blistered/Serum response factor
Tracheal expression of pointed and Serum response factor are targeted by branchless, acting through breathless. To define the role of bnl in later branching events, expression of secondary (pointed) and terminal (Srf) branch genes in bnl loss-of-function mutants were assayed. pnt and Srf fail to be expressed in the tracheal system of bnl mutants. In contrast, in embryos that ectopically express bnl, both markers are activated throughout the tracheal system, and the expressing cells later give rise to secondary and terminal barnches. These results support the hypothesis that bnl expression near the ends of the primary branches not only guides primary branch outgrowth, but also activates the program of secondary and terminal branching in cells at these positions (Sutherland, 1996 and Samakovlis, 1996).
During normal tracheal development, secondary and terminal branching genes are induced at the ends
of growing primary branches by localized expression of Branchless. Because the
ectopic branches in sprouty mutants are formed by the prestalk cells located near the cells that are
normally induced to branch, the extra branches could arise from overactivity of the Bnl pathway. To
test whether sty functions by limiting the Bnl pathway or by preventing branching in some other way, an examination was made of downstream effectors in the Bnl pathway that regulate the later branching events (Hacohen, 1998).
One such effector is pointed (pnt), a downstream target of several receptor tyrosine kinase pathways. pnt expression is induced by Bnl at the ends of
primary branches and promotes secondary and terminal branching. Similarly, the DSRF gene and three other
marker genes (Terminal -2,-3, and -4) are induced at the ends of growing primary branches; all promote terminal branching. In sty mutants, all five downstream effectors are expressed in expanded domains that include the prestalk cells, which later form ectopic branches. The DSRF marker is activated at the same time as in the normal branching cells (Hacohen, 1998).
The transcriptional repressor Yan is another critical target of Bnl signaling. As in other RTK pathways, activation of the Btl receptor leads to MAPK-dependent phosphorylation and degradation of Yan, which is necessary to activate the later programs of tracheal branching. Normally, Yan is degraded only in the tip cells of the outgrowing primary branches. In sty mutants, Yan is degraded in an expanded domain that coincides with the expanded domains of pnt
and DSRF expression. A yan-lacZ transcriptional reporter continues to be
expressed normally, implying that down-regulation of Yan occurs posttranscriptionally as in other RTK pathways. The results show that sty loss of function mutations enhance all known downstream effects in this Bnl pathway. An engineered gain of function condition, in which the sty gene
product is overexpressed during embryonic stages 10-12, severely blocks induction of downstream
effectors and branching by Bnl. The reciprocal is also true: overexpression of Bnl can overcome the opposition of sty and induce secondary and terminal branching throughout the tracheal system. Thus, sty behaves genetically as a competitive inhibitor of the Bnl pathway (Hacohen, 1998).
A study by S. D. Weatherbee (1998) is arguably the best study yet published about how gene regulation differs in homologous structures, and points to future studies for how differential gene regulation will be shown to account for the structural differences between species. The differentiation of the Drosophila haltere from the wing through the action of the Ultrabithorax (Ubx) gene is a classic example of Hox regulation of serial homology. This study reveals several features of the control of haltere development by Ubx which, in principle, are likely to apply to the Hox-regulated differential development of other serially homologous structures in other animals. Specifically, it has been shown that Ubx acts: (1) at many levels of regulatory hierarchies, on long-range signaling proteins and their target genes, as well as genes further downstream; (2) selectively on a subset of downstream target genes of signals common to both wing and haltere, and (3) independently on these diverse targets. This information is presented in terms of the effects of Ubx on gene expression in the three axes of appendage formation, since these axes are to a large extent independently regulated and independent gene regulation in the axes serves to structure the entire wing (Weatherbee, 1998).
In the anterior posterior axis, Ubx represses selected Dpp target genes. The expression pattern of en is essentially the same in the haltere disc as in the wing disc, indicating that Ubx is not regulating haltere identity by altering the expression of this compartmental selector gene. Similarly, the expression of dpp in the developing haltere on the anterior side of the AP compartment boundary resembles that in the wing disc. Because these discs give rise to very different appendages, there may be genes downstream of the Dpp signal that are regulated by Ubx. To identify these, an examination was carried out of how a number of genes involved in the development of specific wing characters are expressed and regulated in the developing haltere (Weatherbee, 1998).
From its cellular site of secretion, Dpp acts as a morphogen to organize wing growth, AP pattern, and to activate target gene expression over a distance. The optomotor blind (omb), spalt (sal), and spalt related (salr) genes are expressed in nested patterns centered on the Dpp stripe and are necessary for proper development of the central wing region, including veins II-IV. The expression of these Dpp target genes was examined in the haltere disc: although omb is expressed in the developing haltere pouch (straddling the Dpp stripe as it does in the wing disc), salr and sal are not expressed in the haltere pouch. These results show that the Dpp signal transduction machinery operates in the haltere disc but that selected wing target genes are not activated by the Dpp signal. To determine whether Ubx represses salr expression in the haltere disc, homozygous Ubx clones were generated. Indeed, salr is derepressed in Ubx clones in the anterior compartment of the haltere disc. As in the wing disc, salr expression in these clones depends on their distance from the Dpp source. To determine whether Ubx is sufficient to repress salr, salr expression was examined in CbxM1/+ wing discs in which Ubx is ectopically expressed along part of the DV boundary. In these wing discs salr expression is repressed in a cell autonomous fashion. Because sal/salr are required for the induction of vein development, the selective repression of salr by Ubx suppresses part of the Dpp-mediated AP wing patterning program in the haltere. As with the spatial patterning of wing veins, the pattern of intervein tissue is also determined by specific regulatory genes and critical for morphogenesis. The Drosophila Serum Response Factor (DSRF, or blistered) gene is expressed in future intervein tissue and required for the adhesion of the dorsal and ventral surfaces of the flat wing. The haltere, however, is more balloon-like; interestingly, DSRF expression is absent from the haltere pouch except for two crescents at the extreme dorsal and ventral edges of the anterior compartment. This difference is caused by Ubx regulation, because in Ubx clones in the haltere disc, repression of DSRF is relieved and a pattern of DSRF expression homologous to that in the wing forms within the boundaries of the clone. Conversely, ectopic expression of Ubx in wing discs extinguishes DSRF expression in a cell-autonomous manner (Weatherbee, 1998).
A small number of major regulatory (selector) genes have been identified in animals that control the development of particular organs or complex structures. In Drosophila, the vestigial gene is required for wing formation and is able to
induce wing-like outgrowths on other structures. Because ectopic expression of Vg in many imaginal discs induces the outgrowth of wing tissue, the expression of various wing patterning genes was examined to see if they are induced in ectopic growths. Vg is expressed in the entire developing wing pouch whereas Sal and
SRF have specific expression patterns within this domain but are not expressed in wild-type
leg discs. Targeted expression of Vg with the Gal4-UAS system induces ectopic expression
of Sal and SRF in developing leg imaginal discs. Similarly,
the nubbin (nub) gene (which is also expressed and required during wing development ) is ectopically induced in leg discs by Vg expression. In each case, only a subset of the cells expressing Vg activate the target gene,
which suggests that additional factors control the expression pattern of each gene. In a first step toward elucidating the molecular mechanism by which Vg regulates gene expression, the response of wing-specific enhancers to ectopic Vg expression was examined. Attention was focused on
both the boundary and quadrant enhancers of the vg gene and the enhancer from the SRF gene
that drives expression specifically in the intervein region between veins three and four. All three enhancers are
induced by ectopic Vg expression in leg and other imaginal discs. Importantly, ectopic expression of Vg
in clones of cells induces the enhancers only within the clones. However, gene
expression is not induced in all cells within clones nor in all clones. In addition, each individual
enhancer is expressed in different regions of these discs that appear to correlate with the spatial
distribution of the different signaling inputs known to be required for activation of these enhancers (Halder, 1998).
Scalloped is required for Vg function. In the notum primordia of the wing disc, the vg enhancers, as well as the sal, SRF, and nub genes
are not induced by ectopic Vg even though the known required extracellular signals are present. Target gene activation could depend then on the function of another
gene(s). One candidate for such a factor is the product of the sd gene, which is expressed in a pattern
similar to Vg in the wing disc and is required for wing formation and the proper expression of Vg and other genes. In other discs, such as the leg and eye discs,
sd is endogenously expressed and is upregulated wherever ectopic Vg is able to induce
wing-specific gene expression and trigger wing development. It is noted, however, that
a sd enhancer trap line and the SRF-intervein C enhancer transgene are also ectopically induced by
Vg in the presumptive notum, although at levels lower than those observed in the wing pouch. This is consistent with the inability of Vg to trigger wing development and induce other wing
patterning genes in the developing notum. Indeed, mis-expression studies show that Sd function is required in parallel with Vg in order for Vg to
exert its wing inducing activity. The three wing-specific enhancers from the SRF and vg genes are activated synergistically
when Sd and Vg are coexpressed in Drosophila S2 cells. Although each individual protein
has some effect on reporter gene expression, this is significantly less than that observed in
the presence of both Vg and Sd. Titration of the amounts of transfected Vg and Sd plasmids
with all enhancers shows that the relative concentration of the two factors is critical and, at any
given Vg concentration, high levels of Sd reduce activation (Halder, 1998).
To define the sequences of the enhancers that respond to Vg/Sd, the activation of smaller
fragments from the 704-bp SRF intervein C enhancer, the 806-bp vg quadrant enhancer, and the
754-bp vg boundary enhancer in tissue culture were analyzed. A 125-bp fragment (SRF-A)
derived from the 5' end of the SRF enhancer is activated, whereas an adjacent 131-bp fragment
(SRF-B) is not activated. A 65-bp fragment from the vg quadrant enhancer (MD2) has
been identified that, when multimerized, produces an expression pattern very similar to the full-length
enhancer in wing discs. When assayed in tissue culture, MD2 is
activated by Vg and Sd. Within the vg boundary enhancer, a 120-bp fragment sufficient to
drive reporter gene expression in the wing pouch (vg-A) as well as a nonoverlapping 90-bp fragment
(vg-B) are also activated synergistically by cotransfection of Vg and Sd. Sd was shown, using mobility shift and DNase I footprinting assays, to bind specifically to essential sites for target gene activation (Halder, 1998).
These results demonstrate that the activation of several genes in the wing field requires Vg/Sd function.
It is also known that for each of the cis-regulatory elements analyzed here, direct input(s) of particular
signaling pathways are also required. Specifically, the activation of the SRF intervein C element
requires both Vg/Sd and Hh signaling; the activation of the vg boundary enhancer requires Vg/Sd and N signaling, and the activation of the vg quadrant enhancer requires Vg/Sd and Dpp signaling. Because these regulatory elements are not expressed in all tissues in which the signals are
active, nor in all wing cells in which Vg/Sd are active, it is deduced that neither the input of various
signals nor of Vg/Sd alone are sufficient for gene activation in vivo. Rather, the results suggest that the
various wing-specific cis-regulatory elements require a combination of direct inputs, comprising the
Vg/Sd selector function, which restricts expression to the wing field, and at least one signal transducer
that mediates signaling inputs and hence, the pattern of gene expression within the wing field. One
prediction of this model is that gene expression patterns within the wing field may be changed by
altering the signal-transducer binding sites within a cis-response element. To test this, the
Suppressor of Hairless [Su(H)] binding site that mediates the N input in the vg boundary enhancer
was changed to sites for the Cubitus interruptus (Ci) protein that transduces Hh signaling.
This switches the pattern of gene expression from a N-induced dorsoventral stripe to a Hh-induced
anteroposterior stripe while retaining the restriction of gene activation to the wing disc (Halder, 1998 and references).
These results demonstrate that the role of the Vg/Sd selector function is to directly regulate wing-specific
cis-regulatory elements that also require particular signaling inputs. The patterns of gene expression
induced in the wing disc are limited to cells in which both the selector genes and specific signaling
pathways are active. The response of the SRF-A, vg boundary, and vg quadrant enhancers to Hh, N, and Dpp signaling are limited to the wing pouch by Vg/Sd and occur in different patterns because of
their direct regulation by the Ci, Su(H), and Mad proteins, respectively. Furthermore, the
finding that the changing of the Su(H) binding site into a Ci binding site in the vg boundary enhancer
switches the pattern from a wing-specific dorsoventral N-regulated stripe to a wing-specific
anteroposterior Hh-regulated stripe suggests that spatial expression patterns are determined by the sites
for individual DNA-binding signal transducers. One corollary of this model is that for any given signaling protein, different selector proteins may be
involved in directing tissue-specific responses in different organs and tissues. For example, other studies have shown that tissue-specific enhancers in the embryo that are regulated by Dpp also require the action of
the Labial/Extradenticle or Tinman selector proteins to
limit expression to the endoderm or mesoderm, respectively. It is suggested that, in general, combinatorial
control by selector proteins and common signal transducers at a cis-regulatory level is required for the
tissue- and organ-specific responses of target genes to widely deployed signaling systems (Halder, 1998 and references).
The secreted Hedgehog (Hh) proteins have been implicated as mediators of positional information in vertebrates and invertebrates. A gradient of
Hh activity contributes to antero-posterior (A/P) patterning of the fly wing. In addition to inducing localised expression of Decapentaplegic (Dpp), which in turn
relays patterning cues at long range, Hh directly patterns the central region of the wing. Short-range, dose-dependent Hh activity is
mediated by activation of the transcription factor Collier (Col). In the absence of col activity, longitudinal veins 3 and 4 (L3 and L4) are apposed and the central
intervein is missing. Hh expression induces col expression in a narrow stripe of cells along the A/P boundary through a dual-input mechanism: inhibition of proteolysis
of Cubitus-interruptus (Ci) and activation of the Fused (Fu) kinase. Col, in cooperation with Ci, controls the formation of the central intervein by activating the
expression of blistered (bs), which encodes the Drosophila serum response factor (D-SRF). D-SRF activity is required for the adoption and maintenance of the
intervein cell fate. Furthermore, col is allelic to knot, a gene involved in the formation of the central part of the wing. This finding completes an understanding of the
sectorial organization of the Drosophila wing. It is concluded that Col, the Drosophila member of the COE family (Col/Olf-1/EBF) of non-basic, helix-loop-helix
(HLH)-containing transcription factors, is a mediator of the short-range organizing activity of Hh in the Drosophila wing. These results support the idea that Hh controls
target gene expression in a concentration-dependent manner and highlight the importance of the Fu kinase in this differential regulation. The high degree of
evolutionary conservation of the COE proteins and the diversity of developmental processes controlled by Hh signaling raises the possibility that the specific genetic
interactions depicted here may also operate in vertebrates (Vervoort, 1999).
Hedgehog (Hh) signaling from posterior (P) to anterior (A) cells is the primary determinant of AP polarity in the limb field in insects and vertebrates. Hh acts in part by inducing expression of Decapentaplegic (Dpp), but how Hh and Dpp together pattern the central region of the Drosophila wing remains largely
unknown. The role played by Collier (Col), a dose-dependent Hh target activated in cells along the AP boundary (the AP organizer in the imaginal wing disc) has been examined. col mutant wings
are smaller than wild type and lack L4 vein, in addition to missing the L3-L4 intervein and mis-positioning of the anterior L3 vein. These phenotypes are linked to col requirement for the local upregulation of both emc and N, two genes involved in the control of cell proliferation,
the EGFR ligand Vein and the intervein determination gene blistered. Attenuation of Dpp signaling in the AP organizer is also col dependent and, in conjunction with Vein upregulation, required for formation of L4 vein. A model recapitulating the molecular interplay between the Hh, Dpp and EGF signaling pathways in the wing AP organizer is presented (Crozatier, 2002).
It has been been proposed that Hh does directly control the position of L3 vein, although the molecular mechanisms of this control have not been firmly established. In both col and mtv mutant clones, the position of L3 vein is shifted posteriorwards. That both col and mtv control the position of L3 vein suggests that this position is defined by Hh signaling through the modulation of Dpp signaling. iro is required for rho activation in the L3 primordium and formation of L3 vein. iro is activated by both Dpp and Hh signaling and its anterior border of expression is under control of sal/salr, a target of Dpp. The patterns of col, iro and rho expression are intimately connected. Both an increased number of cells expressing rho and a posterior shift of the anterior border of iro expression are observed in col1 mutant discs. This posterior shift is interpreted as reflecting a modified range of Dpp signaling relayed, at least in part, by sal/salr activity. The increased number of rho-expressing cells, for its part, indicates that Col is able to antagonize rho activation by iro in cells, which express both iro and Col. This correlates well with the wing phenotype – anteriorwards shift of the L3 vein, together with gaps in its distal region – which results from anterior extension of Col expression, in UAS-Col/dpp-Gal4 wing discs. The distal gaps could reflect the complete absence of rho expression close to the DV border, because of the complete overlap between col and iro expression where iro expression is narrower. From col loss- and gain-of function experiments, it is therefore concluded that the primordium of L3 vein corresponds to cells that express iro but not col. Col thus appears to play a dual role in defining the position and width of L3 vein: activating Blistered and repressing EGFR in the wing AP organizer cells, endows these cells with an intervein fate, while attenuating Dpp signaling indirectly positions the anterior limit of iro expression domain, and L3 vein competence anterior to the AP organizer (Crozatier, 2002).
The stereotyped pattern of Drosophila wing veins is determined by the action of two morphogens, Hedgehog (Hh) and Decapentaplegic (Dpp), which act sequentially to organize growth and patterning along the anterior-posterior axis of the wing primordium. An important unresolved question is how positional information established by these morphogen gradients is translated into localized development of morphological structures such as wing veins in precise locations. In the current study, the mechanism has been examined by which two broadly expressed Dpp signaling target genes, optomotor-blind (omb) and brinker (brk), collaborate to initiate formation of the fifth longitudinal (L5) wing vein. omb is broadly expressed at the center of the wing disc in a pattern complementary to that of brk, which is expressed in the lateral regions of the disc and represses omb expression. A border between omb and brk expression domains is necessary and sufficient for inducing L5 development in the posterior regions. Mosaic analysis indicates that brk-expressing cells produce a short-range signal that can induce vein formation in adjacent omb-expressing cells. This induction of the L5 primordium is mediated by abrupt, which is expressed in a narrow stripe of cells along the brk/omb border and plays a key role in organizing gene expression in the L5 primordium. Similarly, in the anterior region of the wing, brk helps define the position of the L2 vein in combination with another Dpp target gene, spalt. The similar mechanisms responsible for the induction of L5 and L2 development reveal how boundaries set by dosage-sensitive responses to a long-range morphogen specify distinct vein fates at precise locations (Cook, 2004).
Extension of a previous analysis of ab in initiating L5 development has shown that ab functions early in L5 specification. Activation of all known vein genes, including rho, Dl, the caupolican and araucan genes of the Iroquois Complex (IroC), and argos, and repression of the intervein genes
bs (also known as DSRF) and net, is lost in cells corresponding to the L5 primordium in
ab1 mutant wing discs. A determination was also made whether it is critical that ab expression is confined to a narrow stripe for regulating expression of vein or intervein genes. ab was ubiquitously misexpressed in the wing disc using the MS1096-GAL4 driver; such global activation of ab suppresses expression of vein genes, such as rho and Dl. This ab misexpression also caused vein-specific downregulation of the intervein gene bs, in the wing disc, but did not repress expression of other genes, including hh, ptc and dpp. This phenotype may result from unregulated production of a lateral inhibitory signal normally produced by vein cells to suppress vein development in adjacent intervein cells (Cook, 2004).
Polycomb (PcG) and trithorax (trxG) group genes are chromatin regulators
involved in the maintenance of developmental decisions. Although their
function as transcriptional regulators of homeotic genes has been well
documented, little is known about their effect on other target genes or their
role in other developmental processes. The
patterning of veins and interveins in the wing has been used as a model with which to understand the function of the trxG gene ash2 (absent, small or
homeotic discs 2). ash2 is required to sustain the
activation of the intervein-promoting genes net and
blistered (bs) and to repress rhomboid
(rho), a component of the EGF receptor (Egfr) pathway.
Moreover, loss-of-function phenotypes of the Egfr pathway are
suppressed by ash2 mutants, while gain-of-function phenotypes are
enhanced. These results also show that ash2 acts as a repressor of the
vein L2-organising gene knirps (kni), whose expression is
upregulated throughout the whole wing imaginal disc in ash2 mutants
and mitotic clones. Furthermore, ash2-mediated inhibition of kni is independent of spalt-major and spalt-related. Together, these experiments indicate that ash2 plays a role in two processes during wing development: (1) maintaining intervein cell fate, either by activation of intervein genes or inhibition of vein differentiation genes, and (2) keeping kni in an
off state in tissues beyond the L2 vein. It is proposed that the Ash2 complex
provides a molecular framework for a mechanism required to maintain cellular
identities in the wing development (Angulo, 2004).
Loss of ash2 function causes differentiation of ectopic vein
tissue, indicating that ash2 is required for intervein development,
where it functions as an activator of the intervein-promoting genes
net and bs, restricting rho expression to vein
regions. In addition, the loss-of-function phenotypes of Egfr alleles
are rescued in ash2 mutants, while the gain-of-function phenotypes
are enhanced. Furthermore, rho mRNA exhibits an expanded expression
pattern in ash2 mutant tissues. Thus, ash2 promotes the
maintenance of intervein fate, either by activation of net and
bs or by repression of the Egfr pathway. Since rho and
bs/net expression is mutually exclusive, it cannot be determined
whether the Ash2 complex interacts directly with one or all of them. However,
since bs expression is inhibited by the loss-of-function of
ash2 during larval and pupal stages, it can be proposed that
ash2 acts as a long-term chromatin imprint of bs that is
stable throughout development (Angulo, 2004).
The results in adult clones and from analysis of genetic interactions
suggest that ash2 acts principally by maintaining B and D intervein
regions, since the C intervein remains unaltered in ash2 mutants. This
region is under the control of organising genes that respond to the Hh signal. One of
these genes is kn, which prevents vein differentiation in the C
intervein and is required for the expression of bs in this
domain. bs expression is regulated by two enhancer
elements: the boundary enhancer, which is dependent on hh and
controls bs expression in the C intervein region through kn;
and another enhancer dependent on Dpp activity, which controls bs
expression in B and D intervein domains.
Thus, the role of ash2 as a positive regulator of bs is
mainly restricted to regions beyond the kn domain where the Dpp
dependent bs enhancer is active (Angulo, 2004).
Gene expression is regulated in part by protein complexes containing ATP-dependent chromatin-remodelling factors of the SWI/SNF family. In Drosophila there is only one SWI/SNF protein, named Brahma, which forms the catalytic subunit of two complexes composed of different proteins. The protein Osa defines the Bramha associated protein (BAP) complex, and the proteins Polybromo and Bap170 are only present in the complex named PBAP. This work analysed the functional requirements of Osa during Drosophila wing development, and found that osa is needed for cell growth and survival in the wing imaginal disc, and for the correct patterning of sensory organs, veins and the wing margin. Other members of the BAP complex, such as Snr1, Bap55, Mor (Moira) and Brahma, also share these functions of Osa. Focus was placed on the requirement of Osa during the formation of the wing veins. Genetic interactions between osa alleles and mutations affecting the activity of the EGFR pathway suggest that one aspect of Osa is intimately related to the response to EGFR activity. Thus, loss of osa and EGFR signalling results in similar wing vein phenotypes, and osa alleles enhance the loss of veins caused by reduced EGFR activity. In addition, Osa is required for the expression of several targets of EGFR signalling, such as Delta, rhomboid and argos. It is suggested that one role of Osa and Brm in the wing is to establish a chromatin environment in the regulatory regions of EGFR target genes, making them available for both activators and repressors and facilitating transcription in response to EGFR signalling (Terriente-Félix, 2009).
Chromatin structure is critical to modulate gene expression during development, and is affected by a variety of alterations such as histone modification, DNA methylation and changes in conformation. Proteins related to Drosophila Brm, such as yeast SNF2 modify chromatin in an ATP-dependent manner, causing repositioning of nucleosomes along the DNA and re-distribution of histone proteins between nucleosomes. The SWI/SNF complexes are conserved in all eukaryotes, and display specific interactions with distinct transcription factors to regulate different subsets of genes. There are several examples where sequence-specific transcription factors interact specifically with SWI/SNF complexes. For example, the ATPase BRG1 binds Zn-finger proteins and hBRM interacts specifically with CBF-1/Su(H), which recruits hBRM to Notch target promoters such as those of HES1 and HES5 (Terriente-Félix, 2009).
A key aspect in the analysis of Brm function is the identification of targets accounting for the functions of the complex. A necessary step in this analysis is the description of its functional requirements using genetic approaches; which helps to identify the specific processes affected by loss of BAP function. The current data indicate that Osa is required during wing disc development for cell viability, cell proliferation, and for the formation of wing veins and the wing margin. Interestingly, increased expression of Osa in the wing also causes phenotypes related to wing growth and patterning, such as reduced wing size, ectopic sensory organs and hairs and the formation of extra vein tissue in most interveins. This analysis focused mostly on Osa, and this raises the question of whether its requirement reflects the function of the BAP complex. This is the most likely scenario, because the preliminary analysis of other BAP members, such as Snr1, Bap55, Mor and Brm uncovers similar phenotypes in the wing. Thus, lowering Snr1, Bap55 or Mor levels reduces wing size, disrupts the wing epithelium and causes the differentiation of ectopic sensory organs and hairs. These wings also display loss of veins, and in general the overall phenotypes are similar to those of loss of Osa. The phenotype of iRNA expression directed against brm is much milder, perhaps due to a lower efficiency of this construct, but still these wings show a loss of veins phenotype. The reduction of Bap170, a member of the PBAP complex, causes the formation of ectopic veins, which is the opposite phenotype to loss of function in osa and in other members that are present in both the BAP and PBAP complexes. Thus, although Brm is the catalytic subunit in both BAP and PBAP, these complexes could act in opposite manners on the same target genes at least during wing vein formation (Terriente-Félix, 2009).
Some Osa requirements can be explained by modifications in the transcriptional response to the activity of the Wg signalling pathway and by effects on wg expression. The function of Wg is required for the formation of the wing margin, including the development of sensory organs and veins along the anterior wing margin. In the absence of Wg signalling the wing margin does not form, and when Wg signalling is inappropriately activated ectopic sensory organs and hairs differentiate throughout the wing blade. In addition to affecting the response to Wg signalling, Osa is also required for the expression of wg along the dorso-ventral boundary. This requirement might be related to Notch signalling in these cells, and explains why the remnants of wing tissue formed in osa mutant wings do not form the wing margin or ectopic sensory organs (Terriente-Félix, 2009).
This study focused on the characterisation of Osa during the formation of the longitudinal wing veins. This process is independent of Wg signalling, and requires the activities of the Notch, Dpp and EGFR signalling pathways. Osa is needed for the expression of bs in the interveins, because bs is not expressed in cells mutant for osa. The regulation of bs expression involves the activity of Ash2 and the function of the Hh and Dpp pathways. It is suggested that Osa participates in the activation of bs facilitating the availability of its regulatory regions to these activators. This aspect of Osa function does not explain the phenotype of loss of veins characteristic of osa mutant cells, because the loss of Bs expression is normally associated with the differentiation of ectopic veins. The only context where bs mutant cells differentiate as interveins is when the activity of the EGFR pathway is reduced. Therefore, it is suggested that loss of bs expression is accompanied in osa mutant cells by a failure in the response to EGFR activity, leading to the differentiation of intervein tissue. Interestingly, the expression of bs is also severely reduced when Osa is present at higher than normal levels, and in this case loss of Bs is accompanied, as expected, by the formation of ectopic veins. The effects of increased Osa on bs expression can also be explained if Osa facilitates EGFR activity, because this pathway mediates the repression of bs in the proveins. In both cases, the common aspect mediated by Osa might be to regulate bs expression in collaboration with its transcriptional activators and repressors (Terriente-Félix, 2009).
Because the failure of osa mutant cells to differentiate the veins is not due to changes in bs expression, nor to changes in the expression of provein genes such as kni and caup, the search for Osa candidate targets was narrowed to the EGFR pathway. Several results suggest a close relationship between Osa and EGFR signalling in the wing. First, the phenotypes of changing osa expression in the veins are very similar to those resulting from the same manipulation in EGFR activity. Thus, a reduction in any core component of the EGFR pathway eliminates the veins, whereas the increase in EGFR signalling activity causes the formation of extra veins in intervein territories. Second, genetic interactions were observed between osa and several components of the EGFR pathway compatible with a function of Osa promoting EGFR activity in the veins. Finally, the extra veins caused by excess of Osa are suppressed when the activity of EGFR is reduced, indicating that Osa cannot substitute for EGFR activity. The changes in vein and intervein expression patterns are already detected in the wing disc, before other signalling pathways, such as Dpp, act to promote vein formation. Taken together, these observations suggest that Osa facilitates the response to EGFR activity in the wing disc, but cannot promote the transcription of EGFR targets in the absence of EGFR signalling (Terriente-Félix, 2009).
The changes in the expression of EGFR target genes observed in osa mutant cells or in osa gain-of-function experiments are compatible with a direct function of Osa/BAP is the transcriptional regulation of EGFR targets such as Dl, rho and aos. How Osa and the BAP complex are targeted to specific genomic regions is not entirely clear, although it is likely that sequence-specific transcription factors are involved in this process. Transcription in response to EGFR signalling is mediated by proteins belonging to the ETS family, such as Pointed-P2, Pointed-P1 and Yan in Drosophila. However, these genes are not required during wing vein formation, suggesting that other ETS proteins or uncharacterised transcription factors bring about interactions between the regulatory regions of EGFR target genes and the BAP complex (Terriente-Félix, 2009).
It is unlikely that Osa participates in any step of the EGFR pathway previous to the transcription of its target genes. It was noticed, however, that the expression of dP-ERK, a direct read-out of the pathway activity, is also affected in osa mutant cells. Thus, these cells frequently fail to express normal levels of dP-ERK, a result indicating that EGFR activity is reduced. The most likely explanation for this observation is that, in the wing, the EGFR pathway is engaged in a positive feedback loop mediated by the activation of rho expression, which maintains EGFR activity in cells where it has already been activated. Thus, loss of osa leads to a failure to express rho and subsequently to a reduction in the activity of the pathway detected as a loss of dP-ERK expression. There is one experimental situation in which Osa function appears to be dispensable for the expression of EGFR target genes. Thus, when a constitutive active form of Ras, RasV12, is driven in the wing, the augmented expression of Dl and aos, and the accumulation of dP-ERK are not affected by a reduction in Osa levels. It is possible that in this situation of strong and constitutive activity of the pathway, the possible modifications to chromatin structure brought about by Osa/BAP on EGFR target genes are not necessary, perhaps because at this level of EGFR activation the transcriptional repressors antagonising EGFR target gene transcription, such as Cic and Gro, are inactivated by the pathway, and this might make dispensable the function of Osa (Terriente-Félix, 2009).
It is not entirely clear to what extent the link observed between BAP function and EGFR signalling during wing disc development is conserved in other developmental systems and in other organisms. Some phenotypes of osa and brm alleles described in the eye disc, such as the loss of photoreceptor cells, are also observed upon a reduction in EGFR activity. Similarly, the loss of distal growth in the legs is also characteristic of reduced EGFR activity. These data are indicative of a general requirement for Osa in the expression of EGFR target genes at least in imaginal discs. The genetic approach that was used identifies transcription downstream of EGFR signalling as a relevant in vivo function of BAP complexes. Subsequent biochemical analysis should determine whether the functional interactions observed are mediated by direct binding of BAP to the regulatory regions of bs and other EGFR target genes (Terriente-Félix, 2009).
There is a genetic interaction between blistered/Serf and rhomboid suggesting that blistered restricts the expression of rhomboid to vein regions. In blistered mutants rhomboid expression becomes more intense and widens to include regions normally fated to become intervein regions. The increased area of rhomboid anticipates the final vein phenotype of blistered mutants. Mutant blistered wing phenotypes range along a continuum from ectopic venation and a moderate frequency of localized blistering, to abnormally thick posterior veins and a high frequency of blisters, to a complete loss of adhesion between the two wing surfaces resulting in ballooned wings. In rhomboid/blistered double mutants the wing defects associated with blistered/Serf mutations are completely suppressed (Fristrom, 1994).
There is also a genetic interaction between blistered/Serf and the integrin genes inflated and (myospheroid) suggesting that blistered/Serf might regulate integrin expression. There is an increased frequency and severity of blisters in progeny when mutant blistered males are crossed to females carrying inflated or mys. For the most part blistered/integrin combinations do not affect venation even when the blisters are very large (Fristrom, 1994).
Because of the necessity for the concomitant suppression of
the alternative fate when vein or intervein fates are specified,
a system evolved in which Rhomboid determines vein development
by repressing net and later blistered, which in turn specify intervein
fate by repressing vein development in intervein regions. Such
a balanced system is intrinsically labile unless it is stabilized
through feedback loops. Multiple feedback loops
operate at all tiers of vein fate regulation, and tiers are closely
linked and overlap in time, which further enhances the stability
of the system because it generates redundancy. It is proposed that
the functions of Net and Bs are partially redundant because
they both repress rho in intervein regions during overlapping,
though not identical, developmental periods. Thus, while Net
represses rho in all intervein sectors of third instar wing discs
except sector C, Bs begins to repress rho in these regions only
in early prepupal wings. In view of this
hypothesis, it might be less surprising that the wing phenotype
of net null mutants is much weaker than that resulting from
ubiquitous expression of rho, which also represses net
completely, but converts almost the entire wing into vein
material. It is assumed that the lack of
Net function in net minus wing discs is partially compensated by the
activation of bs, whose product represses rho in most of the
intervein regions during the prepupal and pupal stage. This
assumption is consistent with the observations and
with the earlier finding that bs null mutants exhibit a wing
phenotype very similar to that resulting from ubiquitous
expression of rho in the developing wing. The rhove -like phenotype obtained after
ubiquitous expression of Net during wing development is
largely explained by the ability of Net to repress rho. The
partial redundancy of net and bs functions in wing discs is
supported by experiments in which ubiquitous expression of
Net is still able to suppress the strong ectopic vein formation
phenotype of bs mutants (the phenotype is
indistinguishable from that produced in a net1 mutant
background). In addition, bs expression is
reduced in distal portions of net1 wing discs and hence appears
to depend partially on Net, a finding that
is consistent with the observation that LacZ expression of a
bs enhancer trap line is ectopically activated and
enhanced after ectopic expression of Net in
bs wing discs (Brentrup, 2000).
blistered is expressed in the precursors of the terminal tracheal cells and in the future intervein
territories of the third instar wing imaginal disc. Dissection of the blistered regulatory region reveals that a single enhancer element, which is
under the control of the fibroblast growth factor (FGF)-receptor signaling pathway, is sufficient to induce blistered expression in the terminal
tracheal cells. In contrast, two separate enhancers direct expression in distinct intervein sectors of the wing imaginal disc. One element is
active in the central intervein sector and is induced by the Hedgehog signaling pathway. The other element is under the control of
Decapentaplegic and is active in two separate territories, which roughly correspond to the intervein sectors flanking the central sector.
Hence, each of the three characterized enhancers constitutes a molecular link between a specific territory induced by a morphogen signal and
the localized expression of a gene required for the final differentiation of this territory (Nussbaumer, 2000).
A 500 bp enhancer element (TCE) has been isolated whose activity reproduces the blistered expression pattern
in terminal tracheal cells, with respect to its temporal,
spatial and regulatory cues. The TCE is the first enhancer
identified in Drosophila that responds to FGF signaling. It
has been reported that the FGF signaling cascade activates
the MAP kinase pathway and that the Ets-domain containing
protein Pnt is a target for the ERK-MAP kinase. Therefore, the expression of the TCE is likely to be controlled by FGF signaling which, through the ERK-MAP kinase pathway, activates a Pnt-DNA-bound complex
in conjunction with other factors.
Further dissection of the enhancer and the identification of
the individual DNA sites and the relevant transcription
factors should help to elucidate how FGF triggers specific
nuclear responses. Interestingly, during early mesoderm
induction in Xenopus laevis, an Ets-SRF complex has
been implicated in transducing FGF signaling. Therefore, blistered might not only be a target gene whose transcription is activated in response to FGF
signaling, but it might also encode a protein that assembles into a
complex to integrate the FGF signal. However, the TCE
does not require Blistered itself for signal induction since it is
still active in a blistered loss-of-function mutant. Nevertheless, other
putative target genes induced via FGF signaling in the
terminal tracheal cells could require the activation of an
Ets-Blistered-DNA complex (Nussbaumer, 2000).
Is blistered a marker for 'pro-intervein' territories?
Two classes of genes involved in the late differentiation
of the wing have been characterized previously: vein-promoting genes (e.g. ve/rho, vein) and vein-suppressing
genes (e.g. bs, net, plexus). However, it has been proposed that blistered should be considered as an 'intervein-promoting
gene' since its expression in wing cells is required to determine intervein fate. The expression of blistered is negatively regulated in vein territories by the
EGF signaling pathway, whereas ve/rho
expression is restricted to the future veins in the wing pouch
and this expression is indirectly controlled by the long range
organizers of the wing field. Therefore, the simplest
mechanism for establishment of the vein-intervein network
would be to spatially activate vein-specific genes, which,
through their activity, would repress a wing-blade specific
enhancer in the developing vein territories. Surprisingly, promoter analysis reveals that blistered expression in different presumptive intervein territories depends on distinct enhancers, which are controlled by different morphogen
events. Congruent with this observation, it has been
proposed that the vein-intervein network has to be considered on a stripe-by-stripe basis. Since the EGF signaling pathway represses blistered expression in the third instar wing disc, the blistered promoter
could also contain DNA elements negatively regulated by
EGF signaling. In summary, the expression of blistered in
the future intervein cells is triggered by the integration of several signaling events, probably through distinct regulatory elements (Nussbaumer, 2000).
It has been proposed that once the vein and intervein domains have been demarcated independently, gene interactions may occur at vein-intervein boundaries to
maintain and refine their respective domains. During metamorphosis, veins differentiate within the broader provein territories. The blistered expression
domain within the future intervein sector C abuts the A/P
boundary, although at the end of metamorphosis, this
expression expands into adjacent posterior cells. Thus, since the provein territory L4 is posterior and abuts the A/P boundary, blistered in the third instar wing disc is excluded from this provein territory at the A/P boundary. It will be of
great interest to determine whether blistered is excluded
from the other provein domains of the third instar wing
disc. In this case, blistered would have to be considered
as an early marker for 'pro-intervein' territories and its expression would progressively expand during metamorphosis to the final intervein domains of the adult wing (Nussbaumer, 2000).
An enhancer (the boundary enhancer) has been identified
that is activated by Hh signaling in the cells anterior to the
A/P compartment boundary. In agreement with previous
reports demonstrating a direct morphogenic role of Hh in
the central region of the wing, this might indicate that the Hh signaling
is required to trigger intervein differentiation through blistered
expression in the intervein C domain. However, in contradiction to the activity of the boundary enhancer, blistered is
expressed in smo mutant clones analyzed during pupal
development. Noteworthy, the clones that were generated were analyzed during third instar, whereas blistered expression is detected later, 24-36 h
after puparium formation. At this time, gene interactions
between vein- and intervein-specific genes might be sufficient to maintain their respective, mutually exclusive expression domains. Thus, Hh would
be required only for the early setting of blistered expression
as a result of patterning the intervein sector C. Indeed, beta-galactosidase expression directed by the boundary enhancer
is not detected in the wing of newly emerged flies. This indicates that in the presumptive intervein sector C, the early setting of blistered is controlled
through the boundary enhancer, whereas the later expression might recruit another cis-regulatory element. The fact that the expression of blistered is observed in the posterior compartment of pupal wings, whereas the boundary enhancer is restricted to the anterior compartment in third instar discs, further supports this idea (Nussbaumer, 2000).
The boundary enhancer is directly regulated by Vestigial (Vg) and
Scalloped (Sd) which form a complex on a 120-bp DNA
sub-element. The wing-specific Vg-Sd
complex restricts the activation of the boundary enhancer to
the future wing, consistent with the finding that Ci can only
activate it in the pouch region. Hence, the boundary enhancer integrates positional cues from the Vg-Sd transcriptional complex and the Hh signal. The gene knot/collier (kn), which encodes a putative DNA-binding protein acting
downstream of the Hh signaling pathway, has been found to
be required for the expression of blistered in the intervein
sector C. Therefore, the Hh responsiveness of the boundary enhancer may be indirect and mediated by Kn. Alternatively, activation of the enhancer may require a molecular interaction between Ci and Kn. Therefore, it will be of prior interest to determine whether Kn and Ci directly regulate the boundary enhancer and
cooperate for its activation. Further analysis of how the
boundary enhancer integrates input from the Vg-Sd
complex and Hh signaling will contribute to a molecular
understanding of the synergistic activation of enhancers by
signaling input and selector genes, a strategy that may be
widely used to regulate gene expression during development (Nussbaumer, 2000).
In mammalian cells, SRF typically functions in conjunction with an Ets domain containing ternary complex factor such as Elk-1. (Ets containing transcription factors in Drosophila include Pointed and Yan) . The domain of SRF that interacts with ternary complex factors is conserved in the Drosophila SERF protein, suggesting that an Elk-1 like factor may be present in the fly, although none has yet been identified. To test for involvement of a ternary complex factor in terminal branching, a dominant negative form of mammalian Elk-1 protein was expressed constitutively in the fly. The dominant negative form is a C-terminal truncation of Elk-1, that removes the Elk-1 transcriptional activation domain, so that the protein can still interact with SRF and bind DNA but fails to activate transcription. Expression of the dominant negative Elk-1 during the period of terminal branching results in truncated terminal branches similar to those observed in the pruned loss of function mutants. The extra, unregulated branching observed with activated Elk-1 is completely dependent on the function of the endogenous Srf gene. This suggests that an unidentified Elk-1 factor exists in Drosophila (Guillemin, 1996).
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, 2004).
Mutants that cause changes in bristle morphology in Drosophila have been found to encode actin regulatory proteins such as profilin (chickadee) and cofilin phosphatase (slingshot) as well as myosins (crinkled). EP37532, a P element insertion, was identified based on its recessive bristle defects. EP37532 was inserted in the predicted gene CG32296, now renamed mal-d. A stronger allele, mal-dΔ7, was generated by removing the first exon of the gene. mal-dΔ7 mutant flies show a more penetrant bristle phenotype and are in addition female sterile. An antibody directed against MAL-D protein was generated and affinity purified. It showed a single band on a Western blot, which was not detectable in mal-dΔ7 mutant ovaries and was enhanced upon mal-d overexpression. To look at the phenotype on a cellular level, clones of mal-dΔ7 mutant cells were generated within the follicular epithelium, a simple monolayer epithelium in the ovary. The mutant cells proliferate and differentiate properly. However, the basal network of actin filaments prominent in the differentiated epithelial cells is reduced. Other F-actin-rich structures, such as cortical F-actin and ring canals in germline cells, were slightly reduced in mutant ovaries (Somogyi, 2004).
While most follicle cells appeared to function normally without mal-d, border cell migration was severely perturbed in mutant females. In mal-dΔ7 mutant animals, border cell clusters either did not initiate migration at all or migrated very poorly. Clonal analysis showed that this defect is cell autonomous. When migrating, border cells normally display a particularly robust actin cytoskeleton with higher F-actin levels than nonmigrating follicle cells. This enhanced F-actin accumulation is completely absent in mal-dΔ7 mutant border cells. Ubiquitous expression of a mal-d cDNA rescued the mal-dΔ7 mutant phenotypes completely (normal bristles, normal border cell migration), confirming the gene identification. Thus, the mal-d gene product affects F-actin accumulation in multiple cell types and is required for border cell migration (Somogyi, 2004).
MAL-D is related to mammalian MAL/MRTF-A/MKL1/BSAC, MAL16/MRTF-B/MKL2, and Myocardin proteins, with an N-terminal MAL homology domain (MHD) containing three RPEL motifs and a SAP domain, as well as a less well-defined basic region. Mammalian MAL family proteins have been found to interact with SRF and serve as transcriptional cofactors for SRF. Ternary complex factors (TCF), which are ETS domain proteins, represent another type of SRF cofactor in mammalian cells. Cofactors had not been identified for Drosophila SRF; specifically, there was no evidence for a TCF gene in the sequenced Drosophila genome. MAL-D appears to be the only MAL family protein in Drosophila. In transfected Schneider cells, it was found that Drosophila SRF and MAL-D could be coimmunoprecipitated and cooperate to activate transcription from a serum response element (SRE)-containing reporter plasmid. This indicates that MAL-D is a bona fide SRF cofactor in Drosophila. To investigate this in vivo, phenotypes of mal-d and SRF mutants were compared (Somogyi, 2004).
SRF is essential for viability in flies and for proper tracheal and wing development. The mal-dΔ7 mutation removes a 5′ noncoding exon of mal-d that is required for expression in the ovary, and homozygous flies are viable but female sterile. However, the coding region is not altered in mal-dΔ7 mutant flies, and mal-d is still expressed at other times during development. To determine whether mal-d is an essential gene, additional mal-d alleles were generated by ems mutagenesis. Three alleles were used for further analysis: mal-dS9 and mal-dS2 both have a stop codon in the middle of the open reading frame (L659 and Q675 to stop, respectively), and mal-dF2 has a frameshift at position A1364. The mal-d ems alleles were homozygous and transheterozygous early larval lethal, with mal-dF2 larvae surviving longer, suggesting that this might be a hypomorphic allele. The mal-d mutants could be rescued by a mal-d cDNA, expressed ubiquitously under control of an alpha-tubulin promoter. Thus, like Drosophila SRF, mal-d is required for development. Clones of cells mutant for SRF or the new alleles of mal-d showed essentially the same phenotypes as mal-dΔ7 in border cell migration, F-actin accumulation, and bristle morphology. However, none of the mal-d alleles showed blistering when clones were induced in the wing primordium, as found for SRF mutant clones. In patterning the intervein region of wings, SRF may therefore act alone or with a different cofactor. Overall, these results indicate that SRF and MAL-D act together during development to control specific processes that are highly dependent on the actin cytoskeleton. In particular, SRF and MAL-D are required for accumulation of a robust actin cytoskeleton during border cell migration and for this invasive migration event to be effective (Somogyi, 2004).
SRF immunoreactivity is detected in most or all nuclei of both germline and somatic cells. Although a potential transcriptional cofactor, MAL-D protein was detected mainly in the cytoplasm even when highly overexpressed. Mammalian MAL was also found to be cytoplasmic in serum-starved NIH/3T3 cells. Removal of the N terminus of mammalian MAL with the conserved RPEL motifs renders it nuclear and active. The corresponding change in MAL-D (MAL-D-ΔN) had the same effect: MAL-D-ΔN was largely nuclear and highly transcriptionally active in a transfection assay. Expression of MAL-D-ΔN in follicle cells induces excessive F-actin accumulation, an effect opposite that from the mal-d loss-of-function phenotype. Overexpressing high levels of wild-type MAL-D has a similar but milder effect on F-actin. These results indicate that, as for the mammalian MAL, MAL-D protein can accumulate in the cytoplasm, but the nuclear form is the active one. Taken together with the loss-of-function analyses, this indicates that a transcription factor complex consisting of SRF and MAL-D positively regulates genes important for establishing a robust F-actin cytoskeleton (Somogyi, 2004).
To understand why cells in a tissue might need a robust F-actin cytoskeleton, border cell migration, which shows a strong dependence on MAL-D and SRF, was examined. At the initiation of migration, border cells normally produce an actin-rich long cellular extension. Formation of this extension requires proper cell specification, directional signals via the guidance receptors EGFR and PVR, and substrate adhesion via DE-cadherin, but it does not require force generation by myosin, functionally separating these steps. mal-d mutant border cells did produce long cellular extensions, indicating that guidance and adhesion were occurring properly. Subsequently, mal-d mutant border cells showed a unique defect. Large, round cytoplasmic fragments (without nuclei) appeared to 'break off' from the extension. At later stages, the cell fragments were detected progressively further along the normal migratory path. Inspection of intervening confocal sections showed no evidence of any connection between the cytoplasmic fragments and the rest of the cell. The spherical shape also suggested that these fragments were unattached. Thus, failure to augment the cytoskeleton in mal-d mutants led to fragmentation of the long cellular extensions. The border cell fragments continued to move directionally, leaving the cell body and nucleus behind (Somogyi, 2004).
This behavior of mal-d mutant border cells indicates that fragments of invasive, migratory cells have sufficient autonomy to respond to guidance cues and move through a tissue. The fragments appear to move less efficiently than normal border cells (reach the oocyte at a later stage). This could either be due to the cell fragments being fragments and not whole cells or be due to their mutant origin. It has previously been shown that anucleate leukocyte fragments (cytoplasts) can perform chemotaxis in vitro, demonstrating that cytoplasmic fragments can have considerable autonomy from the nucleus with respect to migration in vitro. Specific transcription factors are required for cells to differentiate and acquire migratory/invasive behavior during development. In addition, the MAL-D/SRF complex is required for cells to acquire a robust cytoskeleton and remain intact when performing an invasive migration. However, nuclei and therefore transcriptional changes are apparently not essential for guided movement in vitro or in vivo (Somogyi, 2004).
MAL-D activity might simply be required to stimulate F-actin accumulation and thus contribute to trigger border cell migration. However, expression of constitutively active MAL-D-ΔN in border cells effectively blocks migration, indicating that MAL-D activity needs to be regulated. As a first step in understanding this regulation, when endogenous MAL-D could be detected in the nucleus was investigated as an indication of when MAL-D/SRF might be active. Endogenous MAL-D was detectable in nuclei of some migrating border cells. Nuclear MAL-D can be detected in cells initiating migration or during migration but not when migration is complete (stage 10). About half of the migrating border cell clusters contained one or more nuclei clearly positive for MAL-D, but no specific stage of the migration was always positive. Thus, nuclear MAL-D apparently dies not reflect the cluster position in the egg chamber or developmental stage. During migration, outer border cells could be positive, but the central polar cells were always negative. The polar cells are part of the border cell cluster but are not actively migrating. Both front and rear border cells could be positive. This suggested that MAL-D accumulation is regulated in some dynamic way related to migration. It was noticed that clusters that were elongated or stretched had a high probability of positive nuclei, whereas rounded clusters were less likely to show staining. To quantify this, the length of midmigration clusters was measured as an indicator of stretching. The correlation to MAL-D-positive nuclei was statistically highly significant. Thus, nuclear accumulation of MAL-D correlates with the stretched shape of the migrating cell cluster. Stretching of the cell cluster would be expected to reflect external force application and tension within the cell (Somogyi, 2004).
To further 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).
The conserved protein structure, in particular the conserved RPEL motifs (MHD), as well as the functional interactions with SRF suggested that mammalian and fly MAL proteins might be regulated in similar ways. In a series of interesting experiments, activation of mammalian SRF and nuclear accumulation of MAL have shown to respond to changes in actin dynamics in NIH-3T3 cells. The N-terminal RPEL motifs of MAL were required for this regulation, which has also been called the Rho-actin pathway. One of the strongest activators of MAL/SRF was an activated form of Diaphanous, which acts downstream of Rho. To determine whether MAL-D could be subject to the same regulation, a corresponding activated form of Drosophila Diaphanous (HA-diaCA) was made and overexpressed in border cells. Border cell migration was blocked by HA-diaCA; however, nuclear accumulation of MAL-D was nevertheless stimulated. This effect was observed on endogenous MAL-D but was most obvious when looking at border cell clusters cooverexpressing MAL-D and HA-diaCA. In border cells, as in follicle cells, overexpressed MAL-D was predominantly cytoplasmic. In contrast, when HA-diaCA was present, MAL-D was predominantly nuclear. When both proteins were expressed at high levels, the nuclear pool of MAL-D was still detectable, but MAL-D was mainly cytoplasmic, suggesting that nuclear translocation was saturable. Thus, the ability of the Rho pathway to activate MAL proteins appears to be conserved in Drosophila (Somogyi, 2004).
It is therefore proposed that the transcription factor complex of MAL-D and SRF is responsible for a regulatory mechanism by which physical pulling force upon and tension within an invasively migrating cell induces a compensatory strengthening of its cytoskeleton. Mutant analysis has shown that MAL-D and SRF are required for migrating border cells to build up a robust cytoskeleton and remain intact during invasive migration. Regulation via MAL-D may be particularly critical for cells that perform force-demanding processes such as invasive cell migration. At least, this is the case for border cells. It will be of interest to determine whether this regulation also plays a role in pathologically invasive migrations such as in metastasis. While they migrate, border cells normally display a very robust F-actin cytoskeleton. It is suggested that this F-actin accumulation results from multiple rounds of MAL-D activation during migration. Failure to augment the cytoskeleton leads to fragmentation of the long cellular extensions leading the invasion and production of migrating 'cytoplasts'. Although these fragments are not produced by normal cells, their behavior can be useful in determining what cells can do in vivo without a nucleus. Production of platelets by megakaryocytes is an example of physiological productions of cell fragments -- although not migratory (Somogyi, 2004).
How does MAL-D/SRF regulate the actin cytoskeleton and cell integrity? Studies in mammalian cells give some indications of what the critical target genes might be in Drosophila. Cytoskeletal actin and vinculin genes can be regulated by a feedback mechanism, and these genes are regulated by SRF (and MAL). In mouse ES cells, SRF is important for the production of actin-directed cytoskeletal structures and cell motility. No changes in total actin levels were detected in mal-d mutant tissues. However, actin polymerization and actin filament organization is highly regulated in cells. MAL-D- and SRF-dependent changes in F-actin accumulation could therefore be due to changes in levels of any of the many actin-regulating and actin-interacting proteins, including myosins. Dictyostelium amoebae mutant for myosin II heavy chain display loss of cortical integrity and cell fragmentation when cells migrate under restrictive environments, apparently due to loss of the actin-crosslinking activity of myosin II. Further analysis of transcription profiles is required to pinpoint the exact target genes of MAL-D/SRF. Interestingly, several MAL family proteins as well as SRF are important for muscle-specific gene expression. Also, stretching of mammalian myogenic cells in culture leads to a complex set of trophic and differentiation responses, including increased production of SRF. It is tempting to speculate that the prominent role of MAL/SRF in muscle differentiation is related to its regulatory role in tension-dependent gene expression in nonmuscle cells, muscle being a dedicated actin/myosin-dependent contractile tissue (Somogyi, 2004).
What is the molecular mechanism for MAL-D regulation by tension? Given that the actin cytoskeleton and tension or cell shape changes are interdependent, it is likely that this regulation is related to the regulation of MAL/SRF by actin dynamics (the Rho pathway). Two models were proposed to explain the effect of actin on MAL and SRF. The simplest model is that free G-actin sequesters MAL in the cytoplasm, and depletion of this G-actin pool by actin polymerization results in MAL translocation/activation. Observations in border cells do not fit very well with this simple model. In normal cells, even very highly overexpressed MAL-D is almost exclusively cytoplasmic, indicating practically unlimited capacity in the cytoplasm. Expression of constitutively active Diaphanous, which should 'release' MAL-D by causing actin polymerization, did cause accumulation of MAL-D in the nucleus. But further overexpression of MAL-D led to more protein in the cytoplasm, not in the nucleus as would be expected if G-actin depletion in the cytoplasm (induced by active Diaphanous) were the trigger for nuclear translocation. Finally, even though endogenous MAL-D is expressed at low levels, overexpression of a nonpolymerizable form of actin in border cells did not appear to sequester MAL-D in the cytoplasm. These data seem more consistent with the alternative 'active' model of MAL activation, wherein a subpopulation of actin or an actin protein complex accumulates when actin polymerization is favored, leading to MAL nuclear translocation and activity (Somogyi, 2004).
There are two general ways in which regulation of MAL by actin and by tension might be related. Changes in actin dynamics, as induced by activated Diaphanous, may induce changes in tension, which could then affect MAL. For example, RhoA activation can induce formation of stress fibers, which are contractile structures. Conversely, changes in cell tension could affect RhoA, Diaphanous, and thereby actin dynamics, which then in turn directly regulate MAL. In fact, RhoA and Diaphanous, two of the most potent activators of SRF/MAL, have been shown to be important mediators of mechanosensitive changes at focal adhesions. The physical interaction observed between the conserved N-terminal domain of MAL and unpolymerized forms of actin suggests that regulation of MAL by actin is quite direct and thus supports this type of relationship. Tension applied to cell-matrix attachments or cell-cell interactions may also locally increase actin polymerization by other means and thereby activate MAL. A more speculative link to MAL regulation is offered by actin itself. A specific conformation of actin, or a specific protein complex containing actin, may be induced by tension and serve as the signal that is perceived by MAL. This would be consistent with the idea that a particular subpopulation of actin is responsible for the active regulation of MAL. It would be an elegant way for hard-working migratory cells to regulate strength as needed by the actin cytoskeleton. It is usually thought that actin-myosin supplies force and tension; the MAL/SRF system suggests a role for the complex actin cytoskeleton in force perception as well (Somogyi, 2004).
Serum response factor (SRF) regulates genes involved in cell proliferation, migration, cytoskeletal organization, and myogenesis. Myocardin and myocardin-related transcription factors (MRTFs) act as powerful transcriptional coactivators of SRF in mammalian cells. An MRTF from Drosophila, called DMRTF, is described that shares high homology with the functional domains of mammalian myocardin and MRTFs. DMRTF forms a ternary complex with and stimulates the activity of Drosophila SRF, which has been implicated in branching of the tracheal (respiratory) system and formation of wing interveins. A loss-of-function mutation introduced into the DMRTF locus by homologous recombination results in abnormalities in tracheal branching similar to those in embryos lacking SRF. Misexpression in wing imaginal discs of a dominant negative DMRTF mutant also causes a diminution of wing interveins, whereas overexpression of DMRTF results in excess intervein tissue, abnormalities reminiscent of SRF loss- and gain-of-function phenotypes, respectively. Overexpression of these DMRTF mutants in mesoderm and in the tracheal system also perturbs mesoderm cell migration and tracheal branching, respectively. It is concluded that the interaction of MRTFs with SRF represents an ancient protein partnership involved in cytoplasmic outgrowth and cell migration during development (Han, 2004).
The Drosophila tracheal system is comprised of a network of interconnected epithelial tubes that undergo sequential sprouting. Terminal branches of the tracheal network are formed from individual cells that express high levels of DSRF and extend long cytoplasmic processes toward target tissues. Signaling from fibroblast growth factor (FGF) to SRF has been shown to regulate cytoplasmic outgrowth of terminal cells during tracheal branching. The similarities between the tracheal branching phenotypes of DMRTF and DSRF mutant embryos suggest that DMRTF and DSRF may function together during terminal tracheal branching. The small GTPase Rac, which regulates cell adhesion and actin-based cytoskeletal motility, has also been shown to act in a signaling pathway that interconnects FGF signaling with SRF during tracheal branching. The DMRTF RNAi tracheal phenotype is similar to that of embryos lacking Rac1 and -2, suggesting DMRTF may act in the Rac signaling pathway during tracheal development. Given the role of the mammalian DMRTF ortholog, MRTF-A/MAL, in transduction of growth signals and changes in actin treadmilling to SRF, these findings point to a similar role for DMRTF during cytoplasmic branching of the tracheal system in Drosophila (Han, 2004).
The wings of Drosophila are derived from sheets of epithelial cells that become subdivided into vein and intervein cells, giving rise to a stereotypical pattern of veins that provide structural support to the wings. Intervein cells are required for adherence of the two surfaces of the wing. As in the developing tracheal system, signaling by FGF to SRF has been shown to be required for vein/intervein formation; the absence of either of these factors results in a deficiency of intervein cells (Han, 2004).
Consistent with the proposed role of DMRTF as a coactivator of SRF, DMRTF mutant embryos or embryos expressing dominant negative DMRTF displayed a loss of intervein cells. Conversely, misexpression of hypermorphic DMRTF alleles results in excess intervein cells and the concomitant loss of vein cells. The latter phenotype was also observed in response to misexpression of DSRF and DMRTF together (Han, 2004).
Mesodermal cells form in the ventral region of the Drosophila embryo and migrate in a dorsolateral direction to give rise to a uniform monolayer. This process is essential for the regional specification of different mesodermal derivatives, such as cardiac, somatic, and visceral muscles. Like the dependence of tracheal branching on FGF signaling, the mesoderm-specific FGF receptor Heartless has been shown to be required for mesoderm migration. Recently, a guanyl nucleotide exchange factor, Pebble, was found to be required for mesoderm migration, likely through modification of a small GTPase such as Rho and Rac. Interestingly, the activity of DMRTF, like that of its mammalian orthologue MRTF-A/MAL, is also stimulated by Rho-actin signaling in Drosophila cultured cell assays. Because misexpression of dominant negative and hyperactive forms of DMRTF results in opposite migratory phenotypes, DMRTF may act downstream of heartless and pebble in mesoderm migration (Han, 2004).
The target genes of DMRTF responsible for the diverse DMRTF gain- and loss-of-function phenotypes remain to be determined. It is imagined that many of these target genes are directly regulated by SRF and, indeed, several such genes with multiple SRF-binding sites in their control regions have been identified. However, it is also possible that transcription factors in addition to SRF serve as cofactors for DMRTF (Han, 2004).
SRF integrates diverse signals for cell growth, migration, cytoskeletal organization, and myogenesis via its association with transcriptional cofactors. The mammalian MRTF MAL has been shown to associate with G-actin in the cytoplasm and to translocate to the nucleus in response to growth factor signaling and actin treadmilling. It is intriguing that embryonic stem cells lacking SRF display defects in spreading, adhesion, and migration, all of which correlate with abnormalities in actin stress fibers. Thus, the striking parallels between the roles of SRF and myocardin family members in mammalian cells and Drosophila suggest that these factors comprise an ancient and evolutionarily conserved system for coupling changes in cell shape and extracellular signaling with cell migration during development (Han, 2004).
MIG-10/RIAM/lamellipodin (MRL) proteins link activated Ras-GTPases with actin regulatory Ena/VASP proteins to induce local changes in cytoskeletal dynamics and cell motility. MRL proteins alter monomeric (G):filamentous (F) actin ratios, but the impact of these changes had not been fully appreciated. This study reports here that the Drosophila MRL ortholog, pico, is required for tissue and organismal growth. Reduction in pico levels resulted in reduced cell division rates, growth retardation, increased G:F actin ratios and lethality. Conversely, pico overexpression reduced G:F actin ratios and promoted tissue overgrowth in an epidermal growth factor (EGF) receptor (EGFR)-dependent manner. Consistently, in HeLa cells, lamellipodin was required for EGF-induced proliferation. pico and lamellipodin share the ability to activate serum response factor (SRF), a transcription factor that responds to reduced G:F-actin ratios via its co-factor Mal. Genetics data indicate that mal/SRF levels are important for pico-mediated tissue growth. It is proposed that MRL proteins link EGFR activation to mitogenic SRF signaling via changes in actin dynamics (Lyulcheva, 2008).
The construction of properly sized and functional tissues and organs during animal development requires tight control of cell growth, proliferation, differentiation, and death. Networks of intracellular signal transduction pathways that respond to various secreted ligands and cell surface proteins coordinate these processes. Elucidating the nature of the intracellular signaling networks that connect extracellular stimuli to basic cellular machinery controlling proliferation, growth, and morphology is not only critical for the understanding of tissue size regulation during normal development, but is also important for the identification of aberrant events underlying numerous disease processes, including cancer (Lyulcheva, 2008).
A number of pathways regulating cellular development are initiated by ligation of transmembrane receptor tyrosine kinases (RTKs), such as the epidermal growth factor (EGF) receptor (EGFR). One of the key mediators of RTK signaling is the Ras GTPase, capable of activating proteins harboring Ras association (RA) domains to initiate downstream signaling pathways, such as the mitogen-activated protein kinase (MAPK) cascade, and ultimately resulting in changes in gene transcription. The Ras/MAPK and other canonical RTK signaling pathways have been well characterized, yet they cannot account for all of the observed effects of their respective extracellular signals (Lyulcheva, 2008).
The MIG-10/Rap1-GTP-interacting adaptor molecule (RIAM)/lamellipodin (Lpd) (MRL) proteins are a family of recently identified molecular adaptors, harboring an RA, pleckstrin homology (PH), and several proline-rich domains (Krause, 2004; Lafuente, 2004). Several lines of evidence indicate that MRL proteins act downstream of Ras-like GTPases and transduce extracellular signals to changes in the actin cytoskeleton, cell motility, and adhesion. In particular, Lpd interacts with active Ras and RIAM with active Rap1. Consistent with this, only RIAM is required for Rap1-induced cell adhesion. Lpd also binds to PI(3,4)P2 via its PH domain, which is sufficient for membrane targeting after platelet-derived growth factor stimulation. Both Lpd and RIAM utilize their proline-rich motifs to directly interact with the Enabled (Ena)/vasodilator-stimulated phosphoprotein (VASP) actin regulators, known to regulate lamellipodia formation and cell migration. In addition, Lpd knockdown impairs lamellipodia formation, whereas Lpd overexpression increases speed of lamellipodia protrusion in an Ena/VASP-dependent manner. Finally, both Lpd and RIAM have been shown to alter the cellular ratio between monomeric (G) and filamentous (F) actin, suggesting a wider role in regulating cell metabolism. Indeed, control of the G:F actin ratio is an essential way for cells to regulate gene transcription via the transcription factor serum response factor (SRF), and has been linked to changes in proliferation, migration, and differentiation (Lyulcheva, 2008).
This study reports the characterization of the Drosophila MRL ortholog, which has been named pico on the basis of the retarded growth phenotype resulting from pico knockdown or loss-of-function mutant. Reduction in pico levels results in reduced rates of cell growth and proliferation, whereas ectopic expression of pico promotes coordinated cell growth and proliferation, leading to tissue overgrowth. pico's effect on cell proliferation is conserved in its mammalian ortholog, Lpd. Evidence is presented that pico and Lpd link extracellular signaling to tissue growth via changes in actin dynamics and SRF activation. This is the first time that MRL proteins have been implicated in controlling cell proliferation and tissue growth (Lyulcheva, 2008).
Phylogenetic analysis has shown that pico (CG11940) encodes the only member of the MRL family of proteins in Drosophila. Two transcripts that were identified are generated from alternative transcription start sites of the pico transcription unit: pico and pico-L. pico-L encodes a 1159 amino acid protein that is identical to the protein encoded by pico, except for the presence of an additional 128 N-terminal residues. Both pico proteins contain RA and PH domains and proline-rich Ena/VASP binding sites characteristic of the MRL proteins (Lyulcheva, 2008).
This study shows that pico, which encodes the only Drosophila member of the MRL family of proteins, and its mammalian ortholog, Lpd, have a conserved role in the regulation of cellular proliferation. Reduced pico or Lpd levels result in reduced rates of cellular proliferation, but do not impair cell survival. Too much pico promotes coordinated growth and proliferation, leading to larger tissues with more normal-sized cells. In this respect, the effect of pico is distinct from that of many known Drosophila growth drivers. Growth regulators, such as Drosophila S6K, cause cells to accumulate mass faster than they can divide, primarily due to effects on translation, leading to cellular hypertrophy. Other regulators, such as E2F, can drive cell division without stimulating cell growth, leading to hyperplastic cellular hypotrophy and/or apoptosis (Lyulcheva, 2008).
Attenuating EGFR signaling abrogates the effect of ectopic pico on both F-actin accumulation and tissue growth. pico acts cell autonomously and is therefore unlikely to act upstream of Egfr by affecting the level of EGFR ligands. To rule out that pico regulates levels of EGFR, receptor levels and distribution was examined in wing imaginal discs overexpressing pico or picoIR. EGFR levels and distribution in these genetic backgrounds resembled wild-type. Another possibility is that pico regulates EGFR activity. Although suitable reagents were not available to directly monitor EGFR activity levels in wing discs, effects on extracellular signal-regulated kinase (ERK) activation, which provides a molecular readout for EGFR/Ras/Raf signaling, were measured. Diphosphorylated (dp) ERK levels were not affected by ectopic pico. These data suggest that, rather than being upstream of EGFR, Pico needs to be activated by EGFR or a downstream component of EGFR signaling, such as activated Ras. Consistently, both Lpd and Pico bind to activated, but not wild-type, Ras. Furthermore, pico knockdown partially suppresses the effects of ectopic Egfr and activated Ras; in addition, Lpd knockdown impairs the EGF-induced increase in proliferation. Taken together, these data suggest that pico and Lpd are downstream effectors of EGFR (Lyulcheva, 2008).
Ena/VASP has been reported to act downstream of MRL proteins. Correspondingly, it was found that pico-mediated wing overgrowth and F-actin accumulation are sensitive to the levels of ena. Importantly, ena is also sufficient to cause overgrowth and F-actin accumulation when overexpressed. Changes in actin dynamics induced by Ena/VASP proteins can activate SRF-dependent gene expression in mammalian cells. Similarly, it was found that Pico and Lpd can activate SRF activity. Like pico, ectopic mal or bs/SRF in flies are sufficient to cause wing overgrowth. Pico-mediated overgrowth is sensitive to the levels of bs/SRF, but mal-induced overgrowth could not be suppressed by pico knockdown, suggesting that Mal/SRF may act downstream of pico in flies. Collectively, these data suggest that MRL proteins may exert their mitogenic effects by specifically interacting with Ena/VASP proteins and inducing SRF-responsive transcription. Interactions between EGFR, MRL proteins, Ena/VASP, and Mal may provide a mechanism linking growth factor signaling and Mal-mediated SRF activation (Lyulcheva, 2008).
Are MRL proteins uniquely able to stimulate Mal/SRF-mediated tissue growth? Although other actin regulators are known to activate Mal/SRF (Posern, 2006), there is currently little data to indicate that they play a role in proliferation control. This might be explained if different transcriptional responses occur at different Mal-dependent SRF activation thresholds, leading to diverse cellular outcomes. Alternatively, other actin regulators might influence processes that limit net tissue growth. For instance, Rho activates Mal/SRF in mammalian cells, but increased Rho activity in flies is associated with loss of epithelial integrity and cell extrusion, which may negate any potential mal-mediated growth-promoting effects. These issues warrant further study in both flies and mammals. Future studies are also needed to characterize transcriptional targets of Drosophila SRF and resolve the contribution of SRF targets to MRL-mediated growth and proliferation (Lyulcheva, 2008).
Lpd expression appears to be differentially regulated in cancer compared to normal tissues. The data, showing a conserved role for MRL proteins in proliferation control, may provide a potential mechanistic explanation for these observations. In this regard, it is interesting that loss of pico or Lpd can abrogate the effects of EGFR/Erb signaling, deregulation of which has also been implicated in cancer progression. Collectively, these data suggest that MRL proteins might play a role in the pathogenesis of certain cancers and may therefore represent novel molecular targets for therapeutic intervention (Lyulcheva, 2008).
The transcription cofactor MAL (Myocardin-related transcription factor or Mrtf) is regulated by free actin levels and thus by actin dynamics. MAL, together with its DNA-binding partner, SRF, is required for invasive cell migration and in experimental metastasis. Although MAL/SRF has many targets, this study provides genetic evidence in both Drosophila and human cellular models that actin is the key target that must be regulated by MAL/SRF for invasive cell migration. By regulating MAL/SRF activity, actin protein feeds back on production of actin mRNA to ensure sufficient supply of actin. This constitutes a dedicated homeostatic feedback system that provides a foundation for cellular actin dynamics (Salvany, 2014).
The transcription cofactor MAL is regulated by cellular actin dynamics and confers this regulation on the activity of its DNA-binding partner, SRF. Free G-actin directly binds to MAL via RPEL motifs at the N terminus of MAL and negatively regulates its activity. This regulation is conserved from mammals to insects (Somogyi 2004). In the physiological context of the animal, the function of MAL and related proteins (MRTF-A and MRTF-B in mammals and mal-d/mrtf in Drosophila) appears conserved as well, related to active changes in the cytoskeleton. For example, initiation of invasive cell migration is essentially abolished in the absence of MAL or SRF in border cell migration in the Drosophila ovary (Somogyi 2004) or in mouse bipolar neurons exiting the subventricular zone of the brain and for cancer cells in three-dimensional (3D) invasion assays and experimental metastasis (Medjkane, 2009; Salvany, 2014).
Actin is a very abundant and exquisitely conserved protein in eukaryotic cells. Cycling of actin between G-actin and F-actin pools is controlled by a vast array of regulators, which have been the focus of considerable attention. Actin protein synthesis is also a regulated process. Actin mRNA localization and localized protein synthesis are important for cell migration and axonal growth and guidance. This study presents evidence that regulation of actin gene transcription is itself a key regulatory step in the control of invasive cell migration. Using a genome-wide approach, Actin5C was identified as a major target of the MAL/SRF transcription factor complex. Loss or reduction of MAL activity impairs invasive migration in Drosophila and human cancer cell models. It was found that restoring actin expression can be sufficient to replace the requirement for MAL to support invasive migration in these models. Thus, actin and MAL form a conserved homeostatic feedback system to ensure that actin levels are appropriate to support the actin dynamics required for complex cell behavior (Salvany, 2014).
To understand why MAL is essential for invasive migration and whether the apparent similarity of its role in different organisms reflects a conserved molecular mechanism, attempts were made to identify Drosophila MAL target genes at the genome level. A combination was used of chromatin immunoprecipitation (ChIP) and gene expression analysis and focus was placed on MAL, as SRF has functions independent of MAL/mrtf in mammals and flies. To perform analysis in the relevant tissue context, a mutant in the single Drosophila mal-d gene was used that abolishes expression in the ovary (mal-dΔ7) (Somogyi, 2004). This specifically blocked invasive migration by border cells and caused overall ovary growth defects. Ubiquitous expression of a GFP-tagged version of Mal-d completely rescued the mal-d mutant phenotypes, showing that the fusion protein provides normal Mal-d function. The MAL-GFP transgene also allowed efficient identification of MAL-GFP-bound regions in the Drosophila genome by immunoprecipitation with GFP. Key MAL-GFP-bound regions were confirmed in independent samples, and their recovery was dependent on the presence of the transgene. In parallel, genome-wide expression analysis of wild-type versus mal-d mutant ovaries identified genes whose expression was dependent on MAL. These two complementary data sets allowed genome-wide identification of MAL target genes (Salvany, 2014).
There were two key findings from this genome-wide analysis. First, only a small number of genes qualified as direct MAL targets, with MAL-GFP bound to the regulatory region and a significant decrease of mRNA expression in the mutant: the cytoplasmic Actin5C gene and five other genes, most encoding heat-shock proteins. A few additional genes encoding cytoskeletal proteins or regulators were identified as potential targets in the MAL-GFP-bound set. Second, three of the four most enriched MAL-bound regions in the whole genome were associated with Actin5C. The MAL-bound regions were conserved in other species, suggesting functional importance, and in each case, these sites bracketed gene-free upstream regions of ~10 kb. The latter is noteworthy because the Drosophila genome is dense, with most 'housekeeping genes' closely spaced, and large regulatory regions generally confined to developmental regulators. These findings focused the attention of this study on Actin5C (Salvany, 2014).
The gene expression arrays indicated a modest decrease of Actin5C mRNA levels in the mal-d mutant. Quantitative RT–PCR of carefully matched ovary samples showed a twofold decrease of mature Actin5C mRNA and a threefold to fourfold decrease of primary transcript in mal-d mutants, with no change in the closely related, but less highly expressed, Actin42A gene. In FACS-sorted migratory cells, including border cells, Actin5C levels were fourfold reduced. Analysis of the Actin5C promoter and upstream region in luciferase reporter assays showed robust promoter activity and 200-fold to 600-fold up-regulation by coexpression of SRF and activated MAL (mal-d ΔN -). Conversely, knockdown of MAL or SRF by RNAi gave 50-fold to 100-fold reduction in basal Actin5C expression. The Actin5C regulatory region also conferred responsiveness to drugs affecting actin dynamics, specifically induction by Cytochalasin D and inhibition by Latrunculin B, as observed for mammalian MAL/SRF-regulated genes. This type of reporter assay generally reveals regulatory potential at the transcriptional level. The large magnitude of regulation of the Actin5C promoter/enhancer region by MAL/SRF is consistent with the abundant binding of MAL to this region. In vivo, compensatory mechanisms may contribute to sustaining Actin5C mRNA levels upon loss of MAL activity. Thus, MAL and actin dynamics have the potential to regulate Actin5C transcription over a large dynamic range (Salvany, 2014).
The genomic data raised the possibility that Actin5C might not be just one of many cytoskeletal target genes for MAL regulation but the key target gene. If a transcription factor has one key target gene in vivo, re-expression of this gene should replace the need for the transcription factor. In genetic terms, expression of the target gene should rescue the phenotype of complete loss of function for the transcription factor in specific cells (Salvany, 2014).
To investigate this hypothesis functionally, the severe defect in invasive migration observed in mal-d mutant border cells was examined. Actin5C is the major cytoplasmic actin gene, and, as expected, mutating it perturbs border cell migration. To determine whether Actin5C was the sole required target gene of MAL, whether re-expression of Actin5C in cells that appear to be null for mal-d (mal-d S2) (Somogyi, 2004) could restore invasive migration was tested. Fly strains were used in which the Actin5C gene has a Gal4-responsive transposon, an 'EP element,' in the promoter region. Surprisingly, migration was indeed restored to normal when Actin5C was activated by Gal4 in mal-d mutant cells. Thus, as long as the Actin5C gene is induced at an adequate level, border cells do not need MAL to invade and migrate (Salvany, 2014).
In fully mutant ovaries (mal-d&Delta:7 homozygous females), restricted expression of Actin5C in terminally differentiated outer border cells using slbo-Gal4 provided significant but less efficient rescue of migration despite normal expression levels. This suggested that MAL also acts in other cells, consistent with the general oogenesis phenotype. The ORF of Actin5C in a Gal4-reponsive transgene (UAS-Actin-ORF) also provided some rescue of migration, whereas a construct with stop codons present did not. This confirmed that actin protein expression was responsible for the activity of the Actin5C locus in border cells (Salvany, 2014).
To test whether this finding extended to other tissues, another mal-d mutant phenotype, bent bristles, was examined. Bristles are 'hairs' organized by actin-rich structures and are characteristically defective in mal-d mutants (Somogyi, 2004). This effect of MAL deficiency was also restored to normal by ectopic expression of the Actin5C, accomplished by placing Actin5C cDNA under the control of a heat-shock promoter and rearing at 29°C. Thus, the requirement for MAL in these contexts reflects a specific need for MAL-driven induction of Actin5C expression. Regulation of other targets, direct or indirect, is not required. These findings indicate that the primary role of Drosophila MAL is to regulate actin levels in response to free actin fluctuations, a homeostatic feedback regulation (Salvany, 2014).
It was next asked whether this role of MAL as regulator of actin homeostasis is conserved in mammalian cells. The cytoplasmic β-actin and γ-actin genes are regulated by MAL/SRF in mammalian cells. As for Drosophila Actin5C, β-actin is essential for embryonic development and proper cell migration, whereas the related γ-actin can be compensated for. However, many other genes are more dramatically regulated by SRF and mrtfs, and some of these are important for cell migration and related functions. It is therefore assumed that MAL and SRF exert their function by regulating a battery of cytoskeletal genes. For SRF, the number of direct target genes is estimated at 200-300. For MAL and MRTFs, this is less clear, as systematic ChIP analysis is missing. Based on the findings in the fly model, the hypothesis was tested that a single target gene (β-actin) could be the key effector of mammalian MAL and that its expression could replace the need for MAL-driven gene regulation in an assay of invasive cell migration (Salvany, 2014).
The requirement for MAL is most pronounced in cases of active cell shape change and cytoskeletal challenge, such as tissue or matrix invasion (Somogyi, 2004; Medjkane, 2009; Pinheiro, 2011). Therefore, a simple cellular assay was sought that could test the ability of cells to invade into a confined environment. Migration under agarose provides mechanical resistance to movement and has been used to study migration of eukaryotic cells in a constrained space. The assay can easily be adapted to human tumor cells such as MDA-MB-231 breast cancer cells. MAL activity in these cells is provided by mrtf-a and mrtf-b, and reducing their expression severely reduces invasion in an organotypic assay and in experimental metastasis in mice (Medjkane, 2009). Simultaneous knockdown of mrtf-a and mrtf-b in MDA-MB-231 cells by siRNA reduced β-actin transcript levels. It also produced severe attenuation of migration under agarose, confirming that this assay interrogates MAL-dependent cell movement (Salvany, 2014).
To assess the importance of cytoplasmic actin downstream from MAL, stable cell lines were derived from MDA-MB-231 that allowed the ORF of human β-actin, or an N-terminally Flag-tagged version of β-actin, to be induced from the strong CMV promoter by the TET-ON system. These cell lines showed some baseline expression of the transgenes and six-fold to 10-fold induction upon Tet addition. Under agarose, migration was similar to the parental cell line and not changed by Tet addition. Knockdown of mrtf-a and mrtf-b largely attenuated migration under agarose. Remarkably, ectopic induction of β-actin or Flag-β-actin in the mrtf-a/b-depleted cells rescued migration to control levels. This indicates that human cancer cells require MAL activity to perform invasive migration for the exact same reason that Drosophila border cells do: to regulate cytoplasmic actin gene expression. Regulation of any other potential MAL target genes may not be required for this cell behavior. These experiments rely on siRNA-mediated reduction of mrtf activity; thus, it remains possible that other target genes are important but require only low levels of mrtf activity (Salvany, 2014).
Elegant experiments have shown how activity of SRF and its transcriptional cofactor, MAL, is regulated by the level of free actin and thereby by the dynamics of the actin cytoskeleton. This study has shown that the key role of MAL is to regulate the expression level of cytoplasmic actin. It is suggested that this simple feedback system allows cells to produce sufficient free actin to meet the needs of their changing cellular cytoskeletal dynamics. The extensive MAL-decorated regulatory region displayed by Actin5C may serve to tune actin gene expression sensitively and accurately. This study provides evidence that cytoplasmic actin is the sole critical target gene for invasive migration in Drosophila and possibly also in human cells. It is therefore proposed that this regulation is the ancestral function of the MAL/SRF complex in animal cells. Additional target genes for MAL have been acquired in different species and are likely to contribute to cell fitness. Examples include other cytoskeletal proteins (Medjkane, 2009) as well as genes encoding heat-shock proteins, some of which may interact with actin. While some of these genes appear more dramatically regulated by MAL when considering relative mRNA levels, actin may be the 'most regulated' gene if considering the number of transcripts induced. In any case, identification of actin as the core, essential target of MAL reveals the core of this transcription regulatory 'network' to be a simple and logical feedback system (Salvany, 2014).
Why is the MAL-driven regulation of actin particularly critical for invasively migrating cells? The stimuli inducing a robust F-actin-based cytoskeleton when initiating migration into constrained space is likely to convert free G-actin into F-actin-rich structures. Maintaining the appropriate free actin pool for further cytoskeletal buildup or for other cellular functions then requires new production of actin. Other dramatic shape changes, such as cells rounding up in the stratified epidermal layer of the skin, may induce similar sudden actin pool depletion and therefore require MAL and SRF. It has been suggested that MAL forms part of a mechanical feedback system for invasive cells (Somogyi, 2004) whereby mechanical tension induces MAL activity in order to make 'robust' cells. Related concepts of tension-driven responsiveness have recently been indicated for mammalian MAL/SRF function as well (Connelly, 2010; McGee, 2011). The mechanical feedback logic is fully compatible with the simple molecular feedback system presented in this study. It can be regarded as an alternate point of perturbation impinging on actin homeostasis; namely, stretching or stressing cells to provoke a biomechanical cytoskeletal response. Robust feedback systems such as this one driven by MAL are likely to be well conserved through animal evolution, when the target is a crucial one. The ability of cells to occasionally migrate and invade is a characteristic of animal systems, and the dynamic actin cytoskeleton is central to this behavior (Salvany, 2014).
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