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© 2009 Macmillan Publishers Limited. All rights reserved. NEWS AND VIEWS Breaking a temporal barrier: signalling crosstalk regulates the initiation of border cell migration Dorothea Godt and Ulrich Tepass Correct timing of developmental events is crucial for generating a normal organism. During oogenesis in Drosophila melanogaster, migration of border cells occurs in a defined temporal window and requires Jak/Stat and steroid hormone signalling. The initiation of border-cell migration is now shown to be timed by Jak/Stat-mediated downregulation of the BTB domain transcriptional regulator Abrupt, which acts as a negative regulator of steroid hormone signalling. Multicellular organisms arise from a single cell through a highly orchestrated series of devel- opmental decisions. Developmental events, such as cell division, cell type specification, cell and tissue polarization or cell migration, must be coordinated in both space and time. On page 569 of this issue, Montell and col- leagues 1 clarify the molecular mechanisms that tell border cells in the Drosophila ovar- ian follicle to initiate migration at a specific time-point and no earlier or later. Abnormal timing of developmental events often results in defects that may compro- mise viability or organ function. At the same time, changes in the temporal organization of development may bring about new mor- phologies that can contribute to the evolution of species, a process known as heterochrony. Examples of mechanisms that contribute to the timing of developmental decisions have been described in recent years 2,3 , but in con- trast to the mechanisms underlying spatial patterning, a coherent picture has not yet emerged. Hormone pulses released systemi- cally are major regulatory cues that can coor- dinate developmental events; a well-studied example is the role of the steroid hormone ecdysone in regulating the progression of the Drosophila life cycle 4 . Steroid hormones also regulate the timing of processes associated with reproduction, such as gonad development and gametogenesis, in vertebrates and invertebrates. Drosophila egg follicles consist of an oocyte, nurse cells and a surrounding epithelium of follicle cells. At mid-oogenesis (stage 9), migration of follicle cells and changes in cell shape are initiated concomitantly with rapid growth of the oocyte 5 . In the absence of ecdysone or its receptor (EcR), the transition from stage 8 to stage 9 of oogenesis does not occur and fol- licles degenerate 6,7 . Elevated levels of ecdysone resulting from starvation also cause abortion of follicles at mid-oogenesis 8 . Thus, the tran- sition from stage 8 to 9 represents a tempo- ral boundary that is controlled by ecdysone signalling, adjusting egg production rate to available nutrient reserves. Ecdysone signalling also supports mor- phogenetic movements of follicle cells, such as migration of border cells, which serve as a model to dissect the network of factors that regulate collective cell migration 9,10 . The border cell cluster is composed of 2 non- motile polar cells and 4–8 migratory outer border cells (Fig. 1). It forms at late stage 8 when the polar cells at the anterior end of a follicle secrete the cytokine Unpaired, which activates the Jak/Stat signalling path- way in neighbouring follicle cells, recruit- ing them as outer border cells (Fig. 1). Jak/ Stat signalling activates the expression of the C/EBP transcription factor Slbo in border cells, which regulates several factors that are crucial for migration 9 . Border cells begin to migrate at early stage 9 and reach the oocyte at stage 10 (Fig. 1). They later contribute to the micropyle, which aids sperm entry into the egg. Although border cells can be speci- fied precociously by premature activation of the Jak/Stat pathway, migration will not begin until stage 9, indicating that Jak/Stat signalling is not sufficient for temporal con- trol of migration 1 . When ecdysone signalling is compro- mised, border cells are specified but do not migrate 1,11 . Is it ecdysone signalling then that makes border cells competent to migrate? In support of this hypothesis, a marked increase in 20-hydroxyecdysone, the active form of the steroid, was observed in follicles during mid- oogenesis 12 . Furthermore, EcR-B1, one of the two EcR isoforms that can elicit an ecdysone signalling response in follicles, is enriched in the anterior follicle cells just before border cell migration begins 1 . Importantly, Montell and colleagues show that ecdysone signalling is activated in border cells at early stage 9 and intensifies during this stage (Fig. 1). In con- trast to Jak/Stat signalling, ecdysone signal- ling is not required for expression of Slbo and other tested border cell markers 9,11 , suggest- ing that these two pathways act in parallel to enable border cell migration. A constitutively active form of the EcR co-activator Taiman (Tai), which triggers premature ecdysone signalling, does not affect the timing of migration 1 , indicating that before border cell specification, ecdysone signalling can- not induce migration. However, when both pathways are activated prematurely, either by injecting ecdysone into female flies and early expression of Slbo 11 or by combined early expression of constitutively active forms of Dorothea Godt and Ulrich Tepass are in the Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada. e-mail: [email protected]; [email protected] 536 NATURE CELL BIOLOGY VOLUME 11 | NUMBER 5 | MAY 2009

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Page 1: Breaking a temporal barrier: signalling crosstalk regulates the initiation of border cell migration

© 2009 Macmillan Publishers Limited. All rights reserved.

news and v iews

Breaking a temporal barrier: signalling crosstalk regulates the initiation of border cell migrationDorothea Godt and Ulrich Tepass

Correct timing of developmental events is crucial for generating a normal organism. during oogenesis in Drosophila melanogaster, migration of border cells occurs in a defined temporal window and requires Jak/stat and steroid hormone signalling. The initiation of border-cell migration is now shown to be timed by Jak/stat-mediated downregulation of the BTB domain transcriptional regulator abrupt, which acts as a negative regulator of steroid hormone signalling.

Multicellular organisms arise from a single cell through a highly orchestrated series of devel-opmental decisions. Developmental events, such as cell division, cell type specification, cell and tissue polarization or cell migration, must be coordinated in both space and time. On page 569 of this issue, Montell and col-leagues1 clarify the molecular mechanisms that tell border cells in the Drosophila ovar-ian follicle to initiate migration at a specific time-point and no earlier or later.

Abnormal timing of developmental events often results in defects that may compro-mise viability or organ function. At the same time, changes in the temporal organization of development may bring about new mor-phologies that can contribute to the evolution of species, a process known as heterochrony. Examples of mechanisms that contribute to the timing of developmental decisions have been described in recent years2,3, but in con-trast to the mechanisms underlying spatial patterning, a coherent picture has not yet emerged. Hormone pulses released systemi-cally are major regulatory cues that can coor-dinate developmental events; a well-studied example is the role of the steroid hormone ecdysone in regulating the progression of the Drosophila life cycle4.

Steroid hormones also regulate the timing of processes associated with reproduction, such as gonad development and gametogenesis,

in vertebrates and invertebrates. Drosophila egg follicles consist of an oocyte, nurse cells and a surrounding epithelium of follicle cells. At mid-oogenesis (stage 9), migration of follicle cells and changes in cell shape are initiated concomitantly with rapid growth of the oocyte5. In the absence of ecdysone or its receptor (EcR), the transition from stage 8 to stage 9 of oogenesis does not occur and fol-licles degenerate6,7. Elevated levels of ecdysone resulting from starvation also cause abortion of follicles at mid-oogenesis8. Thus, the tran-sition from stage 8 to 9 represents a tempo-ral boundary that is controlled by ecdysone signalling, adjusting egg production rate to available nutrient reserves.

Ecdysone signalling also supports mor-phogenetic movements of follicle cells, such as migration of border cells, which serve as a model to dissect the network of factors that regulate collective cell migration9,10. The border cell cluster is composed of 2 non-motile polar cells and 4–8 migratory outer border cells (Fig. 1). It forms at late stage 8 when the polar cells at the anterior end of a follicle secrete the cytokine Unpaired, which activates the Jak/Stat signalling path-way in neighbouring follicle cells, recruit-ing them as outer border cells (Fig. 1). Jak/Stat signalling activates the expression of the C/EBP transcription factor Slbo in border cells, which regulates several factors that are crucial for migration9. Border cells begin to migrate at early stage 9 and reach the oocyte at stage 10 (Fig. 1). They later contribute to the micropyle, which aids sperm entry into

the egg. Although border cells can be speci-fied precociously by premature activation of the Jak/Stat pathway, migration will not begin until stage 9, indicating that Jak/Stat signalling is not sufficient for temporal con-trol of migration1.

When ecdysone signalling is compro-mised, border cells are specified but do not migrate1,11. Is it ecdysone signalling then that makes border cells competent to migrate? In support of this hypothesis, a marked increase in 20-hydroxyecdysone, the active form of the steroid, was observed in follicles during mid-oogenesis12. Furthermore, EcR-B1, one of the two EcR isoforms that can elicit an ecdysone signalling response in follicles, is enriched in the anterior follicle cells just before border cell migration begins1. Importantly, Montell and colleagues show that ecdysone signalling is activated in border cells at early stage 9 and intensifies during this stage (Fig. 1). In con-trast to Jak/Stat signalling, ecdysone signal-ling is not required for expression of Slbo and other tested border cell markers9,11, suggest-ing that these two pathways act in parallel to enable border cell migration. A constitutively active form of the EcR co-activator Taiman (Tai), which triggers premature ecdysone signalling, does not affect the timing of migration1, indicating that before border cell specification, ecdysone signalling can-not induce migration. However, when both pathways are activated prematurely, either by injecting ecdysone into female flies and early expression of Slbo11 or by combined early expression of constitutively active forms of

Dorothea Godt and Ulrich Tepass are in the Department of Cell and Systems Biology, University of Toronto, Toronto, Ontario, M5S 3G5, Canada.e-mail: [email protected]; [email protected]

536 nature cell biology VOLUME 11 | NUMBER 5 | MAY 2009

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Tai and Jak1, border cell migration is trig-gered several hours too early. Thus, the pre-cise temporal control of border cell migration requires correct timing of both Jak/Stat and ecdysone signalling.

Montell and colleagues focused on the reg-ulation of ecdysone signalling. Generation of a functional EcR protein complex requires EcR to form a heterodimer with the retin-oid X receptor homologue Ultraspiracle, and the recruitment of Tai into the recep-tor complex, which depends on earlier binding of ecdysone to EcR4,11. As ecdysone signalling can be precociously induced by the expression of constitutively active Tai during stage 8, sufficient hormone must be present at this stage to activate the EcR1, suggesting the existence of a repressor that prevents ecdysone signalling at this time. Through a genetic screen aimed at finding genes that, when overexpressed, block bor-der cell migration and ecdysone signalling, the authors identified Abrupt as a repressor of ecdysone signalling. Loss of Abrupt acti-vates ecdysone signalling ectopically and reduced levels of Abrupt can compensate for reduced ecdysone signalling, rescuing

border cell migration. These data establish Abrupt as a negative regulator of ecdysone signalling1.

Abrupt belongs to a family of transcrip-tional regulators containing a BTB domain and a zinc-finger motif 13. Abrupt is found in all follicle cells before stage 9, but its concentration decreases specifically in bor-der cells at the onset of migration1 (Fig. 1). This is consistent with a model in which removal of an antagonist of EcR signalling can initiate border cell migration. Montell and colleagues then addressed two impor-tant questions. First, they investigated how Abrupt interferes with ecdysone signalling, and second, they examined how removal of Abrupt in border cells is regulated. Their experiments demonstrate that Abrupt binds to Tai directly and that this interaction is essential for suppression of ecdysone signal-ling by Abrupt. This interaction is mediated by the BTB domain of Abrupt and the bHLH domain of Tai1. Further analysis is needed to elucidate whether Abrupt prevents Tai recruitment into the EcR complex or whether Abrupt associates with the EcR complex and interferes with its function.

Abrupt forms a barrier to EcR signalling at stage 8 that begins to break at the transition to stage 9. The demise of Abrupt is caused by a combination of EcR and Jak/Stat signal-ling1. Downregulation of Abrupt requires Jak/Stat signalling, as the amount of Abrupt in border cells remains abnormally high and border cell migration is delayed when Stat function is reduced. In addition, the find-ing that lowered EcR signalling increases the concentration of Abrupt is consistent with a negative-feedback loop between Abrupt and EcR. Given the existence of a mutual nega-tive interaction between EcR signalling and Abrupt, a brief surge in EcR signalling (due to the increased levels of 20-hydroxyecdysone and a pulse of high EcR-B1 expression in bor-der cells; Fig. 1) could be sufficient to trigger a progressive amplification of EcR activity at the expense of Abrupt.

The transient nature of high EcR-B1 expression might be important to prevent excessive ecdysone signalling during migra-tion, when ecdysone levels continue to rise. A hyperactive form of the EcR complex blocks border cell migration1. Thus, reduced and increased ecdysone signalling negatively affects border cell migration, indicating that the level of ecdysone signalling needs to be tightly controlled. It will be interesting to determine whether increasing EcR signal-ling itself causes the decrease of EcR-B1 expression during stage 9. Such a negative-feedback mechanism could stabilize the level of ecdysone signalling even when hormone levels fluctuate (Fig. 1).

Taken together, the results of the study by Montell and colleagues show that tempo-ral control of ecdysone signalling, which is translated into precise temporal control of border cell migration, involves a complex combination of inputs. The rising concen-tration of ecdysone, the transient upregula-tion of EcR-B1 in the anterior follicle cells, and Jak/Stat signalling help loosen the grip of the repressor Abrupt and raise ecdysone signalling to a level that can trigger border cell migration. How these three inputs them-selves are regulated is an open question. It will be interesting to see whether upregulation of EcR-B1 expression is dependent on Jak/Stat signalling, which would make the cooperation between the two signalling pathways even more intertwined. The immediate targets of ecdysone signalling that stimulate border cell migration remain to be identified.

Early stage 8 Early stage 9 Mid stage 9 Early stage 10Late stage 8

Border cellspecification

Anteriorfollicle cells

Start of bordercell migration

Border cellmigration

Border cells reach oocyte

Outerborder cells

Polar cells

Oocyte

Abrupt

Jak/Stat signalling

20-hydroxy ecdysone

Ecdysone signalling

Ecdysone receptor

Figure 1 Border cells develop from anterior follicle cells in response to a Jak/Stat activating signal from polar cells (curved arrows). After delamination, border cells migrate as a cluster through the centre of the follicle until they reach the oocyte. Ecdysone signalling controls the start of border cell migration. Initiation of ecdysone signalling in border cells requires removal of Abrupt, a potent repressor of this signalling pathway. Reduction of Abrupt is triggered by Jak/Stat signalling and reinforced by ecdysone signalling, which seems to become activated by a combination of reduced Abrupt levels, a pulse of high ecdysone receptor expression, and increasing levels of 20-hydroxyecdysone.

nature cell biology VOLUME 11 | NUMBER 5 | MAY 2009 537

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1. Jang, A. C. C., Chang, J. C., Bai, J. & Montell, D. J. Nature Cell Biol. 11, 569–579 (2009).

2. Moss, E. G. Curr. Biol. 17, R425–R434 (2007).3. Lewis, J. Science 322, 399–403 (2008).4. Thummel, C. S. Dev. Cell 1, 453–465 (2001).5. Spradling, A. C. In The Development of Drosophila

melanogaster. (eds Bate, M. & Martinez-Arias, A.) 1–70 (Cold Spring Harbor Laboratory Press, Harbor, New York,1993).

6. Carney, G. E. & Bender, M. Genetics 154, 1203–1211 (2000).

7. Buszczak, M. et al. Development 126, 4581–4589 (1999).

8. Terashima, J., Takaki, K., Sakurai, S. & Bownes, M. J. Endocrinology 187, 69–79 (2005).

9. Montell, D. J. Nature Rev. Mol. Cell Biol. 4, 13–24 (2003).

10. Rørth P. Trends Cell Biol. 17, 575–579 (2007).

11. Bai, J., Uehara, Y. & Montell, D. J. Cell 103, 1047–1058 (2000).

12. Riddiford, L. M. In The Development of Drosophila melanogaster. (eds Bate, M. & Martinez-Arias, A.) 899–929 (Cold Spring Harbor Laboratory Press, New York 1993).

13. Hu, S., Fambrough, D. Atashi, J. R., Goodman, C. S. & Crews, S. T. Genes Dev. 9, 2936–2948 (1995).

smurf1 zaps the talin headDavid R. Critchley

Focal adhesion turnover is essential for cell migration. new results show that the talin head liberated from talin by calpain ii cleavage has a key role in these events, and that its levels are tightly regulated by smurf1-mediated ubiquitylation counteracted by Cdk5-mediated phosphorylation.

The cytoskeletal protein talin has a key role in integrin-mediated cell adhesion, serving to activate integrins and also to couple them to the actomyosin contractile apparatus in the cell1. The domain structure of talin is shown in Fig. 1. The amino-terminal talin head (residues 1–433) contains a FERM domain, which binds to β-integrin cytoplasmic tails through its F3 sub-domain, and significant progress has been made in defining the mechanism by which this leads to integrin activation2. The flexible talin rod also contains an integrin binding site, at least two actin-binding sites and a carboxy-ter-minal helix, which is responsible for dimer formation. In addition, the talin rod contains several vinculin binding sites that are cryptic and are thought to be progressively exposed in response to force exerted on talin by actomyosin contraction3; vinculin recruitment is thought to stabilize the cellular junctions with the extracel-lular matrix (focal adhesions, FAs).

The talin head and rod are joined by an apparently unstructured region, and calpain II-mediated cleavage of talin between residues Gln 433 and Gln 434 is important for FA turno-ver4. Thus, cells expressing a calpain-resistant mutant of talin have more stable FAs and show reduced cell migration. However, the fate of the talin head and rod liberated by calpain II has not been considered, although it has recently been shown that the talin head alone can activate integrins and support cell spreading5. On page

624 of this issue, Huang et al. provide evidence that the stability of the talin head (but not full-length talin) is regulated by an interplay between ubiquitylation catalysed by the Smurf1 E3 ligase, and Cdk5-mediated phosphorylation of Ser 425, which prevents Smurf1 binding6. Blocking phosphorylation of Ser 425 either with a S425A substitution or by inhibition of Cdk5 increases FA disassembly and reduces lamellipodia per-sistence, resulting in reduced cell migration.

The study by Huang et al.6 originated with the painstaking work of Ratnikov et al., who noted that Ser 425 was phosphorylated in platelet talin, and that this was a potential Cdk5 site7. Huang et al.6 confirmed that Cdk5 phosphorylates both the talin head and full-length talin on Ser 425 in vitro and in vivo. As Cdk5 is essential for neuronal cell migration, the authors explored the role of talin Ser 425 phosphorylation in SH-SY5Y neuroblast-oma cells expressing either wild-type EGFP–talin or EGFP–talinS425A. PDGF-stimulated transwell migration of cells expressing the talinS425A mutant was retarded. Inhibition of Cdk5 either with ros-covitine or siRNA-mediated knockdown also inhibited migration. Similar findings were made using a CHO cell wound closure assay.

The significance of these observations began to emerge from studies on FA dynamics— FAs containing EGFP–talinS425A disassembled much more rapidly than those containing wild-type EGFP–talin, although the S425A mutation had little effect on FA assembly. Similarly, inhibiting Cdk5 with roscovitine also increased FA disas-sembly. In addition, cells expressing the talinS425A mutant had a significantly reduced capacity to sustain cell protrusions, although protrusion

velocity was not affected. Taken together, the results indicate that talin phosphorylation on Ser 425 is required to stabilize FAs and when phosphorylation is blocked, either by mutation or by inhibiting Cdk5, FAs and cell protrusions are less stable and as a result, cell migration is reduced.

In searching for mechanisms underlying these effects, Huang et al. explored the possibil-ity that Ser 425 phosphorylation might block the nearby calpain cleavage site, but this was shown not to be the case. The authors then considered the Smurf1 E3 ubiquitin ligase, as it contains an NPxY motif, a sequence that contributes to binding of the cytoplasmic tails of β-integrins to the talin head2. Smurf1 is also implicated in regulating cell migration through ubiquitylation and degradation of RhoA8. Their hunch proved to be correct and Smurf1 was shown to bind to residues 393–433 of the talin head in co-immu-noprecipitation and pulldown assays, although the interaction was independent of the Smurf1 NPxY motif. Significantly, phosphorylation of the talin head by Cdk5 inhibited Smurf1 binding, as did a S425D mutation, whereas the S425A muta-tion increased binding. Remarkably, Smurf1 did not bind to full-length talin, presumably because the binding site for Smurf1 in the linker region between the head and rod is buried.

A role for Smurf1 in regulating the stability of the talin head in cells was confirmed by co-expressing the talin head with Smurf1 in CHO cells. Thus, steady-state levels of the talin head were reduced by about 33% in cells overexpress-ing Smurf1, but not a Smurf1 mutant lacking the ligase domain; this effect was reversed by

David Critchley is in the Department of Biochemistry, University of Leicester, Leicester LE17RH, UK.e-mail: [email protected]

538 nature cell biology VOLUME 11 | NUMBER 5 | MAY 2009