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The ZO-1 protein Polychaetoid as an upstream regulator of the Hippo pathway in Drosophila

['Qingliang Sang', 'Integrative Biomedical', 'Diagnostic Sciences Department', 'School Of Dentistry', 'Oregon Health', 'Science University', 'Portland', 'Oregon', 'United States Of America', 'Gang Wang']

Date: 2022-01 

Abstract The generation of a diversity of photoreceptor (PR) subtypes with different spectral sensitivities is essential for color vision in animals. In the Drosophila eye, the Hippo pathway has been implicated in blue- and green-sensitive PR subtype fate specification. Specifically, Hippo pathway activation promotes green-sensitive PR fate at the expense of blue-sensitive PRs. Here, using a sensitized triple heterozygote-based genetic screening approach, we report the identification of the single Drosophila zonula occludens-1 (ZO-1) protein Polychaetoid (Pyd) as a new regulator of the Hippo pathway during the blue- and green-sensitive PR subtype binary fate choice. We demonstrate that Pyd acts upstream of the core components and the upstream regulator Pez in the Hippo pathway. Furthermore, We found that Pyd represses the activity of Su(dx), a E3 ligase that negatively regulates Pez and can physically interact with Pyd, during PR subtype fate specification. Together, our results identify a new mechanism underlying the Hippo signaling pathway in post-mitotic neuronal fate specification.

Author summary The Hippo signaling pathway was originally discovered for its critical role in tissue growth and organ size control. Its evolutionarily conserved roles in various biological processes, including cell differentiation, stem cell regeneration and homeostasis, innate immune biology, as well as tumorigenesis, have been subsequently found in other species. During the development of the Drosophila eye, the Hippo pathway promotes green- and represses blue-sensitive photoreceptor (PR) subtype fate specification. Taking advantage of this binary PR fate choice, we screened Drosophila chromosomal deficiency lines to seek new regulators of the Hippo signaling pathway. We identified the Drosophila membrane-associated ZO-1 protein Pyd as an upstream regulator of the Hippo pathway to specify PR subtypes. Our results have demonstrated that Pyd represses Su(dx)’s activity in the Hippo pathway to specify PR subtypes. Our results demonstrate a new mechanism underlying the Hippo signaling pathway in post-mitotic neuronal fate specification.

Citation: Sang Q, Wang G, Morton DB, Wu H, Xie B (2021) The ZO-1 protein Polychaetoid as an upstream regulator of the Hippo pathway in Drosophila. PLoS Genet 17(11): e1009894. https://doi.org/10.1371/journal.pgen.1009894 Editor: Claude Desplan, New York University, UNITED STATES Received: March 25, 2021; Accepted: October 19, 2021; Published: November 8, 2021 Copyright: © 2021 Sang et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability: All relevant data are within the paper and its Supporting Information files. Funding: This study was supported by the Whitehall Foundation (#2015-08-72 to BX), the Quanzhou Normal University Outstanding Young Teacher International Program (to QS), the Shandong Outstanding Young Teacher International Training Program (to GW), the National Institutes of Health through grants R01-DE022350, R01-DE 028329 and R01-DE017954 (to HW), a Medical Research Foundation of Oregon award (to DBM) and a Presidential Bridge Fund award (to DBM). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Competing interests: The authors have declared that no competing interests exist.

Introduction Generating neuronal diversity during the development of a sensory organ is a prerequisite for the organ to perceive and discriminate various external stimuli. For example, the perception of color relies on comparing the outputs of multiple light-sensing photoreceptor (PR) subtypes with different spectral sensitivities [1–3]. During development, the fate of sensory neurons is progressively restricted toward terminal differentiation, finally generating diverse neuronal subtypes. Although the role of transcriptional regulations during neuronal terminal differentiation has been extensively studied [4,5], the details of how specific signaling pathways influence this process are not well understood. Here we use the blue- and green-sensitive PR binary fate decisions in the Drosophila eye as a model to understand the role of the Hippo pathway in post-mitotic neuronal terminal differentiation. The Drosophila eye is a powerful model to understand the principles of neuronal development [1,6–8]. The Drosophila compound eye contains ~750 individual units, ommatidia, each of which consists of eight PRs: the outer PRs R1-R6 and the inner PRs R7-R8 (Fig 1A). There are two main subtypes of ommatidia: pale (p) and yellow (y) ommatidia, present in the adult Drosophila eye (Fig 1B). The outer photoreceptors R1-R6 in both p and y ommatidia express the broad spectrum light sensitive opsin Rhodopsin 1 (Rh1) and are responsible for dim light vision and motion detection. However, the inner R7 and R8 cells express Rhodopsins with different spectral sensitivities, making them capable of performing color vision [9]. In p ommatidia, R7s express UV-sensitive Rh3 and R8s express blue-sensitive Rh5, while in y ommatidia, R7s express UV sensitive Rh4 and R8s express green-sensitive Rh6 (Fig 1B). The p and y subtypes are randomly distributed throughout the retina in roughly a 35:65 p:y ratio [10]. The p vs. y fate decision is first made in R7s via the stochastic activation of the transcription factor Spineless in yR7s during mid-pupation [11]. R7s that do not express Spineless (i.e. the pR7s) instruct their underlying R8s to adopt pale R8 (pR8) fate through Activin and BMP signaling [12]. R8s that do not receive the pR7 signals default to yellow R8 (yR8) fate [13,14]. The effectors involved in p vs. y R8 fate in R8s involve two proteins—Melted (Melt) and Warts (Wts) [15] (Fig 1C). melt encodes a pleckstrin homology domain-containing protein [16], while wts encodes a serine/threonine kinase that is a core component in the Hippo signaling pathway [17–19]. melt expression is activated in pR8s by the pR7-driven Activin and BMP signals and its expression leads to the transcriptional repression of wts. Conversely, wts represses melt expression in yR8s by suppressing the activity of the transcriptional coactivator Yorkie (Yki), the downstream effector of the Hippo pathway. Yki is necessary for melt expression in pR8s. Therefore, wts, melt and Yki form a double negative regulatory loop to ensure pR8 vs. yR8 subtype fate decision (Fig 1C) [10]. Yki, together with its DNA-binding partner Scalloped (Sd), regulates the output of the regulatory loop to promote the expression of blue-sensitive Rh5 and prevent the expression of green-sensitive Rh6 [20]. PPT PowerPoint slide

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TIFF original image Download: Fig 1. Tripe heterozygote-based phenotype enhancement assays for R8 subtypes. (A) Schematic of an adult ommatidium with six outer photoreceptors (PRs) (R1-R6) and two inner PRs (R7 and R8). (B) Two main, pale (p) and yellow (y), ommatidial subtypes and corresponding Rhodopsin (Rh) expression patterns. (C) The Hippo pathway and melt specify yR8 (Wts+, Rh6+) vs. pR8 (Melt+, Rh5+) fates. pR7s send instructive pR7-to-pR8 signals (arrow from pink pR7) to activate melt in yR8s, and Melt, together with Yki, represses wts expression. In yR8s, Hippo signaling suppresses Yki activity and represses melt expression. Genes or proteins that are inactive or not expressed are denoted by grey font, while those that are expressed and/or active are represented by blue, green or black font. (D-G) Adult eye cryosections stained for Rh5 (red) and Rh6 (green) from control (D), wtsZn and kib1 heterozygous (E), wtsZn and mer3 heterozygous (F), or wtsZn, kib1 and mer3 triple heterozygous (G) flies. (H) Quantification of Rh5- and Rh6-positive R8s in control flies as well as flies with Hippo pathway heterozygous, double heterozygous or triple heterozygous mutations. Graph presents proportion of R8s (y axis) that express Rh5 (red) or Rh6 (green). Two-tailed, unpaired t test. NS: not significant. **P < 0.001. Error bars represent standard deviation. control: n = 8 retinas, n = 1986 R8s; wtsZn/+: n = 8 retinas, n = 2036 R8s; kib1/+: n = 8 retinas, n = 1796 R8s; savdf/+: n = 7 retinas, n = 1709 R8s; mer3/+: n = 6 retinas, n = 1488 R8s; wtsZn-kib1/+: n = 8 retinas, n = 1867 R8s; wtsZn/savdf: n = 8 retinas, n = 2001 R8s; kib1/savdf: n = 8 retinas, n = 1681 R8s; wtsZn/mer3: n = 6 retinas, n = 1421 R8s; mer3/kib1: n = 6 retinas, n = 1349 R8s; wtsZn-kib1/mer3: n = 6 retinas, n = 1421 R8s; wtsZn-kib1/savdf: n = 6 retinas, n = 1356 R8s. Refer to Supporting Information S1 Text for detail genotypes. Also see S1 Fig. https://doi.org/10.1371/journal.pgen.1009894.g001 The Hippo pathway was originally discovered in Drosophila for its pivotal roles in tissue growth and organ size control [21]. Its critical and conserved roles in mammals have been subsequently identified in a wide range of biological processes, including stem cell regeneration and homeostasis, innate immune biology, cell differentiation, as well as tumorigenesis [19,22–24]. The components of the Hippo pathway can be classified into three categories: the core kinase complex, the downstream effectors and the upstream regulators. The core kinase complex contains the kinases Wts [17,18], Hippo (Hpo) [25–29], and Mob as tumor suppressor (Mats) [30], as well as the scaffold protein Salvador (Sav) [31,32]. Hpo phosphorylates Wts, affiliated by Sav and Mats, and Wts phosphorylates and inhibits the ability of Yki to enter the nucleus [33–35]. Multiple upstream inputs that feed into the core of the Hippo pathway in tissue growth have been identified in recent years. In Drosophila, these upstream inputs include the atypical cadherins Fat and Dachs [36–39], the cell adhesion molecule Echinoid (Ed) [40], the complex formed by the FERM-domain protein Expanded (Ex), Merlin (Mer) [41] and the WW-domain protein Kibra (Kib) [42,43], as well as the cell polarity determinants Crumbs (Crb), Lethal giant larvae (Lgl) and Scribble (Scrib) [44–47]. These upstream inputs act redundantly in tissue growth [41,42]. For example, growth defect in the imaginal disc carrying kib or mer mutations is much weaker compared to those carrying the mutations of the core components of the Hippo pathway. In contrast, double mutations for ex and mer or kib cause severe growth phenotype as demonstrated in hpo or wts mutations [41,42,48]. Interestingly, among these upstream regulators, mer, kib and lgl regulate the Hippo pathway in yR8 fate decision, while fat, dachs and ex are not involved in this process [49]. Additionally, crb is not required for the activation of the Hippo pathway during R8 subtype fate decisions [50]. Therefore, the pale and yellow binary fate assay in the Drosophila retina provides an efficient model with less upstream complication to understand the upstream regulation of the Hippo pathway. Taking advantage of the pR8 and yR8 binary fate assay, we generated and carried out a sensitive and efficient genome-wide screening to identify the new regulators of the Hippo pathway. We identified the Drosophila ZO-1 protein Pyd as a new upstream regulator of the Hippo pathway. Using loss- and gain-of-function studies, we show that Pyd is required and sufficient to promote green-sensitive yR8 fate and repress blue-sensitive pR8 fate. We additionally determined the roles in PR subtype fate specification for pez and suppressor of deltex (su(dx)), the upstream regulators of the Hippo pathway in the Drosophila midgut epithelium [51,52]. Using epistasis analyses, we revealed that Pyd acts upstream of the core components and the upstream regulator Pez in the Hippo pathway, while it may function in parallel to Kib to repress Su(dx)’s activity to specify R8 subtypes. Together, our study identifies a new upstream regulator of the Hippo pathway that functions in post-mitotic neuronal fate specification.

Discussion In this study, we designed a sensitized genetic screen using a triple heterozygote-based PR subtype phenotype enhancement assay to identify novel regulators of the Hippo pathway in the Drosophila eye. Taking advantage of this genome-wide screening, we identified the Drosophila ZO-1 protein Pyd as a new PR subtype fate determinant. We demonstrated Pyd is an upstream regulator of the Hippo signaling pathway and is required for the pathway to promote yR8 and repress pR8 PR subtype fate specification. We also determined the roles of Pez and Su(dx) in R8 subtype fate specification and found they play opposite roles in this process (Fig 8), as they act in intestinal stem cell proliferation. Previous reports have shown that Pyd and Su(dx) can physically interact with each other. We found that Pyd and Su(dx) act antagonistically during R8 subtype fate specification (Fig 8). Further, our pyd and su(dx) double LOF and double GOF results have indicated that the R8 subtype phenotype in pyd LOF retinas depends on the presence of Su(dx), and on the other hand, the overexpressed Pyd represses Su(dx)’s activity to promote pR8 and inhibit yR8 fate specification. Considering that Su(dx) can induce Pez degradation [52], Pyd may be required for Hippo signaling by antagonizing Su(dx)’s activity and therefore stabilizing Pez (Fig 8). Interestingly, it is the WW domain of the Su(dx) protein that interacts with both Pez and Pyd. Therefore, it is possible that Pyd competes with Su(dx) to bind and stabilize Pez. Our data also showed that Kib suppresses Su(dx)’s activity during R8 subtype fate specification, consistent with the previous report that Kib can block Su(dx)-induced Pez degradation [52]. However, Kib was not shown to interact with Su(dx) and it can’t decrease the binding between Su(dx) and Pez [52]. Therefore, Pyd and Kib may use different mechanisms to stabilize Pez: Pyd competes with Su(dx) for Pez binding, while Kib directly binds to Pez. Since loss of pyd or kib lead to significant expansion of pR8s and reduction of yR8s, both of the two mechanisms is required in wild type retinas. However, overexpression of any one of pyd and kib can circumvent loss of another gene (Fig 7), suggesting the two mechanisms might function independently (Fig 8). It will be of interest and important to test this model using biochemical approaches in future studies and explore whether and how Pyd directly competes with Su(dx). PPT PowerPoint slide

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TIFF original image Download: Fig 8. Model: the role of Pyd in R8 subtype fate specification. Pyd represses Su(dx) and may function in parallel to Kib to promote yR8 and repress pR8 subtype fate specification. Pyd may antagonize the Su(dx)-mediated Pez degradation. See Discussion for detail. Genes or proteins that are inactive or not expressed are denoted by grey font, while those that are expressed and/or active are represented by blue, green or black font. Model is based on the data in this study and the previous works [10,15,49,52]. https://doi.org/10.1371/journal.pgen.1009894.g008 The Hippo pathway was originally discovered in Drosophila and its evolutionarily conserved roles in various biological processes have been subsequently found in mammals [19,34,61–68]. However, the regulatory mechanisms upstream of the signaling pathway are less understood. Most of the core components of the Hippo pathway were first isolated as a result of their overgrowth phenotypes in mosaic mutant-based screens [69]. However, this strategy is not efficient to identify the upstream components of the pathway because the overgrowth defects caused by mutations of the upstream genes is much weaker compared to those induced by mutations of the core components due to the redundant roles of the upstream components in tissue growth control [42,43,51,70]. Interestingly, Fat, Expanded as well as Crumbs are not required for the activation of Hippo signaling during R8 subtype fate specification [49], making the upstream regulation of the Hippo pathway during R8 subtype fate decisions less complicated. Additionally, Hippo-dependent R8 subtype fate specification can be precisely quantified. Taking advantage of these features, we generated a sensitive genetic background with double heterozygous wts and kib mutations that affect R8 subtypes modestly, but is able to significantly change pR8 and yR8 subtypes when coupled with one more mutation in a gene of the Hippo pathway. This sensitive genetic tool makes it possible to perform a genome-wide screening by testing the activities of the Drosophila deficiency lines to affect R8 subtypes. Notably, previous studies have demonstrated that Mer physically interacts with Wts and Kib [42,71]. Our results showed the R8 subtype phenotype was enhanced more in heterozygous wtsZn-mer3 or kib1-mer3 flies than in wtsZn-kib1, kib1-savdf or wtsZn-savdf flies. Therefore, the quantitative phenotype enhancement assays for R8 subtypes have a potential application to predict the physical interactions between the components of the Hippo pathway. ZO-1 proteins are crucial for the formation and maintenance of tight junctions in vertebrate cells [72]. While in Drosophila cells, which lack tight junctions [73], Pyd is associated with both adherens and septate junctions [74,75]. ZO-1 proteins are members of the membrane-associated guanylate kinase (MAGUK) family and contain a GUK (guanylate kinase) domain, three PDZ domains, and a SH3 domain [58]. The cellular localization and the presence of multiple protein-protein interaction domains suggest the ZO-1 proteins may play important roles in coupling the extracellular signals to intracellular signaling pathways. A previous study in cultured cells have found that the transiently expressed ZO-1 protein can interact with the carboxy-terminal PDZ binding motif of TAZ, a downstream effector of the Hippo pathway in mammals, via its first PDZ domain [76]. Whether Pyd interacts with Yki, the Drosophila homolog of YAP/TAZ, hasn’t been explored. However, our result that knock-down of wts is sufficient to suppress the phenotype in pyd overexpression retinas suggests the interaction between Yki and Pyd, if there is any, does not play a significant role for the cytoplasmic retention of Yki and thus inhibiting its activity as a transcription co-activator. Additionally, Pyd has been previously implicated in the regulation of the Notch pathway in context-dependent manners [59,77,78]. However, the Notch pathway has not been shown to cell-autonomously regulate R8 subtype fate specification in the Drosophila eye. Our results in this study provide evidence that Pyd is a regulator of the Hippo signaling pathway and functions as an upstream regulator of the pathway for PR subtype fate decisions. Considering its interactions with junctions and cytoskeleton proteins, Pyd might function as a scaffold to organize other components of the Hippo pathway at the plasma membrane to form functional complexes. Furthermore, genetic or direct interactions between Pyd and some transmembrane proteins have been previously reported [79,80]. Given that Pyd functions upstream of the Hippo pathway during R8 subtype fate decisions, it will be of great interest to test the role of these Pyd-interacting transmembrane proteins for R8 subtype fate decisions and investigate whether any of them acts as a transmembrane receptor in Hippo signaling. Notably, Mer plays key roles to recruit the core Hippo components to apical membrane area [81]. Pyd and its transmembrane partner may be required for Mer membrane associations. The R8 terminal differentiation into pR8 or yR8 subtype fate occurs in the late pupal stage and is dependent on the activation and deactivation of the Hippo signaling pathway [82]. As a negative regulator of the Hippo pathway, melt is expressed in a subset of R8s from 40% pupation [10] and is indispensable to transcriptionally repress wts expression and de-activate Hippo signaling [49], allowing these R8s to adopt the pR8 subtype fate. In this study, we determined that the E3 ligase Su(dx) as another negative mediator of the Hippo pathway for R8 subtype fate specification. Su(dx) was shown to degrade Pez and therefore inactivate Hippo signaling in midgut epithelium [52], indicating Su(dx) inactivates Hippo signaling by a different mechanism with Melt-mediated transcriptional repression of wts. It is possible that Su(dx) is present in a subset of R8s at 40–50% APF stage and its presence reduces the default Hippo signaling and thus results in elevated Yki activity which, together with the transcription factors Otd, Traffic jam and Scalloped [10,20], initiates the melt-wts bistable loop to activate melt and repress wts expression, and finally leads to the generation of pR8 subtype.

Materials and methods Drosophila stocks The following fly lines were used: pydex180, pydex147, UAS-GFP-pyd [58], kib1, pez1, pez2, UAS-kib, UAS-pez [43,51], wtsZn (wts-nLacZ) [15,17], meltΔ1 [16], sdΔB [83], lGMR-GAL4 [84], UAS-pydRANi-#450 [79], sensR8-GAL4 [11], ykiB5 [33], UAS-hpo [26], GMR-sav [32], pWIZ-wΔ13 (a white gene RNAi line) [85]. UAS-pydRANi (KK105581), UAS-ykiRNAi (KK109756), UAS-sdRNAi (KK108877), UAS-wtsRNAi (KK101055), UAS-hpoRNAi (KK101704), UAS-savRNAi (KK107562), UAS-matsRNAi (KK100140) and UAS-kibRNAi (KK108510) were from the Vienna Drosophila Resource Center (VDRC). UAS-pydRNAi (HMS00263) UAS-wts, UAS-su(dx), su(dx)2, su(dx)32, sev14, UAS-su(dx)RNAi (HMS05478), UAS-pezRNAi (HMS00862), UAS-kibRNAi (HMC03256), UAS-Dicer2, sev-GAL4, GMR-GAL4 [60], UAS-Luciferase, Df(3R)BSC803 (savdf), Df(3R)ED6096, Df(3R)BSC466, Df(3R)ED5330, Df(3R)Exel6150, Df(3R)BSC478, Df(3R)BSC506, Df(3R)BSC666, Df(3R)Exel6152, and Df(3R)pydB12 were from the Bloomington Drosophila Stock Center (BDSC). p[GawB]NP4419, p[GawB]NP7518, p[GawB]NP0961 and p[GawB]NP4414 were from the Kyoto Stock Center. lGMR-GAL4, pWIZ-wΔ13 and UAS-Dicer2 lines were recombined onto a single chromosome for use in RNAi-mediated knockdown experiments [10]. UAS-pez and pydex180, both on the 3rd chromosome, were recombined and used for misexpression of pez in pyd LOF flies. All flies and crosses were raised on standard cornmeal-molasses media at 25°C with 12 hr:12hr light-dark cycles except that GMR-GAL4>UAS-su(dx) (for Fig 6L and 6M) were in room temperature (22°C). Immunohistochemistry Fly head cryosections, dissection for whole mount retinas, and antibody staining were performed as previously described with modifications [82,86]. Adult fly heads were embedded and frozen in OCT and sectioned (12 μm) using the Cryostat CM1850 (Leica). The samples were then fixed in 4% paraformaldehyde/ PBS, and washed 3x10 min with PBX (PBS + 0.3% Triton X-100), incubated with primary antibodies overnight at 4°C in antibody dilution buffer (PBX + 1% BSA), washed 4x10 min with PBX, and incubated 90 min at room temperature with secondary antibodies diluted in antibody dilution buffer. After 4x10min PBT washes, samples were mounted in anti-fade reagent, and imaged. Antibody dilutions were: rabbit Salm (1:150) [82]; mouse Rh5 (1:1000) [87]; rabbit Rh6 (1:100, this study); chicken LacZ (1:1000, Abcam). Alexa Fluor 488, 555 and 655-conjugated secondary antibodies (1:1500, Invitrogen) were used. Digital images were obtained with an Apotome deconvolution system (Zeiss), and processed with Axiovision 4.5 (Zeiss) and Adobe Photoshop software. Quantifications for the longitudinal sections were performed by counting at least 800 ommatidia from four or more individual flies per genotype, and only sections that include both R7 and R8 layers were counted. Quantifications for the tangential sections use one section for each retina to avoid repeatedly counting the same ommatidia. Retinas for quantifying the whole mount staining are from at least three flies per genotype. Polyclonal antibody production Polyclonal antiserum against Rh6 was generated against a KLH-conjugated peptide from the Rh6 deduced amino acid sequence (CLACGKDDLTSDSRTQAT corresponding to amino acids 344–361). Peptide synthesis, KLH-conjugation, rabbit immunizations and bleeds were performed by GenScript (Piscataway, NJ).

Acknowledgments We thank Sarah Bray, Hugo Stocker, Duojia Pan, Claude Desplan, Jin Jiang, Ruth Johnson, Ross Cagan, Steven Britt, Tiffany Cook, Graeme Mardon, Richard Carthew, the Bloomington Drosophila Stock Center, and the Vienna Drosophila Research Centre (VDRC) for fly stocks and antibody reagents. We thank Tiffany Cook for comments on the manuscript.

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