Ultrabithorax protein is necessary but not sufficient for full activation of decapentaplegic expression in the visceral mesoderm.

To elucidate the mechanisms by which homeotic selector (HOM) genes specify the unique features of Drosophila segments, we have analyzed the regulation of decapentaplegic (dpp), a transforming growth factor (TGF)-beta superfamily member, and have found that the Ultrabithorax (Ubx) HOM protein directly activates dpp expression in parasegment 7 (PS7) of the embryonic visceral mesoderm. Other factors are also required, including one that appears to act through homeodomain protein binding sites and may be encoded by extradenticle (exd). The exd protein binds in a highly co-operative manner to regulatory sequences mediating PS7-specific dpp expression, consistent with a genetic requirement for exd function in normal visceral mesoderm expression of dpp. A second mechanism contributing to PS7 expression of dpp appears not to require Ubx protein directly, and involves a general visceral mesoderm enhancer coupled to a spatially specific repression element. Thus, even in an apparently simple case where visceral mesoderm expression of the dpp target gene mirrors that of the Ubx HOM protein, full activation by Ubx protein requires at least one additional factor. In addition, a distinct regulatory mode not directly involving Ubx protein also appears to contribute to PS7-specific expression.     

Ultrabithorax is a regulator of beta 3 tubulin expression in the Drosophila visceral mesoderm.

beta 3 tubulin expression accompanies the specification and differentiation of the Drosophila mesoderm. The genetic programs involved in these processes are largely unknown. Our previous studies on the regulation of the beta 3 tubulin gene have shown that upstream sequences guide the expression in the somatic musculature, while regulatory elements in the first intron are necessary for expression in the visceral musculature. To further analyse this mode of regulation, which reflects an early embryonic specification program, we undertook a more detailed analysis of the regulatory capabilities of the intron. The results reveal not only a certain degree of redundancy in the cis-acting elements, which act at different developmental stages in the same mesodermal derivatives, but they also demonstrate in the visceral mesoderm, which forms a continuous epithelium along the body axis of the embryo, an early action of regulators guiding gene expression along the anterior-posterior axis of the embryo: an enhancer element in the intron leads to expression in a subdomain restricted along the anterior-posterior axis. This pattern is altered in mutants in the homeotic gene Ultrabithorax (Ubx), whereas ectopic Ubx expression leads to activity of the enhancer in the entire visceral mesoderm. So this element is likely to be a target of homeotic genes, which would define the beta 3 tubulin gene as a realisator gene under the control of selector genes.     

Homeotic genes regulate the spatial expression of putative growth factors in the visceral mesoderm of Drosophila embryos

During Drosophila embryogenesis homeotic genes control the developmental diversification of body structures. The genes probably coordinate the expression of as yet unidentified target genes that carry out cell differentiation processes. At least four homeotic genes expressed in the visceral mesoderm are required for midgut morphogenesis. In addition, two growth factor homologs are expressed in specific regions of the visceral mesoderm surrounding the midgut epithelium. One of these, decapentaplegic (dpp), is a member of the transforming growth factor beta (TGF-beta) family; the other, wingless (wg), is a relative of the mammalian proto-oncogene int-1. Here we show that the spatially restricted expression of dpp in the visceral mesoderm is regulated by the homeotic genes Ubx and abd-A. Ubx is required for the expression of dpp while abd-A represses dpp. One consequence of dpp expression is the induction of labial (lab) in the underlying endoderm cells. In addition, abd-A function is required for the expression of wg in the visceral mesoderm posterior to the dpp-expressing cells. The two growth factor genes therefore are excellent candidates for target genes that are directly regulated by the homeotic genes.     

A Drosophila growth factor homolog, decapentaplegic, regulates homeotic gene expression within and across germ layers during midgut morphogenesis

The decapentaplegic (dpp) gene product, a member of the transforming growth factor-beta family, is required in Drosophila embryos for normal gastrulation and the establishment of dorsal-ventral polarity in the embryo. dpp is also expressed at specific positions in the visceral mesoderm along the developing midgut. We find that mutations that eliminate the visceral mesoderm expression of dpp lead to defects in midgut morphogenesis and alter the spatially localized expression of the homeotic genes Sex combs reduced (Scr), Ultrabithorax (Ubx), and Antennapedia (Antp) in the visceral mesoderm. The extracellular dpp protein migrates from the visceral mesoderm across the apposing endodermal cell layer in a region of the endoderm that expresses the homeotic gene labial (lab). Mesodermal expression of dpp is required for the expression of lab in these endodermal cells indicating that dpp mediates an inductive interaction between the two germ layers. We propose that extracellular dpp protein regulates gut morphogenesis, in part, by regulating homeotic gene expression in the visceral mesoderm and endoderm of the developing midgut.     

The localization and regulation of Antennapedia protein expression in Drosophila embryos

The homeotic Antennapedia (Antp) gene of Drosophila is required for the normal differentiation of the thoracic segments during embryonic development and metamorphosis. Antibodies to a recombinant Antp protein were used to localize the protein in whole mount embryos. Antp is expressed in the nuclei of cells of the thoracic embryonic epidermis and several segments of the ventral and peripheral nervous systems. Analysis of Antp expression in mutant embryos revealed three levels of Antp regulation by genes of the bithorax complex, pleiotropic homeotic loci, and Antp itself. The distributions of the Antp and the Ultrabithorax (Ubx) proteins in doubly-labeled embryos suggest that the Ubx protein may be one direct negative regulator of Antp gene expression.     

Regulation and function of Scr, exd, and hth in the Drosophila salivary gland

Salivary gland formation in the Drosophila embryo is dependent on the homeotic gene Sex combs reduced (Scr). When Scr function is missing, salivary glands do not form, and when SCR is expressed everywhere in the embryo, salivary glands form in new places. Scr is normally expressed in all the cells that form the salivary gland. However, as the salivary gland invaginates, Scr mRNA and protein disappear. Homeotic genes, such as Scr, specify tissue identity by regulating the expression of downstream target genes. For many homeotic proteins, target gene specificity is achieved by cooperatively binding DNA with cofactors. Therefore, it is likely that SCR also requires a cofactor(s) to specifically bind to DNA and regulate salivary gland target gene expression. Here, we show that two homeodomain-containing proteins encoded by the extradenticle (exd) and homothorax (hth) genes are also required for salivary gland formation. exd and hth function at two levels: (1) exd and hth are required to maintain the expression of Scr in the salivary gland primordia prior to invagination and (2) exd and hth are required in parallel with Scr to regulate the expression of downstream salivary gland genes. We also show that Scr regulates the nuclear localization of EXD in the salivary gland primordia through repression of homothorax (hth) expression, linking the regulation of Scr activity to the disappearance of Scr expression in invaginating salivary glands.     

A cis-element mediating Ultrabithorax autoregulation in the central nervous system.

We dissected an upstream control region (a BXD fragment) from the homeotic gene Ultrabithorax (Ubx) of Drosophila which confers a Ubx-like expression pattern in the embryonic ectoderm. We found several distinct enhancer elements spread through the whole BXD fragment each of which is active in transformed embryos, mediating a different pattern of beta-galactosidase expression in the ventral nerve cord. The strongest of these patterns mimics Ubx expression within the Ubx domain. This pattern is strictly dependent on Ubx function. Thus, the BXD control region contains a Ubx response element, suggesting that positive autoregulation of Ubx may occur in the central nervous system of the developing embryo.     

The bx region enhancer, a distant cis-control element of the Drosophila Ubx gene and its regulation by hunchback and other segmentation genes.

The Drosophila homeotic gene Ultrabithorax (Ubx) is regulated by complex mechanisms that specify the spatial domain, the timing and the activity of the gene in individual tissues and in individual cells. In early embryonic development, Ubx expression is controlled by segmentation genes turned on earlier in the developmental hierarchy. Correct Ubx expression depends on multiple regulatory sequences located outside the basal promoter. Here we report that a 500 bp DNA fragment from the bx region of the Ubx unit, approximately 30 kb away from the promoter, contains one of the distant regulatory elements (bx region enhancer, BRE). During early embryogenesis, this enhancer element activates the Ubx promoter in parasegments (PS) 6, 8, 10, and 12 and represses it in the anterior half of the embryo. The repressor of the anterior Ubx expression is the gap gene hunchback (hb). We show that the hb protein binds to the BRE element and that such binding is essential for hb repression in vivo, hb protein also binds to DNA fragments from abx and bxd, two other regulatory regions of the Ubx gene. We conclude that hb represses Ubx expression directly by binding to BRE and probably other Ubx regulatory elements. In addition, the BRE pattern requires input from other segmentation genes, among them tailless and fushi tarazu but not Krüppel and knirps.     

Persistent ectopic expression of Drosophila homeotic genes resulting from maternal deficiency of the extra sex combs gene product

Like other members of the Polycomb group, the extra sex combs gene (esc) is required for the correct repression of loci in the major homeotic gene complexes. We show here that embryos lacking both maternal and zygotic esc+ function display transient, general derepression of both the Ultrabithorax (Ubx) and Antennapedia (Antp) genes during germ band shortening, but Sex combs reduced (Scr) expression is almost normal in the epidermis and lacking in the central nervous system (CNS). In addition, embryos that are maternally esc- but receive two paternal copies of esc+ often are characterized by ectopic expression of the three homeotic genes, especially Ubx and Antp in the CNS. Imaginal discs from these paternally rescued embryos may show discrete patches of expression of Ubx and Scr in inappropriate locations. Thus, lack of esc+ function during a brief period in early embryogenesis results in a heritable change in determined state, even in a genetically wild type animal. Within these ectopic patches, homeotic gene expression may be regulated by the disc positional fields and by cross-regulatory interactions between homeotic genes.     

Synergistic activation of a Drosophila enhancer by HOM/EXD and DPP signaling.

The homeotic proteins encoded by the genes of the Drosophila HOM and the vertebrate HOX complexes do not bind divergent DNA sequences with a high selectivity. In vitro, HOM (HOX) specificity can be increased by the formation of heterodimers with Extradenticle (EXD) or PBX homeodomain proteins. We have identified a single essential Labial (LAB)/EXD-binding site in a Decapentaplegic (DPP)-responsive enhancer of the homeotic gene lab which drives expression in the developing midgut. We show that LAB and EXD bind cooperatively to the site in vitro, and that the expression of the enhancer in vivo requires exd and lab function. In addition, point mutations in either the EXD or the LAB subsite compromise enhancer function, strongly suggesting that EXD and LAB bind to this site in vivo. Interestingly, we found that the activity of the enhancer is only stimulated by DPP signaling significantly upon binding of LAB and EXD. Thus, the enhancer appears to integrate positional information via the homeotic gene lab, and spatiotemporal information via DPP signaling; only when these inputs act in concert in an endodermal cell is the enhancer fully active. Our results illustrate how a tissue-specific response to DPP can be generated through synergistic effects on an enhancer carrying both DPP- and HOX-responsive sequences.     

Identification of Homeotic Target Genes in Drosophila Melanogaster Including Nervy, a Proto-Oncogene Homologue

In Drosophila, the specific morphological characteristics of each segment are determined by the homeotic genes that regulate the expression of downstream target genes. We used a subtractive hybridization procedure to isolate activated target genes of the homeotic gene Ultrabithorax (Ubx). In addition, we constructed a set of mutant genotypes that measures the regulatory contribution of individual homeotic genes to a complex target gene expression pattern. Using these mutants, we demonstrate that homeotic genes can regulate target gene expression at the start of gastrulation, suggesting a previously unknown role for the homeotic genes at this early stage. We also show that, in abdominal segments, the levels of expression for two target genes increase in response to high levels of Ubx, demonstrating that the normal down-regulation of Ubx in these segments is functional. Finally, the DNA sequence of cDNAs for one of these genes predicts a protein that is similar to a human proto-oncogene involved in acute myeloid leukemias. These results illustrate potentially general rules about the homeotic control of target gene expression and suggest that subtractive hybridization can be used to isolate interesting homeotic target genes.     

Hox proteins coordinate peripodial decapentaplegic expression to direct adult head morphogenesis in Drosophila

The Drosophila BMP, decapentaplegic (dpp), controls morphogenesis of the ventral adult head through expression limited to the lateral peripodial epithelium of the eye-antennal disc by a 3.5kb enhancer in the 5' end of the gene. We recovered a 15bp deletion mutation within this enhancer that identified a homeotic (Hox) response element that is a direct target of labial and the homeotic cofactors homothorax and extradenticle. Expression of labial and homothorax are required for dpp expression in the peripodial epithelium, while the Hox gene Deformed represses labial in this location, thus limiting its expression and indirectly that of dpp to the lateral side of the disc. The expression of these homeodomain genes is in turn regulated by the dpp pathway, as dpp signalling is required for labial expression but represses homothorax. This Hox-BMP regulatory network is limited to the peripodial epithelium of the eye-antennal disc, yet is crucial to the morphogenesis of the head, which fate maps suggest arises primarily from the disc proper, not the peripodial epithelium. Thus Hox/BMP interactions in the peripodial epithelium of the eye-antennal disc contribute inductively to the shape of the external form of the adult Drosophila head.