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Targets of DPP in leg morphogenesis

Many of the genes that pattern Drosophila are expressed throughout development and specify diverse cell types by creating unique local environments that establish the expression of locally acting genes. This process is exemplified by the patterning of leg microchaete rows. hairy (h) is expressed in a spatially restricted manner in the leg imaginal disc and functions to position adult leg bristle rows by negatively regulating the proneural gene achaete, which specifies sensory cell fates. While much is known about the events that partition the leg imaginal disc and about sensory cell differentiation, the mechanisms that refine early patterning events to the level of individual cell fate specification are not well understood. In the third instar leg imaginal disc, h is expressed along both the D/V and A/P axes. D/V axis expression appears as a single stripe in the anterior compartment of the disc immediately adjacent to the A/P compartment boundary. A/P axis expression appears as two wedge-shaped blocks in the distal leg segments on either side of the A/P compartment boundary. After disc eversion, the D/V axis stripe forms two of the four longitudinal leg stripes. The A/P axis expression forms five circumferential stripes at the first through fifth tarsal segments. The remaining two longitudinal stripes will be positioned along the A/P axis, intersecting the D/V axis stripes at the distal tip of the leg. These stripes do not appear until 2-3 hours after puparium formation (APF). This paper concerns itself with Hairy expression in the D/V axis (Hays, 1999).

To assess the roles of Dpp and Wg in the regulation of the D-h and V-h enhancer elements, the expression of D-h-lacZ and V-h-lacZ was assayed in legs that were mutant for either dpp or wg. Expression from D-h-lacZ is reduced in leg discs that are mutant for dpp, and is expanded to produce a full D/V axis stripe in discs that are mutant for wg. Conversely, V-h-lacZ expression is severely reduced in wg mutant discs, and duplicated in dpp mutant discs. These findings are in keeping with what has been demonstrated for other Dpp and Wg target genes and suggest that the D-h and V-h enhancers are targets of Dpp and Wg signaling, respectively. The roles of Dpp and Wg in D/V-h regulation were further examined by making somatic clones lacking components of the Dpp and Wg signaling pathways. Given the antagonism between Dpp and Wg in the leg, it was necessary to analyze clones mutant for a component of each pathway. D-h expression was examined in clones mutant for wg and Mothers against dpp (Mad). Mad is a downstream effector in the Dpp signaling pathway that has been shown to bind DNA and transcriptionally regulate some Dpp target genes directly. Dorsal Mad;wg clones that intersect the D-h stripe show loss of h expression, except for variable low level expression in a single row of cells immediately adjacent to the A/P boundary. It can reasonably be concluded that loss of D-h results from the loss of Mad, since there is no duplicated Wg in these clones. These results not only support the finding that D-h is a target of Dpp signaling, it identifies Mad as a potential transcriptional regulator of D-h. Thus it is proposed that D/V-h expression is regulated in a non-linear pathway in which Ci plays a dual role. In addition to serving as an upstream activator of Dpp and Wg, Ci acts combinatorially with them to activate D/V-h expression (Hays, 1999).

Several questions remain open regarding the placement of leg sensory organs. There is much to be learned about the regulation of the eight Ac leg stripes. In the absence of H function, ac is expressed in four broad stripes, which may represent four zones that are competent to express ac. The expression of ac in these zones could be under the control of independent cis-regulatory elements as are D-h and V-h. The D/V axis stripes would likely be regulated by high level Dpp and Wg signaling. The A/P axis stripes could be sensitive to low levels of Dpp and Wg signaling. This may also be the case for the A/P-h stripes, whose regulation is also not yet understood. Alternatively, the whole leg may be competent to express ac with the interstripes established by H and an unknown factor that represses the expression of ac in the four non-H-expressing interstripe regions (Hays, 1999).

The sensory organs of the Drosophila adult leg provide a simple model system with which to investigate pattern-forming mechanisms. In the leg, a group of small mechanosensory bristles is organized into a series of longitudinal rows, a pattern that depends on periodic expression of the hairy gene and the proneural genes achaete and scute. Expression of ac in longitudinal stripes in prepupal leg discs defines the positions of the mechanosensory bristle rows. The ac/sc expression domains are delimited by the Hairy repressor, which is itself periodically expressed. In order to gain insight into the molecular mechanisms involved in leg sensory organ patterning, a Hedgehog (Hh)- and Decapentaplegic (Dpp)-responsive enhancer of the h gene, which directs expression of h in a narrow stripe in the dorsal leg imaginal disc (the D-h stripe) has been examined. These studies suggest that the domain of D-h expression is defined by the overlap of Hh and high-level Dpp signaling. The D-h enhancer consists of a Hh-responsive activation element (HHRE) and a repression element (REPE), which responds to the transcriptional repressor Brinker (Brk). The HHRE directs expression of h in a broad stripe along the anteroposterior (AP) compartment boundary. HHRE-directed expression is refined along the AP and dorsoventral axes by Brk1, acting through the REPE. In D-h-expressing cells, Dpp signaling is required to block Brk-mediated repression. This study elucidates a molecular mechanism for integration of the Hh and Dpp signals, and identifies a novel function for Brk as a repressor of Hh-target genes (Kwon, 2004).

The D-h and V-h stripes are regulated by separate enhancers, which map between 32-38 kb 3' to the h transcription unit. ac stripes are not expressed until 6 hours after puparium formation (APF). The flanking narrow D-h stripe is positioned a few cells anterior to the compartment boundary, allowing expression of two dorsal ac stripes in the anterior compartment. V-h, however, is expressed directly adjacent to the AP boundary so that there is only one ventral ac stripe in the anterior compartment. Expression of each h stripe in its proper register is essential for positioning of the ac stripes and consequently for sensory bristle patterning in the adult leg. Focus was placed on the mechanisms that lead to expression of the D-h stripe in its precise register near the AP boundary (Kwon, 2004).

A question is raised regarding the identity of the repressor(s) that acts through the REPE to refine HHRE-directed expression. A potential candidate, the transcriptional repressor of Dpp target genes, Brk, is suggested by evidence indicating that Dpp is required to override REPE function. In the wing and leg imaginal discs, brk expression is repressed by and is roughly reciprocal to Dpp signaling. Hence, in the leg disc, brk expression is lowest in dorsal-most leg cells. D-h-GFP is expressed within the region of low-level brk expression in leg discs. Furthermore, brk expression expands dorsally in dppd6/dppd12 legs, in which D-h expression is severely reduced (Kwon, 2004).

To determine whether Brk functions as a repressor of D-h expression, D-h-GFP expression was examined in clones lacking brk function. Loss of brk function results in ectopic expression of D-h-GFP on either side of the D-h-GFP stripe. Ectopic expression is observed in clones anterior to the D-h-GFP stripe. However, the expansion is confined to a region two or three cells wide, directly juxtaposed to D-h expression, which presumably corresponds to the HHRE-responsive zone. In addition, ectopic expression is observed in ventral clones. Overexpression of brk along the AP boundary drastically reduces D-h-GFP expression but does not affect HHRE-GFP expression, indicating that Brk acts through the REPE to repress D-h expression. Since D-h expression is activated primarily by the Hh-responsive HHRE, these observations identify Brk as repressor of Hh as well as Dpp target genes (Kwon, 2004).

Genetic data support a hypothesis in which Brk acts through the REPE of the D-h enhancer to modulate activity of the HHRE. If so, it might be expected that the REPE would contain one or more functional Brk-binding sites. Hence, the REPE was examined for the Brk consensus binding site, GGCG(C/T)(C/T), and a potential Brk binding site was identified that overlaps two sequences similar to a consensus binding sites for Mad: GCCGNCGC, and a sequence similar to a cAMP response element (CRE), TGACGTCA. The sequence of overlapping CRE, Brk and Mad sites was designated the CMB element. Site directed mutational studies are consistent with the hypotheses that Brk acts through the CMB to repress D-h expression (Kwon, 2004).

A short sequence in the REPE, the CMB, has been identified that functions to restrict HHRE expression to a narrow dorsal domain. In this study, evidence is provided for the hypothesis that the transcriptional repressor Brk acts through the CMB to repress D-h expression. Although previous studies have shown that brk expression is very low or undetectable in cells near the Dpp source, a genetic requirement has been demonstrated for brk in repression of D-h in this region. In addition, overexpression of brk results in a dramatic reduction of D-h-GFP expression, but only mildly affects expression from a D-h-GFP transgene with a compromised Brk binding site (Kwon, 2004).

Dpp acts through the REPE to block Brk-mediated repression. It is proposed that high-level Dpp signaling defines the domain of D-h expression within the HHRE-response zone. This idea is supported by the observations that D-h-GFP but not HHRE-GFP expression is dependent on Dpp, indicating that Dpp signals through the REPE, and that elevation of Dpp signaling results in expansion of D-h expression along the AP and DV axes, within the domain of HHRE activity. Current studies suggest that the function of Dpp in regulation of D-h expression may be limited to repression of brk. Yet, the presence of Mad-binding sites in the CMB suggests a potentially more direct role for activated Mad (act-Mad), the transcriptional mediator of Dpp signaling. Brk has been shown to be a potent competitor of Mad in vitro for binding to overlapping binding sites in Dpp target enhancers. Hence, a potential role for Mad would be to prevent Brk from binding the CMB, thereby blocking Brk repression in cells receiving high-level Dpp signaling. If this model is correct, one might have expected the Mad1/Mad2 (MM) mutation to compromise D-h expression, which was not the case. However, the destabilization of Brk binding to the MM mutant might have masked a requirement for the Mad sites in blocking Brk repression (Kwon, 2004).

It has recently been shown that an act-Mad/Shn complex represses brk expression by binding a silencer element. Therefore, since mutation of the Mad sites expands D-h expression, it is possible that Mad acts in concert with Brk through the CMB to repress D-h expression. This notion is not inconsistent with genetic evidence, indicating a requirement for Mad in D-h expression, since loss of Mad function elevates Brk levels, which can overcome the requirement for CMB-sequences other than the Brk site. However, if this were the case, a more severe expansion phenotype might be expected with the MM mutant, in which both Brk and Mad binding are compromised. Further analysis is required to determine the role, if any, of the CMB-Mad-binding sites in D-h expression (Kwon, 2004).

This study has identified a novel function for Brk as repressor of Hh-target gene expression. Brk was originally identified as a repressor of Dpp-target genes and a recent study indicates that Brk can block Wg-mediated transcription as well. Brk was shown to antagonize function of a Wg-responsive element in the midgut enhancer of the Ultrabithorax (Ubx). The Ubx midgut enhancer drives Ubx expression in parasegment (ps) 7 of the embryonic midgut. Two elements, one of which is Wg responsive (the WRS) and another Dpp responsive (the DRS), function synergistically to activate Ubx expression in ps 7 expression. In the adjacent ps8, however, Brk binds to the DRS and blocks the activity of the WRS. Curiously, the D-h-CMB and the Ubx-DRS are similarly organized in that each consists of overlapping CRE/Mad and Brk sites. The Ubx-DRS appears to mediate two modes of signal integration which involve: (1) synergistic activation, in which Mad/Med and dTCF act together to activate expression; and (2) activation and refinement, in which there is Wg mediated activation combined with Brk repression, which is blocked by Dpp. In the D-h enhancer, however, the CMB appears to be a component of a dedicated repression element, which appears to mediate only the second mode of signal integration: activation and refinement. The similar organization of the CMB and DRS suggests that it may be possible to predict the structure of enhancers known to be Brk responsive and which integrate Dpp and a second signal (Kwon, 2004).

Despite the similarities, there are important distinctions between the D-h and Ubx-midgut enhancers, suggesting that the mechanisms of Brk-mediated repression might differ in each case. In the Ubx-midgut enhancer, the DRS and WRS are separated by 10 bp, suggesting that Brk acts at short range to inhibit WRS activity. In the D-h enhancer, however, the CMB is positioned at least 1 kb from the HHRE, implying a long-range effect for this element. Furthermore, Brk repression of the WRS depends on Teashirt (Tsh), which binds Brk and acts as a co-repressor. Tsh is unlikely to be required for D-h repression because it is only expressed in proximal leg segments. The current studies suggest the requirement for a second DNA-bound factor, which binds the CRE, in addition to Brk for repression. The DRS-CRE, however, is required in addition to the Mad-binding sites for activation of Ubx in ps 7 (Kwon, 2004).

Together, these observations are consistent with a model in which Ci, acting through the HHRE, activates D-h expression. The domain of HHRE activity can be divided into two zones, 1 and 2. The HHRE has the potential to direct expression in both zones 1 and 2, but its activity is restricted to zone 1 by Brk and perhaps a second factor, X, which binds the CRE. In zone 2 cells, Brk would bind to the CMB and repress HHRE-directed expression. It is proposed that zone 1 is defined by the overlap of Hh and high-level Dpp signaling. Dpp promotes D-h expression by repressing brk expression in zone 1. However, the presence of Mad-binding sites in the CMB suggests the potential for a more direct role for Mad in D-h regulation, perhaps in competing with Brk for binding to the CMB, or in directly mediating repression. Confirmation of a role for the Mad sites awaits further analysis of the D-h enhancer (Kwon, 2004).

Table of contents

decapentaplegic: Biological Overview | Evolutionary Homologs | Transcriptional regulation | Protein Interactions | Post-transcriptional Regulation | Developmental Biology | Effect of mutation | References

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