The Tbx6 Transcription Factor Dorsocross Mediates Dpp Signaling to Regulate Drosophila Thorax Closure.

Movement and fusion of separate cell populations are critical for several developmental processes, such as neural tube closure in vertebrates or embryonic dorsal closure and pupal thorax closure in Drosophila. Fusion failure results in an opening or groove on the body surface. Drosophila pupal thorax closure is an established model to investigate the mechanism of tissue closure. Here, we report the identification of T-box transcription factor genes Dorsocross (Doc) as Decapentaplegic (Dpp) targets in the leading edge cells of the notum in the late third instar larval and early pupal stages. Reduction of Doc in the notum region results in a thorax closure defect, similar to that in dpp loss-of-function flies. Nine genes are identified as potential downstream targets of Doc in regulating thorax closure by molecular and genetic screens. Our results reveal a novel function of Doc in Drosophila development. The candidate target genes provide new clues for unravelling the mechanism of collective cell movement.

Transcriptional repression of the ectodomain sheddase ADAM10 by TBX2 and potential implication for Alzheimer's disease.

BACKGROUND: The ADAM10-mediated cleavage of transmembrane proteins regulates cellular processes such as proliferation or migration. Substrate cleavage by ADAM10 has also been implicated in pathological situations such as cancer or Morbus Alzheimer. Therefore, identifying endogenous molecules, which modulate the amount and consequently the activity of ADAM10, might contribute to a deeper understanding of the enzyme's role in both, physiology and pathology. METHOD: To elucidate the underlying cellular mechanism of the TBX2-mediated repression of ADAM10 gene expression, we performed overexpression, RNAi-mediated knockdown and pharmacological inhibition studies in the human neuroblastoma cell line SH-SY5Y. Expression analysis was conducted by e.g. real-time RT-PCR or western blot techniques. To identify the binding region of TBX2 within the ADAM10 promoter, we used luciferase reporter assay on deletion constructs and EMSA/WEMSA experiments. In addition, we analyzed a TBX2 loss-of-function Drosophila model regarding the expression of ADAM10 orthologs by qPCR. Furthermore, we quantified the mRNA level of TBX2 in post-mortem brain tissue of AD patients. RESULTS: Here, we report TBX2 as a transcriptional repressor of ADAM10 gene expression: both, the DNA-binding domain and the repression domain of TBX2 were necessary to effect transcriptional repression of ADAM10 in neuronal SH-SY5Y cells. This regulatory mechanism required HDAC1 as a co-factor of TBX2. Transcriptional repression was mediated by two functional TBX2 binding sites within the core promoter sequence (- 315 to - 286 bp). Analysis of a TBX2 loss-of-function Drosophila model revealed that kuzbanian and kuzbanian-like, orthologs of ADAM10, were derepressed compared to wild type. Vice versa, analysis of cortical brain samples of AD-patients, which showed reduced ADAM10 mRNA levels, revealed a 2.5-fold elevation of TBX2, while TBX3 and TBX21 levels were not affected. CONCLUSION: Our results characterize TBX2 as a repressor of ADAM10 gene expression and suggest that this regulatory interaction is conserved across tissues and species.

Dpp signaling inhibits proliferation in the Drosophila wing by Omb-dependent regional control of bantam.

The control of organ growth is a fundamental aspect of animal development but remains poorly understood. The morphogen Dpp has long been considered as a general promoter of cell proliferation during Drosophila wing development. It is an ongoing debate whether the Dpp gradient is required for the uniform cell proliferation observed in the wing imaginal disc. Here, we investigated how the Dpp signaling pathway regulates proliferation during wing development. By systematic manipulation of Dpp signaling we observed that it controls proliferation in a region-specific manner: Dpp, via omb, promoted proliferation in the lateral and repressed proliferation in the medial wing disc. Omb controlled the regional proliferation rate by oppositely regulating transcription of the microRNA gene bantam in medial versus lateral wing disc. However, neither the Dpp nor Omb gradient was essential for uniform proliferation along the anteroposterior axis.

The Dorsocross T-box transcription factors promote tissue morphogenesis in the Drosophila wing imaginal disc.

The Drosophila wing imaginal disc is subdivided into notum, hinge and blade territories during the third larval instar by formation of several deep apical folds. The molecular mechanisms of these subdivisions and the subsequent initiation of morphogenic processes during metamorphosis are poorly understood. Here, we demonstrate that the Dorsocross (Doc) T-box genes promote the progression of epithelial folds that not only separate the hinge and blade regions of the wing disc but also contribute to metamorphic development by changing cell shapes and bending the wing disc. We found that Doc expression was restricted by two inhibitors, Vestigial and Homothorax, leading to two narrow Doc stripes where the folds separating hinge and blade are forming. Doc mutant clones prevented the lateral extension and deepening of these folds at the larval stage and delayed wing disc bending in the early pupal stage. Ectopic Doc expression was sufficient to generate deep apical folds by causing a basolateral redistribution of the apical microtubule web and a shortening of cells. Cells of both the endogenous blade/hinge folds and of folds elicited by ectopic Doc expression expressed Matrix metalloproteinase 2 (Mmp2). In these folds, integrins and extracellular matrix proteins were depleted. Overexpression of Doc along the blade/hinge folds caused precocious wing disc bending, which could be suppressed by co-expressing MMP2RNAi.

The transcription factors TBX2 and TBX3 interact with human papillomavirus 16 (HPV16) L2 and repress the long control region of HPVs.

The minor capsid protein L2 of human papillomaviruses (HPVs) has multiple functions during the viral life cycle. Although L2 is required for effective invasion and morphogenesis, only a few cellular interaction partners are known so far. Using yeast two-hybrid screening, we identified the transcription factor TBX2 as a novel interaction partner of HPV type 16 (HPV16) L2. Coimmunoprecipitations and immunofluorescence analyses confirmed the L2-TBX2 interaction and revealed that L2 also interacts with TBX3, another member of the T-box family. Transcription of the early genes during HPV infection is under the control of an upstream enhancer and early promoter region, the long control region (LCR). In promoter-reporter gene assays, we observed that TBX2 and TBX3 repress transcription from the LCR and that this effect is enhanced by L2. Repression of the HPV LCR by TBX2/3 seems to be a conserved mechanism, as it was also observed with the LCRs of different HPV types. Finally, interaction of TBX2 with the LCR was detected by chromatin immunoprecipitation, and we found a strong colocalization of L2 and TBX2 in HPV16-positive cervical intraepithelial neoplasia (CIN) I-II tissue sections. These results suggest that TBX2/3 might play a role in the regulation of HPV gene expression during the viral life cycle.

Optomotor-blind in the development of the Drosophila HS and VS lobula plate tangential cells.

The horizontal system and vertical system cells of the dipteran optic lobes are well understood regarding their physiology and role in visually guided behavior. Little is known, however, about their development. Drosophila optomotor-blind (omb) is required for the development of the HS/VS cells which are lacking in the adult brain of the In(1)omb[H31] regulatory mutant. We have analyzed the omb regulatory region, required for HS/VS development, for enhancers active in the central nervous system. A 1-kb fragment, ombJb, was identified 114 kb downstream of the omb transcription start site, that could drive expression in much of the presumptive embryonic optic lobe anlage. Expression in these cells is lost in In(1)omb[H31] suggesting that they contain the HS/VS precursor cell(s). We used Laser ablation in the embryonic CNS in order to localize the position of the HS/VS precursor cell(s) in this tissue. An omb-Gal4 enhancer trap line, which showed activity in the optic lobe anlage in a pattern similar to ombJb enhancer, was used to drive GFP expression, thus allowing to focus the Laser beam to the relevant area. We identified a small region in the embryonic brain from which the HS/VS cells are likely to develop. Omb encodes a transcription factor of the T-box family. Since loss of omb disrupts HS/VS cell development, we assume that HS/VS ontogeny is controlled by Omb target genes. As a first step toward their identification, we characterized the Omb DNA-binding specificity.

Spatial discontinuity of optomotor-blind expression in the Drosophila wing imaginal disc disrupts epithelial architecture and promotes cell sorting.

BACKGROUND: Decapentaplegic (Dpp) is one of the best characterized morphogens, required for dorso-ventral patterning of the Drosophila embryo and for anterior-posterior (A/P) patterning of the wing imaginal disc. In the larval wing pouch, the Dpp target gene optomotor-blind (omb) is generally assumed to be expressed in a step function above a certain threshold of Dpp signaling activity. RESULTS: We show that the transcription factor Omb forms, in fact, a symmetrical gradient on both sides of the A/P compartment boundary. Disruptions of the Omb gradient lead to a re-organization of the epithelial cytoskeleton and to a retraction of cells toward the basal membrane suggesting that the Omb gradient is required for correct epithelial morphology. Moreover, by analysing the shape of omb gain- and loss-of-function clones, we find that Omb promotes cell sorting along the A/P axis in a concentration-dependent manner. CONCLUSIONS: Our findings show that Omb distribution in the wing imaginal disc is described by a gradient rather than a step function. Graded Omb expression is necessary for normal cell morphogenesis and cell affinity and sharp spatial discontinuities must be avoided to allow normal wing development.

Null mutations in Drosophila Optomotor-blind affect T-domain residues conserved in all Tbx proteins.

The T-box transcription factors TBX2 and TBX3 are overexpressed in many human cancers raising the need for a thorough understanding of the cellular function of these proteins. In Drosophila, there is one corresponding ortholog, Optomotor-blind (Omb). Currently, only two missense mutations are known for the two human proteins. Making use of the developmental defects caused by inactivation of omb, we have isolated and molecularly characterized four new omb mutations, three of them are missense mutations of amino acids fully conserved in all Tbx proteins. We interpret the functional defects in the framework of the known structure of the human TBX3 protein and provide evidence for loss of Omb DNA-binding activity in all three newly identified missense mutations.

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. Here, we show 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. We narrowed down the onset of the IOC defect 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), we identified an enhancer element (ombC) 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.

Functional identity of hypothalamic melanocortin neurons depends on Tbx3.

Heterogeneous populations of hypothalamic neurons orchestrate energy balance via the release of specific signatures of neuropeptides. However, how specific intracellular machinery controls peptidergic identities and function of individual hypothalamic neurons remains largely unknown. The transcription factor T-box 3 (Tbx3) is expressed in hypothalamic neurons sensing and governing energy status, whereas human TBX3 haploinsufficiency has been linked with obesity. Here, we demonstrate that loss of Tbx3 function in hypothalamic neurons causes weight gain and other metabolic disturbances by disrupting both the peptidergic identity and plasticity of Pomc/Cart and Agrp/Npy neurons. These alterations are observed after loss of Tbx3 in both immature hypothalamic neurons and terminally differentiated mouse neurons. We further establish the importance of Tbx3 for body weight regulation in Drosophila melanogaster and show that TBX3 is implicated in the differentiation of human embryonic stem cells into hypothalamic Pomc neurons. Our data indicate that Tbx3 directs the terminal specification of neurons as functional components of the melanocortin system and is required for maintaining their peptidergic identity. In summary, we report the discovery of a key mechanistic process underlying the functional heterogeneity of hypothalamic neurons governing body weight and systemic metabolism.

A fluorescent core-shell dendritic macromolecule specifically stains the extracellular matrix.

The extracellular cell matrix (ECM) surrounds cells and plays important roles in many aspects of cellular fate, including cell migration, stem cell differentiation, and cancer progression. So far, there is no fluorescent dye to directly visualize the ECM network. Here we present a positively charged fluorescent core-shell dendritic macromolecule containing multiple -NH2 groups which specifically binds to highly negatively charged ECM components. Due to its advantageous optical properties and biological specificity, the dye is useful as a routine tool to label the ECM in life science research.

The DrosDel deletion collection: a Drosophila genomewide chromosomal deficiency resource.

We describe a second-generation deficiency kit for Drosophila melanogaster composed of molecularly mapped deletions on an isogenic background, covering approximately 77% of the Release 5.1 genome. Using a previously reported collection of FRT-bearing P-element insertions, we have generated 655 new deletions and verified a set of 209 deletion-bearing fly stocks. In addition to deletions, we demonstrate how the P elements may also be used to generate a set of custom inversions and duplications, particularly useful for balancing difficult regions of the genome carrying haplo-insufficient loci. We describe a simple computational resource that facilitates selection of appropriate elements for generating custom deletions. Finally, we provide a computational resource that facilitates selection of other mapped FRT-bearing elements that, when combined with the DrosDel collection, can theoretically generate over half a million precisely mapped deletions.

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 (omb) 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, we investigated the relevance of individual domains in vitro and in vivo. In cell culture transfection assays, ORG-1 was a strong transcriptional activator, whereas OMB appeared 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 were ectopically expressed during development. Gain-of-function expression of OMB is known to counteract eye formation and resulted in the loss of the arista, whereas ORG-1 had little effect on eye development but caused 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 was 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, we observed, however, a strong influence of the T-domain on ommatidial differentiation. The specificity of chimeric omb/org-1transgenes, thus, depended on the cellular context in which they were 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.

Fold formation at the compartment boundary of Drosophila wing requires Yki signaling to suppress JNK dependent apoptosis.

Compartment boundaries prevent cell populations of different lineage from intermingling. In many cases, compartment boundaries are associated with morphological folds. However, in the Drosophila wing imaginal disc, fold formation at the anterior/posterior (A/P) compartment boundary is suppressed, probably as a prerequisite for the formation of a flat wing surface. Fold suppression depends on optomotor-blind (omb). Omb mutant animals develop a deep apical fold at the A/P boundary of the larval wing disc and an A/P cleft in the adult wing. A/P fold formation is controlled by different signaling pathways. Jun N-terminal kinase (JNK) and Yorkie (Yki) signaling are activated in cells along the fold and are necessary for the A/P fold to develop. While JNK promotes cell shape changes and cell death, Yki target genes are required to antagonize apoptosis, explaining why both pathways need to be active for the formation of a stable fold.

The DrosDel collection: a set of P-element insertions for generating custom chromosomal aberrations in Drosophila melanogaster.

We describe a collection of P-element insertions that have considerable utility for generating custom chromosomal aberrations in Drosophila melanogaster. We have mobilized a pair of engineered P elements, p[RS3] and p[RS5], to collect 3243 lines unambiguously mapped to the Drosophila genome sequence. The collection contains, on average, an element every 35 kb. We demonstrate the utility of the collection for generating custom chromosomal deletions that have their end points mapped, with base-pair resolution, to the genome sequence. The collection was generated in an isogenic strain, thus affording a uniform background for screens where sensitivity to genetic background is high. The entire collection, along with a computational and genetic toolbox for designing and generating custom deletions, is publicly available. Using the collection it is theoretically possible to generate >12,000 deletions between 1 bp and 1 Mb in size by simple eye color selection. In addition, a further 37,000 deletions, selectable by molecular screening, may be generated. We are now using the collection to generate a second-generation deficiency kit that is precisely mapped to the genome sequence.

Putative Breast Cancer Driver Mutations in TBX3 Cause Impaired Transcriptional Repression.

The closely related T-box transcription factors TBX2 and TBX3 are frequently overexpressed in melanoma and various types of human cancers, in particular, breast cancer. The overexpression of TBX2 and TBX3 can have several cellular effects, among them suppression of senescence, promotion of epithelial-mesenchymal transition, and invasive cell motility. In contrast, loss of function of TBX3 and most other human T-box genes causes developmental haploinsufficiency syndromes. Stephens and colleagues (1), by exome sequencing of breast tumor samples, identified five different mutations in TBX3, all affecting the DNA-binding T-domain. One in-frame deletion of a single amino acid, p.N212delN, was observed twice. Due to the clustering of these mutations to the T-domain and for statistical reasons, TBX3 was inferred to be a driver gene in breast cancer. Since mutations in the T-domain generally cause loss of function and because the tumorigenic action of TBX3 has generally been attributed to overexpression, we determined whether the putative driver mutations had loss- or gain-of-function properties. We tested two in-frame deletions, one missense, and one frameshift mutant protein for DNA-binding in vitro, and for target gene repression in cell culture. In addition, we performed an in silico analysis of somatic TBX mutations in breast cancer, collected in The Cancer Genome Atlas (TCGA). Both the experimental and the in silico analysis indicate that the observed mutations predominantly cause loss of TBX3 function.

Optomotor-blind negatively regulates Drosophila eye development by blocking Jak/STAT signaling.

Organ formation requires a delicate balance of positive and negative regulators. In Drosophila eye development, wingless (wg) is expressed at the lateral margins of the eye disc and serves to block retinal development. The T-box gene optomotor-blind (omb) is expressed in a similar pattern and is regulated by Wg. Omb mediates part of Wg activity in blocking eye development. Omb exerts its function primarily by blocking cell proliferation. These effects occur predominantly in the ventral margin. Our results suggest that the primary effect of Omb is the blocking of Jak/STAT signaling by repressing transcription of upd which encodes the Jak receptor ligand Unpaired.

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. Here, we 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 and observed two types of cell motility. 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. Our 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.

Enhancer trap infidelity in Drosophila optomotor-blind.

Reporter gene activity in enhancer trap lines is often implicitly assumed to mirror quite faithfully the endogenous expression of the "trapped" gene, even though there are numerous examples of enhancer trap infidelity. optomotor-blind (omb) is a 160 kb gene in which 16 independent P-element enhancer trap insertions of three different types have been mapped in a range of more than 60 kb. We have determined the expression pattern of these elements in wing, eye-antennal and leg imaginal discs as well as in the pupal tergites. We noted that one pGawB insertion (omb (P4) ) selectively failed to report parts of the omb pattern even though the missing pattern elements were apparent in all other 15 lines. We ruled out that omb (P4) was defective in the Gal4 promoter region or had inactivated genomic enhancers in the integration process. We propose that the Gal4 reporter gene in pGawB may be sensitive to orientation or promoter proximity effects.