Developmental Biology 388 (2014) 57–67

Contents lists available at ScienceDirect

Developmental Biology journal homepage: www.elsevier.com/locate/developmentalbiology

Yap1, transcription regulator in the Hippo signaling pathway, is required for Xenopus limb bud regeneration Shinichi Hayashi, Koji Tamura, Hitoshi Yokoyama n Department of Developmental Biology and Neurosciences, Graduate School of Life Sciences, Tohoku University, Aramaki-Aza-Aoba 6-3, Aoba-ku, Sendai 980-8578, Japan

art ic l e i nf o

a b s t r a c t

Article history: Received 5 September 2013 Received in revised form 21 January 2014 Accepted 24 January 2014 Available online 1 February 2014

The Hippo signaling pathway is conserved from insects to mammals and is important for multiple processes, including cell proliferation, apoptosis and tissue homeostasis. Hippo signaling is also crucial for regeneration, including intercalary regeneration, of the whole body in the flatworm and of the leg in the cricket. However, its role in vertebrate epimorphic regeneration is unknown. Therefore, to identify principles of regeneration that are conserved among bilaterians, we investigated the role of Hippo signaling in the limb bud regeneration of an anuran amphibian, Xenopus laevis. We found that a transcription factor, Yap1, an important downstream effector of Hippo signaling, is upregulated in the regenerating limb bud. To evaluate Yap1's function in limb bud regeneration, we made transgenic animals that expressed a dominant-negative form of Yap under a heat-shock promoter. Overexpression of a dominant-negative form of Yap in tadpoles reduced cell proliferation, induced ectopic apoptosis, perturbed the expression domains of limb-patterning genes including hoxa13, hoxa11, and shh in the regenerating limb bud. Transient expression of a dominant-negative Yap in transgenic tadpoles also caused limb bud regeneration defects, and reduced intercalary regeneration. These results indicate that Yap1 has a crucial role in controlling the limb regenerative capacity in Xenopus, and suggest that the involvement of Hippo signaling in regeneration is conserved between vertebrates and invertebrates. This finding provides molecular evidence that common principles underlie regeneration across phyla, and may contribute to the development of new therapies in regenerative medicine. & 2014 Elsevier Inc. All rights reserved.

Keywords: Hippo pathway Yap1 Limb bud regeneration Intercalation

Introduction The individuals of all animal species are vulnerable to accidental injury, throughout their lifespan and the ability to regenerate injured body parts is essential for some species' survival. All multicellular organisms appear to possess some capacity for regeneration, although the extent of this ability differs among species (Agata and Inoue, 2012; Bonfanti, 2011; Sanchez Alvarado and Tsonis, 2006). Planarians and hydra, for example, have marvelous regeneration abilities, and can even regenerate a whole body from a small fragment. Among vertebrates, only anuran (frog) tadpoles and urodeles (newts and salamanders) can fully regenerate amputated limbs (limb buds). Urodele amphibians have the greatest regenerative ability; they can also reconstruct the brain, optic lens, spinal cord, jaw, and heart (Brockes, 1997; Straube and Tanaka, 2006). In contrast, amniotes (including

n

Corresponding author. E-mail address: [email protected] (H. Yokoyama).

http://dx.doi.org/10.1016/j.ydbio.2014.01.018 0012-1606 & 2014 Elsevier Inc. All rights reserved.

mammals) cannot regenerate any of these organs. In anuran amphibians, such as Xenopus laevis, the capacity for complete limb regeneration is confined to tadpoles at the limb-bud stage or earlier; this ability declines gradually as metamorphosis proceeds (Dent, 1962; Muneoka et al., 1986). Regeneration in animals across tissues and species may involve common mechanisms. These include molecular mechanisms, such as signal transduction and signaling molecules; tissue interactions among the wound epithelium, underlying mesenchyme, and neuronal axons; and patterning mechanisms, such as positional information and intercalation (Agata et al., 2007; Sanchez Alvarado and Tsonis, 2006). Amphibian limb regeneration serves as an ideal model for elucidating such common mechanisms, because this process results in the complete regeneration of multiple tissues and their morphological patterning, thus permitting a comparison of the mechanisms of tissue repair and repatterning among vertebrates and invertebrates. In the amphibian limb regeneration process, a special epithelial sheet, the wound epithelium, promptly covers the amputation plane to protect stump cells from the external environment,

58

S. Hayashi et al. / Developmental Biology 388 (2014) 57–67

thickens, and becomes the AEC (apical epithelial cap), which acts as the signaling center for the regeneration of the limb from the stump tissues (Gardiner et al., 2002; Han et al., 2005; Murawala et al., 2012). This AEC is morphologically and functionally similar to the AER (apical ectodermal ridge) of amniote limb buds (Muneoka and Sassoon, 1992). The AER specifically expresses a secreted protein encoded by fgf8 that is essential for the outgrowth of mesenchyme-derived tissues (Lewandoski et al., 2000; Moon and Capecchi, 2000). Similarly, fgf8 is expressed in the AEC covering the Xenopus limb blastema (Christen and Slack, 1997; Endo et al., 2000), a cluster of highly proliferative and undifferentiated mesenchymal cells derived from the stump. Wnt signaling is also required for the initiation of limb regeneration and is involved in AEC function (Kawakami et al., 2006; Yokoyama et al., 2007). Under the influence of secreted molecules from the AEC, differentiated cells in the stump dedifferentiate to form the blastema. In both Xenopus tadpoles and urodeles, the blastema proceeds to redifferentiate and precisely reconstitutes the same architecture as the original limb, probably guided by anteroposterior (AP) position information determined by Shh (Endo et al., 1997; Imokawa and Yoshizato, 1997; Torok et al., 1999). Certain key events governing regeneration, i.e., formation of a signal center by the wound epithelium and formation of a blastema, are similar between vertebrates and invertebrates. Furthermore, key molecules in vertebrate regeneration, such as Fgf, Wnt (Wingless), and Shh (Hedgehog) signaling components (Stoick-Cooper et al., 2007) are also involved in invertebrate regeneration processes (Cebria et al., 2002; Nakamura et al., 2008; Yazawa et al., 2009). Despite the accumulation of evidence showing that common molecular mechanisms function in organ/ body regeneration across phyla, a full understanding of the machinery governing regeneration, and the extent to which it is conserved, remains elusive. In particular, little information is available on the intracellular signal transduction pathways that regulate the cell behavior and tissue interactions required for organ re-formation, or on how these pathways are regulated. We therefore sought to elucidate the mechanism(s) that regulate cell– cell interactions, cell proliferation, size control, cell differentiation, and pattern formation during regeneration. Here, we focused on the Hippo signaling pathway as a candidate for such a common regulator of organ regeneration. Recently, Hippo signaling was demonstrated to be essential for regeneration of the flatworm body (Demircan and Berezikov, 2013) and the cricket leg (Bando et al., 2009). At the cellular and molecular levels, the Hippo pathway and its functions are highly conserved between invertebrates and vertebrates, and it contributes to tissue growth, cell polarity, mechanotransduction, and tumorigenesis (Dupont et al., 2011; Halder and Johnson, 2011; Wada et al., 2011). Given its known roles in regeneration and multiple functions, we hypothesized that Hippo signaling is a common regulator of regeneration. Yap1 (Yes-associated protein 1), is a key transcriptional regulator of Hippo signaling. It is inactivated when phosphorylated by Mst (a vertebrate homolog of Hippo) and Lats in an intracellular signal kinase cascade (Halder and Johnson, 2011) (see also supplementary material Fig. S1A), and inactive Yap1 is trapped in the cytoplasm (Schlegelmilch et al., 2011). Dephosphorylated (i.e., active) Yap1 is translocated into the nucleus to activate target genes through DNA-binding proteins, such as Tead. yap1 knockout mice show severe defects in yolk sac vasculature, chorioallantoic fusion, and embryonic body axis development (Morin-Kensicki et al., 2006). As in the mouse, yap1 knockdown in Xenopus embryos causes defects in AP axis elongation (Gee et al., 2011). In the mouse, the expression of a Yap1 gain-of-function construct causes the liver size to increase more than 4-fold, and this expansion is reversed when transgene induction is stopped

(Camargo et al., 2007; Dong et al., 2007). Yap1 is also upregulated in and required for regeneration of the mammalian intestine (Barry et al., 2013; Cai et al., 2010). Thus, Yap1 functions downstream of Hippo signaling and plays essential roles in embryogenesis and tissue homeostasis in multiple aspects of vertebrate biology. Classic experiments in appendage regeneration of amphibians and hemimetabolous insects suggest that a common process controls regeneration from invertebrates to vertebrates (Agata et al., 2007; French et al., 1976; Yakushiji et al., 2009b). In vertebrates, the machineries that govern cell–cell interactions, cell proliferation, and organ size are accepted as critical factors for regenerating an amputated limb to its normal size and shape, although the underlying molecular mechanisms remain unclear. Given that the program for phylogenetically conserved regeneration must be robust, the functional requirement for Hippo signaling mediated by Yap1 in cricket and flatworm regeneration coupled with its conserved role in embryogenesis and tissue homeostasis, led us to hypothesize that this pathway also plays a crucial role in vertebrate limb regeneration. To address the functional contribution of the Hippo pathway to vertebrate regeneration, we focused on Yap1, which directly regulates the ON/OFF state of Hippo target gene expression, and analyzed limb bud regeneration in Xenopus, a model system that allows the efficient manipulation of gene expression by transgenesis. Our results showed that Yap1 is essential for Xenopus limb bud regeneration, suggesting that Hippo signaling is a functionally conserved, common mechanism of regeneration in species from invertebrates to vertebrates.

Materials and methods Ethical treatment of animals and animal husbandry The law in Japan (Act on Welfare and Management of Animals) exempts experiments using X. laevis (amphibians) from the requirement for IRB approval. Nonetheless, all surgery was performed under anesthesia, and all efforts were made to minimize suffering. X. laevis frogs were obtained from domestic animal vendors. Conventional methods for raising the animals were used. Xenopus tadpoles were reared at 22–23 1C in dechlorinated tap water and staged according to Nieuwkoop and Faber (1994). The tadpoles were fed powdered barley grass (Odani Kokufun Co., Ltd., Kouchi, Japan). At stage 58, feeding was stopped until metamorphosis was completed. Thereafter, the froglets were fed tubifex every other day. Tadpole surgery Tadpoles were anesthetized by 0.025% ethyl-3-aminobenzoate (Tokyo Chemical Industry, 886-86-2), dissolved in Holtfreter's solution. Ophthalmologic scissors were used to amputate tadpole hindlimb buds at the presumptive knee level, which was identified by comparing visible developmental features with a fate map (Tschumi, 1957). To assess intercalary regeneration in tadpoles, surgical procedures were carried out as described elsewhere (Endo et al., 1997; Shimizu-Nishikawa et al., 2003). In brief, the zeugopod was removed, and the autopod was returned to the stump with a 1801 rotation about the proximo-distal (PD) axis. Operated tadpoles were incubated for 2 h at 4 1C in Holtfreter's solution, and then returned to the rearing container, which was kept at room temperature. RT-PCR Total RNA was extracted from the regenerating limb bud with TRIzol reagent (Ambion, 15596-026). Reverse transcription for

S. Hayashi et al. / Developmental Biology 388 (2014) 57–67

cDNA was carried out using SuperScript III (Invitrogen, 18080044). TaKaRa Taq (TaKaRa, R001) and Power SYBR Green PCR Master Mix (Applied Biosystems, 4367659) were used for conventional PCR and real-time PCR, respectively. Real-time quantitative PCRs were carried out using an ABI StepOnePlus system. Primer sequences are shown in Supplementary Table 1. In situ hybridization A partial sequence of the yap1 long isoform (480 bp) was cloned using the TOPO TA cloning kit (Invitrogen, 45-0640), and then a DIG-RNA probe was synthesized by SP6 RNA polymerase (Roche, 810274). Primer sequences are shown in Supplementary Table 1. Probes for hoxa13 and hoxa11 (Ohgo et al., 2010), shh (Yakushiji et al., 2007), fgf8 (Endo et al., 2000), and mkp3 (Gomez et al., 2005) were synthesized as previously described. Transcripts were detected by in situ hybridization on frozen sections, as previously described (Ohgo et al., 2010), except for the proteinase K treatment (2 mg/ml, 37 1C for 6.5 min) and hybridization temperature (56 1C). Immunohistochemistry All tissue section immunohistochemistry procedures were carried out at room temperature, unless otherwise noted. Tissues were fixed in 4% paraformaldehyde in PBS for 15 min, embedded in OCT compound (Sakura Finetek Japan, 4583), sectioned at a 10-mm thickness on a cryostat, and re-fixed with 4% paraformaldehyde in PBS for 15 min. The sections were then treated sequentially with 0.1% Triton TX-100 (Sigma, T8787) in PBS for 20 min, 0.3% H2O2 (Santoku Chemical Industries, 3041 110823) for 10 min, and 2% FBS in PBS for 1 h. Sections were incubated with an antiYap antibody (1:50 v/v, Cell Signaling Technology, 4912) at 4 1C overnight. A TSA kit (Invitrogen, T20925) was used to amplify the signal. Whole-mount immunohistochemistry was performed as described previously (Schreiber et al., 2001), except that a 2-day incubation with primary antibodies was used. Primary antibodies were diluted 1:100 (v/v) for anti-Myosin heavy chain (Developmental Studies Hybridoma Bank, MF20), anti-acetylated Tubulin (Sigma, 6-11B-1), anti-phosphorylated Histone H3 (Millipore, 06-570), and active Caspase3 (BD Pharmingen, 559565). Cultured cells were immunostained as described previously (Nishioka et al., 2009). Primary antibodies were used at the following dilutions (v/v): anti-Yap antibody, 1:300; anti-Flag antibody (Sigma, F3165), 1:500. Constructs and cell transfections To transfect cultured cells (Fig. S2), X. laevis yap1 was cloned and inserted into Flag-pcDNA3 at the EcoRI and XhoI sites. Cos7 cells (2
104) were plated in 1.7 cm2 on a chamber slide glass (Nunc, 154526) in 10% FBS (HyClone, SV33014.03)/DMEM (Gibco, 11995). After 24 h, the cells were transfected with the expression vector using Lipofectamine 2000 transfection reagent (Invitrogen, 11668-027), following the manufacturer's instructions. After 20 h, the cultured cells were immunostained as described above. Alcian blue and alizarin red staining Cartilage and bone staining was performed as described previously (Yano et al., 2012). Froglets were fixed with either 10% formaldehyde or 4% paraformaldehyde, overnight, at room temperature. To stain the cartilage, samples were dehydrated through a series of ethanol solutions and incubated in Alcian blue solution [0.1 mg/ml Alcian blue 8GX (Sigma, A5268), 80% ethanol, 20%

59

acetic acid] at 37 1C for several hours, until dye deposition was apparent. After rehydration, the samples were treated overnight with 5 mg/ml trypsin (BD Difco, 215240) in 30% saturated NaB4O7/ 70% water at room temperature. For bone staining, samples were incubated in 4% alizarin red (Sigma, A5533) saturated with ethanol, 0.5% KOH at room temperature for several hours until dye deposition was apparent. Pigments were bleached in 0.6% H2O2/1% KO overnight at room temperature. Plasmid constructs and Xenopus transgenesis The dominant-negative form of Yap was amplified by PCR from an expression vector encoding dnYap (Nishioka et al., 2009), which was a kind gift from Dr. Sasaki and Dr. Hirate. A heat-shock inducible expression vector, pHS1 (Wheeler et al., 2000) was combined with the 2 A peptide (Nojima et al., 2010), separable GFP reporter, and tdTomato reporter (Shaner et al., 2004) under control of the 2.2-kb γ-crystallin promoter (Offield et al., 2000). dnYap was inserted in front of the 2 A peptide-GFP in the ClaI site (5') and XhoI/SalI compatible site (3') (Fig. 3A). Primer sequences are shown in Supplementary Table 1. I-SceI meganucleasemediated transgenesis was carried out as previously reported (Ogino et al., 2006a, b; Pan et al., 2006). To induce transgene expression by heat-shock, tadpoles were shifted from the rearing temperature (22–23 1C) into 34 1C tap water, and left for 30 min, as previously described (Beck et al., 2003). They were moved to 18 1C after the heat-shock. Heat-shock treatments were performed 3 h before and 3 days after the amputation.

Results Yap1 is activated in the blastema of the regenerative limb bud Considering its conserved functions in organogenesis and tissue homeostasis, we hypothesized that Hippo signaling plays an important role(s) in limb regeneration. To examine the gene expression of Hippo pathway components in the blastema of the regenerative limb bud, we amputated stage 52 (st52) tadpole limb buds at the presumptive knee level, and extracted total RNA from the blastemas at 5 dpa (days post amputation). At st52, the limb bud has the potential to regenerate completely, and the amputated limb bud generates a cone-shaped blastema by 5 dpa. We performed RT-PCR for core components of the Hippo pathway, mst2, lats1, lats2, yap1, tead1 and tead4 (Halder and Johnson, 2011), using the extracted total RNA as the template. Transcripts for all of these core Hippo components were detected in the regenerative blastema (Fig. 1A; see also Fig. S1A). Two yap1 isoforms were detected. Cloning and sequencing showed that the long isoform included exon4, but the short one did not. We also used RT-PCR to detect Xenopus homologs of the Hippo signaling regulators fat1, fat4, lix1l, wtip, limd1, and agr2, and the Hippo target gene, ctgf (Halder and Johnson, 2011). All of these mRNAs were also expressed in the blastema, but fat3 mRNA was not detected (Fig. S1B). In summary, the main components of the Hippo kinase cascade and transcription factors were expressed in the blastema. Since the product of yap1 directly regulates the ON/OFF state for the transcription of Hippo target genes, we examined the spatial pattern of yap1 mRNA expression in the limb bud and blastema. yap1 mRNA was detected throughout the unoperated st51 limb bud (Fig. 1B). At 3 dpa, yap1 expression was greater in the blastema than in the limb-bud stump or intact limb bud (st53), both of which showed basal levels of expression (Fig. 1C, D). We observed two splicing isoforms of yap1 (with and without exon 4) in the Xenopus limb bud blastema, and it has been reported that

60

S. Hayashi et al. / Developmental Biology 388 (2014) 57–67

Fig. 1. Hippo pathway components expressed in limb bud regeneration. (A) RT-PCR for main components of the Hippo pathway. RNA was extracted from the blastemas of regenerating hindlimb buds amputated at st52, at the presumptive knee level, at 5 dpa (days post amputation). Hippo pathway components were amplified by RT-PCR. rpl8 and ef1a are housekeeping genes used as controls. Asterisk indicates the yap1 splice isoform lacking exon4. We repeated the RT-PCR including total RNA extraction three times and obtained consistent results. (B–D) In situ hybridization for yap1 long isoform mRNA. B. yap1 mRNA was strongly expressed and distributed throughout the st51 limb bud. Dor, dorsal; Ven, ventral; Pro, proximal, Dis, distal. (C) Basal level of yap1 expression was detected in the intact limb bud (st53). (D) yap1 expression was higher in 3-dpa blastemas, but its level was basal in the stump. E. qRT-PCR for yap1 mRNA. mRNA was quantified in the 0-dpa limb bud and in the 4-dpa and 7-dpa blastema. Total RNAs were extracted from the amputated tips of limb buds and from the blastemas (colored regions in the line drawings). Primers were designed to amplify both of isoforms. yap1 mRNA quantity was normalized to ef1a. yap1 expression was higher at 4 and 7 dpa, compared with 0 dpa. We repeated the qRT-PCR including total RNA extraction three times and obtained consistent results. (B–D) Proximal is up, dorsal is left. Scale bar¼ 100 mm. Solid line indicates amputation level. Error bar indicates s.e.m. Experiment was performed in triplicate.

Fig. 2. Distribution of Yap1 protein in limb bud regeneration. (A-F) Immunofluorescent staining with the anti-Yap antibody of an intact limb bud and a 5-dpa blastema from a hindlimb bud amputated at st52 at the presumptive knee level. (A–C) No signal was detected in a control section processed without the primary antibody. (D–F) Yap1 protein was detected throughout the limb bud and in the blastema. (G–L) Intracellular distribution of Yap1. (G–I) Yap1 protein was distributed in the cytoplasm in the intact limb bud. (J–L) Yap1 was localized to nuclei in blastema cells, enabling it to regulate gene transcription. (A–F) All photographs show horizontal sections and proximal is up. Photographs in (G–L) are high-magnification images of the areas shown within the corresponding square in (D–F). Scale bar¼ 200 mm in (A–F) and 10 mm in (G–L). Solid line indicates amputation level.

yap1 promotes cell proliferation, regardless of isoform type, in an expression level-dependent manner (Muramatsu et al., 2011). We therefore quantified the total expression level of yap1 mRNA (both isoforms) by real-time RT-PCR, and found that it was 5.4- and 3.7fold greater at 4 and 7 dpa, respectively, than in the 0 dpa limb bud (st52 limb bud, Fig. 1E). These findings indicate that the transcription of yap1 is upregulated in blastema cells. Next, we performed immunohistochemistry to detect Yap1 protein in the blastema, because Hippo signaling regulates target gene expression via the translocation of Yap1 into the nucleus. We first confirmed that the commercially available anti-Yap antibody (cell signaling), which was produced against human Yap1, cross-reacted with the Xenopus Yap1 homolog in transfected cells

in vitro (Fig. S2). Immunostaining with anti-Yap1 showed that the protein was located in the developing limb bud (Fig. S3), neural tube, and tail muscle (Fig. S4). At 5 dpa, Yap1 was distributed broadly through the intact limb bud and the regenerative blastema (Fig. 2A–F). Since the presence of activated Yap1 protein in the nucleus indicates that the protein is transcriptionally active (Zhao et al., 2007), we used confocal microscopy to evaluate Yap1's presence in 5-dpa blastema cell nuclei. Confocal microscopy allowed us to discriminate between highly fluorescent nuclei and the cumulative fluorescence from overlapping cells. We detected Yap1 protein in the nuclei of 5-dpa blastema cells (Fig. 2J–L), but it was localized to the cytoplasm of cells in the intact limb bud (Fig. 2G–I). These results suggest that Yap1 is

S. Hayashi et al. / Developmental Biology 388 (2014) 57–67

61

Fig. 3. Loss of Yap1 function caused limb bud regeneration defects. (A) Map of the heat-shock-inducible dominant-negative Yap (dnYap) transgene. (B, C) Tg tadpoles were distinguishable from WT by lens-specific tdTomato fluorescence under control of the γ-crystallin promoter. Arrow indicates a tdTomato-positive eye. (D) Experimental schedule. Transgenic F1 tadpoles were heat-shocked 3 h prior to the amputation. The left hindlimb bud was amputated, and the right hindlimb bud kept intact. Tadpoles were heat-shocked again at 3 dpa. (E) Transcript level of a Yap1 target gene, ctgf, was reduced at 10 dpa after dnYap induction. ctgf mRNA quantity was normalized to odc1. n¼ 4 (biological replicates). Asterisk: The difference between WT and Tg was statistically significant at p o 0.05 (Welch's t-test). Error bar indicates s.e.m. (F–I) Examples of limb bud regeneration in WT. (F) GFP reporter expression was not observed after heat-shock in WT. (The limb bud is outlined in white.) (G) Zeugopod and autopod with 5 digits had regenerated at 14 dpa. H, I. Alcian blue and alizarin red staining at 78 dpa. The WT amputated left hindlimb bud had completely regenerated and was equivalent to the intact right hindlimb. (J–M) Examples of limb bud regeneration in dnYap Tg. (J) The GFP reporter was strongly expressed after heat-shock in Tg tadpoles. K. The regenerating limb was regressed, and no obvious primordia of multiple digits were observed at 14 dpa. (L, M) dnYap Tg animals showed regeneration defects: the number of digits was reduced in the regenerated limb, but the intact right hindlimb did not show any obvious developmental defects. N. Percentage of varying degrees of digit number regeneration. (F, G, J, K) Lateral view. (H, I, L, M) Dorsal view. Scale bar¼ 500 mm in (B, C, F, G, J, K) and 5 mm in (H, I, L, M). The dashed and solid lines indicate the amputation plane before and after limb bud amputation, respectively.

activated and translocated into the nucleus of blastema cells during limb bud regeneration. Loss-of-function of Yap1 causes regeneration defects Since Yap1 is activated in limb bud regeneration, we next attempted to reveal the molecular function of Yap1 in limb bud regeneration in transgenic animals. We utilized an available dominant-negative form of Yap (dnYap, Fig. 3A), which was first established in mice (Nishioka et al., 2009), under control of a heatshock promoter to create transgenic Xenopus tadpoles in which inducible overexpression of dnYap allowed us to transiently inhibit endogenous Yap1 function. dnYap is composed of the mouse Yap1 functional domain (without the transactivation domain) and the repressor domain of Drosophila engrailed (Nishioka et al., 2009). We established a stable F1 transgenic line, which was reproduced by cross breeding between a F0 transgenic (Tg) male and a wildtype (WT) female. Transgene integration was confirmed using the γ-crystallin reporter, which drives the expression of fluorescent tdTomato protein in the eyes (Fig. 3B, C). F1 dnYap Tg Tadpoles and their WT siblings were heat-shocked using the schedule shown in Fig. 3D at st52, a stage that permits complete regeneration. This time course of heat-shock was established by preliminary experiments using dnYap F0 Tg animals, with observation of GFP intensity and severity of regeneration defects. Heat-shock once prior to amputation seemed to be insufficient (data not shown), and we therefore added an additional heat-shock at 3 dpa when an early blastema was formed.

To check whether dnYap sufficiently inhibits Yap function, we examined gene expression level of a Yap1 target gene, connective tissue growth factor (ctgf; see also Fig. S1A; (Halder and Johnson, 2011), at 10 dpa by qRT-PCR and found that ctgf transcript was significantly reduced by dnYap induction (Fig. 3E). Three hours after the first heat-shock, GFP fluorescence (the indicator for transgene induction) was detected in the dnYap Tg Xenopus, but not in their WT siblings (Fig. 3F, J). The left hindlimb bud was then amputated at the presumptive knee level. After the second heat-shock, at 3 dpa, the transgene induction diminished gradually, until 14 dpa. In the dnYap Tg froglets, regeneration defects were apparent, and included a reduction in the size and number of skeletal elements (Fig. 3K, L). In contrast, most WT froglets regenerated a complete limb (Fig. 3G, H). Quantification showed that 75% of the WT froglets regenerated a limb with 5 digits, but only 3.8% of the Tg froglets did so (Fig. 3N). The regenerated dnYap Tg limbs exhibited the loss of digits, and the most frequent phenotype was 1 digit (30.8%). All WT animals the regenerated zeugopod, but some dnYap Tg animals could not regenerate it (zeugopod absence, WT: 0%, Tg: 15.9%). In some samples of dnYap Tg, the zeugopod region was regenerated but reduced. This finding indicates that Yap1 is essential for Xenopus limb bud regeneration. Interestingly, no marked defect in skeletal formation was detected in the development of the contralateral intact limb bud in animals with dnYap induction (Fig. 3I, M). Thus, dnYap induction strongly affected limb bud regeneration but not development of the contralateral limb buds in Xenopus. Normal development of

62

S. Hayashi et al. / Developmental Biology 388 (2014) 57–67

the contralateral limb buds excludes the possibility that the dnYap transgene has nonspecific inhibitory effects on limb outgrowth. Next, we investigated whether the loss of Yap1 function affected the re-patterning of the regenerating limb bud, by examining the expression patterns of region-specific genes. During normal Xenopus limb bud development, the expression domains of hoxa13 (in the autopod) and hoxa11 (in the zeugopod) overlap at early stages and then become mutually exclusive (Blanco et al., 1998; Endo et al., 2000; Ohgo et al., 2010). These expression patterns were recapitulated in the regenerating limb bud with regional specificity in WT tadpoles at 5 dpa (Fig. 4A, C). In the Tg tadpoles, the autopod region marked by hoxa13 expression showed markedly slowed growth, and hoxa11 expression partially overlapped with hoxa13 (Fig. 4B, D). shh, which is involved in AP axis formation in Xenopus limb bud regeneration, is normally expressed in the posterior region in the WT tadpole limb bud (Fig. 4E) (Endo et al., 1997; Yakushiji et al., 2007). In Tg tadpoles, the intensity of the shh expression was diminished (Fig. 4F). The expression of Fgf8 in the AER of the developing limb bud mediates intercellular signaling from the epidermis, and mkp3 expression is induced by Fgf signaling in the mesenchyme

(Kawakami et al., 2003). Because we noticed that the AEC and underlying mesenchyme showed morphological regression in the dnYap Tg limbs bud (data not shown), we examined this phenotype to investigate Yap1's role in epithelial–mesenchymal interactions. dnYap induction resulted in the reduction of the fgf8- and mkp3-expressing regions, suggesting that reduced epithelial– mesenchymal interactions mediated by Fgf signaling contributed to the morphological phenotype (Fig. 4G–J). The comparison of gene expression patterns shown in Fig. 4 between WT and dnYap Tg are representative of three independent experiments. The Hippo pathway regulates the cell cycle and apoptosis to maintain tissue homeostasis. To evaluate the contribution of these Hippo functions to limb bud regeneration, we performed wholemount immunohistochemistry at 3 dpa using anti-phosphorylated histone H3 (pH3) as a proliferation marker and anti-active caspase3 (acCas3) as an apoptosis marker. Many pH3-positive cells were present in the WT amputated limb bud (Fig. 5A, n ¼11), but the number of pH3-positive cells in the dnYap Tg blastema and stump was obviously reduced upon visual inspection (Fig. 5B, reduced mitosis: n ¼4/5 animals examined). Apoptosis detected by acCas3 was particularly apparent in the AEC of the WT amputated

Fig. 4. Expression domains of pattern formation genes were reduced or disrupted in dnYap Tg blastemas. st52 tadpoles were heat-shocked and the limb buds were amputated as shown in Fig. 3D. The limb buds were fixed for in situ hybridization at 5 dpa. To ensure the validity of comparing their gene expression levels, the WT and dnYap Tg sections were processed simultaneously. (A, B) hoxa13 was expressed in the autopod in WT regenerating limb buds, but the hoxa13-expressing region showed a regressed pattern in the dnYap Tg ones. (C, D) hoxa11 defined the presumptive zeugopod, and its expression was mutually exclusive with that of hoxa13 in the WT regenerating limb bud. hoxa11 expression in the Tg limb bud partially overlapped with the hoxa13-expressing region. (E, F) shh was expressed in the posterior mesenchyme in the WT limb bud, while its expression level was clearly reduced in the Tg limb bud. (G, H) fgf8 expression, used as an AEC marker, was reduced in the Tg regenerating limb bud, compared with WT. (I, J) mkp3-expressing region in mesenchyme was also regressed in Tg. Posterior is up, proximal is left. Scale bar¼ 200 mm. Solid line indicates amputation level.

Fig. 5. dnYap expression affected cell proliferation, cell death, and cell differentiation in limb bud regeneration. Regenerating limb buds were fixed at 3 dpa (6 h after the second heat-shock, see Fig. 3D), 7 dpa, or 10 dpa, and subjected to whole-mount immunofluorescence staining. (A, B) Phosphorylated Histone H3 (pH3) at 3 dpa, indicating mitotic cells. In dnYap Tg limb buds, the number of pH3-positive cells was obviously reduced in the blastema and stump, compared with the WT regenerating limb buds. (C, D) Active Caspase3 (acCas3) staining at 3 dpa, indicating apoptotic cells. Apoptosis occurred in the AEC of both of WT and Tg regenerating limb buds. Ectopic apoptosis was frequently observed in the stump in Tg limb buds (white arrow). (E, F) Acetylated Tubulin (acTub) staining at 7 dpa to mark neuronal axons. The regenerating limb bud was innervated in WT animals, but innervation was impaired in dnYap Tg animals. (G, H) Sarcomeric Myosin heavy chain (MyHC) staining at 10 dpa with the MF20 antibody. In WT limb buds, some muscle bundles had regenerated. In Tg limb buds, muscle regeneration was reduced, although some muscle bundles were present. Bright-field image was overlaid on fluorescence images to visualize the outline of the regenerative limb bud. All photos are lateral views. Posterior is up, proximal is left. Scale bar ¼200 mm. Solid line indicates amputation level.

S. Hayashi et al. / Developmental Biology 388 (2014) 57–67

limb bud (Fig. 5C, n ¼6), but in dnYap Tg animals, apoptosis occurred ectopically in the amputated stump (Fig. 5D, ectopic apoptosis: n ¼3/5, arrow). These data suggest that Yap1 expression is tightly correlated with the outgrowth of the blastema and the provision of cells from the stump. To estimate the effect of dnYap on nerve and muscle regeneration, we stained regenerating limb buds with antibodies against acetylated alpha tubulin to label axons and against myosin heavy chain to label muscle. Neuronal axons invaded the newly formed tissues in the regenerating WT limb bud at 7 dpa (Fig. 5E, regenerated axon; n ¼6/7), but innervation was impaired in the Tg amputated limb bud, concomitant with blastema regression (Fig. 5F, regenerated axon: n ¼1/6). In amphibian limb regeneration, nerve existence in the limb is important for gene expression, cell proliferation and blastema formation. However, in the limb bud at an early stage, blastema formation and cell proliferation are

63

not dependent on innervation and a limb bud gradually acquires nerve dependency as the nervous system in the limb bud develops (Kumar and Brockes, 2012). For example, hindlimb bud regeneration of the Xenopus tadpole at stage 52 is not dependent on nerve supply (Cannata et al., 2001; Filoni and Paglialunga, 1990). Thus, dnYap effects on gene expression, proliferation and apoptosis are not secondary effects by nerve degeneration observed in dnYap Tg tadpoles. In the regenerating WT limb bud, multiple myosin heavy chain-positive bundles appeared in the presumptive zeugopod region, and muscle fibers began to be visible in the autopod region by 10 dpa (Fig. 5G, muscle formation; n¼ 6/6); however, few regenerating Tg limb buds showed evidence of muscle formation (Fig. 5H, muscle formation; n ¼1/7). In the intact limb bud of the Tg animals, the effect of dnYAP induction on the cell cycle, apoptosis, and tissue formation was much smaller (Fig. S5A–H). In addition, although development of

Fig. 6. Intercalary regeneration was impaired by dnYap1 induction. (A) Schematic drawing of the intercalary regeneration experiment. The limb bud of st52 tadpoles was amputated, the presumptive zeugopod region was removed, and the autopod region was rotated 1801 around the PD axis and returned to the stump. Operated tadpoles were heat-shocked as shown in Fig. 3D. (B–E) Intercalary regeneration with a positional information gap in control tadpoles. (B, C) After heat-shock and implantation, graft survival was confirmed at 3 dpa, and no GFP fluorescence was observed in the WT animals. (D) Zeugopodal regeneration was observed at 28 dpa, along with supernumerary digit formation. (E) Alcian blue and alizarin red stained sample at 56 dpa. Tibio-fibula and tarsi were regenerated along with the compartment containing the cartilaginous joint. Tf: Tibiofibula, T: Tarsus. (F–I) Defective intercalary regeneration in dnYap Tg. (F, G) GFP fluorescence was observed in the graft and stump after heat-shock. (H) Zeugopodal regeneration was impaired, and supernumerary digit formation was reduced. (I) Along the PD axis, the regenerated Tg limb skeleton was compressed, and skeletal elements had fused. No obvious joint compartment was visible. The black bar indicates the part of the limb that was intercalated along the PD axis. (J) Bone length measurement of the tibio-fibula and tarsi. Regenerated zeugopodal bone length was shortened by the compartment defect and bone fusion in dnYap Tg animals. (K) Quantification of supernumerary digit formation. Excessive digit formation was reduced by inhibition of Yap1 function. Dotted line indicates the normal digit number (5) formed from the autopod when there is no gap in the position information. þHS: heat-shocked, –HS: not heat-shocked. Asterisk and double asterisk: The difference between WT and Tg was statistically significant at p o0.05 and po 0.01, respectively (Welch's t-test). Total number of samples is 10 (WT þHS), 11 (Tg–HS) and 11 (Tg þHS), respectively. (B, C, F, G) lateral view. (D, E, H, I) dorsal view. Scale bar ¼500 mm in (B, C, F, G) and 5 mm in (D, E, H, I) Error bar indicates s.e.m.

64

S. Hayashi et al. / Developmental Biology 388 (2014) 57–67

the intact limb bud was transiently delayed during dnYap expression, the morphology of the limb was ultimately unaffected (Fig. S5I and Fig. 3M). dnYap induction reduces intercalary regeneration During limb (leg) regeneration in amphibians and insects, each cell recognizes and assumes its appropriate position between the stump and the blastema, to form a new limb that replicates the original tissue as closely as possible. Experimentally, when distal limb tissues are grafted onto an amputated stump in urodeles, the appropriate intermediate tissues form and are “intercalated” between the stump and the distal organizer. Thus, “intercalation” results in the regenerated cells having to adopt a minimum sequence of positional values (French et al., 1976). Intercalary regeneration has also been reported in Xenopus (ShimizuNishikawa et al., 2003). When the presumptive zeugopod region of a st52 hindlimb bud is surgically removed, and the distal limb bud tip (the autopod) is rotated 180 degrees on the proximodistal (PD) axis and regrafted to the proximal stump (see Fig. 6A), intercalary regeneration compensates for the ablated zeugopod (Shimizu-Nishikawa et al., 2003). To learn whether Yap1 is required for intercalary regeneration in Xenopus, we performed the experiment described above using dnYap Tg tadpoles. dnYap Tg and WT tadpoles were heat-shocked 3 h prior to and 3 days after the transplantation. Engraftment of the distal tissue with blood circulation was confirmed for both the WT and dnYap Tg tadpoles at 3 days post-transplantation (Fig. 6B, F). GFP fluorescence was observed after heat-shock in dnYap Tg (Fig. 6G) but not WT tadpoles, as expected (Fig. 6C). In WT animals, intercalary regeneration along the PD axis was observed 4 weeks after the operation. This process led to supernumerary digits induced by the gap in positional information owing to the 1801 rotation of the autopod tip (Fig. 6D), as previously reported (Shimizu-Nishikawa et al., 2003). Observation of the skeletal pattern 8 weeks after the operation confirmed that very little of the original gap caused by the operation remained along the PD axis, suggesting that the original sequence of positional values had been successfully recapitulated (Fig. 6E). In contrast, in the dnYap Tg tadpoles, intercalary regeneration along the PD axis was impaired by dnYap induction (Fig. 6H, I). The tibio-fibular region (derived from the zeugopod) and tarsi (derived from the basal autopod) were shorter than in WT animals and had fused; the ankle joint was therefore missing (Fig. 6H, I). Complete intercalary PD regeneration occurred in 11 of the 21 control animals and in none of the 11 dnYap Tg animals. Controls included WT animals treated with heat-shock and dnYap Tg animals that were not heat-shocked (5/10 and 6/11 did complete PD intercalation, respectively). The total bone length of the tibio-fibula and tarsi was significantly reduced in the heat-shocked dnYap Tg compared to WT animals (Fig. 6J). Furthermore, the number of supernumerary digits was reduced in the heat-shocked dnYap Tg animals (Fig. 6K): the total number of digits per operated limb ranged from 5 to 15 among control animals and from 4 to 9 in the heatshocked dnYap Tg animals. The total number of digits in a normal Xenopus hindlimb is five and the number will be greater than five if there is any supernumerary digit formation. Supernumerary digits will usually be formed after 180 degree rotation of the limb bud tip as shown in Fig. 6A (Cameron and Fallon, 1997; Muneoka and Murad, 1987; Endo et al., 1997: modified in ShimizuNishikawa et al., 2003) to “intercalate” the positional gap along the AP axis. The reduced range of total number of digits in heatshocked dnYap Tg animals indicates reduced “intercalation” along the AP axis. These results indicate that Yap1 is required for an intercalary response to an experimentally induced positional gap

in the Xenopus limb bud. Furthermore, these results suggest that a molecular link may exist between a normal regeneration and intercalary regeneration as shown by zeugopodal defects both in normal limb bud regeneration and intercalary regeneration after a grafting experiment. To examine this possibility, the spatiotemporal activation patterns of Yap1 and its downstream targets and the temporal requirement of Yap1 function for regeneration should be elucidated and compared in detail both in limb bud regeneration and intercalary regeneration.

Discussion Yap1 activation is important for adequate limb bud regeneration Here we demonstrated that Yap1 activity is upregulated in the regenerating limb bud blastema and that Yap1 plays an essential role in Xenopus limb bud regeneration. yap1 transcript was increased and its protein translocated into the nucleus in the blastema cells (Figs. 1, 2). Loss of Yap1 function caused limb bud regeneration defects (Fig. 3). Yap1 is an essential intercellular mediator of the Hippo signaling pathway and the Hippo pathway transduces signals from cell–cell and cell–ECM (extracellular matrix) contact into changes in cell behavior (Dupont et al., 2011; Wada et al., 2011). Interestingly, this pathway recognizes the different positions of the outer and inner cells in the preimplantation mouse embryo (Nishioka et al., 2009), in which Yap1 is localized to the nuclei of the outer cells, but remains in the cytoplasm of those in the inner cell mass. When an inner cell is isolated from the outer cells, Yap1 translocates into the nuclei, suggesting that the Hippo pathway transduces information about altered cell–cell contacts to change cell behavior. During limb regeneration, the mesenchymal cell contacts with the ECM and/or other cells are reduced at the amputated site, because of the loss of surrounding cells and ECM. Thus, the Hippo pathway may be involved in recognizing the loss of cell/ECM contacts caused by an injury, and in promoting cell proliferation to regenerate the limb. Studies have been conducted on crosstalk between Yap1 and other pathways in organogenesis and tumorigenesis. For example, the β-catenin/TCF4 complex binds to the yap1 enhancer and upregulates its expression downstream of Wnt signaling in colorectal cancer cells (Konsavage et al., 2012). Furthermore, Yap1 interacts with Disheveled (Dvl) in the cytoplasm and cooperates with β-catenin to upregulate common targets in the nuclei in multiple situations (Heallen et al., 2011; Imajo et al., 2012). Wnt signaling is reported to contribute to Xenopus limb bud regeneration (Kawakami et al., 2006; Yokoyama et al., 2007), and the inhibition of Wnt signaling by Dkk1 induction blocks the initiation of limb bud regeneration. Yap1 also interacts with Smad1, in a manner dependent on BMP (Alarcon et al., 2009), and BMP plays an important role in Xenopus limb bud regeneration (Beck et al., 2006). Further investigation of the temporal requirement of Hippo signaling for regeneration processes by dnYap induction at different time points and the further elucidation of molecular crosstalk with other signaling pathways, such as Wnt and BMP signaling, should shed light on the unique role of Hippo signaling. The dissection of signaling crosstalk is an attractive subject for future study aimed at understanding the molecular mechanisms of limb regeneration. Yap1 is critical for re-patterning in limb bud regeneration Our results showed that expressions of patterning genes, the cell cycle, and apoptosis were affected by Yap1 inhibition during Xenopus limb bud regeneration, and that these disruptions

S. Hayashi et al. / Developmental Biology 388 (2014) 57–67

resulted in the reduction of morphological components in the regenerated limb. The changes in patterning genes altered the compartmentalization of the developing limb. In the complete regeneration of a limb from an amputated tadpole limb bud, the hoxa13 and hoxa11 expression domains overlap at first and then separate into the autopod and zeugopod. This separation does not occur in the incomplete limb regeneration of a froglet limb after metamorphosis, which forms only a single cartilaginous spike (Ohgo et al., 2010). In dnYap Tg tadpoles, the hoxa13 and hoxa11 expression domains remained partially overlapping, and the hoxa13-expressing domain regressed (Fig. 4A-D), resembling their pattern in the incomplete limb regeneration of WT froglets. This similarity suggests that appropriate compartmentalization is required for complete regeneration and that Hippo signaling contributes to position reconstruction. The inhibition of Yap1 function also affected shh expression. shh is thought to be involved in pattern formation along the AP axis, in limb regeneration as well as limb development (Endo et al., 1997; Imokawa and Yoshizato, 1997; Torok et al., 1999). Gene expression, cell proliferation and chondrogenesis related to limb regeneration are regulated by Shh signaling (Endo et al., 2000; Yakushiji et al., 2009a., 2007). Here, we showed that Yap1 is required for appropriate shh expression in the regenerative limb bud (Fig. 4E, F). Furthermore, Shh signaling is reported to be required for yap1 transcription in neural precursor cells (Fernandez et al., 2009), suggesting that the Hippo pathway and Shh signaling function in a feedback loop for limb regeneration. Fgf signaling from the apical epithelium also participates in the upregulation of shh expression (Moon and Capecchi, 2000), and our results (Fig. 4G, H) indicated that the AEC, marked by fgf8 expression, was also disrupted by dnYap expression. Thus, the Hippo, Fgf, and Shh signalings may interact tightly with each other to form a network for signaling crosstalk to regulate patterning genes. Conserved functions of the Hippo pathway in bilaterian regeneration Components of the Hippo pathway are conserved among many animal species, suggesting that they have been conserved during evolution and divergence (Halder and Johnson, 2011; Hilman and Gat, 2011). For example, the regulation by the Hippo pathway of organ size in insects and mammals is effected by a conserved mechanism. Yorkie and Scalloped, fly homologs of Yap and Tead, respectively, are involved in the size control of multiple organs (Dong et al., 2007; Zhang et al., 2008; Zhao et al., 2008), as well as mammals (Camargo et al., 2007; Dong et al., 2007; von Gise et al., 2012). The Hippo pathway is also important for whole-body regeneration in the flatworm and leg regeneration in the cricket (Bando et al., 2009; Demircan and Berezikov, 2013) and abnormalities in cell proliferation and defects in regeneration are observed in both animals when the expression of Hippo components is manipulated. Furthermore, the knockdown of Yorkie affects intercalary regeneration, which is caused by a gap in the position information, in leg regeneration in the cricket (Bando et al., 2009). Here, we provide the first data showing that Yap1 regulated by Hippo signaling is essential for vertebrate limb regeneration. These data support the idea that the Hippo pathway function is conserved in limb regeneration between invertebrates and vertebrates, although our results with dnYap1 cannot be directly compared with the downregulation of Yorkie in cricket, because Yorkiespecific RNAi causes nymphal lethality (Bando et al., 2009). Thus, as shown in invertebrates, we observed defects in cell proliferation, skeletal components, and the recognition of positional values in Xenopus dnYap Tg animals. In fact, intercalary regeneration in Xenopus tadpoles was significantly reduced by the loss of Yap1 function (Fig. 6). These parallel findings suggest that

65

there is functional conservation of the Hippo pathway among bilaterians. Regardless of the accumulation of evidence supporting its conserved functions, it is still largely unknown whether the upstream regulators of the Hippo pathway are conserved across bilaterians. In fly and cricket, two large protocadherins, Dachsous and Fat, function as regulators upstream of Hippo signaling (Halder and Johnson, 2011). Opposing gradients of Dachsous and Fat contribute to the recognition of positional values and control organ size by regulating the Hippo signaling activity in leg development and regeneration (Bando et al., 2009). Although vertebrates have Dachsous and Fat homologs and some reports suggest that their signals are activated similarly (Skouloudaki et al., 2009; Van Hateren et al., 2011), how or whether Hippo is regulated by Dachsous/Fat signaling in vertebrates remains unclear (Halder and Johnson, 2011). In the mammalian epidermis, the cell density-dependent regulation of Yap1 is mediated by α-catenin (Schlegelmilch et al., 2011), suggesting that some other mechanism is responsible for transducing the Hippo pathway's function in position recognition. A transcription factor, Meis, is implicated in mediating position information in amphibians (Mercader et al., 2005), and it is a candidate molecule for transducing the Hippo-mediated response to a gap in positional information, since the insect homolog of Meis, Hth (Homothorax), interacts and cooperates with Yorkie (Halder and Johnson, 2011). To fully elucidate the processes that underlie regeneration, both mechanisms that are conserved across species and those that are not must be studied and understood, including the molecular mechanisms that serve as upstream regulators of Hippo signaling. Thus, elucidation of the molecular functions and the signal networks involving the Hippo pathway in vertebrate regeneration will provide comprehensive insight into the conserved machineries of bilaterian regeneration. Such information promises to further preclinical studies of regeneration in model animals, and hopefully, ultimately, in humans.

Acknowledgments We thank Drs. Hajime Ogino and Haruki Ochi for technical advice on I-SceI meganuclease-mediated transgenesis, Drs. Hiroshi Sasaki and Yoshikazu Hirate for providing expression constructs of Hippo components, and Dr. Jose F. de Celis for the mkp3 construct. We thank Drs. Stefan Hoppler, Tim Mohun, Masahiko Hibi, and Roger Tsien for their respective gifts of the Xenopus hsp70 promoter, Xenopus γ-crystalline promoter, 2A peptide, and tdTomato. We thank Yoshiko Yoshizawa-Ohuchi for frog care and Natsume Sagawa for maintaining the frog facility. This work was supported by MEXT and JSPS KAKENHI Grant number 22124005 to HY, JSPS KAKENHI Grant number 25870058 to HY, “Funding Program for Next Generation World-Leading Researchers” [LS007] from the Cabinet Office, Government of Japan to KT, the Kurata Memorial Hitachi Science and Technology Foundation to HY, and the Asahi Glass Foundation to HY.

Appendix. Supplementary material Supplementary data associated with this article can be found in the online version at http://dx.doi.org/10.1016/j.ydbio.2014.01.018. References Agata, K., Inoue, T., 2012. Survey of the differences between regenerative and nonregenerative animals. Dev., Growth Differ. 54, 143–152. Agata, K., Saito, Y., Nakajima, E., 2007. Unifying principles of regeneration I: Epimorphosis versus morphallaxis. Dev., Growth Differ 49, 73–78.

66

S. Hayashi et al. / Developmental Biology 388 (2014) 57–67

Alarcon, C., Zaromytidou, A.I., Xi, Q., Gao, S., Yu, J., Fujisawa, S., Barlas, A., Miller, A. N., Manova-Todorova, K., Macias, M.J., Sapkota, G., Pan, D., Massague, J., 2009. Nuclear CDKs drive Smad transcriptional activation and turnover in BMP and TGF-beta pathways. Cell 139, 757–769. Bando, T., Mito, T., Maeda, Y., Nakamura, T., Ito, F., Watanabe, T., Ohuchi, H., Noji, S., 2009. Regulation of leg size and shape by the Dachsous/Fat signalling pathway during regeneration. Development 136, 2235–2245. Barry, E.R., Morikawa, T., Butler, B.L., Shrestha, K., de la Rosa, R., Yan, K.S., Fuchs, C.S., Magness, S.T., Smits, R., Ogino, S., Kuo, C.J., Camargo, F.D., 2013. Restriction of intestinal stem cell expansion and the regenerative response by YAP. Nature 493, 106–110. Beck, C.W., Christen, B., Barker, D., Slack, J.M., 2006. Temporal requirement for bone morphogenetic proteins in regeneration of the tail and limb of Xenopus tadpoles. Mech. Dev. 123, 674–688. Beck, C.W., Christen, B., Slack, J.M., 2003. Molecular pathways needed for regeneration of spinal cord and muscle in a vertebrate. Dev. Cell. 5, 429–439. Blanco, M.J., Misof, B.Y., Wagner, G.P., 1998. Heterochronic differences of Hoxa-11 expression in Xenopus fore- and hind limb development: evidence for lower limb identity of the anuran ankle bones. Dev. Genes Evol. 208, 175–187. Bonfanti, L., 2011. From hydra regeneration to human brain structural plasticity: a long trip through narrowing roads. Sci. World J. 11, 1270–1299. Brockes, J.P., 1997. Amphibian limb regeneration: rebuilding a complex structure. Science 276, 81–87. Cai, J., Zhang, N., Zheng, Y., de Wilde, R.F., Maitra, A., Pan, D., 2010. The Hippo signaling pathway restricts the oncogenic potential of an intestinal regeneration program. Genes Dev. 24, 2383–2388. Camargo, F.D., Gokhale, S., Johnnidis, J.B., Fu, D., Bell, G.W., Jaenisch, R., Brummelkamp, T.R., 2007. YAP1 increases organ size and expands undifferentiated progenitor cells. Curr. Biol.: CB 17, 2054–2060. Cameron, J.A., Fallon, J.F., 1997. Evidence for polarizing zone in the limb buds of Xenopus laevis. Dev. Biol. 55, 320–330. Cannata, S.M., Bagni, C., Bernardini, S., Christen, B., Filoni, S., 2001. Nerveindependence of limb regeneration in larval Xenopus laevis is correlated to the level of fgf-2 mRNA expression in limb tissues. Dev. Biol. 231, 436–446. Cebria, F., Kobayashi, C., Umesono, Y., Nakazawa, M., Mineta, K., Ikeo, K., Gojobori, T., Itoh, M., Taira, M., Sanchez Alvarado, A., Agata, K., 2002. FGFR-related gene nou-darake restricts brain tissues to the head region of planarians. Nature 419, 620–624. Christen, B., Slack, J.M., 1997. FGF-8 is associated with anteroposterior patterning and limb regeneration in Xenopus. Dev. Biol. 192, 455–466. Demircan, T., Berezikov, E., 2013. The hippo pathway regulates stem cells during homeostasis and regeneration of the flatworm macrostomum lignano. Stem Cells Dev. 22, 2174–2185. Dent, J.N., 1962. Limb regeneration in larvae and metamorphosing individuals of the South African clawed toad. J. Morphol. 110, 61–77. Dong, J., Feldmann, G., Huang, J., Wu, S., Zhang, N., Comerford, S.A., Gayyed, M.F., Anders, R.A., Maitra, A., Pan, D., 2007. Elucidation of a universal size-control mechanism in Drosophila and mammals. Cell 130, 1120–1133. Dupont, S., Morsut, L., Aragona, M., Enzo, E., Giulitti, S., Cordenonsi, M., Zanconato, F., Le Digabel, J., Forcato, M., Bicciato, S., Elvassore, N., Piccolo, S., 2011. Role of YAP/TAZ in mechanotransduction. Nature 474, 179–183. Endo, T., Tamura, K., Ide, H., 2000. Analysis of gene expressions during Xenopus forelimb regeneration. Dev. Biol. 220, 296–306. Endo, T., Yokoyama, H., Tamura, K., Ide, H., 1997. Shh expression in developing and regenerating limb buds of Xenopus laevis. Dev. Dyn.: Off. Publ. Am. Assoc. Anat. 209, 227–232. Fernandez, L.A., Northcott, P.A., Dalton, J., Fraga, C., Ellison, D., Angers, S., Taylor, M. D., Kenney, A.M., 2009. YAP1 is amplified and up-regulated in hedgehogassociated medulloblastomas and mediates Sonic hedgehog-driven neural precursor proliferation. Genes Dev. 23, 2729–2741. Filoni, S., Paglialunga, L., 1990. Effect of denervation on hindlimb regeneration in Xenopus laevis larvae. Differ.: Res. Biol. Diversity 43, 10–19. French, V., Bryant, P.J., Bryant, S.V., 1976. Pattern regulation in epimorphic fields. Science 193, 969–981. Gardiner, D.M., Endo, T., Bryant, S.V., 2002. The molecular basis of amphibian limb regeneration: integrating the old with the new. Semin. Cell Dev. Biol. 13, 345–352. Gee, S.T., Milgram, S.L., Kramer, K.L., Conlon, F.L., Moody, S.A., 2011. Yes-associated protein 65 (YAP) expands neural progenitors and regulates Pax3 expression in the neural plate border zone. PloS one 6, e20309. Gomez, A.R., Lopez-Varea, A., Molnar, C., de la Calle-Mustienes, E., Ruiz-Gomez, M., Gomez-Skarmeta, J.L., de Celis,, J.F, 2005. Conserved cross-interactions in Drosophila and Xenopus between Ras/MAPK signaling and the dual-specificity phosphatase MKP3. Dev. Dyn.: Off. Publ. Am. Assoc. Anat. 232, 695–708. Halder, G., Johnson, R.L., 2011. Hippo signaling: growth control and beyond. Development 138, 9–22. Han, M., Yang, X., Taylor, G., Burdsal, C.A., Anderson, R.A., Muneoka, K., 2005. Limb regeneration in higher vertebrates: developing a roadmap. Anat. Rec. Part B: N Anat. 287, 14–24. Heallen, T., Zhang, M., Wang, J., Bonilla-Claudio, M., Klysik, E., Johnson, R.L., Martin, J.F., 2011. Hippo pathway inhibits Wnt signaling to restrain cardiomyocyte proliferation and heart size. Science 332, 458–461. Hilman, D., Gat, U., 2011. The evolutionary history of YAP and the hippo/YAP pathway. Mol. Biol. Evol. 28, 2403–2417. Imajo, M., Miyatake, K., Iimura, A., Miyamoto, A., Nishida, E., 2012. A molecular mechanism that links Hippo signalling to the inhibition of Wnt/beta-catenin signalling. EMBO. J. 31, 1109–1122.

Imokawa, Y., Yoshizato, K., 1997. Expression of Sonic hedgehog gene in regenerating newt limb blastemas recapitulates that in developing limb buds. Proc. Natl. Acad. Sci. U.S.A. 94, 9159–9164. Kawakami, Y., Rodriguez-Leon, J., Koth, C.M., Buscher, D., Itoh, T., Raya, A., Ng, J.K., Esteban, C.R., Takahashi, S., Henrique, D., Schwarz, M.F., Asahara, H., Izpisua Belmonte, J.C., 2003. MKP3 mediates the cellular response to FGF8 signalling in the vertebrate limb. Nat. Cell Biol. 5, 513–519. Kawakami, Y., Rodriguez Esteban, C., Raya, M., Kawakami, H., Marti, M., Dubova, I., Izpisua Belmonte, J.C., 2006. Wnt/beta-catenin signaling regulates vertebrate limb regeneration. Genes Dev. 20, 3232–3237. Konsavage , W.M., Kyler, S.L., Rennoll, S.A., Jin, G., Yochum, G.S., 2012. Wnt/betacatenin signaling regulates Yes-associated protein (YAP) gene expression in colorectal carcinoma cells. J. Biol. Chem. 287, 11730–11739. Kumar, A., Brockes, J.P., 2012. Nerve dependence in tissue, organ, and appendage regeneration. Trends Neurosci. 35, 691–699. Lewandoski, M., Sun, X., Martin, G.R., 2000. Fgf8 signalling from the AER is essential for normal limb development. Nat. Genet. 26, 460–463. Mercader, N., Tanaka, E.M., Torres, M., 2005. Proximodistal identity during vertebrate limb regeneration is regulated by Meis homeodomain proteins. Development 132, 4131–4142. Moon, A.M., Capecchi, M.R., 2000. Fgf8 is required for outgrowth and patterning of the limbs. Nat. Genet. 26, 455–459. Morin-Kensicki, E.M., Boone, B.N., Howell, M., Stonebraker, J.R., Teed, J., Alb, J.G., Magnuson, T.R., O'Neal, W., Milgram, S.L., 2006. Defects in yolk sac vasculogenesis, chorioallantoic fusion, and embryonic axis elongation in mice with targeted disruption of Yap65. Mol. Cell. Biol. 26, 77–87. Muneoka, K., Holler-Dinsmore, G., Bryant, S.V., 1986. Intrinsic control of regenerative loss in Xenopus laevis limbs. J. Exp. Zool. 240, 47–54. Muneoka, K., Murad, E.H.B., 1987. Intercalation and the cellular origin of supernumerary limbs in Xenopus. Development 99, 521–526. Muneoka, K., Sassoon, D., 1992. Molecular aspects of regeneration in developing vertebrate limbs. Dev. Biol. 152, 37–49. Muramatsu, T., Imoto, I., Matsui, T., Kozaki, K., Haruki, S., Sudol, M., Shimada, Y., Tsuda, H., Kawano, T., Inazawa, J., 2011. YAP is a candidate oncogene for esophageal squamous cell carcinoma. Carcinogenesis 32, 389–398. Murawala, P., Tanaka, E.M., Currie, J.D., 2012. Regeneration: the ultimate example of wound healing. Semin. Cell Dev. Biol. 23, 954–962. Nakamura, T., Mito, T., Bando, T., Ohuchi, H., Noji, S., 2008. Dissecting insect leg regeneration through RNA interference. Cell. Mol. Life Sci.: CMLS 65, 64–72. Nieuwkoop P.D. and Faber J., Normal Table of Xenopus laevis (Daudin), Garland Publishing, New York, 1994. Nishioka, N., Inoue, K., Adachi, K., Kiyonari, H., Ota, M., Ralston, A., Yabuta, N., Hirahara, S., Stephenson, R.O., Ogonuki, N., Makita, R., Kurihara, H., Morin-Kensicki, E.M., Nojima, H., Rossant, J., Nakao, K., Niwa, H., Sasaki, H., 2009. The Hippo signaling pathway components Lats and Yap pattern Tead4 activity to distinguish mouse trophectoderm from inner cell mass. Dev. Cell 16, 398–410. Nojima, H., Rothhamel, S., Shimizu, T., Kim, C.H., Yonemura, S., Marlow, F.L., Hibi, M., 2010. Syntabulin, a motor protein linker, controls dorsal determination. Development 137, 923–933. Offield, M.F., Hirsch, N., Grainger, R.M., 2000. The development of Xenopus tropicalis transgenic lines and their use in studying lens developmental timing in living embryos. Development 127, 1789–1797. Ogino, H., McConnell, W.B., Grainger, R.M., 2006a. High-throughput transgenesis in Xenopus using I-SceI meganuclease. Nat. Protocols 1, 1703–1710. Ogino, H., McConnell, W.B., Grainger, R.M., 2006b. Highly efficient transgenesis in Xenopus tropicalis using I-SceI meganuclease. Mech. Dev. 123, 103–113. Ohgo, S., Itoh, A., Suzuki, M., Satoh, A., Yokoyama, H., Tamura, K., 2010. Analysis of hoxa11 and hoxa13 expression during patternless limb regeneration in Xenopus. Dev. Biol. 338, 148–157. Pan, F.C., Chen, Y., Loeber, J., Henningfeld, K., Pieler, T., 2006. I-SceI meganucleasemediated transgenesis in Xenopus. Dev. Dyn.: Off. Publ. Am. Assoc. Anat. 235, 247–252. Sanchez Alvarado, A., Tsonis, P.A., 2006. Bridging the regeneration gap: genetic insights from diverse animal models. Nat. Rev. Genet. 7, 873–884. Schlegelmilch, K., Mohseni, M., Kirak, O., Pruszak, J., Rodriguez, J.R., Zhou, D., Kreger, B.T., Vasioukhin, V., Avruch, J., Brummelkamp, T.R., Camargo, F.D., 2011. Yap1 acts downstream of alpha-catenin to control epidermal proliferation. Cell 144, 782–795. Schreiber, A.M., Das, B., Huang, H., Marsh-Armstrong, N., Brown, D.D., 2001. Diverse developmental programs of Xenopus laevis metamorphosis are inhibited by a dominant negative thyroid hormone receptor. Proc. Natl. Acad. Sci. U.S.A. 98, 10739–10744. Shaner, N.C., Campbell, R.E., Steinbach, P.A., Giepmans, B.N., Palmer, A.E., Tsien, R.Y., 2004. Improved monomeric red, orange and yellow fluorescent proteins derived from Discosoma sp. red fluorescent protein. Nat. Biotechnol. 22, 1567–1572. Shimizu-Nishikawa, K., Takahashi, J., Nishikawa, A., 2003. Intercalary and supernumerary regeneration in the limbs of the frog, Xenopus laevis. Dev. Dyn.: Off. Publ. Am. Assoc. Anat. 227, 563–572. Skouloudaki, K., Puetz, M., Simons, M., Courbard, J.R., Boehlke, C., Hartleben, B., Engel, C., Moeller, M.J., Englert, C., Bollig, F., Schafer, T., Ramachandran, H., Mlodzik, M., Huber, T.B., Kuehn, E.W., Kim, E., Kramer-Zucker, A., Walz, G., 2009. Scribble participates in Hippo signaling and is required for normal zebrafish pronephros development. Proc. Natl. Acad. Sci. U.S.A. 106, 8579–8584.

S. Hayashi et al. / Developmental Biology 388 (2014) 57–67

Stoick-Cooper, C.L., Moon, R.T., Weidinger, G., 2007. Advances in signaling in vertebrate regeneration as a prelude to regenerative medicine. Genes Dev. 21, 1292–1315. Straube, W.L., Tanaka, E.M., 2006. Reversibility of the differentiated state: regeneration in amphibians. Artif. Org. 30, 743–755. Torok, M.A., Gardiner, D.M., Izpisua-Belmonte, J.C., Bryant, S.V., 1999. Sonic hedgehog (shh) expression in developing and regenerating axolotl limbs. J. Exp. Zool. 284, 197–206. Tschumi, P.A., 1957. The growth of the hindlimb bud of Xenopus laevis and its dependence upon the epidermis. J. Anat. 91, 149–173. Van Hateren, N.J., Das, R.M., Hautbergue, G.M., Borycki, A.G., Placzek, M., Wilson, S. A., 2011. FatJ acts via the Hippo mediator Yap1 to restrict the size of neural progenitor cell pools. Development 138, 1893–1902. von Gise, A., Lin, Z., Schlegelmilch, K., Honor, L.B., Pan, G.M., Buck, J.N., Ma, Q., Ishiwata, T., Zhou, B., Camargo, F.D., Pu, W.T., 2012. YAP1, the nuclear target of Hippo signaling, stimulates heart growth through cardiomyocyte proliferation but not hypertrophy. Proc. Natl. Acad. Sci. U.S.A. 109, 2394–2399. Wada, K., Itoga, K., Okano, T., Yonemura, S., Sasaki, H., 2011. Hippo pathway regulation by cell morphology and stress fibers. Development 138, 3907–3914. Wheeler, G.N., Hamilton, F.S., Hoppler, S., 2000. Inducible gene expression in transgenic Xenopus embryos. Curr. Biol. 10, 849–852. Yakushiji, N., Suzuki, M., Satoh, A., Ide, H., Tamura, K., 2009a. Effects of activation of hedgehog signaling on patterning, growth, and differentiation in Xenopus froglet limb regeneration. Dev. Dyn.: Off. Publ. Am. Assoc. Anat. 238, 1887–1896.

67

Yakushiji, N., Suzuki, M., Satoh, A., Sagai, T., Shiroishi, T., Kobayashi, H., Sasaki, H., Ide, H., Tamura, K., 2007. Correlation between Shh expression and DNA methylation status of the limb-specific Shh enhancer region during limb regeneration in amphibians. Dev. Biol. 312, 171–182. Yakushiji, N., Yokoyama, H., Tamura, K., 2009b. Repatterning in amphibian limb regeneration: a model for study of genetic and epigenetic control of organ regeneration. Semin. Cell Dev. Biol. 20, 565–574. Yano, T., Abe, G., Yokoyama, H., Kawakami, K., Tamura, K., 2012. Mechanism of pectoral fin outgrowth in zebrafish development. Development 139, 2916–2925. Yazawa, S., Umesono, Y., Hayashi, T., Tarui, H., Agata, K., 2009. Planarian Hedgehog/ Patched establishes anterior-posterior polarity by regulating Wnt signaling. Proc. Natl. Acad. Sci. U.S.A. 106, 22329–22334. Yokoyama, H., Ogino, H., Stoick-Cooper, C.L., Grainger, R.M., Moon, R.T., 2007. Wnt/ beta-catenin signaling has an essential role in the initiation of limb regeneration. Dev. Biol. 306, 170–178. Zhang, L., Ren, F., Zhang, Q., Chen, Y., Wang, B., Jiang, J., 2008. The TEAD/TEF family of transcription factor Scalloped mediates Hippo signaling in organ size control. Dev. Cell. 14, 377–387. Zhao, B., Wei, X., Li, W., Udan, R.S., Yang, Q., Kim, J., Xie, J., Ikenoue, T., Yu, J., Li, L., Zheng, P., Ye, K., Chinnaiyan, A., Halder, G., Lai, Z.C., Guan, K.L., 2007. Inactivation of YAP oncoprotein by the Hippo pathway is involved in cell contact inhibition and tissue growth control. Genes Dev. 21, 2747–2761. Zhao, B., Ye, X., Yu, J., Li, L., Li, W., Li, S., Lin, J.D., Wang, C.Y., Chinnaiyan, A.M., Lai, Z. C., Guan, K.L., 2008. TEAD mediates YAP-dependent gene induction and growth control. Genes Dev. 22, 1962–1971.