BIOLOGICAL OVERVIEW

The dorsal/ventral boundary of the developing wing imaginal disc structures the growth of the entire wing. Interacting here are apterous, expressed in the dorsal margin, and the genes scalloped and vestigial (vg), the latter coding for a novel nuclear protein. apterous expression structures the dorsal and ventral expression of the adhesive integrins. Interactions between dorsal and ventral cells in the growing imaginal disc induce vestigial gene expression through a discrete, extraordinarily conserved imaginal disc-specific enhancer. The link between dorsal/ventral compartmentalization and wing formation distinguishes the development of this sheet-like appendage from that of legs and antennae (Williams, 1994).

scalloped is also expressed in the central and peripheral nervous systems of the developing larva, where it is required for the differentiation of sensory organs. The role of scalloped and vestigial in wing and sensory organ morphogenesis recalls the role of hairy in structuring sensory organs in the wing and leg. scalloped interacts with cut, a proneural gene involved at the wing margin (Jack, 1992). Thus scalloped and vestigial are termed patterning genes, because of their effects on wing sensory organ distribution. However, these two genes are responsible as well for a more global pattern, with ramifications for all of wing development.

The scalloped gene is a downstream effector molecule in the wingless pathway of wing imaginal discs. sd is required for early expression of vestigial. In one-good-turn-deserves-another fashion, vg is later required to maintain sd expression. Early expression of vg, and presumably of sd as well, is widespread and unaltered in wingless mutants, but the phenotypic effect of mutations in both genes is the same: a disrupted wing margin.

Both apterous and wingless are required to retain vestigial. Accompanying vestigial is the presumed scalloped expression, flanking the dorso-ventral margin of the wing imaginal disc. Since early scalloped and vestigial expression do not require wingless, sd and vg are the earliest tissue specific pro-wing genes whose appearance reflects the initial specification of the wing. In this model ap and wg are subsequently required for dorso-ventral compartmentalization (Williams, 1993).

The questions continue: what is the role of the homeodomain proteins distal-less and aristaless in regulation of the early expression of scalloped in wing specification? Is scalloped dependent or independent of these organizers of the distal tip of imaginal discs?

Scalloped is required for Vg function: altering Sd and Vg cellular levels relative to one another inhibits wing formation. Whereas Vg expression is normally restricted to the wing and haltere imaginal discs, a subset of cells within almost all imaginal discs normally express sd. Thus, when Vg is ectopically produced a supply of Sd is already present in those tissues. Since Sd is required for formation of the normal wing, a test was performed to see if there is a similar requirement for Sd in the formation of Vg-induced ectopic wings. The induction of wing tissue overgrowths by ectopic Vg is partially suppressed in animals heterozygous for a strong viable allele of sd and is completely suppressed in hemizygotes for the same viable allele. These observations demonstrate that Vg requires Sd to transform cells to wing fates. This requirement does not appear to reflect a role for Sd as a downstream effector of Vg function, because the expression of Sd alone, whether under the control of dpp or other promoters, does not induce the formation of ectopic wing tissue. Instead, these observations suggest that Sd and Vg could act in parallel to induce wing cell fates (Simmonds, 1998).

In vitro, Vg binds directly to both Sd and its human homolog, Transcription Enhancer Factor-1. The interaction domains map to a small region of Vg that is essential for Vg-mediated gene activation and to the carboxy-terminal half of Sd. To map Vg-Sd and Vg-TEF-1 interaction domains, a Far Western blotting assay was used to screen 15 deleted proteins that remove terminal or internal regions of Vg. Only Vg proteins that contain amino acids 279-335 have any significant affinity for Sd. The Vg-Sd interaction appears to be limited to this 56-amino-acid domain, since Sd does not bind to a deleted Vg protein missing only these amino acids, and a construct encoding only this portion of the protein will still bind to Sd. Significantly, a duplicate panel of Vg deletion proteins probed with TEF-1 shows that TEF-1 interacts with Vg via the same protein domain. Affinity columns containing this protein fragment of Vg bind Sd and TEF-1 protein as well as does full-length Vg. This Sd/TEF-1-binding domain of Vg is serine rich and includes putative phosphorylation sites. Phosphorylation of Vg at these sites may potentially modify the Vg-Sd interaction. This region is highly conserved in Vg proteins from Drosophila virilis and Aedes aegypti. Similar sequences also occur in mammalian genomic and expressed sequence tag databases. The amino- and carboxy-terminal portions of Sd were also tested to map which region of Sd interacts with Vg. Previous studies with TEF-1 have demonstrated that regions mediating interaction with cell-specific TIFs are separable from the DNA-binding TEA/ATTS domain. The Vg-binding region of Sd maps to the carboxy-terminal half of the protein, separable from the TEA/ATTS domain in the amino-terminal half. The carboxy-terminal portion of Sd is also highly similar to TEF-1, which is consistent with the observation that TEF-1 binds to Vg with the same affinity as does Sd. To confirm the direct protein-protein interaction between Vg and Sd in a cellular environment, a yeast two-hybrid assay was used. In yeast, Vg and Sd proteins show a specific and reciprocal interaction when fused to either Gal4-binding domain (pGBDU) or Gal4 activation domain (pACT) fusion constructs. Activation of target genes sd and cut by Vg requires the Sd-Vg interaction domain identified in vitro, implying that this activation is Sd dependent. Moreover, a UAS-vg construct with the Sd-binding domain deleted is unable to induce the formation of ectopic wing tissue, consistent with the observation that this induction is sd dependent (Simmonds, 1998).

A wide variety of studies have suggested that TEA/ATTS domain proteins require tissue-specific transcriptional intermediary factors (TIFs), although relatively little progress has been made toward identifying and characterizing these TIFs . According to the definitions established by the analysis of TEF-1, a TIF for Sd would be expected to bind directly to Sd, to show a restricted pattern of expression, and to be required for Sd function in vivo. These observations presented here, together with the analysis of the coordinate regulation of downstream target genes by Sd and Vg, argue that Vg functions as a tissue-specific TIF for Sd. Although it is possible that Vg interacts with proteins other than Sd, genetic studies argue against this, because all vg mutant phenotypes are shared by sd. In contrast, sd is required for the development of other tissues in which vg is not required. Thus, it is likely that there are other trans-acting factors in Drosophila that interact with Sd. Although the DNA target sequence of Sd is as yet uncharacterized, one target of a yeast TEA/ATTS domain protein (TEC-1) is an element in the TEC-1 promoter. Likewise, in flies, one target of Sd-Vg is likely to be the sd promoter itself, since activation of sd during early development is dependent on Vg, and ectopic Vg induces elevated expression of sd. This suggests a model whereby low levels of Sd expression within wing imaginal discs are elevated in the presence of Vg by positive autoregulation. The dependence on sd of elevated levels of Vg in the wing disc suggests that vg is also a target of positive autoregulation, and direct evidence for this now been obtained. The TEA/ATTS domain protein family is involved in developmental processes as diverse as mammalian neuronal and cardiac muscle development to conidial formation in Aspergillus and pseudohyphal growth of Saccharomyces cerevisiae. Although Vg homologs have not yet been identified in these organisms, genes containing sequences related to the Sd/TEF-1 interaction domain of Vg are conserved in mammals; these genes are thus candidate TIFs for mammalian TEF-1-related proteins. One of these candidate TIFs is expressed in fetal heart tissue, which is intriguing, given that gene-targeted mutations in TEF-1 result in cardiac defects. Future challenges will be to determine whether genes encoding these putative Sd-interacting domains actually function as TIFs for TEF-1 or related proteins, and whether distinct regulatory TIFs have evolved that adapt the transcriptional activities of conserved Sd/TEF-1 homologs to specific functions in different tissues in their respective organisms (Simmonds, 1998).

Scalloped interacts with Yorkie, the nuclear effector of the hippo tumor-suppressor pathway in Drosophila

In Drosophila, Scalloped (Sd) belongs to a family of evolutionarily conserved proteins characterized by the presence of a TEA/ATTS DNA-binding domain. Sd physically interacts with the product of the vestigial (vg) gene, where the dimer functions as a master gene controlling wing formation. The Vg-SD dimer activates the transcription of several specific wing genes, including sd and vg themselves. The dimer drives cell-cycle progression by inducing expression of the dE2F1 transcription factor, which regulates genes involved in DNA replication and cell-cycle progression. Yorkie (Yki) is a transcriptional coactivator that is the downstream effector of the Hippo signaling pathway, which controls cell proliferation and apoptosis in Drosophila. This study identified Sd as a partner for Yki. Interaction between Yki and Sd increases Sd transcriptional activity both ex vivo in Drosophila S2 cells and in vivo in Drosophila wing discs and promotes Yki nuclear localization. Yki overexpression induces vg and dE2F1 expression, and proliferation induced by Yki or by a dominant-negative form of Fat in wing disc is significantly reduced in a sd hypomorphic mutant context. Contrary to Yki, Sd is not required in all imaginal tissues. This indicates that Yki-Sd interaction acts in a tissue-specific fashion and that other Yki partners must exist (Goulev, 2008).

Yorkie (Yki) encodes a transcriptional coactivator that is the downstream effector of the Salvador-Warts-Hippo signaling pathway. This pathway consists of the two serine-threonine kinases, Hippo and Warts (Wts, also known as Large Tumor Suppressor), the adaptator molecules Salvador (Sav) and Mob as Tumor Suppressor (MATS), the FERM domain proteins Expanded (Ex) and Merlin (Mer), and the protocadherin Fat (Ft). In this pathway, the Hpo kinase phosphorylates Wts, which in turn phosphorylates and inactivates Yki by excluding it from the nucleus. Mer and Es colocalize at the cell cortex and act upstream of Hpo to regulate the activity of Yki. Fat acts upstream of the Hippo signaling cascade by recruiting Ex to the apical plasma membrane and modulating the abundance of Wts. Inactivation of ft, hpo, wts, and sav, inactivation of both ex and mer, and overexpression of yki all lead to increased proliferation and reduced apoptosis, This pathway acts through Yki to regulate the expression of cyclin E (cycE) and the Drosophila inhibitor of apoptosis 1 (diap-1), which are involved in cell-cycle progression and cell-death inhibition, respectively, and the microRNA bantam, which can promote growth and inhibit apoptosis. The Yki peptide shares significant homology with the mammalian Yes Associated Protein 65 (YAP) (31% identity with human YAP). A particular region near the amino terminus of Yki (amino acids 57-114) displays the highest homology with YAP (56.2% identity). This region in YAP corresponds to a domain that binds all mammalian transcription-enhancer factors (TEFs), which are the homologs of the Drosophila Sd protein (Goulev, 2008).

Conservation between mammalian YAP and Yki of the YAP domain that interacts with TEF suggests that Yki might interact with Sd. To investigate this possibility, glutathione S-transferase (GST)-pull-down experiments were conducted. ³⁵S-labeled Yki specifically binds to GST-Sd beads (Goulev, 2008).

These results are in good accordance with a large two-hybrid screen in which Sd was identified as a binding partner for Yki. To refine the Sd domain that binds Yki and the region of Yki involved in the interaction with Sd, the binding of deleted Sd or Yki peptides was examined. Results that were obtained indicate a requirement of the N terminus of Yki and the C terminus domain of Sd. These results implicate Yki as an auxiliary protein that specifically interacts with Sd and demonstrate that this ability to interact is conserved between TEA factors (TEF-1 and Sd) and YAP factors (YAP and Yki) (Goulev, 2008).

To determine whether Sd, which possesses a putative nuclear localization signal (NLS) within the TEA domain, can modify Yki localization, transfection experiments were performed with plasmids expressing Sd fused to FLAG sequence and Yki fused to hemagglutinin (HA) sequence. In Drosophila S2 cells, Yki is mainly cytoplasmic. S2 cells were transfected with Yki and Sd and it was observed that Yki is exclusively nuclear. The nuclear translocation of Yki when Sd is expressed argues in favor of the idea that Sd and Yki interact ex vivo. Co-transfection experiments indicate that Yki interacts in a cellular context with Sd to stimulate the transcriptional activity conferred by Sd and additional experiments indicate that Vg and Yki do not compete for binding to Sd (Goulev, 2008).

It has been shown that the transcriptional activity of Yki is suppressed by coexpression of upstream components of the Hippo pathway. The transcriptional activity of Sd-Vg complex was analyzed when Hpo was overexpressed. Coexpression of GAL4_db-Sd and Vg results in a powerful induction of luciferase activity. This induction was suppressed when Hpo was overexpressed. Although it cannot be excluded that Hpo used a mechanism other than Yki inhibition, the most likely expanation for these results is that the Hippo pathway, and thus Yki function, is required for Sd-Vg activity (Goulev, 2008).

It has been proposed that, in the Sd-Vg complex, Vg provides the activating component, and that the binding of Vg to Sd switches the target selectivity of Sd. If it is assumed that the Sd-Vg-Yki complex is able to form into a cell, one attractive explanation for the role of interaction between Yki and Sd is that Yki provides the main activating component and Vg undergoes a conformational change in Sd ensuring accurate DNA target selection (Goulev, 2008).

To explore the possibility that Yki function is evolutionarily conserved, the effects of Yki on TEF transcriptional activity was tested in a HeLa human cell line. Yki, similarly to YAP, stimulate TEF1 transcriptional activity. These results reveal an evolutionarily conserved regulatory mechanism. Interestingly, it has recently been shown that YAP is the effector of the mammalian Hippo pathway. It is amplified in mouse tumor models and has several oncogenic properties. Furthermore, YAP can functionally substitute for Yki in Drosophila. The relevance of the YAP-TEF interaction in this pathway, however, remains to be determined (Goulev, 2008).

To determine whether Yki-Sd interaction regulates Sd transcriptional activity in vivo in Drosophila, a sensor transgene, 'hsp70-GAL4_db-sd', expressing a modified Sd protein was used, in which the TEA DNA-binding domain has been replaced by that of the yeast GAL4 DNA-binding domain, under the control of the hsp70 promoter. GAL4::Sd activity, monitored by the UAS-lacZ-responsive reporter transgene, is restricted to the wing pouch, where Sd dimerizes with Vg. A significant reduction in lacZ activity was observed in wing discs heterozygous for the amorphic yki^B5 allele compared to control wing discs. The fact that Sd activity is sensitive to yki dosage in wing imaginal discs supports the idea that Yki is required for Sd transcriptional activity in vivo (Goulev, 2008).

To better understand the functional relationship between Yki and Sd during wing development, genetic interactions between these genes was assessed. The results indicate that reduction of both vg and yki dosage enhances the sd phenotype and support the hypothesis that Yki works together with Sd-Vg during wing development (Goulev, 2008).

It has been shown that binding sites for Sd and Sd-Vg are necessary for regulation of vg expression and thus may regulate by a feedback mechanism vg expression. Results obtained in S2 cells indicate that Yki increases Sd transcriptional activity. This prompted an investigation of whether yki overexpression in imaginal discs enhances Vg-Sd activity, thus regulating vg expression and vgQE activity. yki overexpression was drived by using a UAS-GAL4 system or by flip-out (FLP-FRT) recombination in clones. In both cases, overexpression of yki was shown to induce vg and vgQE expression in a cell-autonomous manner in wing discs and in haltere discs (Goulev, 2008).

Several sets of data argue strongly in favor of an involvement of Vg-Sd in cell proliferation and cell-cycle progression in wing imaginal discs. vg^null cell clones do not proliferate in the wing pouch, whereas they can be recovered in the notum region, where vg is not expressed, indicating that vg is required for the proliferation of wing blade cells or for their survival. In contrast, vg ectopic expression induces wing outgrowths and dE2F1 expression. dE2F1 is the Drosophila homolog of the E2F transcription-factor family that plays a pivotal role during cell-cycle progression in controlling expression of different genes involved in G1-S transition (including cycE, a target gene of the Hippo pathway) and DNA replication. It was found that Yki overexpression with the ptc-GAL4 driver clearly upregulates expression of the dE2F1-LacZ reporter strain in a cell-autonomous manner. This indicates that Yki and Vg-Sd both activate dE2F1 expression and might work together in this process. It can be hypothesized either that Sd-Vg is required for dE2F1 induction by Yki or that Yki induces dE2F1 independently from Vg-Sd and reinforces dE2F1 expression through Sd-Vg induction (Goulev, 2008).

To determine more precisely the relationship between Sd and Yki in cell proliferation, the effect of Yki overexpression was tested in a mutant context for sd. Strong hypomorph alleles of sd are characterized by an absence of wing pouch cells. In contrast, sd^ETX4 corresponds to a weak allele of sd in which a large number of wing pouch cells are still present. This allele was used to exclude the possibility that the observed effect resulted from an absence of cell proliferation in the wing pouch because of a lack of sd expression. Wing discs overexpressing yki with the en-GAL4 driver exhibit clear increases in size of the posterior region, resulting in massive overgrowth. In contrast, the size of the posterior compartment was almost normal in wing discs from flies hemizygous for sd^ETX4 and overexpressing yki with the en-GAL4 driver. This result shows that Yki needs Sd to fully induce cell proliferation. Next, it was asked whether Sd-Yki interaction is acting downstream of the Hippo pathway. To test this, use was made of a dominant-negative form of Ft that lacks the intracellular domain (FTΔICD) . Indeed, Ft is an upstream component of the Hippo pathway, and overexpression of FTΔICD induces overgrowth and the expression of Hippo target genes. A significant reduction was observed of induced overgrowth in sd^ETX4 wing discs. This result strongly suggests that Sd-Yki interaction acts downstream of the Hippo pathway in wing discs (Goulev, 2008).

This paper has addressed the role of Sd-Yki interaction in the wing disc. The role of Sd in other imaginal tissues is poorly understood. Strong alleles of sd are associated with lethality in early larval stage. In the eye disc, clones homozygous for a strong allele of sd die or poorly survived, whereas they are associated with truncated legs when generated in the leg disc, suggesting that Sd should play a role in cell survival in these tissues. In the wing disc, sd clones die in the wing pouch but can be easily recovered in regions that will give rise to the notum, whereas Yki is required in the entire wing disc. This implies that other partners must interact with Yki to promote tissue growth in other structures. However, the induction of vg that was observed in cells overexpressing yki and the results showing that proliferation induced by Yki in the wing disc is significantly reduced in sd mutant context suggest that Sd-Vg is required for Yki function in the wing disc (Goulev, 2008).

The Scalloped and Nerfin-1 transcription factors cooperate to maintain neuronal cell fate

The ability of cells to stably maintain their fate is governed by specific transcription regulators. This study shows that the Scalloped (Sd) and Nervous fingers-1 (Nerfin-1) transcription factors physically and functionally interact to maintain medulla neuron fate in the Drosophila melanogaster CNS. Using Targeted DamID, Sd and Nerfin-1 were found to occupy a highly overlapping set of target genes, including regulators of neural stem cell and neuron fate, and signaling pathways that regulate CNS development such as Notch and Hippo. Modulation of either Sd or Nerfin-1 activity causes medulla neurons to dedifferentiate to a stem cell-like state, and this is mediated at least in part by Notch pathway deregulation. Intriguingly, orthologs of Sd and Nerfin-1 have also been implicated in control of neuronal cell fate decisions in both worms and mammals. These data indicate that this transcription factor pair exhibits remarkable biochemical and functional conservation across metazoans (Vissers, 2018).

When cells differentiate, they must maintain their fate in a stable manner and repress their ability to adopt alternate cell fates. This is essential for the function of differentiated cells and, when aberrant, can result in pathological consequences. The mechanism by which neuronal cell fate is stably maintained is incompletely understood, with only a handful of factors being linked to this process. This study demonstrates that Nerfin-1 maintains the fate of medulla neurons in the optic lobes of the D. melanogaster CNS, in partnership with the TEA domain transcription factor Sd. The data are consistent with the idea that these proteins operate as a transcription factor pair, given that they form a physical complex, and bind to a highly overlapping set of genomic loci. Putative Sd/Nerfin-1 targets were enriched for genes that are functionally associated with the fate of neurons and neuroblasts, cellular metabolism, as well as developmental signaling pathways such as Notch and Hippo. Given that forced activation of Sd target genes induced reversion of medulla neurons to neural stem cells (NSCs), it is hypothesized that aberrant activation of genes that Sd and Nerfin-1 regulate, is the primary driver of neuronal dedifferentiation in their absence (Vissers, 2018).

The Notch pathway was identified as a key target for regulation by Nerfin-1 and Sd, because (1) expression of multiple Notch pathway members was elevated when either Sd or Nerfin-1 function was perturbed; (2) Notch activity was required for dedifferentiation caused by Sd or Nerfin-1 deregulation; and (3) expression of a hyperactive Notch transgene was sufficient to induce dedifferentiation of medulla neurons. Recently, a requirement of Nerfin-1 has been identified in maintaining the differentiated status of neurons in the ventral nerve cord, central brain, and medulla lineages (Froldi, 2015, Xu, 2017). The latter study also demonstrated a requirement for Notch hyperactivity in mediating medulla neuron dedifferentiation following nerfin-1 loss. The current study extend these studies by showing that Nerfin-1 regulates neuronal maintenance in partnership with Sd. Furthermore, this study demonstrates that these transcription factors promote neuronal fate by regulating the expression of multiple Notch pathway genes (Vissers, 2018).

These data further demonstrate that neuronal cell fate is maintained by distinct factors in different regions of the CNS. In contrast to Nerfin-1, which is required to maintain neuronal differentiation in several neuroblast lineages, Sd is specifically required to maintain the fate of medulla neurons but not neurons derived from ventral nerve cord or central brain neuroblast lineages. The CNS region-specific function of Sd, versus the general requirement for Nerfin-1, is reminiscent of that described for Lola and Prospero, where Lola is required to maintain medulla neuronal fate, but acts redundantly with Prospero in other regions of the CNS. Future studies will elucidate the cooperative transcriptional networks that govern neuronal fate maintenance in different regions of the CNS (Vissers, 2018).

Sd function has been best studied in the context of Hippo pathway-dependent tissue growth, where it serves as the key transcription factor of the Yki transcriptional co-activator. Sd has also been linked to regulation of transcription with other proteins, such as Vestigial, Tondu-domain-containing Growth Inhibitor (Tgi), and in this study, Nerfin-1. Interestingly, the Hippo pathway was among the top signaling pathways identified in KEGG analyses on putative Sd/Nerfin-1 target genes. However, Sd promotes medulla neuronal fate independent of Hippo and Yki, as Yki is not obviously expressed in medulla neurons and cannot induce dedifferentiation. Interestingly, this contrasts with the reported role of Hippo, Yki, and Sd in other neurons. For example, these proteins operate together to control the fate of R8 photoreceptor neurons of the D. melanogaster eye. In one class of these light-sensing neurons, Yki and Sd are required to adopt a fate that allows the sensing of blue light, whereas in the other subtype, which senses green light, the Hippo pathway represses Yki and Sd activity (Vissers, 2018).

Orthologs of Sd and Nerfin-1 have been functionally linked in both C. elegans and vertebrates. In C. elegans, EGL-44 and EGL-46 form a physical complex and are both required to specify neuronal cell fate and Q neuroblast differentiation. By characterizing both the biochemical interaction of Sd and Nerfin-1 and their role in maintenance of neuronal fate, this study shows that they cooperate to perform similar functions in flies and worms. The vertebrate orthologs of Sd and Nerfin-1 (TEAD1-4 and INSM1) have also been implicated in various aspects of neural and neuroendocrine development. Murine INSM1 is required for the development of endocrine and neuroendocrine cells of the pancreas, intestine, pituitary, and lung, while in the CNS, it is required for differentiation of neural progenitor cells. Furthermore, zebrafish insm1a has been implicated in dedifferentiation in the context of Müller glia regeneration. Similarly, expression of a TEAD gain-of-function allele caused a marked expansion of the neural progenitor pool in the developing chick neural tube. Interestingly, preliminary studies suggest that the TEAD/INSMI pair might also operate together in vertebrates; a motif corresponding to the TEAD binding site was enriched in INSM1 target genes, as determined by INSM1 ChIP-seq performed in murine pancreatic beta cells. These studies of Sd and Nerfin-1 in the Drosophila CNS, coupled with the finding that human TEAD1 and INSM1 form a physical complex, further strengthen the idea that these proteins represent an evolutionarily conserved transcription factor pair (Vissers, 2018).

Opposing transcriptional and post-transcriptional roles for Scalloped in binary Hippo-dependent neural fate decisions

The Hippo tumor suppressor pathway plays many fundamental cell biological roles during animal development. Two central players in controlling Hippo-dependent gene expression are the TEAD transcription factor Scalloped (Sd) and its transcriptional co-activator Yorkie (Yki). Hippo signaling phosphorylates Yki, thereby blocking Yki-dependent transcriptional control. In post-mitotic Drosophila photoreceptors, a bistable negative feedback loop forms between the Hippo-dependent kinase Warts/Lats and Yki to lock in green vs blue-sensitive neuronal subtype choices, respectively. Previous experiments indicate that sd and yki mutants phenocopy each other's functions, both being required for promoting the expression of the blue photoreceptor fate determinant melted and the blue-sensitive opsin Rh5. This study demonstrates that Sd ensures the robustness of this neuronal fate decision via multiple antagonistic gene regulatory roles. In Hippo-positive (green) photoreceptors, Sd directly represses both melt and Rh5 gene expression through defined TEAD binding sites, a mechanism that is antagonized by Yki in Hippo-negative (blue) cells. Additionally, in blue photoreceptors, Sd is required to promote the translation of the Rh5 protein through a 3'UTR-dependent and microRNA-mediated process. Together, these studies reveal that Sd can drive context-dependent cell fate decisions through opposing transcriptional and post-transcriptional mechanisms (Xie, 2019).

Ensuring that the correct complement of genes remains on or off in any given cell type is an essential feature of multicellular organisms. This is particularly critical in the peripheral nervous system, where exclusive sensory receptor expression is necessary for selective and specific activation of a given sensory neuron. Such exclusion is well-established in the visual system of most animals, where individual photoreceptors (PRs) express a single opsin photopigment and repress the expression of others to prevent sensory overlap. The gene regulatory mechanisms underlying this mutual exclusion, however, are still under investigation (Xie, 2019).

The Drosophila eye has long served as a powerful model to understand the functions and architecture of gene regulatory networks underlying PR subtype cell fate specification. Each of the approximately 750 individual eye units (ommatidia) in the Drosophila compound eye contains 8 PRs. Based on the specific opsin that is expressed in the R8 photoreceptor, two major ommatidial subtypes, pale (p) and yellow (y), are present in the adult eye. Pale ommatidia are primarily defined based on the expression of the blue-sensitive opsin, Rhodopsin 5 (Rh5), while yellow ommatidia express the green-sensitive opsin, Rh6. These ommatidial subtypes are randomly distributed through the eye in a 30:70 blue:green ratio, and are established and maintained through a bistable negative feedback loop between two signaling molecules: the pleckstrin homology-containing protein Melted (Melt) and the Hippo signaling kinase Warts (Wts, aka Lats) (Xie, 2019).

Wts is a core component of the Hippo kinase complex that phosphorylates and inactivates the transcriptional co-activator Yorkie (Yki). Hippo signaling is best understood in the context of growth regulation, where Wts and Yki function in a homeostatic feedback loop: Wts blocks Yki function and Yki initiates its own inactivation by promoting Hippo pathway gene expression. In contrast, in post-mitotic PR fate decisions, Yki promotes the expression of the wts repressor, melt, generating a double-negative 'on/off' feedback loop between wts and Yki that ensures two stably maintained fate choices. In green PRs, Hippo signaling promotes the expression of green fate determinants (wts and Rh6), and prevents the expression of Yki-dependent blue fate determinants (melt and Rh5). In blue PRs, Yki promotes melt, thereby repressing wts and inhibiting Hippo signaling, further promoting Yki-dependent activation of blue fate effectors and suppression of green fate effectors. Thus, Wts-positive (Yki-inactive) cells adopt the default green/wts/Rh6 fate, while Wts-negative (Yki-active) cells acquire the blue/melt/Rh5 fate (Xie, 2019).

Yki, a YES-associated protein (YAP), is a transcriptional co-activator that does not bind DNA itself, but instead requires a DNA-binding partner. The primary binding partners for Yki/Yap factors are members of the TEAD family of transcription factors. In Drosophila, the single TEAD family member is encoded by Scalloped (Sd). Sd/TEAD and Yki/YAP can physically interact and together activate TEAD-site-containing reporter expression in vitro. Furthermore, in ectopic yki conditions, sd/TEAD is essential for yki/YAP to induce tissue overgrowth and activate target gene expression. However, in vivo, sd mutants do not phenocopy yki growth phenotypes and sd mutants do not show changes in yki target gene expression. These data suggest that Sd and Yki use distinct mechanisms to control tissue size. Studies aimed at addressing this conundrum have shown that in developing wing, eye, and follicle cells, Sd functions as a transcriptional repressor under 'Hippo-on' conditions to inhibit cell growth, and that in 'Hippo-off' cells, Yki antagonizes Sd repression to promote growth regulatory genes. This suggests that Sd and Yki can play opposite roles during growth (Xie, 2019).

In post-mitotic PRs, it has been previously shown that sd mutants phenocopy yki's knockdown phenotype in PR subtype fate specification: both sd and yki are necessary to promote blue PR fate and inhibit green PR fate. Combined, these findings suggest that sd and yki function together in this cell fate specification event. This study investigated the molecular basis underlying this interaction. Sd was found to play roles at both the transcriptional and post-transcriptional level to ensure blue vs green PR subtype fate decisions. At the transcriptional level, Sd directly represses blue fate effector gene expression in Hippo (Wts)-positive green PRs, and Yki antagonizes this repression in Hippo (Wts)-negative blue PRs. This is consistent with previously reported antagonism between Sd and Yki. In addition to this function, it was found that Sd promotes blue fate through a post-transcriptional, microRNA (miRNA)-dependent process in Wts-negative blue PRs, revealing a cooperative interaction with Yki in promoting blue PR fate. Together, these new findings elucidate a multi-tiered regulatory network involving the Drosophila TEAD transcription factor that functions at both the transcriptional and post-transcriptional level to precisely specify neuronal subtype fate (Xie, 2019).

The mutually exclusive expression of sensory receptor genes in sense organs is essential to prevent sensory input overlap in the mature organism. This study shows that, in the fly retina, the TEAD factor Sd achieves this in blue and green PRs using two different mechanisms: direct transcriptional repression of the blue fate determinant melt and blue Rh5 opsin genes in green photoreceptors, and relief of post-transcriptional control of the Rh5 mRNA in blue photoreceptors. In addition, Yki, a major Sd cofactor, antagonizes Hippo-specific and Sd-dependent repression of melt and Rh5 to promote blue PR fate. Thus, Sd and Yki play multiple roles to ensure a robust bistable cell fate decision in post-mitotic sensory neurons (Xie, 2019).

The antagonistic relationship between Sd repression and Yki de-repression is similar to the model previously proposed in cell cycle control. Nevertheless, the mechanisms by which Sd represses gene expression in green PRs remains unknown. In cell growth, for instance, repression is mediated in part through Tgi, a Tondu domain containing protein, which Yki competes with to alleviate repression. However, no significant change was detected in Rh5 protein or reporter expression with knockdown of Tgi in PRs, suggesting the existence of another Sd co-repressor in this system. Indeed, a zinc finger protein Nerfin-1 was recently identified as a Tgi-independent Sd co-repressor that participates in Hippo-dependent cell growth and competition during Drosophila eye development (Guo, 2019). Preliminary studies showed that knockdown of nerfin-1 led to an expansion of Rh5-expressing blue PRs at the expense of green PRs, comparable to the expanded expression of Sd site mutants in the melt and Rh5 reporters. Therefore, Nerfin-1 is very likely to be at least one Sd co-repressor during blue- and green PR fate specification in the Drosophila eye. Combined, these findings suggest Sd repression activity is a general mechanism in controlling the output of the Hippo pathway (Xie, 2019).

If the role of Sd in green PRs were solely to repress Rh5 transcription, then Rh5 mRNA levels might be expected to be elevated in sd mutants relative to controls. Instead, a ~50% reduction was observed. This observation could reflect two possibilities, which are not mutually exclusive. First, based on previous and unpublished findings that Otd cooperates with Yki to activate Rh5 in Hippo-negative blue PRs, it is expected that in sd mutants, where all R8s switch to Hippo-positive (and hence Yki-inactive) green PRs, Rh5 activation in green PRs would be reduced. Second, since the current studies suggest a new role for miRNAs in the post-transcriptional control of Rh5, it is possible that Rh5 mRNA stability is affected in sdmutants (Xie, 2019).

In terms of the post-transcriptional control of Rh5, it was demonstrated that the Rh5 3' UTR was required to prevent its co-expression with Rh6 in sd knockdown green PRs. In addition, the simultaneous knockdown of sd and miRNA processing machinery genes led to Rh5 protein de-repression (and co-expression with Rh6) in a substantial subset of green R8 cells. Together, these data suggest miRNA-dependent regulation of Rh5 depends on Sd, either directly or indirectly. It is posited that, as a transcription factor, Sd prevents the transcription of Rh5-directed miRNA genes. However, follow-up studies will be important for defining the complete repertoire of miRNA-dependent events involved in this Hippo-directed cell fate decision. For example, possible differences in an pRh5 reporter and endogenous Rh5 protein were reported in retinas mutant for the transcription factor PvuII-PstI homology 13 (pph13). While this disparity could be due to the rhabdomere defects observed in pph13 mutants, there is potential for a role for Pph13 in Rh5 post-transcriptional regulation. Finally, it is possible that the Rh5 3'UTR recruits other non-coding RNAs or proteins to regulate its expression (Xie, 2019).

Combined, the bimodal functions of Sd in Yki-vs Wts-positive cells form a feedforward regulatory module in post-mitotic PR fate decisions, robustly preventing sensory receptor overlap. Feedforward modules between transcription factors and miRNAs have been previously reported in neuronal differentiation and other biological processes. For example, the proto-transcription factor c-Myc can directly activate E2F1 transcription, but also limit E2F1 translation by activating miR-175p and miR-20a. In contrast to the c-Myc-miRNAs-E2F1 activation module, which fine-tunes a proliferative signal in dividing cells, however, the Sd-miRNA-Rh5 repression module ensures a robust ON-OFF switch in the terminal PR differentiation process. If similar mechanisms take place during Hippo-dependent cell growth remains to be determined (Xie, 2019).

Whether yki is also involved in Sd's post-transcriptional control in blue PRs remains unresolved, as yki itself is essential for blue PR fate, and hence, Rh5-expressing cells. Previous studies have demonstrated that Yki is important for the activation of at least one miRNA to promote cell growth (i.e. bantam). However, in the case of Rh5 regulation, the miRNA must be repressed in Yki-expressing cells, rather than activated. In this context, it is worth noting that the Yki ortholog YAP has been shown to mediate widespread miRNA suppression in tumor cells (Hippo-negative) by sequestering an RNA helicase p72/DDX-17, a regulatory component of microRNA-processing machinery. Comparably, the results suggest that the miRNA(s) is/are inactive in Yki-positive blue PRs in order to allow Rh5 protein expression. These findings raise the possibility that YAP/Yki- and TEAD/Sd-dependent regulation of miRNA biogenesis is a universal mechanism in control of the Hippo signaling pathway in tissue growth and neuronal cell fate decisions (Xie, 2019).

Pits and CtBP control tissue growth in Drosophila melanogaster with the Hippo pathway transcription repressor, Tgi

The Hippo pathway is an evolutionary conserved signalling network that regulates organ size, cell fate control and tumorigenesis. In the context of organ size control, the pathway incorporates a large variety of cellular cues such as cell polarity and adhesion into an integrated transcriptional response. The central Hippo signalling effector is the transcriptional co-activator Yorkie, which controls gene expression in partnership with different transcription factors, most notably Scalloped. When it is not activated by Yorkie, Scalloped can act as a repressor of transcription, at least in part due to its interaction with the corepressor protein Tgi. The mechanism by which Tgi represses transcription is incompletely understood and therefore this study sought to identify proteins that potentially operate together with it. Using an affinity purification and mass-spectrometry approach this study identified Pits and CtBP as Tgi-interacting proteins, both of which have been linked to transcriptional repression. Both Pits and CtBP were required for Tgi to suppress the growth of the Drosophila melanogaster eye and CtBP loss suppressed the undergrowth of yorkie mutant eye tissue. Furthermore, as reported previously for Tgi, overexpression of Pits repressed transcription of Hippo pathway target genes. These findings suggest that Tgi might operate together with Pits and CtBP to repress transcription of genes that normally promote tissue growth. The human orthologues of Tgi, CtBP and Pits (VGLL4, CTBP2 and IRF2BP2) have previously been shown to physically and functionally interact to control transcription, implying that the mechanism by which these proteins control transcriptional repression is conserved throughout evolution (Vissers, 2020).

The Hippo pathway was first discovered in Drosophila melanogaster genetic screens as an important regulator of organ growth. It has subsequently been shown to control the growth of multiple different tissues (epithelial, muscle, neural, and blood) in different species. It also controls cell fate choices in both D. melanogaster and mammals, while mutation of Hippo pathway genes underpins several human cancers. In the context of organ size control, the Hippo pathway responds to cell biological cues and its surrounding environment. For example, the Hippo pathway is regulated by cell-cell adhesion, cell-matrix contacts, cell polarity proteins, and by mechanical forces transmitted by the actin and spectrin cytoskeletons. These signals converge on a core signaling complex consisting of the Hippo (Hpo) and Warts (Wts) kinases and the adaptor proteins Salvador (Sav) and Mats. The central Hippo signaling effector is Yorkie (Yki), which is a transcriptional coactivator. Yki rapidly shuttles between the nucleus and cytoplasm and this is regulated by Wts-mediated phosphorylation at conserved amino acids, which limits access to the nucleus. When nuclear, Yki partners with sequence specific transcription factors to control expression of genes such as DIAP1, bantam, and cyclin E (Vissers, 2020).

The best-characterized Yki-interacting transcription factor is the TEA domain protein Scalloped (Sd). Yki and Sd promote transcription by interacting with chromatin-modifying proteins like the Mediator complex, the SWI-SNF complex, and the Trithorax-related histone methyltransferase complex (Zheng, 2019). The mammalian orthologs of Yki (YAP and TAZ) and Sd (TEAD1-TEAD4) regulate transcription by interacting with similar protein complexes. In human cells, YAP and TEAD regulate gene expression predominantly by binding to enhancers, as opposed to promoters. In addition, they promote transcriptional elongation by recruiting the Mediator complex and Cdk9 (Vissers, 2020).

The mechanism by which Sd and TEADs repress transcription is less well defined. Genetic evidence from tissues such as the D. melanogaster ovary indicates that Sd can act as a default repressor of transcription, and this activity is antagonized by Yki. Sd's default repressor function is mediated in part by the corepressor protein Tondu-domain-containing Growth Inhibitor (Tgi), also known as Sd-Binding Protein (Guo, 2013). To activate gene expression, Yki is thought to compete with Tgi for Sd binding and alleviate repression of target genes. Tgi interacts via its Tondu domains with Sd and via PY motifs with Yki's WW domains (Guo, 2013; Koontz, 2013). This relationship is conserved in mammals between YAP and TAZ, TEAD1-TEAD4, and VGLL4 (the Tgi ortholog). Sd can also regulate transcription together with the Zinc finger domain protein Nerfin-1. These proteins work in partnership to maintain the fate of D. melanogaster medulla neurons (Vissers, 2018) and they also influence cell competition in growing imaginal discs (Guo et al. 2019). Currently, the mechanism by which Tgi and VGLL4 cooperate with Sd/TEADs to control transcription and tissue growth is incompletely understood (Vissers, 2020).

To better understand how Tgi regulates transcription, proteomics approaches were used, and four high-confidence Tgi-interacting proteins were identified, all of which are transcriptional regulatory proteins. In addition to the known Tgi partners Yki and Sd, this study identified two previously unknown Tgi-interacting proteins, CG11138 (also known as protein interacting with Ttk69 and Sin3A, or Pits) and C-terminal binding protein (CtBP), both of which have been linked to transcriptional repression. Both gain and loss of function of pits and CtBP modified tissue growth aberrations caused by Hippo signaling defects. Furthermore, overexpression of Pits reduced expression of well-defined Hippo pathway target genes, thus highlighting the possibility that Pits and CtBP operate with Tgi to limit tissue growth and transcription (Vissers, 2020).

The transcriptional corepressor Tgi has emerged as an important regulator of transcription that is regulated by Yki and Sd, but the mechanism by which it does so is currently unclear. This study set out to address this by identifying Tgi-interacting proteins. Four such proteins were identified with high confidence, all of which have been ascribed functions as transcription regulators: the previously identified Hippo pathway proteins Yki and Sd, as well as Pits and CtBP. Previous structure-function studies showed that Tgi's growth inhibitory function fully depends on its ability to interact with Sd (Guo, 2013; Koontz, 2013), suggesting that Sd is the sole transcription factor that mediates Tgi's influence on transcription. The finding that Sd was the only sequence-specific transcription factor detected by mass spectrometry in Tgi purifications in S2 cells further supports this model. Additionally, biochemical data support the notion that Pits and CtBP function together with the Tgi corepressor to limit tissue growth, although genetic studies fall short of providing conclusive evidence for this. The fact that pits and ctbp were required for Tgi's ability to limit eye growth when overexpressed argues that Tgi requires them to repress gene expression through Sd. Further support for this idea comes from the finding that pits overexpression repressed the expression of the well-defined Yki/Sd/Tgi targets DIAP1 and ban. On the other hand, pits mutant flies were homozygous viable and displayed no obvious gross phenotypic abnormalities, and pits mutant larval eye imaginal discs cells expressed normal levels of DIAP1. However, it should be noted that loss of either sd or tgi in larval eye imaginal discs also has no obvious effect on expression of its target genes, while overexpression does. This suggests that in the growing larval eye imaginal disc Sd does not have a major role in gene repression. Alternatively, in the absence of sd, tgi, or pits, other proteins might compensate for them and regulate expression of their target genes (Vissers, 2020).

Strong genetic evidence that helped identify Tgi as a mediator of Sd's transcriptional repression activity was the finding that loss of tgi partially rescued the undergrowth of yki clones in larval eye imaginal discs (Koontz, 2013). Similarly, in the current study, loss of CtBP partially restored the undergrowth phenotype of yki larval eye clones. Therefore, CtBP might work in partnership with Tgi and Sd to repress target genes that are required for eye growth. Alternatively, CtBP might limit eye overgrowth by acting in parallel to Tgi and possibly also Sd. Indeed, a recent study provided evidence for both of these models with the discovery that CtBP represses transcription of the progrowth microRNA bantam in both Yki-dependent and independent manners. In contrast to CtBP loss, pits loss did not rescue the undergrowth phenotype of yki eye imaginal disc clones, which stands in apparent opposition to its requirement for growth inhibition induced by Tgi overexpression. The reason for this discrepancy is currently unclear. Future studies aimed at identifying the full suite of Yki/Sd/Tgi target genes that are required for mediating eye growth should help to provide clarity on the different roles of Yki, Sd, Tgi, CtBP and Pits on transcription and eye growth, but this genetic program is currently unknown (Vissers, 2020).

The mechanism by which Yki/Sd/Tgi regulate transcription in D. melanogaster appears to be largely conserved in mammals (Guo, 2013; Koontz, 2013; Zhang, 2014). This found that Pits and CtBP bind to Tgi through conserved protein motifs. Therefore, their biochemical relationship is also likely to be conserved in other species. Indeed, the interaction between the human CtBP and Tgi orthologs CTBP2 and VGLL4 has been reported, which inhibits adipogenesis of murine 3T3-L1 cells (Zhang, 2018). In addition, several studies have identified physical interactions between VGLL4 and the human Pits orthologs IRF2BP1, IRF2BP2, and IRF2BPL. A very recent study reported a functional interaction between these genes in the context of cancer: the Pits ortholog IRF2BP2 acted with VGLL4 to suppress liver tumor growth that was caused by YAP hyperactivity and also the expression of YAP-TEAD target genes (Feng, 2019). The D. melanogaster studies imply that Pits can act as a corepressor of Yki/Sd target genes, as opposed to an activator. In support of this, most studies of the three mammalian Pits orthologs IRF2BP1, IRF2BP2, and IRF2BPL indicate that they act as corepressors, although the mechanism of corepression is poorly characterized, and CtBP is generally considered a transcription repressor. Clearly, further studies are required to clarify the mechanism by which Hippo pathway target genes are regulated by Yki, Sd, and Tgi. To explore roles for Pits and CtBP on transcription in the growing eye, it will be important to examine their genome occupancy relative to Tgi and Sd, and also to better define the mechanism by which they regulate transcription. This should shed light on the control of Hippo pathway target genes and could also be valuable for defining the emerging role of the Tgi ortholog VGLL4 in human cancers (Vissers, 2020).

GENE STRUCTURE

Bases in 5' UTR - 596

Exons - 12

Bases in 3' UTR - 1021

PROTEIN STRUCTURE

Structural Domains

The TEA domain, homologous to that of human TEF-1, is a DNA binding domain. (Campbell, 1992). There is an N terminal serine-rich region.

date revised: 10 June 2024

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