The Drosophila eve Insulator Homie Promotes eve Expression and Protects the Adjacent Gene from Repression by Polycomb Spreading

Insulators can block the action of enhancers on promoters and the spreading of repressive chromatin, as well as facilitating specific enhancer-promoter interactions. However, recent studies have called into question whether the activities ascribed to insulators in model transgene assays actually reflect their functions in the genome. The Drosophila even skipped (eve) gene is a Polycomb (Pc) domain with a Pc-group response element (PRE) at one end, flanked by an insulator, an arrangement also seen in other genes. Here, we show that this insulator has three major functions. It blocks the spreading of the eve Pc domain, preventing repression of the adjacent gene, TER94. It prevents activation of TER94 by eve regulatory DNA. It also facilitates normal eve expression. When Homie is deleted in the context of a large transgene that mimics both eve and TER94 regulation, TER94 is repressed. This repression depends on the eve PRE. Ubiquitous TER94 expression is “replaced” by expression in an eve pattern when Homie is deleted, and this effect is reversed when the PRE is also removed. Repression of TER94 is attributable to spreading of the eve Pc domain into the TER94 locus, accompanied by an increase in histone H3 trimethylation at lysine 27. Other PREs can functionally replace the eve PRE, and other insulators can block PRE-dependent repression in this context. The full activity of the eve promoter is also dependent on Homie, and other insulators can promote normal eve enhancer-promoter communication. Our data suggest that this is not due to preventing promoter competition, but is likely the result of the insulator organizing a chromosomal conformation favorable to normal enhancer-promoter interactions. Thus, insulator activities in a native context include enhancer blocking and enhancer-promoter facilitation, as well as preventing the spread of repressive chromatin.     

The convergence of Notch and MAPK signaling specifies the blood progenitor fate in the Drosophila mesoderm

Blood progenitors arise from a pool of pluripotential cells (“hemangioblasts”) within the Drosophila embryonic mesoderm. The fact that the cardiogenic mesoderm consists of only a small number of highly stereotypically patterned cells that can be queried individually regarding their gene expression in normal and mutant embryos is one of the significant advantages that Drosophila offers to dissect the mechanism specifying the fate of these cells. We show in this paper that the expression of the Notch ligand Delta (Dl) reveals segmentally reiterated mesodermal clusters (“cardiogenic clusters”) that constitute the cardiogenic mesoderm. These clusters give rise to cardioblasts, blood progenitors and nephrocytes. Cardioblasts emerging from the cardiogenic clusters accumulate high levels of Dl, which is required to prevent more cells from adopting the cardioblast fate. In embryos lacking Dl function, all cells of the cardiogenic clusters become cardioblasts, and blood progenitors are lacking. Concomitant activation of the Mitogen Activated Protein Kinase (MAPK) pathway by Epidermal Growth Factor Receptor (EGFR) and Fibroblast Growth Factor Receptor (FGFR) is required for the specification and maintenance of the cardiogenic mesoderm; in addition, the spatially restricted localization of some of the FGFR ligands may be instrumental in controlling the spatial restriction of the Dl ligand to presumptive cardioblasts.     

Direct activation of a mouse Hoxd11 axial expression enhancer by Gdf11/Smad signalling

A Hoxd11/lacZ reporter, expressed with a Hoxd11-like axial expression pattern in transgenic mouse embryos, is stimulated in tailbud fragments when cultured in presence of Gdf11, a TGF-β growth/differentiation factor. The same construct is also stimulated by Gdf11 when transiently transfected into cultures of HepG2 cells. Stimulation of the reporter in HepG2 cells is enhanced where it contains only the 332 bp Hoxd11 enhancer region VIII upstream or downstream of a luciferase or lacZ reporter. This enhancer contains three elements conserved from fish to mice, one of which has the sequence of a Smad3/4 binding element. Mutation of this motif inhibits the ability of Gdf11 to enhance reporter activity in the HepG2 cell assay. Chromatin immunoprecipitation experiments show direct evidence of Smad2/3 protein binding to the Hoxd11 region VIII enhancer. The action of Gdf11 upon Hoxd11 in HepG2 cells is inhibited, at least in part, by SIS3, a specific inhibitor of Smad3. SIS3 also produces partial inhibition of Hoxd11/lacZ expression in cultured transgenic tailbuds, indicating that Smad3 may play a similar role in the embryonic expression of Hoxd11. Transgenic mouse experiments show that the Smad binding motif is essential for the axial expression of Hoxd11/lacZ reporter in the embryo tailbud, posterior mesoderm and neurectoderm.     

Definition of germ layer cell lineage alternative splicing programs reveals a critical role for Quaking in specifying cardiac cell fate

Alternative splicing is critical for development; however, its role in the specification of the three embryonic germ layers is poorly understood. By performing RNA-Seq on human embryonic stem cells (hESCs) and derived definitive endoderm, cardiac mesoderm, and ectoderm cell lineages, we detect distinct alternative splicing programs associated with each lineage. The most prominent splicing program differences are observed between definitive endoderm and cardiac mesoderm. Integrative multi-omics analyses link each program with lineage-enriched RNA binding protein regulators, and further suggest a widespread role for Quaking (QKI) in the specification of cardiac mesoderm. Remarkably, knockout of QKI disrupts the cardiac mesoderm-associated alternative splicing program and formation of myocytes. These changes arise in part through reduced expression of BIN1 splice variants linked to cardiac development. Mechanistically, we find that QKI represses inclusion of exon 7 in BIN1 pre-mRNA via an exonic ACUAA motif, and this is concomitant with intron removal and cleavage from chromatin. Collectively, our results uncover alternative splicing programs associated with the three germ lineages and demonstrate an important role for QKI in the formation of cardiac mesoderm.     

Genetic and morphologic variability of annual glassworts (Salicornia L.) from the Gulf of Trieste (Northern Adriatic)

The genetic variability of four pre-determined morphotypes of Salicornia (S. patula, S. emerici, S. veneta and the “saline type”) from 10 locations on the Gulf of Trieste coast were studied by means of ploidy level estimation using flow cytometry and by molecular DNA analysis of ITS regions of nrDNA and cpDNA. Two groups, the diploids and tetraploids, with matching nrDNA sequences, were recognized. Two types of cpDNA emerged among the diploids; one the same as in tetraploids. This incongruence between nrDNA and cpDNA sequences indicates a hybridization with tetraploid maternal progenitors and demonstrates the evidence for reticulate evolution. The morphometry, based on generative morphological traits, did not clearly separate the four morphotypes. However, the most important characters—length of the middle fertile segment, length of the lateral flower, width of the scarious margin of the fertile segment in the floral region, conform to two genetically recognized types: diploid S. patula and the widely distributed tetraploid S. emerici, also comprising the “saline type” and morphotype, known as a charismatic endemic S. veneta, a flagship species for nature conservation. Other discriminative traits for diploid and tetraploid morphotypes are parameters of the flowers (comparison of length of the central vs. lateral flower) and stomatal index. The determination key is also given. The tetraploid S. emerici is by far the most common species of annual glassworts in the area, occupying more extreme habitats than a diploid S. patula, which mostly forms monodominate stands.     

Genetic regulation of engrailed and wingless in Tribolium segmentation and the evolution of pair-rule segmentation

In Drosophila, primary pair-rule genes establish the parasegmental boundaries and indirectly control the periodic expression of the segment polarity genes engrailed (en) and wingless (wg) via regulation of secondary pair-rule genes. Although orthologs of some Drosophila pair-rule genes are not required for proper segmentation in Tribolium, segmental expression of Tc-en and Tc-wg is conserved. To understand how these segment polarity genes are regulated, we examined the results of expressing one or two pair-rule genes in the absence of the other known pair-rule genes. Expression of one or both of the secondary pair-rule genes, Tc-sloppy-paired (Tc-slp) and Tc-paired (Tc-prd), activated Tc-wg in the absence of the primary pair-rule genes, Tc-even-skipped (Tc-eve), Tc-runt (Tc-run) and Tc-odd-skipped (Tc-odd). Tc-eve alone failed to activate Tc-wg or Tc-en, but in combination with Tc-run or Tc-prd activated Tc-en. These results, interpreted within the pair-rule gene expression patterns, suggest separate models for the genetic regulation of the juxtaposed expression of Tc-wg and Tc-en at odd- and even-numbered parasegmental boundaries, respectively. Conserved interactions between eve and prd at the anterior boundary of odd-numbered parasegments may reflect an ancestral segmentation mechanism that functioned in every segment prior to the evolution of pair-rule segmentation.     

Evolutionary plasticity of collier function in head development of diverse arthropods

The insect intercalary segment represents a small and appendage-less head segment that is homologous to the second antennal segment of Crustacea and the pedipalpal segment in Chelicerata, which are generally referred to as “tritocerebral segment.” In Drosophila, the gene collier (col) has an important role for the formation of the intercalary segment. Here we show that in the beetle Tribolium castaneum col is required for the activation of the segment polarity genes hedgehog (hh), engrailed (en) and wingless (wg) in the intercalary segment, and is a regulatory target of the intercalary segment specific Hox gene labial (lab). Loss of Tc col function leads to increased cell death in the intercalary segment. In the milkweed bug Oncopeltus fasciatus, the loss of col function has a more severe effect in lacking the intercalary segment and also affecting the adjacent mandibular and antennal segments. By contrast, col is not expressed early in the second antennal segment in the crustacean Parhyale hawaiensis or in the pedipalpal segment of the spider Achaearanea tepidariorum. This suggests that the early expression of col in a stripe and its role in tritocerebral segment development is insect-specific and might correlate with the appendage-less morphology of the intercalary segment.     

A novel fluorescent timer based on bicistronic expression strategy in Caenorhabditis elegans

Fluorescent timers are useful tools for studying the spatial and temporal cellular or molecular events. Based on the trans-splicing mechanism in Caenorhabditis elegans, we constructed a “fluorescent timer” through bicistronic expression of two fluorescent proteins with different maturation times. When used in vivo, this “timer” changes its color over time and therefore can be used to monitor the activity of the targeted promoters in C. elegans. Using this “timer”, we have successfully traced the time-dependent activity of myo-3 promoter which drives expression in body wall muscle and vulval muscle. We found that the myo-3 promoter started to be active about 7 h after egg-laying and sustained its activity in the following hatching process. We have also determined the myo-3 promoter activity during larval development by this “timer”. We anticipate that more new “fluorescent timers” with variable time-resolution could be designed by bicistronic expression of different fluorescent protein pairs.