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Anemic stress induces the proliferation of stress erythroid progenitors in the murine spleen that subsequently differentiate to generate erythrocytes to maintain homeostasis. This process relies on the interaction between stress erythroid progenitors and the signals generated in the splenic erythroid niche. In this study, we demonstrate that although growth-differentiation factor 15 (Gdf15) is not required for steady-state erythropoiesis, it plays an essential role in stress erythropoiesis. Gdf15 acts at 2 levels. In the splenic niche, Gdf15−/− mice exhibit defects in the monocyte-derived expansion of the splenic niche, resulting in impaired proliferation of stress erythroid progenitors and production of stress burst forming unit-erythroid cells. Furthermore, Gdf15 signaling maintains the hypoxia-dependent expression of the niche signal, Bmp4, whereas in stress erythroid progenitors, Gdf15 signaling regulates the expression of metabolic enzymes, which contribute to the rapid proliferation of stress erythroid progenitors. Thus, Gdf15 functions as a comprehensive regulator that coordinates the stress erythroid microenvironment with the metabolic status of progenitors to promote stress erythropoiesis.

How organ growth is regulated in multicellular organisms is a long-standing question in developmental biology. It is known that coordination of cell apoptosis and proliferation is critical in cell number and overall organ size control, while how these processes are regulated is still under investigation. In this study, we found that functional loss of a gene in Drosophila, named Drosophila defender against apoptotic cell death 1 (dDad1), leads to a reduction of tissue growth due to increased apoptosis and lack of cell proliferation. The dDad1 protein, an orthologue of mammalian Dad1, was found to be crucial for protein N-glycosylation in developing tissues. Our study demonstrated that loss of dDad1 function activates JNK signaling and blocking the JNK pathway in dDad1 knock-down tissues suppresses cell apoptosis and partially restores organ size. In addition, reduction of dDad1 triggers ER stress and activates unfolded protein response (UPR) signaling, prior to the activation of JNK signaling. Furthermore, Perk-Atf4 signaling, one branch of UPR pathways, appears to play a dual role in inducing cell apoptosis and mediating compensatory cell proliferation in this dDad1 knock-down model.

Protein-protein interactions provide a common mechanism for regulating protein functions and also serve as the fundamental step of many biochemical reactions. To accurately determine the involvement and function of protein-protein interactions, it is crucial to detect the interactions with the minimum number of artifacts. In this chapter, we report the method of bimolecular fluorescence complementation (BiFC) in tissue culture and developing tissues of Drosophila, which allows the visualization of subcellular localization of protein-protein interactions in living cells.

Protein-protein interactions provide a common mechanism for regulating protein functions and also serve as the fundamental step of many biochemical reactions. To accurately determine the involvement and function of protein-protein interactions, it is crucial to detect the interactions with the minimum number of artifacts. In this chapter, we report the method of bimolecular fluorescence complementation (BiFC) in tissue culture and developing tissues of Drosophila, which allows the visualization of subcellular localization of protein-protein interactions in living cells.

The considerable amount of experimental evidence has defined the Hippo pathway as a tumor suppressive pathway and increased expression and/or activity of its oncogenic effectors is frequently observed in cancer. However, clinical studies have failed to attribute cancer development and progression to mutations in the pathway. In explaining this conundrum, we investigated the expression and functions of a C-terminally truncated isoform of large tumor suppressor kinase 1 (LATS1) called short LATS1 (sLATS1) in human cell lines and Drosophila. Intriguingly, through overexpression of sLATS1, we demonstrated that sLATS1 either activates or suppresses the activity of Yes-associated protein (YAP), one of the effectors of the Hippo pathway, in a cell type-specific manner. The activation is mediated through inhibition of full-length LATS1, whereas suppression of YAP is accomplished through sLATS1-YAP interaction. In HEK293T cells, the former mechanism may affect the cellular response more dominantly, whereas in U2OS cells and developing tissues in Drosophila, the latter mechanism may be solely carried out. Finally, to find the clinical relevance of this molecule, we examined the expression of sLATS1 in breast cancer patients. The transcriptome analysis showed that the ratio of sLATS1 to LATS1 was increased in tumor tissues comparing to their adjacent normal tissues.

Genome association studies in human and genetic studies in mouse implicated members of the transmembrane protein 132 (TMEM132) family in multiple conditions including panic disorder, hearing loss, limb and kidney malformation. However, the presence of five TMEM132 paralogs in mammalian genomes makes it extremely challenging to reveal the full requirement for these proteins in vivo. In contrast, there is only one TMEM132 homolog, detonator (dtn), in the genome of fruit fly Drosophila melanogaster, enabling straightforward research into its in vivo function. In the current study, we generate multiple loss-of-function dtn mutant fly strains through a polycistronic tRNA-gRNA approach, and show that most embryos lacking both maternal and paternal dtn fail to hatch into larvae, indicating an essential role of dtn in Drosophila reproduction.

In contrast to steady-state erythropoiesis, which generates new erythrocytes at a constant rate, stress erythropoiesis rapidly produces a large bolus of new erythrocytes in response to anemic stress. In this study, we illustrate that Yes-associated protein (Yap1) promotes the rapid expansion of a transit-amplifying population of stress erythroid progenitors in vivo and in vitro. Yap1-mutated erythroid progenitors failed to proliferate in the spleen after transplantation into lethally irradiated recipient mice. Additionally, loss of Yap1 impaired the growth of actively proliferating erythroid progenitors in vitro. This role in proliferation is supported by gene expression profiles showing that transiently amplifying stress erythroid progenitors express high levels of genes associated with Yap1 activity and genes induced by Yap1. Furthermore, Yap1 promotes the proliferation of stress erythroid progenitors in part by regulating the expression of key glutamine-metabolizing enzymes. Thus, Yap1 acts as an erythroid regulator that coordinates the metabolic status with the proliferation of erythroid progenitors to promote stress erythropoiesis.

In recent years, human cancer genome projects provide unprecedented opportunities for the discovery of cancer genes and signaling pathways that contribute to tumor development. While numerous gene mutations can be identified from each cancer genome, what these mutations mean for cancer is a challenging question to address, especially for those from less understood putative new cancer genes. As a powerful approach, in silico bioinformatics analysis could efficiently sort out mutations that are predicted to damage gene function. Such an analysis of human large tumor suppressor genes, LATS1 and LATS2, has been carried out and the results support a role of hLATS1//2 as negative growth regulators and tumor suppressors. Electronic supplementary material The online version of this article (doi:10.1007/s13238-014-0122-4) contains supplementary material, which is available to authorized users.

BRMS1 was first discovered as a human breast carcinoma metastasis suppressor gene. However, the mechanism of BRMS1 in tumor metastasis and its developmental role remain unclear. In this paper, we first report the identification of the Drosophila ortholog of human BRMS1, dBrms1. Through a genetic approach, the role of dBrms1 during development has been investigated. We found that dBrms1 is an essential gene and loss of dBrms1 function results in lethality at early developmental stages. dBrms1mutants displayed phenotypes such as developmental delay and failure to initiate metamorphosis. Further analysis suggests that these phenotypes are contributed by defective ecdysone signaling and expression of target genes of the ecdysone pathway. Therefore, dBrms1 is required for growth control by acting as a modulator of ecdysone signaling in Drosophila and is required for metamorphosis for normal development.

Tissue growth is achieved through coordinated cellular growth, cell division and apoptosis. Hippo signaling is critical for monitoring tissue growth during animal development. Loss of Hippo signaling leads to tissue overgrowth due to continuous cell proliferation and block of apoptosis. As cells lacking Hippo signaling are similar in size compared to normal cells, cellular growth must be properly maintained in Hippo signaling-deficient cells. However, it is not clear how Hippo signaling might regulate cellular growth. Here we show that loss of Hippo signaling increased Akt (also called Protein Kinase B, PKB) expression and activity, whereas activation of Hippo signaling reduced Akt expression in developing tissues in Drosophila. While yorkie (yki) is sufficient to increase Akt expression, Akt up-regulation caused by the loss of Hippo signaling is strongly dependent on yki, indicating that Hippo signaling negatively regulates Akt expression through Yki inhibition. Consistently, genetic analysis revealed that Akt plays a critical role in facilitating growth of Hippo signaling-defective tissues. Thus, Hippo signaling not only blocks cell division and promotes apoptosis, but also regulates cellular growth by inhibiting the Akt pathway activity.

The Yes-associated protein (YAP) transcription coactivator is a candidate human oncogene and a key regulator of organ size. It is phosphorylated and inhibited by the Hippo tumor suppressor pathway. TEAD family transcription factors were recently shown to play a key role in mediating the biological functions of YAP. Here, we show that the WW domain of YAP has a critical role in inducing a subset of YAP target genes independent of or in cooperation with TEAD. Mutation of the WW domains diminishes the ability of YAP to stimulate cell proliferation and oncogenic transformation. Inhibition of YAP oncogenic-transforming activity depends on intact serine residues 127 and 381, two sites that could be phosphorylated by the Hippo pathway. Furthermore, genetic experiments in Drosophila support that WW domains of YAP and Yki, the fly YAP homologue, have an important role in stimulating tissue growth. Our data suggest a model in which YAP induces gene expression and exerts its biological functions by interacting with transcription factors through both the TEAD-binding and WW domains. [Cancer Res 2009;69(3):1089–98]

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The mob as tumor suppressor (mats) family genes are highly conserved in evolution. The Drosophila mats gene functions in the Hippo signaling pathway to control tissue growth by regulating cell proliferation and apoptosis. However, nothing is known about whether mats family genes are required for the normal development of vertebrates. Here we report that zebrafish has three mats family genes. Expression of mats1 is maternally activated and continues during embryogenesis. Through a morpholino-based knockdown approach, we found that mats1 is required for normal embryonic development. Reduction of mats1 function caused developmental delay, a phenotype similar to that of Drosophila mats homozygous mutants. Both cell proliferation and apoptosis were defective in mats1 morphant embryos. Moreover, mats1 morphant cells exhibited a growth advantage in chimeric embryos, similar to mats mutant cells in mosaic tissues in Drosophila. Therefore mats1 plays a critical role in regulating cell proliferation and apoptosis during early development in zebrafish, and the role of mats family genes in growth regulation is conserved in both invertebrates and vertebrates. This work shows that zebrafish can be a good model organism for further analysis of Hippo signaling pathway.

The YAP transcription coactivator has been implicated as an oncogene and is amplified in human cancers. Recent studies have established that YAP is phosphorylated and inhibited by the Hippo tumor suppressor pathway. Here we demonstrate that the TEAD family transcription factors are essential in mediating YAP-dependent gene expression. TEAD is also required for YAP-induced cell growth, oncogenic transformation, and epithelial–mesenchymal transition. CTGF is identified as a direct YAP target gene important for cell growth. Moreover, the functional relationship between YAP and TEAD is conserved in Drosophila Yki (the YAP homolog) and Scalloped (the TEAD homolog). Our study reveals TEAD as a new component in the Hippo pathway playing essential roles in mediating biological functions of YAP.

The Drosophila mats gene plays a critical role in growth control. Using molecular genetic approaches we investigated how mats is regulated in development. A 2236-bp genomic sequence that contains entire mats including upstream and downstream intergenic regions can rescue mats mutant phenotypes, indicating that regulatory elements necessary for proper mats expression are mostly retained. However, constructs without the upstream or downstream intergenic region failed to rescue mats mutants, demonstrating the functional importance of these sequences. Moreover, mats expression is reduced in matse17, a mats allele with over one-third of the downstream intergenic region deleted. Consistent with a model that the downstream intergenic region is critical for mats activity, this sequence contains evolutionarily conserved elements and has enhancer activities.

Studies in Drosophila have defined a new growth inhibitory pathway mediated by Fat (Ft), Merlin (Mer), Expanded (Ex), Hippo (Hpo), Salvador (Sav)/Shar-pei, Warts (Wts)/Large tumor suppressor (Lats), and Mob as tumor suppressor (Mats), which are all evolutionarily conserved in vertebrate animals. We previously found that the Mob family protein Mats functions as a coactivator of Wts kinase. Here we show that mats is essential for early development and is required for proper chromosomal segregation in developing embryos. Mats is expressed at low levels ubiquitously, which is consistent with the role of Mats as a general growth regulator. Like mammalian Mats, Drosophila Mats colocalizes with Wts/Lats kinase and cyclin E proteins at the centrosome. This raises the possibility that Mats may function together with Wts/Lats to regulate cyclin E activity in the centrosome for mitotic control. While Hpo/Wts signaling has been implicated in the control of cyclin E and diap1 expression, we found that it also modulates the expression of cyclin A and cyclin B. Although mats depletion leads to aberrant mitoses, this does not seem to be due to compromised mitotic spindle checkpoint function.

SummaryAppropriate cell number and organ size in a multicellular organism are determined by coordinated cell growth, proliferation, and apoptosis. Disruption of these processes can cause cancer. Recent studies have identified the Large tumor suppressor (Lats)/Warts (Wts) protein kinase as a key component of a pathway that controls the coordination between cell proliferation and apoptosis. Here we describe growth inhibitory functions for a Mob superfamily protein, termed Mats (Mob as tumor suppressor), in Drosophila. Loss of Mats function results in increased cell proliferation, defective apoptosis, and induction of tissue overgrowth. We show that mats and wts function in a common pathway. Mats physically associates with Wts to stimulate the catalytic activity of the Wts kinase. A human Mats ortholog (Mats1) can rescue the lethality associated with loss of Mats function in Drosophila. As Mats1 is mutated in human tumors, Mats-mediated growth inhibition and tumor suppression is likely conserved in humans.

Receptor tyrosine kinase (RTK) signaling plays an instructive role in cell fate decisions, whereas Notch signaling is often involved in restricting cellular competence for differentiation. Genetic interactions between these two evolutionarily conserved pathways have been extensively documented. The underlying molecular mechanisms, however, are not well understood. Here, we show that Yan, an Ets transcriptional repressor that blocks cellular potential for specification and differentiation [1, 2], is a target of Notch signaling during Drosophila eye development. The Suppressor of Hairless (Su[H]) protein of the Notch pathway is required for activating yan expression, and Su(H) binds directly to an eye-specific yan enhancer in vitro. In contrast, yan expression is repressed by Pointed (Pnt), which is a key component of the RTK pathway. Pnt binds specifically to the yan enhancer and competes with Su(H) for DNA binding. This competition illustrates a potential mechanism for RTK and Notch signals to oppose each other. Thus, yan serves as a common target of Notch/Su(H) and RTK/Pointed signaling pathways during cell fate specification.

Abnormal activation of Wnt/β-catenin-mediated transcription is associated with a variety of human cancers. Here we report that LATS2 inhibited oncogenic Wnt/β-catenin-mediated transcription by disrupting the β-catenin/BCL9 interaction. LATS2 directly interacted with β-catenin and to be present on Wnt target gene promoters. Mechanistically, LATS2 inhibited the interaction between BCL9 and β-catenin and subsequent recruitment of BCL9, independent of LATS2 kinase activity. LATS2 was down-regulated and inversely correlated with the levels of Wnt target genes in human colorectal cancers. Moreover, nocodazole, an anti-microtubule drug, potently induced LATS2 to suppress tumor growth in vivo by targeting β-catenin/BCL9. Our results suggest that LATS2 is not only a key tumor suppressor in human cancer, but may also be an important target for anti-cancer therapy.

The role of Large tumor suppressor LATS/Warts in human cancer is not clearly understood. Here we show that hLATS1/2 cancer mutations affect their expression and kinase activity. hLATS1/2 mutants exhibit a decreased activity in inhibiting YAP and tissue growth. Therefore, hLATS1/2 alleles from human cancer can be loss-of-function mutations.

Hippo signaling plays a crucial role in growth control and tumor suppression by regulating cell proliferation, apoptosis, and differentiation. How Hippo signaling is regulated has been under extensive investigation. Over the past three years, an increasing amount of data have supported a model of actin cytoskeleton blocking Hippo signaling activity to allow nuclear accumulation of a downstream effector, Yki/Yap/Taz. On the other hand, Hippo signaling negatively regulates actin cytoskeleton organization. This review provides insight on the mutual regulatory mechanisms between Hippo signaling and actin cytoskeleton for a tight control of cell behaviors during animal development, and points out outstanding questions for further investigations.

The BTB/POZ (broad complex Tramtrack bric-a-brac/Pox virus and zinc finger) domain is an evolutionarily conserved protein-protein interaction motif. Many BTB-containing proteins are transcriptional regulators involved in a wide range of developmental processes. However, the significance of the BTB domain in development has not been evaluated. Here we present evidence that overexpression of the Tramtrack69 (Ttk69) protein not only blocks neuronal photoreceptor differentiation but also promotes nonneuronal cone cell specification in early Drosophila eye development. We show that the BTB domain is essential for Ttk69 function and single amino acid changes in highly conserved residues in this domain abolish Ttk69 activity. Interestingly, the Ttk69 BTB can be substituted by the BTB of the human Bcl-6 protein, suggesting that BTB function has been conserved between Drosophila and humans. We found that the Ttk69 BTB domain is critical for mediating interaction with the Drosophila homolog of C-terminal-binding protein (dCtBP) in vitro, and dCtBP− mutations genetically interact with ttk69. Furthermore, the C-terminal region downstream of the DNA-binding zinc fingers is shown to be essential for Ttk69 function. A dCtBP consensus binding motif in the C terminus appears to contribute to Ttk69 activity, but it cannot be fully responsible for the function of the C terminus.

Gp150 is a transmembrane glycoprotein belonging to the leucine-rich repeat (LRR) superfamily. Here we report the molecular characterization of a Gp150 homolog in Drosophila virilis, which is separated from Drosophila melanogaster by about 60 million years. A silkworm Bombyx mori Gp150 homolog was identified through a sequence database search. Sequence analysis revealed high conservation in the LRRs and cysteine motifs flanking the LRR region in the extracellular domain of Gp150. Using an in vivo assay, we demonstrated that the extracellular domain is essential for Gp150 function. Moreover, structural features unique to the Gp150 proteins were identified that include an incomplete carboxy-flanking cysteine motif, acidic regions on both sides of the LRR region in the extracellular domain, and a short cytoplasmic domain with three putative tyrosine phosphorylation motifs, which might be involved in interaction with SH2 domains. Thus, Gp150 defines a new subfamily of LRR proteins and may be involved in signal transduction. Sequence comparison of the two Drosophila gp150 genes demonstrated a high degree of conservation of genome organization downstream of the gp150 gene. Interestingly, D. virilis gp150 coding region appears to have an additional intron, an uncommon feature in homologs of other genes. The expression pattern of gp150 during embryogenesis in D. melanogaster and D. virilis was found to be identical. gp150 transcripts were localized generally to regions where cells are undergoing dramatic morphogenetic movements. This is further corroborated by the localization of gp150 transcripts in eye imaginal discs in the region spanning the morphogenetic furrow.

Competence for cell fate determination and cellular differentiation is under tight control of regulatory genes. Yan, a nuclear target of receptor tyrosine kinase (RTK) signaling, is an E twenty six (ETS) DNA-binding protein that functions as a negative regulator of cell differentiation and proliferation in Drosophila. Most members of RTK signaling pathways are highly conserved through evolution, yet no yan orthologues have been identified to date in vertebrates. To investigate the degree of yan conservation during evolution, we have characterized a yan homologue from a sibling species of D. melanogaster, D. virilis. Our results show that the organization, primary structure and expression pattern of yan are highly conserved. Both genes span over 20 kb and contain four exons with introns at identical positions. The areas with highest amino acid similarity include the Pointed and ETS domain but there are other discrete regions with a high degree of similarity. Phylogenetic analysis reveals that yan's closest relative is the human tel gene, a negative regulator of differentiation in hematopoetic precursors. In both species, Yan is dynamically expressed beginning as early as stage 4/5 and persisting throughout embryogenesis. In third instar larvae, Yan is expressed in and behind the morphogenetic furrow of the eye imaginal disc as well as in the laminar precursor cells of the brain. Ovarian follicle cells also contain Yan protein. Conservation of the structure and expression patterns of yan genes strongly suggests that regulatory mechanisms for their expression are also conserved in these two species.

The Drosophila yan gene encodes an E twenty-six (ETS) domain nuclear protein with a transcription repressor activity that can be downregulated through phosphorylation by mitogen-activated protein kinase (MAPK). Before photoreceptor precursor cells commit to a particular cell fate, Yan is required to maintain them in an undifferentiated state. We report here identification of tramtrack (ttk) mutations that act as dominant enhancers of yan. We show that ttk synergistically interacts with yan to inhibit the R7 photoreceptor cell fate. Since ttk products are nuclear proteins with zinc-finger DNA-binding motifs, yan and ttk represent two nuclear regulators essential for the control of cellular competence for neural differentiation. Reduction of either yan or ttk activity suppresses eye phenotypes of the kinase suppressor of ras (ksr) gene mutation, which is consistent with the involvement of yan and ttk in the Ras/MAPK pathway.

The evolutionarily conserved Hippo signaling pathway plays an important role in regulating normal development as well as tumorigenesis in animals. How this growth-inhibitory signaling is maintained at an appropriate level through feedback mechanisms is less understood. In this report, we show that bantam microRNA functions to increase the level of the Mob as tumor suppressor protein Mats, a core component of the Hippo pathway, but does not regulate mats at the transcript level. Genetic analysis also supports that bantam plays a positive role in regulating mats function for tissue growth control. Our data support a model that bantam up-regulates Mats expression through an unidentified factor that may control Mats stability.

The Hippo tumor suppressor pathway plays an important role in tissue homeostasis that ensures development of functional organs at proper size. The YAP transcription coactivator is a major effector of the Hippo pathway and is phosphorylated and inactivated by the Hippo pathway kinases Lats1/2. It has recently been shown that YAP activity is regulated by G-protein-coupled receptor signaling. Here we demonstrate that cyclic adenosine monophosphate (cAMP), a second messenger downstream from Gαs-coupled receptors, acts through protein kinase A (PKA) and Rho GTPases to stimulate Lats kinases and YAP phosphorylation. We also show that inactivation of YAP is crucial for PKA-induced adipogenesis. In addition, PKA activation in Drosophila inhibits the expression of Yorki (Yki, a YAP ortholog) target genes involved in cell proliferation and death. Taken together, our study demonstrates that Hippo–YAP is a key signaling branch of cAMP and PKA and reveals new insight into mechanisms of PKA in regulating a broad range of cellular functions.

We have screened a collection of transposable-element-induced mutations for those which dominantly modify the extra R7 phenotype of a hypomorphic yan mutation. The members of one of the identified complementation groups correspond to disruptions of the tramtrack (ttk) gene. As heterozygotes, ttk alleles increase the percentage of R7 cells in yan mutant eyes. Just as yan mutations increase ectopic R7 cell formation, homozygous ttk mutant eye clones also contain supernumerary R7 cells. However, in contrast to yan, the formation of these cells in ttk mutant eye tissue is not necessarily dependent on the activity of the sina gene. Furthermore, although yan mutations dominantly interact with mutations in the Ras1, Draf, Dsor1, and rolled (rl) genes to influence R7 cell development, ttk mutations only interact with yan and rl gene mutations to affect this signaling pathway. Our data suggest that yan and ttk both function to repress inappropriate R7 cell development but that their mechanisms of action differ. In particular, TTK activity appears to be autonomously required to regulate a sina-independent mechanism of R7 determination.

Effective intercellular communication is an important feature in the development of multicellular organisms. Secreted hedgehog (hh) protein is essential for both long- and short-range cellular signaling required for body pattern formation in animals. In a molecular evolutionary study, we find that the vertebrate homologs of the Drosophila hh gene arose by two gene duplications: the first gave rise to Desert hh, whereas the second produced the Indian and Sonic hh genes. Both duplications occurred before the emergence of vertebrates and probably before the evolution of chordates. The amino-terminal fragment of the hh precursor, crucial in long- and short-range intercellular communication, evolves two to four times slower than the carboxyl-terminal fragment in both Drosophila hh and its vertebrate homologues, suggesting conservation of mechanism of hh action in animals. A majority of amino acid substitutions in the amino- and carboxyl-terminal fragments are conservative, but the carboxyl-terminal domain has undergone extensive insertion-deletion events while maintaining its autocleavage protease activity. Our results point to similarity of evolutionary constraints among sites of Drosophila and vertebrate hh homologs and suggest some future directions for understanding the role of hh genes in the evolution of developmental complexity in animals.

Differentiation in the Drosophila eye imaginal disc traverses the disc as a wave moving from posterior to anterior. The propagation of this wave is driven by hedgehog protein secreted by the differentiated cells in the posterior region of the disc. Hedgehog induces decapentaplegic expression at the front of differentiation, in the morphogenetic furrow. We have identified a gene, shortsighted, which is expressed in a hedgehog-dependent stripe in the undifferentiated cells just anterior to the furrow and which appears to be involved in the transmission of the differentiation-inducing signal; a reduction in shortsighted function leads to a delay in differentiation and to a loss of photoreceptors in the adult. shortsighted is also required for a morphogenetic movement in the brain that reorients the second optic lobe relative to the first. shortsighted encodes a cytoplasmic leucine zipper protein with homology to a mouse gene, TSC-22, which is transcriptionally induced in response to TGF-β.

The Drosophila zfh-1 gene encodes an unusual protein with nine Cys2His2 type zinc-finger motifs and one homeodomain that shows a complex pattern of expression in the embryonic mesoderm and nervous system. To study the function of zfh-1, we generated loss-of-function zfh-1 mutations. Phenotypic analysis of zfh-1 mutant embryos reveals that the gene is not required for the initial segregation of the mesoderm or for the differentiation of mesodermally derived tissues. Rather, loss of zfh-1 function results in various degrees of local errors in cell fate or positioning.

Loss-of-function mutations in the yan gene result in the differentiation of supernumerary photoreceptors in the Drosophila eye. The yan gene encodes a protein with an ETS DNA-binding domain that accumulates in the nuclei of undifferentiated cells during the early stages of eye development. Our data suggest that yan functions as a cell-autonomous negative regulator of photoreceptor development; in the presumptive R7 and cone cells, yan appears to act antagonistically to the proneural signal mediated by sevenless and Ras1.

Growth inhibition mediated by Hippo (Hpo) signaling is essential for tissue growth and organ size control in Drosophila. However, the cellular mechanism by which the core components like Mob as tumor suppressor (Mats) and Warts (Wts) protein kinase are activated is poorly understood. In this work, we found that the endogenous Mats is located at the plasma membrane in developing tissues. Membrane targeting constitutively activates Mats to promote apoptosis and reduce cell proliferation, which leads to reduced tissue growth and organ size. Moreover, the ability of membrane-targeted Mats to inhibit tissue growth required the wts gene activity and Wts kinase activity was increased by the activated Mats in developing tissues. Consistent with the idea that Mats is a key component of the Hpo pathway, Mats is required and sufficient to regulate Yki nuclear localization. These results support a model in which the plasma membrane is an important site of action for Mats tumor suppressor to control tissue growth and organ size.

The zfh-1 and zfh-2 genes of D. melanogaster encode novel proteins containing both hemoedomain and C2-H2 zinc-finger DNA-binding motifs. Antisera against these proteins were used to investigate their expression patterns during embryonic development. The zfh-1 gene is expressed in the mesoderm of early embryos and in a number of mesodermally-derived structures of late embryos, including the dorsal vessel, support cells of the gonads, and segment-specific arrays of adult muscle precursors. In addition, zfh-1 is expressed in the majority of identified motor neurons of the developing CNS. The mesodermal zfh-1 expression requires the products of the twist and snail genes. The zfh-2 gene displays a more limited expression pattern, largely restricted to the CNS of late embryos. Ubiquitous zfh-1 expression in transgenic flies bearing an hsp70-zfh-1 construct has specific developmental consequences, including embryonic CNS defects as well as adult eye and bristle abnormalities. The expression patterns of zfh-1 and zfh-2 suggest that both genes may be involved in Drosophila neurogenesis and the zfh-1 may have additional functions in mesoderm development.

Two of the most common DNA-binding motifs found in eukaryotic transcriptional regulatory proteins are the homeodomain and the C2-H2 zinc finger. In Drosophila, homeodomain and zinc-finger proteins have been implicated in a wide variety of developmental processes. Until now, no proteins have been described in which both these DNA-binding motifs are present. We report here the isolation of genes encoding two such Drosophila proteins from a cDNA expression library. The product of the zfh-1 gene (zinc-finger homeodomain protein 1) contains one homeodomain and nine C2-H2 zinc fingers. The product of the zfh-2 gene possesses three homeodomains and sixteen C2-H2 zinc fingers. For zfh-1, antisera raised against nonoverlapping regions of the gene product all recognize a 145 kDa protein on protein immunoblots, suggesting that the different DNA-binding motifs are actually all present in the mature gene product. The novel arrangement of interspersed homeodomain and zinc-finger motifs in the primary sequences of the zfh-1 and zfh-2 gene products may signify an unusual mechanism of transcriptional regulation by these proteins.

The Hippo pathway plays a key role in organ size control by regulating cell proliferation and apoptosis in Drosophila. Although recent genetic studies have shown that the Hippo pathway is regulated by the NF2 and Fat tumor suppressors, the physiological regulations of this pathway are unknown. Here we show that in mammalian cells, the transcription coactivator YAP (Yes-associated protein), is inhibited by cell density via the Hippo pathway. Phosphorylation by the Lats tumor suppressor kinase leads to cytoplasmic translocation and inactivation of the YAP oncoprotein. Furthermore, attenuation of this phosphorylation of YAP or Yorkie (Yki), the Drosophila homolog of YAP, potentiates their growth-promoting function in vivo. Moreover, YAP overexpression regulates gene expression in a manner opposite to cell density, and is able to overcome cell contact inhibition. Inhibition of YAP function restores contact inhibition in a human cancer cell line bearing deletion of Salvador (Sav), a Hippo pathway component. Interestingly, we observed that YAP protein is elevated and nuclear localized in some human liver and prostate cancers. Our observations demonstrate that YAP plays a key role in the Hippo pathway to control cell proliferation in response to cell contact.

Tissue growth and organ size are determined by coordinated cell proliferation and apoptosis in development. Recent studies have demonstrated that Hippo (Hpo) signaling plays a crucial role in coordinating these processes by restricting cell proliferation and promoting apoptosis. Here we provide evidence that the Mob as tumor suppressor protein, Mats, functions as a key component of the Hpo signaling pathway. We found that Mats associates with Hpo in a protein complex and is a target of the Hpo serine/threonine protein kinase. Mats phosphorylation by Hpo increases its affinity with Warts (Wts)/large tumor suppressor (Lats) serine/threonine protein kinase and ability to upregulate Wts catalytic activity to target downstream molecules such as Yorkie (Yki). Consistently, our epistatic analysis suggests that mats acts downstream of hpo. Coexpression analysis indicated that Mats can indeed potentiate Hpo-mediated growth inhibition in vivo. Our results support a model in which Mats is activated by Hpo through phosphorylation for growth inhibition, and this regulatory mechanism is conserved from flies to mammals.

Notch and Delta are required for lateral inhibition during eye development. They prevent a tenfold excess in R8 photoreceptor cell specification. Mutations in two other genes, Scabrous and Gp150, result in more modestly increased R8 specification. Their roles in Notch signaling have been unclear. Both sca and gp150 are required for ectopic Notch activity that occurs in the split mutant. Similar phenotypes showed that sca and gp150 genes act in a common pathway. Gp150 was required for all activities of Sca, including inhibition of Notch activity and association with Notch-expressing cells that occur when Sca is ectopically expressed. Mosaic analysis found that the gp150 and scagenes were required in different cells from one another. Gp150 concentrated Sca protein in late endosomes. A model is proposed in which endosomal Sca and Gp150 promote Notch activation in response to Delta, by regulating acquisition of insensitivity to Delta in a subset of cells.

Yan is a nuclear DNA-binding protein that acts as a general inhibitor of cellular differentiation and proliferation in Drosophila melanogaster. The genetic and biochemical mechanisms required for regulating Yan protein function are well understood, however, the molecular mechanism of yan gene transcriptional regulation has not been fully elucidated. Here we show that the dynamic expression of the yan gene is specified by distinct spatial and temporal cis-acting regulatory elements in embryos and larval tissues. Each of these distinct elements is thus capable of replicating vital aspects of endogenous yan gene expression.