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Gene name - spalt major Synonyms - spalt Cytological map position - 32F2-33A2 Function - transcription factor Keyword(s) - homeotic |
Symbol - salm FlyBase ID:FBgn0004579 Genetic map position - 2-44 Classification - zinc finger Cellular location - nuclear |
spalt major (salm) and forkhead (fkd) both influence the development of the fly's gut. Both are expressed prior to invagination in anterior and posterior anlagen of the gut. The gut of the fly is formed during gastrulation by the invagination or inturning of anterior and posterior gut primordia. Endoderm forms the main tissue of the gut, with some contribution from contiguous ectoderm. In fkh mutants, ectodermal parts of the foregut develop as head structures, while Abdominal-B/salm double mutants develop thoracic structures instead of head structures. Thus fkh and salm have opposing actions in fate determination: fkh changes cell fate to a more posterior morphology while salm influences change to a more anterior morphology. salm appears to have a role in restricting trunk development outside of the mesothorax (Casanova, 1989).
Ectopic expression of Antennapedia induces antenna to leg transformations. Under this circumstance salm expression is entirely repressed by Antp activity, so that leg structures form in place of antennal structures (Wagner-Bernholz, 1991). How does ectopic Antp result in antenna-leg transformation? Current thinking suggests that since Salm represses tsh and ANTP enhances tsh, ectopic Antp provides a double dose, enhancing tsh at the same time it represses salm. This remains conjecture, since these activities have not been observed in imaginal discs (Kühnlein, 1994).
Salm could be both a transcriptional activator and repressor, as it has glutamine - rich regions which are thought to function as activator sequences and proline - and alanine - rich regions required for repressor function.
spalt and Optomotor blind are targets of Dpp, acting through the Dpp receptor Thick veins (Tkv) in the wing imaginal disc. Axis formation in the Drosophila wing depends on the localized expression of the secreted signaling molecule Decapentaplegic. Dpp acts directly at a distance to specify discrete spatial domains, suggesting that it functions as a morphogen. Expression levels of the Dpp receptor thick veins are not uniform along the anterior-posterior axis of the wing imaginal disc. tkv is expressed at low levels in the center of the disc and at higher levels toward the edges of the disc. Although tkv levels are low in the center of the disc, clonal analysis has shown that tkv activity is stringently required in this region for growth and for target gene expression. Receptor levels are low where Dpp induces its targets Spalt and Omb in the wing pouch. Receptor levels increase in cells farther from the source of Dpp in the lateral regions of the disc (Lecuit, 1998).
Evidence is presented that Dpp signaling negatively regulates tkv expression and that the level of receptor influences the effective range of the Dpp gradient. High levels of tkv sensitize cells to low levels of Dpp and also appear to limit the movement of Dpp outside the wing pouch. Thus receptor levels help to shape the Dpp gradient. It was asked whether Dpp signaling regulates tkv expression by examining the effects of clones of cells expressing Dpp at lateral positions in the disc where the level of tkv is normally high. Dpp-expressing clones were marked indirectly by their ability to induce ectopic Spalt expression. tkv transcript levels are reduced where Spalt is misexpressed, suggesting that Dpp can act at a distance to repress tkv expression These results suggest that the reduced levels of tkv transcript in the center of the disc are due to downregulation by Dpp acting at a distance (Lecuit, 1998).
Are the reduced levels of the Tkv expression important for the formation of the Dpp activity gradient? To address this, an examination was made of the effects on the expression of the Dpp-target genes Spalt and Omb in clones of cells that overexpress wild-type Tkv. Tkv-expressing clones well inside the endogenous domains show little effect on either Spalt or Omb expression. Clones near the edge of the endogenous Spalt domain show increased Spalt expression and those near the edge of the Omb domain show elevated Omb expression. Tkv-expressing clones located outside but near the endogenous Spalt domain show ectopic induction of Spalt. Clones located farther from the Spalt domain do not show ectopic activation of Spalt. Together, these observations suggest that overexpressing Tkv can increase the sensitivity of cells to low levels of Dpp (Lecuit, 1998).
Tkv was overexpressed to assess the consequences of broadly elevating Tkv expression levels in the central region of the disc. Wings with elevated Tkv expression are reduced in size. The effect is stronger in the posterior compartment, with the region between veins 4 and 5 being more reduced than the region between veins 2 and 3. The region between veins 3 and 4 is relatively normal, possibly because the size of this intervein region is specified directly by Hedgehog, not by Dpp. These observations suggest that the long-range activity of Dpp in the vein 2-3 and 4-5 regions is compromised by overexpression of the Dpp receptor. Overexpressing Tkv strongly reduces the size of the Spalt domain in the Posterior (P) compartment. The effect on Spalt expression is much stronger in the P compartment than in the A compartment and the Spalt domains in both compartments appear to be less graded at their edges than in wild type. The effective range of the Dpp activity gradient appears to be limited to a few cells in the P compartment in mid- and late-third instar discs. This suggests that overexpression of receptor can limit the spread of Dpp in the P compartment. These observations suggest that high levels of the receptor might sequester ligand and limit its movement across the wing disc. The difference observe between A and P compartments when Tkv is overexpressed probably reflects the fact that cells originating in the Dpp expression domain can contribute to formation of a large part of the anterior compartment, but not to the posterior compartment. Thus cells originating in the Dpp domain could ëcarryí Dpp protein away from the source as they and their progeny are displaced by addition of new cells (the displacement process can be directly visualized by lineage tracing cells originating in the dpp-expression domain). It is concluded that artificially high levels of Thick veins outside the wing pouch appear to limit the spread of Dpp and thereby modulate the shape of the ligand gradient. In addition, the level of Tkv expression modulates the sensitivity of cells to Dpp. Thus regulation of receptor levels by Dpp modulates the shape of the Dpp gradient (Lecuit, 1998).
Spalt and Spalt-related regulate the vein-specific expression of the transcription factors of the knirps and iroquois gene complexes, delimiting their domains of expression in the wing pouch. The effects of spalt/spalt-related mutations on knirps and iroquois expression are cell-autonomous, suggesting that they could be direct. The regulation of iroquois involves transcriptional repression by Spalt and Spalt-related, whereas the regulation of knirps involves a combination of transcriptional activation and repression mediated by the same genes. It is suggested that the regulation of the iroquois and knirps gene complexes by Spalt and Spalt-related translates the Decapentaplegic morphogenetic gradient into precisely spaced pattern elements (de Celis, 2000).
Although the development of the four longitudinal veins of Drosophila (L2-L5) involves the same signaling systems, and vein cells show the identical type of differentiation, there are several characteristics that distinguish one vein from another. Thus, the veins L2 and proximal L4 differentiate predominantly in the ventral wing surface, whereas the veins L3, L5 and distal L4 do so in the dorsal surface. Furthermore, several genes are required for the formation of individual veins, suggesting that each vein is individually specified. Vein-specific genes include the transcription factors of the iroquois gene complex (iro-C), which are only expressed and required in L3 and L5, and the transcription factors of the knirps gene complex (kni-C), which are only expressed and required in L2. Vein-specific genes could be part of a combinatorial code of signals that activate a common vein-differentiation program in different parts of the wing. In addition, vein specific genes could also confer individual qualities to each longitudinal vein (de Celis, 2000 and references therein).
The presumptive region of the wing blade in the wing disc, the wing pouch, is subdivided in alternating vein and intervein territories. The veins correspond to four longitudinal stripes of cells where several genes, such as argos, veinlet and Delta, are expressed. These genes belong to the Egfr (argos and veinlet) and Notch (Delta) signaling pathways, and their activities are required for the formation of veins of appropriate thickness. The interveins are characterized by the expression of blistered (bs), the Drosophila homolog to the human serum responsive factor; bs activity prevents the formation of vein tissue. The expression of E(spl)m beta, a Notch-downstream gene, is present in the wing pouch in broad domains that correspond to most interveins, and it is excluded from the developing veins (de Celis, 2000 and references therein).
The formation of the veins progresses during pupal development, when the expression of the transcription factor ventral veinless is activated in all presumptive vein territories. At this stage, it is possible to distinguish three different domains of expression in each presumptive vein territory, consisting of a central stripe where Egfr signaling is active, and two adjacent stripes where Notch signaling is active. The cells where Egfr signaling is active will differentiate as vein tissue in the adult wing during normal development. The anterior and posterior lateral stripes can be visualized by the expression of the Notch-downstream gene E(spl)m beta and in these cells vein differentiation is prevented. When Notch signaling is compromised, most of these cells differentiate as vein, suggesting that each group of central and associated lateral stripes constitute a vein-competent region (de Celis, 2000 and references therein).
The expressions of Salm and Salr in the wing pouch occur in the same domain, and the Salm and Salr expression territory as Salm/Salr domain. This domain is related to the position of veins. Thus, the limit of detectable Salm protein in the posterior compartment is adjacent to the domain of E(spl)m beta expression and bs downregulation in the L5 territory, and therefore it is placed several cells anterior to the L5 vein. In the anterior compartment, the Salm/Salr expression domain includes a broad territory of low levels of E(spl)m beta expression where the levels of Salm/Salr protein are very low. This territory includes a region from where, based on the expression of other markers, the vein L2 differentiates. The L2 vein can also be recognized because of a subtle but consistent reduction in the levels of bs expression. In the center of the wing pouch high levels of Salm/Salr are detected in two stripes of cells that correspond to the veins L3 and L4, and lower levels are present anterior and posterior to these veins, respectively. In conclusion, the expression of salm/salr in the wing pouch includes the L2 vein and extends to the anterior edge of the L5 territory (de Celis, 2000).
The heterogeneity in the levels of Salm/Salr could have a functional significance during vein patterning, and suggests that other factors, in addition to Dpp, participate in the regulation of salm/salr in the wing pouch. The expressions of Kni (L2) and Iro (L3 and L5) are also related to developing vein regions. Iro proteins are localized in L3 and L5, and are present in the vein and in the associated stripes of E(spl)m beta expression, both during imaginal and pupal development. In the larval disc, Kni is expressed in a domain broader than the vein L2 that corresponds to the region where E(spl)m beta is expressed at low levels. These observations indicate that Iro and Kni are expressed in vein competent regions, and that the loss of L2 and L3/L5 veins in kni and iro mutants, respectively, is due to failures in the specification of the corresponding vein competent region (de Celis, 2000).
The spatial relationships between the distribution of Kni, Iro and Salm were examined directly using appropriate antibodies. Kni is expressed within the anterior edge of the Salm/Salr expression domain, in the region where Salm/Salr are detected at lower levels. This differs from a previous report that placed the limit of Salm expression adjacent to but not overlapping with kni expression. Iro expression in the L5 vein competent region is, in contrast, immediately adjacent to the posterior limit of Salm expression. Thus, each individual vein expresses a unique combination of transcription factors (vein L2: Kni-C + Salm/Salr; veinL3: Iro + Salm/Salr; vein L4: Salm/Salr; L5: Iro) that are required for its formation and could confer individual characteristics to each longitudinal vein (de Celis, 2000).
Large mitotic clones of cell homozygous for a deficiency including salm and salr result in reorganizations to the venation pattern that are manifest both within and outside the salm/salr domain of expression. To characterize more specifically the roles of salm/salr in vein patterning, the effects of small salm and salr double mutant clones on vein development were analyzed. The phenotype of these clones depends on their position in the wing blade. All such clones that span L2 result in the elimination of this vein. However, cells where salm but not salr is mutant form normal L2 tissue. In addition these salm;salr mutant clones can also differentiate ectopic stretches of L2 when they are localized posterior to the normal L2 vein. This result suggests that salm and salr are not equivalent in their roles in L2 development, salm being more effective in suppressing and salr in promoting L2 differentiation. Small salm/salr mutant clones localized between the veins L2 and L3 and between L4 and L5 cause autonomous formation of ectopic vein tissue, irrespective of the wing surface where the clones appear. The differentiation of L3 and L4 is not affected in these clones, but mutant cells close to either of these veins tend to contribute to the vein, resulting in the displacement of L3 and L4. salm mutant clones in the posterior compartment result in similar but weaker phenotypes, suggesting that both salm and salr antagonize some vein promoting factor/s in the L2/L3 and L4/L5 interveins. Clones of salm/salr in the anterior compartment can also differentiate ectopic sensilla characteristic of L3, indicating that these genes repress other attributes typical of the L3 territory. Finally, most salm/salr mutant clones localized in the region between L3 and L4 differentiate normal intervein tissue or displace the L3 vein, suggesting that other factors in addition to salm/salr antagonize vein formation here. A good candidate for a vein-suppressing factor in the L3/L4 intervein is the gene knot, which encodes a transcription factor that is specifically expressed in response to Hh signaling in the L3/L4 intervein (de Celis, 2000).
The contrasting effects of salm/salr on the formation of specific veins (promoting L2 and suppressing L3, L4 and L5) indicate that these genes could both stimulate and antagonize the expression or activity of other vein-promoting genes. Thus, there is no evidence to indicate that salm and salr regulate vein differentiation directly; rather, they appear to influence vein development indirectly through regulating the expression of other genes that define individual veins. This regulation would require low levels of Salm/Salr to promote L2 and higher levels of Salm/Salr to inhibit L3 and L5 development. Two good candidates to be regulated by Salm/Salr are the kni-C and iro-C, because they are expressed in L2 and L3/L5, respectively. Furthermore, the function of kni-C and iro-C is required for the formation of these veins (de Celis, 2000).
To characterize the relationships of salm/salr with kni and iro-C, the effects of salm/salr mutant clones on kni and iro expression were examined. The expression of kni is eliminated in salm/salr clones that overlap the domain of Kni expression. These effects are cell-autonomous and can be observed in very small salm/salr clones. This suggests that, in contrast to the non-autonomous effect of Sal on kni, Salm/Salr regulate kni expression in a cell-autonomous fashion. All salm/salr clones localized between veins L2/L3 and L4/L5 are associated with ectopic expression of Iro proteins. Again, this effect is strictly cell-autonomous, suggesting that the repression of iro-C genes by Salm/Salr could be direct. Ectopic expression of Iro in salm/salr mutant clones is not observed in regions close to the dorso-ventral boundary, presumably because in this region iro-C expression is repressed by wingless. The effects of salm/salr on kni-C and iro-C expression have also been analyzed in experiments in which salm and salr are expressed ectopically using the GAL4 system. Widespread expression of salm or salr in the wing blade eliminates L2 and L5, and prevents expression, respectively, of Kni and Iro in the L2 and L5 territories of the corresponding imaginal discs. The expression of Iro in L3, which depends on Hh activity, is not affected by removal salm/salr functions and only slightly decreased by their ectopic expression. Taken together, these observations indicate that Salm/Salr negatively regulates iro-C expression in cells not exposed to Hh protein, and suggests that precise levels of Salm/Salr proteins are needed to activate kni expression in L2 (de Celis, 2000).
Introns - three
Bases in 3' UTR - 1903
In Spalt are found three widely spaced "double zinc finger" motifs, which have internally conserved sequences; in addition, a single zinc finger motif of a different sequence is found in the central region. Each C-terminal zinc finger of the double zinc fingers contains a stretch of eight conserved amino acids with the sequence KTTKGNLK, similar to the zinc finger sequences of human transcription factor PRDII-BF1 (Kühnlein, 1994).
date revised: 1 Oct 2000
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