slow border cells


DEVELOPMENTAL BIOLOGY

Embryonic

SLBO protein is expressed predominantly during late embryogenesis in the nuclei of a restricted set of differentiating cell types, such as the lining of the gut and epidermis, similar to the mammalian tissues that express C/EBP. The earliest expression is at 9-10 hours postfertilization in cells associated with the posterior spiracles. The highest levels are found at 12-18 hours, first in the salivary gland, and then in the proventriculus and midgut. Lower levels of expression are found in the foregut, hindgut and epidermis (Rorth, 1992).

Adult

Staining in border cell nuclei is evident early in stage 9, just prior to border cell migration. Staining in the leading centripetal follicle cells is also nuclear and is detected at stage 10, just prior to their migration (Montell, 1992).

Effects of Mutation or Deletion

Mutations in the slbo gene cause late embryonic lethality. Embryos that lack slbo die just before or just upon hatching. The lethal phenotype of C/EBP mutants can be rescued with the cloned C/EBP gene introduced by P-element-mediated germ-line transformation (Rorth, 1992).

The involvement of Breathless (a Drosophila FGF receptor tyrosine kinase homolog) in border cell migration has prompted an inquiry as to whether RAS, a downstream effector for receptor tyrosine kinases, contributes to receptor tyrosine kinase-mediated motility. A dominant-negative RAS protein inhibits cell migration when expressed specifically in border cells during the period when these cells normally migrate. When expressed prior to migration, dominant-negative RAS promotes premature initiation of migration. Conversely, expression of constitutively active RAS prior to migration results in a significant delay in the initiation step. Furthermore, the defect in initiation of border cell migration found in slbo1, a mutation at the locus that encodes Drosophila C/EBP homolog, is largely rescued by reducing RAS activity in border cells prior to migration. Taken together, these observations indicate that RAS activity plays two distinct roles in the border cells: (1) reduction in RAS activity promotes the initiation of that migration process and (2) RAS activity is required during border cell migration. The possible involvement of two downstream effectors of Ras in border cell migration was also examined. Raf activity was dispensable to border cell migration while reduced Ra1 activity inhibited initiation. Ra1 is a small GTPase that is activated by RAS. Therefore, RAS plays a critical role in the dynamic regulation of border cell migration via a Raf-independent pathway. It is believed that reducing RAS activity bypasses the normal requirement for SLBO expression for cell migration. The alternative explanation, that SLBO activates the expression of specific receptor tyrosine kinases, is here held to be untenable (Lee, 1996).

During Drosophila oogenesis, spatially restricted activity of the Epidermal growth factor receptor tyrosine kinase first recruits follicle cells adjacent to the oocyte to a posterior cell fate and subsequently, in a later function, specifies dorsal follicle cell fate. Gurken is known to act as the ligand stimulating Egf-r in both instances. Another receptor tyrosine kinase, Breathless, stimulates migration of the anterior follicle cells known as border cells. Since Ras is known to mediate many receptor tyrosine kinase effects, the role of Ras was investigated in follicle cell fate determination, differentiation, and migration throughout oogenesis. Early ectopic Ras activity induces transient expression of posterior follicle cell markers in anterior follicle cells, but does not inhibit anterior differentiation. Among the posterior follicle cell markers is the gene pointed expressed in anterior follicle cells during stages 8 and 9 after being subjected to ectopic Ras during stage 2. Later ectopic Ras activity inhibits anterior follicle cell differentiation but does not induce posterior marker expression. Complete transformation of anterior follicle cells to posterior follicle cells required early ectopic Ras activity in egg chambers where terminal differentiation of anterior cells is inhibited. Although Ras alone is insufficient to completely transform anterior cells into posterior cells, complete transformation of anterior cells to posterior cells does occur when transient Ras activity is induce in a slow border cells (slbo) mutant. Thus Ras and C/EBP play antagonistic roles in the terminal differentiation of border cells (T. Lee, 1997).

slbo targets breathless and is essential for terminal differentiation and migration of the anterior follicle cells known as border cells. Ectopic Ras activity prior to stage 6 of oogenesis impairs border cell migration. An additional copy of slbo is able to rescue the border cell migration defect in egg chambers with elevated ras, indicating that increased C/EBP expression counteracts increased Ras activity. It is concluded that Ras and C/EBP appear to antagonize each other in the terminal differentiation of border cells, and that it does not appear that low Ras activity per se is required for initiation of border cell migration (T. Lee, 1997).

Two other cell populations were studied: dorsal follicle cells and outer follicle cells. It was found that activated Ras is sufficient to specify dorsal follicle cell fate. In addition, a surprising role for border cells was found in the establishment of outer follicle cell fate. During stage 9, follicle cells covering the nurse cells gradually flatten, and, simultaneously, most follicle cells undergo movement from anterior to posterior and finally form a columnar epithelium in contact with the oocyte. These columnar epithelial cells constitute the outer follicle cells of the oocyte. Interestingly, outer follicle cell rearrangement is impaired when border cell fate is suppressed by activated Ras. These results suggest that, in vivo as in vitro, Ras can have diverse effects on different cells, but, in addition, Ras activity can have different effects on the same cells at different stages in their development (T. Lee, 1997).

Cells migrating through a tissue exert force via their cytoskeleton and are themselves subject to tension, but the effects of physical forces on cell behavior in vivo are poorly understood. Border cell migration during Drosophila oogenesis is a useful model for invasive cell movement. This migration requires the activity of the transcriptional factor Serum response factor (SRF) and its cofactor MAL-D and evidence is presented that nuclear accumulation of MAL-D is induced by cell stretching. Border cells that cannot migrate lack nuclear MAL-D but can accumulate it if they are pulled by other migrating cells. Like mammalian MAL, MAL-D also responds to activated Diaphanous, which affects actin dynamics. MAL-D/SRF activity is required to build a robust actin cytoskeleton in the migrating cells; mutant cells break apart when initiating migration. Thus, tension-induced MAL-D activity may provide a feedback mechanism for enhancing cytoskeletal strength during invasive migration (Somogyi

To investigate conditions for MAL-D nuclear accumulation, border cells genetically unable to initiate migration were analyzed. slbo is a transcription factor that is required for border cell migration. None of the clusters in which all cells were mutant for slbo (n = 20 clusters) had nuclear MAL-D, regardless of developmental stage. Thus, border cells that were genetically unable to initiate migration were unable to accumulate nuclear MAL-D (Somogyi, 2004).

To determine whether the lack of nuclear MAL-D in slbo mutant cells was due to cell genotype or due to the physical state of the cell, an in vivo 'pulling experiment' was performed. This experiment takes advantage of the fact that border cells migrate as a cluster of strongly adherent cells and not as individual cells. If nonmigratory slbo mutant cells are found in a border cell cluster with wild-type cells, the mutant cells can be pulled along by the wild-type cells. This 'piggy-back' behavior is observed for a variety of different mutants affecting border cell migration -- in fact, it occurs in all genotypes that have been tested. The slbo mutant cells are always in the rear and delay migration of the border cell cluster in proportion to their abundance. Thus, the mutant cells do not become migratory as such but are pulled along by the actively migrating cells. Remarkably, slbo mutant cells that were pulled into migration by wild-type cells did accumulate nuclear MAL-D. They did so at a frequency similar to that of wild-type migrating cells. Migration of mixed clusters is often delayed and may occur during stage 9 or stage 10. In both situations, nuclear MAL-D accumulation was observed. Finally, even mutant cells that had not (yet) invaded the germline could be positive if attached to migrating wild-type cells. This, together with the observations in wild-type cells, shows that border cell position does not control MAL-D accumulation. Thus, nuclear MAL-D accumulation is not directly dependent on cell genotype, on cell position, or on developmental stage. However, nuclear MAL-D accumulation is only observed in nonmotile mutant border cells if they are being pulled by other cells. These results support the idea that cell deformation or perceived tension regulates MAL-D accumulation (Somogyi, 2004).

Notch signaling links interactions between the C/EBP homolog slow border cells and the GILZ homolog bunched during cell migration

In the follicle cell (FC) epithelium that surrounds the Drosophila egg, a complex set of cell signals specifies two cell fates that pattern the eggshell: the anterior centripetal FC that produce the operculum and the posterior columnar FC that produce the main body eggshell structure. The long-range morphogen DPP represses the expression of the bunched (bun) gene in the anterior-most centripetal FC. bun, which encodes a homolog of vertebrate TSC-22/GILZ, in turn represses anterior gene expression and antagonizes Notch signaling to restrict centripetal FC fates in posterior cells. From a screen for novel targets of bun repression, the C/EBP homolog slow border cells (slbo) has been identified. At stage 10A, slbo expression overlaps bun in anterior FC; by stage 10B they repress each other's expression to establish a sharp slbo/bun expression boundary. The precise position of the slbo/bun expression boundary is sensitive to Notch signaling, which is required for both slbo activation and bun repression. As centripetal migration proceeds from stages 10B-14, slbo represses its own expression and both slbo loss-of-function mutations and overexpression approaches reveal that slbo is required to coordinate centripetal migration with nurse cell dumping. It is proposed that in anterior FC exposed to a Dpp morphogen gradient, high and low levels of slbo and bun, respectively, are established by modulation of Notch signaling to direct threshold cell fates. Interactions among Notch, slbo and bun resemble a conserved signaling cassette that regulates mammalian adipocyte differentiation (Levine, 2007).

bunched refines a DPP activity gradient by antagonizing Notch signaling to establish the posterior edge of the operculum-forming centripetal FC. This study reveals that bunched is part of an intricate switch reliant on Notch activation of slbo to direct alternate FC fates. These observations contribute to a model in which bunched connects long-range morphogen cues to short range, cell contact-dependent signaling. Together with recent work on the bunched homologue GILZ in mammalian cell culture, these data suggest that this family of proteins is part of a conserved signaling cassette regulating cell fate decisions, as detailed below (Levine, 2007).

In different contexts cells migrate either as integrated sheets, such as during convergent extension, or as small groups of cells, such as during neural crest migration. During border cell migration from stages 8-10, a subset of anterior FC transiently loses epithelial polarity, delaminates and rounds into a small semi-polarized cell cluster that migrates through the nurse cell complex. In contrast, during centripetal migration from stages 10-14 a ring of anterior follicle cells changes shape and squeezes through the oocyte/nurse cell complex in a process coordinated with rapid nurse cell dumping. Marker gene expression indicates that the centripetal FC stretch to cover the anterior of the oocyte and retain epithelial contacts with the anterior and posterior nurse cell FC and columnar FC groups, respectively, throughout this mass cell ingression. While unique genetic pathways likely regulate these distinct cell migrations, because both the border cells and the centripetal FC coordinately migrate through the germ line cyst and arrive in the same vicinity at the anterior of the egg, it is unsurprising that common components are involved in both processes. Non-muscle myosin (zipper) and DE-cadherin (shotgun) are expressed and required for migration in both cell types. As well, it has been shown that slbo itself is required for DE-cadherin accumulation during both border cell and centripetal FC migrations, an observation consistent with the role for slbo function in the centripetal FC that are demonstrated in this study. Recently, screens for border cell-specific gene expression have identified many transcripts expressed in both tissues (Levine, 2007).

Comparing the role and regulation of slbo during the centripetal FC sheet and border cell cluster migrations reveals both shared and unique requirements. Weak slbo mutations, which completely block border cell migration, have no discernable effect on centripetal FC migration, which is disrupted only in stronger allelic combinations. While early slbo mutant clones reduced DE-cadherin accumulation in the dorsal anterior FC and in the border cells, late slbo mutant clones in the nurse cell FC and centripetal FC are difficult to recover and properly stage. These clones result in several effects on late stage egg chambers. First, these resulted in increased levels of DE-cadherin and decreased levels of DLG consistent with changes in epithelial polarity and adhesion. Second, large anterior slbo mutant clones are associated with a failure of centripetal FC ingression to coordinate with nurse cell dumping. It is noted that slbo mutant phenotypes are distinct from DE-cadherin shotgun (shg) mutants, which result in ectopic centripetal migration between posterior nurse cells. slbo mutants do resemble dlg mutant phenotypes associated with defects in FC shape and epithelial invasiveness. And third, ectopic slbo-lacZ expression associated with disintegration of the follicular epithelia and egg chamber collapse which are likely connected to defects in epithelial maintenance. Thus previous reports that the strong slbo allele has no effects on centripetal FC migration may result from difficulties recovering and staging these highly aberrant and friable late stage mutant egg chambers (Levine, 2007).

The mechanism of slbo regulation in the border cells and centripetal FC is also distinct. It has been shown that post-transcriptional regulation of slbo protein levels is critical to proper border cell migration but does not occur in the centripetal FC. This study shows that in both cell groups, Notch initiates slbo expression and slbo is necessary and sufficient to repress its own expression as centripetal migration proceeds. SLBO protein can bind to a DNA sequence element located near the start site of its own promoter, and several matches to the canonical C/EBP binding site occur as well in the sequence of the slbo2.6 element that is sufficient to mediate autorepression, so this regulation is likely direct. Thus slbo adopts two strategies to fine-tune its levels: post-transcriptional regulation specifically in the border cell and transcriptional autoregulation in the both cell groups, as shown in this study (Levine, 2007).

It has been shown that DPP establishes the position of the bun expression boundary in the anterior FC and this boundary coincides with the posterior edge of the operculum eggshell structure. This study shows that as this boundary forms, slbo and bun expression patterns initially overlap and subsequently slbo and bun repress each other's expression to resolve respective expression patterns into two distinct cell groups. Notch signaling plays a central role in these interactions: Notch activates slbo expression in the centripetal FC and bun is required to antagonize Notch activation in posterior cells adjacent to the boundary (Levine, 2007).

The position of the boundary is highly sensitive to Notch activity so that increased Notch signaling leads to increased slbo2.6 expression both in the centripetal FC and, surprisingly, in adjacent columnar FC. Ectopic slbo expression in Nintra-expressing columnar FC at stage 10B is not associated with changes in FC proliferation and thus the spread of Notch activity likely relies on cell–cell signaling. This may arise either from (1) Notch activation of slbo expression in a large group of centripetal FC precursors that is not subsequently downregulated to a more narrow domain or (2) a Nintra-dependent activation of Notch signaling in adjacent columnar FC leading to cell contact-dependent posterior spread of slbo expression. The latter explanation is preferred because slbo2.6GAL4 expression expanded to almost all columnar FC in many egg chambers. In this way the position of the DPP-dependent cell fate boundary that defines the operculum is quite flexible but always drawn sharply by Notch activation (Levine, 2007).

While several canonical bun and Suppressor of Hairy [Su(H)] binding sites are located in the slbo2.6 element indicating slbo regulation by bun1 and Notch signaling, respectively, might be direct, several observations indicate slbo regulation at the boundary by bun is likely more complex. It has been noted previously that: (1) high levels of Notch and Notch target gene expression occur in anterior FC, with slightly reduced levels in centripetal FC in contact with bun-expressing cells and (2) increased levels of Notch targets occur in all cells of bun mutant clones at the centripetal FC boundary except those that contact bun+ cells. A parallel relationship is observed between bun and the Notch target slbo: (1) reduced levels of slbo occur in cells adjacent to bun-expressing cells in WT egg chambers, and (2) slbo expression occurs in bun mutant clones located at the centripetal FC boundary, with lower slbo levels in bun cells in contact with bun+ cells. Thus while bun may repress slbo directly, bun also antagonizes Notch activation of slbo in a non-cell autonomous manner. Consistent with this, bun clones removed from the centripetal FC do not lead to increased slbo expression and bun1 is not sufficient to block Nintra activation of slbo2.6 in the centripetal FC (Levine, 2007).

Notch modulation of slbo expression may be indirect as well. Because the Nts; slbo01310/slbo01310 double mutant egg chambers retain strong slbo-lacZ expression throughout the FC compared to Nts; slbo01310/+ egg chambers stained in parallel, it is hypothesized that Notch blocks SLBO protein's ability to repress its own expression. In this scenario, which must be further tested, the rapid reduction in slbo expression as centripetal migration proceeds results from both (1) decreasing Notch activation of slbo via Su(Hw) sites in the slbo promoter and (2) relief of a block on slbo autorepression. Consistent with rapid changes in Notch levels in the migrating centripetal FC, as slbo levels decrease a corresponding increase is seen in the levels of Cut protein, a key target of Notch repression in these cells. Because reduced dorsal appendages and opercula are seen in Nintra-expressing egg chambers, it is likely that rapid reduction in Notch levels is critical to permit the further patterning of anterior structures (Levine, 2007).

Dynamic interactions among bun, slbo and Notch signaling tightly regulate DE-cadherin levels in the centripetal FC. bun mutant clones lead to increased Notch signaling and DE-cadherin accumulation and Nintra is sufficient to increase DE-cadherin levels in the FC. slbo mutant clones lead to loss of DE-cadherin expression early and ectopic DE-cadherin levels late. Thus a recurring theme is that tight modulation of DE-cadherin levels is required in the FC at late oogenesis for epithelial transitions including border cell migration, centripetal FC migration and dorsal appendage elongation (Levine, 2007).

Recently, it has been shown that the bun homolog GILZ antagonizes the ability of C/EBP to activate expression of the key fat cell master regulator gene PPARγ2 (Peroxisome Proliferator Activator γ2) in adipogenic mesenchymal stem cells (Shi, 2003). GILZ binds a promoter element required for C/EBP-mediated activation and recruits HDAC1 (Histone Deacetylase 1) to repress PPARγ2 expression and promote the osteogenic cell fate. GILZ can also directly bind to C/EBP in vitro. Shi (2003) proposes that a balance of GILZ repressor and C/EBP activator in precursor mesenchymal cells regulates levels of PPARγ2, the master fat cell regulator. The similarities between these pathways are striking and it is proposed they constitute a conserved signaling cassette required for cell fate commitment. In support of a role for Notch in both, it has been shown that Notch signaling promotes adipogenesis in tissue culture , although the specific role of Notch in adipogenesis has been questioned. Targets may be conserved as well: expression of a gene homologous to PPARγ2 in the centripetal FC has been noted. While a connection between border cell specification and adipogenesis has been noted, slbo has no role in fly fat body formatio. However, bun expression hduring fat body formation has been detected suggesting that portions of this fly signaling cassette may operate in a general pathway required for storage cell differentiation (Levine, 2007).

HNT mediates its effect on cluster cohesion via JNK and its effect on border cell motility primarily through STAT and SLBO

Cell movements represent a major driving force in embryonic development, tissue repair, and tumor metastasis. The migration of single cells has been well studied, predominantly in cell culture; however, in vivo, a greater variety of modes of cell movement occur, including the movements of cells in clusters, strands, sheets, and tubes, also known as collective cell migrations. In spite of the relevance of these types of movements in both normal and pathological conditions, the molecular mechanisms that control them remain predominantly unknown. Epithelial follicle cells of the Drosophila ovary undergo several dynamic morphological changes, providing a genetically tractable model. This study found that anterior follicle cells, including border cells, mutant for the gene hindsight (hnt) accumulated excess cell-cell adhesion molecules and failed to undergo their normal collective movements. In addition, HNT affected border cell cluster cohesion and motility via effects on the JNK and STAT pathways, respectively. Interestingly, reduction of expression of the mammalian homolog of HNT, RREB1, by siRNA inhibited collective cell migration in a scratch-wound healing assay of MCF10A mammary epithelial cells, suppressed surface activity, retarded cell spreading after plating, and led to the formation of immobile, tightly adherent cell colonies. It is proposed that HNT and RREB1 are essential to reduce cell-cell adhesion when epithelial cells within an interconnected group undergo dynamic changes in cell shape (Melani, 2008).

To explore the mechanisms by which HNT affects cluster cohesion and motility, its effects on known signaling pathways were investigated. In the extraembryonic tissue known as the amnioserosa, hnt is a negative regulator of the JNK signaling cascade. Recently, the JNK pathway was shown to be active in the border cells and to affect border cell migration in clusters with reduced PVR activity. In addition, inhibition of the JNK cascade causes a phenotype that strikingly resembles the cluster dissociation phenotype caused by HNT overexpression, suggesting that HNT could be a negative regulator of the JNK pathway or vice versa. By using phospho-Jun antibody staining as a readout of the JNK signaling cascade, the activity of this pathway was seen to be reduced in border cells overexpressing hnt. In clusters in which JNK was reduced by overexpression of either Puckered (the JNK phosphatase) or a dominant-negative form of Basket (Drosophila JNK), cluster disassembly reminiscent of the hnt gain-of-function phenotype was observed. In addition, HNT was upregulated 1.7- and 1.4-fold, respectively. Together, these results indicate that hnt and JNK repress each other. In the embryo, in which HNT also antagonizes JNK, this pathway is required for the turnover of focal complexes and proper dorsal closure. Therefore, HNT appears to play a general role in remodeling of adhesion complexes to facilitate morphogenesis (Melani, 2008).

Although the cluster-disassembly phenotype of HNT could be attributed to effects on JNK signaling, JNK pathway mutations caused milder border cell motility defects than hnt. To determine whether HNT affected, in addition, one of the known border-cell-motility pathways, the effect of hnt on the activity of STAT and its key target SLBO was examined. STAT activation and nuclear translocation is the most upstream event in the differentiation of the border cells and is also required throughout border cell migration. It was found that, in border cells overexpressing HNT, nuclear accumulation of STAT was reduced though not eliminated. In addition, the levels of slbo were dramatically reduced in border cells overexpressing HNT. Because loss of function of either STAT or SLBO causes a dramatic migration defect, the effects of HNT overexpression on STAT and SLBO can account for the severe effect on motility. However, neither stat nor slbo mutant border cells exhibit a cluster-disassembly phenotype. Therefore, it is concluded that HNT mediates its effect on cluster cohesion via JNK and its effect on border cell motility primarily through STAT and SLBO (Melani, 2008).

Although HNT overexpression affects border cell motility via effects on STAT and SLBO, HNT has general effects on cell adhesion and morphogenesis, whereas SLBO appears to be more specific. For example, the effects of hnt on stretched follicle cells and in embryonic tissues are independent of SLBO because this protein is neither expressed nor required in these other cell types. Therefore, it is proposed that HNT plays a general role in regulating cell adhesion and morphogenesis via JNK signaling and a tissue-specific role in motility through STAT and SLBO. In this way, HNT can cooperate with tissue-specific factors to orchestrate a diverse array of collective cell movements (Melani, 2008).


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slow border cells: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 15 July 2013 

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