gooseberry distal

Promoter Structure

The maintenance of gsb is controlled by the wingless signal. A control element called gsb-late element, responsible for wg-dependent maintenance of gsb expression, is separable from gsb-early element, an element required for the initial activation of gsb by pair-rule transcription factors ( Li, 1993a).

The two Drosophia genes gooseberry (gsb) and gooseberry neuro (gsbn) are closely apposed and divergently transcribed. While gsb is a segment-polarity gene and mainly expressed in the epidermis, gsbn is expressed in the central nervous system. An intriguing question is how their transcriptional specificity arises. This study shows that different non-overlapping enhancer or upstream control elements drive the specific expression of gsb and gsbn. Specificity of these enhancers for their genes is achieved by their inability to activate transcription in combination with the heterologous promoter of the other gene. These results therefore suggest that compatibility between the enhancer and its cognate promoter is a mechanism ensuring transcriptional specificity (Li, 1994b).

Since gsb and gsbn share the same upstream sequence which includes both gsbE (the embryonic gsb enhancer) and gsbnE (the embryonic gsbn enhancer), the question arises as to why these enhancers activate only their own and not also the other gene. gsbE and gsbnE still preserve their distinct regulatory specificities in the corresponding gsb-lacZ and gsbn-lacZ fusion constructs that contain both gsbE and gsbnE. The functions of gsbE and gsbnE are easily distinguished. While gsbnE activates gsbn only in the CNS after stage 10, gsbE activates gsb mainly in the epidermis already much earlier by the successive action of its two elements, GEE and GLE. GEE, the gsb early element, begins to act on gsb during syncytial blastoderm whereas GLE, the gsb late element, takes over gsb activation after stage 10. The 9Z1 lacZ fusion construct is expressed in the epidermis before and after stage 10 in a pattern resembling that of gsb. The additional weak expression of 9Z1 in the CNS does not result from activation by gsbnE since smaller gsb-lacZ constructs that lack gsbnE still display this weak neural expression. Hence, gsbnE is inactive in 9Z1. Similarly, gsbE has no effect in 4Z1 as its expression of gsbn -lacZ remains largely restricted to the CNS and is not detected before stage 10. Therefore, the information restricting the activity of gsbE and gsbnE to their cognate genes is included in the common upstream sequence of gsb and gsbn (Li, 1994b).

This restriction could be explained in several ways. First, gsbE and gsbnE might block each other's action and thus be unable to act on their distal promoters. Second, the activities of gsbE and gsbnE might be dependent on their orientation. Third, a sequence between gsbE and gsbnE may function as a boundary element restricting their action to the gene located on the same side of the boundary. Finally, the gsbE or gsbnE enhancer might be unable to interact with and thus activate the promoter of the other gene (Li, 1994b).

To distinguish between these mechanisms, the embryonic expression patterns of the gsb-lacZ and gsbn-lacZ constructs were analysed. First, whether gsbnE can activate the gsb promoter in the absence of the gsb enhancer was analyzed, by examining the expression of 4n9Z in which gsbE has been removed from 9Z1. 4n9Z is only weakly expressed in a small set of internal cells of either neural or mesodermal origin in each segment after stage 11. This expression pattern differs dramatically from that of gsbn and is not affected by the orientation of gsbnE. Hence, these results suggest that gsbnE cannot act properly on the gsb promoter to activate the gsbn-specific CNS expression although they do not strictly eliminate the possibility of a boundary element located between gsbnE and gsbE. Similarly, gsbnE cannot function properly in combination with the hsp7O instead of the gsb promoter (Li, 1994b).

In analogous experiments, whether gsbE can activate the gsbn promoter was analyzed. When the region between gsbE and the gsbn promoter as well as the first two introns of gsbn were removed from 4Z1, the resulting 9n4Z construct is only weakly expressed in a row of epidermal and underlying neural cells in each segment after stage 11. In addition, the weak epidermal expression of 9n4Z differs from the characteristic barbell-shaped expression pattern of gsb or 9Z1. Few cells of the CNS that normally do not express gsb also express 9n4Z. These results suggest that the elimination of sequences that might block the interaction of the gsb enhancer with the gsbn promoter does not restore its activity in 9n4Z. Neither is the expression pattern of 9n4Z affected by the inversion of gsbE, indicating that orientation is not the cause of its inactivity. Similarly, if gsbE is placed in either orientation upstream of the hsp70 promoter, lacZ expression is never detected in the epidermis at any stage. However, its expression in several neuroblasts or ganglion mother cells in each segment may correspond to part of the normal gsb activity in the CNS. It is concluded that gsbE can activate neither the gsbn nor the hsp7O promoter properly. Taken together, these results clearly show that activation by the gsbE and gsbnE enhancers requires interaction with their cognate promoters (Li, 1994b).

The early expression of the Drosophila segment polarity gene gooseberry is under the control of the pair-rule genes. A 514-bp enhancer, -5.3 to -4.8 kb interval (called fragment IV), has been identified that reproduces the early gsb expression pattern in transgenic flies. The transcription factor Paired (Prd) is the main activator of this enhancer in all parasegments of the embryo. It binds to paired-and homeodomain-binding sites, which are segregated on the enhancer. Using site-directed mutagenesis, sites critical for Prd activity have been identified. Negative regulation of this enhancer is mediated by the Even-skipped protein (Eve) in the odd-numbered parasegments and by the combination of Fushi-tarazu (Ftz) and Odd-skipped proteins in the even-numbered parasegments. The organization of the Prd-binding sites, as well as the necessity for intact DNA binding sites for both the paired- and homeodomain-binding sites, suggests a molecular model whereby the two DNA-binding domains of the Prd protein cooperate in transcriptional activation of gsb. This positive activity appears to be in competition with Eve and Ftz on Prd homeodomain-binding sites (Bouchard, 2000).

Prd, like Gsb, contains a paired domain and a paired-type homeodomain. These domains are able to bind DNA in vitro in an independent as well as in a cooperative manner. This peculiar feature gives the members of this family of transcription factors a great DNA binding versatility. The gsb early transcriptional enhancer studied here presents an interesting case in which Prd uses both DNA-binding domains to interact with homeo- and paired domain recognition sequences segregated on the enhancer. An enhancer fragment lacking either all homeodomain-binding sites (IV.2345M) or a specific paired domain-binding site (IV.7M) loses all activity. These results suggest that the two DNA-binding domains of Paired cooperate for its proper activity on fragment IV. Whether the different sites of fragment IV are bound by the same molecule or by two different molecules is currently unclear. The model involving only one Prd molecule is supported by an experiment showing that a combination of two mutant prd transgenes (under the control of the prd promoter) that mutate in the paired domain and in the homeodomain, respectively, cannot rescue the early expression pattern of gsb in a prd mutant background, whereas a wild-type prd transgene is able to do so (Bouchard, 2000).

The onset of gsb expression first occurs in seven stripes at the cellular blastoderm stage. These stripes appear in a first row of cells at the posterior border of the even-numbered parasegments. The expression rapidly expands to a second row of cells at the anterior border of the odd-numbered parasegments. The gsb stripes coincide with the posterior border of Prd expression in the odd-numbered parasegments. It has been suggested that the early bell-shaped expression of Eve acts as a morphogenetic gradient regulating the posterior border of prd expression. Although the posterior border of gsb expression in the odd-numbered parasegments could be specified by Prd expression alone, it is likely that Eve also acts directly on the gsb control region. Indeed, the Eve-binding sites overlap with some of the Prd-binding sites, suggesting a competition at the DNA-binding level (Bouchard, 2000).

In principle, the consensus sequences identified by DNase I protection with Eve could represent binding sites of other homeodomain proteins regulating gsb expression. However, the fact that Eve and Ftz are the only known homeodomain proteins expressed in a double segment periodicity at the blastoderm stage strongly argues against this. Moreover, a direct action of Eve on gsb regulation is supported by short-pulse heat-shock experiments that favor direct regulatory effects. Using this assay, the ectopic overexpression of prd could override the repression by Eve in the odd-numbered parasegments, while a heat-shock eve could abrogate Prd activation of gsb in all parasegments. Altogether these results suggest that gsb responds to the Eve morphogenetic gradient in the odd-numbered parasegments (Bouchard, 2000).

The endogenous even-numbered gsb stripes appear at stage 6 with a slight delay compared to the odd-numbered stripes. It is now clear that Prd is essential for the activation of these stripes since neither the endogenous gsb transcription nor the fragment IV expression is observed in the trunk during germ-band extension in a prd embryo. Transcriptional activity is also lost in tissue culture assays and in vivo (transgenic lines) upon removal of Prd-binding sites. A similar conclusion concerning the activity of Prd in all parasegments has been reached using ectopic overexpression of Prd. The necessity of Prd for gsb activation does not exclude the potential requirement of another factor such as Odd-paired. Alternatively, it is possible that Opa is involved in the maintenance of gsb by activating wg in the even-numbered stripes. The activity of Opa on gsb through the Wg signal could account for the remnants of even-numbered stripes observed at late stage 11 in a prd transgenic line IV embryo. Indeed, wg is known to depend on prd in the odd-numbered stripes, whereas it depends on opa in the even-numbered stripes (Bouchard, 2000).

The establishment of the posterior border of gsb in the even-numbered parasegments requires an efficient mechanism of repression, since Prd is present throughout all all even-numbered parasegments at the time of gsb initiation. The expression of transgenic line IV-LacZ is derepressed in ftz and odd mutant embryos . Moreover, Prd activity is directly competed by Ftz and Odd in tissue cultured cells. These data identify Ftz and Odd proteins as being responsible for the establishment of gsb expression borders in the even-numbered parasegments (Bouchard, 2000).

In the genetic analysis of fragment IV, it was observed that neither ftz nor odd mutant embryos show a complete derepression in the even-numbered parasegments. An odd embryo shows an anterior widening of the odd-numbered gsb stripes, suggesting a more important role for Odd in the region of low Ftz concentration. This result also indicates that Ftz is a potent repressor in the embryo since it is still able to partially repress gsb, even though Prd levels remain high in the central part of the even-numbered parasegments in an odd embryo, as opposed to its gradual repression in this region in a wild-type background. In a ftz embryo, a posterior widening of two to three cells in the even-numbered stripes is observed. This limited expansion can be explained by the action of Odd in the posteriormost portion of the parasegment combined with the fact that Prd is fading exclusively in this region in a ftz mutant embryo. The true repressor effect of Odd on fragment IV is possibly masked in these genetic experiments by the fact that, in an odd mutant embryo, Ftz is not properly repressed in the posterior portion of the parasegment. In such an embryo, Ftz is thus compensating for the absence of Odd. At the molecular level, the mechanism of action of Odd is unclear. It is possible that Odd binds directly to fragment IV via its zinc-finger domain, but this interaction would have been missed due to insufficient binding activity in vitro. Alternatively, Odd could bind Prd via protein-protein interaction and thereby interfere with its transactivation properties (Bouchard, 2000).

The characterization of the fragment IV early gsb enhancer thus presents a demonstration of the regulation of the segment polarity genes by combinatorial activity of pair-rule proteins. This is also one of the first demonstrations of a cross-regulation between two Pax genes. In Drosophila eye development, it has recently been shown that eyeless gene regulation is under the control of Twin of eyeless, a second Pax6 homolog closely related to ey. Likewise, proper Pax5 expression depends on Pax2 function in the mouse midbrain. Such direct cascades of regulation might prove to be a widespread mode of action of Pax genes during development, as suggested by the coexpression and genetic dependency of many other Pax proteins in different tissues and organisms (Bouchard, 2000).

Transcriptional Regulation

paired and odd-paired are required for the activation of gsb in odd and even numbered stripes, respectively (Li, 1993a). Later ectodermal expression of gsb is activated and maintained in response to a wingless signal (Li, 1993a).

Although gsb and gsb-neuro are closely linked, their regulation is independent. Different non-overlapping enhancer or upstream control elements drive the specific expression of gsb and gsb-n. Specificity of these enhancers for their genes is evidenced by their inability to activate transcription in from the heterologous promoter of the other gene (Li, 1994a).

The response kinetics of known and putative target genes of Ftz has been examined in order to distinguish between direct and indirect Ftz targets. This kinetic analysis was achieved by providing a brief pulse of Ftz expression and measuring the time required for genes to respond. The time required for Ftz to bind and regulate its own enhancer, a well-documented interaction, is used as a standard for other direct interactions. Surprisingly, both positively and negatively regulated target genes respond to Ftz with the same kinetics as autoregulation. The rate-limiting step between successive interactions (<10 minutes) is the time required for regulatory proteins to either enter or be cleared from the nucleus, indicating that protein synthesis and degradation rates are closely matched for all of the proteins studied. The matching of these two processes is likely to be important for the rapid and synchronous progression from one class of segmentation genes to the next. In total, 11 putative Ftz target genes have been analyzed, and the data provide a substantially revised view of Ftz roles and activities within the segmentation hierarchy (Nasiadka, 1999).

Genetic studies have suggested that the segment polarity gene gooseberry (gsb), like wg, may be repressed by Ftz. In ftz mutant embryos, gsb stripes expand into the regions where Ftz is normally expressed, fusing to form seven wide stripes. To test whether this interaction is direct, gsb expression was examined in hsf2 embryos fixed 20 and 35 minutes after a 4 minute heat shock. No response is observed 20 minutes post heat shock. However, changes are detected in embryos fixed 35 minutes post heat shock; stripes expand into the ventral regions of odd-numbered parasegments. Interestingly, this response is a positive one, in contrast to the negative response predicted from expression in ftz minus embryos. This apparent contradiction can be reconciled by postulating that gsb is indirectly regulated by Ftz and that different intermediary factors are involved in each case. The delayed nature of the response in HSFtz embryos is consistent with this interpretation, and a likely intermediary factor is Prd, since gsb, like wg, appears to be activated by Prd. In ftz minus embryos, a likely intermediary activator is wg, since wg also expands in ftz - embryos and appears to function as an activator of gsb. An alternative explanation is that Ftz has the ability to repress gsb directly, but that this effect is spatially limited to regions where ftz is normally expressed. An exception would have to be made, however, in the anterior-most cell of each ftz stripe, where Ftz and gsb normally overlap (Nasiadka, 1999).

In the posterior half of each parasegment Patched protein represses transcription of the wingless gene by an unknown mechanism. In the most posterior row of cells in each parasegment this repression is neutralized by a signal (possibly carried by the product of the hedgehog gene) allowing wg expression. High levels of patched expression might therefore overcome the neutralization by hedgehog and repress wg in all cells. Transient overexpression of patched in all cells has little or no effect on the segmental pattern. Repeated pulses of patched production drastically alter the segment pattern to mimic embryos lacking wg. Repeated overexpression results in repression of wg and transcription of gooseberry in the germband ectoderm but not in the head. Expression of two other segment polarity genes, engrailed and cubitus interruptus, is unaffected. Thus excess patched is capable of overcoming the neutralizing signal presumably carried by hedgehog (Schuske, 1994).

Patched targets gooseberry distal and gooseberry-proximal in neuroblast determination. The RP2 neuron is a motoneuron and innervates muscle number 2 of the dorsal musculature. This neuron originates along with its sibling cell from the first ganglion mother cell derived from NB4-2, and occupies the anterior commissure along with several other RP2 neurons. NB4-2 itself is formed during the second wave of neuroblast delamination in stage 9. Gooseberry and Patched participate in the Wingless-mediated specification of NB4-2 by controlling the response to the wingless signal. In gsb mutants, WG-positive NB5-3 is transformed to NB4-2 in a Wg-dependent manner, suggesting that GSB normally represses the capacity to respond to the wingless signal. In ptc mutants, gsb is ectopically expressed in normally Wg-reponsive cells, thus preventing the response to Wingless and consequently the correct specification of NB4-2 does not take place. The timing of the response to GSB suggests that the specification of neuroblast identities takes place within the neuroectoderm, prior to neuroblast delamination (Bhat, 1996).

Expression of lbe and lbl depends on wingless. Previous studies have shown that ubiquitous expression of gooseberry ectopically activates the endogenous gsb gene in cells located anterior to the wild-type stripe. However, this ectopic induction is not observed in a wingless mutant background (Li, 1993). Heat shock gsb is also able to activate the formation of an ectopic strip of lbe. As for gsb, this phenomenon is wg-dependent and cannot be detected in wg mutants. Therefore, it is likely that wg function is required for both activation and maintenance of lbe and lbl expression, and for that matter, gsb as well. In the dorsal epidermis, both wg and lbe are gsb-independent. It is concluded that whereas ventral epidermal wg expression may require gsb, in the dorsal epidermis, both wg and lbe are gsb-independent (Jagla, 1997).

Targets of Activity

The main function of gsb is the maintenance of wingless expression by a wg-gsb autoregulatory loop after 6 hours of development. The repression of denticles by the wg signal is accomplished by a different pathway from the wingless signaling pathways that activate gsb or en. (Li, 1993b).

Direct wg autoregulation (autocrine signalling) is masked by its paracrine role in maintaining hh, which in turn maintains wg. Zeste-white3 (zw3) and patched mutant backgrounds have been used to uncouple genetically this positive-feedback loop and to study autocrine wg signaling. Direct wg autoregulation differs from wg signaling to adjacent cells by the involvement of fused, smoothened and cubitus interruptus and the lack of involvement of zw3 and armadillo. wg autoregulation during this early hh-dependent phase differs from later wg autoregulation by lack of gooseberry participation (Hooper, 1994).

How is neuroblast-specific gene expression established? This paper's focus was on the huckebein gene, because it is expressed in a subset of neuroblasts and is required for aspects of neuronal and glial determination. hkb is required within the neuroblast 1-1, 2-2 and 4-2 lineages for proper axon pathfinding of interneurons and motoneurons and for proper muscle target recognition by motoneurons. The secreted Wingless and Hedgehog proteins activate huckebein expression in distinct but overlapping clusters of neuroectodermal cells and neuroblasts, whereas the nuclear Engrailed and Gooseberry proteins repress huckebein expression in specific regions of neuroectoderm or neuroblasts. Gooseberry functions to repress hkb expression in row 5 neuroblasts while Engrailed represses hkb expression in rows 6/7 neuroectoderm. Integration of these activation and repression inputs is required to establish the precise neuroectodermal pattern of huckebein, which is subsequently required for the development of specific neuroblast cell lineages (McDonald, 1997).

P-element-mediated transformation with the gooseberry gene has been used to demonstrate that gsb transactivates gsb-n and is sufficient to rescue the gooseberry cuticular phenotype in the absence of gsb-n (Gutjahr, 1993).

Wingless (Wg) and other Wnt proteins play a crucial role in a number of developmental decisions in a variety of organisms. Wg, signaling from row 5 of the ventral nerve cord of the Drosophila embryo is non-autonomously required for the formation and specification of a neuronal precursor cell, NB4-2. NB4-2 gives rise to a well-studied neuronal lineage, the RP2/sib lineage. While the various components of the Wg-signaling pathway are also required for generating NB4-2, the target gene(s) of this pathway in the signal-receiving cell is not known. sloppy paired 1 and sloppy paired 2 play a role in generating the NB4-2 cell, functioning as the downstream targets of Wg signaling. Thus, while the loss-of-function mutations in wg and slp have the same NB4-2 formation and specification defects, these defects in wg mutants can be rescued by expressing slp genes from a heterologous promoter. The fact that slp genes function downstream of the Wg signaling is also indicated by the result that expression of slp genes is lost from the neuroectoderm in wg mutants and that ectopic expression of wg induces ectopic expression of slp. Finally, Gooseberry (Gsb) prevents Wg from specifying NB4-2 identity to the wg-expressing NB5-3. In this paper, it is shown that gsb interacts with slp and prevents Slp from specifying NB4-2 identity in NB5.3. Overexpression of slp overcomes this antagonistic interaction and respecifies NB5-3 as NB4-2. This respecification, however, can be suppressed by a simultaneous overexpression of gsb at high levels. This mechanism appears to be responsible for specifying NB5-3 identity to a row 5 neuroblast and preventing Wg from specifying NB4-2 identity to that neuroblast (Bhat, 2000).

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