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).

Omb used as a marker to study cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs

The evagination of Drosophila imaginal discs is a classic system for studying tissue level morphogenesis. Evagination involves a dramatic change in morphology and published data argue that this is mediated by cell shape changes. The evagination of both the leg and wing discs has been reexamined and it has been found that the process involves cell rearrangement and that cell divisions take place during the process. The number of cells across the width of the ptc domain in the wing and the omb domain in the leg decreases as the tissue extends during evagination and cell rearrangement was observed to be common during this period. In addition, almost half of the cells in the region of the leg examined divided between 4 and 8 h after white prepupae formation. Interestingly, these divisions were not typically oriented parallel to the axis of elongation. These observations show that disc evagination involves multiple cellular behaviors, as is the case for many other morphogenetic processes (Taylor, 2008).

This study established that cell rearrangement takes place during leg and wing evagination and contributes to the thinning and extension of the appendages. These observations are consistent with the pioneering results of Fristrom (1976) on evagination. The current data also established that cell rearrangement takes place throughout the appendage and is not restricted to a particular region along the proximal/distal axis. However, the observations are also consistent with cell rearrangement being non-uniform as some regions appeared to 'thin' more than others. For example, in the wing the width of the ptc domain at position M5 thinned more than at position M4 (refering to neuronal landmarks). The evaginating leg and wing cells retain their epithelial morphology with extensive apical junctional complexes. Rearrangement requires that cells change neighbors and hence must remove old junctions and generate new ones while maintaining tissue integrity. This problem is not restricted to evaginating discs but is a general one for epithelial tissues and is an issue that has concerned developmental/cell biologists for many years. Important insights into how this could be accomplished come from recent observations on germ band elongation in the Drosophila embryo. Several groups have provided evidence that junctional remodeling plays a key role in cell rearrangement in this epithelial tissue. This mechanism also appears to function in the repacking of pupal wing cells. It is suggested that it also plays a role in leg and wing evagination. No clear evidence is seen for the multicellular rosettes that have been implicated in germ band extension. Perhaps this is due to disc evagination being substantially slower than germ band extension (Taylor, 2008).

No evidence was seen of dramatic coordinated changes in cell shape. There was a small but significant increase in the length along the proximal/distal axis of evaginating omb domain tibia cells that should contribute to elongation. However, the change was not large enough to account for leg morphogenesis. No significant change was seen in cell shape in evaginating ptc domain wing cells although there was a hint of a possible small effect. It is worth noting that in these measurements cells from all positions along the relevant part of the proximal/distal axis were included. Casual observation suggested that there might be small regions with consistent changes but these would likely be counterbalanced by changes in shape elsewhere in the domain (Taylor, 2008).

It was not possible to image the earliest stages of leg disc evagination or the disc cells that form ventral thorax. Thus, these observations were not able to distinguish between the two proposed mechanisms of eversion (i.e., spreading vs. invasion hypotheses). Patterned cell death could in principle play an important role in disc evagination. Previous studies have not seen evidence for patterned cell death during wing blade evagination and the current observations support this conclusion. Cell death has been detected in evaginating legs but this is restricted to the regions of the tarsal segments where the leg joints form and hence is unlikely to contribute to the overall thinning of the omb domain of leg segments (Taylor, 2008).

Based on the literature, it was not expected that cell division takes place during evagination, but the current observations showed that it occurred. The most definitive experiments involved generating clones of cells marked by GFP expression and following these in vivo. These experiments provided compelling evidence for cell division. This was only done for the leg but other experiments provided strong evidence for cell division in evaginating wings. The size of wing clones was larger when they were induced at white prepupae than at the formation of the definitive pupae. Cell division was not rare in evaginating legs, and on average about 40% of the cells divided. Indeed, a majority of the cells divided in about 1/3 of clones examined. This amount of cell division is sufficient to account for the thickening of the omb domain that was observed from 6 to 8 h in developing legs. Observations on the size of wing clones suggested a similar fraction of wing cells divided during evagination. A limitation is that the in vivo imaging technique only allowed effective imaging of clones on the leg surface juxtaposed to the pupal case in the basitarsus and tibia (and occasionally tarsal) segments. Thus, data could not be obtained for much of the leg disc derivatives, and hence the overall proportion of evaginating leg cells that divide cannot be confidently estimated. The spindle in these dividing cells was not imaged but it was inferred that the spindle was not oriented parallel to the elongating axis, based on the position of the resulting daughter cells shortly after division. The two daughter cells usually filled up the area taken up by the parental cell prior to division, which helped in assigning a lineage. The leg epidermis is continuous without free 'space'. Hence, that daughter cells would occupy the space of the parental cell is not surprising. A parallel orientation for the spindle might be expected if the cell division plane was tightly linked to the mechanism of elongation. The inferred orientation of the cell divisions was most often between 46^o and 60^o. Thus, they would increase the number of cells both along the proximal/distal and anterior/posterior (and dorsal/ventral) axes. In the second day pupal leg, the width of the omb domain was narrower than it was in the evaginating leg. This could be a reflection of a later stage of convergent extension. However, legs were not followed throughout this period, other possibilities cannot be ruled out. It is interesting to note that cells in the pupal tibia and basitarsus have a spiral arrangement, and this appears to arise from 6 to 8 h after white prepupae. Thus, this arrangement could be at least in part a consequence of the orientation of the cell divisions (Taylor, 2008).

The fraction of dividing cells varied widely from one clone to another. This was not correlated with particular pupae or legs as both clones where a majority of the cells divided and clones where no cells divided were found in the same pupae and on the same leg. One possibility is that the variation is due to region specific differences. For example, cells in one region of the leg might never divide during evagination while a majority of cells in another region might always divide. No evidence is seen for this but the experiments were not compelling on this point. The observations on the omb domain did not examine a majority of leg cells and in the experiments where MARCM clones were followed, it could not be routinely said exactly where on the leg a clone was located. A second possibility is that the variation is due to the clustered distribution of S phase and mitotic cells in wing and leg discs. Any small clone could comprise a cluster (or not contain a cluster) and this could lead to a great deal of variation in observed cell division. The basis for the clustering is uncertain but could simply represent a pseudo-synchronization due to neighboring sister cells having been born at the same time (Taylor, 2008).

The observations suggest that several different factors play a role in evagination. At the start of evagination, the leg and wing discs are folded and some of the initial elongation is due to an unfolding of the tissue that presumably results from changes in the shape of cells along the apical/basal axis. During the period when leg discs evert and present the apical surface of their epithelial cells to the outside, elongation is also taking place and there is active pulsatile movement. This appears to be related to the movement of hemolymph in the prepupae and blood cells can often be seen to move in step with the pulses. This suggests that hydraulic pressure could be playing a role in eversion and elongation. The leg resembles a cylinder closed on one side (distal tip) and open to the body on the other (proximal). Thus, it is expected that hemolymph is pumped by the heart to produce a mechanical force that could help evert and/or elongate the leg. The pulsatile movement starts to decrease at about 4-4.5 h after white prepupae and largely ends by about 5 h. This is around the time of eversion, but the slowing clearly precedes eversion. It is suggested that the hydraulic pressure of the hemolymph helps drive the early stages of evagination, when the leg is short and unfolding of the tissue plays a major role. It is possible that after this time the increased leg length or increased leg stiffness limits the effectiveness of hemolymph hydraulic pressure. Alternatively, it is possible that there is a decline in the hydraulic pressure due to changes in heart pumping or other prepupal events. The lack of hydraulic pressure may be one reason for the less than optimal evagination of discs seen during in vitro culture (Taylor, 2008).

Mutations in many Drosophila genes result in changes in appendage morphology. It is expected that some of these produce their phenotype by interfering with the observed cell rearrangement. A particularly interesting candidate for such a gene is dachsous (ds), which encodes a large protein with many cadherin domains. Mutations in this gene result in shorter fatter wings and legs with an altered distribution of cells (e.g. an increase in the number of cells along the anterior posterior axis of the wing and a decrease in the number of cells along the proximal/distal axis). However, mutations in this gene are known to alter disc patterning and growth and this may be the cause of the altered shape (Taylor, 2008).

Another group of interesting candidate genes for altering cell rearrangement in evaginating legs is the cellular myosin encoded by zipper and the interacting Sqh (myosin regulatory light chain) and RhoA proteins. Mutations in these genes give rise to a crooked leg phenotype that has been interpreted as being due to the mutations altering cell shape. However, myosin has been implicated in the junctional remodeling associated with cell rearrangements in the extending germ band and it is possible that the leg phenotype is also due to an effect on junctional remodeling required for cell rearrangement. One of the interesting properties of extending germ band cells is the planar polarization of membranes so that the anterior/posterior edges of cells are distinct from the dorsal/ventral edges of cells in their content of proteins such as myosin. No evidence was seen for this in prepupal legs and wings but this point deserves further study as it is possible the experimental conditions were not favorable for seeing this (Taylor, 2008).

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).

The orthologous Tbx transcription factors Omb and TBX2 induce epithelial cell migration and extrusion in vivo without involvement of matrix metalloproteinases

The transcription factors TBX2 and TBX3 are overexpressed in various human cancers. This study investigated the effect of overexpressing the orthologous Tbx genes Drosophila optomotor-blind (omb) and human TBX2 in the epithelium of the Drosophila wing imaginal disc; two types of cell motility were observed. Omb/TBX2 overexpressing cells could move within the plane of the epithelium. Invasive cells migrated long-distance as single cells retaining or regaining normal cell shape and apico-basal polarity in spite of attenuated apical DE-cadherin concentration. Inappropriate levels of DE-cadherin were sufficient to drive cell migration in the wing disc epithelium. Omb/TBX2 overexpression and reduced DE-cadherin-dependent adhesion caused the formation of actin-rich lateral cell protrusions. Omb/TBX2 overexpressing cells could also delaminate basally, penetrating the basal lamina, however, without degradation of extracellular matrix. Expression of Timp, an inhibitor of matrix metalloproteases, blocked neither intraepithelial motility nor basal extrusion. These results reveal an MMP-independent mechanism of cell invasion and suggest a conserved role of Tbx2-related proteins in cell invasion and metastasis-related processes (Shen, 2014).

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).

Population genetics of the developmental gene optomotor-blind (omb) in Drosophila polymorpha: evidence for a role in abdominal pigmentation variation

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).

Optomotor-blind expression in glial cells is required for correct axonal projection across the Drosophila inner optic chiasm

In the Drosophila adult visual system, photoreceptor axons and their connecting interneurons are tied into a retinotopic pattern throughout the consecutive neuropil regions: lamina, medulla and lobula complex. Lamina and medulla are joined by the first or outer optic chiasm (OOC). Medulla, lobula and lobula plate are connected by the second or inner optic chiasm (IOC). In the regulatory mutant In(1)omb^H31 of the T-box gene optomotor-blind (omb), fibers were found to cross aberrantly through the IOC into the neuropil of the lobula complex. This study shows that In(1)omb^H31 causes selective loss of OMB expression from glial cells within the IOC previously identified as IOC giant glia (ICg-glia). In the absence of OMB, ICg-glia retain their glial cell identity and survive until the adult stage but tend to be displaced into the lobula complex neuropil leading to a misprojection of axons through the IOC. In addition, adult mutant glia show an aberrant increase in length and frequency of glial cell processes. The onset of the IOC defect was narrowed down to the interval between 48 h and 72 h of pupal development. Within the 40 kb of regulatory DNA lacking in In(1)omb^H31, an enhancer element (ombC) was identified with activity in the ICg-glia. ombC-driven expression of omb in ICg-glia restored proper axonal projection through the IOC in In(1)omb^H31 mutant flies, as well as proper glial cell positioning and morphology. These results indicate that expression of the transcription factor OMB in ICg-glial cells is autonomously required for glial cell migration and morphology and non-autonomously influences axonal pathfinding (Hofmeyer, 2008).

In the developing nervous system, axonal growth cones are guided towards their target region by a variety of cues, which can be presented by several different cell types along the way, such as other neurons or glial cells. Presentation and interpretation of attractive and repellent signals are crucial for correct axonal pathfinding. Proper neuronal connectivity also requires the establishment of confined neuronal compartments, which are separated by glial septa. In Drosophila, glial cells have been shown to be involved in a number of steps in the development of the nervous system, such as axonal guidance, axonal pruning, synapse function and neuronal survival (Hofmeyer, 2008).

In the embryonic CNS, midline glia present guidance cues to commissural axons such as the attractive signal Netrin and the repellent Slit. Longitudinal and segment boundary (SBC) glial cells have been identified as guidepost cells for outgrowing motor axons. However, there is no absolute requirement for these glial cells as indicated by the overall normal axonal patterning in glia cell missing (gcm) mutants where glial cells are transformed into neurons. In contrast, a clear role for glial cells has been established in the development of the adult Drosophila visual system. Lamina glia are required as intermediate targets for R1-R6 photoreceptor axons. Two different kinds of processes have been recognized: (1) lamina glia need to migrate correctly to their final position within the developing lamina, a process which is affected in nonstop and JAB1/CSN5 mutants; (2) lamina glial cells are likely to display signals to the arriving R1-R6 growth cones instructing them to halt their growth. No such signal has been identified so far. While several genes have been identified as being required neuronally for R1-R6 axon targeting to the lamina, nonstop remains the only one reported to play a role in the lamina glial cells (Hofmeyer, 2008).

Altered axonal projection patterns can also be a secondary result of a disruption of neuronal compartment integrity. The four neuronal compartments in the Drosophila optic lobes (lamina, medulla and lobula complex, comprised of lobula and lobula plate) are descendants of two different progenitor regions, the outer and the inner optic anlage. Cell populations derived from different anlagen come to lie in close proximity during development without intermingling, such as the lamina neurons and glia derived from the outer optic anlage and the lobula cortex neurons formed by the inner optic anlage. In certain mutants, the mixing of cells across the border of these two neuropils results in the disorganization of lamina glia correlated with aberrant projections of R1-R6 to the medulla. The border between lamina and lobula cortex was shown to depend on the presence of secreted Slit around lamina glia and expression of the Slit receptor Robo on distal cells of the lobula cortex. Also, the glycosyl transferase Egghead is required in the lobula cortex primordium for compartment border integrity. In egghead mutants, defects in boundary formation can be visualized as disruptions in the curtain between lamina and lobula cortex generated by sheath-like glial cell processes (Hofmeyer, 2008).

In the Drosophila optic lobes (OL), photoreceptor axons and their connecting interneurons are organized in a retinotopic pattern, in which the dorso-ventral and anterior-posterior relations between retinal photoreceptor cells are preserved at the interneuron level throughout the proximal neuropil regions. The relative spatial relationships are retained within the individual neuropils even though the bulk orientations of the medulla and lobula complex are distinctly shifted (by rotation and translation) relative to the coordinates of the retina. The four OL neuropil regions are connected by two chiasmata: the first or outer optic chiasm (OOC) connects lamina and medulla, the second or inner optic chiasm (IOC) connects medulla, lobula and lobula plate (Hofmeyer, 2008).

Currently, little is known about structure and development of the Drosophila IOC. A model of fiber paths connecting the three neuropils, which surround the IOC was proposed based on studies of larger Dipteran flies. According to this model, which is likely to hold also for Drosophila, medulla and lobula plate as well as lobula and lobula plate are connected retinotopically in a horizontal plane by non-inverted connections, whereas medulla and lobula are connected via two types of inverted fiber paths (Hofmeyer, 2008).

Both chiasmata contain glial cells, which can be distinguished from other optic lobe glia by their position, morphology and the expression of specific genetic markers. Because of their large nuclei and cell bodies the IOC glia were termed 'giant' (g), hence ICg-glia. Along the dorso-ventral axis, ICg-glia and fiber bundles that cross the IOC are arranged in an alternating pattern. The ICg-glia, like fiber tract glia in other systems, serve to insulate these tracts from one another by wrapping them with thin cytoplasmic extensions(Hofmeyer, 2008).

In certain combinations of optomotor-blind (omb) alleles, projection defects are observed in the IOC. omb encodes a transcription factor of the T-Box family of DNA-binding proteins. omb was first identified in a screen for genes required for proper large field optomotor response. omb is expressed in several cell types and tissues during embryonic and larval development. In the visual system, OMB is found in many types of glial cells including the three types of lamina glia: satellite, epithelial and marginal glia. omb transcription is under the control of numerous enhancers located up- and downstream of and within the transcription unit. A 45-kb regulatory region downstream of the transcription unit was identified and named 'optic lobe regulatory region' (OLR) due to the fact that deletions of parts or of the entire region lead to various neuroanatomical defects within the optic lobes which are associated with defects in visual behavior. The OLR was dissected into three subregions by use of deletion and inversion mutants removing successively larger parts of the regulatory region, with OLR1 lying closest to and OLR3 farthest away from the transcription unit. In mutants in which OLR2 + 3 are deleted, a disruption of the IOC manifests itself with ectopic fibers invading the adult lobula complex which is not seen in mutants in which OLR3 alone is lacking. The severity of the phenotype is increased when the entire OLR is deleted. Other behavioral and neuroanatomical phenotypes, apparently unrelated to the IOC defect, are seen in these mutants, such as a reduced number of fibers in the anterior optic tract, the loss of the lobula plate giant horizontal and vertical cells and a reduction of the large field response (Hofmeyer, 2008).

Projection defects through the IOC as a result of mutations in other genes have been described before but a glial involvement in patterning this complex structure has not yet been demonstrated. This study reports a requirement of omb expression in ICg-glial cells for IOC integrity. In omb mutants lacking OLR2, the selective loss of omb expression from ICg-glial cells was associated with a disruption of the adult IOC. The structural IOC defect appears to be caused by local mismigration of ICg-glial cells and possibly by the extension of gliopodia of aberrant length (Hofmeyer, 2008).

Heteroallelic In(1)omb^H31/Df(1)rb⁵ flies completely lack the omb optic lobe regulatory region OLR2 and display a disrupted inner optic chiasm in the adult brain. This study shows that this defect is caused by a loss of OMB expression from giant glial cells within the larval inner optic chiasm (ICg-glia). A 6.1-kb OLR2 subfragment, named ombC, was identified which drives expression in ICg-glia as well as in lamina marginal glia. In animals lacking OLR2, only the ICg-glia, not the marginal glia showed a loss of OMB expression. Therefore, OMB expression in marginal glia must be redundantly controlled. Indeed, it was found that a 1.7-kb genomic fragment containing the endogenous omb promoter drove reporter gene expression in a large number of cells, including the marginal glia (Hofmeyer, 2008).

OMB expressed under ombC enhancer control in the OLR2 + 3 mutant background can rescue the adult IOC defect. The ombC enhancer is active in both marginal and ICg-glial cells. Since, in the OLR2 + 3 deficiency mutant, OMB expression is lost only from ICg-glia and not from marginal glia, the latter appear not to be involved in IOC formation. In third instar larval optic lobes of In(1)omb^H31, OMB expression is also lost from a subset of cells in the medulla cortex. None of the genomic DNA fragments derived from OLR1-3 drove reporter gene expression in this group of cells. It is possible that an enhancer active in this expression pattern was destroyed by the particular choice of restriction sites in the dissection of OLR2 or that the relevant enhancer cannot interact with the minimal promoter used in the enhancer reporter constructs. Such incidences of promoter dedication have been described previously. Since the ombC enhancer is not active in medulla cortical cells but can rescue the IOC defect it is assumed that these cells, like the lamina marginal cells, are not required for proper IOC formation. It is therefore concluded that expression of omb in ICg-glia is necessary and sufficient for proper projection of the affected axonal tracts across the inner optic chiasm(Hofmeyer, 2008).

The IOC defect was originally recognized by paraffin histology. In order to analyze this defect in more detail, a small collection of Gal4 enhancer trap lines was screened for expression in neurons that project through the IOC. In this search, the line Mz826 was identified. While it was not possible to determine in which of the known optic lobe neurons Mz826 is expressed, the main direction of information flow through the optic lobes suggests that the incisions of fiber tracts into the lobula neuropil are caused by inappropriate projections from the medulla to the lobula (and not vice versa)(Hofmeyer, 2008).

In the wild-type IOC, neurites from medulla columns are bundled as they emerge from the medulla neuropil and defasciculate again when they enter the neuropil of the lobula complex. One aspect of the In(1)omb^H31 IOC phenotype appears to be a defect in defasciculation of the fiber tracts that cut into the lobula neuropil. In other systems, changes in fasciculation are characterized by glial involvement. In the moth olfactory system, olfactory receptor axons enter the CNS tightly bundled into two nerves. Upon encountering a glia-rich sorting zone, axons defasciculate and reorganize into smaller fascicles that project towards individual glomeruli. Glial cells are essential for this process. In the fly visual system, a cycle of fasciculation, defasciculation and refasciculation occurs between retina and lamina providing the anatomical basis for neural superposition. This reorganization occurs over a distance of only a few cell body diameters in a region characterized by the spatial succession of four glial cell types (Hofmeyer, 2008).

Since the omb IOC defect can be observed by low resolution paraffin histology, it is unlikely that only a minor subpopulation of axon types, which project through each layer of the IOC is affected. There is only indirect evidence regarding the organismic consequences of this local misrouting. In In(1)omb^H31, OLR2 and OLR3 are removed from the omb transcription unit. Loss of OLR3 alone leads to the namesake 'optomotor-blind' behavioral defect which is caused by the lack of ten giant interneurons from the lobula plate (the horizontal and vertical system cells, HS and VS cells). In In(1)omb^H31 mutant animals, the severity of the optomotor defect does not correlate with the severity of the IOC defect. This appears to indicate that the IOC defect does not contribute to this behavioral dysfunction. There are several potential reasons for this finding: First, the misprojecting neurons may not be involved in optomotor behavior. Second, if they are involved, the processing of only a small fraction of the visual input would be disturbed because, in a given fly, misprojection is only seen in a fraction of IOC layers. This should have little influence on large field optomotor behavior. The HS cells, which mediate the response to horizontal visual motion, are downstream in the flow of information with respect to fibers crossing the IOC. The finding that HS cells have response amplitudes that are nearly invariant to stimulus size when a large part of the eye is stimulated indicates the robustness of optomotor behavior. Third, the effect of lack of OLR2 could so far not be measured in isolation but only in combination with loss of OLR3. Since lack of OLR3 causes loss of the HS and VS cells, which are essential for optomotor behavior, this defect is likely to be epistatic to defects caused by the lack of OLR2. Lastly, the affected axons may reach their appropriate target areas and connect to their appropriate postsynaptic partners in spite of their irregular projection through the IOC. In mutants of several other genes in which projection across the outer optic chiasm (OOC) is affected, it was observed that nerve bundles from the lamina were able to reach their correct target area in the medulla in spite of using a widely circuitous route. This is most clearly seen in animals with irregular chiasm C (irreC) mutations. irreC-roughest encodes a homophilic cell adhesion molecule of the immunoglobulin superfamily, which is widely expressed, in particular on recently formed fiber bundles that cross the inner and outer optic chiasm. However, irreC is not expressed in ICg-glia. Irregular connections between lamina and medulla, which circumvent the OOC but appear to project to the normal target area, were also seen in other mutants with more severe brain disorganization (Hofmeyer, 2008).

Projection defects in the region of the IOC have been observed in irreC (which affects both chiasms), in a hypomorphic allele combination of single minded (sim), and in mutants of the Broad Complex and its target gene H217. IOC defects cannot only be caused by defective ICg-glial cells, as in the case of In(1)omb^H31, but also by defects in the interacting bundles of neurons, since neither irreC nor sim are expressed in ICg-glia. In all cases where mutations cause a relatively specific IOC defect, the phenotype is caused by hypomorphic or regulatory amorphic alleles (irreC, sim, omb), indicating that, so far, no genes have been identified that exclusively control IOC development. This confirms conclusions drawn from the analysis of Drosophila behavioral mutants that, in brain development, there are few genes dedicated to the development of just a single structure. In In(1)omb^H31 and in the mutants mentioned above, only a fraction of the IOC layers is disturbed. While penetrance of the IOC defect as assayed by paraffin histology is nearly complete, its expressivity varies. It appears that proper glial positioning is safeguarded by more than one molecular system. Such a redundancy of guidance systems has been demonstrated for the role of EGFR and PVR in Drosophila border cell migration (Hofmeyer, 2008).

In none of the cases cited, the developmental reasons for misprojection across the optic chiasms are understood to a satisfactory degree. In the case of the omb heteroalleles that lack OLR2, it is clear that glial function is essential for the establishment of a normally structured IOC. ICg-glia appear to present a local attractive cue that causes axons to navigate through the IOC in close contact with glial surfaces. In In(1)omb^H31, a fraction of the ICg-glia, which lack OMB, are displaced from the IOC into the neuropil of the lobula complex. If omb mutant ICg-glia aberrantly maintain the presentation of attractive cues, axon fascicles will be drawn to aberrant positions generating the incisions visible in paraffin histology. omb is expressed in several populations of migratory glial cells in the Drosophila visual system but it is not yet known how glial migration is affected by loss of omb. The reduced number of ICg-glia found in In(1)omb^H31 compared to wild-type adult brains suggests that glial migration is already disturbed on the path between the glial proliferation centers and the IOC target area (Hofmeyer, 2008).

At 72 h APF, the earliest time point at which aberrant Mz826-positive fibers were observed cutting into the lobula complex neuropil, displacement of glial cell bodies were seen at levels similar to the adult stage, but no obvious difference was seen in glial cell morphology. Therefore, glial cell mispositioning is likely to be the primary cause of the IOC defect. In Drosophila brain development, much of the adult connectivity is established during metamorphosis. Thus, changes in glial cell positioning that occur in the pupal stage could affect CNS patterning. The change in length and frequency of gliopodia in In(1)omb^H31 mutant must occur between 72 h APF and eclosion. At 72 APF, no hemispheres were observed with strong IOC defects and few with intermediate defects, and it is therefore possible that glial cell morphology influences the severity of the IOC phenotype during the last 24 h of pupal development (Hofmeyer, 2008).

Given the position of ICg-glia between three neuropils, these cells might also be involved in neuropil compartmentalization as described for neuropil glia in the central brain where compartments are separated by extensive glial sheaths. Such a role unlikely for the ICg-glia is considered unlikely because, ultra structurally, ICg-glia are typical fiber tract glia with lamellar extensions wrapping adjacent fiber bundles. Furthermore, in wild type, the cellular extensions produced by ICg-glia are relatively short and highly regular. Lastly, disturbance of ICg-glial function by loss of omb expression apparently did not cause compartmental disruptions of the type described previously (Hofmeyer, 2008).

It is concluded that investigation of the molecular and developmental consequences of OMB loss from ICg-glia appears to be a promising approach to elucidate one of possibly several systems that safeguard chiasmata projections. The availability of ombC-Gal4 with a highly restricted expression pattern in a subpopulations of glia in the developing fly visual system will allow the selective manipulation of gene activity in these cells, for example by RNAi. omb encodes a T-domain transcription factor with yet uncharacterized transactivation and repression functions. Potential target genes could therefore be either activated or repressed in cells where OMB is active. The results only allow to rule out regulation by OMB of three genes (or their enhancers): Repo, 3-66-lacZ (odd-paired) and ombC because (reporter-) gene expression remains active in ICg-glia in the absence of omb function. Only one gene with preferential expression in ICg-glia has been described: argos (aka. giant lens). Argos is a secreted antagonist of EGFR signaling and shows a relatively restricted expression pattern in the developing Drosophila visual system. In argos mutant animals, the visual system is severely disorganized including misrouting defects in the outer optic chiasm. Whether projections across the IOC are also affected in argos has not yet been determined. Such potential IOC defects would be independent of omb since argos remains expressed in ICg-glia in In(1)omb^H31 (Hofmeyer, 2008).

Activity of the ombC enhancer within the ICg-glia is likely to be controlled by a transcription factor, mutations of which should also affect IOC integrity. The DNA binding sites for two glial cell specific candidates, REPO and glide/gcm have been identified. The ombC enhancer sequence does not contain a glide/gcm consensus binding site 5'-AT(G/A)CGGG(T/C)-3'. REPO has been shown to bind a 5'-CAATTA-3' motif in vitro. This 6 nucleotide motif is present 4 times in the 6080-bp ombC sequence, while statistically it should be expected only 1.5× within any 6 kb sequence. It is therefore possible that omb expression in ICg-glia is controlled by REPO through the ombC enhancer. However, the CAATTA motif is also a target site for other homeodomain proteins such as FTZ and EN, and specificity is probably controlled by additional co-factors (Hofmeyer, 2008).

Unlike the Dipteran optic chiasmata, the mammalian optic chiasm (OC) is a midline structure where axons from both eyes have to decide whether to innervate the contralateral hemisphere or whether to stay ipsilateral. The mammalian OC, too, is characterized by a complex glial architecture, including a central palisade of radial glial processes. In spite of their identical names, the fly and mammalian optic chiasmata are apparently non-homologous structures. Given the conserved expression of OMB and its closest vertebrate homologues (Tbx2, 3, 5) in fly and vertebrate eye development, it will nonetheless be of interest to learn whether members of the Tbx2 subfamily are expressed in glial cells of the mammalian visual system (Hofmeyer, 2008).

optomotor-blind suppresses instability at the A/P compartment boundary of the Drosophila wing

Formation and function of the A/P compartment boundary of the Drosophila wing have been studied intensely. The boundary prevents mingling of A and P cells, is characterized by an expression discontinuity of several genes like engrailed, Cubitus interruptus, hedgehog and decapentaplegic and is essential for patterning the wing. Compared with segmental or compartmental boundaries in several other systems which generally manifest as folds or clefts, the wing A/P boundary is morphologically inconspicuous in both the larval and adult stage. This study shows that the Drosophila wing A/P boundary, too, is susceptible to fold and cleft formation, and these processes are suppressed by the T-box transcription factor Optomotor-blind (Omb). Using a targeted deletion encompassing the omb wing enhancer and omb-RNAi to strongly and specifically reduce Omb, it was shown that omb is required in P cells to prevent aberrant apical fold formation at the A/P boundary of the larval wing disc and cleft formation in the adult wing blade. In the larval A/P fold, DE-cadherin-based adherens junctions appeared intact but the apical microtubule web was strongly reduced (Shen, 2008).

The A/P compartment boundary is a lineage restriction that prevents cells from entering the neighbouring compartment. Nonetheless, cells on both sides of the boundary have not been reported to differ morphologically. Cells anterior to the A/P boundary respond to short-range Hh signaling by expressing the long-range morphogen Dpp. Dpp signaling is required for proper morphogenesis in cells of the main disc epithelium. In this tissue, the presence of the apical microtubule web correlates with Dpp signaling. omb which is regulated by both Dpp and Wg in the wing pouch is specifically required in A cells for A/P cell segregation. This study analyzed the developmental consequences of reduced Omb concentration. Evidence is provided that the posterior reduction of Omb causes an apical fold along the A/P boundary; this defect is correlated with a strong reduction of the apical microtubule web (Shen, 2008).

There are numerous enhancer and silencer elements which control the developmental expression pattern of omb. All brain-relevant enhancers identified so far are located downstream of the transcription unit. The omb upstream regulatory region (URR) has been partly characterized and was shown to contain a wing enhancer. This study analyzed the role of the omb URR by creating a precise deletion of a large part of this region (encompassing the wing enhancer) and by characterizing the developmental consequences in hemi- and homozygous mutant animals. Omb expression was strongly reduced in the URR deletion mutant omb^Δ(P2-P3w). This led to the formation of an apical fold along the A/P boundary, more pronounced in female than in male mutant discs. The sexually dimorphic phenotype appears to be due to differential reduction of Omb in male and female wing discs. Omb is involved in the development of sexually dimorphic structures like the genital discs and the differentially pigmented abdominal tergites. Apparently, disturbance of the omb gene structure by insertions or deletions tends to impart sex bias on parts of the expression pattern, which normally are not differentially expressed. In omb^Δ(P2-P3w) and omb^bi, wing size was similarly reduced, overall wing shape being maintained in both cases. Each mutant revealed a characteristic size reduction pattern of the various intervein regions which make up the adult wing blade. This agrees with the notion that the development of the intervein areas is subject to differential gene regulation (Shen, 2008).

In order to answer the question whether a uniform reduction of Omb in both compartments is required or whether reduction of Omb in one compartment is sufficient to induce the morphogenetic defect, the Omb level was reduced in either A or P cells by overexpressing UAS-ombRNAi under the control of dpp-Gal4, ptc-Gal4, or en-Gal4 or by overexpressing UAS-dad under en-Gal4 driver control. The different strategies reduced Omb to similarly low levels in either the A or P compartment. However, formation of the apical fold occurred only when Omb was reduced in P cells, indicating the importance of a normal Omb level in P cells for the regular morphogenesis of cells along the A/P boundary. In en>ombRNAi larvae, no folds were observed in leg or haltere discs in spite of the similar omb expression pattern in haltere and wing discs (Shen, 2008).

Dpp signaling can be monitored via the level of phosphorylated Mad (P-Mad). P-Mad staining across the wing pouch reveals an irregular profile of Dpp pathway activation reflecting the positive and negative influences to which Dpp signaling is subjected. The shape of this profile has a strong influence on how the wing develops. Proper Dpp signaling is required for cellular morphogenesis both in the main epithelium and in the peripodial membrane. Omb reduction causes up-regulation of tkv. When tkv or tkv^QD was overexpressed in A cells along the A/P boundary, a boundary fold was generated along with a posterior reduction of Omb. The posterior decrease in omb expression is due to reduced Dpp signaling in P cells which again is caused by the sequestration of Dpp by anterior Tkv. Overexpression of tkv^QD in P cells did not cause the morphogenetic defect. These results show that omb does not act via tkv. It appears that a certain Dpp signaling profile is required for appropriate omb expression and for proper morphogenesis of cells along the A/P boundary (Shen, 2008).

Reduction of Omb induced an apical morphogenetic defect in both A and P cells with no obvious changes in DE-cadherin level or distribution within the fold. E-cadherin levels do not necessarily correlate with E-cadherin-mediated cell-cell adhesion. The structure of the ectopic A/P fold suggests, however, that the apical adherens junctions are maintained, except at the A/P junction itself where they are likely to become disrupted in the process of cleft formation during pupal development. In the affected cells, the apical microtubule web was strongly reduced. The shape of a cell to a large extent depends on its cytoskeleton made up of actin microfilaments and microtubules. Cells lacking Dpp signaling lose their apical microtubule web. In omb null mutant clones, the apical microtubule web was still present, indicating that loss of Omb is not sufficient to elicit reduction of apically localized microtubules. The latter effect was only observed when posterior Omb reduction abutted the A/P compartment boundary. Reduction of the apical microtubule web was confined to cells within the fold (similar to the increase in apical actin) and did not extend to the entire area of reduced Omb expression. In contrast, changes in tubulin (and actin) localization also occurred in A cells where Omb was not reduced. Thus, Omb cannot directly elicit these changes (Shen, 2008).

During segmental groove formation in the Drosophila embryo, a fold forms at the boundary between En expressing and non-expressing cells. These folds are initiated unilaterally by apical constriction in a single row of En-positive cells. Similarly, the invagination of the Drosophila stomatogastric system is initiated by individual cells which coordinate the morphogenetic changes of the surrounding tissue. It is assumed that under conditions of low posterior Omb concentration a folding program is initiated in a row of cells adjacent to the A/P boundary which is then communicated to adjoining rows. In the well-studied process of ventral furrow formation, the diffusible protein Folded gastrulation (Fog) holds a central role in coordinating the necessary morphogenetic changes. Galindo (2007) has identified Tarsal-less (Tal), an 11 amino acid oligopeptide, as essential for fold formation and tarsal segmentation in Drosophila leg development. Whether Fog and the other proteins involved in ventral furrow formation, or Tal are also required for (ectopic) fold formation in the wing imaginal disc is not yet known (Shen, 2008).

The two regulatory omb mutants (omb^bi and omb^Δ(P2-P3w)) caused a uniform reduction of Omb in the wing pouch. When this reduction was sufficiently strong (influenced by genotype, sex, and temperature), a fold developed along the A/P boundary. What makes this boundary particularly susceptible to fold formation? The A/P boundary is a dynamic structure which requires continuous synthesis of Hh by the En-expressing cells of the P compartment and also responsiveness of the adjoining A cells to the Hh signal. It is not known what property Hh is inducing in A-cells to effect separation of the two cell populations. Omb is required in P-cells to prevent A/P-fold formation. Interestingly, posterior omb clones have no effect on the compartmental properties of the A/P boundary while anterior clones cause its destabilization. The role anterior Omb has in stabilizing the A/P boundary thus appears unrelated to the role posterior Omb plays in suppressing fold formation (Shen, 2008).

In general, segmental or compartmental boundaries eventually become morphologically apparent as folds or clefts. This, clearly, does not hold for the A/P boundary which in the adult wing runs between longitudinal veins L3 and L4 and which only can be recognized by histochemical staining. The A/P boundary, while morphologically inconspicuous, presents a sharp discontinuity in gap junctional communication, in the expression of Ci (in A cells), En (in P cells), and in Dpp signaling activity. Spatial discontinuities in Dpp signaling cause Jun-N-terminal kinase (JNK)-dependent morphogenetic apoptosis and fold formation. omb apparently is required to repress such a process. When Omb was reduced to medium levels, A/P fold formation occurred but was not associated with cell death. Strong reduction of elimination of Omb causes cell death in the centre of the wing blade during pupal development. The insect wing is an intricate and highly conserved biomechanical structure. The central A/P boundary is considered exquisitely apt for patterning the wing imaginal disc but, as outlined above, holds the danger of introducing structural discontinuity into the wing blade which would compromise flight performance. In addition to the roles of Omb in wing development which were identified previously Omb appears to safeguard wing structural integrity. Interestingly, all four closely Omb-related vertebrate Tbx2-subfamily genes are expressed in limb development. Heterozygosity for TBX3, TBX4, and TBX5 causes limb developmental defects in humans suggesting a conserved involvement of Tbx2-subfamily genes in limb development (Shen, 2008).

Umemori (2007) obtained findings similar to these using the weaker omb^bi allele. A/P fold formation in this omb hypomorph was attributed to hyperactivation of the Hh signaling pathway. This mechanistic interpretation at first glance appears at odds with identification of the posterior compartment as the area in which Omb needs to be reduced for fold formation. Hh signaling occurs only in the A compartment because Ci is repressed posteriorly by En. Since anterior Omb reduction does not elicit fold formation, a fold-related role of Omb in signal transduction downstream of the Hh ligand can be ruled out. This can also be concluded from the observation that anterior Mad clones, in which omb is not transcribed, express patched, which is induced by Hh signaling, at normal levels. This leaves the possibility that Omb might be involved in the production of the Hh signal. hh expression is not affected in posterior omb clones. The release of the mature Hh ligand is a carefully controlled process involving autoproteolysis and N- and C-terminal lipid modification in producing cells. The efficiency of Hh as a signaling molecule is affected by these modifications. Omb could impact on Hh signaling by acting at the level of Hh signal production. This is in agreement with the observation of strong A/P fold formation in hh>hh flies. Alternatively, spreading of Hh across the A/P boundary could secondarily be affected by A/P fold formation. In Drosophila eye development, a morphogenetic furrow (MF) sweeps across the eye imaginal disc. The MF is formed as a consequence of synchronized transient apical constriction. Hh is required for MF formation; vice versa, in mutants in which MF formation is disrupted, Hh propagation across the furrow region is altered. Hh signaling prevents the proteolysis of Ci by a microtubule-anchored hetero-oligomeric complex. A second discrepancy between the current work and that of Umemori concerns microtubule and F-actin localization. In the third instar wing pouch microtubules are enriched in an apical web and F-actin, too, (phalloidin staining) is enriched on the apical side. The current study conform to these descriptions. Only in cells within the fold, the apical microtubule web is lost and the apical F-actin is further enriched. Umemori report phalloidin staining and alpha-tubulin immunoreactivity in a largely coinciding distribution predominantly on the basal side of the main disc epithelium, with apical staining only in cells of the fold. The reduction of the apical microtubule web which was observed raises the intriguing possibility that Hh signaling might be affected by changes in the microtubule network. While Hh has an eminent function in A/P compartment boundary formation, it is not its sole determinant. En is required for setting the A/P boundary independent of its role in the production of Hh. Omb could be involved in the latter En function. Further experiments will show at which level Omb acts to suppress the formation of the A/P fold (Shen, 2008).

Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy

Manipulating an exogenous or endogenous gene of interest at a defined level is critical for a wide variety of experiments. The Gal4/UAS system has been widely used to direct gene expression for studying complex genetic and biological problems in Drosophila melanogaster and other model organisms. Driven by a given tissue-specific Gal4, expressing UAS-transgene or UAS-RNAi (RNA interference) could be used to up- or down-regulate target gene expression, respectively. However, the efficiency of the Gal4/UAS system is roughly predefined by properties of transposon vector constructs and the insertion site in the transgenic stock. This study describes a simple way to modulate optomotor blind (omb) expression levels in its endogenous expression region of the wing disc. UAS-omb and UAS-omb-RNAi were co-expressed together under the control of dpp-Gal4 driver which is expressed in the omb expression region of the wing pouch. The repression effect is more sensitive to temperature than that of overexpression. At low temperature, overexpression plays a dominant role but the efficiency is attenuated by UAS-omb-RNAi. In contrast, at high temperature RNAi predominates in gene expression regulation. By this strategy, it was possible to manipulate omb expression levels at a moderate level. It allows manipulation of omb expression levels in the same tissue between overexpression and repression at different stages by temperature control (Zhang, 2019).

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

Apitz, H. and Salecker, I. (2018). Spatio-temporal relays control layer identity of direction-selective neuron subtypes in Drosophila. Nat Commun 9(1): 2295. PubMed ID: 29895891

Arnold, S. J., et al. (2008). The T-box transcription factor Eomes/Tbr2 regulates neurogenesis in the cortical subventricular zone. Genes Dev. 22: 2479-2484. PubMed Citation: 18794345

Aydogdu, N., Rudat, C., Trowe, M. O., Kaiser, M., Ludtke, T. H., Taketo, M. M., Christoffels, V. M., Moon, A. and Kispert, A. (2018). TBX2 and TBX3 act downstream of canonical WNT signaling in patterning and differentiation of the mouse ureteric mesenchyme. Development 145(23). PubMed ID: 30478225

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. PubMed Citation: 10077612

Bedogni, F., et al. (2010). Tbr1 regulates regional and laminar identity of postmitotic neurons in developing neocortex. Proc. Natl. Acad. Sci. 107(29): 13129-34. PubMed Citation: 20615956

Berberoglu M. A., Dong Z., Mueller T. and Guo S. (2009). fezf2 expression delineates cells with proliferative potential and expressing markers of neural stem cells in the adult zebrafish brain. Gene Expr. Patterns 9: 411-422. PubMed Citation: 19524703

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

Bongers, E. M., et al. (2004). Mutations in the human TBX4 gene cause small patella syndrome. Am. J. Hum. Genet. 74: 1239-1248. PubMed Citation: 15106123

Bothe, I., Tenin, G., Oseni, A. and Dietrich, S. (2011). Dynamic control of head mesoderm patterning. Development 138(13): 2807-21. PubMed Citation: 21652653

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. PubMed Citation: 10373308

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. PubMed Citation: 1556634

Büttner, N., Johnsen, S. A., Kügler, S. and Vogel, T. (2010). Af9/Mllt3 interferes with Tbr1 expression through epigenetic modification of histone H3K79 during development of the cerebral cortex. Proc. Natl. Acad. Sci. 107(15): 7042-7. PubMed Citation: 20348416

Calmont, A., et al. (2009). Tbx1 controls cardiac neural crest cell migration during arch artery development by regulating Gbx2 expression in the pharyngeal ectoderm. Development 136(18): 3173-83. PubMed Citation: 19700621

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. PubMed Citation: 8530034

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. PubMed Citation: 10052457

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. PubMed Citation: 9710594

Chapman, D. L., et al. (1996). Tbx6, a mouse T-box ene implicated in paraxial mesoderm formation at gastruation. Dev. Biol. 180: 534-542. PubMed Citation: 8954725

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. PubMed Citation: 9490412

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. PubMed Citation: 10331981

Erives, A. and Levine, M. (2000). Characterization of a maternal T-Box gene in Ciona intestinalis. Dev. Biol. 225: 169-178. PubMed Citation: 10964472

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

Fristrom, D. (1976). The mechanism of evagination of imaginal discs of Drosophila melanogaster: III. Evidence for cell rearrangement. Dev. Biol. 54: 163-171. PubMed Citation: 825402

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

Galindo, M. I., et al. (2007). Peptides encoded by short ORFs control development and define a new eukaryotic gene family. PLoS Biol. 5: e106. PubMed Citation: 17439302

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

Glickstein, S. B., et al. (2009). Cyclin D2 is critical for intermediate progenitor cell proliferation in the embryonic cortex. J. Neurosci. 29(30): 9614-24. PubMed Citation: 19641124

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

Griffin, K. J., et al. (1998). Molecular identification of spadetail: regulation of zebrafish trunk and tail mesoderm formation by T-box genes. Development 125(17): 3379-3388. PubMed Citation: 9693141

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. PubMed Citation: 8599120

Gros, J. and Tabin, C. J. (2014). Vertebrate limb bud formation is initiated by localized epithelial-to-mesenchymal transition. Science 343: 1253-1256. PubMed ID: 24626928

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. PubMed Citation: 9735359

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

Hasegawa, Y., Takata, N., Okuda, S., Kawada, M., Eiraku, M. and Sasai, Y. (2016). Emergence of dorsal-ventral polarity in ESC-derived retinal tissue. Development 143(21): 3895-3906. PubMed ID: 27633992

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

Hofmeyer, K., Kretzschmar, D. and Pflugfelder, G. O. (2008). Optomotor-blind expression in glial cells is required for correct axonal projection across the Drosophila inner optic chiasm. Dev. Biol. 315(1): 28-41. PubMed Citation: 18234176

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. PubMed Citation: 9165118

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, B., Li, X., Tu, X., Zhao, W., Zhu, D., Feng, Y., Si, X. and Chen, J. G. (2018). OTX1 regulates cell cycle progression of neural progenitors in the developing cerebral cortex. J Biol Chem 293(6): 2137-2148. PubMed ID: 29273633

Huh, S. H. and Ornitz, D. M. (2010). Beta-catenin deficiency causes DiGeorge syndrome-like phenotypes through regulation of Tbx1. Development 137(7): 1137-47. PubMed Citation: 20215350

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

Jeong J. Y., et al. (2007). Patterning the zebrafish diencephalon by the conserved zinc-finger protein Fezl. Development 134: 127-136. PubMed Citation: 17164418

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. PubMed Citation: 9367427

Kopp, A., and Duncan, I. (2002). Anteroposterior patterning in adult abdominal segments of Drosophila. Dev. Biol. 242: 15-30. PubMed Citation: 11795937

Kugler, J. E., et al. (2010). Temporal regulation of the muscle gene cascade by Macho1 and Tbx6 transcription factors in Ciona intestinalis. J. Cell Sci. 123(Pt 14): 2453-63. PubMed Citation: 20592183

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. PubMed Citation: 8632795

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). PubMed Citation: 9043075

Logan, M. and Tabin, C. J. (1999). Role of Pitx1 upstream of Tbx4 in specification of hindlimb identity. Science 283(5408): 1736-9. PubMed Citation: 10073939

Lüdtke, T.H., Rudat, C., Wojahn, I., Weiss, A.C., Kleppa, M.J., Kurz, J., Farin, H.F., Moon, A., Christoffels, V.M. and Kispert, A. (2016). Tbx2 and Tbx3 act downstream of Shh to maintain canonical Wnt signaling during branching morphogenesis of the murine lung. Dev Cell 39: 239-253. PubMed ID: 27720610

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. PubMed Citation: 10073939

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

Mora, N., Oliva, C., Fiers, M., Ejsmont, R., Soldano, A., Zhang, T. T., Yan, J., Claeys, A., De Geest, N. and Hassan, B. A. (2018). A temporal transcriptional switch governs stem cell division, neuronal numbers, and maintenance of differentiation. Dev Cell 45(1): 53-66 e55. PubMed ID: 29576424

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

Nishimoto, S., Minguillon, C., Wood, S. and Logan, M. P. (2014). A combination of activation and repression by a colinear hox code controls forelimb-restricted expression of tbx5 and reveals hox protein specificity. PLoS Genet 10: e1004245. PubMed ID: 24651482

Ochiai, W., et al. (2009). Periventricular notch activation and asymmetric Ngn2 and Tbr2 expression in pair-generated neocortical daughter cells. Mol. Cell. Neurosci. 40: 225-233. PubMed Citation: 19059340

Pechmann, M. and Prpic, N. M. (2022). The T-box gene optomotor-blind organizes proximodistal leg patterning in the beetle Tribolium castaneum by repressing dorsal Dpp pathway activity. Dev Biol 482: 124-134. PubMed ID: 34942194

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. PubMed Citation: 2121596

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. Natl Acad. Sci. 89: 1199-1203. PubMed Citation: 1741374

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. PubMed Citation: 1497674

Pilaz, L. J., et al. (2009). Forced G1-phase reduction alters mode of division, neuron number, and laminar phenotype in the cerebral cortex. Proc. Natl. Acad. Sci. 106(51): 21924-9. PubMed Citation: 19959663

Pinto-Teixeira, F., Koo, C., Rossi, A. M., Neriec, N., Bertet, C., Li, X., Del-Valle-Rodriguez, A. and Desplan, C. (2018). Development of concurrent retinotopic maps in the fly motion detection circuit. Cell 173(2): 485-498. PubMed ID: 29576455

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. PubMed Citation: 8492800

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. PubMed Citation: 8325232

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. PubMed Citation: 10235264

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. PubMed Citation: 9569350

Saunders, L. R. and McClay, D. R. (2014). Sub-circuits of a gene regulatory network control a developmental epithelial-mesenchymal transition. Development 141: 1503-1513. PubMed ID: 24598159

Sansom, S. N., et al. (2009). The level of the transcription factor Pax6 is essential for controlling the balance between neural stem cell self-renewal and neurogenesis. PLoS Genet. 5(6): e1000511. PubMed Citation: 19521500

Sessa A., et al. (2008). Tbr2 directs conversion of radial glia into basal precursors and guides neuronal amplification by indirect neurogenesis in the developing neocortex. Neuron 60, 56-69. PubMed Citation: 18940588

Sessa, A., et al. (2010). Tbr2-positive intermediate (basal) neuronal progenitors safeguard cerebral cortex expansion by controlling amplification of pallial glutamatergic neurons and attraction of subpallial GABAergic interneurons. Genes Dev. 24(16): 1816-26. PubMed Citation: 20713522

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

Shen, J., Dorner, C., Bahlo, A., Pflugfelder, G. O. (2008). optomotor-blind suppresses instability at the A/P compartment boundary of the Drosophila wing. Mech. Dev. 125(3-4): 233-46. PubMed Citation: 18171611

Shen, J., Lu, J., Sui, L., Wang, D., Yin, M., Hoffmann, I., Legler, A. and Pflugfelder, G. O. (2014). The orthologous Tbx transcription factors Omb and TBX2 induce epithelial cell migration and extrusion in vivo without involvement of matrix metalloproteinases. Oncotarget [Epub ahead of print]. PubMed ID: 25344916

Shimizu, T., et al. (2010). Zinc finger genes Fezf1 and Fezf2 control neuronal differentiation by repressing Hes5 expression in the forebrain. Development 137(11): 1875-85. PubMed Citation: 20431123

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. PubMed Citation: 9118806

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

Singhvi, A., Frank, C. A. and Garriga, G. (2008). The T-box gene tbx-2, the homeobox gene egl-5 and the asymmetric cell division gene ham-1 specify neural fate in the HSN/PHB lineage. Genetics 179(2): 887-98. PubMed Citation: 18505863

Sivasankaran, R., et al. (2000). Direct transcriptional control of the Dpp target omb by the DNA binding protein Brinker. EMBO J. 19: 6162-6172. PubMed Citation: 11080162

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. PubMed Citation: 11106753

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. PubMed Citation: 9012537

Stennard, F., et al. (1999). Differential expression of VegT and Antipodean protein isoforms in Xenopus. Mech. Dev. 86(1-2): 87-98. PubMed Citation: 10446268

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. PubMed Citation: 10235263

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

Taylor, J. and Adler, P. N. (2008). Cell rearrangement and cell division during the tissue level morphogenesis of evaginating Drosophila imaginal discs. Dev. Biol. 313(2): 739-51. PubMed Citation: 18082159

Torres-Vazquez, J., Warrior, R. and Arora, K. (2000). schnurri is required for dpp-dependent patterning of the Drosophila wing. Dev. Biol. 227: 388-402. PubMed Citation: 11071762

Tsuneizumi, K., et al. (1997). Daughters against dpp modulates dpp organizing activity in Drosophila wing development. Nature 389(6651): 627-631. PubMed Citation: 9335506

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

Umemori, M. et al. (2007). Drosophila T-box transcription factor Optomotor-blind prevents pathological folding and local overgrowth in wing epithelium through confining Hh signal. Dev. Biol. 308: 68-81. PubMed Citation: 17573067

Veien, E. S., et al. (2008). Canonical Wnt signaling is required for the maintenance of dorsal retinal identity. Development 135(24): 4101-11. PubMed Citation: 19004855

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

Huang, T. N., Chuang, H. C., Chou, W. H., Chen, C. Y., Wang, H. F., Chou, S. J. and Hsueh, Y. P. (2014). Tbr1 haploinsufficiency impairs amygdalar axonal projections and results in cognitive abnormality. Nat Neurosci 17: 240-247. PubMed ID: 24441682

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. PubMed Citation: 9585507

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. PubMed Citation: 9502724

Xanthos, J. G., et al. (2001). Maternal VegT is the initiator of a molecular network specifying endoderm in Xenopus laevis. Development 128: 167-180. PubMed Citation: 11124113

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. PubMed Citation: 9693142

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. Biol. 228: 95-105. PubMed Citation: 11087629

Yasuhiko, Y., et al. (2008). Functional importance of evolutionally conserved Tbx6 binding sites in the presomitic mesoderm-specific enhancer of Mesp2. Development 135(21): 3511-9. PubMed Citation: 18849530

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. PubMed Citation: 9012531

Zhang, J., et al. (1998). The role of maternal VegT in establishing the primary germ layers in Xenopus embryos. Cell 94(4): 515-524. PubMed Citation: 9727494

Zhang, X. B., Dong, W., Li, K. X., Wang, J. J., Shen, J., Moussian, B. and Zhang, J. Z. (2019). Flexible manipulation of Omb levels in the endogenous expression region of Drosophila wing by combinational overexpression and suppression strategy. Insect Sci. 27(1):14-21. PubMed ID: 31246335

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: 20 April 2021
 

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.