Dorsocross1, Dorsocross2 and Dorsocross3
Since peak levels of Dpp activity are known to be required for cell fate determination at the dorsal midline, the correlation between Dpp activity and dorsal longitudinal Doc expression during blastoderm stages was examined. Double-staining for Doc mRNA and phosphorylated Mad (PMad) indicates a close correlation between cells containing high levels of PMad and Doc products within the dorsal-longitudinal stripe. In addition, faint Doc signals that are modulated in a pair-rule pattern extend into areas that receive lower Dpp inputs and lack detectable PMad. Both PMad and Doc expression in the dorsal-longitudinal stripe, but not the dorsal-transverse head stripe of Doc expression, are absent in dpp-null mutant embryos. Conversely, in blastoderm embryos with ubiquitous Dpp expression (UAS-dpp activated by maternally provided nanos-GAL4), a significant widening is observed of the dorsal-longitudinal stripes of PMad and Doc expression, during which the correlation between high PMad and Doc mRNA levels is still maintained (Reim, 2003).
The expansion of PMad upon uniform ectopic expression of dpp includes the prospective mesoderm, although not ventrolateral areas of the blastoderm embryo. However, high PMad in the prospective mesoderm does not trigger ectopic Doc expression, suggesting either the presence of a ventral repressor or the requirement for a co-activator in dorsal areas. A candidate for a co-activator is the homeobox gene zerknüllt (zen). Double in situ hybridization shows that the appearance of dorsal Doc mRNAs coincides with the time when zen mRNA levels increase in the areas of the presumptive amnioserosa as a result of high Dpp inputs. When the refinement of zen expression is completed, there is an exact correspondence in the widths of the Doc and zen expression domains -- although Doc expression extends more posteriorly, the activity of zen is necessary for normal levels of Doc expression in the dorsal-longitudinal stripe, because in zen mutant embryos there are only low residual levels of Doc products present in this domain. These observations suggest that Doc expression along the dorsal midline of blastoderm embryos requires the combined activities of dpp and zen (Reim, 2003).
The known distribution of dpp mRNA during its second phase of expression in the dorsolateral ectoderm of stage 9-11 embryos suggests that Doc expression in the dorsolateral ectoderm and mesoderm during these stages is also dependent on Dpp activity. As expected from the known fate map shifts in dpp mutants, these domains of Doc expression are missing in dpp-null mutant embryos. Notably, the exact coincidence between the ventral borders of the domains of dorsolateral Doc expression and high nuclear PMad suggests that Doc expression is directly controlled by Dpp-activated Smad proteins in the ectoderm and mesoderm during this stage. Additional evidence for this hypothesis comes from experiments with ectopic expression of dpp in the ventral ectoderm of the Krüppel domain (by virtue of a modified Kr-GAL4 driver), that results in the concomitant expansion of PMad and the Doc expression stripes towards the ventral midline (Reim, 2003).
In addition to the inputs from dpp, metameric Doc expression in dorsolateral areas of the germ band must depend on the activity of segmental regulators. A direct comparison with the expression of engrailed (en) shows that the clusters of Doc expression straddle the compartmental borders. Although Doc expression overlaps with en in the P compartments, about two-thirds of the Doc expressing cells of each cluster are located in posterior areas of the A compartments. In agreement with this allocation, it has been found that the metameric Doc domains are exactly centered on the stripes of Wingless (Wg) expression. The observed correlation of the segmental registers of Wg and Doc makes wg a good candidate for an upstream regulator of Doc. Dorsolateral Doc expression in the ectoderm and mesoderm is shown to be completely absent if wg is inactive. By contrast, deletion of sloppy paired (slp), a known target of wg in the mesoderm and a wg feedback regulator in the ectoderm, results in a reduction, but not a complete loss of metameric DOC expression. Hence, slp probably affects Doc indirectly through its effect on ectodermal wg expression. Altogether, the data suggest that metameric Doc expression in the ectoderm and mesoderm is triggered by the intersecting activities of Wg and Dpp (Reim, 2003).
The segmental stripes of wg expression in the embryonic trunk segments initially span the entire dorsoventral extent of the ectoderm, but at stage 11 they become interrupted in dorsolateral areas. A comparison of Wg and Doc expression at this stage shows that the positions of the metameric ectodermal domains of Doc expression correspond to the areas in which the Wg stripes become interrupted. Temporally, there is a brief overlap of ectodermal Wg and Doc expression during stage 10 until Wg expression is downregulated within the Doc domains. In contrast to the wild-type situation, the Wg stripes remain continuous in DocA mutant embryos. Similar observations were made with the homeobox gene product Ladybird (Lb=Lbe + Lbl) as a marker. In wild-type embryos after stage 11, Lb is also expressed in striped domains that are interrupted at the positions of the ectodermal Doc domains, whereas in DocA mutant embryos there is ectopic expression in a pattern of continuous stripes. These data show that Doc activity is required for patterning events in the dorsolateral ectoderm, which include the repression of wg and lb expression in these areas (Reim, 2003).
Ectopic expression experiments with Doc genes provide additional evidence for a repressive activity of Doc on wg expression. Upon ectopic expression of Doc2 in all cells of the ectoderm of wild type embryos, the ventral portions of the Wg stripes are lost. However, the dorsal regions of the Wg stripes appear to be under different regulation, because ectopic Doc results in a uniform domain of dorsal Wg along the anteroposterior axis, albeit at lower levels than in wild-type embryos (Reim, 2003).
Ectopic expression experiments with Doc genes in imaginal discs further confirm their ability to repress wg. In third instar larval wing discs, Doc genes are expressed in four distinct areas that do not overlap with the wg expression domains. Specifically, two large Doc expression domains are located in the centers of the dorsal and ventral regions of the prospective wing blades and two smaller domains in prospective dorsal hinge and posterior notal regions, respectively. In leg discs, low levels of Doc expression can be detected in regions of the prospective body wall and proximal leg segments, which also do not express wg. Importantly, ectopic expression of Doc2 within the Dpp domains of imaginal discs causes wg expression to disappear in the corresponding areas. In agreement with the known role of wg in limb development, its repression by ectopic Doc results in the loss of distal structures of wings, legs and antenna of adult animals. Analogous ectopic expression experiments with Doc1 and Doc3 in embryos and discs produce qualitatively similar (although weaker) effects to those of Doc2 (Reim, 2003).
Early heart development in Drosophila and vertebrates involves the specification of cardiac precursor cells within paired progenitor fields, followed by their movement into a linear heart tube structure. The latter process requires coordinated cell interactions, migration, and differentiation as the primitive heart develops toward status as a functional organ. In the Drosophila embryo, cardioblasts emerge from bilateral dorsal mesoderm primordia, followed by alignment as rows of cells that meet at the midline and morph into a dorsal vessel. Genes that function in coordinating cardioblast organization, migration, and assembly are integral to heart development, and their encoded proteins need to be understood as to their roles in this vital morphogenetic process. The Toll transmembrane protein is expressed in a secondary phase of heart formation, at lateral cardioblast surfaces as they align, migrate to the midline, and form the linear tube. The Toll dorsal vessel enhancer has been characterized, with its activity controlled by Dorsocross and Tinman transcription factors. Consistent with the observed protein expression pattern, phenotype analyses demonstrate Toll function is essential for normal dorsal vessel formation. Such findings implicate Toll as a critical cell adhesion molecule in the alignment and migration of cardioblasts during dorsal vessel morphogenesis (Wang, 2005).
At the time dorsal-ventral polarity is established during early Drosophila development, Toll is associated with the plasma membrane around the entire syncytial blastoderm embryo. Thereafter, Toll exhibits zygotic expression on several cell surfaces, including a specific dorsal cell type in late-stage embryos. These were identified at first as leading-edge cells of the two-epidermal sheets moving toward the dorsal midline. Toll expression in dorsal aspects of the embryo has been reevaluated and, to the contrary, it has now been concluded the gene is expressed in cardioblasts of the developing and formed dorsal vessel (Wang, 2005).
Initially, Toll mRNA accumulation was analyzed by in situ hybridization, with gene transcripts first detected in dorsal cell populations in stage 12 embryos and later in two converging rows of cells during the process of dorsal closure. The likelihood of the Toll-positive cells being cardioblasts was strongly implied by the pattern of mRNA accumulation in stage 16 embryos. Toll expression was detected in roughly 50 cell pairs, and the organization of said cells was reminiscent of cardioblasts within structurally identifiable aorta and heart regions of the assembled dorsal vessel. The pattern of Toll protein expression was also investigated, with results comparable to those obtained in the RNA analysis. The transmembrane protein was detected in dorsal cells in late stage 12/early stage 13 embryos. Thereafter, it showed a clear presence on lateral surfaces of all cells aligned within two contiguous rows as they migrate toward the dorsal midline. By stage 16, the Toll-positive cells populate the core of the dorsal vessel, again within defined aorta and heart subregions. Toll was found exclusively on cardioblast surfaces, while organ-associated pericardial, lymph gland, and ring gland cells failed to express the protein. High-resolution analysis by confocal microscopy demonstrated Toll presence at lateral points of contact between all cardioblasts of the mature dorsal vessel (Wang, 2005).
Toll zygotic transcription is complex based on the numerous cell and tissue types that express the gene. Through efforts to identify a regulatory sequence controlling Toll expression in central nervous system (CNS) midline glial cells, Wharton (1993) located three regions upstream of the gene that possessed transcriptional enhancer activity. Relevant to the demonstration of Toll expression in the dorsal vessel, a 6.5-kb DNA was fortuitously found to direct lacZ reporter expression in all cardioblasts, and in pharyngeal and body wall muscles as well. Due to the interest in understanding how this expression might be regulated, the Toll cardioblast enhancer was delimited within the defined upstream region. At first, the analysis involved testing Toll 5'-flanking DNAs for the ability to drive lacZ expression in embryos of transgenic strains. A 7.1-kb region located between ~9.3 and ~2.2 upstream of the gene showed strong enhancer function in all cardioblasts of the dorsal vessel. The DNA was subdivided into five overlapping segments, and only the most distal 1.7-kb DNA maintained cardioblast activity. Subsequently, five fragments spanning this 1.7-kb interval were tested for enhancer function, and dorsal vessel activity was mapped to a 305-bp sequence located between ~8.3 and ~8.0 relative to the Toll transcription start site. Consistent with the timing of Toll mRNA and protein accumulation in cardioblasts, the 305-bp enhancer becomes active during stage 12 and maintains its activity through all subsequent events of dorsal vessel morphogenesis. It is noteworthy that this small DNA also functions in amnioserosa cells from stage 11 through stage 15 (Wang, 2005).
Since Toll encodes a transmembrane protein with leucine-rich repeats in its extracellular domain, a prediction was made that Toll could function as a homophilic cell adhesion molecule, in addition to its well-characterized role as a signal-transducing receptor. In support of this hypothesis, induced expression of the protein in the nonadhesive Schneider 2 cell line causes cellular aggregation, with Toll accumulating at sites of cell-cell interaction. Such a localization property is characteristic of cellular adhesion molecules. Given the highly specialized localization, and structural and functional features of the protein, it is likely that Toll contributes prominently to the molecular environment that aligns and stabilizes cardioblasts on their path toward assembly within the dorsal vessel (Wang, 2005).
The observation of structurally defective dorsal vessels within Toll mutant embryos is consistent with the pattern of Toll expression in cardioblasts. D-MEF2 serves as a marker for all cardioblasts, from their early appearance through their organization within the mature organ. Based on D-MEF2 staining, it appears appropriate numbers of cardioblasts are specified in mutant embryos, but deviations are observed from the normal process of cardioblast alignment and synchronous migration as two contiguous rows of 52 cells. Several other markers for the formed dorsal vessel identified random gaps in the linear organ due to missing and/or abnormally located cardioblasts. Such cardiac phenotypes are reminiscent of those presented by faint sausage (fas) mutant embryos; mutations of the immunoglobulin-like cell adhesion molecule also led to cardioblast alignment problems. Whether Toll and Fas work in combination for the proper alignment and migration of these cells remains to be investigated. Additionally, while structural and phenotypic properties are consistent with its role as a cardioblast adhesion molecule, a function for Toll in mediating signaling events between neighboring cardiac cells cannot be ruled out. So far, no indicators exist for the latter possibility; it was not possible to demonstrate expression of potential Toll transcriptional effectors (Dorsal and Dif) in cells of the dorsal vessel. Either way, these molecular and genetic findings identify Toll as a vital player in dorsal vessel formation (Wang, 2005).
The regulation of Toll expression in cardioblasts was pursued due to an interest in further defining the transcriptional network controlling heart development in Drosophila. The studies demonstrated Toll heart expression is controlled by a 305-bp DNA located 8.0 kb upstream of the transcription start site. This regulatory module contains multiple binding sites for Doc T-box proteins and a single recognition site for the Tin homeodomain protein. The Toll dorsal vessel enhancer contains a single TCAAGTG sequence at nucleotides 163 to 169. The evidence is strong for the transcriptional enhancer being regulated by both of these cardiogenic factors. Doc and Tin are expressed in adjacent but nonoverlapping sets of cardioblasts within segments of the dorsal vessel; together, they make up the complete population of inner cardiac cells. A deletion of the distal part of the Toll 305-bp enhancer that removes the strong Doc-A footprint sequence, which likely binds multiple Doc molecules through T-box domain recognition of GTG motifs, eliminates enhancer function in Svp/Doc cells while maintaining activity in Tin cells. Systematically adding back T-box core binding elements to partially, then fully, reestablish the Doc-A binding site restores enhancer function in the Svp/Doc population (Wang, 2005).
As for Tin, mutation of its recognition element in the Toll 305 DNA leads to decreased and variable enhancer activity in both Tin and Svp/Doc cardioblasts. This result suggests that Tin is required not only for the activation of Toll expression in the four cardioblasts per hemisegment that are Tin positive after stage 12 but also for its initiation in all six cardioblasts in each hemisegment during early stage 12. The residual activity of the mutated Toll 305 DNA may reflect some degree of Tin regulation through cryptic, low-affinity binding sites present in the enhancer. Indeed, perusal of the Toll sequence identifies three candidate Tin elements that match the binding consensus at six of seven nucleotide pairs, and other Tin-regulated enhancers of genes such as D-mef2, ß3-tubulin, and pnr also employ more than one Tin binding site (Wang, 2005).
In contrast to the Toll 305 enhancer element, mutation of the exact Tin site in the Toll 258 DNA completely silenced the enhancer in the normally Tin-active cells. This result strongly implies that Tin, and at least one other factor working through the distal 47 bp of DNA, are required for activating the Toll gene. Candidates for such factors are the Doc T-box proteins, which are initially expressed in all cardioblast progenitors during mid stage 12, as well as products of the T-box genes H15 and Midline (Mid), which are expressed in all cardioblasts from mid stage 12 onward. Mid can bind to the same regions of Toll DNA as Doc, although the relevance of such interactions remains to be investigated. A combinatorial requirement for T-box proteins and Tin during the initiation and/or maintenance of Toll expression is further supported by the observation that derivatives of the enhancer containing only the Doc-A sequences fail to show activity in Svp/Doc cells. Together, these molecular data point to a mechanism wherein T-box proteins, in combination with Tin, initially activate the Toll gene in all cardioblast progenitors. After stage 12, Doc and Tin (perhaps in cooperation with H15 and/or Mid) activate Toll in two complementary subsets of cardioblasts of the dorsal vessel (Wang, 2005).
Unfortunately, a genetic requirement for these two factors in the regulation of the Toll enhancer cannot be proven at this time since Doc and tin mutant embryos fail to produce cardioblasts. Such an analysis could be attempted with the generation of specialized Doc or tin genetic backgrounds that allow for cardioblast specification early on, while lacking protein functions in later stages of dorsal vessel formation. However, forced-expression studies have demonstrated that individual expression of Tin or Doc2 leads to expanded enhancer activity, while simultaneous expression of the cardiac factors results in a robust activation of Toll transcription. These findings convincingly support the model of Doc and Tin being positive transcriptional regulators of the Toll dorsal vessel enhancer (Wang, 2005).
In addition to the demonstration of Doc and Tin as activators of Toll expression in the dorsal vessel, the regulatory analysis has generated important reagents that should facilitate the discovery of novel cardiac-functioning genes of Drosophila. That is, the Toll-cGFP and Toll-nGFP transgenes serve as sensitive markers for assessing distinct aspects of dorsal vessel morphogenesis in living animals. In stage 16 to 17 embryos and thereafter, Toll-cGFP expression can be used to monitor the formation and function of the three pairs of valvelike ostia within the heart region of the dorsal vessel. Likewise, Toll-nGFP can be used to determine the exact number and diversification status of cardioblasts, as larger nuclei are present within Tin-determined cells while smaller nuclei are found in Svp/Doc-determined cells. Such sensitive and easy-to-use reagents will be valuable in genomewide screens to discover new genes involved in Drosophila heart development (Wang, 2005).
Drosophila Groucho, like its vertebrate Transducin-like Enhancer-of-split homologues, is a corepressor that silences gene expression in numerous developmental settings. Groucho itself does not bind DNA but is recruited to target promoters by associating with a large number of DNA-binding negative transcriptional regulators. These repressors tether Groucho via short conserved polypeptide sequences, of which two have been defined: (1) WRPW and related tetrapeptide motifs have been well characterized in several repressors; (2) a motif termed Engrailed homology 1 (eh1) has been found predominantly in homeodomain-containing transcription factors. A yeast two-hybrid screen is described that uncovered physical interactions between Groucho and transcription factors, containing eh1 motifs, with different types of DNA-binding domains. One of these, the zinc finger protein Odd-skipped, requires its eh1-like sequence for repressing specific target genes in segmentation (Goldstein, 2005).
The eh1 Gro recruitment domain was originally defined as a heptapeptide motif that is conserved in members of the En family of homeodomain proteins and their vertebrate homologues. More recently, eh1-dependent binding to Gro has also been demonstrated in vitro for various other Drosophila and mammalian proteins, nearly all of which contain homeodomains. Given that Bowl and Odd, two non-homeodomain ZnF transcription factors, contain this motif and interact with Gro, the possibility was explored that eh1 motifs are prevalent among additional non-homeodomain transcription factor families. Indeed, an unbiased yeast screen for Gro-interacting proteins selected two additional transcriptional regulators that contain eh1-like motifs, namely, Sloppy-paired (Slp; Forkhead related) and Dorsocross (Doc; T box). Alignment of the eh1-like sequences of Bowl, Odd, Slp, and Doc with those of En and Gsc revealed three conserved amino acids: phenylalanine-x-isoleucine-x-x-isoleucine (Phe-x-Ile-x-x-Ile, where x is any amino acid). Subsequent database searches for presumptive Drosophila transcription factors containing this minimal peptide sequence identified a wide range of potential negative regulators belonging to different superfamilies as classified by their distinct DNA-binding domain types. Remarkably, eh1-related motifs have been preserved in many human homologues of these fly proteins, indicating that the ability to bind Gro/TLE has been evolutionarily conserved in human transcriptional regulators and that this sequence may have been widely adopted throughout the proteome as a Gro recruitment domain (Goldstein, 2005).
Several representatives, corresponding to different transcription factor families, were tested for the ability to bind Gro in biochemical assays. Where possible, full-length expressed sequence tags encoding these proteins were obtained; otherwise, single exons containing the eh1-like sequence were PCR amplified from genomic DNA. Each polypeptide was assessed for the ability to pull down radiolabeled Gro in vitro. GST-tagged Slp and Doc (amino acids 254 to 391) readily retain Gro, as do Eyes absent (Eya) and the homeodomain proteins Ventral nervous system defective (Vnd, 1 to 465), Bagpipe (Bap, 1 to 129), BarH1, and Empty spiracles (Ems, 1 to 360), as well as the orphan nuclear hormone receptor DHR96. To confirm that these interactions rely on intact eh1-related sequences, the eh1 motif of one of these, BarH1, was mutated by substituting glutamic acid for Phe at position 1, finding that its binding to Gro is reduced by >60% (Goldstein, 2005).
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