BIOLOGICAL OVERVIEW

escargot is expressed in divergent cells throughout the embryo but it remains primarily ectodermal. Mutations in escargot cause defects in adult structures including the abdominal cuticle, wings and legs (Hayashi, 1993).

The action of ESG as a transcriptional repressor has been documented by measuring the interaction of ESG with E2 boxes, the consensus binding sequence of basic HLH transcription factors. Basic HLH proteins are transcriptional activators and bind very specific nucleotide sequences in gene promoters. ESG binds as a monomer to the E2 box, antagonizing transcriptional activation by Scute and Daughterless (Fuse, 1994). Expression in the neurogenic region and antagonism of Scute and Daughterless suggest that escargot opposes a proneural fate.

ESG regulates the cell cycle in histoblasts. Histoblasts are nests of cells in the abdomen, comprising integral components of the larval epidermis. These cells are precursors of the ectodermal component of the adult abdomen (Hartenstein, 1992).

Abdominal histoblasts in esg mutant larvae undergo endoreplication and become polyploid, preventing the development of adult abdominal epidermis. Endoreplication, which properly occurs in normal development as well as improperly in certain mutations, is the replication of DNA without concommitant cell division. This results in extra thick polytene chromosomes, because the DNA has duplicated beyond the normal diploid amount. Overexpression of esg rescues the abdominal phenotype in esg mutants while at the same time inhibiting normal endoreplication in salivary gland cells (Fuse, 1994).

It has been suggested that ESG represses the activation or somehow restricts the activity of unknown transcription factors responsible for entry into mitosis. Thus Escargot regulates the cell cycle (Fuse, 1994). Subsequent studies reveal that Escargot maintains the level of Dmcdc2, the catalytic kinase subunit of the G2/M cdk heterodimer. In turn, the heterodimer inhibits entry into S and the consequent endoreplication. Dmcdc2 heterodimerizes with cyclin A and cyclin B, both of which are required for entry into mitosis. It would seem that the G2/M cdk heterodimer has two functions: the regulation of entry into mitosis and the prevention of endoreplication (Hayashi, 1996).

Neurogenesis in Drosophila melanogaster starts by an ordered appearance of neuroblasts arranged in three columns (medial, intermediate and lateral) in each side (right and left) of the neuroectoderm. In the intermediate column, the receptor tyrosine kinase Egfr represses expression of proneural genes achaete and scute, and is required for the formation of neuroblasts. Most of the early function of Egfr is likely to be mediated by the Ras-MAP kinase signaling pathway, which is activated in the intermediate column, since a loss of a component of this pathway leads to a phenotype identical to that of Egfr mutants. MAP-kinase activation is also observed in the medial column where escargot (esg) and proneural gene expression are unaffected by Egfr. The homeobox gene ventral nerve system defective (vnd) is required for the expression of esg and scute in the medial column. vnd acts through the negative regulatory region of the esg enhancer that mediates the Egfr signal, suggesting vnd's role is to counteract Egfr-dependent repression. Thus, the nested expression of vnd and the Egfr activator Rhomboid is crucial to subdivide the neuroectoderm into the three dorsoventral domains (Yagi, 1998).

To investigate the involvement of Egfr in neurogenesis, mutant phenotypes of Efgr and its activator rho were examined at various stages of neurogenesis. The dorsoventral subdivision of the neuroectoderm in stage-6 embryos is detectable by expression of esg, which is expressed in the lateral and medial columns but not in the intermediate column. A loss-of-function, temperature-sensitive mutation of Egfr and a null mutation of rhomboid were used for analysis throughout this work. Egfr and rho mutations cause ectopic expression of esg in the intermediate column. Repression of esg in the intermediate column is likely to require a relatively high dose of Egfr signal. To examine the potential role of Egfr in neurogenesis, expression of the proneural genes ac and sc was carried out. These two proneural genes begin expression in the neuroectoderm of stage-7 embryos in a DV pattern of expression similar to that of esg in the previous stage. In Egfr and rho mutant embryos, ac and sc become ectopically expressed in the intermediate column. This phenotype is less penetrant and, occasionally, gaps of ac and sc expression are observed in the intermediate column. Since sc expression was similarly derepressed in Egfr mutant embryos, these phenotypes are likely to represent the near null phenotype of Egfr in the neuroectoderm. These data indicate that, in the intermediate column, the Egfr signal represses not only esg but also proneural genes, which are known to play key roles in neurogenesis. The effect of Egfr on neuroblast formation was monitored by the neuroblast marker Snail. Anti-Sna staining reveals three columns of SI neuroblasts in the control embryo: the intermediate column is distinguishable by the delayed onset of formation and number of Sna-positive cells. In Egfr and rho mutants, Sna-positive neuroblasts in the intermediate position are frequently missing, with a higher frequency of loss in Egfr embryos. In rho mutant embryos, the frequency of the loss of intermediate column neuroblasts is variable among embryos (Yagi, 1998).

To further examine the effect of the loss of Egfr signaling on the late events of neurogenesis, the progeny was traced for one of the intermediate neuroblasts, NB4-2. NB4-2 gives rise to the RP2 motor neurons, which can be identified by the expression of Even-skipped (Eve) and its unique position. Loss of RP2 neurons in stage 13 is observed (over half the cases examined) with the frequency of loss slightly higher in Egfr than in rho mutants, reflecting the earlier defect in neuroblast formation in stage 9. It is known that the Ras-MAPK signaling cascade is the major target of Egfr in many tissues. To understand whether Ras-MAPK signaling also mediates the Egfr signal in the neuroectoderm and to determine the relative contribution of each component of the pathway, the expression of esg and sc was examined in embryos lacking one of the Ras-MAPK signaling components. The phenotype of mutants lacking either Sos, Ras1, Draf or Dsor1 was examined. As in wild-type embryos, embryos mutant for any of the four genes examined express esg in three separate domains: procephalic neurogenic region, amnioserosa and neuroectoderm. In all cases, the anterior limit of the procephalic expression and the posterior limit of neuroectodermal expression are expanded to the terminus, consistent with the fact that Ras-MAPK is required for the terminal fate specification controlled by Torso receptor tyrosine kinase. All mutants exhibit specific defects within the neuroectoderm where esg expression is derepressed in the intermediate column. Essentially the same phenotype is also observed with sc expression, suggesting the loss of Ras-MAPK signaling has the same consequence as the loss of Egfr. All four Ras pathway mutants show, qualitatively, the same phenotype in the neuroectoderm. The neuroectoderm phenotype in Ras1 mutants is not rescued by a paternal copy of the wild-type gene, suggesting that a relatively high dose of the Ras signal is required for repression of esg and sc in the neuroectoderm (Yagi, 1998).

Rhomboid (rho) is initially expressed in the medial half of neuroectoderm, but repression of esg, ac and sc transcription by Egfr and Ras-MAPK occurs only in the intermediate column, posing a question as to whether or not the site of MAPK activation and the site of transcriptional repression exactly correspond. The spatial and temporal pattern of MAPK activation has been described by the use of an antibody that specifically reacts with the phosphorylated and activated form of MAPK (diphospho-MAPK=dpMAPK), which shows that dpMAPK is distributed in a broad domain in the neuroectoderm in stage 5-7 embryos. dpMAPK is distributed in an 8- to 10- cell-wide area in the neuroectoderm in stage-5 embryos and becomes restricted to the ventral region at the end of gastrulation. This rapidly evolving pattern of dpMAPK expression made it difficult to determine the exact correlation between distribution of dpMAPK and the DV subdomains in the neuroectoderm. A protocol was used to double label embryos with dpMAPK and antisense RNA probes to study the spatiotemporal relationship between expression of dpMAPK, its activator Rhomboid (Rho) and its downstream target, esg. Initial expression of dpMAPK overlaps with that of Rho in stage-5 embryos; dpMAPK expression remains in this broad domain when Rho expression became restricted to the medial column at gastrulation in stage 6, and finally narrows down to a 2- to 3-cell-wide stripe abutting the stripe of Rho at stage 7. Comparison with the mesodermal marker sna shows that the ventral border of dpMAPK expression abuts the neuroectoderm-mesoderm border. Examination of histochemically stained material reveals a sharp ventral border of dpMAPK expression, which gradually declines in the dorsal direction, resembling the pattern of Rho expression. In Egfr mutant embryos, dpMAPK staining is not detectable. These results demonstrate that MAPK activation in the neuroectoderm is dependent on Egfr and follows the spatial expression pattern of Rho, but persists for some time after termination of Rho transcription. The latter observation may reflect perdurance of Rho or its target protein, Spitz (Spi). Alternatively, a ligand other than Spi, such as Vein, might be activating Egfr. The dorsal limit of dpMAPK expression was determined relative to the three separate columns of neuroectoderm revealed by esg expression. In stage 5, the dorsal limit of dpMAPK reaches halfway within the intermediate column and subsequently retracts to the medial column in stage 6 and 7. These data indicate MAPK is activated at least in the ventral half of the intermediate column of the neuroectoderm when it is required to repress transcription of esg. It is concluded that transcription of esg is repressed by a marginal level of MAPK activation (Yagi, 1998).

Why does the high level of dpMAPK in the medial column fail to repress transcription of esg, ac and sc? One possibility is that a factor is present in the medial column that antagonizes or overcomes the events downstream of dpMAPK. A candidate for such a gene is vnd, which is expressed in the medial column in late stage 5 and is required for expression of ac. Expression of esg and sc was examined in vnd null mutant embryos: their expression in the medial column was found to be lost. To understand how vnd controls gene expression in the medial column, a target for vnd was sought in the cis-regulatory regions of an esg enhancer. Expression of esg is regulated by the neurogenic enhancer, which can be divided into two regions, the activator region, which mediates activation in the entire neuroectoderm, and the repressor region, which mediates Egfr-dependent repression. Expression of the esg-lacZ fusion genes was examined in the vnd mutant background. The construct esg-lacZ D1 containing the complete neurogenic enhancer reproduces neuroectodermal expression of esg and is regulated by vnd in the same manner as esg. In contrast, the construct esg-lacZ D5 lacks the repressor region for the Egfr-mediated regulation and is expressed in all three columns. Evidence is provided that vnd does not regulate esg-lacZ D5 and that the target site for vnd regulation is included in the repressor region. vnd is also shown not to be involved in activation of esg or Egfr; rather, it works to counteract the negative effect of Egfr (Yagi, 1998).

Given the results of the present work showing that vnd counteracts the negative regulatory effect of Egfr, a model is proposed for the DV structuring of the neuroectoderm. A gradient of nuclear localized Dorsal protein induces expression of dorsoventrally regulated genes such as dpp, sna, and twi, which determine the extent of the neuroectoderm, and the nested expression domains of rhomboid and vnd. rho determines the domain of MAPK activation, which covers the medial and intermediate columns. vnd is expressed in the medial column where it counteracts the Egfr signal to allow expression of esg. Thus the three columns in the stage 5-6 neuroectoderm are distinguished by unique combinations of activated MAPK and vnd expression. In the lateral column, neither of them are activated or expressed, and esg transcription is activated by default. In the intermediate column, MAPK is activated and represses esg transcription. In the medial column, vnd counteracts activated MAPK to allow the default pathway to activate esg transcription. It is possible that proneural genes are also regulated by the same mechanism. Loss of the Egfr signal leaves two domains, one with and the other without expression of vnd, the pattern likely to be reflected in the appearance of only two neuroblast columns in the later stage. Thus it is proposed that the primary role of Egfr signal in this stage is to define the intermediate domain to the neuroectoderm which is otherwise separated into two domains. It is possible that Egfr signal and vnd have later roles in promoting neuroblast formation in the intermediate and medial columns, respectively (Yagi, 1998).

Escargot maintains stemness and suppresses differentiation in Drosophila intestinal stem cells

Snail family transcription factors are expressed in various stem cell types, but their function in maintaining stem cell identity is unclear. In the adult Drosophila midgut, the Snail homolog Esg is expressed in intestinal stem cells (ISCs) and their transient undifferentiated daughters, termed enteroblasts (EB). Loss of esg in these progenitor cells causes their rapid differentiation into enterocytes (EC) or entero-endocrine cells (EE). Conversely, forced expression of Esg in intestinal progenitor cells blocks differentiation, locking ISCs in a stem cell state. Cell type-specific transcriptome analysis combined with Dam-ID binding studies identified Esg as a major repressor of differentiation genes in stem and progenitor cells. One critical target of Esg was found to be the POU-domain transcription factor, Pdm1, which is normally expressed specifically in differentiated ECs. Ectopic expression of Pdm1 in progenitor cells was sufficient to drive their differentiation into ECs. Hence, Esg is a critical stem cell determinant that maintains stemness by repressing differentiation-promoting factors, such as Pdm1 (Korzelis, 2014).

Stem cell identity is controlled by both extrinsic cues from the niche and cell-intrinsic transcriptional programs. Thus far, most studies of the Drosophila midgut have focused on the niche-derived signals that control midgut stem cell self-renewal. This study demonstrates a cell-intrinsic role for the Snail family transcription factor, Escargot, in controlling ISC self-renewal and differentiation. Loss of Esg leads to a rapid loss of all stem/progenitor cells in the midgut, due to their differentiation, whereas Esg overexpression keeps these cells permanently in an undifferentiated state. The dramatic effects of manipulating Esg levels support a central role for this Snail family member in controlling stem cell identity in the fly intestine (Korzelis, 2014).

A transcriptomics analysis indicated that Esg acts as a transcriptional repressor of a large diverse set of differentiation genes. These targets include transcription factors specific to ECs and EEs (Pdm1, Prospero) and genes used in digestion, immunity and cytoarchitectural specialization. Interestingly, one of these transcription factors, Pdm1, plays an important role in EC differentiation: ectopic expression of Pdm1 in progenitor cells was sufficient to trigger EC differentiation, partially mimicking the esg loss of function phenotype. The rapid loss of the Esg-expressing cell population upon Pdm1 overexpression suggests that Pdm1 might repress Esg expression, perhaps directly. In this case, Esg and Pdm1 together would constitute a negative feedback switch that governs EC differentiation (Korzelis, 2014).

Expression analysis also raised the possibility that Esg activates progenitor cell-specific genes in ISCs and EBs. These include the EGF signaling components Cbl, spitz, argos and Egfr as well as the Jak/Stat receptor domeless. Both EGFR and Jak/STAT pathways are crucial for ISC growth and maintenance, and receptivity to these signals is downregulated in differentiated ECs and EEs. While Snail family members are best understood as repressors, the Esg paralog Snail has been reported to function as a context-dependent transcriptional activator (Rembold, 2014), suggesting that an activating role for Esg is also plausible. The function of Esg as either an activator or repressor is likely determined by co-factors and/or other transcription factors acting on the same promoters that are expressed in the ISC and EB population. In the Drosophila embryo, Snail cooperates with Twist at distinct promoters to activate EMT gene expression during mesoderm formation (Rembold, 2014). Snail2 can bind to Sox9 to activate expression from its own promoter during chick neural crest formation. In its role as a repressor, Esg binds the co-repressor CtBP to maintain somatic Cyst stem cells and hub cells in the Drosophila male testis. Future work to unravel the complete transcriptional network within which Esg functions to maintain the stem/progenitor state should prove to be very interesting (Korzelis, 2014).

The data support a model in which Esg acts in a circuit with Delta-Notch signaling to control the switch from stem/progenitor identity to differentiated cell identities. In its simplest form, this circuit might be a bistable switch in which Esg and Notch mutually inhibited each other, with Esg being 'on' and dominant in progenitor cells and Notch signaling 'on' and dominant in their differentiated progeny, the enterocytes. However, the constant presence of a substantial population of intermediate progenitor cells, the enteroblasts (EBs), which express both Esg and Notch reporter genes, indicates that a simple bistable switch is not an accurate conception. Indeed, EBs, defined here as cells positive for both Esg and the Notch reporter Su(H)GBE-LacZ, can persist for many days in the absence of ISC division. Thus, the EB transition state is metastable. In this transition state, Notch is apparently active, but secondary downstream targets that directly affect differentiation, such as Pdm1, brush border Myosin and smooth septate junction proteins, remain repressed. Since these genes are rapidly activated following depletion of Esg, it is suggested that their repression is most likely mediated by Esg binding (Korzelis, 2014).

Two potential explanations are provided for the longevity of the EB transition state. First, it is suggested that the repression of esg transcription by Notch is indirect and that this delays esg silencing. Silencing of Esg is not likely to be mediated by the Notch-regulated transcription factor Su(H) (a transcriptional activator) but by downstream repressors that act only after enterocyte or endocrine differentiation has begun. Pdm1 in ECs and Prospero in EEs are presently the most obvious candidates. Both are specifically induced coincident with Esg silencing, in ECs and EEs, respectively, and Dam-ID assays suggest that Pros has binding sites in the esg locus. The finding that overexpression of Pdm1 caused the rapid differentiation of Esg+ stem/progenitor cells supports the notion that Pdm1 could directly repress Esg expression to control EC differentiation. Furthermore, nubbin/Pdm1 was found to restrict expression of Notch target genes in the Drosophila larval wing disc. Hence, Pdm1 likely triggers EC differentiation by downregulating both Esg and the expression of Notch target genes in the EB. Therefore, Notch is only transiently active in EBs but fully off in mature ECs with high levels of Pdm1 (Korzelis, 2014).

While a delay circuit that controls the silencing of Esg is likely, theoretically it cannot explain how Esg+ EBs can persist for such long periods during times of low gut epithelial turnover and then rapidly differentiate during gut regeneration. Hence, it is speculated that a second input signal acts in combination with Notch-dependent factor(s) to silence Esg. This second signal is likely to be a downstream effector of the growth factor signaling network that also drives ISC division and gut epithelial renewal. Of the transcriptional effectors involved in maintaining gut homeostasis, the most obvious candidate as an indirect mediator of esg repression is Stat92E, which is activated by the highly stress-dependent cytokines, Upd2 and Upd3. Tellingly, the cytokine receptor, Dome, Janus Kinase (hop) and Stat92E are all required for EB maturation into ECs. If the silencing of esg was dependent upon both Notch and Stat92E, and Delta-Notch signaling was irreversible once resolved; then, the Notch+ Esg+ EB transition state should in principle be stable in conditions of low Jak/Stat signaling, as is observed during periods of midgut quiescence. It needs to be noted, however, that ISCs and EBs maintain appreciable levels of Stat-reporter gene expression even during relative quiescence, and so, in this model, it would be Stat activity above some threshold that would combine with Notch signaling to trigger differentiation. Since Jak/Stat signaling also triggers ISC division, a surge in cytokine signaling could coordinately trigger both the differentiation of older EBs and the production of new ones in this model , thus explaining how a significant EB population is maintained even as stem cell activity waxes and wanes (Korzelis, 2014).

Snail family transcription factors have been described as regulators of epithelial-to-mesenchyme transitions (EMT) that occur during development, wound healing and cancer metastasis. In some contexts, notably metastasis, EMT is believed to accompany the acquisition of stem-like properties. Although Esg itself has not been reported to regulate EMT, its paralog in flies (Sna) and homologs in mammals (Snai1, Snai2) do promote EMT. Interestingly, RNA-seq experiments showed that not only Esg, but Snail, Worniu and the Zeb family members Zfh1 and Zfh2 were all expressed in intestinal stem cells and downregulated in ECs and EEs. Thus, these EMT-linked transcription factors may work together to affect different aspects of midgut homeostasis and ISC differentiation. Indeed, Esg-positive ISCs and EBs are morphologically more similar to mesenchymal cells than they are epithelial, whereas Esg-negative EEs and ECs have the pronounced apical-basal polarity typical of epithelial cells. Esg+ cells often make striking lateral projections, suggestive of dynamic behavior, and they have the capacity to multilayer when their differentiation is blocked or they are forced to overproliferate. Furthermore, a number of epithelial-class genes are repressed in Esg+ progenitors and activated upon EC and/or EE differentiation. These include genes encoding the apico-lateral cortical Lgl-Dlg-Scrib-Crb complex, septate junction proteins (e.g., Ssk, Cora, Mesh) and polarity factors including Par3 and Par6. Strikingly, Scrib and Ssk both have Esg-binding sites in their promoters, and their expression is highly regulated by Esg. However, some gene targets that are central to EMT in mammalian cells show opposite trends in the fly's ISC lineage. For instance, Esg+ progenitors express significant levels of integrins, and E-cadherin-typically lost during EMT-is highly upregulated specifically in ISCs and EBs. Thus, the Esg-regulated differentiation of Drosophila ISCs only partially resembles a mesenchymal-to-epithelial transition (MET) (Korzelis, 2014).

Esg's role in ISC maintenance nicely parallels the functions of other Snail family members in Drosophila and mammals. For instance, in Drosophila neuroblasts (neural stem cells), the Snail family member Worniu promotes self-renewal and represses neuronal differentiation. In mice, Snail family members have been associated with the regulation of the stem cell state in both normal and pathological conditions. For instance, mammary stem cells require the Snail family member Slug to retain their MaSC identity. Mouse Snai1 also represses the transition from the stem cell-like mitotically cycling trophoblast precursor cell to the endoreplicating trophoblast giant cell during rodent placental development. This process, which also requires a mitotic-to-endocycle switch upon differentiation, is strikingly similar to the role describe in this study for Esg in EC differentiation and its role during imaginal disc development (Korzelis, 2014).

More interesting yet, mouse Snai1 is specifically expressed and required for stem cell maintenance in the crypts of the mouse intestine and expands the stem cell population when overexpressed. However, few studies highlight the target genes responsible for the function of Snail family members in stem cell maintenance. One example is from mouse muscle progenitors (myoblasts), where Snai1 and Snai2 repress expression from MyoD target promoters and this is required to maintain their progenitor state. The work presented in this study shows that Esg affects many aspects of the differentiation process and that it can form a transcriptional switch with one of the targets it represses (Pdm1) to balance self-renewal and differentiation in this stem cell lineage. Together, these studies suggest that the function of Snail family transcription factors as repressors of differentiation genes is ancient and widespread and may be an essential component in balancing self-renewal with differentiation in diverse animal stem cell lineages (Korzelis, 2014).

GENE STRUCTURE

Bases in 3' UTR - 480

PROTEIN STRUCTURE

Amino Acids 470

Structural Domains

escargot encodes a protein with five zinc finger motifs, four of which are C2-H2 class.

Identification of an α-MoRF in the intrinsically disordered region of the Escargot transcription factor

Molecular recognition features (MoRFs) are common in intrinsically disordered proteins (IDPs) and intrinsically disordered regions (IDRs). MoRFs are in constant order-disorder structural transitions and adopt well-defined structures once they are bound to their targets. Escargot (Esg), a transcription factor in Drosophila melanogaster that regulates multiple cellular functions, consists of a disordered N-terminal domain and a group of zinc fingers at its C-terminal domain. The N-terminal domain of Esg was studied with disorder predictors, and a region of 45 amino acids was identified with high probability to form ordered structures, which was named S2. Through 54 μs of molecular dynamics (MD) simulations using CHARMM36 and implicit solvent (generalized Born/surface area (GBSA)), the conformational landscape of S2 was characterized, and an α-MoRF of ~16 amino acids was found stabilized by key contacts within the helix. To test the importance of these contacts in the stability of the α-MoRF, the effect of point mutations that would impair these interactions was evaluated, running 24 μs of MD for each mutation. The mutations had mild effects on the MoRF, and in some cases, led to gain of residual structure through long-range contacts of the α-MoRF and the rest of the S2 region. As this could be an effect of the force field and solvent model used, the simulation protocol was benchmarked by carrying out 32 μs of MD for the (AAQAA)(3) peptide. The results of the benchmark indicate that the global amount of helix in shorter peptides like (AAQAA)(3) is reasonably predicted. Careful analysis of the runs of S2 and its mutants suggests that the mutation to hydrophobic residues may have nucleated long-range hydrophobic and aromatic interactions that stabilize the MoRF. Finally, this study identified a set of residues that stabilize an α-MoRF in a region still without functional annotations in Esg (Hernandez-Segura, 2020).

date revised: 20 June 2023

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