hunchback, a gene required for segmentation of an anterior and posterior region of the Drosophila embryo

The locus hunchback (hb) is a member of the gap class of segmentation genes of Drosophila. A number of X-ray-induced deletions locate the hb locus at the chromosomal site 85A3-B1, to the right of the pink locus, which maps in the same interval. A total of 14 EMS and 3 X-ray-induced hb alleles have been studied. Homozygous mutant embryos show deletions of segments in two separate regions. In the six strong alleles, the labium and all three thoracic segments are deleted anteriorly while posteriorly the 8th abdominal segment and adjacent parts of the 7th abdominal segment are lacking. The eight weak alleles show smaller deletions both in the thoracic and posterior abdominal region. In the weakest allele only part of the mesothorax is deleted. Three hb alleles produce a homoeotic transformation: superimposed on a strong or weak deletion phenotype, head or thoracic segments are transformed into abdominal segments, respectively. This suggests that hb might also be involved in the regulation of genes in the Bithorax complex (BX-C). Fate mapping of the normal-appearing segments in strong mutant embryos using the UV-laser beam ablation technique (Lohs-Schardin et al., 1979) shows that these segments arise from the normal blastoderm regions. The mutant phenotype can be recognized soon after the onset of gastrulation in a failure to fully extend the germ band. In 6-hr-old mutant embryos, two clusters of dead cells are observed in the thoracic and posterior abdominal region. These observations indicate region specific requirement of hb gene function. The analysis of germ line chimeras by transplantation of homozygous mutant pole cells shows that hb is already expressed during oogenesis. Homozygous mutant embryos derived from a homozygous mutant germ line have a novel phenotype. The anterior affected region is enlarged, including all three gnathal segments and the anterior three abdominal segments. In addition three abdominal segments with reversed polarity are formed between the remaining head structures and the posterior abdomen. Heterozygous mutant embryos derived from a homozygous mutant germ line develop normally, indicating that maternal gene expression is not required for normal development.     

Interactions between fused, a segment-polarity gene in Drosophila, and other segmentation genes.

Fused (fu) is a segment polarity gene whose product is maternally required in the posterior part of each segment. To define further the role of fused and determine how it interacts with other segmentation genes, we examined the phenotypes obtained by combining fused with mutations of pair rule, homeotic and other segment polarity loci. When it was possible, we also looked at the distribution of corresponding proteins in fused mutant embryos. We observed that fused-naked (fu;nkd) double mutant embryos display a phenotypic suppression of simple mutant phenotypes: both naked cuticle and denticle belts, which would normally have been deleted by one of the two mutants alone, were restored. In fused mutant embryos, engrailed (en) and wingless (wg) expression was normal until germ band extension, but partially and completely disappeared respectively during germ band retraction. In the fu;nkd double mutant embryo, en was expressed as in nkd mutant at germ band extension, but later this expression was restricted and became normal at germ band retraction. On the contrary, wg expression disappeared as in fu simple mutant embryos. We conclude that the requirements for fused, naked and wingless activities for normal segmental patterning are not absolute, and propose mechanisms by which these genes interact to specify anterior and posterior cell fates.     

Microevolutionary divergence pattern of the segmentation gene hunchback in Drosophila

To study the microevolutionary processes shaping the evolution of the segmentation gene hunchback (hb) from Drosophila melanogaster, we cloned and sequenced the gene from 12 isofemale lines representing wild-type populations of D. melanogaster, as well as from the closely related species Drosophila sechellia, Drosophila orena, and Drosophila yakuba. We find a relatively low degree of sequence variation in D. melanogaster (theta = 0.0017), which is, however, consistent with its chromosomal location in a region of low recombination. Tests of neutrality do not reject a neutral-evolution model for the whole region. However, pairwise tests with different subregions indicate that there is a relative excess of polymorphic sites in the leader and the intron. Codon usage pattern analysis shows a particularly biased codon usage in the highly conserved regions, which is in line with the hypothesis that selection on translational accuracy is the driving force behind such a bias. A comparison of the expression pattern of hb in different sibling species of D. melanogaster reveals some regulatory changes in D. yakuba, which could be interpreted as changes in the timing of secondary expression domains.     

Comparison of the gap segmentation gene hunchback between Drosophila melanogaster and Drosophila virilis reveals novel modes of evolutionary change.

We have cloned and sequenced a large portion of the hunchback (hb) locus from Drosophila virilis. Comparison with the Drosophila melanogaster hb sequence shows multiple strong homologies in the upstream and downstream regions of the gene, including most of the known functional parts. The coding sequence is highly conserved within the presumptive DNA-binding finger regions, but more diverged outside of them. The regions of high divergence are correlated with regions which are rich in short direct repeats (regions of high 'cryptic simplicity'), suggesting a significant influence of slippage-like mechanisms in the evolutionary divergence of the two genes. Staining of early D.virilis embryos with an hb antibody reveals conserved and divergent features of the spatial expression pattern at blastoderm stage. It appears that the basic expression pattern, which serves as the gap gene function of hb, is conserved, while certain secondary expression patterns, which have separate functions for the segmentation process, are partly diverged. Thus, both slippage driven mutations in the coding region, which are likely to occur at higher rates than point mutations and the evolutionary divergence of secondary expression patterns may contribute to the evolution of regulatory genes.     

Molecular mechanisms of pattern formation by the BRE enhancer of the Ubx gene.

The core activity of the Ubx gene enhancer BRE (bx region enhancer) is encoded within a 500 bp module. bx DNA outside this active module increases the level of expression, expands the expression into ventro-lateral ectoderm and partially stabilizes the late expression pattern. The products of the gap genes hb and tll and of the pair-rule gene ftz bind to the 500 bp BRE module and control directly its initial pattern of expression. ftz enhances expression in even-numbered parasegments within the correct spatial domain whose boundaries are set by hb and tll. In addition, en and twi products activate the enhancer, probably directly. en broadens the parasegmental stripe while twi cooperates with ftz to enhance expression in the mesoderm. Binding sites for the five regulators are closely clustered, often overlapping extensively with one another. In vitro, hb blocks the binding of ftz and can also displace ftz protein pre-bound to an overlapping site, suggesting that competitive binding and/or interference by hb sets the initial boundaries of the domain of expression. Our results also suggest that this interaction is short-range and the long distance interactions among different enhancers may depend on each enhancer's ability to complex with the promoter.     

Modifiers of bx1 alter the distribution of Ubx proteins in haltere imaginal discs of Drosophila.

The bithorax (bx) mutations in the Ultrabithorax (Ubx) gene of Drosophila melanogaster cause homeotic transformations of anterior third thoracic structures (T3a) toward anterior second thoracic structures (T2a) in the adult fly. A corresponding loss of Ubx protein expression in T3a of bx imaginal discs has been observed (White and Wilcox, 1985). We describe two genetic loci which modify the bx-induced transformation. A locus which we map very close to the pink peach (pp) gene suppresses the bx1 phenotype. In contrast, mutations in the suppressor of sable (su(s)) gene enhance the bx1 phenotype. A correlation was observed between patterns of Ubx protein expression and the phenotypic transformations observed.     

The giant gene of Drosophila encodes a b-ZIP DNA-binding protein that regulates the expression of other segmentation gap genes.

The sequence of a cDNA from the giant gene of Drosophila shows that its product has a basic domain followed by a leucine zipper motif. Both features contain characteristic conserved elements of the b-ZIP family of DNA-binding proteins. Expression of the gene in bacteria or by in vitro translation yields a protein that migrates considerably faster than the protein extracted from Drosophila embryos. Treatment with phosphatase shows that this difference is due to multiple phosphorylation of the giant protein in the embryo. Ectopic expression of the protein in precellular blastoderm embryos produces abnormal phenotypes with a pattern of segment loss closely resembling that of Krüppel mutant embryos. Immunological staining shows that giant, ectopically expressed from the hsp70 promoter, represses the expression of both the Krüppel and knirps segmentation gap genes. The analysis of the interactions between Krüppel, knirps and giant reveals a network of negative regulation. We show that the apparent positive regulation of knirps by Krüppel is in fact mediated by a negative effect of Krüppel on giant and a negative effect of giant on knirps. giant protein made in bacteria or in embryos binds in vitro to the Krüppel regulatory elements CD1 and CD2 and recognizes a sequence resembling the binding sites of other b-ZIP proteins.     

Developmental regulation by an enhancer from the Sgs-4 gene of Drosophila

A cis acting regulatory region has previously been identified 300-500 bp upstream of the Drosophila glue protein gene, Sgs-4. The functional capabilities of this region have now been examined by fusing it to the Drosophila Adh gene and determining the pattern of expression from the fused construct after transformation. The results show that the Sgs-4 sequences between −150 and −568 are able to direct Adh expression in late third-instar salivary glands, the appropriate tissue and timing for Sgs-4 expression. In addition, the Sgs-4 sequence elevates Adh expression in the anterior midgut and fat body, despite the fact that Sgs-4 is not normally expressed there. All three regulatory activities, tissue specificity, timing and enhancement, show the positional flexibility of enhancer elements. In addition, the Sgs-4 and Adh regulatory elements combine to direct expression in novel spatial/temporal combinations in which neither would normally be expressed.     

Notch signaling targets the Wingless responsiveness of a Ubx visceral mesoderm enhancer in Drosophila

BACKGROUND: Members of the Notch family of receptors mediate a process known as lateral inhibition that plays a prominent role in the suppression of cell fates during development. This function is triggered by a ligand, Delta, and is implemented by the release of the intracellular domain of Notch from the membrane and by its interaction with the protein Suppressor of Hairless [Su(H)] in the nucleus. There is evidence that Notch can also signal independently of Su(H). In particular, in Drosophila, there is evidence that a Su(H)-independent activity of Notch is associated with Wingless signaling. RESULTS: We report that Ubx(VM)B, a visceral mesoderm-specific enhancer of the Ubx gene of Drosophila, is sensitive to Notch signaling. In the absence of Notch, but not of Su(H), the enhancer becomes activated earlier and over a wider domain than in the wild type. Furthermore, the removal of Notch reduces the requirement for Disheveled-mediated Wingless signaling to activate this enhancer. This response to Notch is likely to be mediated by the dTcf binding sites in the Ubx(VM)B enhancer. CONCLUSIONS: Our results show that, in Drosophila, an activity of Notch that is likely to be independent of Su(H) inhibits Wingless signaling on Ubx(VM)B. A possible target of this activity is dTcf. As dTcf has been shown to be capable of repressing Wingless targets, our results suggest that this repressive activity may be regulated by Notch. Finally, we suggest that Wingless signaling is composed of two steps, a down-regulation of a Su(H)-independent Notch activity that modulates the activity of dTcf and a canonical Wingless signaling event that regulates the activity of Armadillo and its interaction with dTcf.