A gap gene, hunchback, regulates the spatial expression of Ultrabithorax

We have examined the distribution of Ultrabithorax (Ubx) proteins in embryos mutant for the zygotic gap class of segmentation genes. Members of this class include hunchback (hb), knirps (kni), and Krüppel (Kr). All three mutations disrupt segmentation in specific regions of the embryo. Mutations in kni and Kr produce complex alterations in the Ubx expression pattern. In hb mutants Ubx is ectopically expressed both anterior and posterior to its wild-type boundaries. Thus, the hb gene may play an important role in the specification of the boundaries of Ubx expression. Using the Ubx protein distribution as a marker for metameric organization and using Hoechst dye to monitor cell death, we could follow early events that lead to the final gap-segmentation phenotype in the larval cuticle.     

Mutual repression enhances the steepness and precision of gene expression boundaries

Embryonic development is driven by spatial patterns of gene expression that determine the fate of each cell in the embryo. While gene expression is often highly erratic, embryonic development is usually exceedingly precise. In particular, gene expression boundaries are robust not only against intra-embryonic fluctuations such as noise in gene expression and protein diffusion, but also against embryo-to-embryo variations in the morphogen gradients, which provide positional information to the differentiating cells. How development is robust against intra- and inter-embryonic variations is not understood. A common motif in the gene regulation networks that control embryonic development is mutual repression between pairs of genes. To assess the role of mutual repression in the robust formation of gene expression patterns, we have performed large-scale stochastic simulations of a minimal model of two mutually repressing gap genes in Drosophila, hunchback (hb) and knirps (kni). Our model includes not only mutual repression between hb and kni, but also the stochastic and cooperative activation of hb by the anterior morphogen Bicoid (Bcd) and of kni by the posterior morphogen Caudal (Cad), as well as the diffusion of Hb and Kni between neighboring nuclei. Our analysis reveals that mutual repression can markedly increase the steepness and precision of the gap gene expression boundaries. In contrast to other mechanisms such as spatial averaging and cooperative gene activation, mutual repression thus allows for gene-expression boundaries that are both steep and precise. Moreover, mutual repression dramatically enhances their robustness against embryo-to-embryo variations in the morphogen levels. Finally, our simulations reveal that diffusion of the gap proteins plays a critical role not only in reducing the width of the gap gene expression boundaries via the mechanism of spatial averaging, but also in repairing patterning errors that could arise because of the bistability induced by mutual repression.     

Interactions of the Drosophila gap gene giant with maternal and zygotic pattern-forming genes.

The Drosophila gene giant (gt) is a segmentation gene that affects anterior head structures and abdominal segments A5-A7. Immunolocalization of the gt product shows that it is a nuclear protein whose expression is initially activated in an anterior and a posterior domain. Activation of the anterior domain is dependent on the maternal bicoid gradient while activation of the posterior domain requires maternal nanos gene product. Initial expression is not abolished by mutations in any of the zygotic gap genes. By cellular blastoderm, the initial pattern of expression has evolved into one posterior and three anterior stripes of expression. The evolution, position and width of these stripes are dependent on interactions between gt and the other gap genes. In turn, gt activity in these domains affects the expression of the other gap genes. These interactions, typical of the cross-regulation previously observed among gap genes, confirm that gt is a member of the gap gene class whose function is necessary to establish the overall pattern of gap gene expression. After cellular blastoderm, gt protein continues to be expressed in the head region in parts of the maxillary and mandibular segments as well as in the labrum. Expression is never detected in the labial or thoracic segment primordia but persists in certain head structures, including the ring gland, until the end of embryonic development.     

Three hormone receptor-like Drosophila genes encode an identical DNA-binding finger.

The putative finger domain of knirps (kni), a member of the gap class of segmentation genes, was used to isolate two sequence-related genes of Drosophila melanogaster under reduced stringency hybridization conditions. The two kni homologous genes map close to kni in the proximal portion of the third chromosome. One of them is the previously identified gene knirps-related (knrl), kni and knrl are spatially co-regulated in both early and late stages of embryogenesis. Their posterior domains of expression at blastoderm stage are under the control of the maternal pattern organizer gene nanos. In contrast, the expression of the second kni homologous gene is restricted to the late embryonic gonads. Due to its site of expression, we termed this gene 'embryonic gonad' (egon). In addition to the conserved DNA-binding domain, these three genes share an additional sequence of 19 amino acids, the kni-box, adjacent to the finger region. The identical N-terminal Cys/Cys finger encoded by each of the three genes suggests that they code for DNA-binding proteins which might bind to similar (or even identical) target sequences.     

DSP1 interacts with bicoid for knirps enhancement

DSP1 is an HMG-box protein which has been implicated in the regulation of homeotic genes in Drosophila melanogaster. Here we report that DSP1 is also involved in the regulation of the kni gap gene. Analysis of the phenotype of a null mutation of dsp1 (dsp1(1)) reveals that the absence of maternal DSP1 results in A4 segmentation defects that are correlated with a diminution of the kni expression domain. Genetic interaction studies demonstrate that a bcd mutation enhances the A4 defect of dsp1(1). We present in vitro and in vivo evidences for a direct interaction between DSP1 and Bicoid, mediated by the BCD homeodomain and the HMG box of DSP1. Finally, we show by immunoprecipitation of cross-linked chromatin the association of DSP1 with the kni-regulating region and discuss the potential mechanism of DSP1-mediated activation of kni.     

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.     

Control of the initiation of homeotic gene expression by the gap genes giant and tailless in Drosophila

The process of segmentation in Drosophila is controlled by both maternal and zygotic genes. Members of the gap class of segmentation genes play a key role in this process by interpreting maternal information and controlling the expression of pair-rule and homeotic genes. We have analyzed the pattern of expression of a variety of homeotic, pair-rule, and gap genes in tailless and giant gap mutants. tailless acts in two domains, one anterodorsal and one posterior. In its anterior domain tailless exerts a repressive effect on the expression of fushi tarazu, hunchback, and Deformed. In its posterior domain of action, tailless is responsible for the establishment of Abdominal-B expression and demarcating the posterior boundary of the initial domain of expression of Ultrabithorax. giant is an early zygotic regulator of the gap gene hunchback: in giant- embryos, alterations in the anterior domain of hunchback expression are visible by the beginning of cycle 14. giant also regulates the establishment of the expression patterns of Antennapedia and Abdominal-B. In particular, giant is the factor that controls the anterior limit of early Antennapedia expression.     

Gradients of Krüppel and knirps gene products direct pair-rule gene stripe patterning in the posterior region of the Drosophila embryo

Abdominal segmentation of the Drosophila embryo requires the activities of the gap genes Krüppel (Kr), knirps (kni), and tailless (tll). They control the expression of the pair-rule gene hairy (h) by activating or repressing independent cis-acting units that generate individual stripes. Kr activates stripe 5 and represses stripe 6, kni activates stripe 6 and represses stripe 7, and tll activates stripe 7. Kr and kni proteins bind strongly to h control units that generate stripes in areas of low concentration of the respective gap gene products and weakly to those that generate stripes in areas of high gap gene expression. These results indicate that Kr and kni proteins form overlapping concentration gradients that generate the periodic pair-rule expression pattern.     

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.     

Non-canonical functions of hunchback in segment patterning of the intermediate germ cricket Gryllus bimaculatus

In short and intermediate germ insects, only the anterior segments are specified during the blastoderm stage, leaving the posterior segments to be specified later, during embryogenesis, which differs from the segmentation process in Drosophila, a long germ insect. To elucidate the segmentation mechanisms of short and intermediate germ insects, we have investigated the orthologs of the Drosophila segmentation genes in a phylogenetically basal, intermediate germ insect, Gryllus bimaculatus (Gb). Here, we have focused on its hunchback ortholog (Gb'hb), because Drosophila hb functions as a gap gene during anterior segmentation, referred as a canonical function. Gb'hb is expressed in a gap pattern during the early stages of embryogenesis, and later in the posterior growth zone. By means of embryonic and parental RNA interference for Gb'hb, we found the following: (1) Gb'hb regulates Hox gene expression to specify regional identity in the anterior region, as observed in Drosophila and Oncopeltus; (2) Gb'hb controls germband morphogenesis and segmentation of the anterior region, probably through the pair-rule gene, even-skipped at least; (3) Gb'hb may act as a gap gene in a limited region between the posterior of the prothoracic segment and the anterior of the mesothoracic segment; and (4) Gb'hb is involved in the formation of at least seven abdominal segments, probably through its expression in the posterior growth zone, which is not conserved in Drosophila. These findings suggest that Gb'hb functions in a non-canonical manner in segment patterning. A comparison of our results with the results for other derived species revealed that the canonical hb function may have evolved from the non-canonical hb functions during evolution.