seven up

DEVELOPMENTAL BIOLOGY

Embryonic

seven-up is expressed in specific neuroblasts of the fly ventral nervous system. In the Drosophila CNS, early neuroblast formation and fate are controlled by the pair-rule class of segmentation genes. The distantly related Schistocerca (grasshopper) embryo has a similar arrangement of neuroblasts, despite lack of known pair-rule gene function. Four molecular markers have been used to compare Drosophila and Schistocerca neuroblast identity: seven-up, prospero, engrailed, and fushi-tarazu/Dax. In both insect species some early-forming neuroblasts share key features of neuroblast identity (position, time of formation, and temporally accurate gene expression). Thus different patterning mechanisms can generate similar neuroblast fates. In contrast, several later-forming neuroblasts show species-specific differences in position and/or gene expression. These neuroblast identities seem to have diverged, suggesting that evolution of the insect central nervous system can occur through changes in embryonic neuroblast identity (Broadus, 1995).

See Chris Doe's Hyper-Neuroblast map site for information on the expression of seven-up in specific neuroblasts.

For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.

The neuroblasts that give rise to the brain segregate from the procephalic neurectoderm and form three neuromeres: the protocerebrum (P), deuterocerebrum (D) and tritocerebrum (T). The first two neuromeres can each be further subdivided into three regions: the anterior, central and posterior protocerebral domains (Pa, Pc and Pp) and the anterior central and posterior deuterocerebral domains (Da, Dc and Dp). With respect to their position and the expression of the markers asense and seven-up, 23 small groups of neuroblasts consisting of from one to five neuroblasts per group have been identified. In Drosophila there are a total of 75-80 neuroblasts, 19 identified in Pa, 14 in Pc and 18 in Pp. There are 22 identified in the deuterocerebral domain and 6 in the tritocerebrum. The first seven groups of cells that segregate (Pc1 to 4, Dc1 to 3 and Dp1; collectively called SI/II) arise from the central domain of the protocerebral and deuterocerebral neurectoderm, respectively. Later groups form anterior and posterior to the earlier ones, leading to a centrifugal increase in the procephalic neuroblast population. SIII neuroblasts (Pa1 to 4, Pp1 and 2, and Dp2) arise during stage 10. SIV neuroblasts (Pa5 and 6, Pp3 and 4, Da1 and T1 and 2) arise during early stage 11, and SV neuroblasts (Pp5, Pdm) during stage 11 and early stage 12 (Younossi-Hartenstein, 1997).

Neuroblasts delaminate from the procephalic neurectoderm in a stereotyped spatiotemporal pattern that is tightly correlated with the expression of lethal of scute. The pattern of neuroblasts was reconstructed by using the marker asense; similar to its expression in the ventral neuroblasts, asense labels all brain neuroblasts. seven-up, expressed in specific subsets of neuroblasts making up approximately one-third of the total, is also used as a marker. For most, if not all, of these clusters the number of neuroblasts and the time of onset of svp expression are absolutely invariant. Neuroblast groups expressing svp are the following (number of cells in each group is given): Pa1 (1), Pa3 (2), Pa5 (2), Pc1 (1), Pc3 (4-6), Pp1 (1-2), Pp3 (3), Dc1 (1), Dc3 (5-6), Dp1 (2-3), and T1 (2) (Younossi-Hartenstein, 1996).

Seven-up function in heart development

The Drosophila heart is a simple organ composed of two major cell types: cardioblasts, which form the simple contractile tube of the heart, and pericardial cells, which flank the cardioblasts. A complete understanding of Drosophila heart development requires the identification of all cell types that comprise the heart and the elucidation of the cellular and genetic mechanisms that regulate the development of these cells. A new population of heart cells is reported here: the Odd skipped-positive pericardial cells (Odd-pericardial cells). Descriptive, lineage tracing and genetic assays were used to clarify the cellular and genetic mechanisms that control the development of Odd-pericardial cells. Odd skipped marks a population of four pericardial cells per hemisegment that are distinct from previously identified heart cells. Within a hemisegment, Odd-pericardial cells develop from three heart progenitors and these heart progenitors arise in multiple anteroposterior locations within the dorsal mesoderm. Two of these progenitors divide asymmetrically such that each produces a two-cell mixed-lineage clone of one Odd-pericardial cell and one cardioblast. The third progenitor divides symmetrically to produce two Odd-pericardial cells. All remaining cardioblasts in a hemisegment arise from two cardioblast progenitors, each of which produces two cardioblasts. Furthermore, numb and sanpodo mediate the asymmetric divisions of the two mixed-lineage heart progenitors noted above (Ward, 2000).

An enhancer trap in the seven-up gene identifies the two mixed-lineage heart progenitors. Towards the end of the lineage analyses it was discovered fortuitously that an enhancer trap in the gene seven-up labels four heart cells in each abdominal hemisegment. This enhancer trap is referred to as svp-lacZ). Two of these cells reside at the dorsal midline and are cardioblasts since they express Mef2. The other two cells reside just lateral and slightly ventral and anterior to the svp-lacZ cardioblasts. These two cells are Odd-pericardial cells because they express Odd. The relative positioning of the svp-lacZ cardioblasts and Odd-pericardial cells closely resembles that of the sibling cardioblasts and Odd-pericardial cells marked by the mixed lineage heart clones. This suggests that the svp-lacZ heart cells may identify the four progeny of the two mixed lineage heart progenitors that arise in each hemisegment. If the four svp-lacZ heart cells are the progeny of these two progenitors, then loss of sanpodo function should convert all svp-lacZ heart cells to cardioblasts and loss of numb function should convert all svp-lacZ heart cells to Odd-pericardial cells. In sanpodo mutant embryos, all four svp-lacZ cells acquire the cardioblast fate and in numb mutant embryos all four svp-lacZ cells acquire the Odd-pericardial cell fate. The results from these experiments demonstrate that svp-lacZ identifies the progeny of the two mixed lineage heart progenitors and that numb and sanpodo mediate the asymmetric divisions of these mixed-lineage heart progenitors (Ward, 2000).

Present models of heart development suggest that all heart cells arise from dorsal mesodermal cells that reside beneath the transverse stripe of ectodermal cells that express Wg protein. However, Odd-pericardial cells are first detect emerging roughly midway between Eve-pericardial cells, which themselves arise beneath the Wg-expressing ectodermal cells. The initial appearance of Odd expression in pericardial cells at the end of stage 12 precludes its use as a definitive marker of the embryonic origin of these cells. However, since svp-lacZ labels all four progeny of the two mixed lineage heart progenitors an assay was carried out to see whether svp-lacZ is expressed in the mixed lineage heart progenitors. If so, svp-lacZ could be used as a marker to identify the AP origin of mixed lineage heart progenitors (Ward, 2000).

svp-lacZ is first detected in the dorsal mesoderm during stage 11 in two individual cells: one is located beneath the ectodermal Odd stripe midway between Wg-stripes; the other is found immediately anterior to, or just at the anterior edge of, Wg-expressing cells. During early stage 12 these two cells divide and produce four cells, all of which express svp-lacZ and Mef2; at this stage none of these cells express Odd. During stage 12 the four svp-lacZ heart cells congregate together to form a tight four-cell cluster. Using confocal microscopy, it was found that by stage 13, the four svp-lacZ-positive cells could be broken into two groups based on Mef2 expression: two cells express Mef2 at high levels and two cells express Mef2 at low levels. Because cardioblasts retain and Odd-pericardial cells extinguish Mef2 expression, the svp-lacZ heart cells with high-level Mef2 expression were identified as cardioblasts and those with low Mef2 expression as Odd-pericardial cells. These results suggest that the two svp-lacZ heart progenitors arise from two different AP locations in the dorsal mesoderm, at least one of which does not arise from dorsal mesodermal cells located beneath the ectodermal wg stripe, the postulated source of all heart cells. The svp-lacZ molecular marker also allows for distinguishing between svp-lacZ/Odd-pericardial cells and the Odd-pericardial progenitor and its progeny. This is facilitated the identification of the location of the Odd-pericardial progenitor just prior to its division. Odd-expression was first detected in the Odd-pericardial progenitor at stage 12/0. The Odd-pericardial progenitor is located beneath the ectodermal Odd-stripe and divides shortly after stage 12/0 to produce the remaining two Odd-pericardial cells per hemisegment. The Odd-progenitor and its progeny reside adjacent and anterior to the Odd/svp-lacZ-pericardial cells. These results suggest that the Odd-pericardial progenitor is specified from dorsal mesodermal cells located beneath the Odd ectodermal stripe. However, extensive mesodermal rearrangements occur prior to stage 12/0 (Ward, 2000).

The Drosophila dorsal vessel is a linear organ that pumps blood through the body. Blood enters the dorsal vessel in a posterior chamber termed the heart, and is pumped in an anterior direction through a region of the dorsal vessel termed the aorta. The dorsal vessel spans segments thoracic 2 (T2) to abdominal 8 (A8). From T2 to A5 the tube is narrow and is termed the aorta, whereas the posterior portion has a larger bore and is termed the heart. Additionally the heart is perforated by three pairs of valve-like ostia, which serve as inflow tracts for hemolymph. Although the genes that specify dorsal vessel cell fate are well understood, there is still much to be learned concerning how cell fate in this linear tube is determined in an anteroposterior manner, either in Drosophila or in any other animal. The formation of a morphologically and molecularly distinct heart depends crucially upon the homeotic segmentation gene abdominal-A (abd-A). abd-A expression in the dorsal vessel is detected only in the heart, and overexpression of abd-A induced heart fate in the aorta in a cell-autonomous manner. Mutation of abd-A results in a loss of heart-specific markers. abd-A and seven-up co-expression in cardial cells defines the location of ostia, or inflow tracts. Other genes of the Bithorax Complex do not appear to participate in heart specification, although high level expression of Ultrabithorax is capable of inducing a partial heart fate in the aorta. These findings demonstrate a specific involvement for Hox genes in patterning the muscular circulatory system, and suggest a mechanism of broad relevance for animal heart patterning (Lovato, 2002).

A unique characteristic of the Drosophila heart is the presence of inflow tracts, termed 'ostia'. There are three pairs of ostia located at the segmental boundaries of A5/A6, A6/A7 and A7/A8, and each ostium is visible in larvae as a broadening of the width of the heart, at the peak of which are small openings. No ostia form in the aorta during embryonic or larval development. The ostia, which form at each segmental boundary, develop from two pairs of cells expressing the orphan nuclear receptor gene seven-up (svp). The remaining four pairs of cardial cells in each segment express the homeobox-containing gene tinman (tin) and form the heart wall (Lovato, 2002).

Close examination of MHC-stained wild-type hearts from embryos indicated that the wall of the heart curved sharply outwards close to the segment borders, whereas no such broadening occurred in the aorta. At these locations, two cardial cells are morphologically distinct in that they have oval-shaped nuclei, rather than the round nuclei of the remaining cells. Given the locations of these morphologically distinct cells close to the segmental boundary and the similarity of this structure to the organization of the larval heart, it was reasoned that the sharp curves in the outer heart wall corresponded to the locations of the ostia. In support of this, indentations were occasionally seen at the tip of these cell pairs, suggesting that the heart wall is perforated at these locations. To confirm that these cells are ostia, wild-type embryos were double-stained with an antibody to Tin (to identify the heart wall cell nuclei) and with an antibody to muscle MHC (to visualize the shape of the heart). The sharp curves in the heart wall corresponded to the locations of ostia, since ostia are formed by the non-Tin expressing population of cardial cells. In the aorta of wild-type embryos, svp-expressing cells are still detected; however, the wall of the aorta is uniform (Lovato, 2002).

Since ectopic mesodermal expression of abd-A results in ectopic heart formation, these ectopic heart structures were studied for the presence of cells forming ostia. In many cases, sharp curves in the wall of the heart tube in locations more anterior to those found in wild type indicated the presence of ectopic ostia, formed by cells more elongated than their neighbors. Furthermore, by staining these embryos with anti-Tin and anti-MHC, as was done for wild type, these elongated cells were found to precisely correspond to those expressing svp. Although it is difficult to visualize the openings of the ostia, the most likely conclusion from these observations is that ectopic ostia are formed in the presence of ectopic Abd-A. Furthermore, these ostia are positioned appropriately within the segment, only at the coincidence of abd-A expression and svp expression (Lovato, 2002).

To quantify more precisely the alteration in Svp cell morphology upon the induction of ectopic heart structures, the size of each svp-expressing cell was determined by measuring the distance from the luminal surface of the Svp cells to the outer wall of the dorsal vessel. In wild-type embryos there are seven segmentally repeating groups of Svp cardial cells in the dorsal vessel, four cells in each group. To distinguish between groups located at unique positions along the AP axis, the groups are referred to as S1 to S7, from anterior to posterior in the embryo. Thus, the Svp cells of clusters S1 to S4 do not form ostia in wild type, whereas S5 to S7 form the ostia of the heart (Lovato, 2002).

In control embryos, clusters S1 to S4 contained cells measuring approximately 5 µm, whereas the Svp cells of the heart were significantly larger (7-8 µm). Upon overexpression of abd-A in the mesoderm there was a large increase in the sizes of cells in groups S1 to S4, many of which were indistinguishable from those in the wild-type heart. These results clearly show the effects of abd-A expression upon aorta cell fate, transforming Svp cells of the aorta into ostia (Lovato, 2002).

The results of this study also indicate that two distinct patterns of gene expression converge to control the differentiation of the Drosophila dorsal vessel. Superimposed upon the expression of abd-A in the heart segments, is the pattern of tin-expressing versus svp-expressing cells observed in cardial cells in every segment. Formation of the ostia in the heart occurs only at the intersection of abd-A expression and svp expression, and ectopic ostia form in the presence of ectopic AbdA, but only in svp-expressing populations of cells (Lovato, 2002).

Whether svp function is required for ostium formation in Drosophila remains to be determined. A vertebrate homolog of the Svp protein is chick ovalbumin upstream promoter transcription factor II (COUP-TF II), which in mice is expressed in and is required for the formation of the atria and sinus venosus. The atria and sinus venosus carry out functions in the mouse analogous to the ostia in Drosophila, acting as the inflow tracts for blood to enter the heart. It will be interesting to determine whether the homologous expression patterns of svp and COUP-TF II reflect a homologous function in development (Lovato, 2002).

The embryonic dorsal vessel in Drosophila possesses anteroposterior polarity and is subdivided into two chamber-like portions, the aorta in the anterior and the heart in the posterior. The heart portion features a wider bore as compared with the aorta and develops inflow valves (ostia) that allow the pumping of hemolymph from posterior toward the anterior. Homeotic selector genes provide positional information that determines the anteroposterior subdivision of the dorsal vessel. Antennapedia (Antp), Ultrabithorax (Ubx), abdominal-A (abd-A), and Abdominal-B (Abd-B) are expressed in distinct domains along the anteroposterior axis within the dorsal vessel, and, in particular, the domain of abd-A expression in cardioblasts and pericardial cells coincides with the heart portion. Evidence is provided that loss of abd-A function causes a transformation of the heart into aorta, whereas ectopic expression of abd-A in more anterior cardioblasts causes the aorta to assume heart-like features. These observations suggest that the spatially restricted expression and activity of abd-A determine heart identities in cells of the posterior portion of the dorsal vessel. Abd-B, which at earlier stages is expressed posteriorly to the cardiogenic mesoderm, represses cardiogenesis. In light of the developmental and morphological similarities between the Drosophila dorsal vessel and the primitive heart tube in early vertebrate embryos, these data suggest that Hox genes may also provide important anteroposterior cues during chamber specification in the developing vertebrate heart (Lo, 2002).

Since abd-A expression coincides with the heart portion of the dorsal vessel, tests were made to see whether it acts to specify the cardioblasts in which it is expressed to eventually form the heart. In order to distinguish aorta cardioblasts from heart cardioblasts, two different molecular markers were utilized. The first marker was the pattern of ß-Gal derived from the tinCdelta5-lacZ transgene, where the expression of a lacZ gene is controlled by an internally deleted tinman cardiac enhancer element, tinCdelta5. This element drives ß-Gal expression in all the cardioblasts of the aorta, whereas in the heart it is only expressed in three segmentally-spaced double pairs of cardioblasts. These particular cardioblasts correspond to the svp cardioblasts of the heart. The second marker is wingless (wg), which is expressed in these same three double pairs of svp cardioblasts within the heart of the late embryonic dorsal vessel (Lo, 2002).

In abd-A null mutant embryos, the pattern of tinCdelta5-lacZ-derived ß-Gal is continuous in the heart as well as in the aorta of the dorsal vessel. In addition, it appears that the width of the heart is now the same as that of the aorta when compared with a wildtype embryonic dorsal vessel. Similarly, the late expression of Wg in the svp cardioblasts of the heart is not detectable in these mutant embryos. The alterations in the pattern of these two markers strongly suggest that heart cardioblasts have not been specified in the posterior of the dorsal vessel of abd-A null mutant embryos and that these posterior cardioblasts have been transformed instead into aorta cardioblasts. This would indicate that abd-A is necessary for the specification of heart cardioblasts in the posterior portion of the dorsal vessel where it is normally expressed (Lo, 2002).

The pattern of Wg expression in three segmentally repeated double pairs of cardioblasts within the late stage heart is strongly reminiscent of the pattern of svp expression, which suggests that these are the heart svp cardioblasts. Double antibody staining for Wg and ß-Gal in the dorsal vessel of svp-lacZ embryos clearly confirmed that the heart svp cardioblasts express the Wg protein. Since the heart svp cardioblasts eventually form the ostia (inflow valves) of the larval heart and since Wg is a developmentally significant signaling molecule, the regulation of Wg expression in the heart svp cardioblasts during late embryogenesis was more closely examined (Lo, 2002).

Wg expression in heart cardioblasts is dependent on abd-A. Since these Wg-expressing cardioblasts correspond to svp cardioblasts, whether Wg expression is also dependent on svp function was also tested. In homozygous null svp^AE127 mutant embryos, there is no detectable Wg expression in the heart cardioblasts that are marked by svp-lacZ. Therefore, the Wg expression seen in the heart svp cardioblasts of late embryonic dorsal vessels requires both abd-A and svp function. Accordingly, ectopic expression of SvpI in the cardioblasts of the entire dorsal vessel results in wg expression in all cardioblasts of the heart, and ectopic expression of both SvpI and Abd-A in the whole dorsal vessel causes Wg expression in the majority of the cardioblasts of the entire dorsal vessel. These results demonstrate that the combination of abd-A and svp is both necessary and sufficient to activate wg expression in cardioblasts during late dorsal vessel development (Lo, 2002).

Since Wg expression in heart svp cardioblasts initiates toward the end of embryogenesis (stage 16), tests were made to see whether this expression could also be detected in the dorsal vessel during later larval stages when the corresponding cells have formed the ostia. Because of high levels of unspecific background staining with Wg antibodies in larval preparations, wg expression in the dorsal vessel of third instar larvae was indirectly monitored by anti-ß-Gal staining of dissected wg-lacZ animals. Moderate levels of wg-lacZ-derived ß-Gal can indeed be detected in the ostia of the heart, although stronger levels are now present in four separated patches in the aorta that correspond to Tin-negative svp cardioblasts. While this pattern of expression differs from that seen in the late embryonic dorsal vessel, it is clear that wg is expressed differentially and in a temporally regulated manner within the heart and aorta, respectively, of late stage embryos and third instar larvae. These observations suggest a yet undefined role for the signaling molecule in larval dorsal vessel development and/or functioning (Lo, 2002).

The target genes of abd-A that are required for generating functional ostia and for the other heart cells to adopt their characteristic morphology are not yet known. Based on its ostia-specific expression in late stage embryos, wg is a candidate target of abd-A that may function either in an autocrine fashion during ostia differentiation or in a paracrine fashion during the differentiation of the adjacent heart cardioblasts. The activation of the wg gene in the svp cells of the aorta during third instar also precedes ostia formation, in this case of the adult ostia, from these cells. Hence, there is a strong correlation between the initiation of wg expression in svp cardioblasts and their subsequent differentiation into functional ostia (Lo, 2002).

Larval

Ubiquitous expression of seven-up causes transformation of the eye photoreceptor precursor R7 cell to an R1-R6 cell fate. In addition, depending on the timing and spatial pattern of expression, various other phenotypes are produced including the loss of the R7 cell or the formation of extra R7 cells: ubiquitous expression of seven-up close to the morphogenetic furrow interferes with R8 differentiation, resulting in failure to express BOSS protein (the ligand for the sevenless receptor tyrosine kinase), and as a consequence, the R7 cell is lost. Extra R7 cells are formed by recruiting non-neuronal cone cells as photoreceptor neurons independent of sevenless and bride of sevenlessexpression. Thus, the spatiotemporal pattern of seven-up expression plays an essential role in controlling the number and cellular origin of the R7 neuron in the ommatidium. These results also suggest that seven-up controls decisions not only between photoreceptor subtypes, but also between neuronal and non-neuronal fates (Hiromi, 1993).

Expression of seven-up is confined to four of the eight photoreceptor precursor cells: those that will form the R3/R4 and R1/R6 pairs. Misexpression of seven-up in any of the other cell types within the developing ommatidium interferes with their differentiation. Each cell type responds differently to seven-up misexpression. For example, ectopic expression in the non-neuronal cone cells causes the cone cells to take on a neuronal identity. Ectopic expression in R2/R5 causes these neurons to lose aspects of their photoreceptor subtype identity while remaining neuronal. Each cell type appears to have a different developmental time window that is sensitive to misexpressed seven-up. Members of the ras signaling pathway act as suppressors of the cone cell to R7 neuron transformation caused by the double mutation sevenless-seven-up. Suppression of the sev-svp phenotype can be achieved by decreasing the gene-dosage of any of the members of the ras-pathway. This suggests that the function of seven-up in the cone cells requires ras signaling (Kramer, 1995).

The expression patterns of sevenless and seven-up overlap in R3 and R4, the two photoreceptors that in addition to R7 express sevenless at high levels (Mlodzik, 1990).

Mutation of roughex perturbs cell fate determination. Many rux mutant clusters contain multiple boss-expressing cells. In some of these clusters, R8 cells are missing. There is also a reduction in the number of cells expressing bar and Seven-up. This may be due to errors in cell fate determination. Alternatively, the reduced number of cells expressing these markers may reflect cell death. Extensive cell death is seen in rux mutants beginning with the MF and extending to the posterior edge of the disc. In rux mutant discs, neuronal differentiation is delayed by approximately 6 hours of development (Thomas, 1994).

Combinatorial expression of Prospero, Seven-up, and Elav identifies progenitor cell types during sense-organ differentiation in the Drosophila antenna in the pupa

The Drosophila antenna has a diversity of chemosensory organs within a single epidermal field. Three broad categories of sense-organs are known to be specified at the level of progenitor choice. However, little is known about how cell fates within single sense-organs are specified. Selection of individual primary olfactory progenitors is followed by organization of groups of secondary progenitors, which divide in a specific order to form a differentiated sensillum. The combinatorial expression of Prospero, Elav, and Seven-up shows that there are three secondary progenitor fates. The lineages of these cells have been established by clonal analysis and marker distribution following mitosis. High Notch signaling and the exclusion of these markers identifies PIIa; this cell gives rise to the shaft and socket. The sheath/neuron lineage progenitor PIIb, expresses all three markers; upon division, Prospero asymmetrically segregates to the sheath cell. In the coeloconica, PIIb undergoes an additional division to produce glia. PIIc is present in multiinnervated sense-organs and divides to form neurons. An understanding of the lineage and development of olfactory sense-organs provides a handle for the analysis of how olfactory neurons acquire distinct terminal fates (Sen, 2003).

Development of single sensory units have been traced by using enhancer-trap insertions into the neurilized genes (neu^A101 and neu-Gal4). Olfactory progenitor cells delaminate from the epithelium as single isolated cells with apically located nuclei and are arranged in distinct domains in the early antennal disc. By 8 h APF, these progenitors begin association with one to three additional cells forming well-defined clusters. These cell clusters do not arise by division of the olfactory progenitor since the first evidence of cell division as seen by phosophohistone-3 (PH3) immunoreactivity is after 12 h APF. The cells within the cluster are referred to as secondary progenitors, since their division gives rise to all the cells of an individual sense-organ. Most of the clusters divide between 16 and 22 h APF (Sen, 2003).

This analysis was restricted to clusters of secondary progenitors composed of three cells, although two and four cell clusters can also be identified by expression of GFP driven by neu-Gal4 (henceforth referred to as Neu-GFP). At 14 h APF, clusters are oriented in a single plane and have not yet begun cell division. Expression of Pros and Elav was examined by using specific antibodies, while Svp was monitored by following ß-galactosidase activity in the enhancer-trap line svp^P1725. None of these markers express in primary olfactory progenitor cells but appear shortly after formation of groups of secondary progenitors. Double-labeling of 14-h APF discs with anti-Pros and anti-Elav reveals that two of the three cells within a cluster express both of these markers. Pros expression appears prior to that of Elav within the same cell. One of these cells also expresses the Svp reporter (henceforth called Svp-lacZ). The combinatorial expression of genes allowed identification of three progenitor types. PIIa does not express any of the markers and is recognized only by expression of Neu-GFP; PIIb expresses Neu-GFP, Pros, Elav, and SvplacZ, while PIIc expresses Neu-GFP, Pros, and Elav. Clusters composed of only two cells lack the PIIc progenitor and those with four cells contain two PIIc progenitors. Hence, differential expression of genes could provide cells within a single cluster the potential to exhibit independent fates (Sen, 2003).

The distribution of Pros, Elav, and SvplacZ was examined during division of the secondary progenitors. Staining with phenylene-diamine allowed identification of interphase nuclei, while entry and exit from mitosis was monitored by changes in Neu-GFP distribution. During mitosis, only one cell per cluster exhibits asymmetric cortical Pros crescents. The neighboring cell shows either compact or a uniform cytosolic localization. The failure to observe two cortical Pros crescents per cluster even in colcemid-arrested discs suggested either that PIIb and PIIc divide at different times or that Pros is asymmetrically segregated in only one of these cells (Sen, 2003).

By 36 h APF, postmitotic cells of the sensory units occupy positions comparable to those in the adult; the shaft and socket cells are identifiable by their external cuticular projections. Pros is present in sheath and socket cells, while Elav is exclusively neuronal. Clonal experiments have shown that the sheath cell arises from PIIb lineage possibly inheriting Pros asymmetrically from the progenitor. The socket, however, is derived from PIIa, which does not express Pros, indicating de novo synthesis (Sen, 2003).

The majority of peripheral antennal glia arise during development of the coeloconic sensilla. PIIb has been identified as the glial progenitor in a number of gliogenic sense-organs. In the olfactory sense-organs, PIIb can be unequivocally recognized by expression of Pros, Elav, and Svp-lacZ (Sen, 2003).

Clusters in the region of the antenna populated by coleoconic sense-organs were selected for detailed analysis. PIIb divides to produce a large cell that remains within the epithelial layer and a smaller basal cell. The basal cell transiently expresses Pros and low levels of the Svp reporter and also stains with antibodies against the glial cell marker Reverse Polarity. The nascent glial cell loses Pros and Svp expression and rapidly migrates away to become associated with the fasiculating sensory neurons (Sen, 2003).

The gliogenic lineage described above occurs only within the coeloconica sensilla (i.e., ~70 out of 450 sensilla). PIIIb, like PIIb in all other clusters, expresses Pros, Svp, and Elav. Mitosis of all secondary progenitors is completed by 22 h APF, and marker distribution in progeny was examined at 25 h APF. At this time, sensory cells orient along the apicobasal axis resembling positions in the mature sensillum (Sen, 2003).

Pros expression is detected in two subepidermally located accessory cells identified as sheath and socket. Upon division of PIIb, Svp-lacZ is distributed equally to both progeny. One of these is the sheath, which also expresses Pros, while the other stains with the neuron-specific antibody mAb22C10. The expression of ß-galactosidase fades from the sheath cell and is not apparent by 36 h APF. The perdurance of the reporter thus allows for the identification of sheath and neuron as siblings derived from PIIb (Sen, 2003).

The PIIa lineage differs from PIIb by the absence of both Pros and Svp. Misexpression of Pros or Svp using sca-Gal4P309 results in a striking reduction in external cuticular structures on the antennal surface. This is consistent with a lack of PIIa identity. In order to test whether ectopic expression of Pros or Svp could convert PIIa to PIIb/c, 36-h APF antennae were stained with anti-Repo and mAb22C10. The numbers of glia or neurons were not altered. Coexpression of both Pros and Svp was achieved by heat-pulsing P(hs-Pros)/P(hs-Svp) pupae at 32°C for 6 h starting at 10 h APF. This did not result in an alteration in numbers of glia or neurons, although the antennae show a reduction in the numbers of external cuticular structures (Sen, 2003).

These data suggest that, while ectopic expression of either Pros or Svp interfere with PIIa fate, this is insufficient to convert cells to a PIIb identity. This means that N determines secondary progenitors through mechanisms other than and/or in addition to regulating the expression of Pros and Svp (Sen, 2003).

Effects of Mutation or Deletion

The absence of svp function causes a transformation of these cells toward an R7 cell fate, as judged by morphology and expression of an R7-specific marker. This transformation depends in part on the Sevenless gene product (Mlodzik, 1990).

The planar polarity of Drosophila ommatidia is reflected in the mirror-symmetric arrangement of ommatidia relative to the dorso-ventral midline, the equator. This arrangement is generated when ommatidia rotate towards the equator and the photoreceptor R3 displaces R4, creating different chiral forms in each half. Analysis of ommatidia that are mosaic for the tissue polarity gene frizzled (fz) shows that the presence of a single Fz+ photoreceptor cell within the R3/ R4 pair is critical for the direction of rotation and chirality. By analysing clones mutant for seven-up (svp) in which R3/R4 precursors reside in their normal positions and become photoreceptor neurones but fail to adopt the normal R3/R4 fate, it has been found that the R3/R4 photoreceptor subtype specification is a prerequisite for planar polarization in the eye. In mosaic R3/R4 pairs the svp- cell always adopts the R4 position. This bias is reminiscent of what happens in fz mosaic R3/R4 pairs, where the fz- cell also almost always adopts the R4 position. A possible interpretation of the data is that the svp mutant cell is not able to receive the polarity signal or to interpret it (or to communicate with the other cell of the R3/R4 pair). Mutations in the rough gene cause the mis-specification of R2 and R5 and their transformation to an R3/4 pair as seen by their expression of svp and their dependence on svp to develop as outer photoreceptors. In genotypes where too many cells adopt the R3/R4 fate, ommatidial polarity is also disturbed. This defect could arise because, in a situation with too many R3/R4 cells within a cluster, there is crosstalk/competition for the R3 fate between more than two cells that confuses the cluster as a whole. Taken together, these data imply that correct specification of a single R3 cell per ommatidium is a prerequisite for the normal interpretation of the Fz-mediated polarity signal (Fanto, 1998).

The pattern of connections between R1-R6 neurons and their targets in the lamina is one of the most extraordinary examples of connection specificity known. An interwoven set of connections precisely maps R cells in different ommatidia (that 'see' the same point in space) onto the same group of postsynaptic cells, the lamina cartridge. R1-R6 cells that see the same point in space are distributed over six neighboring ommatidia as a consequence of the curvature of the eye and the angular placement of their light-sensing organelles. Conversely, each of the R1-R6 axons from a single ommatidium sees a different point in space and connects to a different set of lamina target neurons arranged in an invariant pattern. Each cartridge is innervated by a complete set of R1-R6 neurons from six different ommatidia (i.e., an R1 from one ommatidium, an R2 from another, and so on). By superimposing multiple inputs from the same point in visual space upon a single synaptic unit, the signal-to-noise ratio of the response to a signal in the visual field is enhanced. This phenomenon is called neural superposition (Clandinin, 2000 and references therein).

The R1-R6 projection pattern develops in two temporally distinct stages. During the third larval stage, R cells extend axons into the brain, where they terminate between two layers of glia, forming the lamina plexus. These glia act as intermediate targets for R1-R6 neurons. R cell axons induce the differentiation and organization of lamina target neurons and glia. At this stage of development, R cell axons from the same ommatidium form a single fascicle. A column of lamina neurons forms above the lamina plexus, in tight association with a single R cell axon fascicle. By the sequential addition of ommatidial bundles and their associated columns of lamina neurons, a precise retinotopic map forms in which fascicles from neighboring ommatidia terminate adjacent to each other. As lamina neurons differentiate, they send axons along the surface of R cell axons through the plexus and fasciculate with R7 and R8 as they project into the medulla. Although lamina neurons are in close association with R cell axons at this early stage, no synaptic contacts are formed (Clandinin, 2000).

In the second phase of development, ~30 hr after reaching the lamina plexus, R cell axons defasciculate from each ommatidial bundle and project across the surface of the lamina to their synaptic partners, making the pattern of connections characteristic of neural superposition. Growth of R cell axons toward their targets occurs approximately simultaneously in all ommatidial bundles and is presaged by an invariant sequence of contacts between R cell growth cones. This reorganization of terminals converts a strictly anatomical retinotopic map that reflects neighbor relationships between ommatidia into a new topographic map that reflects R cell visual response and reconstructs visual space in the first layer of the optic ganglion (Clandinin, 2000 and references therein).

R cell projections from a single ommatidium display two prominent features. (1) Each R cell axon terminates in an invariant position relative to the other axons from the same ommatidial fascicle. (2) The projection is oriented with respect to the dorsoventral midline of the eye (i.e., the equator), with the R3 axon extending toward the equator -- as a result, the projection patterns on opposite sides of the dorsoventral midline of the eye are mirror images. Using mutations that eliminate specific subsets of R cells or alter ommatidial polarity, tests were performed to see whether R cell synaptic specificity requires interactions among neighboring afferent axons or reflects independent navigation of each axon to its target. It has been demonstrated that interactions between specific R cells are required for target selection, and it is proposed that the precise composition of R cell axons within a fascicle plays a critical role in target specificity (Clandinin, 2000).

Neural superposition was first noted 90 years ago and the R1-R6 connection pattern in the lamina was first described using serial reconstruction of electron microscopic images in 1965. This pattern is cited as a classic example of extreme connection specificity. However, mechanistic analysis of this pattern was prevented by the absence of a rapid method for assessing R cell projections. In particular, the complexity of the pattern precludes conventional approaches based on visualizing all R cell axons in the target region, yet the assessment of connection specificity requires visualization of all R cell axons from one ommatidium. A method has been developed to label individual ommatidia with DiI and visualize the projection pattern using confocal microscopy. R1-R6 axons form a single bundle as they project into the brain. They defasciculate, project across the surface of the lamina, and then turn 90° and extend into the lamina cartridge. R cell axons elaborate a complex en passant presynaptic structure with lamina interneurons within the lamina cartridge. The axons of R7 and R8 project through the lamina, into the medulla. The relative positions of lamina targets chosen by each R1-R6 growth cone are invariant between ommatidia. This labeling method facilitates analysis of R1-R6 projections in various genetic backgrounds and creates a unique experimental system in which synaptic partner choices made by identified neurons can be directly assessed (Clandinin, 2000).

Serial electron microscopic reconstruction studies have revealed that, during pupal development, individual R cell axons leave their original bundle and migrate outward, in the precise direction of their final targets. This process was visualized using confocal microscopy. Early in pupal development, each ommatidial bundle forms a compact mass of expanded growth cones in the lamina plexus. This spherical mass then flattens, as distinct filopodial extensions corresponding to individual R cell axons become visible. This pattern of connections forms within a spatially patterned environment containing lamina target neurons and glial cells, as well as R cell axons. Since extension from the bundle is not preceded by extensive filopodial exploration, interactions between axons within ommatidial bundles may specify the initial trajectory of each growth cone. To address whether cell intrinsic mechanisms or interactions between R cell growth cones or both control target specificity, R cell projections were examined in mutant animals lacking specific subsets of R1-R6 cells. R cell axons from single ommatidia were labeled with DiI and visualized by confocal microscopy. In this series of experiments, animals were analyzed in which the eye was genetically mutant and the lamina neurons and glia in the target were wild type. Three mutant backgrounds were examined: (1) phyllopod, in which R1, R6, and R7 are transformed into nonneuronal cone cells; (2) lozenge^sprite, in which R3 and R4 are transformed into R7 cells; and (3) seven-up, in which R1, R3, R4, and R6 are transformed into R7 cells (Clandinin, 2000).

The first step of lamina target innervation is the coordinated defasciculation of R cell axons from bundles comprising axons from the same ommatidium. To determine whether interactions between specific subsets of R1-R6 axons are necessary for this defasciculation, R cell projections were assessed in phyllopod, seven-up, and lozenge^sprite mutants. In all three of the R cell transformation mutants examined, R cell axons migrated outward from the bundle. In particular, 4 R cell fibers in the lamina of 14/15 phyllopod mutant animals (missing R1, R6, and R7) and 20/24 lozenge^sprite mutants (missing R3 and R4) defasciculated from the bundle and projected to local targets. Similarly, in 17/23 seven-up mutants (missing R1, R3, R4, and R6), it was observed that the two remaining R cell axons defasciculated from the ommatidial bundle and innervated separate cartridges. In some cases, additional R cell axons also defasciculated, consistent with the reported incomplete expressivity of cell fate transformations in these mutants. In each case, axons projected to lamina targets in the local environment of the fascicle terminus. It is concluded that each R cell subtype is programmed to initiate a search for targets in a local region of the lamina target, independent of interactions between other R cell subtypes. In the following sections, whether interactions between specific R1-R6 cells regulate target specificity is assessed (Clandinin, 2000).

In phyllopod mutants, R1, R6, and R7 are transformed into nonneuronal cone cells. The remaining R cells made normal projections: a single long projection corresponding to R3 was observed, and R2, R4, and R5 made projections of appropriate relative lengths compared to wild type. In 1 of these 15 phyllopod mutant ommatidia, an additional short R cell projection was also observed, consistent with an incomplete cell fate transformation of either R1 or R6. In phyllopod mutant animals, the pattern of targets chosen by R3 and R4 were invariably normal, while those chosen by R2 and R5 were usually correct. In 4/15 animals, R2 and R5 made projections of the appropriate length, but the targets they chose were misoriented with respect to the equator. Therefore, R3 and R4 do not require R1, R6, and R7 to target correctly, while in some cases R2 and R5 are affected by their loss. These effects are not caused by the loss of R7; a sevenless mutation that specifically eliminates R7 has no effect on R cell targeting in the lamina (Clandinin, 2000).

A gain-of-function mutation, lozenge^sprite, which transforms R3 and R4 into R7 cells was examined. In this mutant, the Lozenge gene product is ectopically expressed in R3 and R4. In such mutant animals, ~73% of ommatidia have both R3 and R4 transformed into R7 cells; in most of the remaining ommatidia (20% of the total), only R4 is transformed; the remaining ommatidia are missing one R cell. Since the reduction in the number of R cells projecting to specific cartridges roughly corresponds to the fraction of R3 and R4 cells transformed into R7, it is presumed that transformation is complete in ommatidia where four fibers are observed in the lamina. In cases in which five R cell axons are observed, it is inferred that R4 but not R3 was transformed into R7. In 20/24 lozenge^sprite ommatidia injected, four R cell projections were observed in the lamina, with R1, R2, R5, and R6 all making projections of appropriate length, while transformed R3 and R4 cells projected through the lamina into the medulla. In the remaining 4/24 cases, five projections were seen in the lamina, one of which was a long projection characteristic of R3. In completely transformed lozenge^sprite ommatidia, the relative positions of the targets chosen by R1, R2, R5, and R6 were frequently highly aberrant. In the remaining 9/20 fully transformed lozenge^sprite animals, the pattern of targets chosen was not grossly distorted, though minor irregularities were seen. The effects seen in lozenge^sprite do not result from defects in ommatidial orientation: ommatidia are normally oriented in this mutant (Clandinin, 2000).

In seven-up mutants, R1, R3, R4, and R6 are frequently transformed into R7, while R2 and R5 are unaffected. Moreover, the transformation of individual seven-up ommatidia is variable and complex, making detailed reconstruction of many ommatidia impossible. However, in the majority of seven-up ommatidia (17/23), two short R cell projections, characteristic of R2 and R5, are seen in the lamina, while the transformed R1, R3, R4, and R6 cells project into the medulla (as R7 cells normally do). The targets chosen by the presumptive R2 and R5 were invariably misoriented with respect to the equator. In 4/23 ommatidia, there were either three or four short R cell projections in the lamina, while the remaining R cells projected into the medulla. In 2/23 cases, a single, relatively long, R3-like projection was observed in the lamina, flanked by either two or three short projections. In summary, these data establish that R2 and R5 project to a local region within the lamina independent of R1, R3, R4, and R6 but require interactions with these neurons to specify their correct targets (Clandinin, 2000).

The defects in R cell projections seen in seven-up and lozenge^sprite animals are not due to effects on the differentiation of neurons in the target region as assessed using multiple markers; lamina neuron differentiation was not assessed in phyllopod. The defects seen in lozenge^sprite and seven-up are also not due to extra R7 cells; a gain-of-function mutation in the Raf gene recruits extra R7 cells to each ommatidium without affecting the differentiation and targeting of R1-R6 neurons. It is possible, however, that the effects seen in these mutants reflect altered composition of axons within the ommatidial fascicle caused by ectopic R7 axons in abnormal positions within the bundle (Clandinin, 2000).

Two models could explain the mechanisms that determine the precise projection of R3 and R4 axons toward the dorsoventral midline and, by extension, the relative orientations of the other R cell axons. The growth cones of R3 and R4 may respond to an orienting cue in the lamina that promotes extension toward the dorsoventral midline. Alternatively, the orientation of R cell bodies in the retina may determine the orientation of R cell growth cones in the lamina, independent of any environmental cues. To assess the role of ommatidial polarity on projection specificity, projections from misoriented ommatidia were assessed (Clandinin, 2000).

If a lamina cue can promote equatorial extension of the R3 and R4 axons, ommatidia that rotate incorrectly should project their axons normally, toward the equator. Alternatively, if ommatidial orientation determines the direction of axon projection in the lamina, incorrectly oriented ommatidia should project their R3 and R4 axons away from the equator (Clandinin, 2000).

In wild-type animals, ommatidia are mirror image reflected about the dorsoventral equator of the eye. R cell projections are also mirror image symmetric about the equator but are rotated 180° with respect to the retina. That is, while the R3 cell body is oriented toward the pole in each ommatidium, its axon projects toward the equator in the lamina. This rotation is generated by a twist in the axon fascicle that occurs between the retina and the lamina (Clandinin, 2000).

To test the effects of large changes in ommatidial orientation, two mutations, spiny legs (in homozygous animals) and frizzled (in somatic mosaic animals in which a mutant eye projects to a wild-type target), were examined. In these mutants, ommatidia frequently adopt orientations that are 180° rotated; that is, the R3 cell body is frequently oriented toward the equator in the eye. In these two mutant backgrounds, the orientation of projections from ommatidia that were correctly oriented was normal. Therefore, neither gene is required for R cell axons to respond to orienting cues in the target. However, almost 90% of the ommatidia that were ~180° misoriented in the eye made projections that were also 180° misoriented in the lamina. Rare, abnormal projections of single R cell axons in both of these mutant backgrounds were observed, irrespective of ommatidial orientation. Therefore, the orientation of R cell projections along the dorsoventral axis of the lamina is largely determined by the orientation of ommatidia in the retina (Clandinin, 2000).

Three exceptional cases, in which misoriented ommatidia projected axons toward the equator, were observed. Thus, a cue in the lamina may reinforce the ommatidial orientation cue to ensure the correct direction of outgrowth along the dorsoventral axis. To test whether such a cue contributes to directionality of R cell projections, a mutation that causes a more moderate defect in ommatidial orientation was examined. In nemo mutant animals, ommatidia are misoriented up to 45°. If ommatidial orientation directly determines the directionality of R cell projections, they would be misoriented 45° with respect to the equator; the angle between ommatidial orientation and the axon projection pattern would remain 180°. However, while ommatidial orientation was disrupted in nemo, R cell projections were normal with respect to the equator. This observation suggests that in addition to ommatidial polarity, a cue in the lamina can influence R cell projection orientation (Clandinin, 2000).

It is concluded that interactions between R cell afferents play a crucial role in target specification, and it is proposed that the spatial relationships between axons within a fascicle influence synaptic specificity. It is hypothesized that the interactions between R cell subtypes that are required for target specificity are mediated by direct contacts between specific growth cones. R3 and R4 are required for the remaining R cell axons to choose their normal targets. R1 and R6 are required for R2 and R5 projections but are not required for the projections of R3 and R4. These interactions could occur between growth cones from the same or neighboring ommatidial bundles. The characteristic morphological changes of these growth cones as revealed through electron microscopic reconstruction studies are consistent with the notion that precise spatial relationships between specific growth cones within the lamina plexus are required for these critical interactions to occur. This sequence of interactions determines the relative positions of targets chosen by R cell axons from the same ommatidium (Clandinin, 2000).

R cell transformation mutants could disrupt these interactions in two ways. First, transformation of specific R cells could directly disrupt the instructive signals between R cell growth cones within the plexus that determine growth cone trajectories. Alternatively, these mutations could affect the interactions indirectly, by disrupting the spatial relationships between the remaining R cell axons. That is, outgrowth trajectory could be determined passively by the position each growth cone occupies as it leaves the ommatidial fascicle. In this view, these mutant backgrounds alter the composition of axons within each ommatidial bundle and, hence, disrupt the precise packing of axons within the fascicle. The differential requirements for particular R cell subtypes would reflect their specific roles in directing the spatial relationships between growth cones within the fascicle, rather than interactions between specific growth cones in the target region (Clandinin, 2000).

The cellular mechanisms described here provide a conceptual framework for understanding the molecular basis of synaptic specificity. While the DiI method facilitates the analysis of R1-R6 specificity on a scale sufficient to analyze many mutants, it is too laborious to accommodate large-scale screening. Hence, a genetic screen based on visual behavior driven specifically by R1-R6 is required to extend these studies to the molecular level. A wealth of visual behaviors have been described in Drosophila, one of which, the optomoter response, is mediated by these cells. Techniques that generate mosaic flies in which only R cells are made homozygous for randomly induced mutations, while the rest of the fly is heterozygous, have recently been described. Currently, projects are underway, combining this specific behavioral screen with genetic mosaics, in order to screen for genes controlling R1-R6 synaptic specificity (Clandinin, 2000).

Mammalian cell culture studies have shown that several members of the nuclear receptor super family such as glucocorticoid receptor, retinoic acid receptor and thyroid hormone receptor can repress the activity of AP-1 proteins (referring to Drosophila Kayak and Jun) by a mechanism that does not require the nuclear receptor to bind to DNA directly, but that is otherwise poorly understood. Several aspects of nuclear receptor function are believed to rely on this inhibitory mechanism, which is referred to as transrepression. This study presents evidence that nuclear receptor-mediated transrepression of AP-1 occurs in Drosophila melanogaster. In two different developmental situations, embryonic dorsal closure and wing development, several nuclear receptors, including Seven up, Tailless, and Eagle antagonize AP-1. The inhibitory interactions with nuclear receptors are integrated with other modes of AP-1 regulation, such as mitogen-activated protein kinase signaling. A potential role of nuclear receptors in setting a threshold of AP-1 activity required for the manifestation of a cellular response is discussed (Gritzan, 2002).

The best understood AP-1-dependent process in Drosophila development is a coordinated cell sheet movement known as dorsal closure. During DC, lateral epidermal cells migrate dorsally and close the epidermis on the dorsal side of the embryo. Failure to undergo DC results in a characteristic dorsal open phenotype, the cuticle of affected embryos displays a dorsal hole. Mutations in genes encoding the Drosophila homologs of JNKK, (JNK, Jun and Fos) all give rise to similar dorsal open phenotypes. Thus, it is thought that DC requires activation of Jun/Fos heterodimers by a JNK-type MAPK cascade. Embryos homozygous for kay¹, a fos null allele are devoid of zygotic Fos activity and DC fails. A large dorsal hole forms and the cuticle collapses. In an embryo homozygous kay², a hypomorphic fos-allele, AP-1 activity is reduced but not eliminated. Correspondingly, the DC phenotype is weaker. The embryo displays a small dorso-anterior hole (Gritzan, 2002).

To test whether Drosophila NRs can antagonize AP-1, a variety of AP-1 constructs were in the embryonic epidermis. Interestingly, expression of some, but not all, NRs tested result in DC phenotypes of different strengths. Expression of Svp in the dorsal epidermis under the control of pnrGal4 results in a DC phenotype reminiscent of that of kay² homozygotes. This finding is consistent with a suppression of AP-1 activity by Svp. Similarly, expression of Tll under the control of a heat shock promoter causes a weak dorsal open phenotype. The differentiation of ventral cells does not seem to be disturbed by Tll expression since the pattern of denticles in this part of the epidermis appears grossly normal. Thus, Tll expression specifically affects the dorsal epidermis where AP-1 activity is required. The expression of Knrl under the control of pnrGal4 elicits stronger DC phenotypes with the dorsal hole frequently extending over several segments (Gritzan, 2002).

Does modulation of AP-1 activity by NRs occur only in situations where AP-1 is regulated by JNK or does this type of regulation also operate in different contexts? A function for Fos downstream of ERK has been demonstrated in the differentiation of wing veins. Extra vein material can result from elevated levels of ERK, as in flies carrying a gain-of-function allele of the rolled gene, which encodes Drosophila ERK. This allele, called rolled^Sevenmaker (rl^Sem), encodes a form of ERK with increased resistance to inactivation by dephosphorylation. Expression of a dominant-negative form of Fos in the wings of rl^Sem flies results in loss of ectopic vein material. Conversely, overexpression of Fos enhances the extra-vein phenotype caused by rl^Sem. 32B Gal4, UAS Sem flies express the Rl^Sem form of ERK in the wing from a UAS-driven transgene. As a consequence of elevated levels of ERK activity, these animals develop ectopic wing vein material. Reducing fos gene dosage in this system strongly suppresses the vein phenotype, consistent with the proposed role of Fos as an ERK effector. Thus, 32B Gal4 UAS Sem flies provide a suitable system to examine how genetic manipulations of AP-1 activity affect vein differentiation. To investigate a potential role of the Drosophila NRs in this process, one copy of kni, eg, tll or svp was removed in 32B Gal4, UAS Sem flies. Reducing kni function does not influence the vein phenotype. However, heterozygosity for any of the other three receptors tested reproducibly leads to a mild enhancement of the ectopic vein differentiation. As an unambiguously scoreable criterion to statistically evaluate phenotypic effects, the presence of ectopic vein material posterior to L5 was chosen. This area of the wing is relatively resistant to the formation of extra vein material. Quantitative analysis clearly shows that whereas the formation of extra vein material posterior to L5 in 32B Gal4 UAS Sem flies is suppressed by reducing fos activity, it is enhanced by a reduction of eg, svp or tll function. These data suggest that all three NRs antagonize AP-1 activity in wing vein differentiation, conceivably in a redundant manner (Gritzan, 2002).

It is speculated that the modulation of AP-1 activity by NRs contributes to what has recently been termed signal consolidation. Cells have to place a value on incoming signals (e.g. EGF-induced ERK activity) such that they are either answered by a biological response (e.g. the execution of a transcriptional program) or disregarded as noise. It is proposed that the modulation of AP-1 activity by NRs facilitates the interpretation of the EGF signal in wing vein differentiation by defining a threshold of ERK activity. Cells in which ERK activity does not reach this threshold do not mount an AP-1-dependent transcriptional response to the EGF signal. When transrepressional control is impaired (as in the svp, tll double mutant clones) the threshold is lowered and more cells than appropriate interpret EGF-induced ERK activity as a consolidated signal. This leads to the formation of ectopic vein material. This model is supported by the finding that the ectopic vein tissue observed in clones of tll and svp mutant tissue did arise close to the position of the endogenous veins and not randomly throughout the clonal area. Thus, the regulation of AP-1 by NRs appears to convey cell-intrinsic information (Gritzan, 2002).

Ahn, M. Y., et al. (1998). Negative regulation of granulocytic differentiation in the myeloid precursor cell line 32Dcl3 by ear-2, a mammalian homolog of Drosophila seven-up, and a chimeric leukemogenic gene, AML1/ETO (MTG8). Proc. Natl. Acad. Sci. 95(4): 1812-1817.

Ahn, J. E., Guarino, L. A. and Zhu-Salzman, K. (2007). Seven-up facilitates insect counter-defense by suppressing cathepsin B expression. FEBS J. 274(11): 2800-14. PubMed citation: 17459103

Armentano, M., et al. (2007). COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas. Nat. Neurosci. 10(10): 1277-86. Medline abstract: 17828260

Beckstead, R., et al. (2001). Bonus, a Drosophila homolog of TIF1 proteins, interacts with nuclear receptors and can inhibit FTZ-F1-dependent transcription. Molec. Cell 7: 753-765. 11336699

Begemann, G., et al. (1995). The Drosophila orphan nuclear receptor seven-up requires the Ras pathway for its function in photoreceptor determination. Development 121: 225-235

Broadus,. J. and Doe, C. Q. (1995). Evolution of neuroblast identity: seven-up and prospero expression reveal homologous and divergent neuroblast fates in Drosophila and Schistocerca. Development 121: 3989-3996

Brodu, V., Elstob, P. R. and Gould, A. P. (2004). EGF receptor signaling regulates pulses of cell delamination from the Drosophila ectoderm. Dev. Cell 7: 885-895. 15572130

Choksi, S. P., et al. (2006). Prospero acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells. Dev. Cell 11(6): 775-89. Medline abstract: 17141154

Clandinin, T. R. and Zipursky, S. L. (2000). Afferent growth cone interactions control synaptic specificity in the Drosophila visual system. Neuron 28: 427-436.

Crew, J. R., Batterham, P. and Pollock, J. A. (1997). Developing compound eye in lozenge mutants of Drosophila: lozenge expression in the R7 equivalence group. Dev. Genes and Evol. 206(8): 481-493

Daga, A., et al. (1996). Patterning of cells in the Drosophila eye by Lozenge, which shares homologous domains with AML1. Genes Dev 10: 1194-1205.

Domingos, P. M., et al. (2004). Spalt transcription factors are required for R3/R4 specification and establishment of planar cell polarity in the Drosophila eye. Development 131: 5695-5702. 15509769

Dressel, U., et al. (1999). Alien, a highly conserved protein with characteristics of a corepressor for members of the nuclear hormone receptor superfamily. Mol. Cell. Biol. 19: 3383-3394. 10207062

Elstob, P. R., Brodu, V. and Gould, A. P. (2001). spalt-dependent switching between two cell fates that are induced by the Drosophila EGF receptor. Development 128: 723-732. 11171397

Fanto, M., Mayes, C. A. and Mlodzik, M. (1998). Linking cell-fate specification to planar polarity: determination of the R3/R4 photoreceptors is a prerequisite for the interpretation of the Frizzled mediated polarity signal. Mech. Dev. 74(1-2): 51-58

Fjose, A., et al. (1993). Functional conservation of vertebrate seven-up related genes in neurogenesis and eye development. EMBO J 12: 1403-14

Fjose, A., Weber, U. and Mlodzik, M. (1995). A novel vertebrate svp-related nuclear receptor is expressed as a step gradient in developing rhombomeres and is affected by retinoic acid. Mech Dev 52: 233-246

Galson, D. L., et al. (1995). The orphan receptor hepatic nuclear factor 4 functions as a transcriptional activator for tissue-specific and hypoxia-specific erythropoietin gene expression and is antagonized by EAR3/COUP-TF1. Mol. Cell. Biol. 15(4): 2135-44. 7891708

Gauchat, D., et al. (2004). The orphan COUP-TF nuclear receptors are markers for neurogenesis from cnidarians to vertebrates. Dev. Biol. 275: 104-123. 15464576

Gritzan, U., Weiss, C., Brennecke, J. and Bohmann, D. (2002). Transrepression of AP-1 by nuclear receptors in Drosophila. Mech. Dev. 115: 91-100. 12049770

Hayashi, T. and Saigo, K. (2001). Diversification of cell types in the Drosophila eye by differential expression of prepattern genes. Mech. Dev. 108: 13-27. 11578858

Herbst, R., et al. (1996). Daughter of sevenless is a substrate of the phosphotyrosine phosphatase Corkscrew and functions during Sevenless signaling. Cell 85(6): 899-909.

Hiromi, Y., et al. (1993). Ectopic expression of seven-up causes cell fate changes during ommatidial assembly. Development 118: 1123-35

Hoshizaki, D. K., et al. (1994). Embryonic fat-cell lineage in Drosophila melanogaster. Development 120: 2489-99

Kanai, M. I., Okabe, M. and Hiromi, Y. (2005). seven-up controls switching of transcription factors that specify temporal identities of Drosophila neuroblasts. Dev Cell. 8(2): 203-13. 15691762

Kerber, B., Fellert, S. and Hoch, M., (1998). Seven-up, the Drosophila homolog of the COUP-TF orphan receptors, controls cell proliferation in the insect kidney. Genes Dev. 12(12): 1781-1786.

Kramer, S., West, S. R. and Hiromi, Y. (1995). Cell fate control in the Drosophila retina by the orphan receptor seven-up: its role in the decisions mediated by the ras signaling pathway. Development 121: 1361-1372

Krishnan, V., et al. (1997). Mediation of Sonic hedgehog-induced expression of COUP-TFII by a protein phosphatase. Science 278(5345): 1947-1950.

Armentano, M., et al. (2007). COUP-TFI regulates the balance of cortical patterning between frontal/motor and sensory areas. Nat. Neurosci. 10(10): 1277-86. Medline abstract: 17828260

Laudet, V. (1997). Evolution of the nuclear receptor superfamily: early diversification from an ancestral orphan receptor. J. Mol. Endocrinol. 19(3): 207-26.

Lo, P. C. H. and Frasch, M. (2001). A role for the COUP-TF-related gene seven-up in the diversification of cardioblast identities in the dorsal vessel of Drosophila. Mech. Dev. 104: 49-60. 11404079

Lo, P. C. H., Skeath, J. B., Gajewski, K. Schulz, R. A. and Frasch, M. (2002). Homeotic genes autonomously specify the anteroposterior subdivision of the Drosophila dorsal vessel into aorta and heart. Dev. Bio. 251: 307-319. 12435360

Lovato, T. L., et al. (2002). The Hox gene abdominal-A specifies heart cell fate in the Drosophila dorsal vessel. Development 129: 5019-5027. 12397110

Lutz, B., et al. (1994). Developmental regulation of the orphan receptor COUP-TF II gene in spinal motor neurons. Development 120(1): 25-36.

McCaffery, P., et al. (1999). Dorsal and ventral retinal territories defined by retinoic acid synthesis, break-down and nuclear receptor expression. Mech. Dev. 82(1-2): 119-130.

Mlodzik, M., et al. (1990). The Drosophila seven-up gene, a member of the steroid receptor gene superfamily, controls photoreceptor cell fates. Cell 60: 211-24

Murray, P. and Edgar, D. (2001). Regulation of laminin and COUP-TF expression in extraembryonic endodermal cells. Mech. Dev. 101: 213-215. 11231078

Pereira, F. A., et al. (1999). The orphan nuclear receptor COUP-TFII is required for angiogenesis and heart development. Genes Dev. 13(8): 1037-49.

Pieri, I., et al. (1999). Regulation of the murine NMDA-receptor-subunit NR2C promoter by Sp1 and fushi tarazu factor1 (FTZ-F1) homologues. Eur. J. Neurosci. 11(6): 2083-92.

Ponzielli, R., et al. (2002). Heart tube patterning in Drosophila requires integration of axial and segmental information provided by the Bithorax Complex genes and hedgehog signaling. Development 129: 4509-4521. 12223408

Qiu, Y., et al. (1997). Null mutation of mCOUP-TFI results in defects in morphogenesis of the glossopharyngeal ganglion, axonal projection, and arborization. Genes Dev. 11(15): 1925-1937.

Rehorn, K.-P., et al. (1996). A molecular aspect of hematopoiesis and endoderm development common to vertebrates and Drosophila. Development 122: 4023-4031

Ryan, K. M., Hendren, J. D., Helander, L. A. and Cripps, R. M. (2007). The NK homeodomain transcription factor Tinman is a direct activator of seven-up in the Drosophila dorsal vessel. Dev Biol. 302(2): 694-702. Medline abstract: 17098220

Sen, A., Reddy, V. and Rodrigues, V. (2003). Combinatorial expression of Prospero, Seven-up, and Elav identifies progenitor cell types during sense-organ differentiation in the Drosophila antenna. Dev. Bio. 254: 79-92. 12606283

Sluder, A. E., Lindbloom, T. and Ruvkun, G. (1997). The Caenorhabditis elegans orphan nuclear hormone receptor gene rhr-2 functions in early embryonic development. Dev. Biol. 184: 303-319

Sudarsan, V., et al. (2002). A genetic hierarchy establishes mitogenic signaling and mitotic competence in the renal tubules of Drosophila. Development 129: 935-944. 11861476

Takamoto, N., et al. (2005). COUP-TFII is essential for radial and anteroposterior patterning of the stomach. Development 132: 2179-2189. 15829524

Thomas, B. J., et al. (1994). Cell cycle progression in the developing Drosophila eye: roughex encodes a novel protein required for the establishment of G1. Cell 77: 1003-1014.

Tripodi, M., et al. (2005). The COUP-TF nuclear receptors regulate cell migration in the mammalian basal forebrain. Development 131: 6119-6129. 15548577

Vlahou, A. and Flytzanis, C. N. (2000). Subcellular trafficking of the nuclear receptor COUP-TF in the early embryonic cell cycle. Dev. Biol. 218: 284-298.

Ward, E. J. and Skeath, J. B. (2000). Characterization of a novel subset of cardiac cells and their progenitors in the Drosophila embryo. Development 127: 4959-4969. 11044409

Weber, U., Siegel. V. and Mlodzik, M. (1995). pipsqueak encodes a novel nuclear protein required downstream of seven-up for the development of photoreceptors R3 and R4. EMBO J. 14: 6247-57

Younossi-Hartenstein, A., et al. (1996). Early neurogenesis of the Drosophila brain. J. Comp. Neur. 370: 313-329

Younossi-Hartenstein, A., et al. (1997). Control of early neurogenesis in the Drosophila brain by the head gap genes tll, otd, ems, and btd. Dev. Biol 182: 270-283

Yu, R. N., Ito, M. and Jameson, J. L. (1998). The murine Dax-1 promoter is stimulated by SF-1 (steroidogenic factor-1) and inhibited by COUP-TF (chicken ovalbumin upstream promoter-transcription factor) via a composite nuclear receptor-regulatory element. Mol. Endocrinol. 12(7): 1010-22.

Zelhof, A. C., et al. (1995a). Seven-up inhibits ultraspiracle-based signaling pathways in vitro and in vivo. Mol Cell Biol 15: 6736-6745

Zelhof, A. C., et al. (1995b). Identification and characterization of a Drosophila nuclear receptor with the ability to inhibit the ecdysone response. Proc. Natl. Acad. Sci. 92 (23): 10477-10481.

Zhou, C., Tsai, S. Y. and Tsai, M. J. (2001). COUP-TFI: an intrinsic factor for early regionalization of the neocortex. Genes Dev. 15: 2054-2059. 11511537

Zhou, H. M. and Walthall, W. W. (1998). UNC-55, an orphan nuclear hormone receptor, orchestrates synaptic specificity among two classes of motor neurons in Caenorhabditis elegans. J. Neurosci. 18(24): 10438-44.

date revised: 16 April 2008

Home page: The Interactive Fly © 1997 Thomas B. Brody, Ph.D.

The Interactive Fly resides on the
Society for Developmental Biology's Web server.