Targets of Activity

sal mutations cause inappropriate expression of homeotic selector gene Ultrabithorax. SAL inhibits teashirt expression in wild-type embryos, thereby preventing trunk development. Thus sal is a transcriptional repressor of teashirt. Since SAL represses tsh and ANTP enhances tsh, it may be that ANTP acts as an antagonist of sal (repressing sal, the tsh repressor) while at the same time enhancing tsh activity (Kühnlein, 1994). sal mutations cause inappropriate expression of Ultrabithorax (Casanova, 1988).

The transcription factor encoded by spalt major gene, which is expressed in the wing disc in a broad wedge centered over the dpp stripe, is one target of Dpp signaling. The anterior edge of the salm expression domain abuts a narrow stripe of rhomboid (rho)-expressing cells corresponding to the L2 longitudinal vein primordium. rhomboid is transcribed in a pattern that matches the future site of vein formation. hedgehog mis-expression along the anterior wing margin induces a surrounding domain of salm expression, the anterior edge of which abuts a displaced rho L2 stripe. salm plays a key role in defining the position of the L2 vein, since loss of salm function in mosaic patches induces the formation of ectopic L2 branches, composed of salm- cells running along clone borders at positions where salm- cells confront salm+ cells. These data suggest that salm determines the position of the L2 vein primordium by activating rho expression in neighboring cells through a locally non-autonomous mechanism. rho then functions to initiate and maintain vein differentiation. The sal expression pattern is determined by dpp signaling: the spacing of the L2 and L5 rhomboid stripes is sensitive to the level of dpp since various forms of dpp misexpression generated discs with an elongated A/P axis and with the L2 and L5 rhomboid stripes shifted away from the A/P border. These data suggest that the positions of the L2 and L5 vein primordia may be determined by threshold responses to DPP produced at the A/P boundary. These data provide the final link connecting the formation of a linear adult structure to the establishment of a boundary by the maternal Bicoid morphogen gradient in the blastoderm embryo (Sturtevant, 1997). It has also been suggested that araucan and caupolican, genes that code for two divergent homeodomain proteins, are involved in establishing the prepattern for rhomboid, which in turn establishes the sites of future veins. It will be of interest to see how Spalt and the two homeodomain proteins interact to establish the wing pattern.

The knirps and knirps-related genes organize the development of the second wing vein in Drosophila. The position of the L2 wing vein is determined by a chain of known developmental events, beginning with the primary subdivision of the wing imaginal disc into anterior versus posterior lineage compartments. The subdivision of body segments such as the wing primordium into anterior and posterior compartments, in turn, can be traced back to early A/P patterning in the blastoderm stage embryo. To summarize these events briefly:

  1. The posterior compartment fate is defined by expression of engrailed (en), which activates expression of the short-range Hedgehog (Hh) signal in posterior compartment cells and prevents posterior compartment cells from responding to Hh.
  2. Secreted Hh travels a short distance (6-8 cells) into the anterior compartment where it initiates a sequence of signaling events, culminating in the activation of several Hh target genes including decapentaplegic.
  3. Dpp synthesized in this narrow strip of cells travels significant distances in both the anterior and posterior directions to activate expression of Dpp target genes, such as the neighboring salm (spalt-major) and spalt-related (salr) genes, in a threshold-dependent fashion.
  4. Juxtaposition of salm expressing and salm non-expressing cells induces expression of the rhomboid (rho) gene in a stripe 1-2 cells wide, corresponding to the L2 vein primordium.
  5. rho then promotes differentiation of all longitudinal veins during late larval and early pupal development by potentiating signaling through the Egf-R/RAS pathway (Lunde, 1998 and references).

Evidence is presented that kni and knrl link A/P positional information in larval wing imaginal discs to morphogenesis of the second longitudinal wing vein (L2). kni and knrl are expressed in similar narrow stripes corresponding to the position of the L2 primordium. The kni and knrl L2 stripes abut the anterior border of the broad central expression domain of the Dpp target gene spalt major (salm). Evidence is provided that radius incompletus (ri), a well-known viable mutant lacking the L2 vein, is a regulatory mutant of the kni/knrl locus. In ri mutant wing discs, kni and knrl fail to be expressed in the L2 primordium. In addition, the positions of molecular breakpoints in the kni/knrl locus indicate that the ri function is provided by cis-acting sequences upstream of the kni transcription unit. Consistent with kni and knrl playing a role in L2 vein formation, kni and knrl are expressed in similar narrow stripes corresponding to the position of the L2 primordium. kni-expressing cells abut the anterior border of strong sal-lacZ expression and express little or no detectable lacZ. For convenience, these kni expressing cells are referred to as salm non-expressing cells. Consistent with the genetic evidence that ri is a regulatory mutant of the kni/knrl locus, the L2 stripes of kni and knrl expression are absent in ri mutant discs. However, outside the wing pouch of ri discs, kni and knrl are expressed normally. In support of the genetic evidence suggesting that ri is a cis-acting regulatory allele of the kni/knrl locus, ri function has been mapped to a region lying immediately upstream of the kni transcription unit (Lunde, 1998).

The salm transcription factor has been shown to function upstream of rho in the L2 primordium; rho expression in L2 has been shown to be induced at the boundary between salm expressing cells and salm non-expressing cells (Sturtevant, 1997). The L2 vein primordium abuts salm-expressing cells but is comprised largely of salm non-expressing cells (Sturtevant, 1997). Like rho, expression of kni in the L2 primordium abuts the anterior edge of the broad salm expression domain in wild-type third instar wing discs, and is displaced along with the anterior border of salm expression in hedgehog Moonrat (hh Mrt) wing discs. In hh Mrt wing discs, the anterior limit of the salm expression domain on the ventral surface is frequently shifted forward, relative to the border on the dorsal surface. Associated with the asymmetry in sal-lacZ expression, the dorsal and ventral components of the kni L2 stripe are driven out of register. The coordinate shift of salm and kni expression is consistent with salm functioning upstream of kni. Strong ectopic expression of either salm or spalt related using the GAL4/UAS system eliminates kni and knrl expression, and leads to the production of small wings lacking the L2 and L5 veins. The loss of kni and knrl expression in discs mis-expressing salm or salr and the subsequent elimination of L2 presumably results from obscuring the sharp boundary of endogenous salm and salr expression. Clonal analysis also indicates that salm acts upstream of kni/knrl. salm - clones generated in the anterior compartment between L2 and L3 induce ectopic forks of the L2 vein, which lie along the inside edge of the salm - clones. In contrast, salm - clones produced in corresponding positions of ri mutant wings never induce L2 forks. However, other phenotypes associated with salm - clones, such as ectopic islands of triple row bristles at the margin, are observed with regularity in an ri background. It is proposed that a short-range diffusible signal X, functioning downstream of salm and salr induces expression of kni/knrl along the anterior border of the salm expression domain (Lunde, 1998).

kni and knrl are likely to maintain and sharpen the anterior salm border through a combination of autoactivation and negative feedback on salm expression. Kni and Knrl may repress salm expression directly or could function indirectly through an intermediate tier of regulation. The ability of ectopic kni or knrl expression to suppress expression of salm as well as vein markers, but not to suppress expression of genes involved in defining the A/P organizing center (i.e. hh, dpp and ptc), is consistent with kni and knrl functioning at the last step in defining positional information required for placement of the L2 primordium. It will be interesting to determine whether there are genes functioning analogously to kni and knrl, that specify the positions of other longitudinal veins along the A/P axis of wing imaginal discs (Lunde, 1998).

The adjacent knirps and knirps-related (knrl) genes encode functionally related zinc finger transcription factors that collaborate to initiate development of the second longitudinal wing vein (L2). kni and knrl are expressed in the third instar larval wing disc in a narrow stripe of cells just anterior to the broad central zone of cells expressing high levels of the related spalt genes. A 1.4 kb cis-acting enhancer element from the kni locus has been identified that faithfully directs gene expression in the L2 primordium. Three independent ri alleles have alterations mapping within the L2-enhancer element; two of these observed lesions eliminate the ability of the enhancer element to direct gene expression in the L2 primordium. The L2 enhancer can be subdivided into distinct activation and repression domains. The activation domain mediates the combined action of the general wing activator Scalloped and a putative locally provided factor, the activity of which is abrogated by a single nucleotide alteration in the ri53j mutant. Misexpression of genes in L2 that are normally expressed in veins other than L2 results in abnormal L2 development. These experiments provide a mechanistic basis for understanding how kni and knrl link AP patterning to morphogenesis of the L2 vein by orchestrating the expression of a selective subset of vein-promoting genes in the L2 primordium (Lunde, 2003).

The L2 stripe of kni/knrl-expressing cells forms along the anterior border of a broad domain of cells expressing high levels of the related and functionally overlapping spalt-major (salm) zinc finger transcription factors. A variety of evidence indicates that central domain cells expressing the patterning genes salm and salr (together referred to as sal) induce their anterior neighbors, which express very low levels of sal, to become the L2 primordium. For example, in wings containing salm-mutant clones, ectopic branches of L2 are induced that track along and inside the salm- clone borders, mimicking the normal situation in which an L2 vein forms just outside the domain of high-level sal-expressing cells. This ability of sal-expressing cells to induce their anterior low-level sal-expressing neighbors to initiate L2 development requires the activity of the kni locus. The induction of kni/knrl expression in the L2 primordium therefore provides an excellent system for studying the transition from spatial patterning to tissue morphogenesis (Lunde, 2003).

Repression also plays a key role in restricting L2 enhancer expression to a narrow stripe of wing disc cells. It has been shown previously that salm and salr, which are expressed strongly in the central region of the wing, repress expression of kni and knrl, although low levels of sal may also be required to activate kni expression. Evidence has been found for repression of L2-enhancer activity in peripheral wing disc cells abutting those expressing high levels of sal. Truncation of the minimal 1.4 kb (fragment EX) L2 enhancer element results in reporter gene expression expanding to fill the anterior and posterior regions of the wing pouch in a pattern complementary to that of sal (Lunde, 2003).

The Drosophila tracheal system arises from clusters of ectodermal cells that invaginate and migrate to originate a network of epithelial tubes. Genetic analyses have identified several genes that are specifically expressed in the tracheal cells and are required for tracheal development. Among them, trachealess (trh) is able to induce ectopic tracheal pits and therefore it has been suggested that it would act as an inducer of tracheal cell fates; however, this capacity appears to be spatially restricted. The expression of the tracheal specific genes in the early steps of tracheal development and their crossinteractions have been examined. There is a set of primary genes including trh and ventral veinless (vvl) whose expression does not depend on any other tracheal gene and a set of downstream genes whose expression requires different combinations of the primary genes. Expression of trh is repressed by sal in the terminal regions leading to the suggestion that this is the mechanism that accounts for the confinement of tracheal placodes to the central segments of the embryo. In contrast, vvl is expressed at the correct positions in segments that normally do not form tracheal placodes, although its expression in those sites is much weaker. Whether sal could also regulate vvl expression was investigated. Indeed, vvl expression is strongly increased in those sites in sal mutant embryos suggesting that vvl is downregulated by sal in the segments that do not form tracheal pits. Similarly, kni expression is also upregulated in the same sites in sal mutant embryos. Repression of vvl and kni by sal could in principle be attributed to the downregulation of trh by sal. However, this seems not to be the case because expression of vvl and early expression of kni in the tracheal placodes does not depend on trh. Therefore, sal seems to independently downregulate trh, vvl and kni in the most anterior and posterior embryonic regions (Boube, 2000).

Because trh and vvl are sufficient to activate btl, additional patches of btl expression were found in sal mutant embryos. dof and rho are expressed in additional patches of cells in sal mutant embryos. Repression of dof and rho by sal could also be attributed to the downregulation of trh and vvl by sal. However, this seems not to be the case because co-expression of trh and vvl in the sal domain is not sufficient to induce either dof or rho expression. Instead, sal could directly repress dof and rho or, alternatively, it could repress an additional factor necessary for their induction. In summary, many tracheal genes appear to be independently downregulated by sal in the terminal regions. Besides, the lack of sal expression does not have the same effect on the tracheal genes. In particular, some of the additional patches of trh expression are much weaker than the normal ones. This difference is not so pronounced in the case of vvl expression in sal mutant embryos. Also, one additional anterior pair of cell clusters for rho and dof expression is observed. Therefore, not all the tracheal placodes are equivalent in sal mutant embryos (Boube, 2000).

Spalt has an important role in specifying cell fate within the tracheal branches. The expression of sal is restricted to the dorsal cells in the developing tracheal tree. By stage 12, sal is expressed only in the dorsal parts of the tracheal metameres, that is, in the cells of the dorsal trunk and the dorsal branches. By stages 14 and 15, sal expression declines in the cells of the dorsal branches, but remains high in the cells of the dorsal trunk. In sal mutant embryos there is ectopic expression of alphaPS1 in the cells of the dorsal trunk and in the dorsal branches, indicating that sal activity is required to inhibit alphaPS1 expression in the dorsal tracheal structures. In summary, these results indicate that alphaPS1 expression is restricted to the cells of the visceral branches by the direct or indirect activities of the transcription factors that spatially subdivide the tracheal placode. The fact that alphaPS1 expression is regulated by the same genes that specify a particular cell fate, rather than independently, suggests that integrin expression is an important part of the cell fate decision (Boube, 2001).

During development of multicellular organisms, cells are often eliminated by apoptosis if they fail to receive appropriate signals from their surroundings. Short-range cell interactions support cell survival in the Drosophila wing imaginal disc. Evidence is presented showing that cells incorrectly specified for their position undergo apoptosis because they fail to express specific proteins that are found on surrounding cells, including the LRR transmembrane proteins Capricious and Tartan. Interestingly, only the extracellular domains of Capricious and Tartan are required, suggesting that a bidirectional process of cell communication is involved in triggering apoptosis. Evidence showing that activation of the Notch signal transduction pathway is involved in triggering apoptosis of cells misspecified for their dorsal-ventral position (Milán, 2002).

In second instar wing discs, the LRR proteins Caps and Tartan are expressed in cells of the dorsal compartment. During third instar, dorsal expression of Caps and Tartan decreases and new lateral expression domains arise. The region of low Caps and Tartan expression in the center of the mature third instar wing disc coincides with the domain in which Dpp signaling induces Spalt expression. The reciprocity of Spalt and Caps/Tartan expression in third instar wing discs suggested that Spalt might repress Caps/Tartan at this stage. spalt mutant clones located medially show ectopic expression of Caps protein and a tartan-lacZ reporter gene. Ubiquitous expression of Spalt in the wing pouch reduces the levels of expression of Caps and Tartan in the lateral wing disc. These results indicate that Spalt restricts expression of caps and tartan to lateral cells in third instar wing discs (Milán, 2002).

Defective proventriculus specifies the ocellar region in the Drosophila head

A pair of the Drosophila eye-antennal disc gives rise to four distinct organs (eyes, antennae, maxillary palps, and ocelli) and surrounding head cuticle. Developmental processes of this imaginal disc provide an excellent model system to study the mechanism of regional specification and subsequent organogenesis. The dorsal head capsule (vertex) of adult Drosophila is divided into three morphologically distinct subdomains: ocellar, frons, and orbital. The homeobox gene orthodenticle (otd) is required for head vertex development, and mutations that reduce or abolish otd expression in the vertex primordium lead to ocelliless flies. The homeodomain-containing transcriptional repressor Engrailed (En) is also involved in ocellar specification, and the En expression is completely lost in otd mutants. However, the molecular mechanism of ocellar specification remains elusive. This study provides evidence that the homeobox gene defective proventriculus (dve) is a downstream effector of Otd, and also that the repressor activity of Dve is required for en activation through a relief-of-repression mechanism. Furthermore, the Dve activity is involved in repression of the frons identity in an incoherent feedforward loop of Otd and Dve (Yorimitsu, 2011).

This study presents evidence that Dve is a new member involved in ocellar specification and acts as a downstream effector of Otd. The results also revealed a complicated pathway of transcriptional regulators, Otd-Dve-Ara-Ci-En, for ocellar specification (Yorimitsu, 2011).

Transcription networks contain a small set of recurring regulation patterns called network motifs. A feedforward loop (FFL) consists of three genes, two input transcription factors and a target gene, and their regulatory interactions generate eight possible structures of feedforward loop (FFL). When a target gene is suppressed by a repressor 1 (Rep1), relief of this repression by another repressor 2 (Rep2) can induce the target gene expression. When Rep2 also acts as an activator of the target gene, this relief of repression mechanism is classified as a coherent type-4 feedforward loop (c-FFL). During vertex development, Ara is involved in hh repression, and the Dve-mediated ara repression is crucial for hh expression and subsequent ocellar specification. However, the cascade of dve-ara-hh seems to be a relief of repression rather than a cFFL, because Dve is not a direct activator of the hh gene. Furthermore, dve RNAi phenotypes were rescued in the ara mutant background, suggesting that a linear relief of repression mechanism is crucial for hh maintenance (Yorimitsu, 2011).

In photoreceptor R7, Dve acts as a key molecule in a cFFL. Dve (as a Rep1) represses rh3, and the transcription factor Spalt (Sal) (as a Rep2) represses dve and also activates rh3 in parallel to induce rh3 expression. Interestingly, Notch signaling is closely associated with the relief of Dve-mediated transcriptional repression in wing and leg disks. These regulatory networks may also be cFFLs in which Dve acts as a Rep1, although repressors involved in dve repression are not yet identified. In wing disks, expression of wg and ct are repressed by Dve, and Notch signaling represses dve to induce these genes at the dorso-ventral boundary. The Dve activity adjacent to the dorso-ventral boundary still represses wg to refine the source of morphogen. In leg disks, Dve represses expression of dAP-2, and Notch signaling represses dve to induce dAP-2 at the presumptive joint region. The Dve activity distal to the segment boundary still represses dAP-2 to prevent ectopic joint formation. Taken together, these results suggest that Dve plays a critical role as a Rep1 in cFFLs in different tissues. In the head vertex region, it is likely that the repressor activity of Dve is repressed in a cFFL to induce frons identity (Yorimitsu, 2011).

The homeodomain protein Otd is the most upstream transcription factor required for establishment of the head vertex. During second larval instar, Otd is ubiquitously expressed in the eye-antennal disk and it is gradually restricted in the vertex primordium until early third larval instar. Expression of an Otd-target gene, dve, is also detected in the same vertex region at early third larval instar. Otd is required for Dve expression, and the Otd-induced Dve is required for repression of frons identity through the Hh signaling pathway in the medial region. However, Otd is also required for the frons identity in both the medial and mediolateral regions (Yorimitsu, 2011).

This regulatory network is quite similar to the incoherent type-1 feedforward loop (iFFL) in photoreceptor R7. Otd-induced Dve is involved in rh3 repression, whereas Otd is also required for rh3 activation. iFFLs have been known to generate pulse-like dynamics and response acceleration if Rep1 does not completely represses its target gene expression. However, the repressor activity of Dve supersedes the Otd-dependent rh3 activation, resulting in complete rh3 repression in yR7. In pR7, Dve is repressed by Sal, resulting in rh3 expression through the Otd- and Sal-dependent rh3 activation. Thus, Dve serves as a common node that integrates the two loops, the Otd-Dve-Rh3 iFFL and the Sal-Dve-Rh3 cFFL (Yorimitsu, 2011).

In the head vertex region, Otd and Dve are expressed in a graded fashion along the mediolateral axis with highest concentration in the medial region. It is assumed that Otd determines the default state for frons development through restricting the source of morphogens Hh and Wg, and also that high level of Dve expression in the medial ocellar region represses the frons identity through an iFFL. It is likely that repression of dve by an unknown repressor X occurs in a cFFL and induces the frons identity in the mediolateral region (Yorimitsu, 2011).

Interlocked FFLs including Otd and Dve appear to be a common feature in the eye and the head vertex. However, other factors are not shared between two tissues. In R7, a default state is the Otd-dependent Rh3 activation, an acquired state is (1) Rh3 repression through the Otd-Dve iFFL and (2) Spineless-dependent Rh4 expression. In the vertex, a default state is Otd-dependent frons formation, an acquired state is (1) frons repression through the Otd-Dve iFFL and (2) Hh-dependent ocellar specification associated with En and Eya activation (Yorimitsu, 2011).

Both Otd and Dve are K50-type homeodomain transcription factors, and they bind to the rh3 promoter via canonical K50 binding sites (TAATCC). The Otd-Dve iFFL in the eye depends on direct binding activities to these K50 binding sites, but the iFFL in the vertex seems to be more complex. Although target genes for frons determination are not identified, the iFFL in the vertex includes some additional network motifs. For instance, in the downstream of Dve, Hh signaling is critically required for repression of the frons identity (Yorimitsu, 2011).

Since iFFLs also act as fold-change detection to normalize noise in inputs, interlocked FFLs of Dve-mediated transcriptional repression may contribute to robustness of gene expression by preventing aberrant activation. It is an intriguing possibility that, in wing and leg disks, Dve also serves as a common node that integrates the two loops as observed in the eye and the vertex. Further characterization of regulatory networks including Dve will clarify molecular mechanisms of cell specification (Yorimitsu, 2011).

Regulation of twin of eyeless during Drosophila development

The Pax-6 protein is vital for eye development in all seeing animals, from sea urchins to humans. Either of the Pax6 genes in Drosophila (twin of eyeless and eyeless) can induce a gene cascade leading to formation of the entire eye when expressed ectopically. The twin of eyeless (toy) gene in Drosophila is expressed in the anterior region of the early fly embryo. At later stages it is expressed in the brain, ventral nerve cord and (eventually) the visual primordium that gives rise to the eye-antennal imaginal discs of the larvae. These discs subsequently form the major part of the adult head, including compound eyes. This study has sought genes that are required for normal toy expression in the early embryo to elucidate initiating events of eye organogenesis. Candidate genes identified by mutation analyses were subjected to further knock-out and misexpression tests to investigate their interactions with toy. The results indicate that the head-specific gap gene empty spiracles can act as a repressor of Toy, while ocelliless (oc) and spalt major (salm) appear to act as positive regulators of toy gene expression (Skottheim Honn, 2016).

spalt: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation | References

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