BIOLOGICAL OVERVIEW zerknüllt is one of approximately 10 zygotically active genes that control the differentiation of the dorsal-ventral (D/V) pattern during early embryogenesis in Drosophila. Genetic analyses suggest that maternal factors repress the expression of zen in ventral regions, thereby restricting zen products to dorsal and dorsal-lateral regions of precellular embryos. Subsequent interactions with other zygotic D/V regulatory genes, especially decapentaplegic (Rushlow, 1990), refine the zen pattern, restricting expression to the dorsal-most ectoderm (Doyle, 1989).

zen is required for only a brief period of Drosophila development, (2- 4 hours after fertilization) but its effects are found later, especially during germ band extention [Images]. In the absence of zen, germ-band extension is abnormal. Normally, the amnioserosa plays a key role in the elongation process and becomes profusely folded where the posterior tip of the germ band contacts the procephalon, and is driven into the proctodeal cavity by the expanding germ band. The absence of the amnioserosa in zen mutants might cause the germ band to be twisted and thrown into folds during an abortive elongation process (Rushlow, 1990).

Analysis of the target genes for ZEN reveal more of its function. Both DNA polymerase and Proliferating cell nuclear antigen are inhibited by ZEN. The inhibition of DNA synthesis in the amnioserosa may have to do with the process of germ-band extention. pannier is associated with dorsal closure, a process that takes place subsequent to germ band extension. Thus the influences of zen are felt long after its expression has terminated.

zerknüllt is part of the Antennapedia cluster in Drosophila and lies between proboscipedia and Deformed. Three of the four Hox clusters of mice possess class three genes in this position, functioning in specification of the antero-posterior axis of the mouse body axis as do Hox genes in Drosophila. zerknüllt fills no such function in Drosophila, but instead is involved in the specification of the amnioserosa. Any gene homologous to vertebrate class 3 would be expected to lie between proboscipedia (a class 2 homolog) and Deformed (a class 4 homolog). Outside the homeobox class 3 genes of vertebrates share sequence motifs with the Drosophila zerknullt and z2 genes, and like zen, are expressed only in extraembryonic membranes. Hoxb3 however is expressed in the hindbrain of mice with an anterior expression limit that maps to the rhombomere 4/5 boundary (Gould, 1997 and references). It would seem that the zen genes of Drosophila derive from a Hox class 3 sequence that once formed part of the common ancestral Hox cluster; in modern insects this (Hox) gene appears to have lost its role in patterning the anterio-posterior axis of the embryo, and has acquired a new function, specification of the amnioserosa. In the lineage leading to Drosophila, the zen genes have diverged with notable rapidity (Falciani, 1996).

In both Drosophila and Xenopus embryos, gradients of Dpp/BMP activity are established that are responsible for patterning along the dorsoventral axis. Dpp activity has its highest levels along the dorsal midline of the cellular blastoderm embryo and declines toward more lateral regions where it is inhibited by the product of the short gastrulation (sog) gene. The high levels determine the cell fate of the amnioserosa in the dorsal-most cells, whereas lower levels specify aspects of the dorsal epidermis in dorsolateral cells. The absence of Dpp activity in ventrolateral regions permits the formation of the neurogenic ectoderm, which gives rise to both the ventral epidermis and the central nervous system (Rushlow, 2001).

How does Dpp specify cell fate in a concentration-dependent manner? It is thought that Dpp signaling in the early embryo regulates the transcription of downstream target genes that are expressed in nested domains centered around the dorsal midline. High-level Dpp targets such as Race and u-shaped (ush) are expressed in the presumptive amnioserosa. pannier (pnr) is expressed in a broader domain that spans the amnioserosa and part of the dorsal ectoderm. Thus, it requires lower levels of Dpp. Finally, low-level targets such as early zen and dpp are expressed in an even broader domain that abuts the ventral ectoderm. A possible molecular mechanism to explain the threshold responses of Dpp target genes is that their promoters have different affinities to Smads and therefore can be induced by different levels of nuclear Smads, similar to the mechanism of differential activation by the Drosophila morphogens Dorsal (Dl) and Bicoid (Bcd). The fact that an additional mechanism is involved came from the characterization of the brinker (brk) gene. brk negatively regulates low-level and intermediate-level target genes. Study of the response elements of these target genes can therefore provide clues about the mechanisms of threshold responses to the Dpp morphogen, as well as the interplay of positive and negative inputs in the expression of target genes (Rushlow, 2001 and references therein). zen has a dynamic pattern of expression in the early embryo. During precellular nuclear division cycles 11-13 and during early cellularization (nuclear cycle 14), zen is expressed in a broad dorsal-on/ventral-off pattern. This pattern is thought to be activated by an unknown ubiquitous activator present throughout the embryo and repressed by the Dl morphogen localized in ventral regions. It is Dpp-independent because early zen expression is normal in dpp null mutants. However, slightly later, during early to mid-cellularization, maintenance of the zen pattern becomes dependent on Dpp because zen transcripts fade away suddenly in dpp null mutants. It also becomes dependent on Brk repression because zen transcripts expand into the ventral ectoderm in brk mutants. Thus, the broad pattern of zen is maintained by Dpp in the dorsal region and repressed by Brk in ventral regions. During mid- to late-cellularization, this pattern undergoes a process of refinement in which zen transcripts are lost from the lateral regions and become restricted to a narrow domain of the dorsal-most cells. Brk plays no role in refinement because in brk mutants, although zen expands ventrally, it refines normally (Rushlow, 2001 and references therein).

zen expression is directed by 1.6 kb of 5' flanking DNA sequences referred to as the zen promoter. The distal part of the promoter between 1.2 and 1.4 kb is responsible for Dl-dependent ventral repression. Sequences required for the initiation, maintenance, and refined expression of zen are located in the proximal 0.7 kb of the promoter, but they are not well-characterized (Rushlow, 2001 and references therein).

The regulation of zen during cellular blastoderm formation has been analyzed. Low levels of the Dpp signal transducer p-Mad (phosphorylated Mad), together with Brinker, define the spatial limits of zen transcription in a broad dorsal-on/ventral-off domain. The subsequent refinement of this pattern to the dorsal-most cells, however, correlates with high levels of p-Mad that accumulate in the same region during late blastoderm. Examination of the zen regulatory sequences reveals the presence of multiple Mad and Brk binding sites, and these results indicate that a full occupancy of the Mad sites due to high concentrations of nuclear Mad is the primary mechanism for refinement of zen. Interestingly, several Mad and Brk binding sites overlap, and it has been shown that Mad and Brk cannot bind simultaneously to such sites. A model is proposed whereby competition between Mad and Brk determines spatially restricted domains of expression of Dpp target genes (Rushlow, 2001).

Examination of p-Mad staining in wild-type embryos indicates that maintenance and refinement require different levels of signaling. Only the highest p-Mad levels in the dorsal-most five to six nuclei are capable of driving zen transcription during late cellularization. The lower levels present in the three to four lateral nuclei to either side are not sufficient to activate zen, although earlier they were sufficient for its maintenance. This indicates that maintenance may involve the contribution of an additional activator, perhaps the same ubiquitous activator that initiates zen earlier (Rushlow, 2001).

Later during refinement, p-Mad at peak levels is sufficient to up-regulate zen. Interestingly, the Dpp target gene ush is expressed in a broader domain than refined zen that includes the three to four lateral nuclei. This indicates that ush can be activated by a lower level of signaling than refined zen and that the high-level class of Dpp target genes can be further subdivided (Rushlow, 2001).

Because the amount of p-Mad depends on the amount of Dpp activity, the simplest explanation is that the zen promoter responds to differences in Dpp activity by measuring the level of nuclear Smads. Such a conclusion is consistent with the presence of multiple Mad/Medea binding sites and mutagenesis analysis. Deletion of only two Mad/Medea sites results in the loss of refined expression; therefore, most if not all of the Smad binding sites are required for this function, as are the peak levels of p-Mad activity (because weak dpp mutants do not refine). However, maintenance is not affected, possibly because several sites remain intact and this function does not require full p-Mad activity. That the zen promoter measures the level of nuclear Smads also explains the broad dorsolateral pattern of both p-Mad immunostaining and zen expression in sog embryos. In the absence of inhibition by Sog, Dpp continues to signal, and p-Mad can accumulate in the dorsolateral region of the embryo and induce zen expression (Rushlow, 2001).

The experiments presented here show that Mad/Medea and Brk regulate zen by binding to separated and overlapping DNA binding sites. There are 10 Mad/Medea and 6 Brk binding sites in the zen promoter, 5 of which are shared, indicating duality in their function. Indeed, the results from mutagenesis of the zen promoter show that the shared sites mediate both Brk and Mad/Medea functions. Five Brk and nine Smad binding sites are clustered in the zen proximal regulatory element over about 600 bp with spacing not exceeding 120 bp. This organization is similar to that of several well-studied enhancers from Drosophila. These enhancers are activated by a variety of transcriptional activators and repressed by short-range repressors such as Snail (Sna), Knirps (Kni), and Krüppel (Kr). All three of these repressors are DNA-binding proteins that can inhibit activator function when they are bound not further than 150 bp away from the activator binding site. It has been shown that they all contain a short stretch of amino acids, P-DLS-K, that is required for recruitment of the corepressor dCtBP. Analysis of zen regulation indicates that Brk also may be a short-range repressor. It is a DNA-binding protein and contains a PMDLSG domain. Preliminary in vitro experiments showed that Brk interacts with dCtBP; however, embryos devoid of dCtBP activity do not ectopically express zen and dpp, indicating that dCtBP is dispensable for Brk repression and other corepressors interact with Brk, or that Brk repression of these targets does not require additional factors (Rushlow, 2001).

What remains illusive is the identity of the ubiquitous transcriptional activator that activates zen in the dorsal ectoderm during precellular stages and early cellularization. It is possible that this activator interacts with Smads to enhance transcription of zen at a time when p-Mad levels are low. Also, Brk represses the ubiquitous activator, because zen becomes ectopically expressed in brk mutants. Thus far, deletion analysis of the zen promoter has not uncovered any sequences that might interact with this putative activator. It is possible that these sequences are redundant and scattered over the entire promoter and may in fact overlap with Smad and/or Brk binding sites (Rushlow, 2001).

In the cellularizing embryo, Dpp and Brk activities overlap in the lateral-most region. Here Dpp and Brk function to set thresholds of response for target genes such as zen and pnr. In this same region, Dpp signaling negatively regulates brk expression. Similarly, in the wing disc, the Brk expression domain overlaps with that of the Dpp target gene omb in the region where activated p-Mad is present. It has been proposed that a dual mechanism whereby Dpp can simultaneously down-regulate Brk repressor levels and antagonize its function on target gene promoters would be very efficient in establishing sharp threshold responses. Based on the experiments described here, a molecular model is proposed to explain mechanistically the antagonizing activities of Brk and Smads. It is proposed that they are involved in direct competition for binding to shared binding sites on target promoters. Thus, it is the balance of their opposing activities that determines the transcriptional state of the target genes. Two sets of experiments support this model: (1) ectopic expression of Brk in eve-stripe 2 abolishes zen expression in those cells. The elevated level of Brk in the stripe was therefore sufficient to repress the zen promoter even in the presence of activated Smads. The possibility that zen is repressed indirectly through Brk-mediated repression of dpp is highly unlikely because there was no delay in zen repression. (2) In vitro competition experiments also support the model. Especially revealing is the fact that the outcome of competition depends on the relative concentrations of both proteins and their binding affinities. Competitive mechanisms have been proposed to operate on many promoters where mutually exclusive DNA-binding factors are involved, and, in some instances, DNA-binding assays similar to the ones used in this study were used to show competition for binding between activator and repressor proteins. For example, bHLH proteins compete with a zinc-finger repressor for E-box binding in the immunoglobulin heavy chain enhancer (Rushlow, 2001 and references therein).

The findings presented here provide a framework for further study of the mechanisms of regulation of Dpp morphogen targets. zen is the only one of the known Dpp target genes that responds to two threshold activities: low (during early to mid-cellularization) and high (during late cellularization). Based on the results presented here and the proposed competition mechanism for activation and repression of the zen promoter, predictions can be made about the organization of the regulatory elements of the other Dpp target genes. High-level targets such as ush strongly depend on high levels of Smads, and their regulatory elements may have many, and possibly closely packed, Smad binding sites. Low-level targets such as omb in the wing imaginal disc may be repressed by Brk binding to their regulatory sequences. The spatial domains of expression of the intermediate targets such as pnr in the embryo and sal in the wing disc, which are dependent on both Dpp signaling and Brk repression, might be determined by the net balance of positive and negative inputs. Interestingly, this type of mechanism can result in expression domains that vary largely in size and may result in even broader domains than the low-level targets. An example is the vg gene. In third-instar imaginal wing discs, vg is expressed in a broader domain than omb. Its expression along the anterior-posterior boundary in the wing pouch is activated by the quadrant enhancer that contains Mad binding sites essential for activation. At the same time, vg is repressed by Brk. However, the essential Smad binding sites do not match the Brk binding sites, like many of the Smad sites in the zen promoter, suggesting that they will have no or low affinity for the Brk protein. Neither are there strong zen-like Brk binding sites in the quadrant enhancer. Its broad expression domain could then be explained if the positive inputs from Smads, enhanced by signals from the dorsoventral boundary, are able to overcome Brk repression far from the Dpp source (Rushlow, 2001).

Further studies of the arrangement, affinities, and numbers of repressor and activator sites in Dpp target promoters will determine to what extent the different thresholds of responses to the Dpp morphogen activity are shaped by a simple balance of positive and negative transcriptional inputs (Rushlow, 2001).

GENE STRUCTURE

zerknüllt is unique among homeo box genes in Drosophila since it is required for the differentiation of the dorsal-ventral pattern, and does not appear to be involved in the process of segmentation. The zen region of the Antennapedia complex (ANTP-C) (between pb and bcd) consists of two closely linked homeo box genes, designated z1 and z2. The z1 and z2 transcription units show essentially identical patterns of expression during early development, which are consistent with the timing and sites of zen gene activity. The putative proteins encoded by z1 and z2 are highly divergent and are related only by virtue of homeo box homology. z1 alone can provide zen gene function, suggesting that the z2 gene might be dispensable (Rushlow, 1987).

Bases in 5' UTR - 50

Exons - two, separated by a 64 nucleotide intron (Rushlow, 1987).

Bases in 3' UTR - 100

PROTEIN STRUCTURE

Amino Acids - 353

Structural Domains

ZEN has a homeodomain, but shares only 60% amino acid identity with members of the Antennapedia Class, 50% identity with Engrailed, and 40% identity with Paired. Z1 and Z2, the pair of genes in this chromosomal region have 75% amino acid identity in the homoeodomain and no significant homology outside the homeodomain (Rushlow, 1987).

date revised: 20 March 2001  

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