twist

Targets of Activity

The first step in the differentiation of the Drosophila mesoderm is the activation of two regulatory genes, twist (twi) and snail (sna), in ventral regions of early embryos. DL and TWI directly activate sna expression. Site-directed mutagenesis of DL- and TWI-binding sites within defined regions of the sna promoter suggest that the two proteins function multiplicatively to ensure strong, uniform expression of sna, particularly in ventral-lateral regions where there are diminishing amounts of DL. These results are consistent with the possibility that the sharp sna borders are formed by multiplying the shallow DL gradient and the steeper TWI gradient (Ip, 1992b).

In mutants of snail or twist, transcription of folded gastrulation is normal in the posterior midgut primordium but almost completely eliminated on the ventral side. Maternal-effect ventralizing mutations that expand the expression of twist and snail also expand the domain of fog transcription. In embryos from torpedoQY mutant mothers, Twist protein expression extends farther laterally, but the ventral furrow is usually split into two narrow ventrolateral invaginations by an unknown patterning mechanism. In this case, fog is transcribed in two separate ventrolateral stripes (Costa, 1994).

During Drosophila gastrulation, morphogenesis occurs as a series of cell shape changes and cell movements that probably involve adhesive interactions between cells. The dynamic aspects of cadherin-based cell-cell adhesion were examined in the morphogenetic events to assess the contribution of such activity to morphogenesis. Shotgun and Cadherin-N show complementary expression patterns in the presumptive ectoderm and mesoderm at the mRNA level. Switching of cadherin expression from the Shotgun to the CadN type in the mesodermal germ layer occurs downstream of the mesoderm-determination genes twist and snail. These dynamic aspects of cadherin-based cell-cell adhesion appear to be associated with the following: (1) initial establishment of the blastoderm epithelium; (2) acquisition of cell motility in the neuroectoderm; (3) cell sheet folding, and (4) epithelial to mesenchymal conversion of the mesoderm. These observations suggest that the behavior of the Shotgun-catenin adhesion system may be regulated in a stepwise manner during gastrulation to perform successive cell-morphology conversions. Also discussed are the processes responsible for loss of epithelial cell polarity and elimination of preexisting Shotgun-based epithelial junctions during early mesodermal morphogenesis (Oda, 1998).

Expression of mef2 is dependent on the mesodermal determinants twist and snail but independent of the homeobox-containing gene tinman, which is required for visceral muscle and heart development (Lilly, 1994).

The function of the Drosophila mef2 gene, a member of the MADS box supergene family of transcription factors, is critical for terminal differentiation of the three major muscle cell types, namely somatic, visceral, and cardiac. During embryogenesis, mef2 undergoes multiple phases of expression, which are characterized by initial broad mesodermal expression, followed by restricted expression in the dorsal mesoderm, specific expression in muscle progenitors, and sustained expression in the differentiated musculatures. Evidence is presented that temporally and spatially specific mef2 expression is controlled by a complex array of cis-acting regulatory modules that are responsive to different genetic signals. Functional testing of approximately 12 kb of 5' flanking region of the mef2 gene shows that the initial widespread mesodermal expression is achieved through a 280-bp twist-dependent enhancer. Subsequent dorsal mesoderm-restricted mef2 expression is mediated through a 460-bp dpp-responsive regulatory module, which involves the function of the Smad4 homolog Medea and contains several binding sites for Medea and Mad. Mef2 expressing cells are coincident with those expressing Tinman. Notably, both Mef2 and Tinman expression are in four of six cardioblasts that are present per hemisegment. The complete overlap between the two expression patterns suggests that the activity of this enhancer element could be dependent on tinman function or under similar regulatory controls as is tinman. The cardiac enhancer that functions at later stages also drives mef2 expression in the caudal visceral mesoderm as well as in the somatic mesoderm. Taken together, the presented data suggest that specific expression of the mef2 gene in myogenic lineages in the Drosophila embryo is the result of multiple genetic inputs that act in an additive manner on distinct enhancers in the 5' flanking region (Nguyen, 1999).

The homeobox-containing gene tinman (msh-2), is expressed in the mesoderm primordium; this expression requires the function of the mesoderm determinant twist. Later in development, as the first mesodermal subdivisions are occurring, expression of tinman becomes limited to the visceral mesoderm and the heart (Bodmer, 1993).

tinman encodes a homeodomain transcription factor required for the development of the dorsal mesoderm and its derivatives in the Drosophila embryo. Genetic analyses indicate that tinman resides downstream of the mesodermal determinant twist, which encodes a basic helix-loop-helix-type transcription factor. However, the regulation of tinman by twist remains poorly understood. Using expression assays in cultured cells and transgenic flies, it has been shown that two distinct clusters of E-box regulatory sequences, present upstream of the tinman gene, mediate tinman expression in the visceral mesoderm. These elements are conserved between the Drosophila melanogaster and Drosophila virilis tinman genes and serve as binding sites for Twist (E1 cluster located from -1134 to -1101) and Tinman (E2 cluster located from -868 to -831) proteins. In cultured cells, Twist and Tinman binding results in the activation of tinman gene expression. In transgenic animals, the E1 and E2 clusters are functionally connected; both elements are required for tinman activation in cells of the visceral mesoderm and also for tinman repression in cells of the somatic musculature. These results demonstrate that tinman is a direct transcriptional target for Twist and its own gene product in visceral mesodermal cells, supporting the idea that twist and tinman function in the subdivision of the mesoderm during Drosophila embryogenesis (Lee, 1997).

The Drosophila tinman homeobox gene has a major role in early mesoderm patterning: it determines the formation of visceral mesoderm, heart progenitors, specific somatic muscle precursors and glia-like mesodermal cells. These functions of tinman are reflected in its dynamic pattern of expression, which is characterized by initial widespread expression in the trunk mesoderm, then refinement to a broad dorsal mesodermal domain, and finally restricted expression in heart progenitors. Each of these phases of expression is driven by a discrete enhancer element, the first being active in the early mesoderm, the second in the dorsal mesoderm and the third in cardioblasts. Surprisingly, all of these elements are located at positions downstream of the transcription start site. Element B(1800 bp) is located in the first intron; a second enhancer element, D (about 350bp), is located about 2 kb downstream of the 3' end of tin and activates gene expression in the dorsal portion of the mesoderm. Element D is active between stage 11 and early stage 12 of embryogenesis. A third element, C (300bp) is active in the dorsal vessel. This element activates expression from stage 12 on, in four out of six cardioblasts per hemisegment. Finally, an element A (about 500 bp), located in the 5' portion of the first intron, activates tin in the anterior tip of the head. After invagination of the stomodeum, the bulk of these tin expressing cells form the roof of the pharynx (Yin, 1997).

The early-active enhancer element is a direct target of twist, a gene necessary for tinman activation that encodes a basic helix-loop-helix (bHLH) protein. This 180 bp enhancer includes three E-box sequences that bind Twist protein in vitro and are essential for enhancer activity in vivo. Ectodermal misexpression of twist causes ectopic activation of this enhancer in ectodermal cells, indicating that twist is the only mesoderm-specific activator of early tinman expression. The 180 bp enhancer also includes negatively acting sequences. Binding of Even-skipped to these sequences appears to reduce twist-dependent activation in a periodic fashion, thus producing a striped tinman pattern in the early mesoderm. In addition, these sequences prevent activation of tinman by twist in a defined portion of the head mesoderm that gives rise to hemocytes. This repression requires the function of buttonhead, a head-patterning gene: buttonhead is necessary for normal activation of the hematopoietic differentiation gene serpent in the same area. The second expression domain, restricting tin mRNA expression in the dorsal mesoderm, is triggered by Dpp-mediated induction events. Together, these results show that tinman is controlled by an array of discrete enhancer elements that are activated successively by differential genetic inputs, as well as by closely linked activator and repressor binding sites within an early-acting enhancer, which restricts twist activity to specific areas within the twist expression domain (Yin, 1997).

Ras signaling elicits diverse outputs, yet how Ras specificity is generated remains incompletely understood. Wingless and Decapentaplegic confer competence for receptor tyrosine kinase-mediated induction of a subset of Drosophila muscle and cardiac progenitors by acting both upstream of and in parallel to Ras. In addition to regulating the expression of proximal Ras pathway components, Wg and Dpp coordinate the direct effects of three signal-activated transcription factors (dTCF, Mad, and Pointed that function in the Wg, Dpp, and Ras/MAPK pathways, respectively) and two tissue-restricted transcription factors (Twist and Tinman) on even-skipped, a progenitor identity gene enhancer. The integration of Pointed with the combinatorial effects of dTCF, Mad, Twist, and Tinman determines inductive Ras signaling specificity in muscle and heart development (Halfon, 2000).

Cell fate specification in the somatic mesoderm of the Drosophila embryo has been examined as a model for dissecting the molecular basis of combinatorial signaling involving receptor tyrosine kinases (RTKs). The somatic musculature and the cells that compose the heart develop from specialized cells called progenitors. Each progenitor divides asymmetrically to produce two founder cells that possess information that specifies individual muscle fate and that seed the formation of multinucleate myofibers. The focus of this study has been a small subset of somatic mesodermal cells that express the transcription factor Even skipped. Eve is expressed in the progenitors and founders of both the dorsal muscle fiber DA1 and a pair of heart accessory cells, the Eve pericardial cells or EPCs. Since eve is the earliest known marker for these cells and is required for their formation, eve is referred to here as a progenitor identity gene (Halfon, 2000).

Given that the eve MHE recapitulates early mesodermal Eve expression, a determination was made of whether this enhancer contains binding sites for candidate signal-dependent and mesoderm-specific transcription factors. Focus was placed on two mesoderm-specific factors, Tin and Twi, as well as the nuclear factors that act downstream of Wg (dTCF), Dpp (Mad) and Ras (Pnt, Yan). A computer-based search of the MHE sequence has suggested the presence of potential binding sites for each of these transcription factors. Gel-shift assays confirm that these putative sites actually bind the relevant factors. This analysis establishes the existence of one binding site for dTCF, six for Mad, two for Twist, and four each for Tin and Pnt. Since Yan binds to each of the Pnt sites, these are referred to as Ets sites (Halfon, 2000).

To ascertain whether these in vitro binding sites have in vivo functional significance, the sites were mutated, both singly and in combination, within the context of the entire MHE. All mutagenesis was by base substitution so as not to affect the spacing between other potential cis-regulatory elements. The ability of the mutated MHEs to drive reporter gene expression was tested in transgenic embryos and this expression was compared to that of endogenous Eve. Of the six Mad sites, only Mad4, 5, and 6 are critical for MHE function when inactivated singly or in combination. Mutation of the single dTCF site or of individual binding sites for Twi, Tin, or the Ets factors also lead to loss of reporter gene expression in some, but not all, Eve-expressing cells, with some mutant sites associated with a more severe loss than others. Of note, both the EPC and DA1 lineages are affected equally by all of the mutations. In addition, the activity level in those Eve-expressing cells that do maintain reporter gene expression is on average lower than that seen with the wild-type MHE. In contrast to the single site mutants, mutation of the two Twi, all four Tin, or all four Ets sites completely eliminate MHE activity. It is concluded that binding sites for two tissue-specific and three signal-responsive transcription factors are required for full activity of the MHE in both the muscle and the heart lineages (Halfon, 2000).

The finding that the three Wg-dependent factors, dTCF, Twi, and Tin, that directly regulate eve could explain why activated Ras is incapable of bypassing Wg in the induction of Eve progenitors. Therefore attempts were made to rescue Eve expression in wg mutant embryos by ectopically expressing Twi and Tin together with activated Ras. However, Eve progenitors were not recovered by this manipulation, perhaps due to the direct requirement of dTCF for eve MHE activity. While activated Arm can supply the missing downstream Wg transcription factor in this rescue experiment, Arm alone is capable of fully rescuing not only the Eve progenitors but also all of the Wg-dependent factors that regulate the MHE, including Twi, Tin, and the RTK/Ras pathway components. Thus, the combined effects of the MHE transcription factors could not be further evaluated in the absence of Wg signaling. Nevertheless, the rescue and enhancer mutagenesis data strongly support the involvement of Wg as a mesodermal competence determinant both upstream of the Ras pathway and directly (via dTCF) as well as indirectly (via Twi and Tin) in the transcriptional response to inductive RTK signaling (Halfon, 2000).

Since mutation of any single transcription factor binding site in the MHE causes only a partial loss of enhancer activity, it was considered whether different sites might function together synergistically. To test this possibility, binding site mutations for two different activators were combined. Simultaneous mutation of the dTCF and Twi1 sites led to reporter gene expression in approximately 5-fold fewer cells than would be expected from the additive independent effects of each mutation. A similar, though slightly less robust, synergy was observed when the dTCF and Ets3 mutations were combined (Halfon, 2000).

An assessment was made of whether ectopic coexpression of individual transcription factors or upstream signals would lead to cooperative effects on endogenous Eve expression. As previously reported, ectopic Wg has no effect on Eve expression at late stage 11, activated Ras1 induces extra Eve progenitors, and ectopic Wg plus activated Ras1 cause a lateral expansion of the progenitor clusters. When Twi is expressed using a twi-Gal4 driver, a few Eve-positive cells develop at ectopic positions. The magnitude of this effect is increased by coexpression of Wg and Twi, and even more so by coexpression of Twi with activated Ras1. The latter effect strikingly resembles that of Wg plus activated Ras1. With the simultaneous ectopic expression of Wg, Twi, and activated Ras1, Eve progenitors form an almost continuous anteroposterior stripe confined to the dorsal mesoderm. These results demonstrate a synergistic induction of Eve progenitors by various combinations of Wg, Twi, and activated Ras1 that parallels the synergistic loss of MHE activity seen by mutating the dTCF, Twi, and Ets binding sites. Taken together, these loss- and gain-of-function findings suggest that dTCF, Twi, and Pnt cooperate at the MHE to synergistically regulate Eve transcription and, by extension, to induce the specification of Eve progenitor fates (Halfon, 2000).

It is concluded that Wg and Dpp coordinate a series of signal-activated (dTCF and Mad) and mesoderm-specific (Twi and Tin) transcription factors in a temporal and spatial pattern that facilitates cooperation with the Ras transcriptional effector Pnt. The synergistic integration of these five transcription factors by a single enhancer generates a specific developmental response to Ras/MAPK signaling. Moreover, Wg and Dpp exert proximal effects in this signaling network by enabling Ras/MAPK activation through the regulated localized expression of upstream components of the RTK signal transduction machinery. A model governing the acquisition of developmental competence, signal integration and response specificity in this system is presented. Wg and Dpp provide competence through the regulation of tissue-specific transcription factors (Tin and Twi), signal-responsive transcription factors (Mad and dTCF), and proximal components of the RTK/Ras pathways (Htl, Hbr, and Rho). The Ras signaling cascade leads to activation of the inductive transcription factor, Pnt, and inactivation of the Yan repressor. While a direct role for Mad in regulating Tin expression has been demonstrated, Wg regulation of Tin, Twi, Htl, Hbr, and Rho may be either direct or indirect. Dpp has additional effects on the proximal RTK factors. The five transcriptional activators assemble at and are integrated by the MHE, where they function synergistically to promote eve expression. Specificity of the response to inductive RTK/Ras signaling derives from the combinatorial effects of the tissue-restricted and signal-activated transcription factors that converge at the MHE. In the absence of inductive signaling, Yan would repress eve by binding to the Ets sites. Since eve is a muscle and heart identity gene, the regulatory mechanisms are inferred to have a more general function in determining progenitor fates. Additional complexity attendant upon the control of RTK activity in this system derives from positive feedback regulation of the Ras/MAPK cascade and from reciprocal regulatory interactions between the Ras and Notch pathways (Halfon, 2000).

The ventral nervous system defective/NK-2 gene may be repressed at several sites, by different genes: in the mesodermal anlage by Snail, in mesectodermal cells by Single-minded, and in the lateral neuroectodermal and/or dorsal epidermal anlagen, repression may be mediated indirectly by Decapentaplegic. Twist either activates vnd gene in the posterior portion of the embryo or is a coactivator with Dorsal (Mellerick, 1995).

Primary neurogenesis in the central nervous system of insects and vertebrates occurs in three dorsoventral domains on either side of the neuroectoderm. Among the three dorsoventral domains of the Drosophila neuroectoderm, the medial and lateral columns express the zinc-finger gene escargot (esg), whereas the intermediate column does not. esg expression was examined as a probe to investigate the mechanism of neuroectoderm patterning. The effect of dorsoventral patterning genes on esg expression was studied. decapentaplegic, snail and twist repress esg expression outside the neuroectoderm. The expression of esg in the intermediate column is normally repressed, but is de-repressed when Egfr activity is either elevated or reduced. A neurogenic enhancer of esg was identified, and shown to be separable into a distal region that promotes ubiquitous expression in the neuroectoderm and a proximal region that represses the intermediate expression. It is concluded that decapentaplegic, snail, twist and an activator all act through the distal region to initiate transcription of esg in the neuroectoderm. It is proposed that the combination of opposing gradients of Egfr and its ligand creates a peak of Egfr activity in the intermediate column, where Egfr represses esg transcription through the proximal repressor region. These two kinds of regulation establish the early esg expression that prefigures the neuroectoderm patterning (Yagi, 1997).

The fibroblast growth factor (FGF)/receptor system is thought to mediate various developmental events in vertebrates. DFR1, known as Heartless, and Breathless proteins contain two and five immunoglobulin-like domains, respectively, in the extracellular region, and a split tyrosine kinase domain in the intracellular region. In early embryos, DFR1 mRNA expression, requiring both twist and snail proteins, is specific to mesodermal primordium and invaginated mesodermal cells. At later stages, putative muscle precursor cells and cells in the central nervous system (CNS) express DFR1. breathless expression occurs in endodermal precursor cells, CNS midline cells and certain ectodermal cells such as those of trachea and salivary duct. FGF-receptor homologs in Drosophila would thus appear essential for generation of mesodermal and endodermal layers, invaginations of various types of cells, and CNS formation (Shishido, 1993).

rhomboid (rho) encodes a putative transmembrane receptor that is required for the differentiation of the ventral epidermis. Dorsal acts in concert with basic helix-loop-helix (b-HLH) proteins, possibly including Twist, to activate rhomboid in both lateral and ventral regions. Expression is blocked in ventral regions (the presumptive mesoderm) by Snail, which is also a direct target of the DL morphogen (Ip, 1992a).

The twist gene is a good candidate for regulating expression of buttonless. buttonless is expressed in dorsal median cells, mesodermal cells that are arranged as a single pair within each segment along the dorsal midline, just above the central nervous system. There are a total of 6 consensus binding sites for TWI within a 300 base pair region of genomic sequence immediately upstream of the btn structural gene (Chiang, 1994).

In an attempt to identify genes that are involved in Drosophila embryonic cardiac development, a gene was cloned whose function is required late in embryogenesis to control heart rate and muscular activity. This gene has been named held out wings (how) because hypomorphic mutant alleles produce adult animals that have lost their ability to fly; they keep their wings on the horizontal, at a 90° angle from the body axis. In contrast to the late phenotype observed in null mutants, the How protein is expressed early in the invaginating mesoderm; this expression is apparently under the control of twist. When the different mesodermal lineages segregate, the expression of How becomes restricted to the myogenic lineage, including the cardioblasts and probably all the myoblasts. Antibodies directed against the protein demonstrate that How is localized to the nucleus. how encodes a protein containing one KH-domain that has been implicated in binding RNA. how is highly related to the mouse quaking gene that plays a role in myelination and that could serve to link a signal transduction pathway to the control of mRNA metabolism. The properties of the how gene described herein suggest that this gene participates in the control of expression of as yet unidentified target mRNAs coding for proteins essential to cardiac and muscular activity (Zaffran, 1997).

Zygotic expression of modifier of variegation modulo depends on the activity of genes which pattern the embryo along dorsoventral and anteroposterior axes and specify diversified morphogenesis. dorsal and the mesoderm-specific genes twist and snail direct modulo expression in the presumptive mesoderm. The homeotic genes Sex combs reduced and Ultrabithorax positively regulate the gene in the ectoderm of parasegment 2 and abdominal mesoderm (Graba, 1995).

Drosophila has a single MEF2 gene, DMEF2, that is alternatively spliced to produce different transcripts; it is expressed in the mesodermal primordium before gastrulation. The mechanisms responsible for the subsequent subdivision of the mesoderm are unknown. However, DMEF2 may play a role in this important event. Experiments show that it is a downstream target for twist and that its early expression pattern modulates as the mesoderm organizes into cell groupings with distinct fates. DMEF2 is also expressed in both the segregating primordia and the differentiated cells of the somatic, visceral and heart musculature. It is the only known gene expressed throughout differentiation in these three main types of muscle (Taylor, 1995).

MEF2 is a MADS-box transcription factor required for muscle development in Drosophila. The bHLH transcription factor Twist directly regulates Mef2 expression in adult somatic muscle precursor cells via a 175-bp enhancer located 2245 bp upstream of the transcriptional start site. Within this element, a single evolutionarily conserved E box is essential for enhancer activity. Twist protein can bind to this E box to activate Mef2 transcription. Ectopic expression of twist results in ectopic activation of the wild-type 175-bp enhancer. Twist activation of Mef2 transcription via this enhancer is required for normal adult muscle development; reduction in Twist function results in phenotypes similar to those observed in Mef2 mutant adults. The requirement of Twist for adult muscle development is seen in defective development of dorsal longitudinal indirect flight muscles (DLMs). When Twist function is reduced during the larval stage, Mef2 is not expressed in the adult muscle precursor cells, resulting in abnormal patterning of the adult somatic muscles, most strikingly in the DLMs. Hypomorphic Mef2 mutant adults show very similar defects, indicating that this phenotype, resulting from loss of twist expression, likely occurs through a requirement to maintain normal Mef2 levels. The 175-bp enhancer is also active in the embryonic mesoderm, indicating that this enhancer functions at multiple times during development, and that its function is dependent on the same conserved E box. In embryos, a reduction in Twist function also strongly reduces Mef2 expression. Since there is only weak expression from the 175-bp enhancer early in embryogenesis, Twist alone cannot be responsible for activating Mef2 early on. It is likely that the genomic region governing the earliest expression of Mef2 is either outside this enhancer or overlapping it. The activity of the 175-bp enhancer increases during embryogenesis until stage 12, where it is expressed in segmentally repeating groups of cells corresponding to the unfused and undifferentiated myoblasts of the larval somatic muscles. At this stage, twist and Mef2 are coexpressed in these cells. These findings define a novel transcriptional pathway required for skeletal muscle development and identify Twist as an essential and direct regulator of Mef2 expression in the somatic mesoderm (Cripps, 1998a).

The basic helix-loop-helix transcription factor Twist is required for normal development of larval and adult somatic muscles in Drosophila. Adult flies normally have six pairs of dorsal longitudinal indirect flight muscles (DLMs), whereas when Twist function is reduced, only three pairs of DLMs are formed. Although twist is expressed in precursors of adult muscles throughout the larval and early pupal stages, it is demonstrated that Twist function is required only during the late larval stage for DLM patterning. In wild-type flies, this is just prior to the time when three pairs of persistent larval muscle fibers split longitudinally to form templates for the six pairs of DLMs. These larval muscle fibers do not express twist, but the adult muscle precursors to which they fuse are found to express twist. Thus Twist function in the adult muscle precursor cells is required for splitting to occur in the muscles to which the myoblasts fuse. By examining sections at various times during pupal development, it has been found that splitting of the larval muscles does not occur in twist mutants, indicating that Twist function is required to induce major changes in the larval templates prior to differentiation. The function of Twist in larval muscle splitting is likely mediated by myocyte enhancer factor-2 (MEF2) since in Mef2 hypomorphic mutants splitting is also reduced and Mef2 expression is dependent on Twist. These findings define specific roles for Twist and MEF2 during pupal myogenesis and demonstrate that these transcription factors function in adult muscle precursor cells to regulate downstream factors controlling muscle cell splitting and morphogenesis (Cripps, 1998b).

Both twist and snail are required for the mesodermal activation of the novel zinc-finger homeodomain gene zfh-1. ZFH-1 contains one homeodomain and nine C2H2 zinc fingers. In twist mutants, all the early mesodermal anti-AFH-1 staining posterior to the cephalic furrow is lost, although expression anterior to this furrow, as well as the later expression in the developing CNS is unaffected. A similar result is obtained in snail mutant embryos except that the early mesodermal staining anterior to the the cephalic furrow is also lost. In both mutants, the later sites of presumptive mesodermal zfh-1 expression are also devoid of staining, although this may be due to the failure of mutant embryos to reach later developmental stages (Lai, 1991).

The transcription factor Twist initiates Drosophila mesoderm development, resulting in the formation of heart, somatic muscle, and other cell types. Using a Drosophila embryo sorter, enough homozygous twist mutant embryos were isolated to perform DNA microarray experiments. Transcription profiles of twist loss-of-function embryos, embryos with ubiquitous twist expression, and wild-type embryos were compared at different developmental stages. The results implicate hundreds of genes, many with vertebrate homologs, in stage-specific processes in mesoderm development. One such gene, gleeful/lame duck, related to the vertebrate Gli genes, is essential for somatic muscle development and sufficient to cause neural cells to express a muscle marker (Furlong, 2001).

Formation of muscles during embryonic development is a complex process that requires coordinate actions of many genes. Somatic, visceral, and heart muscle are all derived from mesoderm progenitor cells. The Drosophila twistgene, which encodes a bHLH transcription factor, is essential for multiple steps of mesoderm development: invagination of mesoderm precursors during gastrulation, segmentation, and specification of muscle types. The role of twist in mesoderm development has been conserved during evolution, perhaps because it controls conserved regulatory mesoderm genes. For example, tinman and dMef2 are regulated by Twist in flies and are highly conserved in sequence and function in vertebrates (Furlong, 2001).

In Drosophila, somatic muscle forms from progenitor cells that divide to become muscle founder cells. Founder cells acquire unique identities controlled by transcription factors including Krüppel, S59, vestigial, and apterous. Each of the 30 body wall muscles in an abdominal hemisegment is initiated by a single founder cell and has unique attachments and innervations. To further clarify mechanisms underlying founder cell specification, myoblast fusion, and muscle patterning, Drosophila mutants together with microarrays of cDNA clones were used (Furlong, 2001).

twist mutant embryos develop no mesoderm. The population of mRNA species isolated from twist homozygous embryos were compared with that of stage-matched wild-type embryos. Drosophila lethal mutations are maintained as heterozygotes, in trans to balancer chromosomes. A twist mutation was established in trans to a balancer chromosome carrying a transgene encoding green fluorescent protein (GFP). Embryos were collected from wild-type and twist/GFP-balancer fly stocks at specific developmental stages. The twist/GFP-balancer collections contain a mixed population of embryos: one-quarter twist homozygotes lacking GFP; half heterozygotes with one copy of GFP, and one-quarter homozygous for the balancer chromosome with two copies of GFP. Homozygous twist mutant embryos were separated from their siblings using an embryo sorter. Putative homozygous twist embryos were assessed by immunostaining with an antibody to dMef2. More than 99% of the selected embryos had the twist phenotype (Furlong, 2001).

Three different periods of mesoderm development were analyzed: stages 9-10, 11, and 11-12. During stage 9-10, the earliest time GFP is detectable in the balancer embryos, mesoderm cells migrate dorsally and become specified as somatic, visceral, cardiac, and fat body mesoderm. twist and its direct targets tinman and dMef2 are expressed throughout stage 9-10 mesoderm. The middle period contains stage 11 embryos and is a transition between the first period (stage 9-10) and the third period (late stage 11-12). During late stage 11 and stage 12, myoblast fusion begins and twist expression remains prominent in only a subset of the somatic muscle cells (Furlong, 2001).

For each developmental period, three independent embryo collections, embryo sortings, and microarray hybridizations were conducted. The microarrays used for the analysis contained over 8500 cDNAs corresponding to 5081 unique genes plus a variety of controls. Each embryonic RNA sample was compared with a reference sample, which contains RNA made from all stages of the Drosophila life cycle and allows direct comparisons among all the experiments (Furlong, 2001).

To determine how transcription was affected by the twist mutation, SAM (significance analysis of microarrays) analysis was used. Genes that are normally highly expressed in mesoderm should have lower transcript levels in twist homozygotes. Genes in other tissues whose expression depends on signals from the mesoderm might also have reduced expression. Transcripts of 130 genes, the 'Twist-low' group, were significantly lower in twist mutants than in wild type. Conversely, cells that would have formed mesoderm may take on other fates in the absence of twist, such as neuroectoderm; therefore, many transcript levels could increase in twist mutants. Genes whose transcription is repressed by signals from the mesoderm would also be enriched in twist mutants. One hundred fifty genes, called the 'Twist-high' group, have increased levels of RNA in twist mutant embryos (Furlong, 2001).

In total, 280 of ~5000 genes had significant changes in transcript levels, with 10 false positives. The genes on the array include 15 previously characterized mesoderm-specific genes, all of which were significantly reduced in twist mutant embryos. The arrays also contain genes known to be transcribed in both mesoderm and other cell types. Significant changes in expression were detected for many of these genes (Furlong, 2001).

To ectopically express twist, a dominant gain-of-function mutation of the maternal gene Toll (Toll^10B) was used. Activated Toll induces the expression of twist and snail in early embryos and of immune response genes in older embryos. Thus, the effects of Toll^10B on gene expression reflect the activities of Twist as well as Snail and Dorsal, or their combined actions. Toll^10B embryos are essentially bags of mesoderm; epidermal structures are absent or greatly reduced, and they have been used successfully in subtractive hybridization screens to identify mesoderm genes. The gene transcription profile of Toll^10B embryos was compared with that of wild-type embryos during four periods of development, using the reference sample to normalize experiments. The earliest period, stage 5, is when twist is initially expressed in presumptive mesoderm. The other three periods analyzed were those used in the twist mutant analysis: stages 9-10, 11, and 11-12 (Furlong, 2001).

Data from loss- and gain-of-function experiments, combined with careful staging, yield a useful picture of genes that are likely to be required for mesoderm specification and muscle differentiation. Of 360 identified mesoderm genes, 273 have not been the focus of developmental studies. The predicted proteins encode transcription factors, signal transduction molecules, kinases, and pioneer proteins. The stage at which each gene is active is one criterion for assigning possible functions. Another key criterion will be finding a mutant phenotype. As a pilot, this additional step was undertaken for the gene CG4677 (LD47926). Changes in CG4677 transcript levels were also observed in a Toll^10B subtractive hybridization screen (Furlong, 2001).

CG4677 is transcribed in the visceral mesoderm at stages 10-13 and the somatic mesoderm during stages 11-13. This gene encodes a C₂H₂ zinc finger transcription factor with high sequence similarity to vertebrate Gli proteins, the gene has been named gleeful (gfl). Mammalian Gli proteins act downstream of Hedgehog signaling proteins to control target gene transcription (Furlong, 2001).

The role of gfl in mesoderm development was assessed by disrupting its function using RNA interference. Injection of a double-stranded RNA (dsRNA) control sequence had no effect on mesoderm development. In contrast, gfl dsRNA injection causes severe loss and disorganization of somatic muscle cells, whereas heart and visceral muscle were unaffected. A similar phenotype was seen in Df(3R)hh homozygous embryos: the deficiency removes gfl but not the nearby hedgehog gene (Furlong, 2001).

To determine whether gfl can induce muscle cell development, a UAS-gfl transgenic fly strain was generated. Ectopic expression of gfl using an en-GAL4 driver results in lethality and induction of ectopic dMEF2 expression in the ventral nerve cord. Remarkably, Gfl is sufficient to induce expression of a muscle gene in neuronal cells. Previous studies have shown an essential role for Sonic hedgehog signaling in the formation of slow muscle in avian and zebrafish embryos. gfl may be performing a similar role in Drosophila somatic muscle development (Furlong, 2001).

Determining the position of ventro-lateral neuroectoderm versus dorsal non neural ectoderm is controlled by maternal (dorsal) and zygotic genes (dpp, sog, brk, sna, twi). SoxNeuro (SoxN) expression is specifically affected in these mutants. dl mutants lack early SoxN expression. Embryos mutants for dpp show a dorsal expansion of SoxN expression, as also observed when misexpressing sog by means of the Gal4 system. Inversely, misexpressing dpp early in embryogenesis leads to severe reduction of SoxN expressing-cells, as observed in sog mutants and sog, brk double mutants. Finally, twi mutants are characterized by a ventral expansion of SoxN expression into the presumptive mesoderm. These experiments are consistent with a role for the D/V patterning genes in the control of SoxN expression, with SoxN being negatively regulated dorsally and ventrally by dpp and mesoderm genes, and positively by sog and brk in the neuroectodermal region. A similar situation has been reported in Xenopus, with SoxD, an essential mediator of neural induction, being negatively regulated by BMP4 and positively by chordin (the vertebrate homologs of dpp and sog, respectively) (Cremazy, 2000).

Organization of developmental enhancers in the Drosophila embryo

Most cell-specific enhancers are thought to lack an inherent organization, with critical binding sites distributed in a more or less random fashion. However, there are examples of fixed arrangements of binding sites, such as helical phasing, that promote the formation of higher-order protein complexes on the enhancer DNA template. This study investigated the regulatory 'grammar' of nearly 100 characterized enhancers for developmental control genes active in the early Drosophila embryo. The conservation of grammar is examined in seven divergent Drosophila genomes. Linked binding sites are observed for particular combinations of binding motifs, including Bicoid-Bicoid, Hunchback-Hunchback, Bicoid-Dorsal, Bicoid-Caudal and Dorsal-Twist. Direct evidence is presented for the importance of Bicoid-Dorsal linkage in the integration of the anterior-posterior and dorsal-ventral patterning systems. Hunchback-Hunchback interactions help explain unresolved aspects of segmentation, including the differential regulation of the eve stripe 3 + 7 and stripe 4 + 6 enhancers. Evidence is presented that there is an under-representation of nucleosome positioning sequences in many enhancers, raising the possibility for a subtle higher-order structure extending across certain enhancers. It is concluded that grammar of gene control regions is pervasively used in the patterning of the Drosophila embryo (Papatsenko, 2009).

Nearly 100 characterized enhancers and ~30 associated binding motifs control the patterning of the early Drosophila embryo, probably the best understood developmental process. These enhancers and sequence-specific TFs regulate the expression of ~50 genes controlling AP and DV patterning, including segmentation and gastrulation. The known TFs controlling embryogenesis represent less than ~10% of all TFs in the Drosophila genome. Thus, this analysis of regulatory grammar was restricted to the ~100 AP and DV enhancers and their ~30 TF inputs (Papatsenko, 2009).

Inspection of aligned enhancer sequences among all 12 Drosophila species revealed strong conservation within the D. melanogaster subgroup (D. melanogaster, D. simulans, D. seichellia, D. yakuba and D. erecta) and also within the D. obscura group (D. pseudoobscura and D. persimilis). In order to focus on evolutionary changes in these enhancers the seven most divergent Drosophilids were analyzed: D. melanogaster, D. ananassae, D. pseudoobscura, D. willistoni, D. mojavensis, D. virilis and D. grimshawi. The remaining five species contain conservation patterns that are similar to those present in D. melanogaster or D. pseudoobscura (Papatsenko, 2009).

Short-range TF-binding linkages (0-80 bp) were examined in the collection of 96 enhancers from seven species for homo- and heterotypic pairs of binding motifs. Binding sites for the 30 most reliable TF motifs (see the Berkeley online resource) were mapped in enhancers using position weight matrices with match probability cutoff values set to ~2E-04. Distance histograms were generated for distances smaller than 80 bp, measured between the putative centers of each pair of neighboring site matches. Periodic signals were identified in the distance histograms using Fourier analysis, and statistical significance was estimated by bootstrapping positions of site matches in each enhancer sequence (Papatsenko, 2009).

Fourier analysis has identified helical phasing (~11 bp spacing) for several different homotypic activator-activator motif pairs. Such periodic signals were found in the distributions of Bcd-binding sites. Weaker helical-phasing signals were also identified for Caudal (Cad) and Dl-binding sites. Periodic signals close to two DNA turns (~20-22 bp) were found for Twi, Hb and Kruppel. Such helical phasing raises the possibility of direct protein-protein interactions (Papatsenko, 2009).

A weaker, ~11.4-bp periodic signal was detected in the distribution of heterotypic activator-activator site pairs, including Dl-Twi and Bcd-Cad. In contrast, there is a significant reduction in helical phasing signatures for activator-repressor motif pairs, and in fact, an over-representation of site pairs with 'anti-helical' spacing (15.2 bp). A similar 15.2 bp anti-helical signal was detected in distributions of all possible pair-wise combinations of the 30 binding motifs examined in this study. Thus, it would appear that any two randomly chosen binding sites are more likely to occupy the opposite sides of the DNA duplex as compared with helical phasing. This observation raises the possibility that most TFs function either additively or antagonistically to one another and just a special subset of TFs function in a synergistic fashion as reflected by helical phasing of the associated binding sites (Papatsenko, 2009).

The preceding analysis considered 'short-range' organizational constraints, involving linked binding sites separated by <25-30 bp. The possibility of 'long-range' constraints were also considered. The 96 enhancers under study possess characteristic 'unit lengths' of ~500 bp to 1.5 kb (300 bp minimum). The minimal/maximal sizes of the functional enhancers and the 'optimal' site densities can be determined by the amount of encoded information (pattern complexity), mechanisms of TF-DNA recognition such as lateral diffusion, or structural chromatin features like nucleosome positioning (Papatsenko, 2009).

Differential distance histograms reveal an over-representation of short-range linkages (<50 bp), but a depletion in mid-range distances (100-500 bp). These observations raise the possibility that TFs are distributed in a non-uniform manner across the length of the enhancer. That is, there may be sub-clusters, or 'hotspots', of binding sites within a typical enhancer. Such hotspots are observed in the prototypic eve stripe 2 enhancer, whereby 8 of the 12 critical binding sites are observed within two ~50-bp fragments located at either end of the minimal 480 bp enhancer. Homotypic motifs display the greatest propensity for such sub-clustering. Homotypic clusters (38) usually contain 3-5-binding sites distributed over 50-100 bp. Heterotypic activator-activator motif pairs also demonstrate sub-clustering, but these clusters are smaller (<25-30 bp) and usually contain just a pair of heterotypic sites. Heterotypic activator-repressor pairs show moderate enrichment over a distance of 50-70 bp, which is in agreement with the well-documented phenomenon of 'short-range repression'. Depletion of mid-range spacing constraints (around ~200 bp) is especially striking in the case of heterotypic motif pairs. Thus, activator synergy is like short-range repression: it appears to depend on closely linked binding sites (Papatsenko, 2009).

A possible explanation for this depletion of mid-range spacing is the occurrence of positioned nucleosomes, which might separate functionally distinct regions within an enhancer, and also separate neighboring enhancers. To test this hypothesis, nucleosome formation potential was compared with the distributions of TF-binding motifs in enhancers using the 'Recon' program. Three of the four eve enhancers that were examined (eve 1+5, eve 2 and eve 4+6) display a clear negative correlation between potential nucleosome formation and the distribution of TF-binding sites. This observation is consistent with the depletion of nucleosomes near TF-binding sites in vertebrates. This anti-correlation is especially striking in the case of the bipartite eve stripe 1+5 enhancer, where two enhancer regions (stripe 1 and stripe 5) are separated by a 400 bp 'spacer' DNA (in positions 600-1000), which might promote positioning of two nucleosomes and associated linker sequences (Papatsenko, 2009).

To investigate nucleosome positioning further, nucleosome-forming potential was measured in two sets of sequences, previously identified based on clustering of Dl sites and tested in vivo for enhancer activity. One set of sequences functioned as bona fide enhancers and produced localized patterns of gene expression across the DV axis of early embryos. The other set produced no expression in transgenic embryos, despite the presence of the same quality Dl-binding site clusters. The nucleosome-forming potential of the enhancers (true positives) was lower than that of the non-functional sequences (false-positives). These observations raise the possibility that the false Dl-binding clusters fail to function due to the formation of inactive nucleosomal structures (Papatsenko, 2009).

All 465 possible pairwise motif combinations for the 30 relevant binding motifs were tested for conservation in divergent drosophilids. Only linked binding sites, separated by a distance with small variations (max. distance bin = five bases) were considered. In the case of motif pairs, statistical significance was evaluated by bootstrapping columns in the binding motif alignments, thus preserving patterns of conservation. Pairs of homotypic motifs strongly prevailed in this type of analysis (28% of total pairs versus 6.5% expected), suggesting that homotypic interactions are important and pervasive in embryonic patterning. The strongest linkages were found for Bcd, Cad and Hb homotypic pairs. Each of these pairs was shared by five to six different enhancers and conserved in four to seven species. Among the identified heterotypic motif pairs, the most interesting were Bcd-Dl, Bcd-Cad and Dl-Twi (Papatsenko, 2009).

To identify cases of binding site pairs organized in a more flexible fashion, significant motif combinations were extracted using large distance bins or large distance variations. Along with the previously identified motif pairs, this analysis revealed several additional combinations, mainly involving the 'TAG-team' sequence motif, which is recognized by Zelda, a ubiquitous zinc finger TF. Zelda participates in the activation of the early zygotic genome and regulates a wide range of critical patterning genes. Indeed, significant combinations were identified for the TAG motif and Bcd, Dl and Hb. However, all of these TAG-X combinations exhibit spacing variability in different Drosophilids (Papatsenko, 2009).

It is conceivable that these results represent an underestimate of significantly linked motif combinations since very conservative cutoff values were used for statistical evaluation. A database of shared and/or conserved motif pairs, including those below the selected significance cutoff P = 0.03 is available from the Berkeley online resource (Papatsenko, 2009).

Conserved Bcd-Dl-binding site pairs were identified in the enhancers of several AP- and DV-patterning genes, including sal (AP), brk and sog (DV). The sites were found at similar distances, in the same orientation and were conserved in all seven species. It was suggested that the Bcd sites in the brk enhancer might augment gene expression in anterior regions, but this possibility was not directly tested. In wild-type embryos, both brk and sog exhibit significantly broader patterns of gene expression in anterior regions. This expanded pattern is lost in bcd mutants (Papatsenko, 2009).

Highly conserved Hb tandem repeats were detected in the regulatory regions of pair-rule genes, in the gap gene Kruppel, and in the Notch-signaling gene nubbin. Most of the homotypic Hb-Hb site pairs fall into two major groups, separated by either 6-8 or 13-15 bases. Some of the pair-rule enhancers selectively conserve either the 'short' or 'long' arrangement. For example, the eve stripe 4 + 6 enhancer contains two short Hb elements, while the stripe 3 + 7 enhancer contains a single long element. The odd 3 + 6 enhancer contains both short and long elements with various degrees of conservation. The hairy stripe 2,6,7 enhancer contains a single short element. Among the known gap genes, the long and short Hb elements were widely present in the enhancers of Kruppel, and in the blastoderm enhancer of nubbin, but not in any of the known knirps enhancers. It is conceivable that the distinct Hb site arrangements are important for the differential regulation of pair-rule genes by the Hb gradient (Papatsenko, 2009).

In conclusion, the systematic analysis of TF-binding sites in AP and DV patterning enhancers suggests a much higher degree of grammar, or fixed arrangements of binding sites, than is commonly believed. Developmental enhancers are thought to be highly flexible, with randomly distributed binding sites sufficing for the integration of multiple TFs. The results suggest that a large number of enhancers contain conserved short-range arrangements of pairs of binding sites. For instance, virtually all of the enhancers that respond to intermediate and low levels of the Dl gradient contain conserved arrangements of Dl-binding sites along with recognition sequences for other critical DV determinants, such as Twist and Zelda. Cooperating pairs of Bcd sites are found in enhancers responding to low Bcd concentrations, such as Knirps. Finally, distinctive arrangements of Hb-binding sites might influence whether the associated target genes are activated or repressed by high or low levels of the Hb gradient (Papatsenko, 2009).

Wingless targets twist in muscle progenitors

The patterning of the Drosophila mesoderm requires Wingless. Little is known about how Wg provides patterning information to the mesoderm, which is neither an epithelium nor contains the site of Wg production. By studying specification of muscle founder cells as marked by the lineage-specific transcription factor Slouch, it was asked how mesodermal cells interpret the steady flow of Wg. Through the manipulation of place, time and amount of Wg signaling, it has been observed that Slouch founder cell cluster II is more sensitive to Wg levels than the other Slouch-positive founder cell clusters. To specify Slouch cluster I, Wg signaling is required to maintain high levels of the myogenic transcriptional regulator Twist. However, to specify cluster II, Wg not only maintains high Twist levels, but also provides a second contribution to activate Slouch expression. This dual requirement for Wg provides a paradigm for understanding how one signaling pathway can act over time to create a diverse array of patterning outcomes (Cox, 2005).

In wg mutant embryos, the heart and approximately half the body wall muscles are lost. One subset of these Wg-dependent body wall muscles can be visualized using an antibody to the NK-homeodomain protein Slouch (S59). Slouch expression arises in a precise, stereotypic pattern during embryonic development. It is first expressed in a single progenitor cell during early stage 11 of embryonic development; this cell divides to give rise to two founder cells (Ia and Ib) which together form cluster I (cI). During late stage 11, two additional Slouch-positive progenitors appear at a different ventral location and divide sequentially to form four founder cells that make up cluster II. Still later, at stage 12, a single progenitor arises dorsally and divides to give rise to cluster III. These muscle founder cells contain all the information needed to create a particular subset of muscles and contribute to the stereotypic set of larval muscles in each abdominal segment. After stage 12, Slouch expression is maintained in a subset of these founder cells that give rise, in the final muscle pattern, to muscle VT1 (from cI), VA2 (from cII) and DT1 (from cluster III). Maintenance of Slouch expression in these founder cells is crucial to the development of these muscles; removal of slouch leads to complete (VT1) and partial muscle transformations (VA2; DT1). In this study, focus was placed on the role of Wg in patterning the Slouch muscle founder cells. For simplicity, focus was placed solely on two ventral Slouch clusters (I and II), which develop independently and arise in a similar position along the dorsoventral axis but have different anterior-posterior positions within each abdominal hemisegment (Cox, 2005).

Through the manipulation of the amount and time of exposure to Wg signaling in the Drosophila mesoderm, it is shown that Slouch founder cell cII requires more Wg signaling than its neighbor, cI. Because cII arises in the mesoderm beneath the source of Wg signal, it was initially thought that the sensitivity that was detected would be due to Wg acting as a classic morphogen. Specifically, during stage 11, Wg would directly elicit concentration-dependent responses, leading to Slouch cI specification at low levels and cII at higher levels. Instead, the data suggest an alternative mechanism underlying this sensitivity. For Slouch cI, Wg signaling through Twist is sufficient for fate specification. However, for Slouch cII, a second, Twist-independent Wg signal is also necessary (Cox, 2005).

It has been shown that wg mutants fail to maintain high levels of Twist. Overexpression of Twist leads to expanded somatic mesodermal fates at the expense of other mesodermal fates, such as heart and gut muscle. Conversely, decreasing Twist levels leads to a reduction in somatic mesodermal fate, while heart and gut muscle remain largely unaffected. These findings underscore the importance of high Twist levels for the proper implementation of somatic muscle fate. Because loss of high Twist levels leads to loss of muscle founder cells, including all Slouch-positive clusters of founder cells, it has always appeared that each Slouch cluster requires the same amount of Wg signal (relayed through Twist) to assume its particular fate. In this study, the requirement for Wg in maintaining high Twist levels was uncoupled from the later role of Wg in specifying cII fate. The fact that Twist specifically rescues Slouch cI in a wg mutant background suggests that Slouch cII requires an additional, Twist-independent contribution from Wg for proper patterning. Consistent with these results, wg hypomorphs were found that provided sufficient signaling to maintain high Twist levels during early mesoderm development and therefore pattern cI, but that do not pattern cII. Temperature-shift experiments using wg temperature-sensitive alleles have shown that Slouch cII specification and engrailed expression in the ectoderm require Wg expression at later stages of embryonic development. Thus, the absence of Slouch cII in the different wg alleles, in hh mutant embryos and in a Twist rescued wg mutant embryo, all suggest that proper patterning requires not only an earlier Wg-dependent regulation of Twist, but also an additional Wg contribution to specify its identity (Cox, 2005).

Manipulations of Wg signaling also revealed two additional aspects of Wg signaling to the mesoderm. (1) It was found that the mesoderm, in general, has a different threshold for Wg signaling when compared with the ectoderm. Conditions that completely rescue the ventral ectoderm and epidermis (wg^PE6 at the permissive temperature) fail to completely rescue the mesoderm. (2) It was found that different mesodermal targets respond differently to Wg signaling. For example, expression of the DeltaNTcf (dominant negative form of Pangolin) has mild effects on Twist but significant effects on Slouch cII. Although it is predicted that TCF binds slouch regulatory regions directly, it was found that Wg regulates Twist both directly through TCF and indirectly through the pair-rule gene sloppy-paired. Whether or not the difference in Wg regulation of twist and slouch is due to the structure of the regulatory regions, additional factors that integrate on these promoters in these contexts and the activity of the Arm/dTCF complex remains to be uncovered (Cox, 2005).

This study also underscores the contribution that other factors make to position the Slouch clusters: ectopic Wg expression in the mesoderm does not produce uniform Slouch expression. This aspect of Wg signaling is reflected in other tissues such as the epidermis. The size of Slouch cII could not be further enlarged beyond that seen when Wg signaling was initially increased. This suggests a prepatterning mechanism, perhaps involving the activity of the pair-rule genes that have been shown to be responsible for segmentation of the mesoderm, as well as the integration of other signal transduction pathways, such as EGF/FGF and Notch signaling. The data suggest that Wg signaling then works on this prepattern to regulate the domain of Slouch expression (Cox, 2005).

The effect of Wg on muscle patterning is similar to that described for even-skipped muscle progenitor specification; that is, Wg signaling (in collaboration with such signals as Decapentaplegic) is first required to set up a region of 'competence' through activation of mesoderm-specific factors such as Twist and Tinman. Wg then later cooperates with these intrinsic factors to induce the expression of even-skipped in dorsal muscle progenitors, much as would be suggested for Slouch cII. However, the observations suggest an important variation of Wg signaling in mesodermal patterning. In the case of Slouch patterning, Wg creates temporal as well as spatial diversity, while in patterning eve it only acts temporally. Wg signaling contributes to the expression of Slouch in its two discrete ventral patches by two distinctive mechanisms: through the regulation of an upstream transcription regulator (Twist), which is sufficient for one domain of expression; and through the cooperation of this factor with a second, temporally distinct Wg input for the second domain of expression. The expression of the same gene but at two different times and places, through two Wg-dependent means, gives insight into how an organism may generate diverse tissues in response to the same signal (Cox, 2005).

Work carried out in the wing imaginal disc suggests that Wg acts as a morphogen. In this tissue, Wg protein was visualized in a graded distribution and it appears to activate multiple target genes directly, in a concentration-dependent manner. Based on these criteria, Wg has been labeled as a classical morphogen. However, careful inspection of the molecular mechanisms underlying Wg activation of both short- and long-range targets in the wing have revealed that the pattern of Wg expression changes during wing imaginal disc development, and that Wg collaborates with other pathways to set up the expression of these genes. These studies have cast doubt on whether Wg is a true morphogen in this tissue (Cox, 2005).

Investigating the molecular mechanisms that govern patterning of the embryonic mesoderm, similarly suggests that Wg does not act on Slouch clusters I and II as a classical morphogen. Wg does not activate cI directly, but instead maintains high levels of Twist, which sets up a somatic mesodermal competency domain that is sufficient to create cI. Additional Wg is then required later to pattern cII. It can be argued that Wg acts as a morphogen to regulate Twist expression (at low levels), and then to control Slouch expression (at high levels) within cells of cII. However, the precise regulation and dependence of Slouch clusters I and II on Wg within both the dorsoventral and anteroposterior axes suggest that there must be additional patterning information available to properly place these two cell types. As more putative morphogens are held up to the lens of molecular biology, it will be interesting to see whether there are unexpected, new twists in the molecular underpinnings of morphogens (Cox, 2005).

WntD is a target and an inhibitor of the Dorsal/Twist/Snail network in the gastrulating embryo

The maternal Toll signaling pathway sets up a nuclear gradient of the transcription factor Dorsal in the early Drosophila embryo. Dorsal activates twist and snail, and the Dorsal/Twist/Snail network activates and represses other zygotic genes to form the correct expression patterns along the dorsoventral axis. An essential function of this patterning is to promote ventral cell invagination during mesoderm formation, but how the downstream genes regulate ventral invagination is not yet known. wntD (FlyBase name: Wnt8) is shown to be a member of the Wnt family. The expression of wntD is activated by Dorsal and Twist, but the expression is much reduced in the ventral cells through repression by Snail. Overexpression of WntD in the early embryo inhibits ventral invagination, suggesting that the de-repressed WntD in snail mutant embryos may contribute to inhibiting ventral invagination. The overexpressed WntD inhibits invagination by antagonizing Dorsal nuclear localization, as well as twist and snail expression. Consistent with the early expression of WntD at the poles in wild-type embryos, loss of WntD leads to posterior expansion of nuclear Dorsal and snail expression, demonstrating that physiological levels of WntD can also attenuate Dorsal nuclear localization. The de-repressed WntD in snail mutant embryos contributes to the premature loss of snail expression, probably by inhibiting Dorsal. Thus, these results together demonstrate that WntD is regulated by the Dorsal/Twist/Snail network, and is an inhibitor of Dorsal nuclear localization and function. The closest homologs of Drosophila WntD, vertebrate Wnt8 proteins, regulate mesoderm patterning, neural crest cell induction, neuroectoderm patterning, and axis formation (Hoppler, 1998; Lekven, 2001; Lewis, 2004; Popperl, 1997). These vertebrate Wnt8 proteins may transmit the signal through the canonical pathway, but the exact mechanism remains unclear. So far, the downstream mediators of Drosophila WntD signaling are not known (Ganguly, 2005).

A second study (Gordon, 2005) confirms and extends Ganguly (2005) by inducing a mutation in wntD by homologous replacement. The Gordon study shows that WntD acts as a feedback inhibitor of the NF-kappaB homologue Dorsal, during both embryonic patterning and the innate immune response to infection. wntD expression is under the control of Toll/Dorsal signalling, and increased levels of WntD block Dorsal nuclear accumulation, even in the absence of the IkappaB homologue Cactus. The WntD signal is independent of the common Wnt signalling component Armadillo. By engineering a gene knockout, this study shows that wntD loss-of-function mutants have immune defects and exhibit increased levels of Toll/Dorsal signalling. Furthermore, the wntD mutant phenotype is suppressed by loss of zygotic dorsal (Gordon, 2005).

To identify novel components in the dorsoventral pathway, a microarray assay was carried out using embryos derived from gain-of-function and loss-of-function mutants of the Toll pathway. Among the novel genes identified, the expression and function of wntD was analyzed because the Wnt family of secreted proteins regulates patterning, cell polarity and cell movements. The results show that wntD is activated by Dorsal and Twist but repressed by Snail. Increased expression of WntD in wild-type early embryos inhibits ventral invagination. Thus, wntD is the first Snail target gene shown to have an interfering function in mesoderm invagination. The overexpressed WntD blocks invagination by inhibiting Dorsal nuclear localization. Loss-of-function analyses also show that physiological levels of WntD can attenuate Dorsal nuclear localization and function. Therefore, wntD is a novel downstream gene of the Dorsal/Twist/Snail network and can feed back to inhibit Dorsal (Ganguly, 2005).

The dynamic pattern of wntD expression in the early embryo is a combined result of activation by Dorsal/Twist and repression by Snail. Overexpressed WntD negatively regulates Dorsal nuclear localization, leading to an inhibition of ventral cell invagination. Physiological levels of WntD can also negatively regulate Dorsal, since loss of WntD leads to detectable expansion of both Dorsal nuclear localization and snail expression in the posterior regions. Furthermore, de-repressed WntD expression in the ventral region of snail mutant embryos can also attenuate Dorsal function. However, the loss of WntD could not rescue the invagination defect of the snail mutant embryo, suggesting that in the snail mutant embryo there are other de-repressed genes that can interfere with ventral invagination (Ganguly, 2005).

The wntD loss-of-function phenotype correlates with the expression of wntD at the poles of pre-cellular blastoderms. wntD is also expressed a bit later in the mesectoderm, and weakly in the mesoderm. Because WntD can inhibit Dorsal, one speculation is that WntD in the early mesectoderm may help to establish the sharp snail expression at the mesectoderm-neuroectoderm boundary. However, no changes were detected in the Dorsal protein gradient or snail pattern in the trunk regions of the Df(3R)l26c embryos. It is speculated that the timing of early expression of wntD, which may have additional input from the Torso pathway at the poles, is important for the feedback inhibition of Dorsal. By the time of cellularization, the Dorsal protein gradient is well established. This well-established Dorsal gradient activates the wntD gene in the trunk regions, but the subsequently translated WntD protein may not be capable of exerting a strong negative-feedback effect on the already formed Dorsal gradient. This timing argument is supported by the results of WntD-overexpression experiments. The use of maternal nanos-Gal4 caused a strong inhibition of Dorsal nuclear localization and of ventral invagination, whereas the use of zygotic promoters did not result in a significant phenotype (Ganguly, 2005).

Snail acts as a transcriptional repressor for at least 10 genes in the ventral region where mesoderm arises. In snail mutant embryos, all of these target genes are de-repressed in the ventral cells, concomitant with severe ventral invagination defects. However, no direct evidence has been reported on whether these de-repressed genes interfere with invagination. This study showed for the first time that a target gene of Snail, namely wntD, can block ventral invagination when overexpressed. If de-repressed WntD is solely responsible for inhibiting ventral invagination, it would be expected that, in the snail;Df(3R)l26c double-mutant embryos, ventral invagination would appear again. No rescue of ventral invagination was detected in the double-mutant embryos, suggesting that wntD is not the only de-repressed target gene that inhibits invagination. Nonetheless, the de-repressed WntD can attenuate Dorsal function, and may contribute to the ventral invagination defect (Ganguly, 2005).

Mesodermally expressed Drosophila microRNA-1 is regulated by Twist and is required in muscles during larval growth

Although hundreds of evolutionarily conserved microRNAs have been discovered, the functions of most remain unknown. This study describes the embryonic spatiotemporal expression profile, transcriptional regulation, and loss-of-function phenotype of Drosophila miR-1 (DmiR-1). DmiR-1 RNA is highly expressed throughout the mesoderm of early embryos and subsequently in somatic, visceral, and pharyngeal muscles, and the dorsal vessel. The expression of DmiR-1 is controlled by the Twist and Mef2 transcription factors. DmiR-1^KO mutants, generated using ends-in gene targeting, die as small, immobilized second instar larvae with severely deformed musculature. This lethality is rescued when a DmiR-1 transgene is expressed specifically in the mesoderm and muscle. Strikingly, feeding is what triggers DmiR-1^KO-associated paralysis and death; starved first instar DmiR-1^KO larvae are essentially normal. Thus, DmiR-1 is not required for the formation or physiological function of the larval musculature, but is required for the dramatic post-mitotic growth of larval muscle (Sokol, 2005).

MiR-1 is an evolutionarily conserved miRNA whose tissue-specific expression pattern also appears to be phylogenetically conserved. The worm and fly genomes each possess a single miR-1 gene (Cel-miR-1 and DmiR-1, respectively) while the zebrafish, mouse, and human genomes each contain two miR-1 loci (Dre-miR-1-1 and Dre-miR-1-2, Mmu-miR-1-1 and Mmu-miR-1-2, and Hsa-miR-1-1 and Hsa-miR-1-2, respectively). Northern blot analysis indicates that miR-1 is expressed specifically in mouse and human heart and skeletal muscle. This result has been further confirmed using three different in vivo techniques to examine miRNA expression; a murine Mmu-miR-1-1 and Mmu-miR-1-2 'sensor' transgene is repressed specifically in the adult heart; promoter fusion constructs to Mmu-miR-1-1 and Mmu-miR-1-2 are both expressed in cardiac and skeletal muscle precursor cells; in situ hybridization using locked nucleic acid (LNA) probes detected Dre-miR-1-1 and Dre-miR-1-2 expression in muscle. Two recent studies suggest that miR-1 might possess a variety of in vivo functions. Microarray analysis of HeLa cells transfected with Hsa-miR-1 indicates that miR-1 functions to maintain muscle cell identity by repressing the expression of nonmuscle genes. Overexpression analysis of Mmu-miR-1 indicates that it may regulate the proliferation of cardiomyoctes by controlling the expression of the Hand2 transcription factor (Sokol, 2005 and references therein).

To directly assess the in vivo function of miR-1, the expression pattern, transcriptional regulation, and loss-of-function phenotype of Drosophila miR-1 (DmiR-1) were each examined. As in zebrafish, mice, and humans, DmiR-1 is specifically expressed in muscle cells. DmiR-1 expression is regulated by the promesodermal transcription factor Twist and the promyogenic transcription factor Mef2. Muscles form normally in DmiR-1^KO mutant embryos and function normally in first instar DmiR-1^KO mutant larvae. However, when larval growth is initiated by feeding, DmiR-1^KO mutant larvae become paralyzed, arrest their growth and ultimately die as small, second instar larvae with massively disrupted somatic musculature (Sokol, 2005).

In situ hybridization was performed using probes to detect the full-length primary transcript (pri-DmiR-1) from which the DmiR-1 21mer is processed. Pri-DmiR-1 is initially detected ventrally in the presumptive mesoderm of stage 5 embryos in a pattern similar to that of the snail and twist gene products. Direct comparison of the pri-DmiR-1 expression pattern along the anterior-posterior axis with the snail and twist mRNA pattern in the cellular blastoderm indicates that pri-DmiR-1 shares the sharp posterior border of snail RNA but its anterior boundary is posterior to that of both twist and snail RNA. Pri-DmiR-1 is expressed in the ventral-most cells of the cellular blastoderm, in a swath that is 18-20 cells wide. During gastrulation and after invagination of the ventral cells of the cellular blastoderm, pri-DmiR-1 is expressed exclusively in mesodermal cells. After subdivision of the mesoderm, pri-DmiR-1 expression is observed in the cephalic mesoderm and the primordia of the visceral and somatic musculature. After germ-band retraction, pri-DmiR-1 was detected in the pharyngeal and visceral musculature, the myocardial cells of the dorsal vessel and in the segmentally repeated clusters of mesodermal cells that give rise to the somatic muscles. Notably, pri-DmiR-1 was restricted to muscle progenitors and was not expressed in other mesodermal derivatives such as the fat body, gonadal mesoderm, and midline glia (Sokol, 2005).

The expression pattern of the mature 21-nt form of DmiR-1 was directly assayed using a 21-nt digoxigenin-labeled LNA oligonucleotide probe complementary to DmiR-1. The similarity between the two expression patterns confirmed that DmiR-1 RNA is expressed specifically in the mesoderm and its muscle cell derivatives. However, there were two key differences between the expression patterns. (1) While pri-DmiR-1 is detected in a punctate, nuclear-staining pattern, DmiR-1 21mer is cytoplasmic. This is consistent with the rapid processing of miRNA transcripts into mature miRNAs in conjunction with transport to the cytoplasm. (2) While robust expression in the presumptive mesoderm is detected with the pri-DmiR-1 probes, the DmiR-1 21mer was first faintly detected later at gastrulation. This could reflect stage-specific differences in probe accessibility. Alternatively, pri-DmiR-1 may be transcribed beginning at the cellular blastoderm stage but not processed until gastrulation (Sokol, 2005).

Four lines of evidence are presented that DmiR-1 is essential for myofiber function during larval development: (1) consistent with the muscle- and heart-specific expression of miR-1 in zebrafish, mouse, and humans, zygotically expressed DmiR-1 is expressed in most, if not all, the myogenic cells of the larval muscle system; (2) the genetic removal of zygotic DmiR-1 results in a highly penetrant and temporally specific locomotion defect -- first instar DmiR-1^KO larvae become increasingly lethargic and they eventually die as small immobilized, second instar larvae; (3) the body wall muscles of second instar DmiR-1^KO mutant larvae are massively disrupted, presumably causing the locomotion defec; (4) the larval DmiR-1^KO mutant phenotypes, gradual paralysis, and death, can be rescued when DmiR-1 transcript is expressed specifically in muscle cells (Sokol, 2005).

Strikingly, the DmiR-1^KO larval phenotype is only manifest after feeding. Newly hatched DmiR-1^KO larvae that have not yet begun to feed are nearly wild type in their body wall contractions, dorsal vessel contractions, and excretion. Furthermore, first instar DmiR-1 mutant larvae that are cultured with only sucrose for an energy source but no nutritional supplement to support growth, and consequently do not proceed through larval development to the second instar, are essentially wild type in their movement and longevity. This growth-dependent larval muscle phenotype of DmiR-1^KO animals is in contrast to previously described mutations that more generally disrupt larval muscle function. For example, mutations in the alpha-actinin and ryanodine receptor genes, both of which are expressed throughout the larval musculature, cause growth-independent phenotypes; muscle cell function is strongly compromised even in newly hatched larvae. From this, it is concluded that DmiR-1 function and hence the regulation of gene expression by the DmiR-1 miRNA, is critical for maintaining muscle integrity during the dramatic, post-mitotic growth in muscle mass of wild-type larvae (Sokol, 2005).

Wild-type newly hatched Drosophila larvae encounter a developmental decision: Larvae that are fed a sucrose-only diet arrest development as first instars and can remain so, vigorously searching for food, for up to 2 weeks. By contrast, larvae that are fed a nutritional food source grow dramatically, increasing their body mass ~200-fold in 4 d. For most terminally differentiated larval-specific tissues, including the gut, epidermis, fat body, trachea and salivary glands, larval growth is accomplished by expansion of cell size rather than by cell proliferation. For these cells, feeding triggers a specialized cell cycle, the endocycle, in which cells undergo rounds of DNA replication without division. In contrast, the precursors of adult structures, including imaginal disc cells and neuroblast cells, grow via conventional diploid cell cycles (Sokol, 2005).

Little is known about the cell biology of larval muscle growth. While neither cell division nor nuclear division takes place, each myofiber expands at least 100-fold in size, presumably also involving endocyclic DNA replication. The data do not distinguish whether the DmiR-1^KO mutation disrupts entry into the endocycle or some other step in muscle cell growth. Genes that act throughout the larva to control entry into the endocycle have been identified as mutations that cause growth arrest after feeding. But unlike DmiR-1^KO, endocycle mutants display wild-type larval behavior and locomotion. Hence the DmiR-1^KO phenotype cannot be explained as simply a consequence of muscle cells failing to enter the endocycle (Sokol, 2005).

Evidence is presented that places DmiR-1 within the established mesodermal and myogenic transcriptional networks. Twist and Dorsal are known to coregulate a number of mesodermal genes and two weak Dorsal-binding sites have been observed in the DmiR-1 enhancer/lacZ fusion transgenes tested here. However, the finding that ectopic Twist in a gastrulation defective mutant background is sufficient to direct DmiR-1 expression indicates that Twist alone, without Dorsal, is sufficient for DmiR-1 expression. Although Twist activates DmiR-1 transcription throughout the embryonic mesoderm it is nevertheless found that embryos entirely depleted of DmiR-1 appear normal. The expression of DmiR-1 in larval muscle, where it carries out its critical function, is likely to be maintained by factors downstream of Twist, particularly Mef2. So, what might be the role for Twist-mediated embryonic expression of DmiR-1? The early expression of DmiR-1 throughout the mesoderm could be gratuitous for the embryo per se but nevertheless could reflect a role for Twist-activated DmiR-1 at a later developmental stage. For example, Twist expression is maintained during larval development in clusters of undifferentiated cells that will give rise to adult myoblasts and ultimately to adult muscles. Interestingly, the Notch signaling pathway down-regulates Twist expression and thereby promotes the formation of embryonic somatic muscle founder cells at the expense of adult myoblast progenitor cells. Perhaps Twist-activated expression of DmiR-1 counteracts Notch signaling and hence helps maintain adult myoblast progenitor cells during embryogenesis and larval development (Sokol, 2005).

Spatial regulation of microRNA gene expression in the Drosophila embryo: The 8-miR enhancer is regulated by the localized Huckebein repressor, whereas miR-1 is activated by Dorsal and Twist

MicroRNAs (miRNAs) regulate posttranscriptional gene activity by binding to specific sequences in the 3' UTRs of target mRNAs. A number of metazoan miRNAs have been shown to exhibit tissue-specific patterns of expression. This study investigated the possibility that localized expression is mediated by tissue-specific enhancers, comparable to those seen for protein-coding genes. Two miRNA loci in Drosophila melanogaster are investigated, the mir-309–6 polycistron (8-miR) and the mir-1 gene. The 8-miR locus contains a cluster of eight distinct miRNAs that are transcribed in a common precursor RNA. The 8-miR primary transcript displays a dynamic pattern of expression in early embryos, including repression at the anterior and posterior poles. An 800-bp 5' enhancer was identified that recapitulates this complex pattern when attached to a RNA polymerase II core promoter fused to a lacZ-reporter gene. The miR-1 locus is specifically expressed in the mesoderm of gastrulating embryos. Bioinformatics methods were used to identify a mesoderm-specific enhancer located ~5 kb 5' of the miR-1 transcription unit. Evidence is presented that the 8-miR enhancer is regulated by the localized Huckebein repressor, whereas miR-1 is activated by Dorsal and Twist. These results provide evidence that restricted activities of the 8-miR and miR-1 miRNAs are mediated by classical tissue-specific enhancers (Biemar, 2005).

The 8-miR complex is located between two predicted protein-coding genes, CG15125 and CG11018, in the 56E region on the right arm of chromosome 2. To determine the approximate transcription start site of the 8-miR transcription unit, 5' RACE was used. Several independent experiments were carried out, and RACE products corresponding to two different start sites were isolated several times. Consensus sequences for both an initiator and a TATA box are appropriately spaced upstream of the identified start sites. The alignment of this genomic interval with the corresponding regions of the most divergent Drosophilids indicates strong conservation of each of the individual miRNAs within the 8-miR complex (Biemar, 2005).

A digoxigenin-labeled 8-miR antisense RNA probe was hybridized to staged embryos to determine the expression profile of the precursor transcript during development. Expression is initially detected in all of the nuclei of precellular embryos. As expected, staining is restricted to nuclei and not seen in the cytoplasm. The first indication of differential spatial regulation occurs at the midpoint of cellularization, when 8-miR transcripts are lost at the posterior pole. By the completion of cellularization, this loss in staining expands and there is also reduced expression in anterior regions. Staining persists at the anterior tip but is lost from subterminal regions of the anterior pole (Biemar, 2005).

During gastrulation there is both dorsal-ventral and anterior-posterior modulation of the 8-miR-staining pattern. Staining is first lost from the presumptive mesoderm and neurogenic ectoderm in ventral and lateral regions. There are transient stripes of 8-miR expression in the dorsal ectoderm, but they rapidly give way to a single band of staining in central regions. By the onset of the rapid phase of germband elongation, staining is essentially lost except for residual expression at the anterior tip and dorsal ectoderm (Biemar, 2005).

The early loss of staining at the posterior pole suggests that Huckebein (Hkb) might repress 8-miR transcription in the early embryo. To investigate this possibility, colocalization assays were done with snail, which is selectively expressed in the presumptive mesoderm of cellularizing and gastrulating embryos. The posterior border of the snail pattern is established by the localized Hkb repressor. The 8-miR pattern displays a similar posterior border, and there is an expansion of both the snail and 8-miR patterns in hkb^-/hkb^- mutant embryos (Biemar, 2005).

Further evidence for repression by Hkb was obtained by analyzing torso dominant (tor^D) mutants. tor encodes a receptor tyrosine kinase that is normally activated only at the poles, where it is required for the localized expression of tailless (tll) and hkb. tor^D encodes a constitutively activated form of the receptor tyrosine kinase that results in expanded expression of hkb and tll at the poles. This expansion in Hkb causes a severe shift in the posterior border of both the snail and 8-miR expression patterns. The identification of a sequence-specific transcriptional repressor, Hkb, as a likely regulator of 8-miR expression suggests that the dynamic staining pattern is probably controlled at the level of de novo transcription (Biemar, 2005).

Direct support for this possibility was obtained by the identification of an 8-miR enhancer. An ~800-bp genomic DNA fragment extending from the miR-3 region of the 8-miR complex to the predicted start site of CG11018 was attached to a lacZ-reporter gene containing the minimal eve promoter sequence. The resulting fusion gene recapitulates most aspects of the endogenous 8-miR expression pattern. In particular, lacZ transcripts are initially detected throughout precellular embryos but sequentially lost from the posterior pole and anterior regions during cellularization. At the onset of gastrulation, expression is diminished in ventral regions, and the staining detected in the dorsal ectoderm exhibits segmental modulation. Thus, the 5' 8-miR enhancer contains repression elements that mediate silencing by Hkb (and possibly Tll) at the termini in response to Tor signaling (Biemar, 2005).

The preceding analysis provides evidence that cell-specific enhancers regulate miRNA gene expression, as seen for protein coding genes. Further support was obtained by analyzing a second miRNA that displays localized expression in the early Drosophila embryo, miR-1. The mir-1 gene is highly conserved in different animal groups and displays localized expression in a variety of mesodermal lineages, including cardiac mesoderm in vertebrates. The Drosophila mir-1 gene is first expressed in the presumptive mesoderm during the final phases of cellularization. Expression persists in differentiating mesodermal tissues during gastrulation, germband elongation, and segmentation. Mutant embryos that contain the constitutively activated Toll^10B receptor display ubiquitous expression of miR-1, concomitant with the transformation of all of the tissues into mesoderm (Biemar, 2005).

Whole-genome tiling arrays were used to obtain an estimate of the miR-1 transcription unit. These high-density oligonucleotide arrays contain 25-nt oligomers spaced on average every 36 bp and cover the entire nonrepetitive Drosophila genome, from one end of each chromosome to the other. Total RNA was extracted from three different mutant strains. Embryos derived from pipe^-/pipe^- females lack Toll-signaling activity and thereby lack a Dorsal nuclear gradient. As a result, genes normally activated by high, intermediate, and low levels of the gradient are silent, and there is a loss of mesoderm and neurogenic ectoderm. Instead, genes that are repressed by the Dorsal gradient, and normally restricted to the dorsal ectoderm, are now expressed throughout the embryo, causing the transformation of mesoderm and neurogenic ectoderm into dorsal ectoderm. Previous microarray assays have shown that genes expressed in the dorsal ectoderm are overexpressed in mutant embryos derived from pipe^-/pipe^- embryos. As expected, such mutants display little or no expression of the miR-1 transcription unit. Similarly, embryos derived from Toll^rm9/Toll^rm10 mutants contain weak Toll signaling and low levels of nuclear Dorsal everywhere. These low levels are insufficient for the activation of mesoderm genes, but are sufficient for the activation of neurogenic genes and the repression of dorsal ectoderm genes. Again, these mutants fail to express miR-1. Toll^10B embryos contain strong, ubiquitous Toll signaling and high levels of Dorsal, which activate mesoderm genes throughout the embryo. These embryos display strong expression of the miR-1 transcription unit. The tiling array suggests that the gene is ~2.9 kb in length. The mature, processed miRNA is located roughly in the center of the inferred transcription unit (Biemar, 2005).

The early expression of the miR-1 primary transcript in the mesoderm raises the possibility that the gene might be regulated by the Dorsal gradient. Approximately one-half of all Dorsal-target enhancers also contain binding sites for the basic helix-loop-helix Twist activator. A 50-kb interval encompassing the miR-1 locus was surveyed for clusters of Dorsal and Twist binding sites. The best cluster was identified ~5 kb upstream of the miR-1 start site. There are a total of three Dorsal- and four Twist-binding sites contained over an interval of ~1.1 kb in this distal 5' region (Biemar, 2005).

A genomic DNA fragment encompassing these sites was attached to a lacZ-reporter gene and expressed in transgenic embryos. The reporter gene exhibits localized expression in the ventral mesoderm, beginning at the onset of gastrulation. Expression persists during germband elongation. These observations suggest that miR-1 is directly activated by Dorsal and Twist. However, lacZ transcripts expressed from the miR-1::lacZ transgene are detected somewhat later than the endogenous miR-1 primary transcript, which first appears before the completion of cellularization. It is conceivable that the miR-1 locus contains a second enhancer that directs earlier expression (Biemar, 2005).

The preceding analysis provides evidence that dynamic patterns of miRNA gene expression are controlled by tissue-specific enhancers, and not by the differential processing of miRNA precursor RNAs. Both the 8-miR and miR-1 enhancers produce authentic patterns of lacZ-reporter gene expression when attached to the core promoter region of the eve gene. The 8-miR enhancer appears to be regulated by the Hkb repressor, whereas miR-1 is activated by Dorsal and Twist (Biemar, 2005).

The miR-1 enhancer is somewhat unusual among 'type 1' Dorsal target enhancers, in that it contains a large number of Snail repressor sites. Type 1 enhancers are activated by high levels of the Dorsal gradient in the ventral mesoderm. Previous studies have identified six such enhancers. They all contain multiple low-affinity Dorsal binding sites, but essentially lack Snail repressor sites. The general absence of Snail sites permits activation of type 1 genes in the ventral mesoderm where there are high levels of the repressor. An exception is the type 1 intronic enhancer that regulates Heartless (Htl), one of the two FGF receptor genes in the Drosophila genome (Biemar, 2005).

The htl intronic enhancer is ~800 bp in length and contains two low-affinity Dorsal binding sites and two optimal Twist sites. Each Twist site overlaps a Snail repressor site, but the enhancer nonetheless activates lacZ-reporter gene expression in the presumptive mesoderm before the completion of cellularization. The htl enhancer fails to mediate expression in the neurogenic ectoderm because it lacks the arrangement of optimal Dorsal and Twist sites required for activation by intermediate levels of the Dorsal gradient (type 2 enhancers) (Biemar, 2005).

The miR-1 enhancer contains three weak Dorsal sites, four optimal Twist sites (CACATGT; Kate Senger, unpublished results cited in Biemar, 2005), and five Snail repressor sites (three of the sites overlap the optimal Twist sites and two occur at separate sites). Perhaps the relative increase in the number of Snail repressor sites in the miR-1 enhancer (vs. the htl enhancer) causes late onset of miR-1::lacZ transgene expression. The Snail repressor is transiently expressed in the ventral mesoderm during cellularization but disappears after invagination. It is during the time when Snail levels subside that the miR-1 enhancer first becomes active (Biemar, 2005).

Previous studies have emphasized the importance of the Snail repressor in defining spatially localized patterns of gene expression. Dorsal target genes activated by intermediate (type 2) and low (type 3) levels of the gradient contain Snail repressor sites that keep the genes off in the ventral mesoderm and restricted to the neurogenic ectoderm. The present identification of the distal miR-1 enhancer raises the possibility that Snail also influences the timing of gene expression (Biemar, 2005).

The similarities in miR-1 and Htl regulation raise the possibility that the miR-1 miRNA attenuates the activity of one or more components of the FGF-signaling pathway. FGF is essential for the migration of the invaginated mesoderm along the inner surface of the neurogenic ectoderm. It is also important for the activation of cardiac genes in the dorsal-most mesoderm that forms the heart. miR-1 might attenuate one or more target mRNAs engaged in mesoderm migration and/or heart induction. The mammalian miR-1 miRNA has been shown to attenuate Hnd2 expression, which is essential for the differentiation of ventricular cardiomyocytes (Zhao, 2005). Despite the conservation of the miR-1 miRNA sequence, and a potential role in suppressing heart formation in both flies and mice, it would appear that distinct mechanisms of regulation are used in the two systems: Dorsal and Twist activate miR-1 in flies, whereas distinct regulatory factors, SRF and MyoD, activate miR-1 in the mouse embryo. It is possible however, that later phases of miR-1 expression depend on nautilus (nau), the Drosophila homolog of MyoD (Biemar, 2005).

Transcription of Myocyte enhancer factor-2 in adult Drosophila myoblasts is induced by the steroid hormone ecdysoneL Expression of Mef2 is delayed in twist-expressing adult myoblasts until the end of the third larval instar

The steroid hormone 20-hydroxyecdysone (ecdysone) activates a relatively small number of immediate-early genes during Drosophila pupal development, yet is able to orchestrate distinct differentiation events in a wide variety of tissues. This study demonstrates that expression of the muscle differentiation gene Myocyte enhancer factor-2 (Mef2) is normally delayed in twist-expressing adult myoblasts until the end of the third larval instar. The late up-regulation of Mef2 transcription in larval myoblasts is an ecdysone-dependent event that acts upon an identified Mef2 enhancer, and enhancer sequences have been identified required for up-regulation. Evidence is presented that the ecdysone-induced Broad Complex of zinc finger transcription factor genes is required for full activation of the myogenic program in these cells. Since forced early expression of Mef2 in adult myoblasts leads to premature muscle differentiation, these results explain how and why the adult muscle differentiation program is attenuated prior to pupal development. A mechanism is proposed for the initiation of adult myogenesis, whereby twist expression in myoblasts provides a cellular context upon which an extrinsic signal builds to control muscle-specific differentiation events, and the general relevance of this model for gene regulation in animals is discussed (Lovato, 2005).

Since a 175-bp Mef2 enhancer recapitulates the pattern of gene expression seen for Mef2 during larval development, attempts were made to identify the factors that might be interacting with this sequence. No consensus binding sites for the ecdysone receptor were found, nor for the transcription factors encoded by the immediate-early gene E74. Therefore, to identify cis-acting elements involved in enhancer activation, deletion analysis of the 175-bp enhancer was performed. Deletion of 20 bp from the 5′ end of the enhancer to generate a 156-bp Mef2 enhancer has a dramatic impact upon transgene expression in adult myoblasts. Lines carrying the wild-type enhancer showed strong β-galactosidase activity in the adult myoblasts, whereas the 156-bp enhancer was barely active in all lines tested. This result indicates that the 5′ portion of the enhancer is essential for its activity in adult myoblasts and identifies this region as a possible target for ecdysone-dependent gene regulation (Lovato, 2005).

The 175-bp enhancer is also active at the embryonic stage in skeletal muscle precursors; therefore, the embryonic activities of the 175-bp and 156-bp enhancers were compared. Interestingly, both enhancers are strongly active in embryos, although there was a slightly reduced activity of the 5′ deleted enhancer compared to the full-length. This observation suggested that the 20 bp deleted from the 175-bp enhancer to generate the 156-bp construct contains specific response elements for activation of Mef2 in adult myoblasts, rather than a general factor necessary for enhancer activation in all contexts (Lovato, 2005).

Within the 20 bp of DNA deleted in the above experiment were sequences that weakly resembled the binding sites for the zinc finger transcription factors of the BR-C. Since the BR-C has been shown to mediate gene activation in response to ecdysone, the BR-C gene products were considered to be excellent candidates for direct regulation of Mef2. Therefore, the accumulation of MEF2 was studied in the imaginal discs of control wild-type siblings and br^npr-3 mutants which lack BR-C function. There was a significant, but not complete, reduction in the level of MEF2 in mutants. However, there were no effects upon Mef2 expression of mutations affecting any of the Z1, Z2, or Z3 BR-C isoforms individually, perhaps due to the documented functional redundancy between many of these products (Lovato, 2005).

These results indicate that although the 5′ enhancer sequence is required for enhancer activity in myoblasts, it is not likely to be a direct target of proteins of the BR-C. This is because removal of the 5′ sequence ablates enhancer activity almost completely, whereas removal of BR-C function has an incomplete effect upon Mef2 expression. In further support of this conclusion, although antibody stains have indicated that isoforms Z1, Z2, and Z4 are detected in myoblasts of larvae and young pupae, it has not been possible to demonstrate binding of any of these three factors to the Mef2 enhancer sequence using in vitro DNA binding assays. It is therefore concluded that while the BR-C influences ecdysone-dependent Mef2 expression, it does so indirectly and not through direct binding to the 175-bp enhancer (Lovato, 2005).

The steroid hormone ecdysone functions broadly in Drosophila to control molting and metamorphosis during the life cycle, and much research has concentrated upon the mechanisms of its action. Ecdysone is known to induce a number of immediate-early genes, which mediate transcriptional responses to hormone levels. However, since ecdysone is a systemic signal, and since many of the immediate-early genes are expressed in multiple tissues, a central challenge in the field has been to understand how widespread activation of immediate-early genes can have specific effects in different target tissues. This study shows that temporal expression of Mef2 in adult myoblasts occurs as a result of the ecdysone pathway. The finding that Mef2 expression in adult myoblasts is low prior to the onset of pupariation is consistent with the known role of Mef2 in muscle differentiation. Since the adult myoblasts do not initiate differentiation until after puparium formation, this might account for why Mef2 expression is absent in young myoblasts. In fact, early expression of Mef2 in adult myoblasts causes premature differentiation of these cells. While the possibility cannot be excluded that this premature differentiation results from the demonstrated ability of high levels of Drosophila Mef2 to inappropriately induce myogenesis, this seems unlikely given the profound myogenesis which was observed in the imaginal discs (Lovato, 2005).

Sustained expression of twist in the adult myoblasts prevents normal muscle differentiation, and Twist levels must decline during the pupal stage for normal adult muscle development to occur. Forced expression of Mef2 in the discs can induce muscle differentiation; however, it is not known if such Mef2 expression attenuates Twist levels, or if the myogenesis observed occurs concurrently with twist expression (Lovato, 2005).

Since the BR-C is expressed in many tissues late during larval development, the myoblast-specific activation of Mef2 must also depend upon an additional factor(s). This factor is likely to be the myoblast marker Twist, which is expressed in the adult myoblasts throughout the larval stage and which has been shown to be an essential activator of Mef2 transcription. Furthermore, the temporal and tissue-specific signals are integrated at the genome level via the 175-bp Mef2 enhancer (Lovato, 2005).

It is therefore proposed that systemic signals such as ecdysone and immediate-early gene activation have specific effects in distinct tissues via interpretation of the systemic signals by a tissue-specific factor: Twist in the case of the adult myoblasts. These findings are analogous to the activation of Fbp1 in fat body cells at the late third larval instar. Fbp1 activation results from the combined effects of ecdysone/EcR complex and the tissue-specific factor dGATAb. The current findings support this model of the specificity of hormone action and extend them to apply to the formation of the adult musculature. The interpretation of systemic hormone signals by cell-autonomous factors is a powerful mechanism to control gene expression. It has recently been shown that gut epithelial cell-specific response to the nuclear hormone receptor PPAR-gamma requires the tissue-specific co-activator Hic-1 (Lovato, 2005).

The data also provide a novel mechanism for regulating Mef2 expression in the animal. This is the first demonstration that Mef2 levels in vivo can be regulated by hormone action, and this may be a broadly relevant paradigm. Indeed, it has been shown that in cultured mammalian myotubes, mef2 mRNAs are induced by treatment with nonapeptide Arg-8 vasopressin. This mechanism is also similar to that demonstrated in vertebrates where Mef2 and thyroid hormone receptor interact with each other and synergistically activate the alpha cardiac myosin heavy-chain gene. These results, and the the results presented in this study, support an important role for hormones in impacting muscle development and underline the utility of the Drosophila system for defining these important mechanisms (Lovato, 2005).

To date, it has not been possible to identify how the effects of ecdysone are directly mediated on the Mef2 gene. There is a requirement for the function of the BR-C, and involvement of this gene complex is attractive given both the expression of BR-C isoforms in the developing adult muscles and a demonstrated role of the Z1 and Z4 isoforms in controlling indirect flight muscle development. Several studies have identified complex cross-regulatory interactions among ecdysone immediate-early genes. This complexity may explain the partial requirement of the BR-C for Mef2 activation and suggests that the direct regulation of Mef2 in adult myoblasts might be complex. Nevertheless, these studies define how the onset of adult myogenesis is orchestrated and also define the Mef2 enhancer as an ecdysone-responsive element. Identification of the factors that interact with the Mef2 enhancer will ultimately provide important insight into the mechanisms of hormone-induced gene regulation and differentiation (Lovato, 2005).

Computational models for neurogenic gene expression in the Drosophila embryo

The early Drosophila embryo is emerging as a premiere model system for the computational analysis of gene regulation in development because most of the genes, and many of the associated regulatory DNAs, that control segmentation and gastrulation are known. The comprehensive elucidation of Drosophila gene networks provides an unprecedented opportunity to apply quantitative models to metazoan enhancers that govern complex patterns of gene expression during development. Models based on the fractional occupancy of defined DNA binding sites have been used to describe the regulation of the lac operon in E. coli and the lysis/lysogeny switch of phage lambda. This study applies similar models to enhancers regulated by the Dorsal gradient in the ventral neurogenic ectoderm (vNE) of the early Drosophila embryo. Quantitative models based on the fractional occupancy of Dorsal, Twist, and Snail binding sites raise the possibility that cooperative interactions among these regulatory proteins mediate subtle differences in the vNE expression patterns. Variations in cooperativity may be attributed to differences in the detailed linkage of Dorsal, Twist, and Snail binding sites in vNE enhancers. It is proposed that binding site occupancy is the key rate-limiting step for establishing localized patterns of gene expression in the early Drosophila embryo (Zinzen, 2006a).

Evolution of the ventral midline in insect embryos; sim regulation by Twist

The ventral midline is a source of signals that pattern the nerve cord of insect embryos. In dipterans such as the fruitfly Drosophila melanogaster (D.mel.) and the mosquito Anopheles gambiae (A.gam.), the midline is narrow and spans just 1–2 cells. However, in the honeybee, Apis mellifera (A.mel.), the ventral midline is broad and encompasses 5–6 cells. slit and other midline-patterning genes display a corresponding expansion in expression. Evidence is presented that this difference is due to divergent cis regulation of the single-minded (sim) gene, which encodes a bHLH-PAS transcription factor essential for midline differentiation. sim is regulated by a combination of Notch signaling and a Twist (Twi) activator gradient in D.mel., but it is activated solely by Twi in A.mel. It is suggested that the Twi-only mode of regulation—and the broad ventral midline—represents the ancestral form of CNS patterning in Holometabolous insects (Zinzen, 2006b).

Dorsoventral (DV) patterning of the D.mel. embryo is initiated by a nuclear gradient of the Dorsal (Dl) transcription factor, which differentially regulates at least 50 target genes in a concentration-dependent manner. Most of these genes encode sequence-specific transcription factors and components of cell signaling pathways that control gastrulation. Genetic analyses, microarray screens, and DNA-binding assays with defined DV enhancers have elucidated a gene network of functional interconnections among 40 Dl target genes. The goal of this study is to use this information to understand the evolution of DV patterning among divergent insects (Zinzen, 2006b).

In D.mel., the Dl gradient leads to localized activation of Notch signaling in single rows of cells straddling the presumptive mesoderm (Bardin, 2006; De Renzis, 2006). This localized Notch signal works together with the bHLH factor Twi to activate sim expression. After invagination of the ventral furrow, the sim-expressing cells converge at the ventral midline, and the bHLH-PAS Sim transcription factor activates target genes required for midline differentiation (Zinzen, 2006b).

The ventral midline is a source of localized signals that help pattern the nerve cord. For example, a transmembrane protease encoded by rhomboid (rho) produces a secreted source of the EGF ligand Spitz. Sim also leads to the expression of slit, which encodes a secreted repellant that binds the Roundabout receptor and inhibits the growth of axonal projections across the midline (Zinzen, 2006b).

Sim target genes are highly conserved in A.mel., and in situ hybridization assays reveal that they are similarly expressed in the ventral midline of the developing honeybee nerve cord. However, their expression is significantly broader in A.mel. than in D.mel., 5–6 cells versus 1–2 cells, respectively. Evidence is presented that this broader midline is due to divergent regulation of sim expression. In A.mel., sim is regulated solely by Twi and does not depend on Notch signaling, whereas Notch is responsible for restricting sim to single rows of cells in the early D.mel. embryo (e.g., Bardin, 2006; De Renzis, 2006). It is proposed that the acquisition of Notch dependence at the sim locus is sufficient to account for restricted expression of sim and the narrow midline in D.mel (Zinzen, 2006b).

The ventral midline in D.mel. embryos encompasses just 1–2 cells that express signaling molecules such as rho and slit. In contrast, orthologous genes are expressed in 5–6 cells in the honeybee embryo. Notably, the initial expression pattern of A.mel. sim is expanded, and the sim-staining pattern remains broad after convergence of the midline following the spreading of neurogenic ectoderm over the mesoderm. In addition to expression in the ventral midline, sim staining is also detected in more lateral clusters of cells exhibiting segmental periodicity in A.mel. embryos; these might be neurons or glial cells migrating away from the midline (Zinzen, 2006b).

Previous studies suggest that sim functions as a 'master control gene' to direct differentiation of the ventral midline in D.mel. To determine whether the expanded sim pattern in honeybees can account for the broadening of the midline, whether ectopic sim expression is sufficient to induce transcription of target genes such as slit and rho. The D.mel. sim-coding sequence was placed under the control of the eve stripe 2 enhancer (eve.2) and expressed in transgenic embryos. There is transient sim expression in the stripe 2 domain of early (stages 5–7) embryos in addition to the endogenous pattern (mesectoderm) in the presumptive ventral midline (Zinzen, 2006b).

The initial sim expression pattern is established by a distal 5′ enhancer that contains linked Dl-, Twi-, and Suppressor of Hairless [Su(H)]-binding sites. Expression is maintained by a separate autoregulatory enhancer containing Sim/Tango-binding sites; Tango is a ubiquitous bHLH-PAS transcription factor that forms heterodimers with Sim. Though the eve stripe 2 enhancer mediates transient activation, autoregulation maintains expression of the endogenous sim gene in the ventral neurogenic ectoderm of advanced-stage embryos, but not in the mesoderm or dorsal ectoderm (Zinzen, 2006b).

Ectopic sim expression leads to the induction of various target genes, including rho, slit, sog, and the transcription factor otd. These results provide evidence that ectopic sim expression is sufficient to expand the ventral midline in D.mel. In principle, the altered midline seen in the honeybee embryo could be explained by a change in sim regulation. The distal 5′ enhancer that establishes sim expression is the most likely site of change, since the autoregulatory enhancer merely maintains expression within the limits of the established pattern (Zinzen, 2006b).

To determine the basis for the distinct sim expression patterns in flies and honeybees, it was necessary to isolate the early sim enhancer from A.mel. However, the identification of homologous enhancers is complicated by the rapid turnover of noncoding DNA sequences in insect genomes. For example, the 5′ flanking regions of the sim loci in D.mel. and A.gam. lack simple sequence homology, even though they belong to the same order (Diptera). Nonetheless, it was possible to identify the early sim enhancer in A.gam. based on the clustering of Dl-, Twi-, and Su(H)-binding sites. The D.mel. and A.gam. enhancers are located in similar positions relative to the sim transcription unit (Zinzen, 2006b).

A.mel. is a member of the order Hymenoptera and is highly divergent from D.mel. Computational methods used for the in silico identification of the A.gam. sim enhancer were further developed to ensure the accurate identification of the sim enhancer in A.mel. The current method (ClusterDraw2) employs position-weighted matrices (PWMs) to identify binding motif clusters (Zinzen, 2006b).

The efficacy of the method was tested by surveying ~50 kb genomic intervals encompassing the sim loci of D.mel. and A.gam.. PWMs of Dl, Twi, Snail, and Su(H) were used in various combinations and individually. The best binding site clusters coincide exactly with the known sim enhancers (Zinzen, 2006b).

ClusterDraw2 was used to survey a ~50 kb genomic DNA interval encompassing the sim locus of A.mel.. The best prediction occurs in the 5′ flanking region of the gene, similar to the locations of the fly and mosquito enhancers. However, while the D.mel. and A.gam. sim enhancers contain several optimal Su(H)-binding sites, the A.mel. cluster lacks such sites, but contains several high-scoring Twi sites. This is consistent with the possibility that A.mel. sim is regulated by Twi alone, rather than by the combination of Twi+Notch (Zinzen, 2006b).

A 2.2 kb genomic DNA fragment encompassing the predicted A.mel. sim enhancer directs lateral stripes of lacZ expression in transgenic D.mel. embryos. A similar pattern was obtained with a 471 bp fragment containing the predicted Twi-binding sites. This pattern encompasses 3–4 cells on either side of the presumptive mesoderm, similar to the expression of the endogenous A.mel. sim gene, but distinct from the single-row sim patterns in D.mel. and A.gam. (Zinzen, 2006b).

The fly, honeybee, and mosquito sim enhancers were crossed into various genetic backgrounds to determine the basis for their distinct expression patterns. The D.mel. m5/8 enhancer was also examined. It is located within the Enhancer of split (E(spl)) complex, where it controls the expression of the m5 and m8 genes within the mesectoderm. The m5/8 enhancer directs lacZ expression in a pattern that is virtually identical to that produced by the D.mel. sim enhance (Zinzen, 2006b).

Transgenic D.mel. embryos carrying an eve.2::N^ICD fusion gene exhibit ectopic Notch signaling in the eve stripe 2 domain. The m5/8-lacZ transgene is strongly induced in the neurogenic ectoderm and dorsal ectoderm, but not in the mesoderm, where the Sna repressor is present. The D.mel. sim-lacZ transgene displays only modest ectopic induction by the eve.2::N^ICD transgene; this induction appears as a 'pyramid' limited to ventral regions of the neurogenic ectoderm. This pyramid coincides with the intersection of ectopic Notch signaling and the endogenous Twi gradient. The different patterns ('pyramid' versus 'column') seen for the sim and m5/8 enhancers appear to reflect activation by Notch+Twi or regulation by Notch alone, respectively. The m5/8 enhancer contains an SPS (Su(H) Paired Site) motif, and it has been suggested that the endogenous m8 gene is activated solely by Notch signaling (Zinzen, 2006b).

The A.mel. sim enhancer is not activated by the eve.2::N^ICD transgene, consistent with the absence of Su(H) sites in this enhancer. To determine whether it is activated by Twi, the lacZ fusion gene was crossed into embryos carrying an hsp83::twi-bcd-3′UTR transgene that produces high levels of Twi transcripts at the anterior pole. The resulting ectopic anteroposterior Twi protein gradient induces intense expression of the lacZ reporter gene directed by the A.mel. sim enhancer. In contrast, neither the D.mel. sim enhancer nor the m5/8 enhancer is induced by this ectopic gradient. Finally, the D.mel. sim enhancer is inactive in mutant embryos derived from germline clones lacking Su(H) activity, whereas the honeybee sim enhancer is fully active. Thus, unlike the D.mel. sim enhancer, the A.mel. enhancer does not rely on Notch signaling (Zinzen, 2006b).

The preceding analysis suggests that the D.mel. sim enhancer is activated by Twi and Notch signaling, whereas the A.mel. sim enhancer is activated solely by Twi. These distinct modes of regulation are reflected by the composition of binding sites in the different enhancers. The A.mel. enhancer contains several optimal Twi sites, but it lacks unambiguous Su(H) sites. In contrast, the D.mel. enhancer contains several optimal Su(H) sites, but just one optimal Twi site. Both enhancers contain binding sites for the Sna repressor, which inhibits expression in the mesoderm (Zinzen, 2006b).

sim regulation was examined in the mosquito, A.gam., to determine whether the midline of ancestral dipterans might have been regulated solely by Notch signaling, as seen for the fly m5/8 enhancer. The A.gam. genome contains a clear ortholog of the sim gene, expressed in a single row of cells in the mesectoderm, similar to the pattern seen in D.mel. The A.gam. sim enhancer directs sporadic expression within the mesectoderm of transgenic D.mel. embryos, but it is strongly induced by the eve.2::N^ICD transgene. This response is similar to that obtained with the D.mel. m5/8 enhancer, but it is distinct from the 'pyramid' pattern seen for the D.mel. sim enhancer (Zinzen, 2006b).

To determine whether the sim loci of other drosophilids are regulated by Twi+Notch, as seen in D.mel., or Notch alone, sim enhancers from D. pseudoobscura (D.pse.) and D. virilis (D.vir.) were tested in transgenic eve.2::N^ICD D.mel. embryos. Surprisingly, these enhancers behave like the A.gam. sim enhancer: they are expressed throughout the neurogenic ectoderm and dorsal ectoderm (“column”) in response to Notch signaling, rather than the “pyramid” pattern indicative of Notch+Twi regulation. These observations suggest that the evolution of sim regulation is highly dynamic, although there is no obvious difference in the number or quality of Su(H) and Twi sites in the different drosophilid enhancers. Perhaps a subtle shift in the organization of binding sites distinguishes regulation by Notch alone versus Notch+Twi (Zinzen, 2006b).

collier transcription in a single Drosophila muscle lineage: nautilus and twist contribute to the combinatorial control of muscle identity

Specification of muscle identity in Drosophila is a multistep process: early positional information defines competence groups termed promuscular clusters, from which muscle progenitors are selected, followed by asymmetric division of progenitors into muscle founder cells (FCs). Each FC seeds the formation of an individual muscle with morphological and functional properties that have been proposed to reflect the combination of transcription factors expressed by its founder. However, it is still unclear how early patterning and muscle-specific differentiation are linked. This question was addressed using Collier (Col; also known as Knot) expression as both a determinant and read-out of DA3 muscle identity. Characterization of the col upstream region driving DA3 muscle specific expression revealed the existence of three separate phases of cis-regulation, correlating with conserved binding sites for different mesodermal transcription factors. Examination of col transcription in col and nautilus (nau) loss-of-function and gain-of-function conditions showed that both factors are required for col activation in the 'naive' myoblasts that fuse with the DA3 FC, thereby ensuring that all DA3 myofibre nuclei express the same identity programme. Together, these results indicate that separate sets of cis-regulatory elements control the expression of identity factors in muscle progenitors and myofibre nuclei and directly support the concept of combinatorial control of muscle identity (Dubois, 2007).

col belongs to the class of Drosophila regulatory genes with numerous introns, large amounts of flanking sequence and multiple expression sites. During embryogenesis, col is expressed in the MD2/PS0 head region, the somatic DA3 muscle, precursor cells of the lymph gland, a small set of multidendritic (md) neurons of the peripheral nervous system and specific neurons of the central nervous system (CNS). A lacZ reporter transgene (P{5col::lacZ}, abbreviated P5cl, contains 5 kb of col upstream DNA, which faithfully reproduced col transcription both in the MD2/PS0 and the DA3 muscle, starting at the progenitor stage and not in promuscular cluster(s). To identify the missing cis-regulatory information, a longer construct was tested containing the entire 9 kb region separating col from CG10200, the next predicted upstream gene. In addition to the head and DA3 muscle, P9cl expression reproduced col expression in md neurons and a subset of neurons in the CNS. A DNA fragment located further upstream, between CG10200 and the next predicted gene CG10202, was independently shown to drive col expression in the anteroposterior organiser of the wing imaginal disc (Hersh, 2005). However, neither this construct nor P9cl reproduced Col expression in promuscular clusters. The col transcription unit is immediately flanked at its 3' end by another gene, BEAF32, making rather unlikely the presence of cis-regulatory elements within this region. However, it contains ten different introns, of total length around 30 kb, the cis-regulatory content of which remains to be assessed (Dubois, 2007).

To delineate more precisely the CRM driving col expression in the DA3 muscle, a series of constructs was tested containing 2.6, 2.3, 1.6 and 0.9 kb of DNA upstream of the col transcription start site, respectively. P2.6cl retained the information necessary for col expression in MD2/PS0 and the DA3 progenitor and muscle, although it was noted that P2.6cl expression in muscle progenitors was less robust than P9cl. P2.3cl was also activated in MD2/PS0 at stage 6 and the DA3 muscle. However, unlike P9cl or P2.6cl, P2.3cl was not activated in the DA3/DO5 progenitor but only at the FC stage; ectopic lacZ expression was observed in clusters of neuroectodermal cells at embryonic stage 11). This difference indicated that cis-regulatory elements required for col expression in the DA3/DO5 progenitor reside between positions -2.6 and -2.3 and act separately from those required for expression in the DA3 FC and muscle. P1.6cl was active only in MD2/PS0, whereas no expression at all could be detected with P0.9cl. Together, expression data from this series of reporter constructs allowed the mapping of the CRM required for col-specific expression in the DA3/DO5 muscle progenitor and DA3 FC/myofibre to a DNA fragment between positions -2.6 and -1.6 upstream of the col transcription start (Dubois, 2007).

Advantage was taken of the recently available genome sequences of several Drosophila species to search for conserved motifs in the col upstream DNA, as it has often proven to be effective to identify functionally important cis-regulatory elements. Among these species, D. virilis (D. vir) is the most distant from D. melanogaster (D. mel). It was first verified that Col expression in D. vir was similar to that in D. mel embryos and could infer from this that the regulatory information controlling col transcription in the DA3 muscle lineage has been conserved. Sequence comparison of 9 kb of the col upstream region between D. mel, D. vir and four other Drosophila species, D. yakuba, D. ananassae, D. pseudoobscura and D. mojavensis revealed numerous stretches of high sequence conservation, of sizes up to 100 bp. Ten conserved motifs of size >20 bp, numbered 1 to 10 from 5' to 3', were found in the same order and at the same relative position between position -2.6 and the start of transcription in all six Drosophila species. To test the relevance of this conservation, lacZ reporter constructs were created containing either D. vir or D. mel DNA (Dubois, 2007).

P.3.4cl^vir corresponds to D. mel P2.6cl, whereas P3.4-1.3cl^vir and P2.6-0.9cl are truncated versions covering motifs 1 to 10. All four reporter genes showed expression in the DA3 muscle, starting at the progenitor stage, confirming the evolutionary conservation of a DA3-muscle-specific CRM. A Gal4 driver line containing only the -2.6 to -1.6 region (P2.6-1.6cG), harbouring only motifs 1 to 7, was also specifically expressed in the DA3 muscle. This confirmed that the DA3 muscle CRM is located between positions -2.6 and - 1.6. It was noticed, however, that expression of P2.6-1.6cG was weaker and more sporadic than P2.6-0.9cl, suggesting the existence of cis-regulatory element(s) between positions -1.6 and -0.9 contributing to robust DA3 muscle expression. The conserved motifs 1 to 10 were searched for consensus binding sites of known TFs that could account for col activation in the DA3 muscle. This identified a binding site for the mesodermal basic helix-loop-helix (bHLH) protein Twi (within motif 2), correlating well with the position of the muscle progenitor cis-element and a potential EBF/Col-binding site within motif 7. Further visual inspection of the sequence alignments identified other conserved TF-binding sites, including one Mef2-binding site within the -1.6 to -0.9 fragment and one consensus binding site for Nau (Huang, 1996; Kophengnavong, 2000). In contrast, the position of the Mef2 site correlated well with the requirement of the -1.6 to -0.9 fragment for robust DA3 muscle expression. The presence of a Nau-binding site was particularly intriguing since Nau is required for DA3 muscle formation. Potential binding sites for other TFs could be found in the DA3 CRM, but the annotation to the conserved sites. The relative paucity of known TF-binding sites in the conserved sequence motifs found in the DA3 muscle CRM leaves largely open the question of the roles of these motifs in col regulation (Dubois, 2007).

Functional dissection of the DA3 muscle CRM present in the col upstream region showed that col expression in the DA3 FC can be separated from its expression in the DA3/D05 progenitor and the promuscular cluster. It thus revealed the existence of three steps in the transcriptional control of muscle identity. That col expression in the DA3/D05 progenitor could be uncoupled from that in promuscular clusters was in apparent contradiction with the previous conclusion from pioneering studies on Eve expression in dorsal muscle progenitors that this expression issued from Eve activation in promuscular clusters. Restriction of Eve expression to progenitors was considered a secondary step, mediated by N-signalling during progenitor selection by lateral inhibition. To reconcile these data and this model, it is proposed that the muscle DA3 CRM is active only in the DA3/D05 progenitor because it lacks some positively acting cis-elements necessary to counteract N-mediated repression of col transcription. It has been shown that col transcription is repressed by N during the progenitor selection process. It is also noted that a Twi-binding site is present in the 'progenitor' subdomain of the DA3 CRM. The functional importance of this site is supported by its in vivo occupancy in 4- to 6-hour-old embryos when selection of the DA3/DO5 progenitor takes place (Sandmann, 2007). Together, Twi in vivo binding and the col/P2.6cl/P2.3cl expression data suggest that Twi activity contributes to col expression in the DA3/DO5 progenitor but may not be sufficient to override N repression of col transcription before progenitor selection. Additional binding sites for Twi present in the col upstream region, between positions -8.7 and -8.3, are also bound by Twi in vivo (Sandmann, 2007) and probably contribute to the robustness of P9cl expression in progenitor cells, but the question of which cis-regulatory elements mediate col activation in promuscular clusters remains open. From Eve expression studies, a computational framework has been developed to identify other FC-specific genes (Estrada, 2006; Philippakis, 2006). This framework, named Codefinder, integrates transcriptome data and clustering of combinations of binding sites for five different TFs (Pnt, dTCF, Mad, Twi and Tin). col/kn was selected by Codefinder owing to the presence of five clusters of binding sites, four of which are located within introns (Philippakis, 2006). It remains to be determined which of these could be responsible for col activation in promuscular clusters, but it is interesting to note that another in vivo Twi-binding site in 4-6-hour-old embryos correlates with the 3'-most cluster (Sandmann, 2007). In addition to Twi, conserved binding sites for Nau and Mef2 are found within the DA3 CRM. The Mef2 binding site is located in a region required for robust DA3-muscle expression of a reporter gene. A direct control of col transcription by Mef2 during the muscle fusion process is further supported by the recent finding (Sandmann, 2006) that Mef2 binds in vivo to the col upstream region between 6 and 8 hours of embryonic development (Dubois, 2007).

Detailed analysis of col auto-activation revealed a reiterative, two-step process: import of pre-existing Col protein in the fusion competent myoblast nuclei that incorporate into the growing DA3 myofibre precedes activation of col transcription. This process ensures that all incorporated FCM nuclei acquire the same identity. Nau is required for maintaining col transcription in the DA3 muscle precursor and this control is probably direct. The presence of a putative EBF-binding site in the DA3 muscle CRM also correlates with the Col requirement for maintaining its own transcription beyond the FC stage. Thus, despite the failure to detect strong Col binding to this site in vitro, it appears to be essential for col auto-regulation in vivo. This suggests that in vivo binding is potentiated by one or more specific co-factor(s) present in the DA3 muscle. One co-factor is probably Nau, as the ability of Col to activate its own transcription in newly recruited fusion competent myoblasts is dependent upon Nau activity. Nau is not sufficient, however, as many muscles containing both Nau and Col proteins do not activate col transcription. Interestingly, mouse EBF (also known as Ebf1 and Olf1 - Mouse Genome Informatics) and E2A (Tcfe2a - Mouse Genome Informatics), a bHLH protein of the same subgroup as MyoD, have been shown to act on the same target promoter and synergistically upregulate transcription of B-lymphocyte-specific genes, although no direct physical interaction between EBF and E2A could be found in vitro. This suggested that functional interaction of EBF and E2A, similar to Col and Nau, requires yet another factor. Taking into account the restricted pattern of ectopic col activation in hs-col conditions, it is hypothesised that Vg could be another component of the DA3 combinatorial identity. However, we found that Vg is not required for DA3 muscle specification, leaving open the question of which factor may bridge Col and Nau functions (Dubois, 2007).

Unlike col or P2.6cl, P2.3cl is expressed in the DA3 FC and muscle precursor but not the DA3/DO5 progenitor, showing that col transcription in the progenitor and muscle precursor is under separate control. These two phases of col regulation are intimately linked, however, as Col is required for activating its own transcription in the nuclei of FCM recruited by the DA3 FC. This regulatory cascade may explain how pre-patterning of the somatic mesoderm and muscle identity are transcriptionally linked in the Drosophila embryo. As discussed above, the ability of Col to auto-regulate depends upon the presence of Nau, another muscle identity TF. Col and Nau act as obligatory co-factors fo maintenance/activation of Col expression in all nuclei of the DA3 muscle, thus bringing to light a clear case of combinatorial coding of muscle identity (Dubois, 2007).

Specificity of Notch pathway activation: twist controls the transcriptional output in adult muscle progenitors

Cell-cell signalling mediated by Notch regulates many different developmental and physiological processes and is involved in a variety of human diseases. Activation of Notch impinges directly on gene expression through the Suppressor of Hairless [Su(H)] DNA-binding protein. A major question that remains to be elucidated is how the same Notch signalling pathway can result in different transcriptional responses depending on the cellular context and environment. This study investigated the factors required to confer this specific response in Drosophila adult myogenic progenitor-related cells. This analysis identifies Twist (Twi) as a crucial co-operating factor. Enhancers from several direct Notch targets require a combination of Twi and Notch activities for expression in vivo; neither alone is sufficient. Twi is bound at target enhancers prior to Notch activation and enhances Su(H) binding to these regulatory regions. To determine the breadth of the combinatorial regulation Twi occupancy was mapped on a genome-wide level in DmD8 myogenic progenitor-related cells by chromatin immunoprecipitation. Comparing the sites bound by Su(H) and by Twi in these cells revealed a strong association, identifying a large spectrum of co-regulated genes. It is concluded that Twi is an essential Notch co-regulator in myogenic progenitor cells and has the potential to confer specificity on Notch signalling at over 170 genes, showing that a single factor can have a profound effect on the output of the pathway (Bernard, 2010).

To determine whether the Twi co-regulation could be extrapolated to a broad spectrum of Notch targets in muscle progenitors, whether there was a significant association between Su(H) and Twi binding in the muscle progenitor-related DmD8 cells was assessed. Comparison of the binding regions genome-wide revealed a strong association of Twi and Su(H) among these targets: 71% of Su(H) peaks directly overlapped with Twi peaks. This association was highly significant based on random models that constrained the positions of peaks across the genome to take account of the non-random distribution of transcription factor binding sites and in comparison to several other ChIP data sets. Expression of putative Notch-Twi targets in myogenic precursors was dependent on Twi, as predicted by the association. Together, these data indicate that one transcription factor, Twi, has the potential to co-ordinate the expression of a broad cross-section of Notch targets in muscle progenitors (84% of previously assigned Notch targets are associated with a Twi peak) and thus to confer a specific context on the Notch response (Bernard, 2010).

Further evidence in support of the instructive role of Twi comes from its ability to confer Notch responsiveness on some muscle precursor targets when expressed in a heterologous cell line. Twi itself was found to occupy sites on the target enhancers prior to Notch activation, and in the heterologous cells it was accompanied by increased Su(H) binding after Notch activation. This suggests that Twi binding precedes Su(H) recruitment. However, the co-regulation does not appear to require the Twi partner Da [the E47 (TCF3) homologue], which was previously reported to contact Notch. Furthermore, there does not appear to be any specific organisation or spacing of Twi and Su(H) motifs among the co-regulated targets, in contrast to the conserved motif, consisting of paired Su(H) sites closely linked to an A-class bHLH binding site, that is thought to underlie Notch-proneural bHLH synergy. Nevertheless, in most of the co-regulated targets, the Su(H) and Twi mid-peaks are separated by less than 500 bp, suggesting that the interaction operates over a limited range. The Twi-Su(H) co-regulation appears, therefore, more in keeping with models in which binding sites for transcription factors are flexibly disposed and act independently with targets in the basal transcriptional machinery (the so-called 'billboard' model). However, mutation of a single Twi binding motif in the aos enhancer is sufficient to compromise activity, despite the fact that there are several matches to the Twi consensus site, suggesting that only a subset of the possible binding motifs are crucial. In addition, as the characterised Notch-Twi-dependent enhancers do not have identical patterns of expression, it is likely that their activity is further constrained by others factors. This is most evident for E(spl)m6, which is only expressed in a small patch of the AMPs but nevertheless responds very robustly to Twi and NICD. What are the likely characteristics conferred on cells by the Twi-Notch combination (Bernard, 2010)?

The AMPs have the capacity for self-renewal and are not committed to a particular muscle lineage, characteristics similar to those of mammalian muscle satellite cells. The genes regulated by the combination of Twi and Notch might therefore be important for maintaining these cells as progenitors with myogenic potential. Normally, Twi expression declines as the muscles differentiate. Interfering with this regulation by persistent expression of Twi or Notch inhibits the development of mature fibres. Conversely, ablating Notch results in premature differentiation. The genes regulated by the combination of Twi and Notch include those with proven roles in myogenic regulation that are relevant to the maintenance of muscle progenitors. These include twi itself, Him [an inhibitor of Mef2 (Liotta, 2007)] and zfh1 [a repressor of myogenesis. As Notch signalling and Twi homologues also inhibit vertebrate myogenic differentiation, and overexpression of Twi in terminally differentiated myotubes can induce reversal of cell differentiation, it will be interesting to test whether homologues of the identified co-regulated targets of Notch and Twi are similarly regulated (Bernard, 2010).

The Notch-Twi combination might also be required to confer properties on the adult myogenic precursors, such as differential adhesion, migration and proliferation. Besides the genes with proven roles in myogenic regulation, many of the Notch-Twi co-regulated genes are implicated in morphogenesis. These include the Ig-domain proteins Roughest, Kirre and Dscam, the netrin receptor Unc-5 and leucine repeat protein Capricious. Notch and Twi have both been found to contribute to the regulation of the epithelial-mesenchymal transition (EMT), and Twi is proposed to affect malignant progression by inducing EMT and suppressing the senescence response. Therefore, it is possible that the co-regulated genes might also confer specialised behaviours that are required in the adult precursors and in cells undergoing EMT (Bernard, 2010).

In conclusion, this study found that Notch and Twi potentially co-regulate a broad spectrum of genes required for the maintenance of muscle progenitors. This suggests that a single co-regulatory relationship can account for a significant component (>170 genes) of the Notch output in one cell type (Bernard, 2010).

HOT regions function as patterned developmental enhancers and have a distinct cis-regulatory signature

HOT (highly occupied target) regions bound by many transcription factors are considered to be one of the most intriguing findings of the recent modENCODE reports, yet their functions have remained unclear. This study tested 108 Drosophila melanogaster HOT regions in transgenic embryos with site-specifically integrated transcriptional reporters. In contrast to prior expectations, 102 (94%) were found to be active enhancers during embryogenesis and to display diverse spatial and temporal patterns, reminiscent of expression patterns for important developmental genes. Remarkably, HOT regions strongly activate nearby genes and are required for endogenous gene expression, as was shown using bacterial artificial chromosome (BAC) transgenesis. HOT enhancers have a distinct cis-regulatory signature with enriched sequence motifs for the global activators Vielfaltig, also known as Zelda, and Trithorax-like, also known as GAGA. This signature allows the prediction of HOT versus control regions from the DNA sequence alone (Kvon, 2012).

Taken together, these data show that Drosophila HOT regions function as cell type-specific transcriptional enhancers to up-regulate nearby genes during early embryo development. In contrast to prior expectations, HOT enhancers display diverse spatial and temporal activity patterns, which are reminiscent of expression patterns of important developmental genes. It was further found that the activity of many HOT enhancers appears to be unrelated to the expression of the bound transcriptional activators, suggesting that neutral TF binding to HOT regions is frequent. Interestingly, for Twi, Kr, and five additional TFs, it was found that HOT enhancers with functional footprints of the TFs are significantly enriched in the TFs' motifs compared with HOT enhancers to which the TFs seem to bind neutrally (e.g., 2.2-fold for Twi). This supports previous suggestions that the recruitment of TFs to HOT regions might be independent of the TFs' motifs and mediated by protein-protein interactions or nonspecific DNA bindin. This seems to be particularly true for (HOT) regions to which the TFs bind neutrally without impact on the regions' transcriptional enhancer activity (Kvon, 2012).

By uncovering a distinct cis-regulatory signature that is characteristic and predictive of HOT regions, computational analysis establishes a link between HOT regions, early embryonic enhancers (EEEs), and maternal TFs that are ubiquitously present in the early Drosophila embryo. Specifically, the results suggest that ZLD might be more generally important for the establishment of regulatory elements in the early embryo, while GAGA appears to be a distinguishing feature of HOT regions. This is supported by an analysis of genome-wide data on ZLD and GAGA binding in early Drosophila embryos: While 71.4% of HOT regions and 75.0% of EEEs are bound by ZLD (compared with 42.2% and 13.0% of control WARM and COLD regions), GAGA binds to 53.4% of HOT regions but only 20.0% of EEEs (compared with 28.3% and 7.8% for WARM and COLD regions). Even when considering only regions that are functioning as transcriptional enhancers in the early embryo (all EEEs from CAD and this study combined), GAGA binds to significantly more HOTenhancers than to enhancers that are not HOT. An instructive role for ZLD in defining chromatin that is open and accessible to other factors is further supported by its unusual property to bind to the majority (64%) of all occurrences of its sequence motif in the Drosophila genome. ZLD might thus be a prerequisite for both HOTregions and EEEs more generally. Similarly, a role for GAGA in nucleating or promoting the formation of TF complexes is consistent with its ability to self-oligomerize via its BTB/POZ domain and also form heteromeric complexes with the TF Tramtrack and potentially other BTB/POZ domain- containing TFs (e.g., Abrupt, Bric-a-brac, Broad complex, and others). GAGA, with its ability to recruit other TFs by protein-protein interactions, might contribute to HOT regions independent of the specific cellular or developmental context. Interestingly, C. elegans HOT regions are also strongly enriched in the GAGA motifs, and the motif is the most important sequence feature when classifying C. elegans HOT versus control regions. GAGA-like factors or their putative homologs or functional analogs across species might be a conserved feature of metazoan HOT regions (Kvon, 2012).

A conserved role for Snail as a potentiator of active transcription

The transcription factors of the Snail family are key regulators of epithelial-mesenchymal transitions, cell morphogenesis, and tumor metastasis. Since its discovery in Drosophila approximately 25 years ago, Snail has been extensively studied for its role as a transcriptional repressor. This study demonstrate that Drosophila Snail can positively modulate transcriptional activation. By combining information on in vivo occupancy with expression profiling of hand-selected, staged snail mutant embryos, 106 genes were identified that are potentially directly regulated by Snail during mesoderm development. In addition to the expected Snail-repressed genes, almost 50% of Snail targets showed an unanticipated activation. The majority of 'Snail-activated' genes have enhancer elements cobound by Twist and are expressed in the mesoderm at the stages of Snail occupancy. Snail can potentiate Twist-mediated enhancer activation in vitro and is essential for enhancer activity in vivo. Using a machine learning approach, it was shown that differentially enriched motifs are sufficient to predict Snail's regulatory response. In silico mutagenesis revealed a likely causative motif, which this study demonstrates to be essential for enhancer activation. Taken together, these data indicate that Snail can potentiate enhancer activation by collaborating with different activators, providing a new mechanism by which Snail regulates development (Rembold, 2014).

The function of Snail in distinguishing mesodermal from ectodermal fates has been traditionally seen as a repressor of the ectodermal differentiation program. This study demonstrates that Drosophila Snail can also activate part of the program specific for the mesoderm. The role of Snail in gastrulation is thus dual and involves a balance of repression and activation (Rembold, 2014).

One of the functions of Snail is to enable the formation of the ventral furrow together with Twist. Whereas the target genes of Twist that mediate furrow formation are known, it is completely unclear which genes act downstream from Snail. Only one such gene has been identified so far. The gene bearded, which is repressed in the mesoderm by Snail, is partly responsible for allowing adherens junctions in the mesoderm to be relocalized, but this is not sufficient for furrow formation. Therefore, there must be other genes that fulfill essential functions in gastrulation downstream from Snail. The hypomorphic sna^V2 mutant might give some hints of what genes these might be, since it is still able to make a furrow, although many Snail target genes are misregulated. Stepwise reduction of only the repressive activity of Snail by mutation of one or two corepressor-binding sites results in a stepwise increase in the strength of the gastrulation phenotype. Thus, the repressive activity of Snail is certainly required. However, the 60% of Snail-dependent genes that are not or are only weakly affected in the sna^V2 mutant (i.e., those most likely to be responsible for mediating furrow formation) do not fall into a uniform category; they contain both up-regulated and down-regulated genes. This might be an indication that misregulation of a larger set of both repressed and activated genes leads to the failure in furrow formation. This is also consistent with the fact that simply reducing the level of Snail by half leads to a delay in gastrulation (Rembold, 2014).

In summary, this study revealed a direct activator role for Drosophila Snail, a function that is seemingly conserved from flies to humans and places the Snail family of proteins in the category of dually acting TFs (Rembold, 2014).

Protein Interactions

Synergistic interactions between the maternal regulatory factor Dorsal (DL) and basic helix-loop-helix (bHLH) activators, are essential for initiating differentiation of the mesoderm and neuroectoderm in the early Drosophila embryo. DL-bHLH interactions mediating gene expression in the neuroectoderm and mesoderm are fundamentally distinct. Sharp on/off patterns of gene expression in the presumptive mesoderm do not require linkage of Dorsal and other bHLH binding sites. Analysis of minimal and synthetic promoter elements suggests that DL and bHLH activators, such as Twist, might interact with different rate-limiting components of the transcription complex. (Szymanski, 1995).

The establishment of mesoderm and neuroectoderm in the early Drosophila embryo relies on interactions between the Dorsal morphogen and basic-helix-loop-helix (bHLH) activators. Dorsal and the bHLH activator Twist synergistically activate transcription in cell culture and in vitro from a promoter containing binding sites for both factors. Somewhat surprisingly, a region of Twist outside the conserved bHLH domain is required for the synergy. The two N-terminal Gln-rich regions of Twist appear to mediate synergistic activation by Dorsal and Twist and are also required for binding to Dorsal in vitro. In Dorsal, the rel homology domain appears to be sufficient for synergy. The interaction between Dorsal and Twist does not appear to be of sufficient strength to yield cooperative binding to DNA. It is suggested that the interaction betweein Dorsal and Twist induces a conformational change in one of the factors that enables it to efficiently activate transcription (Shirokawa, 1997).

The basic helix-loop-helix transcription factor Twist regulates a series of distinct cell fate decisions within the Drosophila mesodermal lineage. These twist functions are reflected in its dynamic pattern of expression, which is characterized by initial uniform expression during mesoderm induction, followed by modulated expression at high and low levels in each mesodermal segment, and finally restricted expression in adult muscle progenitors. Two distinct partner-dependent functions for Twist were found that are crucial for cell fate choice. Twist can form homodimers and heterodimers in vitro with the Drosophila E protein homolog, Daughterless. Using tethered dimers to assess directly the function of these two particular dimers in vivo, it has been shown that Twist homodimers specify mesoderm and the subsequent allocation of mesodermal cells to the somatic muscle fate. Misexpression of Twist-tethered homodimers in the ectoderm or mesoderm leads to ectopic somatic muscle formation overriding other developmental cell fates. In addition, expression of tethered Twist homodimers in embryos null for twist can rescue mesoderm induction as well as somatic muscle development. Loss of function analyses, misexpression and dosage experiments, and biochemical studies indicate that heterodimers of Twist and Daughterless repress genes required for somatic myogenesis. It is proposed that these two opposing roles explain how modulated Twist levels promote the allocation of cells to the somatic muscle fate during the subdivision of the mesoderm. Moreover, this work provides a paradigm for understanding how the same protein controls a sequence of events within a single lineage (Castanon, 2001).

At stage 10, in response to transcriptional regulators such as Sloppy paired and Even skipped, as well as signals from the overlying ectoderm such as Wingless, the uniform expression of Twist modulates into regions of high and low expression within each segment. Da is expressed uniformly in the mesoderm at this time. The region that maintains high Twist levels subsequently gives rise to somatic muscles whereas the region that has lower Twist levels gives rise to tissues such as visceral muscle, fat body, gonadal mesoderm and some glia cells. The heart is derived from the region that initially expresses high levels of Twist; however these cells lose Twist expression, an event necessary for the execution of heart fate. Expressing high Twist levels in cells destined to become visceral muscle, for example, blocks visceral muscle differentiation and promotes somatic muscle. Reduction of Twist levels in cells normally expressing high Twist levels blocks somatic myogenesis (Castanon, 2001 and references therein).

Several possible mechanisms are provided to explain these observations and illustrate the in vivo roles for the two opposing activities of Twist homodimers and Twist/Da heterodimers. Regions that normally express lower Twist levels do not form somatic muscles owing to higher concentrations of Twist/Da heterodimers as compared to Twist homodimers. These heterodimers repress transcription of pro-muscle genes, such as l’sc as well as founder cell genes such as Kr, thereby prohibiting somatic muscle development. Other differentiation programs for visceral muscle or fat body development can proceed unaffected. No evidence is found that Twist/Da heterodimers promote visceral mesoderm or fat body fate through the direct activation of targets such as Fas III. Regions that normally express higher Twist levels do form somatic muscle owing to higher concentrations of Twist homodimers as compared to Twist/Da heterodimers. Dimer competition, then, restricts the developmental potential of mesodermal cells, by not allowing Twist homodimers to convert all mesodermal cells into somatic muscle (Castanon, 2001).

These conclusions are consistent with the observations that increasing Twist/Da levels, either by overexpression of Da or the tethered Twist-Da heterodimer, repress the earliest steps in somatic myogenesis. These are the same steps that are activated by Twist homodimers. For example, L’sc expression, which marks clusters of equipotential cells that segregate the muscle founder cells, is drastically reduced or absent upon an increase of Twist/Da heterodimers. This indicates an early failure in the somatic muscle program. Likewise failure in subsequent steps is seen; for example, few founder cells as well as few identifiable muscles are detected. These failures in muscle development are interpreted as an outcome of the initial block in the differentiation pathway. The possibility that overexpression of Da or of Twist-Da could directly repress these subsequent steps is not eliminated. Gal4 lines that drive expression at later stages of muscle development or in particular subsets of muscle cells (i.e., the S59-expressing founder cells) could provide insight into this alternative (Castanon, 2001).

The MADF-BESS domain factor Dip3 potentiates synergistic activation by Dorsal and Twist

The transcription factors Dorsal and Twist regulate dorsoventral axis formation during Drosophila embryogenesis. Dorsal and Twist bind to closely linked DNA elements in a number of promoters and synergistically activate transcription. A novel protein named Dorsal-interacting protein 3 (Dip3) has been identified that may play a role in this synergy. Dip3 functions as a coactivator to stimulate synergistic activation by Dorsal and Twist, but does not stimulate simple activation of promoters containing only Dorsal or only Twist binding sites. In addition, Dip3 is able to bind DNA in a sequence specific manner and activate transcription directly. Dip3 possesses an N-terminal MADF domain and a C-terminal BESS domain, an architecture that is conserved in at least 14 Drosophila proteins, including Adf-1 and Stonewall. The MADF domain directs sequence specific DNA binding to a site consisting of multiple trinucleotide repeats, while the BESS domain directs a variety of protein-protein interactions, including interactions with itself, with Dorsal, and with a TBP-associated factor. The possibility is assessed that the MADF and BESS domains are related to the SANT domain, a well-characterized motif found in many transcriptional regulators and coregulators (Bhaskar, 2002).

A regulatory code for neurogenic gene expression in the Drosophila embryo involves Dorsal, Twist and Su(H)

Bioinformatics methods have identified enhancers that mediate restricted expression in the Drosophila embryo. However, only a small fraction of the predicted enhancers actually work when tested in vivo. In the present study, co-regulated neurogenic enhancers that are activated by intermediate levels of the Dorsal regulatory gradient are shown to contain several shared sequence motifs. These motifs permit the identification of new neurogenic enhancers with high precision: five out of seven predicted enhancers direct restricted expression within ventral regions of the neurogenic ectoderm. Mutations in some of the shared motifs disrupt enhancer function, and evidence is presented that the Twist and Su(H) regulatory proteins are essential for the specification of the ventral neurogenic ectoderm prior to gastrulation. The regulatory model of neurogenic gene expression defined in this study permitted the identification of a neurogenic enhancer in the distant Anopheles genome. The prospects for deciphering regulatory codes that link primary DNA sequence information with predicted patterns of gene expression are discussed (Markstein, 2004).

Previous studies identified two enhancers, from the rho and vnd genes, that are activated by intermediate levels of the Dorsal gradient in ventral regions of the neurogenic ectoderm. The present study identified a third such enhancer from the brk gene. This newly identified brk enhancer corresponds to one of the 15 optimal Dorsal-binding clusters described in a previous survey of the Drosophila genome. Although one of these 15 clusters has been shown to define an intronic enhancer in the short gastrulation (sog) gene, the activities of the remaining 14 clusters were not tested. Genomic DNA fragments corresponding to these 14 clusters were placed 5' of a minimal eve-lacZ reporter gene, and separately expressed in transgenic embryos using P-element germline transformation. Four of the 14 genomic DNA fragments were found to direct restricted patterns of lacZ expression across the dorsoventral axis that are similar to the expression patterns seen for the associated endogenous genes (Markstein, 2004).

The four enhancers respond to different levels of the Dorsal nuclear gradient. Two direct expression within the presumptive mesoderm where there are high levels of the gradient. These are associated with the Phm and Ady43A genes. The third enhancer maps ~10 kb 5' of brk, and is activated by intermediate levels of the Dorsal gradient, similar to the vnd and rho enhancers. Finally, the fourth enhancer maps over 15 kb 5' of the predicted start site of the CG12443 gene, and directs broad lateral stripes throughout the neurogenic ectoderm in response to low levels of the Dorsal gradient. In terms of the dorsoventral limits, this staining pattern is similar to that produced by the sog intronic enhancer (Markstein, 2004).

The remaining ten clusters failed to direct robust patterns of expression and are thus referred to as 'false-positives'. Since analysis of spacing and orientation of the Dorsal sites alone did not reveal features that could discriminate between the false positives and the enhancers, whether additional sequence motifs could aid in this distinction was examined. A program called MERmaid was developed that identifies motifs over-represented in specified sets of sequences. MERmaid analysis identified a group of motifs, which was largely specific to the brk, vnd and rho enhancers, suggesting that the regulation of these coordinately expressed genes is distinct from the regulation of genes that respond to different levels of nuclear Dorsal (Markstein, 2004).

The rho, vnd and brk enhancers direct similar patterns of gene expression. The rho and vnd enhancers were previously shown to contain multiple copies of two different sequence motifs: CTGNCCY and CACATGT. A three-way comparison of minimal rho, vnd and brk enhancers permitted a more refined definition of the CTGNCCY motif (CTGWCCY), and also allowed for the identification of a third motif, YGTGDGAA. The CACATGT and YGTGDGAA motifs bind the known transcription factors, Twist and Suppressor of Hairless [Su(H)], respectively. All three motifs are over-represented in authentic Dorsal target enhancers directing expression in the ventral neurogenic ectoderm, as compared with the 10 false-positive Dorsal-binding clusters. Some of the false-positive clusters contain motifs matching either Twist or CTGWCCY; however, none of the false-positive clusters contain representatives of both of these motifs. The rho enhancer is repressed in the ventral mesoderm by the zinc-finger Snail protein. The four Snail-binding sites contained in the rho enhancer share the consensus sequence, MMMCWTGY; the vnd and brk enhancers contain multiple copies of this motif and are probably repressed by Snail as well (Markstein, 2004).

The functional significance of the shared sequence motifs was assessed by mutagenizing the sites in the context of otherwise normal lacZ transgenes. Previous studies have suggested that bHLH activators are important for the activation of rho expression, since rho-lacZ fusion genes containing point mutations in several different E-box motifs (CANNTG) exhibited severely impaired expression in transgenic embryos. However, it was not obvious that the CACATGT motif was particularly significant since it represents only one of five E-boxes contained in the rho enhancer. Yet, only this particular E-box motif is significantly over-represented in the rho, vnd and brk enhancers. vnd-lacZ and brk-lacZ fusion genes were mutagenized to eliminate each CACATGT motif, and analyzed in transgenic embryos. The loss of these sites causes a narrowing in the expression pattern of an otherwise normal vnd-lacZ fusion gene. By contrast, the brk pattern is narrower in central and posterior regions, but relatively unaffected in anterior regions. The brk enhancer contains two copies of an optimal Bicoid-binding site, and it is possible that the Bicoid activator can compensate for the loss of the CACATGT motifs in anterior regions (Markstein, 2004).

Similar experiments were performed to assess the activities of the Su(H)-binding sites (YGTGDGAA) and the CTGWCCY motif. Mutations in the latter sequence cause only a slight reduction and irregularity in the activity of the vnd enhancer, whereas similar mutations nearly abolish expression from the brk enhancer. Thus, CTGWCCY appears to be an essential regulatory element in the brk enhancer, but not in the vnd enhancer. Mutations in both Su(H) sites in the brk enhancer caused reduced staining of the lacZ reporter gene, suggesting that Su(H) normally activates expression. Further evidence that Su(H) mediates transcriptional activation was obtained by analyzing the endogenous rho expression pattern in transgenic embryos carrying an eve stripe 2 transgene with a constitutively activated form of the Notch receptor (Notch^IC). rho expression is augmented and slightly expanded in the vicinity of the stripe2-Notch^IC transgene. A similar expansion is observed for the sim expression pattern (Markstein, 2004).

To determine whether the shared motifs would help identify additional ventral neurogenic enhancers, the genome was surveyed for 250 bp regions containing an average density of one site per 50 bp and at least one occurrence of each of the four motifs for Dorsal, Twist, Su(H) and CTGWCCY. In total, only seven clusters were identified. Three of the seven clusters correspond to the rho, vnd and brk enhancers. Two of the remaining clusters are associated with genes that are known to be expressed in ventral regions of the neurogenic ectoderm: vein and sim. Both clusters were tested for enhancer activity by attaching appropriate genomic DNA fragments to a lacZ reporter gene and then analyzing lacZ expression in transgenic embryos. The cluster associated with vein is located in the first intron, about 7 kb downstream of the transcription start site. The vein cluster (497 bp) directs robust expression in the neurogenic ectoderm, similar to the pattern of the endogenous gene. The cluster located in the 5' flanking region of the sim gene (631 bp) directs expression in single lines of cells in the mesectoderm (the ventral-most region of the neurogenic ectoderm), just like the endogenous expression pattern. These results indicate that the computational methods define an accurate regulatory model for gene expression in ventral regions of the neurogenic ectoderm of D. melanogaster (Markstein, 2004).

To assay the generality of these findings, genomic regions encompassing putative sim orthologs from the distantly related dipteran Anopheles gambiae were scanned for clustering of Dorsal, Twist, Su(H), CTGWCCY and Snail motifs. One cluster located 865 bp 5' of a putative sim ortholog contains one putative Dorsal binding site, two Su(H) sites, three CTGWCCY motifs (or close matches to this motif), a CACATG E-box and several copies of the Snail repressor sequence MMMCWTGY. A genomic DNA fragment encompassing these sites (976 bp) was attached to a minimal eve-lacZ reporter gene and expressed in transgenic Drosophila embryos. The Anopheles enhancer directs weak lateral lines of lacZ expression that are similar to those obtained with the Drosophila sim enhancer. These results suggest that the clustering of Dorsal, Twist, Su(H) and CTGWCCY motifs constitutes an ancient and conserved code for neurogenic gene expression (Markstein, 2004).

This study defines a specific and predictive model for the activation of gene expression by intermediate levels of the Dorsal gradient in ventral regions of the neurogenic ectoderm. The model identified new enhancers for sim and vein in the Drosophila genome, as well as a sim enhancer in the distant Anopheles genome. Five of the seven composite Dorsal-Twist-Su(H)-CTGWCCY clusters in the Drosophila genome correspond to authentic enhancers that direct similar patterns of gene expression. This hit rate represents the highest precision so far obtained for the computational identification of Drosophila enhancers based on the clustering of regulatory elements. Nevertheless, it is still not a perfect code (Markstein, 2004).

Two of the seven composite clusters are likely to be false-positives: they are associated with genes that are not known to exhibit localized expression across the dorsoventral axis. It is possible that the order, spacing and/or orientation of the identified binding sites accounts for the distinction between authentic enhancers and false-positive clusters. For example, there is tight linkage of Dorsal and Twist sites in each of the five neurogenic enhancers. This linkage might reflect Dorsal-Twist protein-protein interactions that promote their cooperative binding and synergistic activities. Previous studies identified particularly strong interactions between Dorsal and Twist-Daughterless (Da) heterodimers. Da is ubiquitously expressed in the early embryo and is related to the E12/E47 bHLH proteins in mammals. Dorsal-Twist linkage is not seen in one of the two false-positive binding clusters (Markstein, 2004).

The regulatory model defined by this study probably fails to identify all enhancers responsive to intermediate levels of the Dorsal gradient. There are at least 30 Dorsal target enhancers in the Drosophila genome, and it is possible that 10 respond to intermediate levels of the Dorsal gradient. Thus, half of all such target enhancers might have been missed. Perhaps the present study defined just one of several 'codes' for neurogenic gene expression (Markstein, 2004).

The possibility of multiple codes is suggested by the different contributions of the same regulatory elements to the activities of the vnd and brk enhancers. Mutations in the CTGWCCY motifs nearly abolish the activity of the brk enhancer, but have virtually no effect on the vnd enhancer. Future studies will determine whether there are distinct codes for Dorsal target enhancers that respond to either high or low levels of the Dorsal gradient. Indeed, it is somewhat surprising that the sog and CG12443 enhancers essentially lack Twist, Su(H) and CTGWCCY motifs, even though they direct lateral stripes of gene expression that are quite similar (albeit broader) to those seen for the rho, vnd and brk enhancers (Markstein, 2004).

This study provides direct evidence that Twist and Su(H) are essential for the specification of the neurogenic ectoderm in early embryos. The Twist protein is transiently expressed at low levels in ventral regions of the neurogenic ectoderm. SELEX assays indicate that Twist binds the CACATGT motif quite well. The presence of this motif in the vnd, brk and sim enhancers, and the fact that it functions as an essential element in the vnd and brk enhancers, strongly suggests that Twist is not a dedicated mesoderm determinant, but that it is also required for the differentiation of the neurogenic ectoderm. However, it is currently unclear whether the CACATGT motif binds Twist-Twist homodimers, Twist-Da heterodimers or additional bHLH complexes in vivo. Su(H) is the sequence-specific transcriptional effector of Notch signaling. The restricted activation of sim expression within the mesectoderm depends on Notch signaling; however, the rho, vnd and brk enhancers direct expression in more lateral regions where Notch signaling has not been demonstrated. Nonetheless, mutations in the two Su(H) sites contained in the brk enhancer cause a severe impairment in its activity. This observation raises the possibility that Su(H) can function as an activator, at least in certain contexts, in the absence of an obvious Notch signal (Markstein, 2004).

The Dorsal gradient produces three distinct patterns of gene expression within the presumptive neurogenic ectoderm. It is proposed that these patterns arise from the differential usage of the Su(H) and Dorsal activators. Enhancers that direct progressively broader patterns of expression become increasingly more dependent on Dorsal and less dependent on Su(H). The sog and CG12443 enhancers mediate expression in both ventral and dorsal regions of the neurogenic ectoderm, and contain several optimal Dorsal sites but no Su(H) sites. By contrast, the sim enhancer is active only in the ventral-most regions of the neurogenic ectoderm, and contains just one high-affinity Dorsal site but five optimal Su(H) sites. The reliance of sim on Dorsal might be atypical for genes expressed in the mesectoderm. For example, the m8 gene within the Enhancer of split complex may be regulated solely by Su(H). The Anopheles sim enhancer might represent an intermediate between the Drosophila sim and m8 enhancers, since it contains optimal Su(H) sites but only one weak Dorsal site. This trend may reflect an evolutionary conversion of Su(H) sites to Dorsal sites, and the concomitant use of the Dorsal gradient to specify different neurogenic cell types. A testable prediction of this model is that basal arthropods use Dorsal solely for the specification of the mesoderm and Su(H) for the patterning of the ventral neurogenic ectoderm (Markstein, 2004).

Control of Drosophila gastrulation by apical localization of adherens junctions and RhoGEF2; T48 is a target of Twist

A hallmark of epithelial invagination is the constriction of cells on their apical sides. During Drosophila gastrulation, apical constrictions under the control of the transcription factor Twist lead to the invagination of the mesoderm. Twist-controlled G protein signaling is involved in mediating the invagination but is not sufficient to account for the full activity of Twist. A Twist target was identified, the transmembrane protein T48, which acts in conjunction with G protein signaling to orchestrate shape changes. Together with G protein signaling, T48 recruits adherens junctions and the cytoskeletal regulator RhoGEF2 to the sites of apical constriction, ensuring rapid and intense changes in cell shape (Kolsch, 2007).

Apical constriction of cells can contribute to the invagination of epithelia, such as during gastrulation or organogenesis, and the closure of wounds. In the Drosophila embryo, apical constrictions occur along the ventral side of the blastoderm epithelium, leading to the formation of the ventral furrow and the invagination of the mesoderm. Proteins necessary for the mechanics of these cell shape changes include the Rho guanosine 5'-triphosphate-exchange factor RhoGEF2 and a heterotrimeric G protein. Whereas RhoGEF2 is essential for furrow formation, disruption of the heterotrimeric G protein, such as by loss of its α subunit Concertina (Cta), leads to a delay but no lasting defects in mesoderm morphogenesis. These maternally supplied proteins must be activated under the control of the zygotic genome in the embryo (Kolsch, 2007).

Twist is the zygotic transcriptional activator that is essential for the cell shape changes that produce the ventral furrow. One of its targets is the transcriptional repressor Snail, which is also essential for mesodermal morphogenesis (Kolsch, 2007).

However, the cell biological events responsible for the cell shape changes must ultimately be regulated by targets that are not transcription factors. Of the known Twist targets, only one, folded gastrulation (fog), is involved in mediating shape changes. Mutants in fog, which codes for a secreted peptide, show the same defects as embryos lacking Cta. Fog is therefore thought to act in the same pathway as Cta, which is referred to as Fog/Cta signaling (Kolsch, 2007).

Fog/Cta signaling is thought to cause changes in the actin cytoskeleton in conjunction with RhoGEF2. Recruitment of myosin from basal to apical in constricting ventral cells is partly dependent on Fog/Cta and absolutely dependent on RhoGEF2. Furthermore, the mammalian homologs of RhoGEF2 and Cta interact. Finally, binding of Drosophila RhoGEF2 to microtubules by means of EB1 is disrupted by activated Cta. Given that myosin recruitment and apical constriction are reduced but not abolished in the absence of Fog/Cta, there must be other factors regulated by Twist that explain its effects on apical constriction (Kolsch, 2007).

In a screen for genes that mediate the zygotic control of gastrulation, the region uncovered by the chromosomal deficiency Df(3R)Tl^P was found to be necessary for the proper formation of the ventral furrow. Phenotypic analysis and molecular mapping of a set of overlapping deficiencies identified the gene T48 as being responsible for the defects seen in Df(3R)Tl^P. T48 is expressed in the mesoderm. It codes for a predicted protein with a signal peptide and a potential transmembrane domain. When an internally hemagglutinin-tagged T48 protein (T48HA) was expressed in embryos, it localized at the peripheries of blastoderm cells, consistent with a close association with or insertion into the plasma membrane. Optical cross-sections showed that T48HA is targeted to the apical membrane (Kolsch, 2007).

No other structural motifs are recognizable in the protein. However, the C-terminal amino acid sequence -Ile-Thr-Thr-Glu-Leu (-ITTEL) conforms to the class I consensus for peptides that interact with PDZ domains. T48 has no obvious human ortholog but shows some similarity to the intracellular part of Fras1, which also has a PDZ-binding motif. To find candidates for PDZ domains that might interact with T48, the putative PDZ-binding sequence was analyzed with an algorithm designed to determine the PDZ domains that show the optimal fit for any given peptide. Of the predicted interactors, RhoGEF2 was particularly interesting in view of its role in ventral furrow formation. Furthermore, the mammalian ortholog of RhoGEF2 has been shown to bind to Plexin-B1 by means of a PDZ-binding motif (-Val-Thr-Asp-Leu) very similar to that of T48 (Kolsch, 2007).

Whether the C terminus of T48 is indeed able to interact with RhoGEF2 was tested. A ³⁵S-labeled C-terminal peptide of T48 preferentially coprecipitated with the PDZ domain of RhoGEF2 rather than those of other PDZ domain-containing proteins, in contrast to Crumbs, which was used as a control and which preferentially coprecipitated with PDZ domains from its physiological interaction partner Stardust, as well as Bazooka. In Schneider S2 cells, a green fluorescent protein (GFP)-tagged RhoGEF2 PDZ domain or full-length RhoGEF2 was localized in the cytoplasm or formed intracellular aggregates when expressed alone, but localized to the plasma membrane when coexpressed with T48. In both assays, the interaction required the presence of the -ITTEL motif and was not seen with other PDZ domains. Thus, T48 interacts with RhoGEF2 by means of its PDZ-binding motif and is able to enrich RhoGEF2 to the plasma membrane (Kolsch, 2007).

To understand the function of T48 during gastrulation, the subcellular localization of RhoGEF2 and its dependence on T48 were studied in the developing embryo. Before gastrulation, the apical surfaces of the blastoderm epithelium are dome shaped and the developing adherens junctions are located subapically. RhoGEF2 is associated with the basally located furrow canals, whereas Armadillo is found just below this site and at a subapical position of the lateral cell membranes (Kolsch, 2007).

After cellularization was completed, these distributions changed specifically in ventral cells. Even before morphological changes occurred, RhoGEF2 and Armadillo disappeared from the basal ends. Subsequently, Armadillo disappeared from its subapical site and accumulated apically. A weak association of RhoGEF2 with the apical plasma membrane was seen at this stage (Kolsch, 2007).

As cells begin to flatten apically, high levels of both RhoGEF2 and Armadillo accumulate apically. Although they concentrated in the same region of the cell, Armadillo was restricted to the cell junctions, whereas RhoGEF2 was often more enriched between these sites. Notably, movement of the adherens junctions occurred not only in constricting cells but also in the more lateral mesodermal cells that flattened and became stretched on their apical sides (Kolsch, 2007).

To examine whether these processes depend on T48, stage-selected T48 mutant embryos were stained. Loss of RhoGEF2 and Armadillo from the basal side was unaffected in these embryos, as was the apical concentration of Armadillo. The cells flatten apically and lengthen, but the absence of constrictions results in a thick placode rather than an indentation. Localization of RhoGEF2 to the apical membrane is slightly delayed and possibly reduced. T48 therefore contributes to but is not essential for the recruitment of RhoGEF2 to the apical membrane. This is consistent with the observation that furrow formation is not completely abolished, but only delayed or weakened. Therefore other mechanisms were examined that might participate in RhoGEF2 localization (Kolsch, 2007).

As in the case of T48, mutations in the Fog/Cta pathway delay but do not abolish apical constriction and furrow formation. It was therefore considered whether Fog/Cta signaling might cooperate with T48 to recruit RhoGEF2. In embryos lacking Cta, the recruitment of RhoGEF2 was weakened. Combining mutations in cta and T48 resulted in much more notable effects. These cta,T48 embryos failed to make a furrow; the lack of apical constrictions was mirrored by a failure to accumulate RhoGEF2 apically. Thus, T48 and Fog/Cta signaling act in parallel to concentrate RhoGEF2 apically (Kolsch, 2007).

Severe defects were also observed in the behavior of the adherens junctions in the double-mutant embryos. Armadillo staining disappeared from its tight subapical localization but did not reaccumulate apically. Thus, movement of the junctions is not simply mediated by a tensile force from the constricting actin cytoskeleton: an independent step of at least partial disassembly must occur. It is speculated that this might be controlled by Snail, which regulates the disassembly of cell junctions in vertebrates. It was found that the disassembly of Armadillo from the subapical position was indeed blocked in snail (but not in twist) mutant embryos. Thus, Snail acts in parallel to Twist to direct the disassembly of subapical junctions, a process to which currently unknown Twist targets may also contribute (Kolsch, 2007).

Having observed that T48 and Fog/Cta activation are required for the apical localization of RhoGEF2 and Armadillo, whether T48, like Fog/Cta signaling, was able to trigger their relocalization in other cells was also tested. Ubiquitous expression of T48 in the embryo led to a concentration of RhoGEF2 at the apical membranes of lateral cells. Armadillo localization in ectodermal cells was no longer restricted to a distinct subapical domain but extended to the apical end of the lateral membranes in many cells. When T48 was coexpressed with activated Cta, this effect was slightly enhanced, and some embryos showed morphological defects (Kolsch, 2007).

With T48, a missing factor has been found in the control cascade from transcriptional regulation by Twist to the cell biological mediators of furrow morphogenesis. Two Twist targets, Fog and T48, appear to act in separate pathways that converge on RhoGEF2, which integrates the signal to activate myosin and modify the actin cytoskeleton. This model shows the maternally supplied RhoGEF2 is largely attached to microtubules by means of EB1. The onset of Twist expression has two effects. Fog is synthesized, which triggers the activation of Cta. This in turn releases RhoGEF2 from the microtubules that, by analogy to its vertebrate homologs, may bind to Cta through its RGS domain, allowing some myosin activation and constriction. In parallel, T48 is synthesized and targeted to the apical membrane, where it acts to concentrate RhoGEF2 through its PDZ-binding motif. In the absence of Fog-mediated displacement of RhoGEF2 from EB1, T48 can probably still recruit sufficient freely diffusible RhoGEF2 to allow slow constriction. Only when both mechanisms fail are the downstream events of constriction and junction reassembly abolished completely (Kolsch, 2007).

The utilization of Gα12/13 proteins and a microtubule-bound RhoGEF have also been reported in vertebrate gastrulation. The absence of an obvious homolog of T48 in vertebrates might suggest that this element of the control mechanism is unique to Drosophila gastrulation. However, the PDZ-binding motif in Plexin-B1 is similar to that of T48 and acts during neuronal growth cone remodeling by recruiting PDZ-RhoGEF. Therefore, this mechanism of controlling cell shape may operate in a variety of systems (Kolsch, 2007).

Whole-genome ChIP-chip analysis of Dorsal, Twist, and Snail suggests integration of diverse patterning processes in the Drosophila embryo

Genetic studies have identified numerous sequence-specific transcription factors that control development, yet little is known about their in vivo distribution across animal genomes. This study determined the genome-wide occupancy of the dorsoventral (DV) determinants Dorsal, Twist, and Snail in the Drosophila embryo using chromatin immunoprecipitation coupled with microarray analysis (ChIP-chip). The in vivo binding of these proteins correlate tightly with the limits of known enhancers. This analysis predicts substantially more target genes than previous estimates, and includes Dpp signaling components and anteroposterior (AP) segmentation determinants. Thus, the ChIP-chip data uncover a much larger than expected regulatory network, which integrates diverse patterning processes during development (Zeitlinger, 2007).

ChIP-chip assays were performed with antibodies directed against Dorsal, Twist, or Snail on Toll^10b mutant embryos, aged 2-4 h. These embryos contain a constitutively activated form of the Toll receptor, which results in high levels of nuclear Dorsal protein and uniform expression of Twist and Snail throughout the embryo. The high levels of Dorsal, Twist, and Snail cause all cells to form derivatives of the mesoderm at the expense of neurogenic and dorsal ectoderm. Thus, these embryos represent a uniform cell type with respect to DV fate (Zeitlinger, 2007).

The whole-genome ChIP-chip experiments reveal several hundred strong binding clusters of Dorsal, Twist, and Snail with up to 40-fold ChIP enrichment, most of which span regions of ~1 kb in length. To identify the binding patterns of bona fide target enhancers of the Dorsal regulatory network, known enhancers were analyzed. The 22 known enhancers fall into three classes: type 1, type 2, and type 3, based on which levels of nuclear Dorsal regulate their expression (Zeitlinger, 2007).

The 10 type 1 enhancers (associated with twi, sna, miR-1, htl, hbr, mes3, CG12177, ady43A, tin, and Phm) are activated by peak levels of Dorsal in the presumptive mesoderm, and are all constitutively activated in Toll^10B mutant embryos. The ChIP-chip experiments identify strong binding peaks (greater than fivefold enrichment) of Dorsal, Twist, and Snail (DTS) within five of the 10 enhancers (twi, sna, miR-1, CG12177 and Phm). Another three enhancers, those associated with htl, tin, and ady43A, show significant but lower (less than fivefold) binding peaks restricted to Twist and Snail (TS) binding. This observation is consistent with earlier studies indicating that these enhancers might be primarily activated by Twist. Hence, eight of the 10 known type 1 enhancers exhibit significant in vivo occupancy by Twist and Snail (Zeitlinger, 2007).

An even greater correspondence between known enhancers and in vivo occupancy is seen for the type 2 [sim, E(spl), vn, rho, vnd and brk] and type 3 enhancers (ths, sog, ind, dpp, zen and tld), which are regulated by intermediate and low levels of the Dorsal gradient, respectively. All 12 enhancers are silenced in Toll^10B mutant embryos due to constitutive expression of the Snail repressor. Remarkably, every enhancer exhibits strong DTS or TS peaks with greater than fivefold enrichment in the ChIP-chip assays. Thus, ChIP-chip assays correctly identified 20 of the 22 known Dorsal target enhancers (Zeitlinger, 2007).

Most known DV enhancers are associated with overlapping binding clusters of Dorsal, Twist, and Snail regardless of whether they mediate activation or repression. Moreover, 17 of the 20 binding clusters at known enhancers display greater than fivefold enrichment of Twist and/or Snail. Using these binding criteria, 428 high-confidence DTS regions and 433 high-confidence TS regions were identified across the genome (Zeitlinger, 2007).

To confirm these regions through independent evidence, sequence analysis on these regions was performed using the known consensus binding motifs of Dorsal, Twist, and Snail. As expected, the identified regions are highly enriched in all three binding motifs. Moreover, a large fraction of the motifs is conserved across the 12 sequenced Drosophila species providing evidence that the discovered regions are functionally important. Finally, when motifs that are enriched in these regions were identified de novo, the known binding motifs can be rediscovered. Hence, the regions identified represent putative target gene enhancers of the DV network (Zeitlinger, 2007).

To show that newly identified regions indeed function as enhancers in vivo, putative enhancers were selected of primary DV genes; i.e., those genes that are expressed as localized stripes across the DV axis. In addition to the 22 known DV enhancers, 47 new putative enhancers were identified , some of which appear to regulate the same gene, were identified. By attaching the genomic sequence to a lacZ reporter and expressing the construct in transgenic embryos, seven of these enhancers were shown to be bona fide DV enhancers and that regulation by multiple enhancers occurs (Zeitlinger, 2007).

The wntD gene is expressed in portions of the presumptive mesoderm where it mediates feedback inhibition of Toll signaling. A cluster of DTS-binding peaks was identified in the 5'-flanking region, and the corresponding genomic DNA fragment mediates lacZ expression in the same region of the mesoderm as the endogenous gene. Similar results were obtained with the DTS-binding cluster located in the 5'-flanking region of mes5/mdr49 (Zeitlinger, 2007).

The vnd locus contains a well-documented intronic enhancer that mediates expression in the neurogenic ectoderm and recapitulates the spatial and temporal expression pattern of the endogenous gene. The ChIP-chip analysis detected this enhancer but also revealed two novel clusters further upstream. When tested for lacZ reporter activity, these novel genomic sequences directed lacZ expression in a pattern resembling that of the endogenous gene over different time periods: One directs early vnd expression in the presumptive ventral neurogenic ectoderm (vNE) while the other directs later expression in the medial column (mc) of the developing nervous system. All three enhancers contain evolutionarily conserved binding sites for Dorsal, Twist, and Snail, suggesting that the enhancers are not redundant but may function to fine-tune the vnd expression pattern. Overlapping enhancer activity was also observed for multiple miR-1 enhancers. Overall, as many as a third of all DV genes have multiple binding clusters, and thus might be subject to similar regulatory control (Zeitlinger, 2007).

Several of the occupied regions are associated with Dpp target genes expressed in the dorsal ectoderm. When the tup and pnr intronic sequences are tested in transgenic embryos, both fragments function as authentic enhancers and direct localized expression in the dorsal ectoderm, comparable to the endogenous tup and pnr expression patterns. These results suggest that the Dorsal patterning network directly regulates the expression of Dpp target genes (see below) (Zeitlinger, 2007).

It was noticed that many of the new DTS/TS clusters are associated with AP genes involved in segmentation. Although classical genetic studies argue that AP and DV patterning of the early embryo are controlled by separate maternal genetic programs, it is conceivable that the expression of AP target genes is modulated by the DV network. Indeed, DV modulation of segmentation gene expression has been observed previously (Zeitlinger, 2007).

The gap gene orthodenticle (otd) is expressed in two stripes across the AP axis in the early embryo. The anterior stripe shows diminished expression on the ventral side. Previous studies identified a 5' enhancer that recapitulates the normal expression pattern, including Dorsal-dependent suppression in ventral regions. ChIP-chip identified a strong DTS cluster within the limits of this enhancer. A similar DV bias in the expression pattern was found for the gap gene tailless (tll) and the pair-rule genes runt and hairy. In each case, the regions identified by ChIP-chip overlap or map close to known regulatory regions and contain several Dorsal-binding motifs (Zeitlinger, 2007).

At the gap gene knirps, a DTS-binding cluster was found in a region distinct from the known Bicoid-dependent enhancer. This newly identified genomic region functions as a bona fide enhancer directing expression in the anteroventral domain like endogenous knirps. Thus, the ChIP-chip analysis identified novel AP regulatory regions modulated by DV activity (Zeitlinger, 2007).

In summary, many segmentation genes contain DTS/TS-binding clusters, and at least some of these regions modulate gene expression across the DV axis, particularly in anterior regions of the embryo. It is concluded that the Dorsal gradient does not only regulate primary DV target genes, but rather appears to fine-tune a large number of genes that do not contribute to DV axis formation themselves, at least based on their known genetic function (Zeitlinger, 2007).

Many DTS/TS-binding clusters are also found at genes encoding signal transduction components. Analysis of the network formed by these pathways suggests that the Dorsal gradient controls the expression of many target genes by multiple regulatory pathways (Zeitlinger, 2007).

Dorsal directly represses Dpp expression in the mesoderm and neuroectoderm, leading to localized Dpp signaling in the dorsal ectoderm. Dpp activates a variety of genes, including tup and pnr. Accurate identification of intronic tup and pnr enhancers suggests that these genes are directly regulated by the Snail repressor, in addition to indirect regulation by the Dorsal gradient via Dpp signaling. zen is another well-known target gene of Dorsal in the dorsal ectoderm, and its product, a homeodomain transcription factor, functions synergistically with Dpp signaling. Target genes of Zen also appear to be subject to additional regulation by the Dorsal gradient. In the dorsal ectoderm, Dorsal may regulate gene expression by two mechanisms: direct repression, and indirect repression via Snail (Zeitlinger, 2007).

Similar network configurations regulate gene expression in the neuroectoderm. High levels of Dorsal repress the expression of rho via Snail in the mesoderm, thereby blocking EGF signaling in Toll^10b mutant embryos. ChIP-chip data suggest that the Dorsal network regulates additional genes encoding EGF signaling components as well as EGF target genes such as pnt, aop/yan, and argos. In the case of Notch signaling, it is known that the Dorsal network represses Notch target genes such as sim in Toll^10B mutant embryos through Snail. The Dorsal network may also regulate Notch signaling more directly, by suppressing genes encoding components of the signaling pathway including Notch itself (Zeitlinger, 2007).

Although repression of neuroectodermal target genes is likely to occur predominantly through Snail, Dorsal also induces the expression of a number of microRNAs in Toll^10b mutant embryos, including miR-1. Some of the neuroectodermal genes repressed by Snail are also predicted targets of these microRNAs. Hence, there may be multiple tiers of repression in the DV system, similar to the activities of the gap repressors in the AP system (Zeitlinger, 2007).

In summary, the present ChIP-chip study revealed an unexpectedly broad distribution of binding peaks for Dorsal, Twist, and Snail in the genome, and suggests extensive integration of the Dorsal regulatory network with additional patterning processes, such as Dpp signaling in the dorsal ectoderm and segmentation across the AP axis. In addition to the observed tight correlation between binding peaks and known enhancers, two lines of evidence suggest that a significant fraction of the newly identified regions is functional: First, the bound regions are highly enriched in evolutionarily conserved Dorsal, Twist, and Snail sequence motifs; and, second, several of the identified enhancers were experimentally confirmed by lacZ reporter gene expression in transgenic embryos. Thus, while genetic studies identified core sets of regulators for each developmental process in Drosophila, gene regulation integrates information more widely from several different systems. It is likely that integration of diverse patterning processes will also apply to mammalian development, including stem cell differentiation (Zeitlinger, 2007).

Daughterless dictates Twist activity in a context-dependent manner during somatic myogenesis

Somatic myogenesis in Drosophila relies on the reiterative activity of the basic helix-loop-helix transcriptional regulator, Twist (Twi). How Twi directs multiple cell fate decisions over the course of mesoderm and muscle development is unclear. Previous work has shown that Twi is regulated by its dimerization partner: Twi homodimers activate genes necessary for somatic myogenesis, whereas Twi/Daughterless (Da) heterodimers lead to the repression of these genes. This study examined the nature of Twi/Da heterodimer repressive activity. Analysis of the Da protein structure revealed a Da repression (REP) domain, which is required for Twi/Da-mediated repression of myogenic genes, such as Dmef2, both in tissue culture and in vivo. This domain is crucial for the allocation of mesodermal cells to distinct fates, such as heart, gut and body wall muscle. By contrast, the REP domain is not required in vivo during later stages of myogenesis, even though Twi activity is required for muscles to achieve their final pattern and morphology. Taken together, evidence is presented that the repressive activity of the Twi/Da dimer is dependent on the Da REP domain and that the activity of the REP domain is sensitive to tissue context and developmental timing (Wong, 2008).

This study explores the regulation of Twi activity through mesoderm development and somatic myogenesis in Drosophila . Focus was placed on how Twi is modulated by its dimer partner, Da. The examination of Twi/Da dimers revealed that the activity of these dimers is acutely sensitive to their tissue environment: both between germ layers (the ectoderm versus the mesoderm), and within cell lineages (early mesoderm versus somatic muscle). This sensitivity is determined, in part, by the activity of the Da REP domain, which is critical for Twi/Da activity during mesodermal subdivision and FC specification, but is not required for the later activity of Twi/Da during muscle differentiation. This work provides insight to the mechanism of Twi/Da activity and calls attention to the effect of tissue context and developmental timing on bHLH protein regulation (Wong, 2008).

One of the most striking aspects of this study is the role of the Da REP domain in switching Twi/Da behaviour between a repressor and an activator function. This 'switchable' behaviour of Twi/Da activity was initially observed by its ability to inhibit myogenesis in the mesoderm, but activate myogenesis in the ectoderm. Notably, the deletion of the REP domain from Da has little effect on Da activity in the absence of Twi, as demonstrated by cell culture transcriptional assays. However, the activity of Twi-Da^Δ tethered dimers has a distinct effect on the mesoderm. Overexpression of these dimers had the greatest effect on somatic myogenesis during the process of mesodermal subdivision. The detection of increased numbers of founder cells (FCs), which appear to be specified normally, indicated an increased number of mesodermal cells being allocated to a somatic muscle fate at the expense of cardiac and visceral mesoderm (Wong, 2008).

An outstanding question is how the Da REP domain functions to modulate Twi/Da activity. Since Twi/Da dimers bind DNA and therefore may actively regulate the transcriptional state of a target gene, it was initially postulated that the REP domain must directly interact with transcriptional corepressors or factors that were expressed solely in the mesoderm and therefore were required for the repressive activity of Twi/Da in that tissue context. Exhaustive studies were conducted to identify these factors but no protein that satisfies all necessary criteria has been identified (Wong, 2008).

Deletion analysis of the E protein Rep domain suggested that this domain is required for the repression of the E protein activation domains, AD1 and AD2. Like the Da REP domain, the E protein Rep domain has specific activities depending on its dimer partner and tissue context (Markus, 2002). Informed by this work, the current data was interpreted to suggest that the Da REP domain is a cis-acting repressor, which functions to repress both Da AD1 and AD2 when Da is dimerized to Twi and bound to myogenic enhancers. Moreover, the effect of the Da REP domain is not restricted to the E protein/Da protein family. This work suggests that the Da REP domain also represses Twi's activation domains, Twi-AD1 and Twi-AD2, in Twi/Da dimers. It is proposed that the Da REP domain acts to mask the activation domains in both Twi and Da. Therefore, the net effect of the Da REP domain results in the recruitment of corepressors to myogenic enhancers by Twi/Da dimers. Alternatively, Twi/Da dimers may not actively repress target myogenic genes: instead, these dimers could compete for myogenic E boxes or transcriptional cofactors and machinery. In this model of passive repression, the Da REP domain could function to stabilize interactions with Twi or other factors that are required to properly mediate repression of myogenic target genes. These aspects of Da REP domain repression are currently being evaluated (Wong, 2008).

To date, various transcriptional regulators have been shown to have different activities and target genes in different tissues and be modulated by dimerization partners. Recently, ChIP-on-chip analyses have identified almost 500 direct Twi targets throughout mesodermal development. This study, however, is one of the first that focuses on how Twi activity is dynamically modulated through multiple developmental stages of a specific cell lineage, and how this regulation affects expression of Twi target genes (Wong, 2008).

One gene that is regulated by Twi dimers throughout somatic myogenesis is Dmef2. Dmef2 protein is expressed throughout and necessary for all stages of myogenesis. Dmef2 coordinates multiple processes necessary for proper somatic myogenesis. Moreover, it has been suggested that Dmef2 is required in combination with Twi to regulate the expression of a subset of Twi target genes in a feed-forward mechanism. The current data support these arguments, since mesodermal phenotypes were observed in Twi/Da^Δ (activation) or Twi/Da (repression) overexpressing embryos that mirror those of embryos overexpressing Dmef2 or in Dmef2 mutant embryos, respectively. For example, increased Dmef2 reporter gene expression and increased numbers of FCs were observed in embryos that overexpress Twi/Da^Δ panmesodermally. Consistent with these observations, Dmef2 has been shown to regulate components of the Ras/MAPK and Notch pathways, which are both required for the proper specification of FCs, and the expression of a subset of FC identity genes. Dmef2 has also been shown to regulate a subset of genes that are required for myoblast fusion and muscle attachment, processes required for proper muscle morphogenesis. This study found that Twi/Da and Twi/Da^Δ dimers disrupt myoblast fusion and muscle differentiation, which is likely due to these dimers repressing Dmef2 expression. In agreement with this observation, muscle analysis revealed that embryos overexpressing Twi-Da and Twi-Da^Δ dimers have muscle phenotypes that are similar to those observed in Dmef2⁴²⁴ hypomorph embryos and Dmef2^22.21 null embryos that have been partially rescued by UAS-Dmef2 transgenes. Taken together, these results supported conclusions of the pivotal regulation of Dmef2 activity by Twi dimers throughout myogenesis (Wong, 2008).

Another notable question is how the Da REP domain is required for Twi/Da mediated transcriptional repression during mesodermal subdivision, but not during muscle morphogenesis. One possibility is that during somatic muscle differentiation, the repressive activity of Twi/Da relies on a different protein domain. Another possibility includes the changes in Twi/Da target genes through the course of somatic myogenesis. Studies conducted on chromatin remodeling have emphasized the specificity involved with the transcriptional regulation of a single gene. Therefore, it is likely that the regulation of multiple sets of genes through time would rely on the modular nature of transcriptional regulators. The Da REP domain may be required for the repression of a subset of Twi/Da target genes, whereas other target genes are unresponsive to this domain's repressive activity (Wong, 2008).

In summary, these results suggest that the regulation of Dmef2 by Twi/Da throughout myogenesis and the subsequent feed-forward mechanism by which Dmef2 and Twi regulate myogenic genes is critical for the coordination of the various disparate processes-mesodermal subdivision, FC specification, and muscle differentiation-necessary for somatic myogenesis (Wong, 2008).

Twi proteins are conserved across species [mouse, chicken, C. elegans, and jellyfish] and have been shown to dimerize with Da homologs, suggesting that REP domain regulation of Twi activity is conserved. Similarly to flies, Mouse Twi1 (MTwi1) heterodimerizes with E proteins to compete with MyoD/E proteins for binding sites on myogenic enhancers. In this manner, MTwi1/E protein heterodimers act like Twi/Da dimers to repress myogenesis. In other tissues, however, MTwi1/E protein heterodimers have been identified as an activator of targets, such as thrombospondin-1 during cranial suture formation. Therefore, like Twi/Da, MTwi1/E protein heterodimers are sensitive to tissue contexts. Of particular interest would be the examination of the E protein Rep domain in vivo. The function of this domain has been studied in mammalian cell culture, but not yet investigated in developmental processes. Moreover, the function of the E protein Rep domain has not been addressed in MTwi1/E protein dimers (Wong, 2008).

Notably, Twi proteins have also been implicated in a variety of tumourigenic processes, such as the inhibition of apoptosis and the coordination of metastasis. Mouse models and correlative data from human tumour samples suggest that MTwi1 and human Twi1 (HTwi1), respectively, direct epithelial-to-mesenchymal transitions (EMT) during breast cancer metastasis. The involvement of Twi1 in the complex process of cancer has many similarities to the developmental processes that Twi directs in the fly mesoderm, which include cell proliferation and cell migration, processes that have been recently revealed to be directly regulated by Twi. The role of the Da REP domain in directing Twi/Da transcriptional repression, and the tissue specificity of this domain's activity has illuminated various aspects of Twi regulation. It is anticipated that these findings will shed light on mammalian Twi1 activity and the Twi family of proteins in development and disease (Wong, 2008).

High resolution mapping of Twist to DNA in Drosophila embryos: Efficient functional analysis and evolutionary conservation

Cis-regulatory modules (CRMs) function by binding sequence specific transcription factors, but the relationship between in vivo physical binding and the regulatory capacity of factor-bound DNA elements remains uncertain. This relationship was investigated for the well-studied Twist factor in Drosophila embryos by analyzing genome-wide factor occupancy and testing the functional significance of Twist occupied regions and motifs within regions. Twist ChIP-seq data efficiently identified previously studied Twist-dependent CRMs and robustly predicted new CRM activity in transgenesis, with newly identified Twist-occupied regions supporting diverse spatiotemporal patterns. Some, but not all, candidate CRMs require Twist for proper expression in the embryo. The Twist motifs most favored in genome ChIP data (in vivo) differed from those most favored by Systematic Evolution of Ligands by EXponential enrichment (SELEX) (in vitro). Furthermore, the majority of ChIP-seq signals could be parsimoniously explained by a CABVTG motif located within 50 bp of the ChIP summit and, of these, CACATG was most prevalent. Mutagenesis experiments demonstrated that different Twist E-box motif types are not fully interchangeable, suggesting that the ChIP-derived consensus (CABVTG) includes sites having distinct regulatory outputs. Further analysis of position, frequency of occurrence, and sequence conservation revealed significant enrichment and conservation of CABVTG E-box motifs near Twist ChIP-seq signal summits, preferential conservation of ±150 bp surrounding Twist occupied summits, and enrichment of GA- and CA-repeat sequences near Twist occupied summits. The results show that high resolution in vivo occupancy data can be used to drive efficient discovery and dissection of global and local cis-regulatory logic (Ozdemir, 2011).

Thirty-one new candidate Twist CRMs drawn from the entire ChIP-seq set were tested in a standard reporter gene assay. Of the 31 test regions, 23 (74%) supported expression; 21 supported expression in a classic dorso-ventral pattern or a subregion thereof, and 2 supported distinct patterns (i.e., ubiquitous or purely anterior-posterior). The 23 new CRMs were distributed throughout the ChIP-seq signal range. Peaks near genes Cyp310a1, Traf4, mirror (mirr), and Mef2 were clearly defined by the ChIP-seq data (see Twist occupancy supported by Twist ChIP-seq identifies functional CRMs), while the equivalent ChIP-chip data in these regions was much broader and, in some cases, gave multiple peaks, making the location of a candidate CRM ambiguous. While Twist ChIP-seq data led to a high recovery rate of CRM detection, surprisingly, only ~25% of the associated genes including Cyp310a1, Asph, and emc (i.e., 3 of 12 assayed) actually required Twist to support expression in embryos. For instance, mirr, Traf4, and Mef2 expression was unaffected in twist mutants, even though their Twist-ChIP-seq signals were equally prominent and numerous (Ozdemir, 2011).

High conservation of transcription factor binding and evidence for combinatorial regulation across six Drosophila species

The binding of some transcription factors has been shown to diverge substantially between closely related species. This study shows that the binding of the developmental transcription factor Twist is highly conserved across six Drosophila species, revealing strong functional constraints at its enhancers. Conserved binding correlates with sequence motifs for Twist and its partners, permitting the de novo discovery of their combinatorial binding. It also includes over 10,000 low-occupancy sites near the detection limit, which tend to mark enhancers of later developmental stages. These results suggest that developmental enhancers can be highly evolutionarily constrained, presumably because of their complex combinatorial nature (He, 2011; full text of article).

It was asked which, if any, of the 10 possible E-box recognition motifs (counting reverse complements as the same motif) are selectively concentrated within 50 bp of called ChIP-seq signal summits. CA and GA core E-boxes were most prominent, while GC, TA, and CG were relatively minor. Compared with regions sampled from ChIP-seq control data or from the entire non-repeat genome, only CA, TA, CG, and GA core E-boxes were statistically enriched in Twist-occupied regions. When larger radii from the ChIP signal summits were interrogated, the number of E-boxes of all types increased, and the specific enrichment trend was less apparent (i.e., enrichment of CA, TA, CG, and GA core E-boxes). In contrast, when ChIP-chip regions were similarly examined, no specific enrichment of any motif was detected at any radius from the called Twist peaks. Overall, the enrichment and resolution results suggest that the ChIP-seq data could be used to model individual binding domains and causal motif instances in them (He, 2011).

Uncovering cis-regulatory sequence requirements for context specific transcription factor binding: The activity of Twist-bound enhancers and Twist binding itself depends on Vielfaltig motifs

The regulation of gene expression is mediated at the transcriptional level by enhancer regions that are bound by sequence specific transcription factors (TFs). Recent studies have shown that the in vivo binding sites of single TFs differ between developmental or cellular contexts. How this context-specific binding is encoded in the cis-regulatory DNA sequence has however remained unclear. This study computationally dissected context-specific TF binding sites in Drosophila, C. elegans, mouse, and human and finds distinct combinations of sequence motifs for partner factors, which are predictive and reveal specific motif requirements of individual binding sites. It is predicted that TF binding in the early Drosophila embryo depends on motifs for the early zygotic TFs Vielfaltig (also known as Zelda) and Tramtrack. The activity of Twist-bound enhancers and Twist binding itself depends on Vielfaltig motifs, suggesting that Vielfaltig is more generally important for early transcription. The finding that the motif-content can predict context-specific binding and that the predictions work across different Drosophila species suggests that characteristic motif combinations are shared between sites, revealing context-specific motif codes (cis-regulatory signatures), which appear to be conserved during evolution. Taken together, this study establishes a novel approach to derive predictive cis-regulatory motif requirements for individual TF binding sites and enhancers. Importantly, the method is generally applicable across different cell-types and organisms to elucidate cis-regulatory sequence determinants and the corresponding trans-acting factors from the increasing number of tissue- and cell-type specific TF binding studies (Yanez-Cuna, 2012).

Recent ChIP experiments revealed that in vivo TF binding sites differ between different cell-types (or more generally cellular contexts), consistent with the frequent re-use of TFs in different cellular or developmental contexts and their context-specific functions. However, whether and how context-specific TF binding is encoded in the cis-regulatory sequences and the relation between the DNA sequence and in vivo binding has remained unclear (Yanez-Cuna, 2012).

This study used binding sites of a single TF in different contexts as pivots to study the sequence determinants of in vivo binding. By systematically comparing the binding site sequences, it was shown that they contain motifs for other TFs that are characteristic for each context and allow the prediction of context-specific binding. The motif-based predictions were sufficiently strong to pinpoint cis-regulatory requirements for individual binding sites, providing specific testable hypotheses, which were validated experimentally (Yanez-Cuna, 2012).

This finding has important implications for transcriptional regulation: First, it argues that context-dependent TF binding is determined by the cis-regulatory sequence, consistent with the sufficiency of enhancer sequences to recapitulate their endogenous chromatin state (i.e. histone modifications and DNA methylation and activity in different contexts. Second, in vivo binding appears to be determined by combinations of TF motifs rather than a single TF’s motif, therefore substantially increasing the information content and specificity of in vivo binding. Individual motifs are often only 4-6 nucleotides long and would therefore occur every 256 to 4096 nucleotides by chance (i.e. in random DNA sequences - even when motif degeneracy is not taken into account). Second, as different motif combinations are functional, a single TF can have context-specific binding sites and target genes depending on both, the cis-regulatory sequence that contains a certain combination of motifs and the cell-type that expresses the corresponding TFs. For example, Twist motifs in the vicinity of motifs for Snail, Dorsal, or Vielfaltig are preferentially bound early while those near motifs for Tinman (TIN) or Chorion factor 2 (CF2) are preferentially bound late, when these TFs are present, respectively (Yanez-Cuna, 2012).

Twist binding correlates with the binding of other mesodermal TFs (e.g. early with Myocyte enhancer factor 2 (MEF2) and TIN with Pearson correlation coefficients of 0.2 and 0.4, respectively) and ChIP-chip data for other mesodermal TFs are predictive of Twist binding using cross-validation, suggesting that partner TFs might assist each others binding in a correlated fashion (Yanez-Cuna, 2012).

In general, the action of partner TFs might be direct, e.g. mediated by direct protein-protein interactions (suggested e.g. for 'condition-altered binding' or passive e.g. by opening or otherwise preparing chromatin for TF binding. Some of the uncharacterized motifs might for example recruit chromatin remodeling factors and one of them indeed correlates with nucleosome-depleted open chromatin. It is conceivable that chromatin-mediated functions might be temporally decoupled such that partner TFs could act sequentially rather than simultaneously (Yanez-Cuna, 2012).

The TF Vielfaltig’s TAGteam motif appears to be a key determinant of early Twist binding: It is enriched in early binding sites, required to successfully classify them in a predictive framework, required for function of four early enhancers with diverse activity patterns, and necessary for Twist binding. Similarly, the early binding sites of other factors are enriched in TAGteam motifs (e.g. early MEF2 binding sites), suggesting that it is a general determinant of early binding and enhancer function. Interestingly, Vielfaltig is maternally deposited and has been shown to bind to the TAGteam motifs, a set of motifs that are enriched in regulatory regions of early blastodermal genes (Yanez-Cuna, 2012).

Vielfaltig is sufficient to activate enhancers that contain TAGteam motifs and required for early gene expression and cellularization. It has further been shown that Vielfaltig binds to about 60% of all genomic instances of the TAGteam motif. The finding that Vielfaltig is a key determinant of early binding is intriguing and suggests that Vielfaltig might help to open (or keep open) chromatin and allow TFs to access their binding sites on DNA thereby defining early enhancers (Yanez-Cuna, 2012).

The motif for Tramtrack is important for early Twist binding. Maternal Tramtrack has been proposed to repress zygotic transcription of early patterning genes in a concentration dependent manner, thereby explaining the timing of zygotic activation. Due to the overlap of different motifs, the 38% and 52% early Twist binding sites that depend on Vielfaltig and Tramtrack are conservative estimates, and both factors are likely important for additional binding sites. This study suggests that Vielfaltig and Tramtrack play an important regulatory role in the early embryo, preparing and/or regulating enhancers of a broad set of genes (Yanez-Cuna, 2012).

The finding that context-specific TF binding can be predicted using cross-validation indicates that the motif combinations extracted from training sequences are sufficiently general to correctly predict previously unseen test sequences. This means that different sites share characteristic sequence features and might function by similar means. In fact, similar patterns of motifs enriched in binding sites for different TFs are found in the same context (e.g. the early Drosophila mesoderm), suggesting that different cell-types have specific gcodesh that are indicative of binding for different TFs. This is generally true for all datasets studied in species as diverse as human, mouse, C. elegans, and Drosophila. In its ability to discover which cis-regulatory motifs (and the corresponding TFs) are relevant for different functionally defined sets of sequences (e.g. those active or bound in defined cellular contexts), this approach is similar to recent k-mer based enhancer predictions in mammals (Lee, 2011). It is complementary to recent thermodynamic models of gene expression in the early Drosophila embryo. Here, all relevant TFs, their motifs, and their cellular protein-concentrations are known, and the models predict enhancer activity for selected DNA sequences in order to gain insights into mechanistic aspects of transcriptional regulation, e.g. the importance of weak binding or homo- and heterotypic TF-TF interactions (Yanez-Cuna, 2012).

This study shows that motif-analyses of context-specific binding sites can identify the precise cis-regulatory sequence requirements and the trans-acting factors for individual genomic sites. This has important implications for the many TFs such as Hox factors or TFs downstream of signaling pathways, which are broadly expressed but regulate certain genes specifically in some tissues but not in others: it is foreseem that the recent increase in cell-type specific ChIP analyses will reveal specific cis-regulatory requirements and the corresponding transacting factors that define the regulatory state for many cell-types. As TF-binding has been shown to be predictive of cell-type specific enhancer activity, this will bridge the gap between the sequence, TF binding, and enhancer/CRM function and will ultimately reveal how cell-type specific regulatory information is encoded in the DNA sequence (Yanez-Cuna, 2012).

Akirin links twist-regulated transcription with the Brahma chromatin remodeling complex during embryogenesis

The activities of developmentally critical transcription factors are regulated via interactions with cofactors. Such interactions influence transcription factor activity either directly through protein-protein interactions or indirectly by altering the local chromatin environment. Using a yeast double-interaction screen, a highly conserved nuclear protein, Akirin, was identified as a novel cofactor of the key Drosophila melanogaster mesoderm and muscle transcription factor Twist. Akirin interacts genetically and physically with Twist to facilitate expression of some, but not all, Twist-regulated genes during embryonic myogenesis. akirin mutant embryos have muscle defects consistent with altered regulation of a subset of Twist-regulated genes. To regulate transcription, Akirin colocalizes and genetically interacts with subunits of the Brahma SWI/SNF-class chromatin remodeling complex. The results suggest that, mechanistically, Akirin mediates a novel connection between Twist and a chromatin remodeling complex to facilitate changes in the chromatin environment, leading to the optimal expression of some Twist-regulated genes during Drosophila myogenesis. It is proposed that this Akirin-mediated link between transcription factors and the Brahma complex represents a novel paradigm for providing tissue and target specificity for transcription factor interactions with the chromatin remodeling machinery (Nowak, 2012).

The Twist transcription factor controls many key processes in the establishment and patterning of the mesoderm, including organogenesis of the somatic body wall musculature during Drosophila embryonic development. As Twist activity regulates a large number of genes and processes over the course of mesodermal development, specific Twist functions are likely conferred through secondary and tertiary proteins that interact with Twist. In a screen for such interacting proteins, the highly conserved nuclear protein Akirin was identified. Akirin mutants show defects in myogenesis consistent with a role in regulating Twist activity, in particular at the Dmef2 gene. Akirin accomplishes this regulation through interactions with core subunits of the Brahma chromatin remodeling complex. Finally, Twist, Akirin and a BRM complex subunit were all found to co-occupy the Dmef2 enhancer during early embryogenesis, and this occupancy of a core BRM subunit requires the presence of Akirin. These data suggest that Akirin is required for optimal expression of this Dmef2 enhancer element. By contrast, the Twist-regulated eve MHE regulatory element does not require Akirin for its expression. Together, these results suggest that Akirin is an accessory protein that links Twist and the BRM chromatin remodeling complex for activation of specific Twist-regulated enhancers during Drosophila embryonic development. It is proposed that this Akirin-mediated link between Twist and the Brahma complex represents a novel paradigm for providing tissue and target specificity for Twist transcription factor activity with chromatin remodeling machinery during embryogenesis (Nowak, 2012).

Establishment of the somatic musculature during embryogenesis requires precisely regulated Twist activity. The muscle phenotype of akirin mutants and twist and akirin double heterozygotes indicates a functional interaction between Twist and Akirin during myogenesis. This interaction is direct, as Akirin and Twist physically bind. Additionally, both Twist and Akirin co-occupy the Dmef2 enhancer during embryogenesis, and their association is important for robust expression from the Dmef2 enhancer. Dmef2 is a critical regulator of myogenesis and is expressed throughout this process. Dmef2 coordinates multiple processes necessary for proper somatic myogenesis and regulates, in combination with Twist, a subset of Twist-regulated genes in a feed-forward manner. The missing, misattached, or duplicated muscle phenotypes that was observed in akirin mutants are similar to the phenotypes of embryos in which Dmef2 or twist expression levels are modified. For example, missing and misattached muscles were reported in incompletely rescued Dmef2 mutant embryos, while muscle duplications have been reported as a result of RNAi-mediated knockdown of twist. Therefore, akirin mutant muscle phenotypes likely reflect a perturbation in Twist activity, resulting in an early alteration in Dmef2 expression levels during embryogenesis. There are other elements in the Dmef2 enhancer that are bound and controlled by transcription factors such as Pannier, Medea and Tinman, that regulate Dmef2 expression during embryogenesis. It is unknown whether Akirin interacts with these factors at other Dmef2 control elements for their optimal expression or whether interaction with just one factor is sufficient. These questions are subjects for further study (Nowak, 2012).

Whole genome ChIP-on-chip experiments have identified almost 500 cis-regulatory elements that are bound by Twist during mesodermal development. Further, Twist co-regulates a number of targets together with Dorsal, Dmef2 and Tinman in a feed-forward manner. Interestingly, the data would argue that Twist does not require Akirin for optimal expression of all Twist-dependent enhancers. First, analysis of polytene chromosomes indicates that Akirin colocalizes with Twist at only 54% of Twist binding sites. Second, chromatin immunoprecipitation experiments indicate that Akirin is enriched at the Dmef2 enhancer during the first 10 hours of embryonic development, but not at the even-skipped MHE enhancer at the same time. Accordingly, no noticeable decrease in Eve expression, a reduction in Eve-positive clusters, or were defects observed in patterning of the DA1 muscle in akirin mutants. What could explain the different requirements for Akirin for optimal expression of Dmef2 versus eve? On one level, there are inherent differences in the complexities of these enhancers: While both the eve MHE and Dmef2 enhancers contain E-boxes bound and regulated by Twist, the eve MHE element also contains multiple DNA elements bound by a large array of factors from the Wingless, Decapentaplegic, and RTK-Ras-MAPK pathways. This greater complexity of regulation may obviate the need for Akirin to achieve optimal expression. On another level, the difference in Akirin regulation of these enhancers could be reflected in the local chromatin environment of these enhancers, and whether the individual enhancer needs to be remodeled by SWI/SNF activity for optimal expression. Analysis of deposited Modencode data suggests that the histone modification profile of the eve MHE remains largely static during the same temporal window as the chromatin immunoprecipitation experiment, while the environment surrounding the Dmef2 enhancer element changes during this same time. Further studies are required to identify the basis of this different requirement for Akirin at these Twist targets. Polytene analysis indicates that a large number (>200) of Akirin-positive bands colocalize with Twist in the genome. Identifying the complete list of Twist target genes that require Akirin will likely yield further clues as to the regulatory logic of Akirin together with chromatin remodeling during Twist target expression (Nowak, 2012).

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