bifid

DEVELOPMENTAL BIOLOGY

Embryonic

omb expression is first observed in the optic lobe anlagen, It later expands to a larger part of the developing embryonic brain and to the gnathal lobes [Image]. Cells in the central nervous system and peripheral nervous system begin to express omb after completion of germ band extention [Image]. Later in embryonic development, expression declines and only persists in the antennomaxillary complex and in part of the brain hemispheres (Poeck, 1993a).

During embryogenesis, omb expression is first observed in the optic lobe anlagen. It later expands to a larger part of the developing larval brain and to the gnathal lobes. Cells in the ventral and peripheral nervous systems begin to express omb after completion of germ band extension. Later in embryonic development, expression declines and only persists in the antennomaxillary complex and in part of the brain hemispheres. During the larval and pupal stages, omb expression in the brain is confined to the developing optic lobes and contiguous regions of the central brain. At these stages, only a few cells show expression in the ventral ganglion. In the eye imaginal disc, transcript accumulation is most conspicuous in a group of presumptive glia precursor cells posterior to the morphogenetic furrow and in the optic stalk. In the adult brain, expression is prominent in several regions of the optic lobe cortex and along the border between central brain and optic lobes (Poeck, 1993b).

Larval

During larval stage, omb expression in the brain is confined to the developing optic lobes and contiguous regions of the central brain. Only a few cells show expression in the ventral ganglion. In the eye imaginal disc, transcript accumulation is most conspicuous in a group of presumptive glia precursor cells posterior to the morphogenetic furrow and in the optic stalk (Poeck, 1993a).

The arrival of retinal axons in the Drosophila brain triggers the assembly of glial and neuronal precursors into a neurocrystalline array of lamina synaptic cartridges. Retinal axons arriving from the eye imaginal disc trigger the assembly of neuronal and glial precursors into precartridge ensembles in the crescent-shaped lamina target field. In the eye disc, photoreceptor cells assemble into ommatidial clusters behind the morphogenetic furrow (mf) as it moves to the anterior. The ommatidial clusters project their axon fascicles into the crescent-shaped lamina. Neuronal precursor cells of the lamina (LPCs) are incorporated into the axon target field at its anterior margin, which is demarcated by a morphological depression known as the lamina furrow. Glia precursor cells (GPCs) are generated in two domains that lie at the dorsal and ventral anterior margins of the prospective lamina. These glial precursors migrate into the lamina along an axis perpendicular to that of LPC entry. Postmitotic LPCs within the lamina axon target field express the nuclear protein Dac, as revealed by anti-Dac antibody staining. Like the eye, lamina differentiation occurs in a temporal progression on the anterioposterior axis. Axon fascicles from new ommatidial R-cell clusters arrive at the anterior margin of the lamina (adjacent to the lamina furrow) and associate with neuronal and glia precursors in a vertical lamina column assembly. At the anterior of the lamina, at the trough of the lamina furrow, LPCs await a retinal axon-mediated signal in G1-phase and enter their terminal S-phase at the posterior margin of the furrow. Postmitotic (Dac-positive) LPCs assemble into columns at the posterior margin of the furrow. In older columns at the posterior of the lamina, a subset of postmitotic LPCs express definitive neuronal markers as they become specified as the lamina neurons L1-L5. Lamina neurons L1-L4 form a stack in a superficial layer, while L5 neurons reside in a medial layer near the R1-R6 axon termini. These neurons arise at cell-type specific positions along the column's vertical axis. Lamina glial cells take up cell-type positions in the precartridge assemblies. Epithelial (E-glia) and marginal (Ma-glia) glia are located above and below the R1-R6 termini, respectively. Satellite glia are interspersed among the neurons of the L1-L4 layer. The Ma-glia and E-glia layers, both located ventral to the neuronal precursor column, sandwich the R1-R6 axon termini. The medulla neuropil serves as the target for R7/8 axons and is separated from the lamina by the medulla glia, situated just below the Ma-glia (Huang, 1998 and references).

Hedgehog, a secreted protein, is an inductive signal delivered by retinal axons for the initial steps of lamina differentiation. In the development of many tissues, Hedgehog acts in a signal relay cascade via the induction of secondary secreted factors. Lamina neuronal precursors respond directly to Hedgehog signal reception by entering S-phase, a step that is controlled by the Hedgehog-dependent transcriptional regulator Cubitus interruptus. The terminal differentiation of neuronal precursors and the migration and differentiation of glia appear to be controlled by other retinal axon-mediated signals. Thus retinal axons impose a program of developmental events on their postsynaptic field utilizing distinct signals for different precursor populations (Huang, 1998).

A number of markers distinguish glial and neuronal precursor cells from the corresponding mature cell types. The expression of optomotor-blind (omb) labels both glial precursors in the dorsal and ventral anlagen and mature glia that have migrated into the lamina target field. The glia cell marker Repo and the enhancer-trap lacZ insertion 3-109 are expressed by glia once they have entered the lamina target field. Cubitus interruptus (Ci), a transcriptional mediator of Hh signaling is expressed by LPCs anterior of the lamina furrow and by the postmitotic neuronal precursors within the lamina. The nuclear protein Dachshund is expressed only by neuronal precursors that have begun terminal differentiation and lie posterior to the lamina furrow. Thus, Omb and Ci label the glial and neuronal precursors, respectively, while the mature cells, following their interaction with retinal axons, additionally express Repo and Dac. In the lamina target field of eyeless mutants (mutants that project no neurons toward the optic disc), such as eyes absent (eya) or sine oculis (so), Dac expression is not detected and Repo expression is greatly diminshed. The migration and early differentiation of lamina glia are independent of Hh. Enhanced transcription of the putative Hh receptor, patched (ptc) is a universal characteristic of Hh signal reception. All classes of glia in the lamina region upregulate ptc expression in an hh-dependent fashion. These cells are thus Hh-responsive. All three classes of lamina glia, as well as medulla glia, that express a ptc-lacZ reporter construct are in close proximity to Hh-bearing retinal axons. Glia cell ptc reporter gene expression is not observed in hh- animals. This raises the question of whether Hh signal reception is responsible for the migration and/or subsequent maturation of glia cells. To determine whether the migration of glial precursors into the lamina target field is Hh-dependent, the distribution of Omb-positive cells was examined in hh- animals. In the wild type, a trail of Omb-positive cells delineates a path of glia migration from the dorsal and vental anlagen. Is glia precursor migration Hh-dependent? This was investigated by examining the distribution of Omb-positive cells in hh¹ mutant animals. hh¹ is a regulatory mutation that specifically affects hh expression in the visual system. In hh¹ animals, approximately 12 columns of ommatidia initiate differentiation in the eye imaginal disc before the anterior progression of the morphogenetic furrow ceases. hh¹ retinal axons lack Hh immunoreactivity by the time they reach the lamina target field and thus the Hh-dependent steps of LPC maturation fail to occur in hh¹ animals. Omb staining reveals a relatively normal number of glia precursors in the lamina target field of hh¹ animals, despite the absence of Dac induction. The Omb-positive cells are distributed uniformly along the dorsoventral axis among the retinal axon fascicles, but appear more closely spaced than in the wild type. A likely explanation for this spacing defect is the absence of the neuronal precursors that would constitute the majority of lamina cells at this point in development. To determine whether the glial precursors that enter the lamina target field in hh- animals express a retinal innervation-dependent marker, their expression of Repo was examined. In hh¹ animals, the Omb-positive cells within the lamina also express Repo. Moreover, the Repo-positive cells occupy proper layers above and below the R1-R6 axon termini expected for satellite, marginal and epithelial glia, though the lack of markers specific for these three glia types precludes an unambiguous determination of glial cell type. The presence of marginal and epithelial glia is consistent with the observation that R1-R6 growth cones terminate in their proper positions between these layers in hh- animals. The ectopic expression of Hh in the brains of `eyeless' animals is sufficient to induce the initial steps of LPC maturation in the absence of retinal axons. However, neither Hh nor the Hh-mediated events of LPC maturation are sufficient for glia cell migration and maturation (Huang, 1998).

The activities of a number of Hh signal transduction pathway components are now well characterized. Mutations at these loci have been shown to either mimic or block Hh signal reception in a cell-autonomous fashion. Examining the cellular requirements for these genes in mosaic animals should help illuminate the cellular circuitry that mediates the Hh-dependent events of lamina development. The seven-pass transmembrane protein encoded by smoothened (smo) acts as a positive effector of Hh signal reception, downstream of the Hh receptor Patched. If Hh exerts its effects directly on LPCs, it would be expected that loss of smo function should block the entry of G1-phase LPCs into S-phase and/or prevent the expression of Hh-dependent markers of lamina differentiation such as Dac. Inducing smo mutant clones reveals that with respect to lamina differentiation, smo acts cell autonomously. smo clones that extended to the posterior of the lamina are rare. It is possible that LPCs that cannot respond to Hh are not readily incorporated into the lamina and displaced by smo+ LPCs. LPCs that are unable to respond to Hh might be eliminated by cell death (Huang, 1998).

Pupal

The cuticle of the adult abdomen of Drosophila is produced by nests of imaginal histoblasts, which proliferate and migrate during metamorphosis to replace the polyploid larval epidermal cells. In this report, a detailed description is presented of the expression of four key patterning genes, engrailed (en), hedgehog (hh), patched (ptc), and optomotor-blind (omb), in abdominal histoblasts during the first 42 h after pupariation, a period in which the adult pattern is established. In addition, the expression is described of the homeotic genes Ultrabithorax, abdominal-A, and Abdominal-B, which specify the fates of adult abdominal segments. The results indicate that abdominal segments develop in isolation from one another during early pupal stages, and that some patterning events are independent of hh, wg, and dpp signaling. Pattern and polarity in a large anterior portion of the segment are specified without input from Hh, and evidence is presented that abdominal tergites possess an underlying symmetric pattern upon which patterning by Hh is superimposed. The signals responsible for this underlying symmetry remain to be identified (Kopp, 2002).

The dorsal cuticle of a typical abdominal segment contains a stereotyped sequence of pattern elements. At the anterior edge of each segment is the acrotergite, a narrow strip of naked sclerotized cuticle (a1). The remainder of the tergite is covered by trichomes, and can be subdivided into four regions. From anterior to posterior these regions are: a lightly pigmented region with no bristles (a2 fate); a lightly pigmented region that contains two to three rows of microchaetes (a3); a darkly pigmented region with one to two rows of microchaetes (a4); and a darkly pigmented region with a single row of macrochaetes (a5). The tergite is followed by the unpigmented posterior hairy zone (PHZ), which is composed of both anterior (a6) and posterior (p3) compartment cells. All trichomes and bristles in the segment are oriented uniformly from anterior to posterior. Finally, at the posterior edge of the segment is a zone of thin, naked intersegmental membrane (ISM), which can be subdivided into anterior smooth (p2) and posterior crinkled (p1) regions (Kopp, 2002).

The adult abdominal pattern is established in the first 2 days of pupal development, concurrent with the proliferation and migration of histoblasts and the destruction of the larval epidermal cells (LECs.) The spatial and temporal evolution of en, hh, ptc, and omb expression is followed during this critical period. The cuticle of each abdominal hemisegment is formed by three major histoblast nests. The anterior dorsal nest (aDHN) is composed of anterior compartment histoblasts and produces the tergite and part of the PHZ (a1-a6), whereas the posterior dorsal nest (pDHN) is composed of posterior compartment cells and produces the intersegmental membrane and the remainder of the PHZ (p1-p3). The ventral histoblast nest, which produces the sternite and pleura, contains both anterior and posterior compartment cells. en, hh, ptc, and omb are expressed in similar patterns in dorsal and ventral histoblasts, and the description is limited to the dorsal abdomen (Kopp, 2002).

en-lacZ and hh-lacZ are expressed throughout the pDHN, but are not expressed in the aDHN. hh-lacZ is expressed in a gradient within the pDHN, with expression highest at the anterior edge. A similar gradient can be detected in understained preparations of en-lacZ. ptc-lacZ expression is present in only a few cells at the posterior edge of the aDHN. omb-GAL4 expression is seen in the posterior of the aDHN and the anterior of the pDHN. omb-GAL4 expression is highest near the compartment boundary and decreases symmetrically in both anterior and posterior directions. By 20-24 h APF, the aDHN and pDHN fuse to form a combined dorsal histoblast nest (DHN). The gradients of en-lacZ and hh-lacZ expression within the posterior compartment become more pronounced at this stage. ptc-lacZ is expressed in a narrow stripe in the middle of the DHN, which is presumably located just anterior to the compartment boundary. The posterior border of this stripe is sharply defined, whereas a short gradient forms in the anterior direction; no ptc-lacZ expression can be detected at the anterior edge of the DHN at this time. omb-GAL4 is expressed in a wide, double-sided gradient in the middle of the DHN. Double labeling for ß-galactosidase and En protein in omb-GAL4²/UAS-lacZ pupae shows that omb-GAL4 is expressed in both compartments (Kopp, 2002).

To test whether Hh signaling is required for ptc and omb expression, homozygous hh^ts2 individuals were grown at 29°C for 48 h prior to dissection. Under these conditions, ptc-lacZ expression was completely eliminated at all stages. However, the effect on omb-GAL4 expression was different, depending on the stage of development. In early pupae, the symmetric expression of omb-GAL4 about the compartment boundary was only slightly reduced, while expression in the LECs appeared normal. In contrast, the later asymmetric expression of omb-GAL4 in the anterior compartment was virtually eliminated. No change was seen in the expression of en-lacZ or En protein in hh^ts2 pupae raised at 29°C, suggesting that the gradients of en expression in the posterior compartment are established independently of Hh function (Kopp, 2002).

Ubiquitous expression of omb causes double-posterior patterning of the tergite (a6-a5-a4-a4-a5-a6), whereas loss of omb function can cause reciprocal, double-anterior patterning (a2-a3-a3-a2). Ubiquitous expression of omb driven by the gain-of-function allele Qd^Fab has no effect on expression of en-lacZ, hh-lacZ, hh transcript, or the omb-GAL4² enhancer trap. Moreover, pupae hemizygous for the null allele omb²⁸² show normal expression of hh-lacZ, en-lacZ, and En protein (Koop, 2002).

These observations indicate that omb does not regulate the expression of hh , en, or omb. ptc-lacZ expression is also unaffected in omb²⁸² pupae, indicating that omb is not required for Hh signaling. However, Omb may potentiate Hh signaling: in Qd^Fab, the level of ptc-lacZ expression is increased relative to that of wild-type at both edges of the anterior compartment, although the timing of ptc-lacZ activation is not affected (Koop, 2002).

There are two main conclusions which may be drawn from the work to define Hh requirements in abdominal patterning: (1) Hh signaling is not required to specify pattern or polarity in the a2 and a3 regions, which comprise most of the anterior tergite; (2) abdominal tergites possess an underlying mirror-symmetric patterning that is specified independently of Hh. The phenotypes of hh^ts2 and omb² mutants, in which the a2 and a3 regions are often duplicated in mirror image, imply that a single patterning system is responsible for specifying both the a2 and a3 regions and the underlying mirror symmetry of the tergite. The identity of this system remains to be determined (Koop, 2002).

Several observations suggest that posterior compartments in the abdomen are organized in much the same way as anterior compartments. Ectopic expression of omb transforms the entire posterior compartment to PHZ (p3 fate) that has clear mirror-symmetry: trichomes in the anterior region orient toward the posterior, while those in the posterior region orient toward the anterior. Thus, anterior and posterior compartments in the abdomen may be organized in a similar fashion and patterned by similar mechanisms (Koop, 2002).

Adult

In the adult brain, expression is prominent in several regions of the optic lobe cortex and along the border between central brain and optic lobes (Poeck, 1993a).

Effects of Mutation or Deletion

optomotor blind was initially described as bifid, an alteration in longitudinal vein morphology at the base of the wing. Hemizygous mutant males develop to pharate adults that only rarely eclose but can be rescued from the pupal case. These animals show a severe maldevelopment of the optic lobes. In addition they have only rudimentary wings as well as a variant abdominal pigmentation. These three anatomical defects, in eye, wing and abdominal pigmentation are responsible for the three names that have been ascribed historically to this gene, optomotor blind, bifid and Quadroon (Pflugfelder, 1992a). The optomotor blind phenotype mutants lack a subset of lobula plate giant neurons which are thought to mediate optomotor behavior. These cells have been shown to collect information from large parts of the visual field and to relay it to the central brain, in particular to descending neurons of the cervical connectives (Pflugfelder, 1990).

brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila

The stereotyped pattern of Drosophila wing veins is determined by the action of two morphogens, Hedgehog (Hh) and Decapentaplegic (Dpp), which act sequentially to organize growth and patterning along the anterior-posterior axis of the wing primordium. An important unresolved question is how positional information established by these morphogen gradients is translated into localized development of morphological structures such as wing veins in precise locations. In the current study, the mechanism has been examined by which two broadly expressed Dpp signaling target genes, optomotor-blind (omb) and brinker (brk), collaborate to initiate formation of the fifth longitudinal (L5) wing vein. omb is broadly expressed at the center of the wing disc in a pattern complementary to that of brk, which is expressed in the lateral regions of the disc and represses omb expression. A border between omb and brk expression domains is necessary and sufficient for inducing L5 development in the posterior regions. Mosaic analysis indicates that brk-expressing cells produce a short-range signal that can induce vein formation in adjacent omb-expressing cells. This induction of the L5 primordium is mediated by abrupt, which is expressed in a narrow stripe of cells along the brk/omb border and plays a key role in organizing gene expression in the L5 primordium. Similarly, in the anterior region of the wing, brk helps define the position of the L2 vein in combination with another Dpp target gene, spalt. The similar mechanisms responsible for the induction of L5 and L2 development reveal how boundaries set by dosage-sensitive responses to a long-range morphogen specify distinct vein fates at precise locations (Cook, 2004).

The ab gene, which encodes a zinc finger protein containing a BTB/POZ domain, is required for L5 development as revealed by viable alleles such as ab¹, which bypass the early embryonic requirement for this gene in motor neuron axon guidance and result in distal truncation of the L5 vein (Hu, 1995). Four additional viable ab alleles have been recovered in a genome-wide screen for new wing vein mutants, one of which results in a somewhat stronger phenotype in which the L5 vein is consistently truncated proximal to the posterior cross-vein. Expression of ab in the wing disc was examined; it is expressed as a single stripe in the posterior compartment. The viable ab¹ allele is likely to be a regulatory mutation, since ab expression is greatly reduced in ab¹ mutant wing discs. ab expression is similarly reduced or undetectable in the other four independently isolated viable ab alleles. Double-label experiments with the vein marker Delta (Dl), which is expressed in L1 and L3-L5, reveal that ab is co-expressed with Dl in the L5 primordium (Cook, 2004).

Extension of a previous analysis of ab in initiating L5 development (Biehs, 1998; Sturtevant and Bier, 1995) has shown that ab functions early in L5 specification. Activation of all known vein genes, including rho, Dl, the caupolican and araucan genes of the Iroquois Complex (IroC), and argos, and repression of the intervein genes bs (also known as DSRF) and net, is lost in cells corresponding to the L5 primordium in ab¹ mutant wing discs. A determination was also made whether it is critical that ab expression is confined to a narrow stripe for regulating expression of vein or intervein genes. ab was ubiquitously misexpressed in the wing disc using the MS1096-GAL4 driver; such global activation of ab suppresses expression of vein genes, such as rho and Dl. This ab misexpression also caused vein-specific downregulation of the intervein gene bs, in the wing disc, but did not repress expression of other genes, including hh, ptc and dpp. This phenotype may result from unregulated production of a lateral inhibitory signal normally produced by vein cells to suppress vein development in adjacent intervein cells (Cook, 2004).

Whether restricted expression of ab in small clones is sufficient to induce vein development was also investigated. The flip-out misexpression system was used to generate clones of cells ectopically expressing ab in the wing disc; these cells (identified by Ab or ß-Gal expression) ectopically express the vein marker Dl and downregulate expression of the intervein marker Bs in a cell-autonomous fashion when located anywhere within the wing pouch. Adult wings containing small ab-expressing clones marked with forked also produce ectopic vein material cell autonomously. These results demonstrate that ab is necessary to control known gene expression in the L5 primordium, and is sufficient to induce vein development when expressed in a restricted number of cells. These data are consistent with ab acting in a vein-organizing capacity to direct L5 development (Cook, 2004).

The L2 primordium forms along the anterior boundary of the sal expression domain, in cells expressing low levels of sal and facing those expressing high levels of sal. The symmetrical disposition of the L2 and L5 veins, and the positioning of both of these veins by Dpp rather than Hh signaling, suggests that the L5 vein might form along the posterior border of the sal expression domain in much the same way that L2 is induced along its anterior border. However, two lines of evidence indicate that sal is not likely to be directly involved in determining the position of L5. (1) The posterior border of the sal expression domain is located several cells anterior to the L5 primordium (Sturtevant, 1997). (2) Although salm^- clones do occasionally result in the formation of ectopic posterior veins, they do so non-autonomously at a distance of several cell diameters from the clone border (Sturtevant, 1997). This phenotype is entirely different from the ectopic L2 veins that form at high penetrance immediately within the borders of anterior sal^- clones, located between the L2 and L3 veins (Sturtevant, 1997). Clones of a deficiency removing both salm and the related salr gene also result in the production of an ectopic vein, but this vein forms within the interior of such clones between L4 and L5, in a position corresponding to a cryptic vein, or paravein, which has a latent tendency to form along the posterior border of the sal domain (Cook, 2004).

Since the L5 primordium forms approximately four to six cell diameters posterior to the sal expression domain (Sturtevant, 1997), the expression was examined of other BMP target genes, omb and brk, relative to the L5 primordium. The borders of these gene expression domains are known to form posterior to that of the sal domain. Previous studies revealed that the domains of cells expressing high levels of omb and brk are largely reciprocal, although these genes are co-expressed at lower levels in cells along the border. Therefore the relative positions of the border of high level omb/brk expression was determined with respect to vein primordia marked by Dl (L1, and L3-L5) and Kni (L2). These experiments revealed that the L5 stripe of Dl expression forms inside and along the posterior border of the domain expressing high levels of omb, whereas the anterior border of the omb domain extends well beyond the L2 primordium. A complementary pattern was observed in wing discs of brk-lacZ flies double stained for ß-Gal and Dl, in which the L5 Dl stripe runs outside and along the border of the high level brk expression domain. Similar results were obtained using ab as a marker for the L5 primordium, in which the stripe of ab-expressing cells was found to lie within the omb domain, adjacent to high level brk-expressing cells. These expression studies reveal that omb and brk are expressed in the right location to play a role in positioning the L5 primordium (Cook, 2004).

As a first step in determining whether omb or brk play a role in L5 development, genetic interactions between these genes and ab were tested. Several viable or lethal ab alleles were crossed to stocks carrying the brk^m68 allele or a deficiency of brk, and trans-heterozygous brk^-/+;ab^-/+ F1 flies were examined for L5 phenotypes. None of the combinations of brk and ab alleles tested resulted in any dominant vein-loss phenotype in trans-heterozygotes. In addition, no enhancement of the homozygous ab¹/ab¹ L5 truncation phenotype was observed in brk^-/+; ab¹/ab¹ flies. By contrast, when trans-heterozygous interactions between ab and omb alleles were tested, consistent genetic interactions were observed. For example, omb¹/+; ab¹/+ flies exhibit truncations in the distal portion of L5 (with 3% penetrance, whereas neither ab¹/+ nor omb¹/+ heterozygotes ever show any L5 phenotype. Moreover, the omb¹ allele, which causes notching of the wing margin when homozygous but has no associated L5 phenotype, strongly enhances the ab¹/ab¹ L5 truncation phenotype. This interaction is evident in omb¹/+; ab¹/ab¹ females, and is very pronounced in omb¹/omb¹;ab¹/ab¹ double homozygous females or hemizygous omb¹/Y; ab¹/ab¹ males. These results suggest that omb and ab function in concert to promote L5 formation (Cook, 2004).

Additional detailed experiments have shown that (1) misexpression of omb and brk shifts or eliminates the L5 and L2 veins; (2) omb is required cell autonomously for L5 development; (3) brk is required for the production of an L5 inductive signal, and (4) ab acts downstream of brk in L5 development (Cook, 2004).

Thus, this study examined the role of two Dpp target genes, brk, which is expressed in a domain abutting the L5 primordium, and omb, which is expressed in a domain just including the L5 primordium, in establishing the position of this vein. The results suggest a model for how the BMP activity gradient induces formation of the L5 primordium in the posterior compartment of the wing. According to this model, L5 development is initiated within the posterior region of the wing where brk and omb are expressed in adjacent domains with a sharp border between them. Since brk^- clones induce vein development within the clone along the border with brk⁺-neighboring cells, it is suggested that brk-expressing cells produce a short-range vein-inductive signal, Y, to which they cannot respond. This signal acts on neighboring omb-expressing cells to initiate vein development. The additional cell-autonomous requirement for Omb activity to respond to this Brk-derived signal suggests that the intracellular effector of the vein inductive signal Y must act in combination with Omb to induce vein formation. Because Brk is a repressor of omb expression, the combined requirement for the short-range Brk-derived vein-inductive signal and Omb activity within responding cells constrains L5 initiation to omb-expressing cells adjacent to brk-expressing cells. In this scheme, Brk plays at least two distinct roles in L5 induction. First, as a repressor of omb, Brk defines the border between the brk and omb expression domains, and, second, brk-expressing cells are the source of a vein-inductive signal required to initiate L5 development within adjacent omb-expressing cells (Cook, 2004).

A key mediator of L5 induction is the Ab transcription factor, which is expressed in a narrow stripe along the brk/omb border, just within the omb expression domain. ab is required for expression of all known vein genes and for downregulation of intervein genes in the L5 primordium (Biehs, 1998). Similarly, the ability of brk^- clones to induce an ectopic posterior vein depends on ab function. In addition, localized misexpression of ab in small flip-out clones leads to induction of vein markers in wing imaginal discs and to the formation of ectopic patches of vein material. The vein-organizing activity of ab depends on its being expressed in a localized pattern, since ubiquitous expression of ab suppresses vein development throughout the wing disc. This effect of ubiquitous ab misexpression is similar to that observed previously for ubiquitous expression of kni or knrl, in which all distinctions between vein and intervein regions are lost although expression of other genes in the wing disc are not perturbed. One explanation for this vein-erasing phenotype is that kni/knrl and ab control the expression of a lateral inhibitory signal. Consistent with this possibility, small ab flip-out clones autonomously express the lateral inhibitory signal Dl. According to the model, establishment of the L5 primordium requires input from both omb (cell autonomous) and brk (cell non-autonomous), which collaborate to initiate ab expression in a narrow stripe along their borders (Cook, 2004).

A curious phenotype associated with some brk^- clones generated in an ab¹/ab¹ background is the formation of diffuse wandering veins within the interior of the clone. A similar disorganized ectopic vein phenotype is also observed in a fraction of omb^- brk^- double mutant clones. This phenotype may reflect the lack of a lateral inhibitory factor (e.g. Dl) produced by ab-expressing cells to suppress vein formation in neighboring cells. The observation that ubiquitous expression of ab suppresses vein formation throughout the wing disc is consistent with this possibility. It is also possible that omb plays a role in promoting intervein development as well as in activating ab expression. Additional analysis will be needed to address this question (Cook, 2004).

Previous analysis of L2 initiation lead to a model in which sal-expressing cells produce a short-range vein-inductive signal (X) to which they cannot respond (Sturtevant, 1997). In response to signal X, neighboring cells outside of the sal domain express the L2 vein-organizing genes kni and knrl. In addition, analysis of an L2-specific cis-regulatory element of the kni/knrl locus provided indirect evidence for negative regulation by a repressor, possibly Brk, expressed in peripheral/lateral regions of the wing disc (Cook, 2004).

An interesting question regarding veins forming within more anteriorly located brk^- clones is whether they have an L2- or an L5-like identity. In one case, these veins express kni, but not Dl, suggesting that they have an L2-like identity. In the other, the ectopic veins induced anteriorly by brk^- clones require omb function, as do L5-like veins generated in the posterior compartment of the wing. This latter observation suggests that the brk^- border in anterior regions acts as it does in posterior regions of the wing disc, but that its effect may be mediated by the L2 organizing kni/knrl locus rather than the L5 organizing gene ab. This hypothesis might provide an explanation for why ectopic veins that form in various mutant backgrounds tend to form along a line running between the L2 vein and the margin (which is referred to as the P2 paravein) (Sturtevant, 1997). This sub-threshold vein promoting position may be defined by the anterior border of brk and omb expression. Further analysis of the identity of these ectopic veins will be required to resolve this question (Cook, 2004).

Since the L2 and L5 veins form at similar lateral positions within the anterior and posterior compartments of the wing, respectively, it is informative to compare the mechanisms by which positional information is converted into vein initiation programs in these two cases. The positions of these two veins are determined by precise dosage-sensitive responses to BMP signaling emanating from the center of the wing; these responses are mediated by the borders of the broadly expressed, Dpp signaling target genes sal and omb. Brk also plays a role in initiating both L2 and L5 development. In the posterior compartment, Brk leads to the production of a hypothetical vein-promoting signal Y, which has a function and range similar to the putative L2 vein-inducing signal X, produced by sal-expressing cells. It is not clear whether the signals X and Y are the same or different; however, an important difference between L2 and L5 initiation is that only L5 has an additional requirement for omb function. This dual requirement for omb function within the L5 vein primordium and a short-range inductive signal in neighboring brk-expressing cells provides a stringent constraint on where the L5 primordium forms. Brk may also directly repress expression of the vein-organizer gene ab in cells posterior to the L5 primordium, analogous to the proposed role as a repressor of kni/knrl anterior to L2. One possible rationale for induction of the L5 vein depending on inputs from both omb and brk is that these genes are expressed in partially overlapping patterns and neither pattern may carry sufficiently detailed information to specify the position of the L5 primordium alone. Although the omb and brk expression levels fall off relatively steeply (i.e. over a distance of six to eight cells), these borders are not as sharp as the anterior sal border (two to three cells wide), which alone is sufficient to induce the L2 primordium (Cook, 2004).

A final similarity between the initiation of L2 and L5 formation is that induction of both veins is mediated by a vein-organizing gene that regulates vein and intervein gene expression in the vein primordium. Although kni and ab are members of different subfamilies of Zn-finger transcription factors, they are both expressed in a narrow stripe of cells along their respective inductive borders, and ubiquitous misexpression of either gene results in elimination of vein pattern in the wing disc. Thus, the L2 and L5 veins are induced by remarkably similar mechanisms and principles of organization. Further comparison of the mechanisms of these developmental programs should provide insights into the degree to which general and specific vein processes define the L2 versus the L5 vein identity (Cook, 2004).

Induction of Drosophila wing veins at borders between adjacent gene expression domains provides a simple model system for studying how information provided by morphogen gradients is converted into the stereotyped pattern of wing vein morphogenesis. Each of the four major longitudinal veins (L2-L5) is induced by a for-export-only mechanism in which cells in one region of the wing produce a diffusible signal to which they cannot respond. In the case of L3 and L4, an EGF-related signal (Vein) is produced between these veins in the central organizer where expression of the EGF receptor is locally downregulated. With respect to L2, response to the vein-inductive signal X is repressed in Sal-expressing cells that produce the hypothetical signal X. Finally, the L5 vein-inductive signal produced by brk-expressing cells depends on omb, the expression of which is repressed by Brk (Cook, 2004).

For-export-only mechanisms also underlie the induction of boundary cell fates in many other developmental settings. In the well-studied Drosophila wing, the earliest and most rigorously defined boundaries are the AP and DV borders, which are determined by Hh and Notch signaling, respectively. These compartmental borders define domains of non-intermixing groups of cells, and function as organizing centers by activating expression of the long-range morphogens Dpp and Wingless (Wg), respectively. In both cases, cells in one compartment produce a signal to which they cannot respond. This signal is constrained to act only on neighboring cells in the adjacent compartment. Other well-studied examples of for-export-only signaling include: induction of the mesectoderm in blastoderm stage Drosophila embryos by a likely cell-tethered Notch ligand expressed in the mesoderm; induction of parasegmental expression of stripe via Wg, Hh and Spi signaling in gastrulating Drosophila embryos; induction of mesoderm in Xenopus embryos by factors produced in the endoderm under the control of VegT, and formation of the DV border of leaves in plants controlled by the PHANTASTICA gene. The similar but distinct mechanisms for inducing the L2 and L5 vein primordia offers a well-defined system for examining these relatively simple cases in depth. These inductive events take place at the same developmental stage but within separate compartments of a single imaginal disc, and should provide general insights into the great variety of mechanisms that can be co-opted to accomplish for-export-only signaling (Cook, 2004).

The relative role of the T-domain and flanking sequences for developmental control and transcriptional regulation in protein chimeras of Drosophila OMB and ORG-1

optomotor-blind and optomotor-blind related-1 (org-1) encode T-domain DNA binding proteins in Drosophila. Members of this family of transcription factors play widely varying roles during early development and organogenesis in both vertebrates and invertebrates. Functional specificity differs in spite of similar DNA binding preferences of all family members. Using a series of domain swap chimeras, in which different parts of Omb and Org-1 were mutually exchanged, the relevance of individual domains were investigated in vitro and in vivo. In cell culture transfection assays, Org-1 is a strong transcriptional activator, whereas Omb appears neutral. The main transcriptional activation function was identified in the C-terminal part of Org-1. Also in vivo, Omb and Org-1 showed qualitative differences when the proteins are ectopically expressed during development. Gain-of-function expression of Omb is known to counteract eye formation and results in the loss of the arista, whereas Org-1 has little effect on eye development but causes antenna-to-leg transformations and shortened legs in the corresponding gain-of-function situations. The functional properties of Omb/Org-1 chimeras in several developmental contexts is dominated by the origin of the C-terminal region, suggesting that the transcriptional activation potential can be one major determinant of developmental specificity. In late eye development, however, a strong influence of the T-domain on ommatidial differentiation is observed. The specificity of chimeric omb/org-1 transgenes, thus, depends on the cellular context in which they are expressed. This suggests that both transcriptional activation/repression properties as well as intrinsic DNA binding specificity can contribute to the functional characteristics of T-domain factors (Porsch, 2004).

org-1, a Drosophila homologue of mammalian TBX1, is most strongly expressed during embryogenesis where it appears to play a role in the patterning of the visceral mesoderm (Lee, 2003). Expression in imaginal discs is poorly detectable. No org-1 mutants are known. Ubiquitous induction of org-1 RNA interference causes pupal lethality. Strong RNAi expression during imaginal development revealed an org-1 requirement predominantly in thorax and distal wing development but no apparent involvement in eye-antennal or leg discs. RNAi constructs from two different parts of the org-1 cDNA yielded the same phenotype indicating specificity of the interference experiment (Porsch, 2004).

The T-domain was originally defined by a statistical analysis of the Omb protein sequence and by homology to the mouse Brachyury protein. Further work has revealed the Brachyury T-domain as a sequence specific DNA binding domain, C-terminally flanked by a set of transactivation and repression domains. In vitro target site selection analyses indicate preferential Brachyury binding to a 20 bp degenerate palindrome made up of two closely related half sites. Numerous in vitro selection and in vivo transcription experiments, and the characterization of actual target genes identified the Brachyury consensus half site as a common target of all T-domain proteins investigated, so far. This also holds for Omb and Org-1. Within the enhancers of T-domain protein-controlled genes, half sites generally occur in small groups of two or more members. The half sites are variably spaced, occur in all conceivable relative orientations, and can show considerable deviation from the consensus sequence (Porsch, 2004 and references therein).

The majority of T-box factors investigated to date, are transcriptional activators. ORG-1 also functions as an activator. The only known transcriptional repressors in the T-box gene family are TBX2 and TBX3/ET, the closest vertebrate homologs of Omb. t OMB, too, is a member of this functional subgroup. Furthermore, T-domain proteins can physically interact with other DNA bound factors to control target gene expression. On natural promoters, closely related T-domain proteins can replace one another while more distantly related family members are inactive or exert different effects. However, even in the same cellular context, T-domain proteins of the same subfamily can differ in developmental specificity. An example for this is provided by the role of Tbx4 and Tbx5 in limb development. Tbx4 and Tbx5 code for closely related T-domain proteins. In higher vertebrates, they are expressed in nearly complementary patterns during limb formation: Tbx5 is exclusively expressed throughout the forelimb bud, whereas Tbx4 mRNA is predominantly found in the hindlimb bud. In the chick, misexpression of Tbx5 in the presumptive hindlimb region causes a partial transformation of the leg into wing, resulting in wing/leg mosaic limbs. Conversely, ectopic Tbx4 in the developing wing promotes the growth of leg-like structures. The mechanisms underlying T-domain protein functional specificity are therefore far from understood (Porsch, 2004 and references therein).

This study addresses the question about the relative role of different parts of T-domain proteins (T-domain versus flanking regions) for their biological activity. Two distantly related Drosophila T-domain proteins (Omb and Org-1 share 61% of residues in the T-domain but are highly diverged outside) cause distinct phenotypes when ectopically expressed during fly development. This provides the basis for a domain swap experiment. Chimeras were created in which the T-domain or the T-flanking regions were exchanged between the two proteins and whether a given phenotypic effect was associated with a particular protein domain was determined. The chimeric genes were expressed in cultured cells and in transgenic flies. In transfected cells, Omb and Org-1 show a clear-cut difference in transcriptional activation potential which can be attributed largely to the origin of the C-terminal region. Also in vivo, in several tissues, the developmental consequences of chimeric gene expression were dominated by the origin of the C-terminal domain. In one developmental context (retinal differentiation), however, the developmental outcome was affected by the origin of all parts of the chimeric protein. The results demonstrate the importance of protein sequences lying both within and outside of the T-domain for the functional specificity of these T-domain proteins and show that distinct parts of Omb and Org-1 are required for their specific effects in different cellular contexts (Porsch, 2004).

Expression of omb or org-1 during imaginal disc development can interfere with normal imaginal development or promote the development of novel adult features. The phenotypic consequences of ectopic expression differed profoundly for Omb and Org-1. This observation provided the basis for an in vivo assay to investigate the significance of the T-domain versus the N- and C-terminally flanking regions in Omb and Org-1 for developmental specificity (Porsch, 2004).

The data obtained with Omb/Org-1 chimeras indicate that regions outside of the T-domain can govern chimera developmental specificity. In dpp-Gal4 driven expression, the C-terminal domains of Omb or Org-1 are sufficient to endow Omb/Org-1 chimeras with Omb-like or Org-1-like character in the early development of both eye and antenna. These data cannot be interpreted as signifying that differences in DNA binding specificity are irrelevant for the different developmental roles of the two T-domain proteins. They do suggest, however, that the intrinsic (i.e., T-domain autonomous) DNA binding specificity is not the decisive factor. Rather, the C-domains of the two proteins could differ in activation/repression potential or could specify DNA binding extrinsically by interaction with cofactors. In cell culture assays it has been shown that Omb and Org-1 drastically differ in activation potential, Org-1 being a strong activator while Omb could not activate transcription from the hsp70 minimal promoter. Due to the low basal transcription rate of the reporter gene, these experiments could not provide direct evidence for a repressive role of Omb. Two observations suggest, however, that in particular the Omb C-domain is able to act as a repressor. (1) The Omb C-terminal region contains several runs of homopolymeric alanine. This has been noted as a characteristic feature in several transcriptional repressors. (2) The low transcriptional activation by the Org-1 N-domain in the presence of the Omb C-domain and the apparent activational synergism between the Org-1 N- and C-domains suggest repressive qualities of the Omb C-domain (Porsch, 2004).

This difference in transcription activation function correlates with the dominant character of the C-domains in the Omb/Org-1 chimeras. This suggests that, in early eye-antenna development, the effects of Omb and Org-1 are governed by their distinct transcriptional activation potentials. These results do not rule out a more specific role of the C-terminal domains. This question can now be addressed using heterologous activation/repression domains. In early eye development, the phenotypic consequences of ectopic omb and org-1 expression appear as antagonistic, omb leading to a reduction, org-1 to an increase in ommatidial number. One explanation for this antagonism is that, in this tissue, Omb and Org-1 can bind to the same set of target genes, whose activation leads to an increase in ommatidial number, while their repression causes the opposite effect. There is precedence for the antagonistic action of T-domain proteins in developmental decisions. Tbx5 and Tbx2 bind to the ANF gene promoter in mammalian heart development either activating or repressing its transcription. In zebrafish mesoderm development, the T-domain proteins Ntl and Tbx6 compete for common target sites. Comparable to Org-1 and Omb, NTL is a transcriptional activator and Tbx6 has no activation potential. In a transphylum domain-swap experiment between Brachyury homologs, a quantitative determinant of mesoderm induction could also be localized to the C-terminus. In this case, it was not determined whether there was a correlation with the transactivation potential. In the same experiments, a determinant for endoderm specification was identified in the N-terminus. The relevance of the T-domain flanking domains is also apparent from the analysis of TBX5 mutations in Holt-Oram syndrome (HOS) patients (Fan, 2003). HOS can be elicited by mutations in the flanking domains, which do not affect DNA binding in vitro but completely abolish synergistic interaction with Nkx2-5 (Porsch, 2004 and references therein).

All chimeras containing the Org-1 T-domain cause an unusual hairy eye phenotype when expressed under GMR-Gal4 control indicating an involvement of T-domain DNA binding specificity. GMR-Gal4 drives gene expression in all cells of the eye disc epithelium posterior to the morphogenetic furrow. In this cell population, omb is expressed and required only at the dorso-ventral margins while org-1 appears to play no role in normal development and has not been detected. GMR-Gal4, therefore, is active in a tissue where neither gene is normally expressed. It is difficult to conceive of retinal degeneration and loss of interommatidial bristles as the two alternatives of one developmental decision effected by the antagonistic regulation of one set of target genes. In this case, it appears more likely that Omb and Org-1 ectopically bind and regulate different target genes (Porsch, 2004).

In a previous study on T-box specificity, Smith and colleagues (Conlon, 2001) investigated the relevance of the T-domains of Xbra, VegT, and Eomesodermin for determination of target gene specificity by expressing T-domain fusion proteins in early Xenopus embryos. In this case, the specificity of the three investigated proteins was determined to a large extent, but not exclusively, by the T-domain. As outlined in the introduction, all T-domain proteins are able to interact with (groups of) half sites as originally defined by Brachyury binding studies. Individual T-domain proteins can, however, differ in other binding characteristics such as dimerization tendency or the preference for certain arrangements of binding sites with regard to spacing or orientation. The replacement of a single presumably DNA binding amino acid of VegT and Eomesodermin with the corresponding amino acid of Xbra is sufficient to change the target gene expression profile of VegT and Eomesodermin to resemble that of Xbra (Conlon, 2001). This suggests that differences in DNA binding can be crucial for target gene specificity of T-domain proteins in vivo. As stated above, intrinsic DNA binding specificity as exclusive determinant of developmental specificity is not compatible with the overriding influence of the C-terminal domain which was found in some experiments (Porsch, 2004).

The phenotype of a given Omb/Org-1 chimera could be Omb-like or Org-1-like depending on the Gal4 driver under whose control it was expressed. The clearest example for this observation was provided by chimera org-1N+ombT+ombC, which, when expressed under dpp-Gal4 control, unambiguously showed omb-specific phenotypes in eye and antenna, but caused an org-1-like character in ommatidia when activated by GMR-Gal4. It is concluded from these findings that different protein domains contribute to Omb or Org-1 function in the undifferentiated eye/antennal disc versus differentiating retina cells. The C-domains of Omb and Org-1 are the main specificity determinants in Omb/Org-1 chimeras in early eye/antennal development, whereas generally all three Omb domains contribute to Omb specificity during ommatidial differentiation. This suggests that the specificity of Omb/Org-1 chimeras is not solely intrinsic to their protein sequences, but also depends on the cellular context in which they are expressed. In T-domain proteins, a cell-type dependence was described for the nucleo-cytoplasmic distribution (Collavoli, 2003) and for activation/repression properties (Stennard, 2003). The situation with T-domain proteins is thus comparable to that observed with homeodomain DNA binding proteins. In various proteins and cellular contexts a domineering influence of both the homeodomain and flanking protein domains on developmental specificity has been described. The different phenotypes of Omb/Org-1 chimeras in different cell types and developmental stages will aid in the identification of tissue- and stage-specific cofactors (Porsch, 2004 and references therein).

It was noted that certain Omb/Org-1 domain compositions give rise to novel phenotypes that are not observed with the parental proteins Omb or Org-1. For example, misexpression of org-1N+ombT+org-1C or ombN+org-1T+ombC induces caudal expansions of the eye. In the antenna, third antennal segments showed bulbous outgrowths upon expression of various chimeras. Certain omb/org-1 chimeras induced abundant ectopic microchaetae in the eye field when misexpressed with GMR-Gal4. The loss of interommatidial bristles, as caused by org-1 overexpression, is a rather common phenotype which can, for example, be observed upon changes in the signaling activity of the Notch and wingless pathways. The ectopic formation of microchaetae on the facet eye has not been described previously, in Drosophila melanogaster. However, in Drosophila robusta, the isolation of a spontaneous hairy eyed mutant has been reported which may have been phenotypically similar (Porsch, 2004).

Possible explanations for the potency of certain chimeras to produce novel phenotypes include an altered regulation of Omb or Org-1 target genes and/or new target gene specificities due to inappropriate protein-protein interactions. In the metazoan T-box proteins, the N- and C-terminal domains flanking the T-domain generally are poorly conserved, even between closely related species. This lack of constraint may explain the great versatility of T-box genes which evolved to control a wealth of biological processes (Porsch, 2004 and references therein).

The developmental gene optomotor-blind (omb) encodes a T-box-containing transcription factor that has multiple roles in Drosophila development. Previous genetic analyses established that omb plays a key role in establishing the abdominal pigmentation pattern of Drosophila melanogaster. In this report patterns of omb nucleotide variation were examined in D. polymorpha, a species that is highly polymorphic for the phenotype of abdominal pigmentation. Haplotypes at this locus fall into two classes that are separated by six mutational steps; five of these mutational events result in amino acid changes. Two lines of evidence are consistent with a role for omb in the abdominal pigmentation polymorphism of D. polymorpha: (1) it was found that haplotype classes of omb are correlated with abdominal pigmentation phenotypes, as are microsatellite repeat numbers in the region; (2) tests of selection reveal that the two haplotype classes have been maintained by balancing selection. Within each class there is a significantly low amount of diversity, indicative of previous selective sweeps. An analysis including D. polymorpha's closest relatives (members of the cardini group) provides evidence for directional selection across species. Selection at this locus is expected if omb contributes to variation in abdominal pigmentation, since this trait is likely of ecological importance (Brisson, 2004).

At the distantly related species level (i.e., across most of the Drosophila phylogeny) nucleotide variation is so high in the N-terminal region of omb that it is unalignable. The amino acid sequences encoded also have few alignable regions. However, it is notable that despite this large amount of variation, two domains, at the beginning and end of the sequence considered in this study, are nearly perfectly conserved and therefore likely to be of functional importance. A transcription repression domain lies upstream of the T-domain of Tbx2, the mouse homolog of omb. Whether or not a similar repression domain exists in omb will require functional studies. Despite showing extensive amino acid and length variation, the remaining portion of the N-terminal region studied retains similar estimated isoelectric points. In marked contrast, there was very little variation among distantly related species in the T-domain. All nucleotide variation in this region produced synonymous codons. This result is not surprising, given the functional significance of this region (Brisson, 2004).

The N-terminal domain contains within it a number of microsatellite runs. Microsatellite runs are common in the coding regions of important developmental genes, especially in the motifs flanking the DNA-binding domain. Greater that 80% of Drosophila proteins with multiple runs seem to function in the regulation of transcription. Among these are such well-known genes as Ultrabithorax, period, mastermind, and nonA. Further, alanine, serine, and glutamine account for a significant proportion of the runs. At the omb locus, runs of these three amino acids are seen as well as a threonine and three asparagine runs. These runs are in close proximity to the DNA-binding region, the T-domain. Length variation of these homopolymers may cause differences in structure that allow plasticity in protein-protein interactions. The multiple asparagine (N) runs are particularly interesting, since N runs avoid the secondary structures of alpha-helices and ß-strands and may provide binding sites for protein-protein interactions (Brisson, 2004).

Genetic studies have led to the expectation that omb might contribute to variation in the width of the abdominal pigment band among individuals of Drosophila. Indeed, a significant association is found between the A and B haplotype classes and other haplotype clades and abdominal pigmentation variation. There is also a significant association between microsatellite variation within the N-terminal omb coding region and the pigmentation phenotype. Seven of eight variable microsatellites in this region had repeat numbers that were nonrandomly distributed across the three phenotypic categories of light, medium, and dark pigmentation. These correlations indicate that omb (or a regulatory region of omb or another locus in linkage disequilibrium with this region of omb) may play a role in natural variation in this trait. Further, because omb haplotypes do not correlate with geography as determined by phylogeographic nested clade analysis, the correlation observed here is not a by-product of geographic sampling (Brisson, 2004).

The association between omb haplotypes and pigmentation variation would be greatly strengthened by linkage information from omb haplotypes segregating in an F₂ generation. Unfortunately, the only lines available in the laboratory are light lines that have a dominant light allele and a single dark line. These light lines were established from flies collected in the northern part of D. polymorpha's range, the only region in which the dominant light allele is known to exist. Therefore, these are not appropriate for examining the polymorphism that was detected in flies from southern Brazil, where the dominant light allele is not present. And, any results from these types of analyses would not be able to illuminate whether omb is the major locus underlying pigmentation variation for this species, because a cross between a light line with a dominant light allele and a dark line would produce only light or dark flies, never flies with intermediate levels of pigmentation. Therefore the amount of phenotypic variation explained by omb haplotypes on the basis of the data presented here must be estimated. The average phenotype of an individual bearing an A-class allele is 6.0, while the average phenotype of an individual bearing a B-class allele is 6.9. The phenotypic differences between the two classes are thus on the order of one phenotypic class, suggesting omb may be a modifier locus (Brisson, 2004).

Variation at omb is best described as biallelism, with two main haplotype clusters, referred to in this study as allele class A and allele class B. Allele class A is associated with very little variation (fewer haplotypes) compared to allele class B. These haplotype counts argue that allele class B is the older of the two classes. In contrast, the amino acid states found in allele class A are identical to those found in the majority of the other species of the cardini group examined here. Thus, if placed within a phylogenetic context, it is apparent that allele class A is the ancestral state. However, the wide geographic spread (all populations sampled) and haplotype diversification observed in this study for allele class B indicate that it has been in existence for some time. In fact, it was the only allele class found in 13 of the 19 populations, although the sparse sampling makes it possible that both alleles are found in the majority of the populations. It is not known whether the replacement substitutions that separate the two allele classes result in a functional change in the omb protein or whether they are in linkage disequilibrium with other changes in either the regulatory regions or the protein-coding region that are in some manner selectively advantageous (Brisson, 2004).

Because molecular variation can be shaped by both demographic and selective processes, it is important to consider the demographic history of D. polymorpha. Certain scenarios could explain the biallelic structure observed in the N-terminal domain of omb. (1) If D. polymorpha is fragmented into several smaller populations, genetic drift can cause populations to diverge from one another, resulting in a situation with very little polymorphism within populations compared to polymorphisms among populations. (2) The breakdown of a previous barrier between divergent populations or introgression from a related species may result in biallelism (Brisson, 2004).

Insight into these scenarios is aided by previous work on the phylogeography of the species. D. polymorpha is a widespread species found throughout much of South America in a wide diversity of habitats and is therefore not prone to inbred, fragmented populations. The only structure among populations of the species from south and central Brazil (the same individuals used in this study), as determined by both a mitochondrial locus (cytochrome b and flanking regions) and a nuclear locus (pgd), is that of restricted gene flow via isolation by distance and one inference of a contiguous range expansion. Therefore, population fragmentation can be excluded as a causative factor in the maintenance of biallelism at this locus. Further, omb haplotypes from all the closely related species to D. polymorpha display nucleotide differences significantly different from D. polymorpha haplotypes, making it unlikely that introgression from a related species is contributing to variation at the omb locus in D. polymorpha (Brisson, 2004).

Finally, a biallelic structure can be maintained by a chromosomal inversion. Indeed, previous work on D. polymorpha indicates that inversion polymorphisms are widespread in this species. To test whether the A or B alleles are associated with inversions, heterozygotes were created for allele classes A and B and their chromosomal morphology was examined. Inversions were observed on the X chromosomes, but not on the autosomes. In this analysis, omb was determined to not be on the X chromosome in D. polymorpha (wild-caught males display two unique haplotypes), as it is in D. melanogaster. Therefore, it is concluded that an inversion polymorphism is not responsible for maintenance of the two haplotype classes of omb in D. polymorpha (Brisson, 2004).

Because omb is a candidate for abdominal pigmentation variation, the signature of selection at this locus was sought within a single species, D. polymorpha. The pattern of a significantly large number of singleton mutations (rare haplotypes) was observed in comparison to the neutral expectation. This pattern implies that a diversity-reducing event occurred in the past. In contrast, a contingency test of neutrality indicates balancing selection was acting to preserve both haplotype classes. Therefore, the history of selection at this locus within D. polymorpha appears to be selective sweeps within each allele class, while both classes are maintained by balancing selection (Brisson, 2004).

The source of the balancing selection at omb within D. polymorpha, if it is indeed involved in the production of the pigmentation phenotype, could be the habitat of these generalist fruit flies. In southern Brazil, populations of D. polymorpha found in forested habitats have, on average, lighter pigmented abdomens than populations found in open, drier habitats. Therefore, alternative alleles could be selected in different environments and thus in different populations, maintaining a balanced polymorphism across the species (Brisson, 2004).

The contingency test also revealed a higher number of fixed (interspecific) amino acid replacements relative to intraspecific tip amino acid replacements, suggesting that directional selection is favoring amino acid changes among species. These results, combined with the intraspecific selection tests, suggest the following picture: within D. polymorpha nucleotide replacement variation at omb has occurred with some frequency, but has been reduced by purifying selection within each allele class while both allele classes are maintained by balancing selection. In contrast, interspecific comparisons suggest that directional selection has been responsible for fixing differences between species. This behavior is consistent with that expected for a gene underlying an ecologically relevant trait such as abdominal pigmentation. A closer examination of omb variation in different species of the cardini group will elucidate the history of selection at this locus (Brisson, 2004).

In the T-domain, the lack of amino acid variation among divergent species indicates purifying selection. Within D. polymorpha, no nucleotide variation at all was observed in the T-domain coding region. This total lack of nucleotide polymorphism could be indicative of codon bias or functional constraint at the nucleotide level (Brisson, 2004).

The role of Dpp signaling in maintaining the Drosophila wing anteroposterior compartment boundary: Omb regulates the segregation behavior of cells at the A/P boundary

The subdivision of the developing Drosophila wing into anterior (A) and posterior (P) compartments is important for its development. The activities of the selector genes engrailed and invected in posterior cells and the transduction of the Hedgehog signal in anterior cells are required for maintaining the A/P boundary. Based on a previous study, it has been proposed that the signaling molecule Decapentaplegic (Dpp) is also important for this function by signaling from anterior to posterior cells. However, it has not been known whether and in which cells Dpp signal transduction is required for maintaining the A/P boundary. The role of the Dpp signal transduction pathway and the epistatic relationship of Dpp and Hedgehog signaling in maintaining the A/P boundary has been analyzed by clonal analysis. A transcriptional response to Dpp involving the T-box protein Optomotor-blind is required to maintain the A/P boundary. Further, Dpp signal transduction is required in anterior cells, but not in posterior cells, indicating that anterior to posterior signaling by Dpp is not important for maintaining the A/P boundary. Finally, evidence is provided that Dpp signaling acts downstream of or in parallel with Hedgehog signaling to maintain the A/P boundary. It is proposed that Dpp signaling is required for anterior cells to interpret the Hedgehog signal in order to specify segregation properties important for maintaining the A/P boundary (Shen, 2005).

For many years, it was thought that En and Inv regulated the segregation of A and P cells by specifying a P-type cell segregation in a cell-autonomous fashion. Recent work has challenged this view by showing that a unidirectional Hh-mediated signal from P to A cells is required to specify the A-type segregation behavior of A cells and that the role of En and Inv is mainly to control Hh signaling. Based on the findings that A cells signal back to P cells via Dpp and that wings from flies hypomorphic for dpp have a distorted A/P boundary, it has been proposed that A to P signaling by Dpp might also be important to maintain the A/P boundary. However, whether Dpp signal transduction is required for the maintenance of the A/P boundary and in which cells the Dpp signal is required remained unknown. By analyzing clones mutant for tkv, mad, and omb, several independent lines of evidence are provided that Dpp signal transduction is required to maintain the A/P boundary and that it is only required in A cells, but not in P cells. Thus, the results do not support the hypothesis that A to P signaling by Dpp is required to maintain the A/P boundary. Instead, the results suggest that Dpp signaling within Dpp-producing A cells is required to maintain the A/P boundary (Shen, 2005).

Through analysis of mutant clones located at the A/P boundary lacking the activity of the type I Dpp receptor Tkv, evidence is provided that the reception of the Dpp signal in A cells is required to maintain the A/P boundary. When generated in the P compartment, a few tkv⁻bsk⁻ clones displace the A/P boundary to a small extent: this is attributed to the unusual round shape of these clones. However, the majority of P tkv⁻bsk⁻ clones do not displace the A/P boundary, suggesting that the reception of the Dpp signal is not required in P cells to maintain the A/P boundary. In contrast, mutant clones generated in the A compartment at the A/P boundary displace the position of the A/P boundary toward P, indicating that the reception of the Dpp signal is required in A cells to maintain the A/P boundary (Shen, 2005).

How does the reception of the Dpp signal control cell segregation at the A/P boundary? Although the molecular basis is unknown, a cell's segregation behavior presumably depends on its cytoskeletal or surface properties (cell affinity). Members of the TGFβ superfamily have been observed in other systems to be able to activate regulators of the actin cytoskeleton independently of Mad/Smad transcription factors, raising the possibility that Dpp reception could control cell segregation by directly altering structural components of the responding cells. Alternatively, Dpp could control the segregation of cells by regulating the transcription of one or several target genes. To distinguish between these possibilities, the role of downstream components of the Dpp signal transduction pathway were analyzed. Three independent lines of evidence is provided that a transcriptional response to the Dpp signal is required to maintain the A/P boundary. (1) The segregation behaviors of mad⁻bsk⁻ and tkv⁻bsk⁻ clones are indistinguishable. Like tkv⁻bsk⁻ clones, A mad⁻bsk⁻ clones displace the A/P boundary toward P, indicating a role for the transcription factor Mad in A cells to maintain the A/P boundary. (2) mad⁻brk⁻ clones respect the A/P boundary, indicating that repression of brk transcription by Mad is important for normal A/P cell segregation. (3) A omb⁻ clones displace the A/P boundary toward P. The frequency and extent of the boundary displacement of A omb⁻, tkv⁻bsk⁻, and mad⁻bsk⁻ clones is comparable, suggesting that the Dpp target gene omb is the main mediator of this aspect of the Dpp signal. In contrast to omb⁻ clones, most A clones mutant for the Dpp target gene sal do not displace the A/P boundary, indicating that sal does not play an important role in maintaining the A/P boundary. Together, these data suggest that the transduction of the Dpp signal controlling the maintenance of the A/P boundary bifurcates at the level of the Dpp target genes (Shen, 2005).

Cells of tkv⁻bsk⁻, mad⁻bsk⁻, and omb⁻ clones displacing the A/P boundary do not appear to intermingle well with P cells. In fact, within the entire wing disc pouch, these mutant clones have a round shape and smooth borders, suggesting that these mutant cells in general do not intermingle freely with wild-type cells. Similar clone shapes have been reported upon mutation or misexpression of several genes, including mutants in the Dpp target gene sal and misexpression of a constitutively active form of Tkv. The round shapes and smooth borders of clones have been attributed to differences in the affinity of clone cells for their neighbors, suggesting that Tkv, Mad, and the Dpp target genes omb and sal may affect some aspects of wing pouch cell affinity. Therefore, the inability of A tkv⁻bsk⁻, mad⁻bsk⁻, and omb⁻ clones displacing the A/P boundary to intermingle well with P cells is attributed to this particular role (Shen, 2005).

Taken together, this analysis indicates two roles for Dpp signal transduction: (1) it provides some aspects of the cell affinity of both A and P wing pouch cells; (2) it is required in A cells to specify an A cell affinity important for maintaining the A/P boundary. These two roles of Dpp signal transduction could either be related or distinct. The finding that the Dpp target gene sal is required for the first role, but not the second, provides a first indication that these two roles are implemented by partially distinct molecular mechanisms (Shen, 2005).

How might Omb regulate the segregation behavior of cells at the A/P boundary? Recent work has shown that Omb has at least two roles during the patterning of the Drosophila wing. First, Omb is required for the expression of two Dpp target genes sal and vestigial (vg) (del Alamo Rodriguez, 2004). Since sal mutant clones do respect the A/P boundary, the role of Omb in maintaining the A/P boundary cannot depend on sal induction. Since Vg is required for wing cell proliferation, its role in maintaining the A/P boundary cannot be tested. Second, Omb is involved in shaping the expression pattern of tkv along the A/P axis of the wing disc (del Alamo Rodriguez, 2004). The expression of tkv is reduced in Dpp-producing A cells along the A/P boundary. This reduction of tkv expression is mediated by the transcription factor Master of thickveins (Mtv, also known as Brakeless and Scribbler, which is expressed in these cells in response to the Hh signal. Since both tkv and mtv are upregulated in omb mutant clones, it has been proposed that Omb is required for Mtv to repress tkv (del Alamo Rodriguez, 2004). However, reduction of tkv transcription in A cells does not seem to be important for the segregation of cells at the A/P boundary, because A clones either mutant for mtv, in which tkv levels are increased, or overexpressing tkv, respect the A/P boundary. Thus, neither the role of Omb in repressing tkv nor in activating sal transcription appears to be important for Omb's function in maintaining the A/P boundary. Therefore, other target genes of Omb must exist that mediate Omb's function in maintaining the A/P boundary (Shen, 2005).

Anterior cells at the A/P boundary have been shown to require Hh signal transduction to segregate from P cells. Evidence is provided that A cells in addition need to transduce the Dpp signal for normal segregation. What is the epistatic relationship between Hh and Dpp signaling? The activity of the Hh transduction pathway is not affected in either tkv⁻bsk⁻ or mad⁻bsk⁻ clones as monitored by the expression of the Hh target gene ptc, indicating that Hh signal transduction does not require Dpp signal transduction components for its activity. However, the Dpp target gene omb appears to be important for A cells to interpret the Hh signal because the ability of Ci to specify A-type segregation properties depends, in part, on the activity of Omb. Thus, Dpp signaling acts either downstream of or in parallel with Hh signaling in maintaining the A/P boundary (Shen, 2005).

Previously, three transcription factors, a transcriptional activator form of Ci (hereafter referred to as Ci[act]), En, and Inv, have been shown to be required for the segregation of A and P cells. Evidence exists for the involvement of a fourth transcription factor, the T-box protein Omb. Omb is further shown to act downstream of or in parallel with Ci. How could these four transcription factors regulate the segregation of A and P cells? In a simple model, Ci[act], En, Inv, and Omb could regulate the segregation of A and P cells by controlling the transcription of the same set of target genes that may encode cell affinity molecules or regulate the activity of cell affinity molecules. Omb is activated in both A and P cells in a broad domain centered around the A/P boundary by Dpp signaling where Omb may upregulate the expression of this putative target gene(s). The activity of Ci[act] is restricted to Hh-responding A cells along the A/P boundary. In these A cells, the target gene(s) would be further induced. En and Inv expressions are mainly confined to P cells in which they are known to act as repressors of transcription. Thus, En and Inv would repress the putative target gene(s) in P cells. The abrupt difference in the expression of putative target gene(s) would contribute to the segregation of A and P cells. Anterior clones (but not P clones) of cells lacking Omb would displace the A/P boundary because normally the putative target gene would be highly expressed in A cells, but not in P cells, where it would be repressed by En and Inv. Omb may therefore provide a basal affinity to cells in the center of the wing disc that is modified by Ci[act], En, and Inv to create a sharp difference of this affinity in cells on both sides of the A/P boundary. In an alternative model, Omb, Ci[act], En, and Inv would regulate distinct sets of genes. To distinguish among these models, it will be necessary to identify the Ci[act], En, Inv, and Omb target genes mediating cell segregation (Shen, 2005).

The precise position and shape of the Dpp organizer along the A side of the A/P boundary are important for normal growth and patterning of the wing. It has been proposed that the segregation of cells at the A/P boundary contributes to maintain this precise position and shape of the Dpp organizer in the growing wing disc epithelium. It is intriguing to notice that the Dpp-organizing activity itself plays a role in the segregation of A and P cells, suggesting that the Dpp-organizing activity contributes to maintain its own position. It will be interesting to investigate whether other organizers associated with compartment boundaries have similar functions (Shen, 2005).

Reference names in red indicate recommended papers.

Agarwal, P., et al. (2003). Tbx5 is essential for forelimb bud initiation following patterning of the limb field in the mouse embryo. Development 130: 623-633. 12490567

Bachiller, D., et al. (2003). The role of chordin/Bmp signals in mammalian pharyngeal development and DiGeorge syndrome. Development 130: 3567-3578. 12810603

Basson, C. T., et al. (1999). Different TBX5 interactions in heart and limb defined by Holt-Oram syndrome mutations. Proc. Natl. Acad. Sci. 96(6): 2919-24.

Biehs, B., Sturtevant, M. A. and Bier, E. (1998). Boundaries in the Drosophila wing imaginal disc organize vein-specific genetic programs. Development 125: 4245-4257. 9753679

Brisson, J. A., Templeton, A. R. and Duncan, I. (2004). Population genetics of the developmental gene optomotor-blind (omb) in Drosophila polymorpha: evidence for a role in abdominal pigmentation variation. Genetics 168(4): 1999-2010. 15611170

Bruneau, B. G., et al. (1999). Chamber-specific cardiac expression of Tbx5 and heart defects in Holt-Oram syndrome. Dev. Biol. 211(1): 100-8.

Bruneau, B. G., et al. (2001). A murine model of Holt-Oram syndrome defines roles of the T-box transcription factor Tbx5 in cardiogenesis and disease. Cell 106(6): 709-21. 11572777

Brunner, A., Wolf, R., Pflugfelder, G.O., Poeck, B. and Heisenberg, M. (1992). Mutations in the proximal region of the optomotor-blind locus of Drosophila melanogaster reveal a gradient of neuroanatomical and behavioral phenotypes. J. Neurogenet. 8: 43-55

Campbell, C., et al. (1995). Cloning and mapping of a human gene (TBX2) sharing a highly conserved protein motif with the Drosophila omb gene. Genomics 28: 255-260

Campbell, G. and Tomlinson, A. (1999). Transducing the Dpp morphogen gradient in the wing of Drosophila: regulation of Dpp targets by brinker. Cell 96(4): 553-62.

Carreira, S., et al. (1998). Brachyury-related transcription factor Tbx2 and repression of the melanocyte-specific TRP-1 promoter. Mol. Cell. Biol. 18(9): 5099-108.

Chapman, D. L., et al. (1996). Tbx6, a mouse T-box ene implicated in paraxial mesoderm formation at gastruation. Dev. Biol. 180: 534-542.

Chapman, D. L. and Papaioannou, V. E. (1998). Three neural tubes in mouse embryos with mutations in the T-box gene Tbx6. Nature 391(6668): 695-697.

Chapman, D. L., et al. (2003). Critical role for Tbx6 in mesoderm specification in the mouse embryo. Mech. Dev. 120: 837-847. 12915233

Clements, D., Friday, R. V. and Woodland, H. R. (1999). Mode of action of VegT in mesoderm and endoderm formation. Development 126: 4903-4911.

Collavoli, A., et al. (2003). TBX5 nuclear localization is mediated by dual cooperative intramolecular signals, J. Mol. Cell. Cardiol. 35: 1191-1195. 14519429

Conlon, F. L., et al. (2001). Determinants of T box protein specificity. Development 128: 3749-3758. 11585801

Cook, O., Biehs, B. and Bier, E. (2004). brinker and optomotor-blind act coordinately to initiate development of the L5 wing vein primordium in Drosophila. Development 131: 2113-2124. 15073155

Croce, J., Lhomond, G. and Gache, C. (2003). Coquillette, a sea urchin T-box gene of the Tbx2 subfamily, is expressed asymmetrically along the oral-aboral axis of the embryo and is involved in skeletogenesis. Mech. Dev. 120: 561-572. 12782273

del Alamo Rodriguez, D., Terriente Felix, J., Diaz-Benjumea, F. J. (2004). The role of the T-box gene optomotor-blind in patterning the Drosophila wing. Dev. Biol. 268(2): 481-92. 15063183

Dheen, T., et al. (1999). Zebrafish tbx-c functions during formation of midline structures. Development 126(12): 2703-2713.

Erives, A. and Levine, M. (2000). Characterization of a maternal T-Box gene in Ciona intestinalis. Dev. Biol. 225: 169-178.

Fan, C., Liu, M. and Wang, Q. (2003). Functional analysis of TBX5 missense mutations associated with Holt-Oram syndrome, J. Biol. Chem. 278: 8780-8785. 12499378

Firnberg, N. and Neubuser, A. (2002). FGF signaling regulates expression of Tbx2, Erm, Pea3, and Pax3 in the early nasal region. Dev. Bio. 247: 237-250. 12086464

Fong, S. H., Emelyanov, A., Teh, C. and Korzh, V. (2005). Wnt signalling mediated by Tbx2b regulates cell migration during formation of the neural plate. Development. 132(16): 3587-96. 16033799

Friedman, R. A., et al. (2005). Eya1 acts upstream of Tbx1, Neurogenin 1, NeuroD and the neurotrophins BDNF and NT-3 during inner ear development. Mech. Dev. 122: 625-634. 15817220

Galceran, J., Sustmann, C., Hsu, S. C., Folberth, S. and Grosschedl, R. (2004). LEF1-mediated regulation of Delta-like1 links Wnt and Notch signaling in somitogenesis. Genes Dev. 18(22): 2718-23. 15545629

Garg, V., et al. (2003). GATA4 mutations cause human congenital heart defects and reveal an interaction with TBX5. Nature 424(6947): 443-7. 12845333

Gorfinkiel, N., Sanchez, L. and Guerrero, I. (1999). Drosophila terminalia as an appendage-like structure. Mech. Dev. 86(1-2): 113-23. 10446270

Griffin KJ., et al. (1998). Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm formation by T-box genes. Development 125(17): 3379-3388.

Grimm, S. and Pflugfelder, G. O. (1996). Control of the gene optomotor-blind in Drosophila wing development by decapentaplegic and wingless. Science 271: 1601-1604

Gross, J. M., et al. (2003). LvTbx2/3: a T-box family transcription factor involved in formation of the oral/aboral axis of the sea urchin embryo. Development 130: 1989-1999. 12642501

Habets, P. E. M. H., et al. (2002). Cooperative action of Tbx2 and Nkx2.5 inhibits ANF expression in the atrioventricular canal: implications for cardiac chamber formation. Genes Dev. 16: 1234-1246. 12023302

Haerry, T. E., et al. (1998). Synergistic signaling by two BMP ligands through the SAX and TKV receptors controls wing growth and patterning in Drosophila. Development 125(20): 3977-3987

Harrelson, Z., et al. (2004). Tbx2 is essential for patterning the atrioventricular canal and for morphogenesis of the outflow tract during heart development. Development 131: 5041-5052. 15459098

Hasson, P., et al. (2001). Brinker requires two corepressors for maximal and versatile repression in Dpp signalling. EMBO J. 20: 5725-5736. 11598015

Hasson, P., Del Buono, J. and Logan, M. P. O (2007). Tbx5 is dispensable for forelimb outgrowth. Development 134: 85-92. Medline abstract: 17138667

Hilton, E., Rex, M. and Old, R. (2003). VegT activation of the early zygotic gene Xnr5 requires lifting of Tcf-mediated repression in the Xenopus blastula. Mech. Dev. 120: 1127-1138. 14568102

Hofmann, M., et al. (2004). WNT signaling, in synergy with T/TBX6, controls Notch signaling by regulating Dll1 expression in the presomitic mesoderm of mouse embryos. Genes Dev. 18(22): 2712-7. 15545628

Hoogaars, W. M. H., et al. (2007). Tbx3 controls the sinoatrial node gene program and imposes pacemaker function on the atria. Genes Dev. 21: 1098-1112. Medline abstract: 17473172

Horb, M. E. and Thomsen, G. H. (1997). A vegetally localized T-box transcription factor in Xenopus eggs specifies mesoderm and endoderm and is essential for embryonic mesoderm formation. Development 124: 1699-1709

Hu, S., Fambrough, D., Atashi, J. R., Goodman, C. S. and Crews, S. T. (1995). The Drosophila abrupt gene encodes a BTB-zinc finger regulatory protein that controls the specificity of neuromuscular connections. Genes Dev. 9: 2936-2948. 7498790

Hu, T., Yamagishi, H., Maeda, J., McAnally, J., Yamagishi, C. and Srivastava, D. (2004). Tbx1 regulates fibroblast growth factors in the anterior heart field through a reinforcing autoregulatory loop involving forkhead transcription factors. Development 131(21): 5491-502. 15469978

Huang, Z. and Kunes, S. (1998). Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly. Development 125: 3753-3764

Hug, B., Walter, V. and Grunwald, D. J. (1997). tbx6 a Brachyury-related gene expressed by ventral mesendodermal precursors in the Zebrafish embryo. Dev. Biol. 183: 61-73.

Hyde, C. E. and Old, R. W. (2000). Regulation of the early expression of the Xenopus nodal-related 1gene, Xnr1. Development 127: 1221-1229.

Isaac, A., et al. (1998). Tbx genes and limb identity in chick embryo development. Development 125(10): 1867-1875.

Jazwinska, A., et al. (1999). The Drosophila gene brinker reveals a novel mechanism of Dpp target gene regulation. Cell 96(4): 563-73.

Kofron, M., et al. (1999). Mesoderm induction in Xenopus is a zygotic event regulated by maternal VegT via TGFbeta growth factors. Development 126: 5759-5770.

Koshiba-Takeuchi, K., et al. (2000). Tbx5 and the retinotectum projection. Science 287: 134-137.

Knezevic, V., Santo, R. D. and Mackem, S. (1997). Two novel chick T-box genes related to mouse Brachyury are expressed in different, non-overlapping mesodermal domains during gastrulation. Development 124: 411-419

Knezevic, V., De Santo, R. and Mackem, S. (1998). Continuing organizer function during chick tail development. Development 125(10): 1791-1801.

Kokubo, H., et al. (2007). Hesr1 and Hesr2 regulate atrioventricular boundary formation in the developing heart through the repression of Tbx2. Development 134: 747-755. Medline abstract: 17259303

Kopp, A. and Duncan, I. (1997). Control of cell fate and polarity in the adult abdominal segments of Drosophila by optomotor-blind. Development 124(19): 3715-3726.

Kopp, A., and Duncan, I. (2002). Anteroposterior patterning in adult abdominal segments of Drosophila. Dev. Bio. 242: 15-30

Law, D. J., et al. (1995). Identification, characterization, and localization to chromosome 17q21-22 of the human TBX2 homolog, member of a conserved developmental gene family. Mamm. Genome 6: 793-797

Lawrence, P. A., Casal, J. and Struhl, G. (2002). Towards a model of the organisation of planar polarity and pattern in the Drosophila abdomen. Development 129: 2749-2760. 12015301

Lecuit, T., et al. (1996). Two distinct mechanisms for long-range patterning by Decapentaplegic in the Drosophila wing. Nature 381: 387-393

Lee, H. H., Norris, A., Weiss, J. B. and Frasch, M. (2003), Jelly belly protein activates the receptor tyrosine kinase Alk to specify visceral muscle pioneers. Nature. 425(6957): 507-12. 14523446

Li, H., et al. (1997). A single morphogenetic field gives rise to two retina primordia under the influence of the prechordal plate. Development 124: 603-615 (1997)

Logan, M. and Tabin, C. J. (1999). Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity. Science 283(5408): 1736-9.

Lustig, K. D., et al. (1996). Expression cloning of a Xenopus T-related gene (Xombi) involved in mesodermal patterning and blastopore lip formation. Development 122: 4001-4012

Manning, L., et al. (2007). Regional morphogenesis in the hypothalamus: A BMP-Tbx2 pathway coordinates fate and proliferation through Shh downregulation. Dev. Cell 11(6): 873-85. Medline abstract: 17141161

Marlow, F., et al. (2004). No tail co-operates with non-canonical Wnt signaling to regulate posterior body morphogenesis in zebrafish. Development 131: 203-216. Medline abstract: 14660439

Maves, L. and Schubiger, G. (1998). A molecular basis for transdetermination in Drosophila imaginal discs: interactions between wingless and decapentaplegic signaling. Development 125(1): 115-124

Moraes, A., et al. (2005). Tbx1 is required for proper neural crest migration and to stabilize spatial patterns during middle and inner ear development. Mech. Dev. 122: 199-212. 15652707

Naiche, L. A. and Papaioannou, V. E. (2003). Loss of Tbx4 blocks hindlimb development and affects vascularization and fusion of the allantois. Development 130: 2681-2693. 12736212

Naiche, L. A. and Papaioannou, V. E. (2007). Tbx4 is not required for hindlimb identity or post-bud hindlimb outgrowth. Development 134: 93-103. Medline abstract: 17164415

Nellen, D., et al. (1996). Direct and long-range action of a DPP morphogen gradient. Cell 85: 357-369

Pflugfelder, G.O., Schwartz, H., Roth, H., Poeck, B., Sigl, A., Kerscher, S., Jonschker, B., Pak, W.L. and Heisenberg, M. (1990). Genetic and molecular characterisation of the optomotor-blind gene locus in Drosophila melanogaster. Genetics 126: 91-104

Pflugfelder, G.O., Roth, H., Poeck, B., Kerscher, S., Schwartz, H., Jonschker, B. and Heisenberg, M. (1992a). The lethal (1) optomotor-blind gene of Drosophila melanogaster is a major organizer of optic lobe development: isolation and characerization of the gene. Proc. Nat'l Acad. Sci. 89: 1199-1203

Pflugfelder, G.O., Roth, H. and Poeck, B. (1992b). A homology domain shared between Drosophila optomotor blind and mouse Brachyury is involved in DNA binding. Biochem. Biophys. Res. Commun. 186: 918-25

Poeck, B., Balles, J. and Pflugfelder, G.O. (1993a). Transcript identification in the optomotor-blind locus of Drosophila melanogaster by intragenic recombination mapping and PCR-aided sequence analysis of lethal point mutations. Mol. Gen. Genet. 238: 325-32

Poeck, B., Hofbauer, A. and Pflugfelder, G. O. (1993b). Expression of the Drosophila optomotor-blind gene transcript in neuronal and glial cells of the developing nervous system. Development 117: 1017-29

Porsch, M., Sauer, M., Schulze, S., Bahlo, A., Roth, M. and Pflugfelder, G. O. (2004). The relative role of the T-domain and flanking sequences for developmental control and transcriptional regulation in protein chimeras of Drosophila OMB and ORG-1. Mech. Dev. 122: 81-96. 15582779

Prpic, N. M., Janssen, R., Damen, W. G. and Tautz, D. (2005). Evolution of dorsal-ventral axis formation in arthropod appendages: H15 and optomotor-blind/bifid-type T-box genes in the millipede Glomeris marginata (Myriapoda: Diplopoda). Evol. Dev. 7(1): 51-7. 15642089

Raft, S., et al. (2004). Suppression of neural fate and control of inner ear morphogenesis by Tbx1. Development 131: 1801-1812. 15084464

Rallis, C., et al. (2003). Tbx5 is required for forelimb bud formation and continued outgrowth. Development 130: 2741-2751. 12736217

Rodriguez-Esteban, C., et al. (1999). The T-box genes Tbx4 and Tbx5 regulate limb outgrowth and identity. Nature 398(6730): 814-8.

Ruvinsky, I., Silver, L. M. and Ho, R. K. (1998). Characterization of the zebrafish tbx16 gene and evolution of the vertebrate T-box family. Dev. Genes Evol. 208(2): 94-9.

Shen, J. and Dahmann, C. (2005). The role of Dpp signaling in maintaining the Drosophila anteroposterior compartment boundary. Dev. Biol. 279(1): 31-43. 15708556

Simon, H.-G., et al. (1997). A novel family of T-box genes in urodele amphibian limb development and regeneration: candidate genes involved in vertebrate forelimb/hindlimb patterning. Development 124, 1355-1366

Singh, M. K., et al. (2005). Tbx20 is essential for cardiac chamber differentiation and repression of Tbx2. Development 132(12): 2697-707. 15901664

Sivasankaran, R., et al. (2000). Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker. EMBO J. 19: 6162-6172.

Song, Y., Chung, S. and Kunes, S. (2000). Combgap relays Wingless signal reception to the determination of cortical cell fate in the Drosophila visual system. Mol. Cell 6: 1143-1154.

Stennard, F., Carnac, G. and Gurdon, J. G. (1996). The Xenopus T-box gene, Antipodean, encodes a vegetally localised maternal mRNA and can trigger mesoderm formation. Development 122: 4179-4188

Stennard, F., et al. (1999). Differential expression of VegT and Antipodean protein isoforms in Xenopus. Mech. Dev. 86(1-2): 87-98.

Stennard, F. A., et al. (2003), Cardiac T-box factor Tbx20 directly interacts with Nkx2-5, GATA4, and GATA5 in regulation of gene expression in the developing heart. Dev. Biol. 262: 206-224. 14550786

Stennard, F. A., et al. (2005). Murine T-box transcription factor Tbx20 acts as a repressor during heart development, and is essential for adult heart integrity, function and adaptation. Development 132(10): 2451-62. 15843414

Sturtevant, M. A. and Bier, E. (1995). Analysis of the genetic hierarchy guiding wing vein development in Drosophila. Development 121: 785-801. 7720583

Sturtevant, M. A., Biehs, B., Marin, E. and Bier, E. (1997). The spalt gene links the A/P compartment boundary to a linear adult structure in the Drosophila wing. Development 124: 21-32. 9006064

Suzuki, T., et al. (2004). Tbx genes specify posterior digit identity through Shh and BMP signaling. Dev. Cell 6: 43-53. 14723846

Szeto, D. P. and Kimelman, D. (2004). Combinatorial gene regulation by Bmp and Wnt in zebrafish posterior mesoderm formation. Development 131: 3751-3760. 15240553

Takeuchi, J. K., et al. (1999). Tbx5 and Tbx4 genes determine the wing/leg identity of limb buds. Nature 398(6730): 810-4.

Takeuchi, J. K., et al. (2003a). Tbx5 and Tbx4 trigger limb initiation through activation of the Wnt/Fgf signaling cascade. Development 130: 2729-2739. 12736216

Takeuchi, J. K., et al. (2003b). Tbx5 specifies the left/right ventricles and ventricular septum position during cardiogenesis. Development 130: 5953-5964. 14573514

Torres-Vazquez, J., Warrior, R. and Arora, K. (2000). schnurri is required for dpp-dependent patterning of the Drosophila wing. Dev. Bio. 227: 388-402.

Tsuneizumi, K., et al. (1997). Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature 389(6651): 627-631.

Tümpel, S., et al. (2002). Regulation of Tbx3 expression by anteroposterior signalling in vertebrate limb development. Dev. Bio. 250: 251-262. 12376101

Vitelli, F., et al. (2002). A genetic link between Tbx1 and fibroblast growth factor signaling. Development 129: 4605-4611. 12223416

Weatherbee, S. D., et al. (1998). Ultrabithorax regulates genes at several levels of the wing-patterning hierarchy to shape the development of the Drosophila haltere. Genes Dev. 12(10): 1474-1482.

White, P. H., et al. (2003). Defective somite patterning in mouse embryos with reduced levels of Tbx6. Development 130: 1681-1690. 12620991

Winter, S. E. and Campbell, G. (2004). Repression of Dpp targets in the Drosophila wing by Brinker. Development 131: 6071-6081. 15537684

Wisotzkey, R. G. (1998). Medea is a Drosophila Smad4 homolog that is differentially required to potentiate DPP responses. Development 125: 1433-1445

Xanthos, J. G., et al. (2001). Maternal VegT is the initiator of a molecular network specifying endoderm in Xenopus laevis. Development 128: 167-180

Xu, H., et al. (2004). Tbx1 has a dual role in the morphogenesis of the cardiac outflow tract. Development 131: 3217-3227. 15175244

Xu, H., Cerrato, F. and Baldini, A. (2005). Timed mutation and cell-fate mapping reveal reiterated roles of Tbx1 during embryogenesis, and a crucial function during segmentation of the pharyngeal system via regulation of endoderm expansion. Development 132(19): 4387-95. 16141220

Yamamoto, A., et al. (1998). Zebrafish paraxial protocadherin is a downstream target of spadetail involved in morphogenesis of gastrula mesoderm. Development 125(17): 3389-3397.

Yamada, M., et al. (2000). Expression of chick Tbx-2, Tbx-3, and Tbx-5 genes during early heart development: Evidence for BMP2 induction of Tbx2. Dev. Bio. 228: 95-105.

Yoon, Y. J. and Mowry, K. L. (2004). Xenopus Staufen is a component of a ribonucleoprotein complex containing Vg1 RNA and kinesin. Development 131: 3035-3045. 15163628

Zhang, J. and King, M. L. (1996). Xenopus VegT RNA is localized to the vegetal cortex during oogenesis and encodes a novel T-box transcription factor involved in mesodermal patterning. Development 122: 4119-4129

Zhang, J., et al. (1998). The role of maternal VegT in establishing the primary germ layers in Xenopus embryos. Cell 94(4): 515-524.

Zhang, Z., et al. (2005). Tbx1 expression in pharyngeal epithelia is necessary for pharyngeal arch artery development. Development 132(23): 5307-15. 16284121

Zuber, M. E., et al. (2003). Specification of the vertebrate eye by a network of eye field transcription factors. Development 130: 5155-5167. 12944429

date revised: 25 August 2007

 

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