicalis for the screening enhancer was provided by the Amphibian Research Center, Hiroshima University, with partial support from the National Bio-Resource Project of AMED, Japan.

Ethics

Animal experimentation: This study was performed in strict accordance with the recommendations in the Act on Welfare and Management of Animals in Japan and the Guide for the Care and Use of Laboratory Animals of the National Institutes of Health. All of the animals were handled in accordance with the Animal Research Guidelines at Yamagata University. The protocol was approved by the Committee on the Ethics of Animal Experiments of Yamagata University.

Senior and Reviewing Editor

  1. Marianne E Bronner, California Institute of Technology, United States

Reviewer

  1. Iain Drummond

Publication history

  1. Received: October 27, 2018
  2. Accepted: December 18, 2018
  3. Version of Record published: January 8, 2019 (version 1)

Copyright

© 2019, Suzuki et al.

This article is distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use and redistribution provided that the original author and source are credited.

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