midline represses Dpp signaling and target gene expression in Drosophila ventral leg development.

Ventral leg patterning in Drosophila is controlled by the expression of the redundant T-box Transcription factors midline (mid) and H15. Here, we show that mid represses the Dpp-activated gene Daughters against decapentaplegic (Dad) through a consensus T-box binding element (TBE) site in the minimal enhancer, Dad13. Mutating the Dad13 DNA sequence results in an increased and broadening of Dad expression. We also demonstrate that the engrailed-homology-1 domain of Mid is critical for regulating the levels of phospho-Mad, a transducer of Dpp-signaling. However, we find that mid does not affect all Dpp-target genes as we demonstrate that brinker (brk) expression is unresponsive to mid. This study further illuminates the interplay between mechanisms involved in determination of cellular fate and the varied roles of mid.

The selector genes midline and H15 control ventral leg pattern by both inhibiting Dpp signaling and specifying ventral fate.

mid and H15 encode Tbx20 transcription factors that specify ventral pattern in the Drosophila leg. We find that there are at least two pathways for mid and H15 specification of ventral fate. In the first pathway, mid and H15 negatively regulate Dpp, the dorsal signal in leg development. mid and H15 block the dorsalizing effects of Dpp signaling in the ventral leg. In loss- and gain-of-function experiments in imaginal discs, we show that mid and H15 block the accumulation of phospho-Mad, the activated form of the Drosophila pSmad1/5 homolog. In a second pathway, we find mid and H15 must also directly promote ventral fate because simultaneously blocking Dpp signaling in mid H15 mutants does not rescue the ventral to dorsal transformation in most ventral leg structures. We show that mid and H15 act as transcriptional repressors in ventral leg development. The two genes repress the Dpp target gene Dad, the laterally expressed gene Upd, and the mid VLE enhancer. This repression depends on the eh1 domain, a binding site for the Groucho co-repressor, and is likely direct because Mid localizes to target gene enhancers in PCR-ChIP assays. A mid allele mutant for the repressing domain (eh1), mideh1, was found to be compromised in gain-of-function assays and in rescue of mid H15 loss-of-function. We propose that mid and H15 specify ventral fate through inhibition of Dpp signaling and through coordinating the repression of genes in the ventral leg.

The expression of the T-box selector gene midline in the leg imaginal disc is controlled by both transcriptional regulation and cell lineage.

The Drosophila Tbx20 homologs midline and H15 act as selector genes for ventral fate in Drosophila legs. midline and H15 expression defines the ventral domain of the leg and the two genes are necessary and sufficient for the development of ventral fate. Ventral-specific expression of midline and H15 is activated by Wingless (Wg) and repressed by Decapentaplegic (Dpp). Here we identify VLE, a 5 kb enhancer that drives ventral specific expression in the leg disc that is very similar to midline expression. Subdivision of VLE identifies two regions that mediate both activation and repression and third region that only mediates repression. Loss- and gain-of-function genetic mosaic analysis shows that the activating and repressing regions respond to Wg and Dpp signaling respectively. All three repression regions depend on the activity of Mothers-against-decapentaplegic, a Drosophila r-Smad that mediates Dpp signaling, and respond to ectopic expression of the Dpp target genes optomoter-blind and Dorsocross 3. However, only one repression region is responsive to loss of schnurri, a co-repressor required for direct repression by Dpp-signaling. Thus, Dpp signaling restricts midline expression through both direct repression and through the activation of downstream repressors. We also find that midline and H15 expression are both subject to cross-repression and feedback inhibition. Finally, a lineage analysis indicates that ventral midline-expressing cells and dorsal omb-expressing cells do not mix during development. Together this data indicates that the ventral-specific expression of midline results from both transcriptional regulation and from a lack of cell-mixing between dorsal and ventral cells.

The Tbx20 homolog Midline represses wingless in conjunction with Groucho during the maintenance of segment polarity.

The regulation of the segment polarity gene wingless is essential for the correct patterning of the Drosophila ectoderm. We have previously shown that the asymmetric activation of wingless downstream of Hedghog-signaling depends on the T-box transcription factors, midline and H15. Hedgehog activates wingless anterior to the Hedgehog domain. midline/H15 are responsible in part for repressing wingless in cells posterior to the Hedgehog expressing cells. Here, we show that Midline binds the Groucho co-repressor directly via the engrailed homology-1 domain and requires an intact engrailed-homology-1 domain to repress wingless. In contrast, the regulation of Serrate, a second target of midline repression, is not dependent on the engrailed-homology-1 domain. Furthermore, we identify a midline responsive region of the wingless cis-regulatory region and show that Midline binds to sequences within this region. Mutating these sequences in transgenic reporter constructs results in ectopic reporter expression in the midline-expression domain, consistent with wingless being a direct target of Midline repression.

The Tbx20 homologs midline and H15 specify ventral fate in the Drosophila melanogaster leg.

Regional fates in the developing limbs of Drosophila melanogaster are controlled by selector gene transcription factors. Ventral fate in the fly leg is specified by the expression of the ligand Wingless. We present evidence that midline and H15, members of the Tbx20 class of T-box transcription factors, are key mediators of the Wingless signal in the formation of the ventral region of the fly leg. midline and H15 are restricted to identical ventral domains of expression through activation by Wingless and repression by the dorsal signal Decapentaplegic. midline and H15 function redundantly and cell autonomously in the formation of ventral-specific structures. Conversely, midline is sufficient to induce ventral fate. Finally, the induction of ectopic ventral fate by mid is compromised when Wingless signaling is attenuated, suggesting that Wingless acts both upstream and in parallel with midline/H15 to specify ventral fate. Based on these results, we propose that midline and H15 may be considered as the selector genes for ventral leg fate.

dAP-2 and defective proventriculus regulate Serrate and Delta expression in the tarsus of Drosophila melanogaster.

The segmentation of the proximal-distal axis of the Drosophila melanogaster leg depends on the localized activation of the Notch receptor. The expression of the Notch ligand genes Serrate and Delta in concentric, segmental rings results in the localized activation of Notch, which induces joint formation and is required for the growth of leg segments. We report here that the expression of Serrate and Delta in the leg is regulated by the transcription factor genes dAP-2 and defective proventriculus. Previous studies have shown that Notch activation induces dAP-2 in cells distal and adjacent to the Serrate/Delta domain of expression. We find that Serrate and Delta are ectopically expressed in dAP-2 mutant legs and that Serrate and Delta are repressed by ectopic expression of dAP-2. Furthermore, Serrate is induced cell-autonomously in dAP-2 mutant clones in many regions of the leg. We also find that the expression of a defective proventriculus reporter overlaps with dAP-2 expression and is complementary to Serrate expression in the tarsal segments. Ectopic expression of defective proventriculus is sufficient to block joint formation and Serrate and Delta expression. Loss of defective proventriculus results in localized, ectopic Serrate expression and the formation of ectopic joints with reversed polarity. Thus, in tarsal segments, dAP-2 and defective proventriculus are necessary for the correct proximal and distal boundaries of Serrate expression and repression of Serrate by defective proventriculus contributes to tarsal segment asymmetry. The repression of the Notch ligand genes Serrate and Delta by the Notch target gene dAP-2 may be a pattern-refining mechanism similar to those acting in embryonic segmentation and compartment boundary formation.

The Drosophila melanogaster T-box genes midline and H15 are conserved regulators of heart development.

The Drosophila melanogaster genes midline and H15 encode predicted T-box transcription factors homologous to vertebrate Tbx20 genes. All identified vertebrate Tbx20 genes are expressed in the embryonic heart and we find that both midline and H15 are expressed in the cardioblasts of the dorsal vessel, the insect organ equivalent to the vertebrate heart. The midline mRNA is first detected in dorsal mesoderm at embryonic stage 12 in the two progenitors per hemisegment that will divide to give rise to all six cardioblasts. Expression of H15 mRNA in the dorsal mesoderm is detected first in four to six cells per hemisegment at stage 13. The expression of midline and H15 in the dorsal vessel is dependent on Wingless signaling and the transcription factors tinman and pannier. We find that the selection of two midline-expressing cells from a pool of competent progenitors is dependent on Notch signaling. Embryos deleted for both midline and H15 have defects in the alignment of the cardioblasts and associated pericardial cells. Embryos null for midline have weaker and less penetrant phenotypes while embryos deficient for H15 have morphologically normal hearts, suggesting that the two genes are partially redundant in heart development. Despite the dorsal vessel defects, embryos mutant for both midline and H15 have normal numbers of cardioblasts, suggesting that cardiac cell fate specification is not disrupted. However, ectopic expression of midline in the dorsal mesoderm can lead to dramatic increases in the expression of cardiac markers, suggesting that midline and H15 participate in cardiac fate specification and may normally act redundantly with other cardiogenic factors. Conservation of Tbx20 expression and function in cardiac development lends further support for a common ancestral origin of the insect dorsal vessel and the vertebrate heart.

Drosophila T box proteins break the symmetry of hedgehog-dependent activation of wingless.

BACKGROUND: Segmentation of the Drosophila embryo is a classic paradigm for pattern formation during development. The Wnt-1 homolog Wingless (Wg) is a key player in the establishment of a segmentally reiterated pattern of cell type specification. The intrasegmental polarity of this pattern depends on the precise positioning of the Wg signaling source anterior to the Engrailed (En)/Hedgehog (Hh) domain. Proper polarity of epidermal segments requires an asymmetric response to the bidirectional Hh signal: wg is activated in cells anterior to the Hh signaling source and is restricted from cells posterior to this signaling source. RESULTS: Here we report that Midline (Mid) and H15, two highly related T box proteins representing the orthologs of zebrafish hrT and mouse Tbx20, are novel negative regulators of wg transcription and act to break the symmetry of Hh signaling. Loss of mid and H15 results in the symmetric outcome of Hh signaling: the establishment of wg domains anterior and posterior to the signaling source predominantly, but not exclusively, in odd-numbered segments. Accordingly, loss of mid and H15 produces defects that mimic a wg gain-of-function phenotype. Misexpression of mid represses wg and produces a weak/moderate wg loss-of-function phenocopy. Furthermore, we show that loss of mid and H15 results in an anterior expansion of the expression of serrate (ser) in every segment, representing a second instance of target gene repression downstream of Hh signaling in the establishment of segment polarity. CONCLUSIONS: The data we present here indicate that mid and H15 are important components in pattern formation in the ventral epidermis. In odd-numbered abdominal segments, Mid/H15 activity plays an important role in restricting the expression of Wg to a single domain.