BIOLOGICAL OVERVIEW

Gastrulation is a process of tissue involution that creates three germ layers from one. In Drosophila, mesoderm and endoderm are created by different invagination events. Mesoderm is created as a result of ventral furrow formation, one of the three invagination events taking place during gastrulation [Images]. Invagination is initiated by two regulatory genes, twist (twi) and snail (sna). sna is sufficient to initiate the invagination of the ventral-most embryonic cells in the absence of twist gene activity, but without twist the invaginated cells fail to express mesodermal genes under the control of twist, such as tinman and bagpipe. Low levels of SNA that fail to repress neuroectoderm determinants in the presumptive mesoderm are nonetheless able to promote invagination (Ip, 1994a).

snail is a transcriptional repressor. It acts to restrict neuroectoderm and neural fate in the invaginating mesoderm. snail mutants display a massive derepression of mesectodermal genes such as single-minded and rhomboid (Kasai, 1992 and Ip, 1992a).

The opposing actions of twist and snail can best be observed by examining the extent of altered mitotic domains in mutant embryos. The first 13 cleavage divisions of a wild type embryo are synchronous. The cells divide in uninterruped waves, like continuously cheering fans in a stadium. The cell membranes that will later separate individual cells have not yet formed, so each cell does exactly what its neighbors do.

Following cellularization, groups of cells enter mitosis 14 in an established non-synchronous spatial and temporal sequence. In twist mutants, the position of dividing cells in the mesectoderm is shifted closer to the ventral midline, indicating a smaller presumptive mesodermal domain. In snail mutants, groups of dividing cells that have been delayed in the first non-synchronous division are seen in the mid-ventral region, suggesting that they represent an expansion of the neuroectoderm. What had been presumptive mesoderm has been transformed into neuroectoderm (Aurora, 1992). This demonstrates that snail, in its capacity as a transcriptional repressor, assures the extent and integrity of the mesodermal domain.

Gastrulation and neuroectodermal gene expression are subject to differential regulation by Snail. In snail mutants, the ventral cell invagination during gastrulation is blocked and no mesoderm-derived tissue is formed. One of the functions of Snail is to repress neuroectodermal genes and restrict their expressions to the lateral regions. The derepression of the neuroectodermal genes into the ventral region in snail mutants is a possible cause of defects in gastrulation and in mesoderm differentiation. To investigate such a possibility, a series of snail mutant alleles was analyzed. Different neuroectodermal genes respond differently in various snail mutant backgrounds. Due to the differential response of target genes, one of the mutant alleles, V2, which manifests reduced Snail function, also shows an intermediate phenotype. In V2 embryos, neuroectodermal genes, such as single-minded and rhomboid, are derepressed while ventral invagination proceeds normally. However, the differentiation of these invaginated cells into mesodermal lineage is disrupted. The results suggest that the establishment of mesodermal cell fate requires the proper restriction of neuroectodermal genes, while the ventral cell movement is independent of the expression patterns of these genes. The expression of some ventral genes disappear in snail mutants. Snail function is required for activation of genes such as dGATAb (serpent) and zfh-1. It is proposed that Snail may repress or activate another set of target genes, including folded gastrulation, that are required specifically for gastrulation (Hemavathy, 1997).

snail has a second function later, as a regulator of neurogenesis. Its role here is independent of daughterless/achaete-scute, and it acts in both the central and peripheral nervous systems. The nature of snail's neural actions are uncharacterized. It may act to repress non neural fates. If so, then its role in the nervous system is the reverse of its role in the mesoderm (Ip, 1994b).

snail is also involved is the specification of the wing disc. In this role, snail is expressed in the ectoderm. escargot and snail are required for the maintenance of vestigial expression in the wing disc (Fuse, 1996).

The mesoderm determinant Snail collaborates with related zinc-finger proteins to control Drosophila neurogenesis

By the time of neuroblast delamination, Sna is present in most of the neuroblasts that have segregated from the ectoderm. Despite the extensive expression in the neuroblasts, prior to this study, Sna had no known function in the developing nervous system. The neuroblast pattern of sna resembles that of a group of genes called pan-neural genes. One of these genes, scratch (scrt), encodes a protein that has sequence similarity to Sna in the zinc-finger domain. Mutations of scrt have no obvious phenotype except that viable escapers have morphological defects in the eyes. Furthermore, no nervous system defect can be seen in sna scrt double mutants. However, the scrt dpn double mutants exhibit some defects in nervous system development. deadpan (dpn) is another pan-neural gene that encodes a basic helix-loop-helix protein. Therefore, scrt does have a function in the central nervous system (CNS), but the function of sna, if any, in the nervous system does not overlap with that of scrt (Ashraf, 1999 and references therein).

Escargot (Esg) is another protein that contains five zinc fingers with sequences highly homologous to those of Sna. The expression of esg is rather dynamic during embryonic development. The gene is expressed in the epidermis, neuroectoderm and imaginal precursor cells. The Esg protein probably acts through the cdc2 kinase to maintain the proper cell cycle in larval imaginal disc cells; in esg mutant larvae the imaginal disc cells lose their diploidy as they re-enter the S phase without going through mitosis. Moreover, esg and sna are both expressed in the embryonic wing imaginal disc primodia and the two genes have redundant functions in this tissue; the vestigial marker gene expression in the disc is lost in esg sna double mutants. Despite a clear demonstration of the redundant requirements of sna and esg in the wing disc, the double mutant has been reported to have no significant embryonic CNS phenotype. Thus, the function of sna in nervous system development has remained a mystery (Ashraf, 1999 and references therein).

Evidence is provided that CNS expression of Snail is required for nervous system development. The neural function of snail is masked by two closely linked genes, escargot and worniu. worniu (pronounced war-niu, Chinese for 'snail') encodes a protein with a zinc-finger domain highly homologous to those of Sna and Esg; it has been identified from the Berkeley Drosophila Genome Project database. RNA in situ hybridization reveals extensive expression of worniu in the developing nervous system. wor is located between esg and sna, ~100 kb apart, in the 35D region of the second chromosome. Although not affecting expression of early neuroblast markers, the deletion of the region containing all three genes correlates with loss of expression of CNS determinants including fushi tarazu, pdm-2 and even-skipped. Transgenic expression of each of the three Snail family proteins efficiently rescues the fushi tarazu defects, and partially rescues the pdm-2 and even-skipped CNS patterns. These results demonstrate that the Snail family proteins have essential functions during embryonic CNS development, around the time of ganglion mother cell formation (Ashraf, 1999).

The putative Wor protein sequence contains a C-terminal domain with six zinc fingers that are very similar to those of Sna and Esg, even though those proteins contain only five fingers. The N-terminal halves of these proteins have rather divergent sequences, except that they all contain a conserved basic motif very close to the N-termini. The function of this motif is not known. Moreover, the proteins contain two P-DLS-K motifs. The P-DLS-K domains in Sna have been shown to interact with the Drosophila C-terminal binding protein (dCtBP) and to play important roles in transcriptional repression. Since all three Sna family proteins contain highly homologous corepressor-interacting and DNA-binding domains, and can bind to similar DNA sequences, it is possible that they bind to promoters of overlapping sets of target genes and repress transcription (Ashraf, 1999 and references therein).

While there is no maternal RNA deposition of wor, zygotic expression can be detected first at the onset of neurogenesis. At a late stage 8, WOR transcript can be observed in two small patches of cells in the dorsal head region anterior to the cephalic furrow, representing precursor cells of the developing brain. At stage 9 wor expresses in the first wave of delaminating neuroblasts along either side of the midline, as well as in cells in the head region. Later in the germ band-extended embryo, most of the neuroblasts contain WOR mRNA. This pattern greatly resembles that of sna at this stage of development, except that sna expression in some of the centrally located neuroblasts in each hemisegment is at lower levels. In later stages, wor continues to express in the brain and part of the ventral nerve cord. No expression of wor is detected in any other embryonic tissue (Ashraf, 1999).

There is no extensive expression of esg in the neuroblasts similar to that shown for wor or sna. However, it has been demonstrated that esg RNA is expressed in the ventral neuroectoderm. Careful examination of the expression reveals that esg transcript is probably present in the CNS, albeit at variable levels. Based on the expression analyses, it is hypothesized that the newly identified wor might serve a redundant function with that of sna or esg during neural development. This would explain why neither single nor double mutants of sna and esg show severe defects in the nervous system (Ashraf, 1999).

To test the hypothesis that the Sna family proteins function redundantly in the developing nervous system, the neural phenotype associated with a deletion that uncovers all three genes was examined. wor is located between esg and sna, ~100 kb apart, in the 35D region of the second chromosome. Advantage of the close proximity of these genes and the phenotypes of a deficiency mutant are examined. Since high levels of Sna and Wor are present in the neuroblasts, the expression of the proneural gene achaete, which marks a subset of early delaminating neuroblasts was examined. This expression is not affected in the osp29 deficiency mutants. The expression patterns of additional neuroblast markers including hunchback, dpn, scrt and lethal of scute also are similar in wild-type and mutant embryos. Therefore, the early waves of neuroblast delamination are normal in the absence of the Snail family proteins (Ashraf, 1999).

The CNS patterns of GMC markers ftz, pdm-2 and eve were examined. ftz is expressed in a number of midline precursor cells and extensively in GMC. In contrast to the neuroblast markers, the ftz expression is almost abolished in the mutant embryo. The pdm-2 gene is also expressed in some neuroblasts and GMC. The early neuroblast expression of pdm-2 in the mutant is nearly normal, while the expression in later staged embryos is highly defective. eve gene products are present in a number of GMC and postmitotic neurons during normal development. All the eve CNS expression is absent in homozygous osp29 deletion mutant embryos. Taken together, the deletion mutant that uncovers the three sna family genes shows severe defects in CNS development (Ashraf, 1999).

To confirm the function of these three proteins in neural development, transgenic rescue plasmids were constructed in which individual genes (esg, wor or sna) were placed under the control of a sna promoter, containing an enhancer element that directs expression in the neuroblasts. The transgenic flies obtained were then crossed with the osp29 strain and analyzed for CNS development. In the presence of any one of the three constructs the ftz expression is restored significantly. Analysis of the rescued pattern under higher magnification reveals that part of the ftz staining is clearly absent. However, more detailed analysis is required to pinpoint the exact cell lineages that are missing. Nevertheless, the results demonstrate that each of the three sna family genes can perform essential functions in the CNS in the absence of the other two. The rescue by the transgenes of the expression of pdm-2 and eve, both of which are defective in the osp29 mutant, was also examined. While all three sna family genes clearly can rescue the expression of pdm-2 , the effect is not as extensive compared with that of ftz. For eve RNA, the transgenes rescue the expression in a significant number of cells when compared with the total loss of expression in the parental osp29 mutant. The rescue of eve, again, is not as extensive as that of ftz. Later stage CNS morphology in the rescued embryos was also monitored by BP102 staining. The embryos carrying the transgenes have slightly better overall CNS axonal morphology, but they are still highly abnormal when compared with the wild type (Ashraf, 1999).

Pairwise recombination of the transgenes were constructed and a test was performed to see whether they could achieve better rescue. By staining embryos obtained from stable lines that are homozygous for two transgenes, the constructs were found to give slightly direct the expression of ftz slightly better. Meanwhile, the eve and BP102 antigen expression in the presence of two transgenes reveals only minor improvement of the axonal morphology. These results suggest that the three proteins may have some collaborative function. It is also possible that the promoter used has some limitation in driving the rescue transgenes or that there are additional genes involved for the severe CNS phenotype (Ashraf, 1999).

Increasing numbers of sna-related genes have been identified in diverse species. These proteins have been assigned to the Sna family based mostly on the similarity of the sequences in the zinc-finger domains. The expression patterns and some functional studies of the vertebrate proteins suggest a role in regulating cell movement. However, gene knock-out experiments have demonstrated that mutating a mouse Slug homolog does not lead to a detectable cell movement defect. Such a result suggests a possible redundant function provided by other genes, similar to this report. If the vertebrate homologs do have a function in controlling cell movement, it would be reminiscent of the control of cell movement during gastrulation by Drosophila Sna. However, the expression of vertebrate Sna proteins in developing CNS has not been demonstrated. A careful examination of the expression and function in the CNS is needed to reveal the importance of Sna expression. The analysis of the functions of Sna, Esg and Wor in Drosophila CNS development will certainly provide a foundation for similar analysis in other species (Ashraf, 1999 and references therein).

Mitosis-associated repression in development

Transcriptional repression is a pervasive feature of animal development. This study employed live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. The study proposes that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail (Esposito, 2016).

The enhancers tested in this study were derived from two different dorsal-ventral patterning genes: brk and sog; both encode inhibitors of BMP signaling. The brk enhancer is located ~10 kb upstream of the transcription start site, while the sog enhancer is located within the first intron of the transcription unit, ~1.5 kb downstream from the start site. Each enhancer was placed immediately upstream of its cognate promoter and attached to a yellow reporter gene containing 24 MS2 stem-loops within the 5' untranslated region (UTR). Nascent transcripts were visualized in living embryos using a maternally expressed MCP-GFP fusion protein (Esposito, 2016).

Both transgenes recapitulate the expression profiles of the endogenous genes; namely, they are activated throughout the presumptive mesoderm and neurogenic ectoderm and then repressed in the mesoderm. Prior studies with fixed embryos suggest that these enhancers respond to different levels of the Snail repressor (e.g., see Bothma et al. 2011). The brk 5' enhancer appears to be more efficiently repressed by Snail as compared with the sog intronic enhancer. Both transgenes were examined in living embryos to determine whether they exhibit distinctive repression dynamics (Esposito, 2016).

The btk>MS2 transgene exhibits an expression profile that is similar to that seen for the endogenous locus based on conventional in situ hybridization methods. The main difference is that the transgene produces a slightly narrower pattern due to the absence of the 3' 'shadow' enhancer (Esposito, 2016).

There is broad activation of the btk>MS2 transgene in both ventral and lateral regions during nuclear cleavage cycles 10-13 (nc10-nc13). btk>MS2 nascent transcripts are lost during the general silencing of transcription at each mitosis. Interestingly, upon reactivation of the transgene at the onset of nc14, a sudden loss of de novo transcription was observed in the mesoderm. Transcripts are restricted to the neurogenic ectoderm, suggesting that the mature brk expression pattern is established immediately following mitosis (Esposito, 2016).

In an effort to quantify the dynamics of this repression, individual nuclei were partitioned within the presumptive mesoderm and ectoderm and the fraction of active nuclei was calculated in these regions throughout nc13-nc14. Active nuclei are defined as those exhibiting nascent RNA signals in at least one z-series (20 sec). No significant variation was observed in the fraction of active nuclei between the mesoderm and lateral ectoderm during nc13. However, at the onset of nc14, ~90% of the nuclei in the mesoderm are silenced, while expression persists in the lateral ectoderm (Esposito, 2016).

To obtain more detailed information on the dynamics of repression in the ventral mesoderm, the fluorescence of individual transcription foci was quantified, since previous studies have shown that it scales with the number of RNA polymerase II (Pol II) complexes engaged in active transcription. Quantitative analysis of the btk>MS2 transgene reveals that the ventral-most nuclei in the mesoderm exhibit ~25% reduction in signal intensity during nc13 as compared with active nuclei in the lateral ectoderm. The majority of nuclei that display nascent transcripts at the onset of nc14 is located in the ectoderm, and the few active nuclei in the mesoderm show a substantial reduction in signal intensity (~60% reduction). Thus, Snail begins to attenuate the brk 5' enhancer during nc13, and this repression appears to be strongly reinforced during mitosis. It is suggested that mitotic silencing augments the activity of the localized Snail repressor (Esposito, 2016).

sog is regulated by two enhancers with overlapping activities: a distal enhancer located ~20 kb upstream of the sog promoter and an intronic enhancer located ~1.5 kb downstream from the transcription start site. The distal 5' enhancer contains high-affinity Snail-binding sites and exhibits repression dynamics similar to that of the btk>MS2 transgene following mitosis. The intronic enhancer contains weak Snail-binding sites and shows modest repression in the ventral mesoderm of wild-type embryos. Most nuclei are reactivated following mitotic silencing but display a significant reduction (more than twofold) in expression (Esposito, 2016).

To explore the dynamics of sog repression, embryos carrying three copies of the snail locus were examined. They exhibit significantly more complete repression of the sog>MS2 transgene during the onset of nc14 as compared with wild-type embryos. Fewer than half the nuclei in the mesoderm reactivate sog>MS2 expression at the onset of nc14 (Esposito, 2016).

The preceding results suggest a clear correlation between mitotic silencing and repression of the btk>MS2 transgene and attenuation of sog>MS2. There is an approximately twofold reduction in the levels of sog>MS2 expression in the mesoderm following mitotic silencing in wild-type embryos and a substantial reduction in the number of nuclei that reactivate the transgene at the onset of nc14 in embryos containing three copies of snail. Nonetheless, it is not clear whether the ~8-min interval during mitosis is a more effective period of Snail-mediated repression than a comparable interphase period (Esposito, 2016).

Additional support stems from the analysis of a rare haploid embryo. Following fertilization, the paternal and maternal haploid pronuclei sometimes fail to fuse, and development proceeds with successive divisions of the maternal pronucleus. Due to the reduced amount of DNA, these haploid nuclei undergo an additional, 14th mitotic division. As expected, the resulting nc15 nuclei are half the volume of normal nc14 diploid nuclei due to the extra division cycle. Strikingly, the sog>MS2 transgene exhibits a dramatic loss of expression in mesoderm nuclei at the onset of nc15. There is only a modest loss in the number of nuclei that exhibit repression in the mesoderm during nc14 (~10%). This is followed by an ~10-fold reduction in the number of nuclei that reactivate the transgene at the onset of nc15. This loss in expression following mitosis is similar to that seen for the btk>MS2 transgene in wild-type embryos. Unfortunately, given the scarcity of spontaneous haploid embryos, it was not possible to perform these measurements on only one embryo (Esposito, 2016).

These observations reinforce the correlation between mitosis and repression. Additional evidence was obtained by extending the normal period of mitosis by lowering the temperature of developing embryos. A temperature-controlled microfluidic chamber was used to produce a transient reduction in temperature-from 22°C to 17°C during the 13th mitosis while maintaining the temperature at 22°C during interphases 13 and 14. This treatment diminishes the rate of embryonic development and extends the time of mitosis from ~8 to ~15 min. There is a more pervasive loss of expression in the mesoderm upon reactivation of the sog>MS2 transgene at the onset of nc14. Less than half the mesoderm nuclei exhibit expression of the sog>MS2 transgene upon reactivation at the onset of nc14 (Esposito, 2016).

It is not believed that the correlation between mitosis and repression is a peculiarity of the Snail repressor. Mitotic silencing might influence gap repressors such as Kruppel, since there is a substantial refinement in the eve stripe-2 expression pattern at the onset of nc14 (Esposito, 2016).

The preceding analyses employed heterozygous embryos carrying a single copy of the brk or sog transgenes. Next homozygous embryos were examined to determine whether the two alleles of a locus display coordinated or uncoupled patterns of repression. The btk>MS2 transgene exhibits a sharp transition from active to inactive mesoderm nuclei following mitosis. This study therefore focused analysis on the regulation of sog>MS2 homozygotes (Esposito, 2016).

There is no detectable repression of either sog>MS2 allele during nc13. At the onset of nc14, most of the daughter nuclei reactivate expression following mitotic silencing but produce fewer transcripts than the nuclei in the ectoderm. Moreover, mesoderm nuclei exhibit asymmetric reactivation of the two alleles. One of the alleles exhibits expression that is comparable with each allele in the ectoderm, whereas the other allele is either silent or exhibits very weak and transient expression. These observations suggest that the diminished levels of expression that are observed for sog>MS2 at the onset of nc14 are mainly due to the repression of one of the alleles. Thus, Snail represses sog expression in the mesoderm one allele at a time. Instead of diminishing the levels of both alleles, there is a clear trend to silence one of the alleles. The basis for this 'digital' mode of repression is uncertain, but it is possible that it reflects the exact time when each homolog is silenced and then decondensed following mitosis (Esposito, 2016).

This study presents evidence that transcriptional repression is intimately linked to the cell cycle, and it is proposed that Snail exploits the general silencing of transcription that occurs during mitosis. Mitotic silencing offers an opportunity to reset the balance between transcriptional activators and repressors. It is possible that Snail outcompetes the Dorsal activator during mitosis. Indeed, immunohistochemical localization assays suggest that the Snail repressor remains associated with the apical cytoplasm during mitosis, whereas Dorsal becomes distributed throughout the cytoplasm. This might give Snail 'the jump' on Dorsal after the completion of mitosis. It is also possible that the balance between the Dorsal activator and the Snail repressor is influenced by 'titration' of Dorsal due to increases in chromosomal templates arising from replication (Esposito, 2016).

It is proposed that Snail and other developmental repressors exploit natural discontinuities in transcription. In addition to mitotic silencing, many genes exhibit transcriptional bursts. It is possible that repressors like Snail get the upper hand during the refractory periods between bursts. Indeed, inhibition between successive bursts of sog>MS2 expression was observed in the mesoderm following mitotic silencing. Approximately 80% of mesoderm nuclei exhibit a single burst of expression before falling silent, and a similar trend in the repression of the btk>MS2 transgene was observed. The Snail repressor may be more effective in maintaining the off state following a burst (or mitotic silencing) than inhibiting a gene at the peak of its activity. Mitotic silencing and transcriptional bursting might represent intrinsic mechanisms that foster dynamic repression of gene expression during development (Esposito, 2016).

Myosin-dependent remodeling of adherens junctions protects junctions from Snail-dependent disassembly

Although Snail is essential for disassembly of adherens junctions during epithelial-mesenchymal transitions (EMTs), loss of adherens junctions in Drosophila melanogaster gastrula is delayed until mesoderm is internalized, despite the early expression of Snail in that primordium. By combining live imaging and quantitative image analysis, the behavior of E-cadherin-rich junction clusters were tracked, demonstrating that in the early stages of gastrulation most subapical clusters in mesoderm not only persist, but move apically and enhance in density and total intensity. All three phenomena depend on myosin II and are temporally correlated with the pulses of actomyosin accumulation that drive initial cell shape changes during gastrulation. When contractile myosin is absent, the normal Snail expression in mesoderm, or ectopic Snail expression in ectoderm, is sufficient to drive early disassembly of junctions. In both cases, junctional disassembly can be blocked by simultaneous induction of myosin contractility. These findings provide in vivo evidence for mechanosensitivity of cell-cell junctions and imply that myosin-mediated tension can prevent Snail-driven EMT (Weng, 2016).

This study shows that during Drosophila gastrulation, subapical junctions are repositioned toward the apical surface and are strengthened as the cortical tension increases. Both these phenomena follow apical myosin activation and thus may reflect a mechanosensitive response of junctional complexes to the tension generated by this activation of myosin. The junctional responses occur on the time scale of individual myosin pulses and are temporally correlated with those pulses. Such junctional changes depend on myosin activity but do not require Sna, given that ectopic myosin activation recapitulates similar junctional responses in Sna-negative tissues. This phenomenon may not be restricted to Drosophila embryos. The increased contractile actomyosin on the apical cortex of human cell lines deficient for the cortex actin regulator Merlin is associated with a condensation of adherens junctions toward the apical surface, suggesting that the response of adherens junctions to cortical tension can be of general significance (Weng, 2016).

The changes in junction mass and density suggest that, rather than being simple passive anchors for contractile actomyosin filaments, adherens junctions respond to the contractile actomyosin by restructuring and repositioning themselves, potentially involving aggregation and rearrangement of E-Cad molecules within the plasma membrane or vesicle-based redistribution of E-Cad. Indeed, actomyosin organization has been shown to be critical in the lateral clustering of E-Cad molecules. The change in E-Cad clustering is considered an active mechanosensitive mechanism to strengthen the adhesion. Alternatively, the adhesion can also be remodeled through the vesicle-based mechanisms, and endocytosis of E-Cad has been shown to be up-regulated when junctions are under actomyosin-generated stress. The repositioning could also arise through restructuring rather than passive dragging, if for example recycling and turnover rates in the basal regions of the junctions differ from apical regions. Overall, regardless of the underlying mechanism, this mechanosensitivity may be advantageous, providing a direct self-corrective mechanism that allows junctions to adjust their localization and intensity to match the mechanical force they experience (Weng, 2016).

Although the molecular mechanism for the junction strengthening requires further investigation, the data suggest that it is resistant to the posttranscriptional disassembly of adherens junctions downstream of Sna. The phenotype of myosin knockdown in this study resembles that previously described for cta; T48 double mutants, in which apical actomyosin cannot be activated and junctions are lost only in the ventral mesodermal cells. In all scenarios in which Sna expression is associated with junction loss (ventral cells in cta; T48 mutants, ventral cells in myosin knockdown mutants, and ectodermal cells with ectopic Sna expression), Sna is expressed in cells in the absence of myosin contractility. Maintenance of adherens junctions ultimately relies on the balance between assembly and disassembly rates of junctional components. Thus mechanical force likely modulates the assembly/disassembly balance and therefore remains in a homeostatic relationship with the junctions bearing the force (Weng, 2016).

In the early stages of embryogenesis analyzed in this study, E-Cad is maternally provided and thus not subject to direct transcriptional repression. The disassembly of junctions in the absence of myosin contraction must therefore reflect a posttranscriptional regulation on junctions, likely performed by one or several of Sna’s transcriptional targets. Much effort has been invested in identifying transcription targets of Sna, but it is not known which, if any, of its known targets might play such a role. One mesodermally expressed gene, Traf4, is required for fine-tuning junction morphology, but its expression appears to depend on the other mesodermal determinant, Twist, rather than Sna. One gene repressed by Sna in Drosophila mesoderm, bearded, is required for the subapical positioning of adherens junctions in cells not expressing Snail. It is not clear, however, whether Bearded plays a direct role in junction disassembly or a more general role in apical polarity or the apical myosin contractility that drives repositioning. The posttranscriptional regulation of adherens junction disassembly may allow more rapid and effective EMT than a disassembly relying on transcriptional down-regulation of junctional components such as E-Cad. Identifying and characterizing the relevant Sna targets in Drosophila may provide insights into the underlying mechanism for this disassembly, especially with respect to its apparent sensitivity to externally exerted tension. The force-dependent resistance to this Sna function may help in dissecting the underlying molecular functions. Further exploration of Sna’s posttranscriptional effect on junctions and how myosin contraction antagonizes Sna will shed light on understanding of EMT processes (Weng, 2016).

Differential roles of the Drosophila EMT-inducing transcription factors Snail and Serpent in driving primary tumour growth

Several transcription factors have been identified that activate an epithelial-to-mesenchymal transition (EMT), which endows cells with the capacity to break through basement membranes and migrate away from their site of origin. A key program in development, in recent years it has been shown to be a crucial driver of tumour invasion and metastasis. However, several of these EMT-inducing transcription factors are often expressed long before the initiation of the invasion-metastasis cascade as well as in non-invasive tumours. Increasing evidence suggests that they may promote primary tumour growth, but their precise role in this process remains to be elucidated. To investigate this issue this study has focused studies on two Drosophila transcription factors, the classic EMT inducer Snail and the Drosophila orthologue of hGATAs4/6, Serpent, which drives an alternative mechanism of EMT; both Snail and GATA are specifically expressed in a number of human cancers, particularly at the invasive front and in metastasis. Thus, this study recreated conditions of Snail and of Serpent high expression in the fly imaginal wing disc and analysed their effect. While either Snail or Serpent induced a profound loss of epithelial polarity and tissue organisation, Serpent but not Snail also induced an increase in the size of wing discs. Furthermore, the Serpent-induced tumour-like tissues were able to grow extensively when transplanted into the abdomen of adult hosts. The differences between Snail and Serpent correlate with the genetic program they elicit; while activation of either results in an increase in the expression of Yorkie target genes, Serpent additionally activates the Ras signalling pathway. These results provide insight into how transcription factors that induce EMT can also promote primary tumour growth, and how in some cases such as GATA factors a 'multi hit' effect may be achieved through the aberrant activation of just a single gene (Campbell, 2018).

EMT transcription factors are often found upregulated in human tumours. Given their role in driving a transition from a polarised static epithelial cell to a migratory invasive cell state, much focus has been put on the pro-invasive and metastatic implications of their aberrant expression. However, tumour progression involves the progressive acquisition of many other biological capabilities including sustained proliferation, evasion of growth suppressors and resistance to cell death, and there is increasing evidence suggesting that EMT transcription factors contribute to these earlier stages of tumour progression. This study shows that the Drosophila EMT-inducers Sna and Srp drive not only EMT, but also over-proliferation in a well-established epithelial tumour model. Sna-driven proliferation is accompanied by extensive cell death and a decrease in cell size, and thus the overall effects of aberrant Sna expression on tissue size are negligible. In contrast, Srp drives an increase in cell size as well as cell proliferation, but not cell death, leading to a profound overall increase in the size of the tissue, which is particularly evident upon transplantation, when the tissue has more time to grow. This study finds that both Sna and Srp repress crb transcription, which has previously been shown to induce a repression of the Hippo pathway and thus drive Yki-dependent overproliferation. Indeed, this study shows that both Sna and Srp activate Yki activity, which has previously been shown to drive excess proliferation in the wing disc. However, in addition to this, Srp was also found to activate the mitogenic Ras pathway, which has recently been shown to act synergictically with Yki to promote hyperproliferation and tumour development in the Drosophila wing disc. Studies in breast cancer models and oesophageal epithelial cells have shown that the EMT transcription factors Twist and Zeb contribute to primary tumour growth through the activation of programs that prevent cells from undergoing oncogene-induced senescence and apoptosis. Taken together with the current results, this suggests that EMT transcription factors can contribute to the multistep process of tumour progression through the activation of different onco-promoting cell biological processes, and that this is both transcription factor and tumour dependent (Campbell, 2018).

EMT transcription factors drive a loss of epithelial cell polarity, which has been shown to activate cell death pathways in a number of contexts. For example, scribble (scrib) mutant clones are completely eliminated from wild type discs through programmed cell death pathways. Intriguingly, while overexpression of both Srp and Sna drives a loss of cell polarity, an increase in cell death is only seen with Sna, whereas Srp appears to correlate with an increase in cell survival. This is despite the fact thatan increase was found in the transcription of the key apoptosis inhibitor Diap1 in both scenarios. Diap1 functions as an E3-ubiquitin ligase that protects cells from unwanted death by blocking the activity of the caspase DRONC and the Drosophila apoptotic protease-activating factor-1 (Apaf-1) homolog, Dark, and the relative levels of Diap1, Dronc and Dark are important in determining the outcome ie. cell survival vs cell death. As a lot of cell death is seen in the wing disc upon overexpression of Sna, despite a clear increase in Diap1 expression, this suggests that the levels of Diap1 induced are not sufficient to block the level of cell death induced by Sna. While a comparable increase is seen in the levels of Diap1 upon Srp overexpression, Srp also activates Ras, which has been reported to protect cells with mutations in cell polarity genes from death. However, reducing Ras signalling does not lead to an increase in cell death when Srp is overexpressed. It has been previously seen that ectopic Srp also induces expression of Forkhead (Fkh), which has been reported to act as a survival factor in a number of Drosophila systems, including the midgut. These results therefore suggest that cells expressing ectopic Srp evade death through the upregulation of multiple cell survival factors (Campbell, 2018).

Intriguingly, so-called EMT transcription factors such as Sna, Srp, Twist and Zeb proteins often activate many developmental pathways and processes of which a loss of cell polarity and EMT is only a part. They are all expressed in multiple tissues during development and play pleotrophic roles, depending on the context and time window in which they are activated. The cell context dependence of the activity of these genes is emphasized, suggesting that other genes may collaborate to the Srp and Sna induced transformations. For example, in Drosophila Sna activates an EMT in mesoderm cells during early stages of embryonic development, but later on it plays distinct roles during central nervous system and peripheral eye development. Srp is required for EMT in the Drosophila midgut, but also for midgut cell specification, and additionally plays multiple roles during specification and maturation of the haemocytes. Hence, it is not surprising that activation of such transcription factors outside the normal controls imposed during development can impinge on multiple cell features and signalling programs in addition to EMT, and thus play key roles in the initiation and development of primary tumours, rather than being limited to the steps of cancer cell invasion and metastatic spread. Additionally, it is worth noting that the effect of Srp activity on tissue overgrowth in the wing disc is due, at least in part, to the ectopic triggering of effector genes normally elicited by Srp in the midgut, one of its regular domains of expression (Campbell, 2018).

A previously investigated the effects of triggering ectopic sna and srp in the Drosophila embryo, by driving their expression in ectodermal epithelial cells in which they are never normally expressed. While a previous study found that sna had no effect on ectoderm cell behaviour (Campbell, 2015), a more recent study showed that when sna was expressed at high levels using a maternal driver, it triggers adherens junction disassembly in ectodermal cells, and in rare cases, the movement of some cells to inside the embryo (Weng, 2016). Similarly, ectopic srp drives a loss of apicobasal polarity and junction disassembly, although with srp there is a profound migration of cells into the embryo. Remarkably, in the embryo no proliferation is seen in these circumstances. Conversely, overproliferation is seen in wing discs, but very little cell migration. Intriguingly, a 'Go or Grow' hypothesis has been proposed which postulates that cell division and cell migration are temporally exclusive events and that tumor cells defer migration to divide and vice versa. The results suggest that EMT transcription factors can drive migration or proliferation, but tend to favour one over the other at any given time. Given the fact that EMT transcription factors are increasingly associated with cancer stem cells, it will be important to unravel when and how EMT promotes one over the other (Campbell, 2018).

The transformation of a healthy cell into a cancerous one requires multiple mutations and cooperation between different oncogenic/tumor suppressor mutations. Not only can EMT transcription factors accelerate tumour progression by the activation of multiple biological processes, this can also be exacerbated through cooperative effects of the different pathways. For example, in breast cancer models cooperation between Twist and an active form of RAS is sufficient to trigger transformation of mammary epithelial cells into malignant cells exhibiting all the characteristic features of claudin-low tumors. Similarly the current results suggest that ectopic Sna in combination with situations where cells become resistant to cell death may have catastrophic effects. Remarkably, over-expression of Srp alone activates both the EGFR/Ras and Yki signalling pathways. Of note, over-activation of the Ras pathway in situations of compromised cell polarity often leads to dramatic tissue overgrowth, for example when oncogenic Ras is combined with a scribble mutation. Furthermore, it has previously been shown that loss of Drosophila cell polarity regulators such as Scribble promotes epithelial tissue overgrowth and cooperation with the Ras pathway through impaired Hippo pathway signaling. Thus the profound effects seen upon Srp activation are likely due to cooperation between these two pathways. GATA factors are increasingly found deregulated in human tumours, both at the invasive front and in primary lesions and are receiving increasing attention as onco-promoting genes. The current work suggests that GATA factors could be activating multiple tumour promoting pathways, that act cooperatively both in early stages of primary tumour growth and later in driving invasion and metastasis (Campbell, 2018).

GENE STRUCTURE

Bases in 5' UTR - 163

Bases in 3' UTR - 340

PROTEIN STRUCTURE

Amino Acids - 390

Structural Domains

There are five C terminal zinc fingers (Boulay, 1987).

date revised: 10 June 2023

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