Amotl2a interacts with the Hippo effector Yap1 and the Wnt/β-catenin effector Lef1 to control tissue size in zebrafish

During development, proliferation must be tightly controlled for organs to reach their appropriate size. While the Hippo signaling pathway plays a major role in organ growth control, how it senses and responds to increased cell density is still unclear. In this study, we use the zebrafish lateral line primordium (LLP), a group of migrating epithelial cells that form sensory organs, to understand how tissue growth is controlled during organ formation. Loss of the cell junction-associated Motin protein Amotl2a leads to overproliferation and bigger LLP, affecting the final pattern of sensory organs. Amotl2a function in the LLP is mediated together by the Hippo pathway effector Yap1 and the Wnt/β-catenin effector Lef1. Our results implicate for the first time the Hippo pathway in size regulation in the LL system. We further provide evidence that the Hippo/Motin interaction is essential to limit tissue size during development. DOI: http://dx.doi.org/10.7554/eLife.08201.001


Introduction
Control of cell number is a critical process during the development of an organism. While proliferation is essential for organs to reach a correct size, failure to tightly regulate proliferation can lead to organ overgrowth and tumor formation. Proliferation must therefore be tightly controlled and coordinated with other developmental processes so that organs reach their proper final size, but do not exceed it. How this is achieved, however, is still largely unknown.
In the past 15 years, the Hippo signaling pathway has been identified as a major regulator of organ size during development and homeostasis, by promoting cell death and differentiation and inhibiting proliferation. First identified in Drosophila (Pan, 2007), the Hippo signaling pathway is highly conserved in vertebrates (Halder and Johnson, 2010;Pan, 2010). When Hippo signaling is active, the Hippo pathway effectors YAP1 (Yes-associated protein 1) and TAZ (transcriptional co-activator with a PDZ domain), the vertebrate homologs of the Drosophila Yorkie, are phosphorylated by a cascade of kinases leading to their sequestration in the cytoplasm and/or their degradation. In contrast, when the Hippo signaling pathway is inactive, YAP/TAZ can translocate into the nucleus and mediate transcription of genes that promote proliferation and inhibit apoptosis Yu and Guan, 2013;Bossuyt et al., 2014).
Recent studies have shown that, in addition to changes in cell density, YAP/TAZ responds to changes in cell shape, tension forces, and substrate stiffness. This, however, seems largely independent of the canonical Hippo kinase cascade but depends on actin (Dupont et al., 2011;Wada et al., 2011;Halder et al., 2012;Aragona et al., 2013). The actin cytoskeleton indeed plays an important role in integrating and transmitting upstream signals to the Hippo pathway effectors YAP and TAZ (Gaspar and Tapon, 2014). Yet, how this is achieved is not well understood.
Several recent reports suggest that the Motin family of junction-associated proteins could play a central role here. AMOT, AMOTL1, and AMOTL2 are scaffold proteins associated with tightjunctions, required for tight junction integrity (Bratt et al., 2002;Sugihara-Mizuno et al., 2007;Zheng et al., 2009) and endothelial cell migration (Troyanovsky et al., 2001;Bratt et al., 2005;Aase et al., 2007;Hultin et al., 2014;Moleirinho et al., 2014). Recently, Motin proteins have further been shown to interact with YAP and TAZ via their PPxY motifs and the WW motifs of YAP and TAZ (Chan et al., 2011;Hirate et al., 2013;Hong, 2013;Lucci et al., 2013;Yi et al., 2013). In most cases, this physical interaction leads to the inhibition of YAP/TAZ via cytoplasmic retention, similar to, but independent of the canonical Hippo pathway. Interestingly, there is a competition between YAP/TAZ and F-actin to bind to Motin proteins (Mana-Capelli et al., 2014). Motin proteins have thus been proposed to mediate the response of YAP/TAZ to changes in the actin cytoskeleton downstream of mechanical signals. Yet, whether Motin proteins play such a central role in regulating organ growth in vivo in developing organisms is still largely unknown.
The posterior lateral line (pLL) system in zebrafish provides an excellent model system to address this question. The pLL is a sensory system comprised of mechanosensory organs, the neuromasts, eLife digest How do organs and tissues know when to stop growing? A cell communication pathway known as Hippo signaling plays a central role as it can tell cells to stop dividing. It is activated when cells in developing tissues come into contact with each other and causes a protein called Yap1 to be modified, which prevents it from entering the cell nucleus to activate genes that are involved in cell division.
In a zebrafish embryo, an organ called the lateral line forms from a cluster of cells that migrate along the embryo's length. At regular intervals, the cluster deposits small bunches of cells from its trailing end. The resulting loss of cells from the cluster is balanced by cell division at the front of the cluster, which is triggered by another signaling pathway called Wnt signaling. A protein of the 'Motin' family called Amotl2a is present in this migrating cluster. Motin proteins form junctions between cells and inhibit the activity of Yap1, but it is not known whether they are involved in regulating the size of organs.
Here, Agarwala et al. used the lateral line as a model to study the control of organ size in zebrafish embryos. The experiments show that when Amotl2a is absent, the migrating cell cluster becomes larger, with the highest levels of cell division occurring at its trailing end. Yap1 and a protein involved in Wnt signaling called Lef1 are also present in the cluster and are required for it to be normal in size. In zebrafish that lack Amotl2a, the additional loss of Yap1 prevents this cluster from becoming too large. From these and other results, it appears that Amotl2a regulates the size of the lateral line cell cluster by restricting the ability of Yap1 and Lef1 to promote cell division.
Agarwala et al.'s findings demonstrate a role for Amotl2a in controlling the size of organs. A future challenge is to understand the details of how it restricts the activities of Yap1 and Lef1.
scattered on the surface of the body. The pLL primordium (pLLP) consists of about 100 progenitors that delaminate from a cranial placode and migrate posteriorly towards the tip of the tail (Metcalfe et al., 1985;Ghysen and Dambly-Chaudiere, 2004). As the pLLP migrates, small groups of cells within its trailing region undergo cell shape changes to assemble into rosette-like structures, called proneuromasts. This assembly requires Fibroblast Growth Factor (FGF) signaling and the downstream effectors Shroom3 and Rock2a to activate non-muscle myosin and apical-constriction (Lecaudey et al., 2008;Nechiporuk and Raible, 2008;Ernst et al., 2012;Harding and Nechiporuk, 2012). Once assembled, proneuromasts are deposited behind the migrating pLLP and differentiate into functional neuromasts.
Proneuromast deposition comes with a significant loss of cells for the migrating pLLP. Wnt/ β-catenin signaling partially compensates for this loss by promoting proliferation in the leading region (Gamba et al., 2010;Aman et al., 2011;McGraw et al., 2011;Valdivia et al., 2011;Matsuda et al., 2013). Hereafter, we use the term 'leading region' for the cells in the posterior part of the pLLP that do not assemble into rosettes. In contrast, with 'trailing region', we refer to the part of the pLLP where cells change their shape to assemble into rosettes and where FGF signaling is active.
Here, we use the pLLP to address the mechanisms required to control proliferation and tissue size during organ development. We focus on the role of the Motin protein Angiomotin-like 2a (Amotl2a) that has been reported to be expressed in the migrating pLLP, to be a target of FGF signaling (Huang et al., 2007), to inhibit Wnt/β-catenin signaling during early zebrafish development (Li et al., 2012), and is a potential candidate to interact with the Hippo signaling pathway. Loss of Amotl2a function results in a significant increase in pLLP size due to overproliferation. In a yeast two-hybrid (Y2H) screen, we identified the zebrafish Hippo pathway effectors Yap1 and Taz as strong interacting partners of Amotl2a. We show that Yap1 is required for the pLLP to reach its correct size and that reducing the level of Yap1 suppresses the overproliferation in amotl2a mutant pLLP. Finally we show that, in addition to Yap1, the Wnt/β-catenin pathway effector Lef1 also mediates the hyperplasia phenotype of amotl2a mutants. This leads us to propose that Amotl2a, possibly by forming a ternary complex with Yap1 and β-catenin, limits proliferation in the trailing part of the pLLP. Here, we report the first mechanism that limits proliferation in the pLLP. In addition, we implicate the Hippo effector Yap1 in size control in the pLLP for the first time. Altogether, our results strongly suggest that Motin proteins play a major role in limiting organ growth during development by negatively regulating Yap/Taz and Wnt/β-catenin activities in vivo.

Results
amotl2a is expressed in the pLLP and localizes at cell-cell junctions amotl2a has been shown to be expressed in the pLLP (Huang et al., 2007). By combining in situ hybridization (ISH) with Green Fluorescent Protein (GFP) immunostaining in cldnb:gfp transgenic embryos, we showed that amotl2a was expressed throughout migration ( Figure 1A-C) in most of the pLLP with reduced or no expression in the most leading and trailing domains ( Figure 1D-I). The other motin genes amot, amotl1, and amotl2b were not expressed in the pLLP (Figure 1-figure supplement 1A-C). To determine the intracellular localization of Amotl2a in the pLLP, we fused Amotl2a to the red fluorescent protein TdTomato (TdT). Amotl2a-TdT was present in the cytoplasm and was strongly concentrated at the most apical, constricted part of the cells in assembling proneuromasts ( Figure 1J-K′). In addition, it appeared as an apical ring in deposited neuromasts ( Figure 1L,L′).
We next asked if amotl2a expression in the pLLP also required FGF signaling as it has been reported during early development (Huang et al., 2007). amotl2a expression was specifically lost in the pLLP of fgf3 −/− ;fgf10 −/− double mutants ( Figure  Amotl2a is not essential for tight junction and proneuromast assembly in the pLLP To investigate the function of Amotl2a in the pLLP, we used a previously published translationblocking morpholino (Amotl2aMo) (Huang et al., 2007;Li et al., 2012). At low doses, Amotl2aMo did not lead to any obvious morphological defects ( Since Motin proteins are necessary for tight junction integrity in several contexts (Bratt et al., 2002;Sugihara-Mizuno et al., 2007;Zheng et al., 2009), we performed ZO1 and phalloidin staining to label tight junctions and actin, respectively. While mature rosettes had similarly strong apical actin and ZO1 staining in morphant and control embryos (white arrows in Figure 2C-F), the distance between the tip of the pLLP and the most recent fully assembled rosette (yellow arrows), appeared increased in amotl2a morphants (double arrowhead in Figure 2A,B). Yet, neuromasts were deposited normally ( Figure 2G,H). This result suggested that rosette assembly was slightly delayed in amotl2a morphants but that Amotl2a was neither essential for tight junction assembly nor for neuromast formation and deposition.
In addition, the pLLP of amotl2a morphant embryos migrated 35% slower than controls ( Figure 2I-M). Yet, all primordia reached the tip of the tail ( Figure 2G,H). Expression of the two G-protein-coupled receptors cxcr4b and cxcr7b was not detectably affected in morphants ( Figure 2-figure supplement 2), suggesting that a deregulation of their expression is unlikely to account for the decrease in migration speed. Altogether, these results suggested that while rosette assembly and migration are delayed in amotl2a morphants, the pLLP deposits neuromasts and eventually migrates to the tip of the tail.
Amotl2a is essential to restrict the number of cells in the pLLP, and thus to limit its size Interestingly, the size of amotl2a morphant pLLP appeared obviously increased throughout the migration process ( Figure 3A-C, also Figure 2I,J). To determine whether this was due to an increase in cell number, we developed an algorithm to automatically count cells in the pLLP based on the  . Cell counts were done in pLLP that had reached the middle of the yolk extension in both controls and amotl2a morphants (thereafter referred to as 'mid-migration', red arrowhead in Figure 2-figure supplement 1, see also 'Materials and methods'). Cell counts indicated that the number of cells in amotl2a morphant pLLP was increased by 35-38% as compared to either uninjected or p53Mo-injected embryos ( Figure 3E, p = 1.58E-40 and p = 9.53E-31, respectively). To confirm the specificity of the morphant phenotype, we co-injected Amotl2aMo with amotl2a m , an RNA that was insensitive to the morpholino. Co-injection of Amotl2aMo with amotl2a m RNA partially rescued the morphant phenotype ( Figure 3D,F). While overexpression of amotl2a at the concentration used for the rescue experiment did not significantly reduce the number of cells in the pLLP ( Figure 3F), injection at a higher concentration induced a moderate, but significant decrease in cell number (Figure 3-figure supplement 2, −15%, p = 5.20E-07). Altogether, these results indicated that Amotl2a was essential to limit the number of cells in the migrating pLLP.
Amotl2a is required to limit the proliferation rate in the trailing region of the pLLP To determine the cause of the increase in cell counts in the pLLP of amotl2a morphants, we quantified proliferation rates using EdU to label proliferating cells ( Figure 3G-I′′). There was a significant increase in the EdU index of about 15% in the pLLP of amotl2a morphants as compared to controls ( Figure 3J, p < 0.05). Since amotl2a was not expressed in the most leading part of the pLLP, we determined the proliferation rates separately in the leading vs trailing region of the primordium. While there was no difference in the proliferation index in the leading region between morphants and controls, there was a 34% increase in the trailing region ( Figure 3K, p < 0.0001). Altogether, these results indicated that Amotl2a is required to restrict the number of cells in the pLLP by limiting proliferation in the region where rosettes assemble.

TALEN-induced non-sense mutations in amotl2a phenocopy the amotl2a knock-down phenotype
To confirm the specificity of the Amotl2Mo-induced phenotype, we generated amotl2a mutants using the transcription activator-like effector nuclease (TALEN) technique. Among several identified amotl2a alleles, two were further analyzed that introduced a frame-shift and a premature STOP codon in the third exon ( Figure 4A). The putative resulting truncated proteins would lack most of the coiled-coil domain and the PDZ-binding domain ( Figure 4B). Both alleles had identical phenotypes. amotl2a −/− homozygous mutants were morphologically indistinguishable from their siblings (Figure 4C-F) but had larger primordia ( Figure 4G,H). Cell count analyses revealed a significant increase in cell numbers of 21% in amotl2a −/− mutants as compared to their siblings ( Figure 4I). In addition and as in morphants, pLLP migrated slower   Homozygous mutants were viable, fertile, and generated maternal-zygotic (MZ) mutants that were also morphologically normal, viable, and fertile. Since amotl2a is expressed maternally (Huang et al., 2007), we wanted to determine whether maternal RNA and/or proteins also contributed to limit the number of cells in the pLLP. pLLP cell counts were further increased to 40% in MZamotl2a −/− mutants as compared to controls ( Figure 4J-L, p = 1.18E-21), an increase similar to that quantified in amotl2a morphants ( Figure 3E,F).
We then asked where these additional cells ended up later in development. Neither deposited neuromasts nor interneuromast chains comprised more cells at 48 hpf in morphants as compared to controls ( ). Yet, there was on average one additional neuromast deposited in morphants (not shown) and mutants ( Figure 4M-O) as compared to control embryos. Altogether, these results confirmed that Amotl2a is required to limit the number of cells in the pLLP and thus its size. Also, loss of Amotl2a does not affect the size of mature neuromasts but increases their number.

Zebrafish Amotl2a physically interacts with zebrafish Yap1 and Taz
To dissect the mechanisms by which Amotl2a was limiting the number of cells in the pLLP, we performed a Y2H screen to identify Amotl2a-interacting partners. The screen was performed with a full-length (FL) cDNA of amotl2a as bait and a cDNA library of 18-20 hpf zebrafish embryos as prey library (Hybrigenics, France). 25 proteins were identified as potential Amotl2a interaction-partners and were given a score ranging from A (very high confidence in the interaction) to D (moderate confidence in the interaction). The Hippo pathway effectors Yap1 and Taz were among the 10 strongest identified partners with an A and B score, respectively. Further analysis showed that in both cases, the amino acid (aa) sequence shared by all prey fragments (Selected Interaction Domain) included the evolutionarily conserved WW motifs (2 for Yap1 and 1 for Taz, Figure 5A).
To further test if the WW motifs were required for the physical interactions, we mutated both and tested the interactions with Amotl2a in a Y2H assay. The Y2H assay first confirmed the interaction between zebrafish Amotl2a and zebrafish Yap1 and Taz (red in Figure 5B). Furthermore, mutations of the WW motifs of Yap1 and Taz abolished the corresponding interaction with Amotl2a (pink in Figure 5B). Conversely, mutations of the LPTY and PPEY motifs of Amotl2a, but not of either of them alone, abolished the interaction with WT Yap1 and Taz (green and yellow, respectively, in Figure 5B). This result indicated that, similar to their human orthologs, zebrafish Yap1 and Taz physically interact with zebrafish Amotl2a and that this interaction is mediated by the WW motifs of Yap1/Taz and the LPTY/PPEY motifs of Amotl2a. We therefore hypothesized that the increase in proliferation upon loss of Amotl2a could result from an upregulation of Yap1/Taz activity.

Yap1 activity is required for the pLLP to reach its normal size
To test whether an upregulation of Yap1/Taz activity could account for the increased cell number in the pLLP upon loss of Amotl2a function, we first assessed the expression of zebrafish yap1 and taz. Both genes were broadly expressed during development including in the pLLP (Figure 5-figure supplement 1A,B). We then asked whether Yap1 or Taz was implicated in controlling pLLP cell number using morpholino-based knockdown. Injection of a previously published translation-blocking Mo against Taz (Hong et al., 2005) did not cause obvious differences in pLLP size (data not shown). In contrast, embryos injected with a previously published, splice-blocking Yap1 morpholino (Yap1Mo) (Skouloudaki et al., 2009;Fukui et al., 2014) were morphologically normal but had smaller pLLP than controls and often a rounder shape ( Figure 5C,D). Cell counts indicated that the pLLP of yap1 morphant embryos had about 15% less cells than uninjected controls (p = 2.65E-12) ( Figure 5E). Yet, they completed migration and deposited neuromasts. In order to confirm this essential role of Yap1 in controlling cell number in the pLLP, we generated yap1 mutants using TALEN-mediated mutagenesis. Like for amotl2a, we further analyzed two yap1 alleles with a frame shift leading to a premature STOP codon in the second exon ( Figure 5F). The putative resulting truncated proteins would lack most of their functional domains including the Transcriptional Enhancer Activator Domain (TEAD) binding domain, the 2 WW motifs and the PDZ-binding domains ( Figure 5G). Both alleles had identical phenotypes. yap1 −/− homozygous mutants embryos were morphologically normal ( Figure 5H,I) with smaller and at times rounder pLLP ( Figure 5J,K), a phenotype identical to that of the yap1 morphants. Cell counts confirmed that yap1 −/− pLLP also showed a decrease in cell number of 19% (p = 1.25E-05) ( Figure 5-figure supplement 1C). Interestingly, these embryos again developed into normal looking and fertile adult fish. MZyap1 −/− mutants also developed normally and were morphologically indistinguishable from controls. To determine at which stage Yap1 activity was required in the pLLP, we performed cell counts in MZyap1 −/− mutants when the pLLP starts to migrate (24 hpf), when it reaches the middle of the yolk extension (30 hpf) ( Figure 5L,M), and at the end of the yolk extension (36 hpf). At 24 hpf, the pLLP of MZyap1 −/− mutants was already 15% smaller than related controls (p = 4.12E-08). This difference continued to increase with time to reach 21% at 30 hpf (p = 2.15E-10) and 27% at 36 hpf (p = 2.41E-14) ( Figure 5N-P). These results showed that Yap1 is required both before and during migration to establish the correct number of cells in the pLLP.
A recent study reported that yap1 morphants show a reduced number of neuromasts (Loh et al., 2014). Given the reduced number of cells in the pLLP of yap1 morphants and mutants, we expected a similar result. Intriguingly, neither our morphants (not shown) nor our MZyap1 −/− mutants showed a decreased number of neuromasts ( Figure 5-figure supplement 1D). Furthermore, these neuromasts did not contain fewer cells ( Figure 5-figure supplement 1E). These results strongly suggested that compensatory mechanisms kick-in in the pLLP of yap1 −/− mutants to allow them to migrate to the tip of the tail while depositing a normal number of neuromasts consisting of a normal number of cells despite their smaller size. It was also reported that neuromasts of yap1 morphants contain fewer hair cells due to loss of prox1 expression in these embryos (Li et al., 2012). mutants. These discrepancies are discussed below. Altogether, our results indicated that Yap1 activity is required for the pLLP to reach its normal cell number and size. This further supported the idea that the increase in proliferation upon loss of Amotl2a activity could be due to an increase of Yap1 activity.

Amotl2a inhibits Yap1 activity to limit proliferation in the pLLP
We reasoned that if an increase of Yap1 activity was the cause of the hyperplasia in the pLLP of amotl2a morphants and mutants, we should be able to suppress this phenotype by reducing Yap1 levels. The pLLP size of embryos co-injected with Yap1Mo and Amotl2aMo was indeed smaller than that of amotl2a morphants alone and was comparable to that of control embryos ( Figure 6A-D). Automated cell counts revealed that the number of cells in the pLLP of amotl2a;yap1 double morphants was significantly reduced as compared to amotl2a morphants (34% reduction, p = 1.53E-16) and was not significantly different from controls (p = 0.098) ( Figure 6E). To confirm this result, we generated MZamotl2a −/− ;MZyap1 −/− double mutants. These mutants were morphologically indistinguishable from related controls. Here also, the increase in pLLP size in MZamotl2a −/− was suppressed in the MZ double mutant embryos ( Figure 6-figure supplement 1A-E). These observations were confirmed by automated cell counts: the 37% increase in MZamotl2a −/− pLLP (p = 4.62E-11) was suppressed in MZamotl2a −/− ;MZyap1 −/− double mutants and comparable to controls (p = 0.06). MZyap1 −/− mutants were, however, still lower than double mutants (−27%, p = 1.09E-11) ( Figure 6-figure supplement 1E). Together, these results demonstrated that the increased pLLP cell counts that resulted from the loss of Amotl2a function could be suppressed by an additional loss of Yap1 activity. This strongly suggested that the overproliferation in amotl2a −/− mutants pLLP was in part mediated by Yap1.
Therefore, we directly tested if the increased proliferation rate of amolt2a morphants was similarly suppressed in double morphants. As previously observed, the proliferation rate in amotl2a morphants was significantly increased in the trailing region as compared to controls (control vs Amotl2aMo: 0.27 ± 0.01 and 0.40 ± 0.01, p < 0.0001); and this increase was partially suppressed in embryos co-injected with Amotl2aMo and Yap1Mo (0.32 ± 0.02, p = 0.003) ( Figure 6F-K). Altogether, these results strongly suggest that Amotl2a is limiting proliferation and cell number in the pLLP, at least in part, by inhibiting Yap1 activity.
Finally, we wanted to test whether the physical interaction between Amotl2a and Yap1 was necessary for Amotl2a function. For this purpose, we attempted to rescue the amotl2a morphant phenotype with amotl2a m (wild-type amotl2a modified to be insensitive to the Mo) and with  Figure 6. continued on next page amotl2a mLP , which was mutated in the LPTY and PPEY motifs and thus could not interact with Yap1. While amotl2a m could partially rescue the increase in cell counts of amotl2a morphants as shown above (p = 6.36E-08), amotl2a mLP could not (p = 0.18) ( Figure 6L-P). We also checked that the stability of the two proteins were comparable. Since there was no antibody recognizing zebrafish Amotl2a, we took advantage of the myc-tagged versions of the proteins that we used in our Y2H assays. The two proteins had a comparable stability in yeast ( Figure 6-figure supplement 1F). Altogether, these results suggested that the physical interaction between Amotl2a and Yap1 is required for the proliferation-limiting activity of Amotl2a.

Amotl2a suppresses Lef1 activity to limit proliferation in the pLLP
Our results so far indicated that the loss of Yap1 suppressed the increase in cell number in amotl2a mutants, possibly via a physical interaction between Amotl2a and Yap1. Yet, pLLP cell counts in MZamotl2a −/− ;MZyap1 −/− double mutants were still higher than in MZyap1 −/− suggesting that additional factors mediated the hyperplasia in amotl2a mutant pLLPs. Given that Wnt/β-catenin signaling, via Lef1, promotes proliferation in the leading region of the pLLP (Gamba et al., 2010;Aman et al., 2011;McGraw et al., 2011;Valdivia et al., 2011;Matsuda et al., 2013) and that Amotl2a has been shown to physically interact with β-catenin and inhibit Wnt/β-catenin signaling during zebrafish gastrulation (Li et al., 2012), we tested if Wnt/β-catenin signaling could also mediate the increase in cell number upon loss of Amotl2a activity.
We first injected Amotl2aMo in tg(7xTCFXla.Siam:nlsmCherry) ia5 embryos that carry a Wnt reporter (Valdivia et al., 2011;Moro et al., 2012). Although the intensity of red fluorescence was not significantly increased upon Amotl2aMo injection (  (Figure 7-figure supplement 1K-N). This suggested that an increase in Wnt/β-catenin/Lef1 activity in the trailing region of the pLLP could also partially mediate the increase in cell number. We thus tested if suppressing Lef1 activity in amotl2a mutants would suppress this phenotype. For this purpose, we injected MZamotl2a −/− mutant embryos with a previously published lef1 morpholino (Lef1Mo) and performed cell counts at midmigration (Valdivia et al., 2011). While MZamotl2a −/− mutants showed bigger primordia as expected (36%, p = 2.89E-09) ( Figure 7G, see 'a' in N), lef1 morphants at this stage did not show a significant reduction in pLLP cell counts (p = 0.1) (Figure 7F,J and 'b' in N). In contrast, cell counts in Lef1Mo-injected MZamotl2a −/− mutants were significantly reduced as compared to MZamotl2a −/− mutants (−23%, p = 5.32E-08, Figure 7N 'c') and equivalent to controls (p = 0.28, Figure 7N 'd'). Yet, they were still slightly higher than lef1 morphants (15%, p = 0.01) ( Figure 7G,K and 'e' in N). These results suggested that the increased cell number in amotl2a −/− mutants was Lef1-dependent. To confirm this, we tested if the difference in cell number in MZamotl2a −/− ;MZyap1 −/− and MZyap1 −/− (see above) was due to Lef1 activity. Indeed when we injected MZamotl2a −/− ;MZyap1 −/− with Lef1Mo, the number of cells in the pLLP was significantly lower than in MZamotl2a −/− ;MZyap1 −/− double mutants (−19%, p = 0.001, Figure 7N 'f') and was not significantly different from that of MZyap1 −/− mutants ('g' in Figure 7N, p = 0.07). Altogether, these results strongly suggested that Amotl2a limits the number of cells and thus the size of the pLLP by repressing both Yap1 and Lef1 proliferation-promoting activities (Figure 8), possibly by physically interacting with Yap1 (our data) and β-catenin, as previously shown in zebrafish (Li et al., 2012).

Discussion
In the present study, we describe a novel mechanism underlying control of tissue size in developing vertebrate embryos. We show that the cell junction-associated Motin protein Amotl2a is required to limit proliferation in the pLLP and thus to control the size of this tissue during migration. We further show that hyperplastic pLLP do not generate bigger neuromasts but more neuromasts. Our results indicate that Amotl2a exerts its function by negatively regulating both the Hippo pathway effector Yap1 and the Wnt/β-catenin effector Lef1. Our data point to a new mechanism by which Motin proteins limit tissue size by repressing the Wnt/β-catenin pathway. In addition, while Motin proteins are known to physically interact with, and inhibit YAP/TAZ, this is to our knowledge the first in vivo report showing that Motins control tissue size by interacting with the Hippo pathway effector Yap1 in a living organism.

Control of proliferation and tissue size in the zebrafish pLLP
Embryonic development is associated with high proliferation rates ensuring proper organ formation. Yet, proliferation must remain under tight spatiotemporal control to ensure that a sufficient number of cells is produced, but not exceeded. In the pLLP, Wnt/βcatenin signaling is required to maintain proliferating progenitors in the leading region to compensate for the loss of cells resulting from neuromast deposition (Gamba et al., 2010;Aman et al., 2011). This is essential for the pLLP to deposit a complete set of LL organs (McGraw et al., 2011;Valdivia et al., 2011). However, no mechanism has been proposed that restricts proliferation and growth in the pLLP. We show here that Amotl2a is essential to limit proliferation of the cells that assemble into proneuromasts in the trailing region of the pLLP. This leads us to propose that the size of the pLLP is controlled throughout migration by a balance between Wnt/βcatenin-dependent mitogenic signals from the leading region and Amotl2a-dependent proliferation-restricting signals from the trailing region (Figure 8).
We further show that amotl2a is expressed downstream of FGF signaling in the trailing region of the pLLP. Yet, embryos lacking FGF signaling in the pLLP do not have larger primordia (Aman et al., 2011, and our own observations). This suggests that FGF has a mitogenic function in the pLLP in addition to promoting amotl2a expression, and thus, limiting proliferation. This is not surprising given that FGF signaling promotes proliferation in several contexts during vertebrate development  (Thisse and Thisse, 2005;Aman et al., 2011). We propose that in FGF-deficient embryos, an increase in Yap1 and Lef1 activity resulting from decreased amotl2a expression might partially compensate for the absence of FGF-dependent proliferation.
The increase in cell number in the pLLP of amotl2a mutants does not lead to bigger neuromasts, but to one additional neuromast. While a recent study reported that the size of differentiated neuromasts depends on the canonical Wnt pathway at later stages (Wada et al., 2013), the mechanisms that dictate how many cells participate in forming a new rosette in the migrating pLLP are not known. Intriguingly, our results suggest that this number does not depend on the size of the pLLP itself. This is in agreement with the fact that smaller primordia, for example in lef1 mutants, also deposit fewer, but apparently not smaller, neuromasts (McGraw et al., 2011;Valdivia et al., 2011).
Finally, we show that rosette assembly is slightly delayed in amotl2a morphants and mutants. At least two mechanisms could account for this delay. First, Motin proteins have been implicated in the formation of tight junctions in different contexts (Bratt et al., 2002;Sugihara-Mizuno et al., 2007;Zheng et al., 2009), although tight junctions do form in the absence of Amotl2a, their assembly might be challenged, leading to a delay in epithelial rosette assembly. Second, we show that lef1 expression is expanded upon loss of Amotl2a function. Interestingly, upon loss of Lef1 activity, rosettes assemble closer to the pLLP tip (McGraw et al., 2011;Matsuda et al., 2013). This has been attributed to the loss of the FGF pathway inhibitor Dusp6 downstream of Lef1 leading to a premature incorporation of leading cells into epithelial rosettes (McGraw et al., 2011;Matsuda et al., 2013). The expansion of lef1 expression into the trailing region upon loss of Amotl2a function could conversely explain the delay in rosette assembly. That this delay does not result in an increased distance between deposited neuromasts could be explained by the fact that one additional neuromast form in this context due to the increase in cell number.

Yap1 mutant pLL primordia are smaller
We show for the first time that the Hippo signaling pathway effector Yap1 is required for the pLLP to contain a sufficient number of cells. Since Yap1 is already required before migration starts, we propose that one of Amotl2a's functions is to maintain low Yap1 activity as rosettes assemble in the trailing region. In amotl2a mutants, Yap1 activity would fail to be repressed and would remain higher for a longer period of time leading to overproliferation. Our data do not allow us to determine, however, whether the canonical Hippo pathway is active in the pLLP to additionally regulate Yap1 activity. More work will be required to answer this interesting question.
A paper recently reported that morpholino-mediated knock-down of Yap1 in zebrafish leads to a reduction of the number of neuromasts but not of the size of the pLLP (Loh et al., 2014). They also reported a failure of hair cell differentiation that they attributed to a reduction of expression of the Wnt-target gene prox1. Intriguingly, both our yap1 morphants and mutants showed significantly different phenotypes with a reduction of the size of the pLLP but not of the number of neuromasts deposited. In addition, neither prox1 expression nor the numbers of hair cells were affected in yap1 −/− mutants. One possible explanation for these discrepancies is that our yap1 mutation does not affect all yap1 transcripts. This is, however, unlikely since it lies in the TEAD-binding domain, which is probably essential to Yap1 function. On the other hand, the authors also reported increased apoptosis and activation of the p53 pathway, two processes that have been shown to lead to a reduction in the number of neuromasts (Aman et al., 2011). This could explain the discrepancies of phenotypes between the previously reported yap1 morphants and our newly generated yap1 −/− mutants.
How is amotl2a inhibiting Yap1 activity in the pLLP?
Our findings that Amotl2a physically and genetically interacts with the Hippo pathway component Yap1 to control proliferation in the pLLP is in agreement with several studies showing that Motin proteins physically interact with YAP and TAZ, sequester them in the cytoplasm, and inhibit their proliferation-promoting transcriptional activity Chan et al., 2011;. Our results provide the first evidence that a similar mechanism could be involved in vivo during vertebrate development to control tissue and organ size. Antibodies recognizing Yap1 will be necessary to determine its localization in the pLLP cells. The commercial Yap1 antibodies we have tested did not cross-react with the zebrafish protein. Intriguingly, none of the known YAP/TAZ target genes we looked at, including ctgfs, cyr61, myc, cyclins, and prox1, showed differences in expression level in amotl2a morphants (data not shown).
Alternatively, rather than sequestering Yap1 into the cytoplasm, Amotl2a could promote Yap1 degradation. Motins have indeed been shown to promote the phosphorylation of YAP/TAZ by LATS1/ 2, which would not only lead to their cytoplasmic retention but also to their degradation (Paramasivam et al., 2011). Although there is currently no evidence that the canonical Hippo pathway is active in the pLLP, Amotl2a could be involved in controlling Yap1 stability.
What is the link between amotl2a, Yap1, and Lef1 in the pLLP?
Our results indicate that Amotl2a limits the number of cells and thus the size of the migrating pLLP by repressing the activity of both Lef1 and Yap1. This is in agreement with the fact that both Lef1 (Gamba et al., 2010;McGraw et al., 2011;Valdivia et al., 2011;Matsuda et al., 2013) and Yap1 (this paper) are required for establishing and maintaining the correct number of cells in the pLLP.
To our knowledge, a direct link between Motin proteins and the Wnt/β-catenin pathway has been described only once, with a physical interaction between zebrafish Amotl2a and β-catenin (Li et al., 2012). Although we did not detect β-catenin in our Y2H screen nor identified an interaction between Amotl2a and β-catenin in our Y2H assay (not shown), Amotl2a might be physically interacting with β-catenin in vivo in the pLLP cells thereby limiting its access to the nucleus.
Our data suggest that Yap1 and Lef1 function at least in part independently downstream of Amotl2a since the pLLP of both yap1 single mutants and lef1 morphants has less cells than amotl2a; yap1 double mutants and Lef1Mo-injected amotl2a mutants, respectively. Yet, it is also possible that both pathways are partially interconnected downstream of Amotl2a. Several recent studies have indeed reported cross-regulations between Yap/Taz and Wnt/β-catenin signaling Fish et al., 2011;Heallen et al., 2011;Azzolin et al., 2012;Imajo et al., 2012;Azzolin et al., 2014;Piccolo et al., 2014). In particular, Yap1 and β-catenin have been shown to physically interact and mutually repress each other in the cytoplasm while rather promoting each other's activity in the nucleus. Further work will be required to precisely dissect potential cross-talks between Yap1 and Wnt/β-catenin in the pLLP.

Is Amotl2a linking proliferation to morphogenesis?
One particularly interesting property of YAP/TAZ is their capacity to sense and respond to changes in biophysical properties of cells including cell density, cell polarity, cell shape, tension forces, and substrate stiffness (Dupont et al., 2011;Halder et al., 2012;Aragona et al., 2013;Gaspar and Tapon, 2014;Rauskolb et al., 2014). Our results raise the question whether Yap1 responds to such changes in the pLLP, and to which extent the response depends on Amotl2a function. We show that the repressive function of Amotl2a on Yap1 coincides with the assembly of cells into rosettes. Interestingly, these cells undergo a number of changes including epithelialization, acquisition of a columnar shape, and apical constriction via acto-myosin contraction (Lecaudey et al., 2008;Ernst et al., 2012). In principle, Yap1 could respond to any of these changes and Amotl2a could act as a mediator. First, tight junction-associated Amotl2a in newly formed rosettes could recruit and inhibit Yap1. In addition, Motins, including Amotl2a (Hultin et al., 2014), can bind actin and this interaction has been shown to compete with the binding of Motin to YAP (Ernkvist et al., 2006;Chan et al., 2013;Dai et al., 2013;Gaspar and Tapon, 2014). It is thus tempting to propose that Amotl2a could link actomyosin-based cell shape changes such as columnarization and/or apical constriction during rosette assembly to Yap1 activity and proliferation.
Finally, at low density in culture, cells are flat and spread and this morphology promotes nuclear YAP accumulation (Wada et al., 2011). In contrast at high density, cells are compact and tall and this is associated with cytoplasmic localization of YAP. In the pLLP, cells in the leading region are rather flat while cells in the trailing region, which assemble into rosettes, are more columnar (Lecaudey et al., 2008). Thus, in the primordium, more than the cell density, the assembly of cells into rosettes and the associated changes in cell shape could regulate the cytoplasmic localization of Yap1 in an Amotl2a-dependent manner. Such a mechanism would allow to couple proliferation rate to organogenesis. Upon loss of Amotl2a, in contrast, this coupling would be lost so that proneuromasts assemble but proliferation is not restricted, leading to hyperplastic LLP and additional sensory organs.
Cloning of FL cDNA and mutant form of amotl2a, yap1, and taz FL amotl2a, amotl2aΔSTOP, MoBS_Amotl2a, yap1, and taz were amplified by PCR from zebrafish embryo cDNA using the primer pairs indicated in the Supplementary file 1B. PCR products were further cloned into pCS2, pCS2-gfp, pCS2-TdT, pGADT7, or pGBKT7 vectors as indicated in the Supplementary file 1C.

TALEN-mediated mutagenesis and screening
The mutant lines amotl2a fu45 , amotl2a fu46 , yap1 fu47 , and yap1 fu48 were generated using TALEN. The TALE repeat array plasmid kit was a gift from Daniel Voytas and Adam Bogdanove obtained via Addgene (kit #1000000024). TALE repeat arrays were assembled following the Golden Gate TALEN assembly protocol originally described in Cermak et al. (2011), modified by the Voytas lab and available on the Addgene website (https://www.addgene.org/static/cms/filer_public/98/5a/985a6117-7490-4001-8f6a-24b2cf7b005b/golden_gate_talen_assembly_v7.pdf). Target sites and the corresponding repeat-variable diresidue (RVD) sequences were chosen using the online tool MoJo Hand (http:// talendesign.org/) in exon 2 and 3 of yap1 and amotl2a genes, respectively. The array plasmids were fused to the Fok1 endonuclease in the GoldyTALEN backbone. After linearization, mRNAs were transcribed using the T3 mMessage mMachine Kit (Ambion by Life Technologies GmbH, Darmstadt, Germany) according to the manufacturer's instructions. The two mRNAs corresponding to the left and right arms were then mixed in equal quantities and injected into embryos at the one-cell stage. 20-30 embryos at 48 hpf were collected from each clutch and gDNA was extracted. The target sites were amplified using primers generating a 350-600 bp long PCR product (Supplementary file 1E). Efficiency of the TALEN pair was estimated by digesting the PCR product with the restriction site present in the spacer of the target site. If a significant amount of uncut PCR product was observed, the rest of the injected embryos were further grown to adulthood. The resulting mosaic adult fish were out-crossed and genomic DNA was prepared from 50 embryos to identify potential mutations using the same PCR and digestion as described above. F1 fish were finally genotyped by fin clipping. The uncut band (carrying the mutation) was further amplified and sent to sequence.
To obtain MZ mutants, heterozygous carriers were incrossed and the progeny were raised to adulthood. Homozygous wild-type and mutants were identified by fin-clipping. In all experiments with MZ mutants, control embryos originate from incrosses of these homozygous wild-type and are thus related to the MZ mutants in a way that they share the same 'grand-parents'.
EdU (5-ethynyl-2′-deoxyuridine) treatment 28-30 hpf embryos were dechorionated, incubated with 10 mM EdU solution (Click-iT EdU, Life Technologies GmbH) for 20 min on ice. After washing, embryos were incubated at 28.5˚C for 1 hr to allow EdU incorporation and treated with the Click-iT reaction solution following manufacturer's instructions.

Y2H screen and assay
The Y2H screen was performed by Hybrigenics (Paris, France) with the FL coding sequence of zebrafish amotl2a (aa 1-721). 80 millions interactions were analyzed. The Y2H assay was performed using the Matchmaker system and the AH109 yeast strain (Clontech) according to the manufacturer's instructions. amotl2a was fused to the Gal4 DNA-binding domain in the pGBKT7 vector. Zebrafish yap1, yap1ΔWW1,2 (WQDP and WLDP motifs mutated to AQDA and ALDA), taz and tazΔWW (WHDP motif mutated to AHDA) were fused to the Gal4 activation domain in the pGADT7 vector. All constructs fused to the DNA-binding domain were tested for autoactivation. To check for protein interactions, colonies were scraped off the plates, diluted to an OD 600 of 0.4, patched on selective plates lacking histidine, and grown for 3-4 days.

Yeast cell lysates and Western blot
Yeast cell lysates were prepared according to the protocol from Kushnirov with some modifications (Kushnirov, 2000). Yeast cultures were grown to a logarithmic stage to reach an OD 600 0.4-0.8. Cultures of 2 ml were harvested by centrifugation. After discarding the supernatant, the pellets were resuspended in 500 μl of 0.1 M NaOH, transferred to 2 ml eppendorf tubes, and incubated at room temperature for 10 min followed by centrifugation at 14,000 rpm for 2 min at room temperature. After carefully discarding the supernatant, the pellet was resuspended in 1× sample buffer slightly modified from standard Laemmli (25 mM Tris pH6.8, 30% glycerol, 5% Sodium Dodecyl Sulfate (SDS), 1% bromophenol blue, and 5% β-mercaptoethanol) and dissolved by full speed shaking on a Thermoblock set at 37˚C for 20-30 min. The samples were then boiled at 95˚C for 5 min. The cell debris was removed by centrifugation at 14,000 rpm for 5 min at room temperature. The supernatant was transferred to a fresh eppendorf tube and stored at −20˚C until loading.

Live imaging, image analysis, and statistical analysis
Imaging and image analysis were performed as previously described (Ernst et al., 2012).
Quantifications of data are presented as boxplot in which the central line is the median of the group and the edges of the box are the 25th and 75th percentiles. The black vertical lines extend to the most extreme data points not considered outliers. In all cell count boxplots, each blue dot corresponds to one data point (one primordium). Statistical analyses were performed with GraphPad Prism6 and Matlab using the Welch's t-test. p values are indicated on each figure and/or in the result part. Differences in cell counts between groups are differences between mean values (and not between median values shown in the boxplots) and are expressed in percentage.
Image enhancement, cell segmentation, and automated cell counting Automated cell counting was done using the GFP channel of cldnb:gfp transgenic embryos (Figure 3-figure supplement 1A). To exclude that the delayed migration in amotl2a morphants would impact on the pLLP cell number, cell counts were done when the primordium had reached the middle of the yolk extension ('mid-migration', Figure 2-figure supplement 1A,C) both in control embryos (about 30 hpf) and in amotl2a morphants and mutants.
Because the cell membrane is a plane-like structure in three dimensions, we used anisotropic diffusion (Weickert, 1998) to enhance local consistency in structure and suppress noise (Figure 3-figure supplement 1B). The diffusion tensor, which enhances plane-like structures, was constructed from the local Hessian matrix. We then computed the smallest eigenvalue of the local Hessian matrices, which is a good indicator of the cell membrane (Sato et al., 2000). The cell segmentation was done by watershed segmentation with h-minima transform (Soille, 2003) on the eigenvalue image. Finally, very small segments were filtered out: their regions are merged into neighboring segments by applying a second watershed segmentation (Figure 3-figure supplement 1C). A number of publicly available tools, for example, 'Icy' (de Chaumont et al., 2012) or Matlab programs, can be used for these image-processing steps.
To generate a segmentation mask of the pLLP, the 3D images were smoothed with a small Gaussian kernel. An optimal threshold was estimated for converting the smoothed intensity image to a binary image (e.g., using the 'graythresh' function in Matlab), so that the intraclass variance of the two regions '0' and '1' is minimized. Based on this pLLP segmentation mask (Figure 3-figure  supplement 1D), we obtained the cell numbers by counting each segment that had more than 75% of its volume inside the mask. Visual monitoring of two experiments validated the accuracy of the segmentation (Figure 3-figure supplement 1).