Interacting insulators from the Drosophila melanogaster bithorax complex can form independent expression domains

Regulatory region of three bithorax complex genes, Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) can be divided into nine iab domains, capable of directing expression of one of the genes in certain abdominal parasegment of Drosophila. In the Abd-B regulatory region, three insulators were identified, including Fab-7 and Fab-8, which flanked the iab-7domain, and Mcp, which separated the Abd-B and abd-A regulatory regions. It was suggested that boundary insulators formed a barrier between active and repressed chromatin. In the present study, using the yellow and white reporter genes and different combinations of known insulators, Mcp, Fab-7, and Fab-8, it was demonstrated that only specific interaction of two insulators was capable of isolation of active and repressed chromatin, i.e., the formation of independent expression domains.     

Studies on transvection at the bithorax complex in Drosophila melanogaster

Summary We have studied the influence of some mutations in the bithorax complex on the observed synapsis dependent phenotype of the genotypes Cbx ¹Ubx¹/+ and bx ^34e/Ubx¹. The effect of these mutations is similar to that introduced by disruption of pairing or by the z ^a mutation. Among the bx mutations, we find that bx ⁸ behaves differently from most other bx mutations in its influence on the synapsis dependent phenotype. This observation induced us to map the position of bx ⁸ with respect to other bx mutations; we find that it maps between bx ^34e and bx ³. We show how some of the observations reported here can be fitted into a model of activation of the bithorax complex proposed by one of us.     

The M/SAR elements of the bithorax complex in Drosophila melanogaster

The bithorax (BX) complex of Drosophila is a complex polygenic region with a multifactorial system of regulation. One of the levels of the regulatory system of the BX complex is its association with the nuclear skeleton structures through a specific interaction of the M/SAR DNA with the nuclear matrix proteins. In the present work, M/SAR elements were mapped on the molecular-genetic map of the region. All of the elements examined were found to colocalize with regulatory elements and form clusters that restrict/bracket the genetically active domains. All M/SAR DNA revealed was shown to bins specifically to the purified Drosophila melanogaster lamin.     

Determination of the shape of the operculum by the bithorax complex in Drosophila melanogaster

Summary We have studied the course of the operculum line in the larval hypoderm of several bithorax complex mutants of Drosophila melanogaster. The bifurcation of the line, a characteristic of the first abdominal segment in wild-type (A₁), can also appear in the metathoracic (T₃) and other abdominal segments (A₂, A₃) depending on mutations within the bithorax complex. Therefore, we concluded that the course of the operculum line and thus the shape of the operculum is not determined by a suprasegmental gradient of positional information but by the functional state of the genes of the bithorax complex in each metamere. The dorsal and ventral branches of the operculum line react differently, the dorsal branch being more sensitive to the effect of loss of function mutations (bxd, iab-2 ^k), the ventral branch more affected by gain of function mutations (Hab). In some cases the effects of the mutations on the operculum line differed from those in the adult, suggesting a difference in sensitivity of larval hypodermal cells and histoblast cells to the functional gene products of the bithorax complex.     

Lethal bithorax complex mutations of Drosophila melanogaster show no germ line maternal effects

Three Ultrabithorax (Ubx) alleles and three different deficiencies of the bithorax complex (BX-C) of Drosophila melanogaster have been tested for maternal effects in the germ line. The dominant female sterile technique was used. The Ubx alleles and a deletion of the abdominal region of the BX-C are homozygous viable in germ line clones and show no maternal effects. Two deletions which lack the proximal portion of the BX-C are lethal in the female germ line indicating either that these deficiencies lack genes apart from the BX-C that are necessary for fertility or that there are BX-C genes that are essential for normal maternal germ line function. The significance of the bias in the isolation of only zygotic mutations at the BX-C are discussed with respect to these results.     

Study of the enhancer-blocking activities of new boundaries in the bithorax complex of Drosophila melanogaster

It was shown earlier that the Mcp, Fab-7, and Fab-8 boundaries of the bithorax complex contain insulators that effectively block the enhancers of the yellow and white genes. Other boundaries have not been studied so far. The recent mapping of binding sites for the insulator protein dCTCF in the regulatory regions of the bithorax complex genes permitted the Fab-3, Fab-4, and Fab-6 boundaries to be localized. Here, we showed despite the presence of dCTCF-binding sites fragments of the Fab-3, Fab-4, and Fab-6 boundaries do not exhibit the properties of insulators in the model system with the yellow and white genes. Moreover, in some regions of the genome the Fab-4 and Fab-6 boundaries display the properties of silencers.     

Mutants of the bithorax complex and determinative states in the thorax of Drosophila melanogaster.

Homoeotic mutations of the bithorax complex cause segmental transformations. The genes in which these mutations occur are good candidates for genes that are involved in determination. The determination system in imaginal discs must have at least two functions. One is a cell heredity function that is responsible for maintaining the determined state during growth and development. A second is the expression of the determined state (e.g., different imaginal discs have different morphologies). The homoeotic mutations of the bithorax complex could be affecting either of these two functions. I have found that when posterior haltere disc cells, that are transformed by the mutation postbithorax so that they form wing cuticle in situ, regenerate anterior structures, these structures are anterior wing. This is the same result as that seen when wild-type posterior-wing disc cells regenerate anterior structures. On the other hand, when anterior haltere disc cells transformed by the mutation bithorax³, so that they produce wing cuticle in situ, regenerate, they produce posterior haltere structures. This is unlike wild-type anterior-wing disc cells, which regenerate posterior-wing structures. From these results, I conclude that bithorax³ affects the expression of the determined state and postbithorax affects the cell heredity of determination.     

Heat shock induced phenocopies of dominant mutants of the bithorax complex in Drosophila melanogaster.

Summary Heat shock of 37°C applied to Drosophila embryos at blastoderm stage induces phenocopies of some dominant mutants of the bithorax complex. Heat shock might shut down functions of cis regulator genes involved in embryonic pattern formation.     

The mcp element from the Drosophila melanogaster bithorax complex mediates long-distance regulatory interactions.

In the studies reported here, we have examined the properties of the Mcp element from the Drosophila melanogaster bithorax complex (BX-C). We have found that sequences from the Mcp region of BX-C have properties characteristic of Polycomb response elements (PREs), and that they silence adjacent reporters by a mechanism that requires trans-interactions between two copies of the transgene. However, Mcp trans-regulatory interactions have several novel features. In contrast to classical transvection, homolog pairing does not seem to be required. Thus, trans-regulatory interactions can be observed not only between Mcp transgenes inserted at the same site, but also between Mcp transgenes inserted at distant sites on the same chromosomal arm, or even on different arms. Trans-regulation can even be observed between transgenes inserted on different chromosomes. A small 800-bp Mcp sequence is sufficient to mediate these long-distance trans-regulatory interactions. This small fragment has little silencing activity on its own and must be combined with other Polycomb-Group-responsive elements to function as a "pairing-sensitive" silencer. Finally, this pairing element can also mediate long-distance interactions between enhancers and promoters, activating mini-white expression.     

Maternal Nanos and Pumilio regulate zygotic vasa expression autonomously in the germ-line progenitors of Drosophila melanogaster embryos

vasa (vas) is transcribed earliest among reported genes expressed in the germ-line progenitors, or pole cells, in Drosophila melanogaster embryos. Its expression is detected in the germ-line cells throughout their development, making vas expression a useful marker for the establishment of germ-line fate. In the present report, it is shown that maternal Nos and Pum are required for normal expression of vas in pole cells. First, expression of enhancer-trap marker BC69, which reflects vas expression, is promoted by maternal Nos and Pum. Second, expression of vas mRNA in pole cells is promoted by maternal Nos and Pum. Third, pole cell transplantation experiments reveal that maternal Nos and Pum are required autonomously in pole cells for proper expression of vas. Finally, Nos and Pum are dispensable for vas expression in oogenesis, although they are expressed zygotically in adult ovaries. These observations show that germ-line-specific vas expression is promoted by autonomous function of maternal Nos and Pum in the germ-line progenitors during embryogenesis, and is regulated differentially in embryogenesis and oogenesis.     

Gonad formation and development requires the abd-A domain of the bithorax complex in Drosophila melanogaster.

The abdominal-A (abd-A) gene, a member of the bithorax complex, is required for the correct identity of parasegments (PS) 7 through 13. Mutations in iab-4, one of the cis-regulatory regions of abd-A, transform epidermal structures of PS 9 and also cause loss of gonads in adult flies. Here, we describe a developmental and molecular analysis of the role of iab-4 functions in gonadal development. In flies homozygous for a strong iab-4 allele, gonadogenesis is not initiated in the embryo because the mesodermal cells fail to encapsulate the pole cells. Flies homozygous for weaker iab-4 mutations sometimes form ovaries. The ovary-oviduct junctions are abnormal, however, and egg transfer from the ovary to the uterus is blocked in the adult. To localize the sites that require iab-4 function, we have analyzed animals chimeric for the mutant and wild-type cells. These chimeras were generated by three kinds of transplantation experiments: pole cells, embryonic somatic nuclei or larval ovaries. Our results suggest that iab-4 is required in the somatic cells of the gonadal primordia, but not the germ line. In addition, the formation of functional ovary-oviduct junctions and egg transfer also requires iab-4 functions in the somatic cells of the ovary and in at least one additional somatic tissue.     

Control of the expression of the bithorax complex genes abdominal-A and abdominal-B by cis-regulatory regions in Drosophila embryos.

The abdominal-A (abd-A) and Abdominal-B (Abd-B) genes of the bithorax complex (BX-C) specify the identity of most of the Drosophila abdomen. Six different classes of infraabdominal (iab) mutations within the BX-C transform a subset of the parasegments affected by the lack of these two genes. It is thought that these mutations define parasegmental cis-regulatory regions that control the expression of abd-A and Abd-B. By staining embryos mutant for different iab mutations with anti-abd-A and anti-Abd-B antibodies I show here that the expression of Abd-B (and probably also abd-A) exhibit a parasegmental regulation. I have also studied the significance of the chromosomal order of parasegmental iab regulatory sequences, and the possible presence of chromosomal 'boundaries' between them, by looking at the expression of abd-A and Abd-B in embryos carrying the Uab and Mcp mutations. These data are discussed in the light of models of parasegmental-specific regulatory regions within the BX-C.     

Protein products of the bithorax complex in Drosophila

A sequence from the Ubx 5' exon in the bithorax complex of Drosophila melanogaster was expressed as a fusion protein in bacteria. This protein was used to raise rabbit antisera and monoclonal antibodies. These antibodies detect antigens that, on protein blots and by immunofluorescence on whole mounts of imaginal discs, show the predicted segmental distribution of Ubx products. These products are predominantly, if not totally, localized in the cell nucleus. In the embryonic nervous system nuclei are labeled from the second thoracic segment to the eighth abdominal segment. There is no labeling in homozygous Df bxd100 embryos.     

Quantitative analysis of ventral denticular patterns of Drosophila melanogaster larvae and the regulation of the bithorax complex

A quantitative model of the effect of the bithorax complex on segmentation is presented which could explain the known data of the spatiotemporal regulation of key gene complex during early Drosophila development, in relation to their effects on some of the segmentation landmarks. The model tries to put together the two different genetic levels, the genotypic and the phenotypic. At the genotypic level, a minimal cross-regulatory network of the different genes involved, Antp, Ubx, abd-A and Abd-B which explains the reported levels of expressions of these genes. At the phenotypic level, the pattern of the ventral denticle belts across the larva which are characteristics of the different segments have been compared by calculating a value of the degree of similarity in the case of the wild-type and several mutant combinations. Finally the two parts of the model are combined, showing that a satisfactory agreement between the two can be achieved. Therefore, this work is a first attempt to develop a method which will provide an explanatory solution of the old question in morphogenesis of how the phenotype is directed by the genotype of a cell or organism.     

Autonomous cellular differentiation of homoeotic bithorax mutants of Drosophila melanogaster

Various mixtures of imaginal disc cells from wild-type and from homoeotic bithorax mutants have been studied in an in vivo Drosophila cell culture system. These mutants effect specific types of segmental transformations, e.g., bithorax-3 (bx³) transforms the anterior region of the metathorax (MT) into a region resembling the anterior mesothorax (MS), while postbithorax (pbx) transforms the posterior MT into a posterior MS-like region. In cell mixtures, wild-type haltere-disc cells segregate from wild-type wing-disc cells. On the other hand, bx³ and pbx haltere-disc cells integrate with wild-type cells derived from anterior and posterior regions, respectively, of wing discs. The behavior of these and other tested mutants of the bithorax series indicates in all cases studied that (1) the effects of the mutants are cell-autonomous, and (2) cellular affinities are determined by the genetic constitution rather than the segmental origin of the cells.