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.

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 svpAE127 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 (neuA101 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 svpP1725. 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).

Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila

Locomotion in adult Drosophila depends on motor neurons that target a set of multifibered muscles in the appendages. This study describes the development of motor neurons in adult Drosophila, focusing on those that target the legs. Leg motor neurons are born from at least 11 neuroblast lineages, but two lineages generate the majority of these cells. Using genetic single-cell labeling methods, the birth order, muscle targeting, and dendritic arbors for most of the leg motor neurons were analyze. The results reveal that each leg motor neuron is born at a characteristic time of development, from a specific lineage, and has a stereotyped dendritic architecture. Motor axons that target a particular leg segment or muscle have similar dendritic arbors but can derive from different lineages. Thus, although Drosophila uses a lineage-based method to generate leg motor neurons, individual lineages are not dedicated to generate neurons that target a single leg segment or muscle type (Baek, 2010).

To study the development of the Drosophila leg motor neurons, a clonal analysis was performed using a modified version of mosaic analysis with a repressible cell marker (MARCM) method. The Vglut-Gal4 (also called OK371-Gal4) driver was used to positively label clones. This Gal4 driver, which is inserted into the Vglut gene, is expressed in all neurons that use glutamate as a neurotransmitter, including all motor neurons. As can be seen in adult leg preparations in which Vglut-Gal4 was used to express a membrane-tagged version of green fluorescent protein (CD8GFP), motor neurons innervating all of the muscles in the coxa (co), trochanter (tr), femur (fe), and tibia (ti) were labeled by this driver. In addition, a subset of sensory neurons, whose cell bodies reside in the tibia and tarsal segments, were labeled by Vglut-Gal4. Except for the tarsus, each leg segment has a stereotyped set of multifibered muscles that are labeled by the MHC-tauGFP reporter gene. This reporter gene was used to identify each of the muscles innervated by the leg motor neurons. In the adult CNS, Vglut-Gal4 labeled groups of neurons in each thoracic hemisegment. In addition to motor neuron cell bodies, the dendritic arbors of these neurons were observed in densely packed neuropils in each thoracic hemisegment. This study focused on the motor neurons innervating the first thoracic (T1) legs. The axons of these motor neurons fasciculate and exit the CNS through a large nerve that extends into the ipsilateral leg (Baek, 2010).

Drosophila NBs are born during embryogenesis and undergo two waves of neurogenesis, one during embryogenesis and one during larval development. During the first, embryonic wave of NB divisions, the majority of the embryonically born neurons are dedicated to larval motor and sensory functions and die during metamorphosis. To determine how many independent NB lineages give rise to the leg motor neurons, positively labeled MARCM clones were induced during embryogenesis and analyzed in the adult. Because these clones were generated infrequently and early in development, entire NB lineages were labeled. These data revealed that the leg motor neurons are derived from at least 11 independent lineages. Strikingly, two of these lineages, Lin A and Lin B, give rise to the majority of the leg motor neurons. Embryonically induced clones of Lin A innervated the muscles of the femur and tibia but did not include any motor neurons that targeted the coxa or trochanter. Moreover, the tibia is only targeted by Lin A-derived motor neurons. Thus, Lin A motor neurons generally target distal, but not proximal, leg segments (Baek, 2010).

The second major lineage defined by these experiments is Lin B, which gives rise to seven leg motor neurons. In contrast to Lin A, Lin B motor neurons target the three most proximal leg segments, the coxa, trochanter, and femur, but does not generate any motor neurons that target the tibia. Thus, Lin B motor neurons generally target proximal, rather than distal, leg segments (Baek, 2010).

Embryonically induced MARCM clones revealed that another 12 Vglut-Gal4+ leg motor neurons are generated from nine additional lineages, Lin C to Lin K. These 12 motor neurons target the coxa (six), the trochanter (one), and the femur (five), but not the tibia. In contrast to Lin A and Lin B, these lineages give rise to only one or two Vglut-Gal4-expressing leg motor neurons. Lin E is distinctive because, in addition to generating a single motor neuron targeting the coxa, it also gives rise to ~25 Vglut-Gal4-expressing interneurons. Five of these lineages (C to G) were labeled frequently, by both embryonic and postembryonic heat shocks. In contrast, four of these lineages, Lin H to Lin K, were labeled infrequently and only by embryonic heat shocks. These findings suggest that these motor neurons, which target the coxa (one) and femur (five), are born during embryogenesis and persist to the adult stage in which they contribute to the adult leg nervous system (Baek, 2010).

In total, 53 neurons were identified, derived from 11 independent NBs, that innervate the T1 leg. Two of these lineages give rise to 35 of these 53 motor neurons. By characterizing individually labeled motor neurons, the birth dates, muscle targets, and dendritic arbors for most of these motor neurons were determined. These results show that, although each motor neuron is born from a specific lineage, and at a specific time during development, individual lineages give rise to motor neurons that target multiple leg segments and multiple muscles within these leg segments (Baek, 2010).

Accurate motor neuron development in the fly requires that axons target the correct muscles along the PD axis of the leg. This axis has several levels of refinement. The first level is the global PD axis of the leg. Lin A only generates motor neurons that target the two more distal leg segments, the tibia and the femur. In addition, Lin A is the only lineage that produces motor neurons that target the tibia. In contrast, the seven Lin B motor neurons target all leg segments except the tibia. Thus, there is a PD bias built into these lineages (Baek, 2010).

A second level of refinement within the PD axis is targeting the correct muscle in individual leg segments. Among the Lin A-derived motor neurons, a PD bias was observed within the tibia and within the femur that correlates with birth date: the first half of the motor neurons born from Lin A have a strong bias for targeting proximal positions in these segments, whereas the later-born half of the motor neurons target distal muscles in these segments (Baek, 2010).

Third, for muscles that are targeted by multiple motor neurons (e.g., ltm1 in the tibia), it was found that more distal projecting motor neurons are born before those that target more proximal positions in the same muscle. The differential targeting of axons to unique positions within the same muscle suggests the existence of high-resolution topographic maps that match specific motor neurons to specific muscle compartments, as has been observed in mouse skeletal muscles (Baek, 2010).

Most of the leg motor neurons are born within a narrow window of development. The NB that gives rise to Lin A, for example, switches into a phase that is dedicated to generating leg motor neurons at ~50 h AEL. At that time, this NB begins to produce its 28 motor neurons for the next ~40 h. Presumably, this NB gives rise to nonmotor neuron progeny before this time and possibly after it completes this motor neuron generating phase. This scenario shares some similarities with the lineages that give rise to postembryonic neurons in the fly brain. For example, the entire mushroom body of Drosophila, the portion of the fly brain used in olfactory learning and memory, is derived from only four NBs that each give rise to one of four nearly identical anatomical units. Interestingly, there is a temporal switch in the types of neurons that these NBs generate at specific times of development. Thus, like Lin A, mushroom body NBs switch the type of neuron they generate at specific times. However, unlike the leg motor neuron NBs, those that generate the mushroom body are dedicated to forming this brain structure. In contrast, it was found that functionally related leg motor neurons, for example those that target a specific leg segment, muscle, or muscle type, are often derived from several different NB lineages. This logic is reminiscent of that used to generate olfactory projection neurons in the fly, in which three neuroblasts each give rise to different numbers and types of projection neurons (Baek, 2010).

The temporal control of NB identity in Drosophila is directed by transcription factors that are sequentially expressed as NBs age. During embryogenesis, progeny postmitotic neurons inherit the transcription factor expressed in the NB at the time it was born. This temporal information works in combination with positional information that makes each NB unique, providing progeny neurons their individual identities. Although the specific factors are not yet known, a similar transcription factor code may exist for leg motor neurons. Two of the temporal control genes that are used during Drosophila embryogenesis, seven-up (svp) and castor (cas), are also important for controlling postembryonic neural fates. Interestingly, some NBs switch from expressing cas to svp at ~50 h AEL, similar to the time that the leg NBs begin to generate their leg motor neuron progeny. It will be interesting to determine whether this or other changes in transcription factors are responsible for initiating the production of leg motor neurons in the lineages defined here (Baek, 2010).

These results demonstrate that adult motor neurons in the fly come from identifiable lineages that give rise to stereotyped progeny with defined birth dates. Importantly, however, of the 11 lineages that give rise to leg motor neurons in the fly, only one of these, Lin A, appears to be dedicated to producing these neurons. Even this restriction only occurs during the ~50 to ~90 h AEL time window. Although most of the progeny produced by the other lineages were not marked in these experiments (except for Lin E, which generates ~25 Vglut-Gal4+ interneurons), it is likely that these lineages also produce nonmotor neuron progeny. Thus, although seemingly invariant lineages are used in the fly, the closest relatives for many leg motor neurons are not other leg motor neurons. This conclusion is similar to the picture that emerged from lineage analyses performed in the vertebrate spinal cord showing that cell lineages are not dedicated to the production of motor neurons. As in the fly, closely related cells in the spinal cord may have distinct fates. Conversely, although adult fly motor neurons are born from stereotyped lineages, position within the CNS determines NB identities and, consequently, the progeny they generate. Although C. elegans has a more extreme version of a lineage-based mechanism, even in this case cell-cell signaling plays an important role in specifying identities. These considerations blur the distinction between lineage and position-based mechanisms and suggest that both play a role in vertebrates and invertebrates (Baek, 2010).

Consistent with the idea that lineage may play a role in vertebrates, the transcription factor Coup-TF acts as a temporal switch between neurogenesis and gliogenesis in the vertebrate brain. Interestingly, Coup-TF is a relative of Drosophila svp, which encodes one of the temporal transcription factors used in postembryonic fly neuroblasts. The use of Coup-TF/Svp for executing a temporal switch in both flies and vertebrates suggests the existence of a conserved molecular mechanism for controlling developmental timing in neural lineages (Baek, 2010).

Because motor neurons receive complex inputs from interneurons and sensory neurons, the architecture of their dendritic arbors is critical for forming the circuitry that is required for locomotion. An initial analysis of the dendritic arbors of the leg motor neurons suggests that, as in other systems, they exhibit a functional organization in the thoracic neuromere. For example, nine leg motor neurons, targeting two different reductor muscles in different leg segments (coxa and femur), have overlapping dendritic arbors. That these nine motor neurons have similar dendritic architectures suggests that they share presynaptic inputs, perhaps allowing these two reductor muscles to contract in synchrony. Similarly, all eight motor neurons that have dendrites that cross the midline of the CNS, and thus probably make contacts with neurons in the contralateral neuromere, send their axons to one of two long tendon muscles, one in the tibia and one in the femur. These two examples suggest that the organization of motor neuron dendrites may be important for muscle synergies as described in vertebrate locomotion (Baek, 2010).

In vertebrate motor systems, motor neuron cell bodies are organized in columns and pools that correlate with their muscle targets. This organization implies that many of the presynaptic inputs into the motor neurons within individual pools will be similar. Consistently, in some cases, the dendritic arbors of motor neurons have been shown to correlate with motor neuron targeting. In these examples, the arborization patterns are controlled by the transcription factor Pea3, which requires a specific Hox code to be activated, but is only induced after motor axons invade the limb target. In contrast, the myotopic map exhibited by the dendrites of the fly larval motor neurons does not need target muscles to form. In the fly olfactory system, the dendrites of projection neurons form a map in the antennal lobe before the arrival of olfactory receptor neurons (ORNs), suggesting that this map forms independently of ORNs. It remains unclear whether the characteristic dendritic arbors of the fly's leg motor neurons require muscle targeting or whether they form independently of their targets using local cues in the CNS and the identities they acquire at birth (Baek, 2010).

Integration of temporal and spatial patterning generates neural diversity

In the Drosophila optic lobes, 800 retinotopically organized columns in the medulla act as functional units for processing visual information. The medulla contains over 80 types of neuron, which belong to two classes: uni-columnar neurons have a stoichiometry of one per column, while multi-columnar neurons contact multiple columns. This study shows that combinatorial inputs from temporal and spatial axes generate this neuronal diversity: all neuroblasts switch fates over time to produce different neurons; the neuroepithelium that generates neuroblasts is also subdivided into six compartments by the expression of specific factors (see The OPC neuroepithelium is patterned along its dorsal-ventral axis). Uni-columnar neurons are produced in all spatial compartments independently of spatial input; they innervate the neuropil where they are generated. Multi-columnar neurons are generated in smaller numbers in restricted compartments and require spatial input; the majority of their cell bodies subsequently move to cover the entire medulla. The selective integration of spatial inputs by a fixed temporal neuroblast cascade thus acts as a powerful mechanism for generating neural diversity, regulating stoichiometry and the formation of retinotopy (Erclik, 2017).

The optic lobes, composed of the lamina, medulla and the lobula complex, are the visual processing centres of the Drosophila brain. The lamina and medulla receive input from photoreceptors in the compound eye, process information and relay it to the lobula complex and central brain. The medulla, composed of ~40,000 cells, is the largest compartment in the optic lobe and is responsible for processing both motion and colour information. It receives direct synaptic input from the two colour-detecting photoreceptors, R7 and R8. It also receives input from five types of lamina neuron that are contacted directly or indirectly by the outer photoreceptors involved in motion detection (Erclik, 2017).

Associated with each of the ~800 sets of R7/R8 and lamina neuron projections are 800 medulla columns defined as fixed cassettes of cells that process information from one point in space. Columns represent the functional units in the medulla and propagate the retinotopic map established in the compound eye. Each column is contributed to by more than 80 neuronal types, which can be categorized into two broad classes. Uni-columnar neurons have arborizations principally limited to one medulla column and there are thus 800 cells of each uni-columnar type. Multi-columnar neurons possess wider arborizations, spreading over multiple columns. They compare information covering larger receptor fields. Although they are fewer in number, their arborizations cover the entire visual field (Erclik, 2017).

The medulla develops from a crescent-shaped neuroepithelium, the outer proliferation centre (OPC). During the third larval instar, the OPC neuroepithelium is converted into lamina on its lateral side and into medulla neuroblasts on its medial side. A wave of neurogenesis moves through the neuroepithelial cells, transforming them into neuroblasts; the youngest neuroblasts are closest to the neuroepithelium while the oldest are adjacent to the central brain. Neuroblasts divide asymmetrically multiple times to regenerate themselves and produce a ganglion mother cell that divides once more to generate medulla neurons. Recent studies have shown that six transcription factors are expressed sequentially in neuroblasts as they age: neuroblasts first express Homothorax (Hth), then Klumpfuss (Klu), Eyeless (Ey), Sloppy-paired 1 (Slp1), Dichaete (D) and Tailless (Tll). This temporal series is reminiscent of the Hb --> Kr --> Pdm --> Cas --> Grh series observed in Drosophila ventral nerve cord neuroblasts that generates neuronal diversity in the embryo. Indeed, distinct neurons are generated by medulla neuroblasts in each temporal window. Further neuronal diversification occurs through Notch-based asymmetric division of ganglion mother cells. In total, over 20 neuronal types can theoretically be generated using combinations of temporal factors and Notch patterning mechanisms. However, little is known about how the OPC specifies the additional ~60 neuronal cell types that constitute the medulla (Erclik, 2017).

To understand the logic underlying medulla development, late larval brains were stained with 215 antibodies generated against transcription factors and 35 genes were identifiied that are expressed in subsets of medulla progenitors and neurons. The OPC neuroepithelial crescent can be subdivided along the dorsal-ventral axis by the mutually exclusive expression of three homeodomain-containing transcription factors: Vsx1 is expressed in the central OPC (cOPC), Rx in the dorsal and ventral posterior arms of the crescent (pOPC), and Optix in the two intervening 'main arms' (mOPC). These three proteins are regionally expressed as early as the embryonic optic anlage and together mark the entire OPC neuroepithelium with sharp, non-overlapping boundaries. Indeed, these three regions grow as classic compartments: lineage trace experiments show that cells permanently marked in the early larva in one OPC region do not intermingle at later stages with cells from adjacent compartments. Of note, Vsx1 is expressed in cOPC progenitor cells and is maintained in a subset of their neuronal progeny whereas Optix and Rx are not expressed in post-mitotic medulla neurons. The OPC can be further subdivided into dorsal (D) and ventral (V) halves: a lineage trace with hedgehog-Gal4 (hh-Gal4) marks only the ventral half of the OPC, bisecting the cOPC compartment. As hedgehog is not expressed in the larval OPC, this dorsal-ventral boundary is set up in the embryo. Thus, six compartments (ventral cOPC, mOPC and pOPC and their dorsal counterparts) exist in the OPC. The pOPC compartment can be further subdivided by the expression of the wingless and dpp signalling genes. Cells in the wingless domain behave in a very distinct manner from the rest of the OPC, and have been described elsewhere (Erclik, 2017).

The Hth --> Klu --> Ey --> Slp1 --> D --> Tll temporal progression is not affected by the compartmentalization of the OPC epithelium; the same neuroblast progression throughout the entire OPC. Thus, in the developing medulla, neuroblasts expressing the same temporal factors are generated by developmentally distinct epithelial compartments (Erclik, 2017).

To test whether the intersection of the dorsal-ventral and temporal neuroblast axes leads to the production of distinct neural cell types, focus was placed on the progeny of Hth neuroblasts, which maintain Hth expression. In the late third instar, Hth neurons are found in a crescent that mirrors the OPC (see Distinct neuronal cell types are generated along the dorsal-ventral axis of the OPC). The NotchON (NON) progeny of Hth+ ganglion mother cells express Bsh and Ap, and they are distributed throughout the entire medulla crescent. In contrast, the NotchOFF (NOFF) progeny, which are BshHth+ neurons, express different combinations of transcription factors, and can be subdivided into three domains along the dorsal-ventral axis: (1) in the cOPC, NOFFHth+ neurons express Vsx1, Seven-Up (Svp) and Lim3; (2) in the pOPC, these neurons also express Svp and Lim3, but not Vsx1; (3) in the ventral pOPC exclusively, these neurons additionally express Teashirt (Tsh). NOFFHth+ cells are not observed in the mOPC. Rather, Cleaved-Caspase-3+ cells are intermingled with Bsh+ neurons. When cell death is prevented, Bsh+Hth+ cells become intermingled with neurons that express the NOFF marker Lim3, confirming that the NOFFHth+ progeny undergo apoptosis in the mOPC (Erclik, 2017).

It was therefore possible to distinguish three regional populations of Hth neurons (plus one that is eliminated by apoptosis) and a fourth population that is generated throughout the OPC. The neuronal identity of each of these populations was identified, as follows. (1) Bsh is a specific marker of Mi1 uni-columnar interneurons that are generated in all regions of the OPC. (2) To determine the identity of Hth+NOFF cOPC-derived neurons, Hth+ single cell flip-out clones were generated (using hth-Gal4) in the adult medulla. The only Hth+ neurons that are also Vsx1+Svp+ are Pm3 multi-columnar local neurons. (3) For Hth+NOFF pOPC-derived neurons, 27b-Gal4 was used; it drives expression in larval pOPC Hth+NOFF neurons and is maintained to adulthood. Flip-out clones with 27b-Gal4 mark Pm1 and Pm2 neurons, as well as Hth- Tm1 uni-columnar neurons that come from a different temporal window. Both Pm1 and Pm2 neurons (but not Tm1) express Hth and Svp. Pm1 neurons also express Tsh, which only labels larval ventral pOPC neurons (Erclik, 2017).

Thus, in addition to uni-columnar Mi1 neurons generated throughout the OPC, Hth neuroblasts generate three region-specific neuronal types: multi-columnar Pm3 neurons in the cOPC; multi-columnar Pm1 neurons in the ventral pOPC; and multi-columnar Pm2 neurons in the dorsal pOPC (Erclik, 2017).

To determine the contribution of the temporal and spatial factors to the generation of the different neuronal fates, the factors were mutated them and whether neuronal identity was lost was examined. To test the temporal axis, hth was mutated. As previously reported, Bsh expression is lost in hth mutant clones. Loss of hth in clones also leads to the loss of the Pm3 marker Svp without affecting expression of Vsx1, indicating that Vsx1 is not sufficient to activate Svp and can only do so in the context of an Hth+ neuroblast. Hth is also required for the specification of Pm1 and Pm2 in the pOPC as Svp and Tsh expression is lost in hth mutant larval clones. Ectopic expression of Hth in older neuroblasts is not able to expand Pm1, 2 or 3 fates (on the basis of the expression of Svp) into later born neurons, although it is able to expand Bsh expression. Thus, temporal input is necessary for the specification of all Hth+ neuronal fates but only sufficient for the generation of Mi1 neurons (see Temporal and spatial inputs are required for neuronal specification in the medulla. ) (Erclik, 2017).

Next, whether regional inputs are necessary and/or sufficient to specify neuronal fates in the progeny of Hth+ neuroblasts was determined. In Vsx1 RNA interference (RNAi) clones, Svp expression is lost in the cOPC but Bsh is unaffected. Additionally, Hth+Lim3+ cells are absent, suggesting that NOFF cells undergo apoptosis in these clones. Conversely, ectopic expression of Vsx1 leads to the expression of Svp in mOPC Hth+ neurons but does not affect Bsh expression. Therefore, Vsx1 is both necessary and sufficient for the specification of Pm3 fates in the larva. However, unlike the temporal factor Hth, Vsx1 does not affect the generation of Mi1 neurons (Erclik, 2017).

In Rx whole mutant larvae and in mutant clones, Svp+Lim3+Hth+ larval neurons (that is, Pm1 and Pm2 neurons) in the pOPC are lost. Additionally, the Pm1 marker Tsh is lost in ventral pOPC Hth+ cells. Consistent with the Vsx1 mutant data, larval Bsh expression is not affected by the loss of Rx. In adults, the Pm1/Pm2 markers (Svp, Tsh and 27b-Gal4) are lost in the medulla (Erclik, 2017).

Ectopic expression of Rx leads to the activation of Svp in mOPC Hth+ neurons, but does not affect the expression of Bsh. It also leads to the activation of Tsh, but only in the ventral half of mOPC Hth+ neurons, suggesting that a ventral factor acts together with Rx to specify ventral fates. Taken together, the above data show that Rx is both necessary and sufficient for the specification of Pm1/2 neurons but (like Vsx1) does not affect the generation of Mi1 neurons (Erclik, 2017).

Finally, the role of the mOPC marker Optix in neuronal specification was examined. In Optix mutant clones, Svp is ectopically expressed in the mOPC, but Bsh expression is not affected. Of note, these ectopic Svp+ neurons fail to express the region-specific Pm markers Vsx1 or Tsh (in ventral clones), which suggests that they assume a generic Pm fate. Conversely, ectopic expression of Optix leads to the loss of Svp expressing neurons in both the cOPC and pOPC but does not affect Bsh. These NOFF neurons die by apoptosis as no Lim3+ neurons are found intermingled with Bsh+Hth+NON neurons. When apoptosis is prevented in mOPC-derived neurons, Svp is not derepressed in the persisting Hth+NOFF neurons, which suggests that Optix both represses Svp expression and promotes cell death in Hth+NOFF neurons (Erclik, 2017).

The above data demonstrate that input from both the temporal and regional axes is required to specify neuronal fates. The temporal factor Hth is required for both Mi1 and Pm1/2/3 specification. The spatial genes are not required for the specification of NON Mi1 neurons, consistent with the observation that Mi1 is generated in all OPC compartments. The spatial genes, however, are both necessary and sufficient for the activation (Vsx1 and Rx) or repression (Optix) of the NOFF Pm1/2/3 neurons. Thus, Hth+ neuroblasts generate two types of progeny: NOFF neurons that are sensitive to spatial input (Pm1/2/3) and NON neurons that are refractory to spatial input (Mi1). Vsx1 expression in the cOPC is only maintained in Hth+NOFF neurons, suggesting that spatial information may be 'erased' in Mi1, thus allowing the same neural type to be produced throughout the OPC (Erclik, 2017).

Do spatial genes regulate each other in the neuroepithelium? In Vsx1 mutant clones, Optix (but not Rx) is derepressed in the cOPC epithelium. Conversely, ectopic Vsx1 is sufficient to repress Optix in the mOPC and Rx in the pOPC. Similarly, Optix, but not Vsx1, is derepressed in Rx mutant clones in the pOPC epithelium and ectopic Rx is sufficient to repress Optix in the mOPC (but not Vsx1 in the cOPC). In Optix mutant clones, neither Vsx1 nor Rx are derepressed in the mOPC epithelium, but ectopic Optix is sufficient to repress both Vsx1 in the cOPC and Rx in the pOPC. The observation that Optix is not necessary to suppress Vsx1 or Rx in the mOPC neuroepithelium is surprising because Svp is activated in a subset of Hth+ neurons in the mOPC in Optix mutant clones. Nevertheless, when cell death in the mOPC is abolished, the ectopic undead NOFF neurons express Lim3 but not Svp, which confirms that Optix represses Svp expression in mOPC neurons. Taken together, these results support a model in which Optix is sufficient to repress Vsx1 and Rx, to promote the death of Hth+NOFF neurons and to repress Pm1/2/3 fates (see Spatial genes cross-regulate each other in the OPC neuroepithelium). Vsx1 and Rx act to promote Pm3 (Vsx1) or Pm1/2 (Rx) fates but can only do so in the absence of Optix (Erclik, 2017).

These results suggest that multi-columnar neurons are generated at specific locations in the medulla crescent. However, since these neurons are required to process visual information from the entire retina in the adult medulla, how does the doral-ventral position of neuronal birth in the larval crescent correlate with their final position in the adult? Lineage-tracing experiments were performed with Vsx1-Gal4 to permanently mark neurons generated in the cOPC and with Optix-Gal4 for mOPC neurons, and the position of the cell bodies of these neurons was analyzed. In larvae, neurons from the cOPC or from the mOPC remain located in the same dorsal-ventral position where they were born. However, in adults, both populations have moved to populate the entire medulla cortex along the dorsal-ventral axis (see Neuronal movement during medulla development is restricted to multi-columnar cell types). The kinetics of cell movement during development was analyzed by following cOPC neurons. Neurons born in the cOPC remain tightly clustered until 20 h after puparium formation (P20), after which point the cell bodies spread throughout the medulla cortex. By P30 the neurons are distributed over the entire dorsal-ventral axis of the medulla cortex. In the adult, most neurons derived from the cOPC neuroepithelium are located throughout the cortex although there is an enrichment of neurons in the central region of the cortex (Erclik, 2017).

To determine whether these observed movements involve the entire neuron or just the cell body, the initial targeting of cOPC or mOPC-derived neurons in larvae was examined before the onset of cell movement. In larvae, both populations send processes that target the entire dorsal-ventral axis of the medulla neuropi. Therefore, medulla neurons first send projections to reach their target columns throughout the entire medulla. Later, remodelling of the medulla results in extensive movement of cell bodies along the dorsal-ventral axis, leading to their even distribution in the cortex (Erclik, 2017).

What is the underlying logic behind why some neurons move while others do not? Markers were studied for the Mi1 (Bsh), Pm2 (Hth+Svp+), Pm1 (Hth+Svp+Tsh+), and Pm3 (Vsx1+Svp+Hth+) populations of neurons through pupal stages and up to the adult. Mi1 neurons are generated evenly throughout the larval OPC and remain regularly distributed across the dorsal-ventral axis at all stages. The lineage-tracing experiment was repeated with Vsx1-Gal4 to follow Mi1 neurons produced by the cOPC. These Mi1 neurons remain exclusively in the centre of the adult medulla cortex, demonstrating that they do not move. In contrast, Pm3 neurons remain tightly clustered in the central region until P20, at which point they move to occupy the entire cortex (Erclik, 2017).

However, not all multi-columnar neurons have cell bodies that move to occupy the entire medulla cortex. Unlike Mi1 and Pm3, adult Pm1 and Pm2 cell bodies are not located in the adult medulla cortex but instead in the medulla rim, at the edges of the cortex. Pm1 and Pm2 markers remain clustered at the ventral (Pm1) or dorsal (Pm2) posterior edges of the medulla cortex throughout all pupal stages. In the adult, both populations occupy the medulla rim from where they send long horizontal projections that reach the entire dorsal-ventral axis of the medulla neuropil. The pOPC may be a specialized region where many of the medulla rim cell types are generated. Even though most of cOPC-derived neurons move during development, a cOPC-derived multi-columnar neuron (TmY14) was identified that sends processes targeting the entire dorsal-ventral length of the medulla neuropil but whose cell bodies remain in the central medulla cortex in the adult (Erclik, 2017).

Thus, the four populations of Hth neurons follow different kinetics: Mi1 neurons are born throughout the OPC and do not move; Pm3 neurons are born centrally and then move to distribute throughout the entire cortex; and Pm1/Pm2 neurons are born at the ventral or dorsal posterior edges of the OPC and occupy the medulla rim in adults (Erclik, 2017).

It is noted that uni-columnar Mi1 neurons, whose cell bodies do not move, reside in the distal cortex whereas multi-columnar Pm3 neurons, which move, reside in the proximal cortex. The hypothesis was thus tested that neurons whose cell bodies are located distally in the medulla cortex represent uni-columnar neurons generated homogeneously throughout the OPC that do not move. In contrast, proximal neurons, which are fewer in number and are generated in specific subregions of the medulla OPC, would be multi-columnar and move to their final position (Erclik, 2017).

It was first confirmed that neurons that move have their cell bodies predominantly in the proximal medulla cortex. The cell body position of neurons born ventrally that have moved dorsally was analyzed using the hh-Gal4 lineage trace: in the adult, the cell bodies found dorsally are mostly in the proximal medulla cortex, whereas the cell bodies in the ventral region are evenly distributed throughout the distal-proximal axis of the ventral cortex. They probably represent both distal uni-columnar neurons that did not move as well as proximal multi-columnar neurons that remained in the ventral region (Erclik, 2017).

Next the pattern of movement of Tm2 uni-columnar neurons from the ventral and dorsal halves of the OPC was analyzed using the hh-based lineage-trace. The cell bodies of Tm2 neurons are located throughout the dorsal-ventral axis in the adult medulla cortex but are co-labelled with the hh lineage marker only in the ventral half. Thus, like Mi1, Tm2 uni-columnar neurons do not move. Furthermore, uni-columnar Tm1 neurons, labelled by 27b-Gal4, are born throughout the dorsal-ventral axis of the OPC crescent with distal cell bodies in the adult, suggesting that they also remain where they were born (Erclik, 2017).

Conversely, it was asked whether neurons that are specified in only one region, such as the Vsx+ neurons of the cOPC, are multi-columnar in morphology. By sparsely labelling cOPC-derived neurons using the Vsx1-Gal4 driver, 13 distinct cell types were characterized that retain Vsx1 expression in the adult medulla. Strikingly, all are multi-columnar in morphology, further supporting the model that it is the multi-columnar neurons that move during pupal development (Erclik, 2017).

Finally, MARCM clones were generated in the OPC neuroepithelium and visualized using cell-type-specific Gal4 drivers in the adult medulla. Two classes of adult clone distribution were observed: clones in which neurons are tightly clustered, and clones in which neurons are dispersed. Consistent with the model, the clustered clones are those labelled with uni-columnar neuronal drivers, whereas the dispersed clones are those labelled with a multi-columnar driver (Erclik, 2017).

Taken together, these data demonstrate that neurons that do not move are uni-columnar (with cell bodies in the distal cortex), whereas most multi-columnar neurons (with cell bodies in the proximal cortex) move (Erclik, 2017).

This study shows that combinatorial inputs from the temporal and spatial axes act together to promote neural diversity in the medulla. Previous work has shown that a temporal series of transcription factors expressed in medulla neuroblasts allows for a diversification of the cell types generated by the neuroblasts as they age. This study now shows that input from the dorsal-ventral axis leads to further diversification of the neurons made by neuroblasts; at a given temporal stage, neuroblasts produce the same uni-columnar neuronal type globally as well as smaller numbers of multi-columnar cell types regionally. This situation is reminiscent of the mode of neurogenesis in the Drosophila ventral nerve cord, in which each neuroblast also expresses a (different) temporal series of transcription factors that specifies multiple neuronal types in the lineage. Spatial cues from segment polarity, dorsal-ventral and Hox genes then intersect to impart unique identities to each of the lineages. However, neuroblasts from the different segments give rise to distinct lineages to accommodate the specific function of each segment. In contrast, in the medulla, the entire OPC contributes to framing the repeating units that form the retinotopic map. It is therefore likely that each neuroblast produces a common set of neurons that connect to each pair of incoming R7 and R8 cells, or L1-L5 lamina neurons. This serves to produce 800 medulla columns with a 1:1 stoichiometry of medulla neurons to photoreceptors. The medulla neurons that are produced by neuroblasts throughout the dorsal-ventral axis of the OPC are thus uni-columnar The production of the same neuronal type along the entire OPC could be achieved by selectively 'erasing' spatial information in uni-columnar neurons, as observed in Mi1 neurons (Erclik, 2017).

Regional differences in the OPC confer further spatial identities to neuroblasts with the same temporal identity, and lead to specific differences in the lineages produced in the compartments along the dorsal-ventral axis of the medulla. These differences produce smaller numbers of multi-columnar neurons whose stoichiometry is much lower than 1:1. The majority of these neurons move during development to be uniformly distributed in the adult medulla cortex. This combination of regional and global neuronal specification in the medulla presents a powerful mechanism to produce the proper diversity and stoichiometry of neuronal types and generate the retinotopic map (Erclik, 2017).

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) lozengesprite, 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 lozengesprite 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 lozengesprite 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, lozengesprite, 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 lozengesprite 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 lozengesprite 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 lozengesprite animals, the pattern of targets chosen was not grossly distorted, though minor irregularities were seen. The effects seen in lozengesprite 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 lozengesprite 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 lozengesprite 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 rolledSevenmaker (rlSem), encodes a form of ERK with increased resistance to inactivation by dephosphorylation. Expression of a dominant-negative form of Fos in the wings of rlSem flies results in loss of ectopic vein material. Conversely, overexpression of Fos enhances the extra-vein phenotype caused by rlSem. 32B Gal4, UAS Sem flies express the RlSem 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).

Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila

The timing mechanisms responsible for terminating cell proliferation toward the end of development remain unclear. In the Drosophila CNS, individual progenitors called neuroblasts are known to express a series of transcription factors endowing daughter neurons with different temporal identities. This study shows that Castor and Seven-Up, members of this temporal series, regulate key events in many different neuroblast lineages during late neurogenesis. First, they schedule a switch in the cell size and identity of neurons involving the targets Chinmo and Broad Complex. Second, they regulate the time at which neuroblasts undergo Prospero-dependent cell-cycle exit or Reaper/Hid/Grim-dependent apoptosis. Both types of progenitor termination require the combined action of a late phase of the temporal series and indirect feedforward via Castor targets such as Grainyhead and Dichaete. These studies identify the timing mechanism ending CNS proliferation and reveal how aging progenitors transduce bursts of transcription factors into long-lasting changes in cell proliferation and cell identity (Maurange, 2008).

Initially investigated was whether distinct temporal subsets of neurons are generated throughout the larval CNS. Chinmo and Broad Complex (Br-C), two BTB-zinc finger proteins known to be expressed in the postembryonic CNS, are distributed in complementary patterns in the central brain, thoracic, and abdominal neuromeres at the larval/prepupal transition stage at 96 hr (timings are relative to larval hatching at 0 hr). Chinmo is expressed by early-born neurons located in a deep layer, whereas Br-C marks later-born neurons in a largely nonoverlapping and more superficial layer. The deep Chinmo+ layer comprises most/all neurons born in the embryo plus an early subset of those generated postembryonically. Thoracic postembryonic neuroblasts undergo the Chinmo --> Br-C switch at ~60 hr such that they have each generated an average of 15 Chinmo+ cells expressing little or no Br-C and 39 Chinmo- Br-C+ cells by 96 hr. The Chinmo+ and Br-C+ neuronal identities can be recognized as distinct cell populations on the basis of an ~2-fold difference in cell-body volume. This equates to an average cell-body diameter for Chinmo+ neurons of 4.5 μm, compared to only 3.6 μm for Br-C+ neurons. Plotting cell diameter versus deep-to-superficial position within postembryonic neuroblast clones reveals an abrupt decrease in neuronal size at the Chinmo --> Br-C transition. Together, these results provide evidence that most, if not all, postembryonic neuroblasts sequentially generate at least two different populations of neurons. First they generate large Chinmo+ neurons and then they switch to producing smaller Br-C+ neurons (Maurange, 2008).

To begin dissecting the neuronal switching mechanism, the functions of Chinmo and Br-C were investigated, but neither factor was found to be required for the transition in cell identity and cell size. Next it was asked whether a temporal transcription factor series related to the embryonic Hb --> Kr --> Pdm --> Cas sequence might be involved. Cas is known to be expressed in the larval CNS, and this study shows that many different thoracic neuroblasts progress through a transient Cas+ phase during the 30-50 hr time window. Thoracic neuroblasts transiently express another member of the embryonic temporal series, Svp, during a somewhat later time window, from ~40 to ~60 hr. These results indicate that postembryonic Cas and Svp bursts are observed in many, but probably not all, thoracic progenitors and that their timing varies from neuroblast to neuroblast (Maurange, 2008).

To determine Svp function, thoracic neuroblast clones were generated homozygous for svpe22, an amorphic allele. In ~53% of svpe22 neuroblast clones induced in the early larva (at 12-36 hr), the Br-C+ neuronal identity is completely absent, all neurons express Chinmo, and there is no sharp decrease in neuronal size. The proportion of lineages failing to generate Br-C+ neurons rises to ~70% when clones are induced in the embryo and falls to only ~7% with late-larval (65-75 hr) induction. This is consistent with a previous finding that Svp bursts are asynchronous from neuroblast to neuroblast. The expression and clonal analyses together demonstrate that a progenitor-specific burst of Svp is required in many lineages for the switch from large Chinmo+ to small Br-C+ neurons (Maurange, 2008).

Thoracic neuroblast lineages homozygous for a strong cas allele, cas24, show no obvious defects in the Chinmo --> Br-C transition when induced at 12-36 hr. However, since Cas is expressed in many postembryonic neuroblasts before their first larval division, it can only be removed by inducing clones during embryonic neurogenesis. Such cas24 clones generate supernumerary Chinmo+ neurons and completely lack Br-C+ neurons at 96 hr, although this switching phenotype is restricted to only ~16% of thoracic neuroblasts. Constitutively expressing Cas blocks the Chinmo --> Br-C switch in a similar manner, with a frequency dependent upon whether thoracic UAS-cas clones are induced during embryogenesis (~47%), at early-larval (~10%) or at late-larval (0%) stages. This indicates that the response to Cas misexpression decreases as neuroblasts age. Together, the expression and loss- and gain-of-function analyses demonstrate that Chinmo and Br-C are negative and positive targets, respectively, of Cas and Svp. They also strongly suggest that progression through transient Cas+ and Svp+ states permits many postembryonic neuroblasts to switch from generating large to small neurons (Maurange, 2008).

To investigate whether Cas and Svp regulate neural proliferation as well as neuronal fates, the effector mechanism ending neurogenesis in the central brain and thorax was identified. In these regions, most neuroblasts cease dividing in the pupa at ~120 hr. Correspondingly, neurogenesis in all regions of the wild-type CNS ceases before the adult fly ecloses such that no adult neuroblasts are detected. In contrast to the central abdomen, blocking cell death by removing Reaper, Hid, and/or Grim (RHG) activity in the central brain and thorax does not prevent or delay pupal neuroblast disappearance. However, time-lapse movies of individual thoracic neuroblasts at ~120 hr reveal an atypical mitosis that is much slower than at ~96 hr, producing two daughters of almost equal size. This is largely accounted for by a reduction in the average diameter of neuroblasts from 10.4 μm at 96 hr to 7 μm at 120 hr, as GMC size does not vary significantly during this time window. The end of this atypical progenitor mitosis temporally correlates with reduced numbers of Mira+ cells and disappearance of the M phase marker phosphorylated-Histone H3 (PH3), indicating that it marks the terminal division of the neuroblast (Maurange, 2008).

Next whether late changes in basal complex components might underlie loss of neuroblast self-renewal was addressed. At 120 hr, it was found that Mira becomes delocalized from the cortex to the cytoplasm and nucleus of many interphase neuroblasts. In metaphase neuroblasts, Mira fails to localize to the basal side of the cortex, although it does selectively partition into one daughter during telophase. This late basal restoration resembles the 'telophase rescue' associated with several apical complex mutations. Pros, the Mira-binding transcription factor and GMC-determinant, is not detectable in the neuroblast nucleus at 96 hr, but at 120 hr a burst of Pros was observe in the nucleus of many Mira+ cells of intermediate size indicative of neuroblasts in the interphase preceding the terminal mitosis. Clones lacking Pros activity contain multiple Mira+ neuroblast-like cells. It was found that they do not respect the ~120 hr proliferation endpoint and even retain numerous dividing Mira+ progenitors into adulthood. In addition, GAL80ts induction was used to induce transiently the expression of a YFP-Pros fusion protein well before the normal ~120 hr endpoint. Under these conditions, YFP-Pros can be observed in the nucleus of neuroblasts, most Mira+ progenitors disappear prematurely, and neural proliferation ceases much earlier than normal. Together, these results provide evidence that most neuroblasts terminate activity in the pupa via a nuclear burst of Pros that induces cell-cycle exit. These progenitors are referred to as type I neuroblasts to distinguish them from the much smaller population of type II neuroblasts that terminate via RHG-dependent apoptosis (Maurange, 2008).

Whether the postembryonic pulses of Cas and Svp in type I neuroblasts are implicated in scheduling their subsequent cell-cycle exit was tested. Remarkably, it was observed that many svpe22 clones induced at early-larval stages retain a single Mira+ neuroblast at 7 days into adulthood. The persistent adult neuroblasts in a proportion of these clones also express the M phase marker, PH3, indicating that they remain engaged in the cell cycle and, accordingly, they generate approximately twice the normal number of cells by 3 days into adulthood. Furthermore, superficial last-born neurons in these over proliferating svpe22 adult clones are Chinmo+ Br-C- indicating a blocked Chinmo --> Br-C transition. Similar phenotypes are obtained in some UAS-cas and cas24 clones. This analysis demonstrates that stalling the temporal series not only inhibits the late-larval switch to Br-C+ neuronal identity but also prevents the pupal cell-cycle exit of type I neuroblasts (Maurange, 2008).

To test the regulatory relationship between Pros and the temporal series, svpe22 clones were examined at pupal stages. It was found that mutant interphase neuroblasts fail to switch on nuclear Pros at 120 hr, although svpe22 GMCs express nuclear Pros as normal. This likely accounts for why adult clones lacking Svp retain only a single neuroblast, whereas those lacking Pros contain multiple neuroblast-like progenitors. Importantly, these results demonstrate that nuclear Pros acts downstream of the temporal series in type I neuroblasts. Together, the genetic and expression analyses of Svp and Pros show that the temporal series triggers a burst of nuclear Pros in type I neuroblasts, thus inducing their cell-cycle exit (Maurange, 2008).

To determine how the temporal series is linked to the cessation of progenitor divisions, two transcription factors expressed in neuroblasts in a temporally restricted manner were examined. Dichaete (D), a member of the SoxB family, is dynamically expressed in the early embryo and is required for neuroblast formation. Consistent with the previous studies, it was observed that most or all embryonic neuroblasts progress through a transient D+ phase, but those in the lateral column of the ventral nerve cord initiate expression after their medial and intermediate counterparts. Dichaete subsequently becomes repressed in ~85% of neuroblasts during late-embryonic and postembryonic stages. Grainyhead (Grh) is first activated in neuroblasts in the late embryo and is required for regulating their mitotic activity during larval stages. Blocking early temporal series progression in the embryo, either by persistent Hb or loss of Cas activity, prevents most neuroblasts from downregulating D and also from activating Grh at late-embryonic and postembryonic stages. As forcing premature Cas expression leads to precocious D repression and Grh activation, both factors are likely to be regulated by Cas rather than by a later member of the temporal series. These results demonstrate that transient embryonic Cas activity permanently switches the expression of Grh on and D off. They also identify Grh and D as positive and negative targets, respectively, of the temporal series in many neuroblasts (Maurange, 2008).

Loss of Grh activity in thoracic neuroblasts (here defined as type I neuroblasts) leads to their reduced cell-cycle speed and disappearance during larval stages. At 96 hr, it was observed that 65% of grh370 type I neuroblasts are smaller than normal (~6.4 μm in diameter), delocalize Mira from the cortex to the cytoplasm and nucleus, and strongly express Pros in the nucleus. These events, reminiscent of the 120 hr terminal cell cycle of wild-type progenitors, show that Grh is required to prevent the premature cell-cycle exit of type I neuroblasts. Given this finding, and that Cas is required to activate Grh, the question arises as to how some neuroblasts lacking embryonic Cas activity are able to continue dividing into adulthood. However, cas24 neuroblasts persisting in adults all retain Grh, suggesting that they may derive from clones that lacked only the last of the two Cas pulses observed in some embryonic neuroblasts, perhaps retaining enough Cas activity to support Grh activation but not later progression of the temporal series (Maurange, 2008).

To determine if late temporal series inputs, after embryonic Cas, are also required to maintain the long-lasting postembryonic expression of Grh, svpe22 clones were induced in early larvae. Although type I neuroblasts in these mutant clones have a stalled temporal series, they retain postembryonic Grh expression through to adult stages. Thus, two sequential inputs from the temporal series are required for type I neuroblasts to undergo timely Pros-dependent cell-cycle exit. First, embryonic Cas activity switches on sustained Grh expression, inhibiting premature nuclear Pros and permitting continued mitotic activity. Second, a late postembryonic input, requiring Svp, counteracts this activity of Grh by triggering a pupal burst of nuclear Pros (Maurange, 2008).

Despite undergoing premature cell-cycle exit, it was noticed that grh mutant neuroblasts can still generate both Chinmo+ and Br-C+ neurons. Thus, Grh is not required for the neuronal Chinmo --> Br-C switch. Conversely, neither Chinmo nor Br-C appears to be required postembryonically for neuroblast cell-cycle exit. In summary, the properties of both neuroblasts and neurons are regulated by downstream targets of the temporal series (Maurange, 2008).

Next whether the temporal series and its targets also function in type II neuroblasts, which terminate via RHG-dependent apoptosis rather than Pros-dependent cell-cycle exit, was tested. Focus was placed on one identified type II neuroblast in the central abdomen, called dl, which undergoes apoptosis at 70-75 hr and produces only a small postembryonic lineage of ~10 neurons. It was observed that the dl neuroblast expresses bursts of Cas (~45 to ~60 hr) and Svp (~62 to ~65 hr) and sequentially generates Chinmo+ (~7 deep) and Br-C+ (~3 superficial) neurons. Loss of Cas or Svp activity, or prevention of temporal series progression in several other ways all lead to a blocked Chinmo --> Br-C transition, a failure to die at 70-75 hr, and the subsequent generation of many supernumerary progeny. These results show that the temporal series performs similar functions in type I and type II neuroblast lineages, regulating both the Chinmo --> Br-C neuronal switch and the cessation of progenitor activity. Next, the mechanism linking the temporal series to RHG-dependent death of type II neuroblasts was tested. As for type I neuroblasts, dl progenitors in cas24 clones, induced in the embryo, fail to activate Grh and repress D. Moreover, if grh activity is reduced, or if D is continuously misexpressed, dl progenitors persist long after 75 hr. Therefore, both of these early Cas-dependent events are essential for subsequent type-II neuroblast apoptosis. However, in contrast to members of the temporal series themselves, persistent misexpression of their target D does not block the Chinmo --> Br-C switch in the dl lineage. Thus, the D- Grh+ state of type I and type II neuroblasts, installed via an embryonic Cas pulse, appears to be necessary for progenitor termination but not for Chinmo --> Br-C switching. Nevertheless, dl neuroblasts stalled at a postembryonic stage in svpe22 and UAS-cas clones retain the D- Grh+ code yet still fail to undergo apoptosis. Therefore, as with type I cell-cycle exit, timely type II apoptosis requires both embryonic Cas-dependent and postembryonic Svp-dependent inputs from the temporal series (Maurange, 2008).

Finally, the regulatory relationship between the temporal series and AbdA, a Hox protein transiently expressed in postembryonic type II (but not type I) neuroblasts and required for their apoptosis was dissected. dl neuroblasts lacking postembryonic Svp activity or persistently expressing Cas still retain AbdA expression yet do not die. This suggests that AbdA is unable to kill neuroblasts unless they progress, in a Svp-dependent manner, to a late Cas- temporal state. To test this prediction directly, use was made of the previous finding that ectopic AbdA is sufficient to induce neuroblast apoptosis, albeit only within a late time window. Constitutive AbdA-induced apoptosis is efficiently suppressed by persistent Cas, not only in type II but also in type I neuroblasts. This result demonstrates that, in order to terminate, type II neuroblasts must progress to a late Cas- state, thus acquiring a D- Grh+ Cas- AbdA+ code. It also suggests that AbdA is sufficient to intercept progression of the temporal series in type I neuroblasts, inducing an early type II-like termination (Maurange, 2008).

This study has found that the Drosophila CNS contains two distinct types of self-renewing progenitors: type I neuroblasts terminate divisions by cell-cycle withdrawal and type II neuroblasts via apoptosis. Despite these different exit strategies, both progenitor types use a similar molecular timer, the temporal series, to shut down proliferation and thus prevent CNS overgrowth. These findings demonstrate that the temporal series does considerably more than just modifying neurons; it also has multiple inputs into neural proliferation. The identification and analysis of several pan-lineage targets of the temporal series also begins to shed light on the mechanism by which developmental age modifies the properties of neuroblasts and neurons. Two targets, Chinmo and Br-C, are part of a downstream pathway temporally regulating the size and identity of neurons. Two other temporal series targets, Grh and D, function in neuroblasts to regulate Prospero/RHG activity, thereby setting the time at which proliferation ends. The temporal series regulates both cell proliferation and cell identity; a feedforward mechanism is proposed for generating combinatorial transcription factor codes during progenitor aging (Maurange, 2008).

This study has found that the temporal series regulates a widespread postembryonic switch in neuronal identity. Most, if not all, type I and type II neuroblasts first generate a deep layer of large Chinmo+ neurons and then switch to producing a superficial layer of small Br-C+ neurons. Two lines of evidence argue that this postembryonic neuronal switch is likely to be regulated by a continuation of the same temporal series controlling early/late neuronal identities in the embryo. First, the postembryonic Chinmo --> Br-C neuronal switch is promoted by the transient redeployment of two known components of the embryonic temporal series, Cas and Svp. Second, this switch remains inhibitable by misexpression of the other embryonic temporal factors such as Hb. Since both Cas and Svp are expressed somewhat earlier than the neuronal-size transition, it is likely that they promote bursts of later, as yet unknown, members of the temporal series that more directly regulate Chinmo and Br-C. Although neuronal functions for both BTB zinc-finger targets have yet to be characterized, a progressive early-to-late decrease in postmitotic Chinmo levels is known to regulate the temporal identities of mushroom-body neurons. The current results now suggest that this postmitotic gradient mechanism may be linked to, rather than independent from, the temporal series (Maurange, 2008).

Type I neuroblasts in clones lacking postembryonic Cas/Svp activity or retaining an early temporal factor, fail to express nuclear Pros during pupal stages and thus continue dividing long into adulthood. These overproliferating adult clones each contain only a single neuroblast, sharply contrasting with adult clones lacking Brat or Pros, in which there are multiple neuroblast-like progenitors. Hence, manipulations of the temporal series and its progenitor targets offer the prospect of immortalizing neural precursors in a controlled manner, without disrupting their self-renewing asymmetric divisions (Maurange, 2008).

This study demonstrates that type I and type II neuroblasts must progress through at least two critical phases of the temporal series in order to acquire the D- Grh+ Cas- combinatorial transcription factor code that precedes Pros/RHG activation. The early phase corresponds to embryonic Cas activity switching neuroblasts from D+ Grh- to D- Grh+ status. The equally essential, but less well-defined, late postembryonic phase of the temporal series requires transition to a Cas- state and a late Svp burst. For type I neuroblasts, Grh and a late Cas- temporal identity are both required for timely expression of nuclear Pros and subsequent cell-cycle withdrawal. For type II neuroblasts, these two inputs are also necessary for RHG-dependent apoptosis, with the additional requirement that D must remain repressed. Although the temporal series and its targets are similarly expressed in type I and type II neuroblasts, only the latter progenitors undergo a larval burst of AbdA. This AbdA expression is likely to be the final event required to convert the D- Grh+ Cas- state, installed by the temporal series, into the D- Grh+ Cas- AbdA+ combinatorial code for RHG-dependent apoptosis. This code prevents type II neuroblasts in the abdomen from reaching the end of the temporal series and accounts for why they generate fewer progeny and terminate earlier than their type I counterparts in the central brain and thorax (Maurange, 2008).

The data in this study support an indirect feedforward model for neuroblast aging. Key to this model is the finding that, although members of the temporal series are only expressed very transiently, some of their targets can be activated or repressed in a sustained manner, as observed for Chinmo/Br-C in neurons and also for Grh/D in neuroblasts. In principle, this indirect feedforward allows aging progenitors to acquire step-wise the combinatorial transcription factor codes modulating cell-cycle speed, growth-factor dependence, competence states, and neural potential. Like Drosophila neuroblasts, isolated mammalian cortical progenitors can sequentially generate neuronal fates in the correct in vivo order. These studies suggest that it will be important to investigate whether the transcription factors controlling this process also regulate cortical proliferation and whether their targets include BTB-zinc finger, Grh, SoxB, Prox, or proapoptotic proteins. Some insect/mammalian parallels seem likely, since it is known that Sox2 downregulation and Prox1 upregulation can both promote the cell-cycle exit of certain types of vertebrate neural progenitors (Dyer, 2003, Graham, 2003). Thus, although insect and mammalian neural progenitors do not appear to use the same sequence of temporal transcription factors, at least some of the more downstream components identified in this study might be functionally conserved (Maurange, 2008).

Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons

Specificity of sensory neurons requires restricted expression of one sensory receptor gene and the exclusion of all others within a given cell. In the Drosophila retina, functional identity of photoreceptors depends on light-sensitive Rhodopsins (Rhs). The much simpler larval eye (Bolwig organ; see The Extraretinal Eyelet of Drosophila: Development, Ultrastructure, and Putative Circadian Function) is composed of about 12 photoreceptors, eight of which are green-sensitive (Rh6) and four blue-sensitive (Rh5). The larval eye becomes the adult extraretinal 'eyelet' composed of four green-sensitive (Rh6) photoreceptors. This study shows that, during metamorphosis, all Rh6 photoreceptors die, whereas the Rh5 photoreceptors switch fate by turning off Rh5 and then turning on Rh6 expression. This switch occurs without apparent changes in the programme of transcription factors that specify larval photoreceptor subtypes. It was also shown that the transcription factor Senseless (Sens) mediates the very different cellular behaviours of Rh5 and Rh6 photoreceptors. Sens is restricted to Rh5 photoreceptors and must be excluded from Rh6 photoreceptors to allow them to die at metamorphosis. Finally, Ecdysone receptor (EcR) was shown to function autonomously both for the death of larval Rh6 photoreceptors and for the sensory switch of Rh5 photoreceptors to express Rh6. This fate switch of functioning, terminally differentiated neurons provides a novel, unexpected example of hard-wired sensory plasticity (Sprecher, 2008).

The adult Drosophila eyelet comprises approximately four photoreceptors located between the retina and the optic ganglia. It directly contacts the pacemaker neurons of the adult fly, the lateral neurons. In conjunction with the compound eye and the clock-neuron intrinsic blue-sensitive receptor cryptochrome it helps shift the phase of the molecular clock in response to light. All eyelet photoreceptors express green-sensitive Rh6, and are derived from photoreceptors of the larval eye that mediate light avoidance and entrainment of the molecular clock by innervating the larval lateral neurons (Sprecher, 2008).

Larval photoreceptors develop in a two-step process during embryogenesis. Primary precursors are specified first and develop as the four Rh5-subtype photoreceptors. They signal through Epidermal growth factor receptor (EGFR) to the surrounding tissue to develop as secondary precursors, which develop into the eight Rh6-subtype photoreceptors. Two transcription factors specify larval photoreceptor subtypes. Spalt (Sal) is exclusively expressed in Rh5 photoreceptors, where it is required for Rh5 expression. Seven-up (Svp) is restricted to Rh6 photoreceptors, where it represses sal and promotes Rh6 expression. A third transcription factor, Orthodenticle (Otd), expressed in all larval photoreceptors, acts only in the Rh5 subtype to promote Rh5 expression and to repress Rh6 (Sprecher, 2008 and references therein).

To address the relation between the larval Rh5 and Rh6 photoreceptors and the adult eyelet, they were tracked through metamorphosis. To permanently label them, UAS-Histone2B::YFP, which is stably incorporated in the chromatin, and thus remains detectable in post-mitotic neurons throughout pupation, was used. Surprisingly, all Rh6 photoreceptors degenerate and disappear during early phases of metamorphosis. In contrast, Rh5 photoreceptors can be followed throughout pupation. Expression of Rh5 ceases during early stages of pupation and, at mid-pupation, neither Rh5 nor Rh6 can be detected. About four cells are still present, however, and can be identified by rh5-Gal4/UAS-H2B::YFP or GMR-Gal4/UAS-H2B::YFP. Eyelet photoreceptors only express Rh6, even though H2B::YFP driven by rh5-Gal4 is detectable in those cells. Therefore, the four larval Rh5 photoreceptors must switch rhodopsin expression at metamorphosis to give rise to the four eyelet Rh6 photoreceptors. The remaining eight Rh6 photoreceptors die, their axon becoming fragmented before disappearing. A 'memory experiment' (rh5-Gal4/UAS-Flp;Act-FRT > STOP > FRT-nlacZ) also showed that eyelet Rh6 photoreceptors did express Rh5 earlier (Sprecher, 2008).

The death of Rh6 photoreceptors and transformation of Rh5 photoreceptors was further verified by three independent sets of experiments (Sprecher, 2008).

(1) Rh5 photoreceptors were ablated by expressing pro-apoptotic genes rpr and hid (rh5-Gal4/UAS-rpr,UAS-hid). This results in the absence of larval Rh5 photoreceptors and the complete absence of the eyelet. Conversely, preventing cell death of the Rh6 subtype by expressing the apoptosis inhibitor p35 (rh6-Gal4/UAS-p35) leads to an eyelet that consists of 12 photoreceptors, all expressing Rh6 (Sprecher, 2008).

(2) Larval Rh6 photoreceptors development was blocked by expressing a dominant negative form of EGFR (so-Gal4/UAS-H2B::YFP; UAS-EGFRDN). The eyelet of these animals is not affected and three or four cells express Rh6 normally. This shows that larval Rh6 photoreceptors do not contribute to the eyelet (Sprecher, 2008).

(3) The expression of Sal (Rh5-subtype specific) and Svp (Rh6-subtype specific) was analyzed in the adult eyelet: eyelet photoreceptors still express Sal, but not Svp even though these photoreceptors now express Rh6. Rh5 requires Sal expression in the Bolwig organ, but Otd function is also necessary to activate Rh5 and to repress Rh6. In otd mutants, larval Rh5 photoreceptors marked by Sal express Rh6 and lack Rh5 expression, thus mimicking the switch at metamorphosis. Thus, Rh6 could be expressed in Rh5 photoreceptors if otd function were lost in the eyelet. However, Otd expression does not change during the transition from the Bolwig organ to eyelet although it might be inactive in the eyelet (Sprecher, 2008).

What is the trigger that controls the switch from rh5 to rh6? Ecdysone controls many developmental processes during metamorphosis. EcR is expressed during the third larval instar and pupation in all larval photoreceptors and surrounding tissues. To evaluate EcR activity, a reporter line was used in which lacZ is under the control of multimerized ecdysone response elements (7XEcRE-lacZ). The expression of lacZ is absent until late third instar and prepupation, whereas thereafter all larval photoreceptors (and surrounding tissue) express 7XEcRE-lacZ. EcR expression decreases during late pupation and is no longer detectable by the time Rh6 expression starts in the eyelet (Sprecher, 2008).

To test the role of ecdysone, a dominant negative form of EcR was expressed specifically in larval Rh5 photoreceptors, while permanently labelling these cells (rh5-Gal4/UAS-H2B::YFP;UAS-EcRDN). This causes no disruption of larval photoreceptor fate, but the eyelet of these animals now consists of four photoreceptors that all express Rh5 instead of Rh6. A comparable phenotype is observed after expression of an RNA interference (RNAi) construct for EcR (rh5-Gal4/UAS-H2B::YFP;UAS-EcRRNAi). Therefore, loss of EcR function prevents larval photoreceptors from switching to Rh6 expression. In both cases, larval Rh6 photoreceptors still degenerate and are not observed in the eyelet (Sprecher, 2008).

The dominant negative form of EcR was also expressed in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP; UAS-EcRDN). In this case, the Bolwig organ is not affected but the resulting adult eyelet consists of about 12 photoreceptors, all expressing Rh6. This presumably results from Rh6 photoreceptors not undergoing apoptosis whereas larval Rh5 photoreceptors still switch expression to Rh6 in the eyelet. Expression of UAS-EcR-RNAi in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-EcRRNAi) leads to the same results (Sprecher, 2008).

Although EcR could directly control the switch of rhodopsin expression through binding to the promoters of rh5 and rh6, these promoters contain no potential EcR binding sites. Moreover, as no EcR expression is detectable when Rh6 starts to be expressed, this would make it unlikely for EcR to control directly the switch to Rh6. Finally, only allowing expression of the dominant negative form of EcR starting at mid-pupation (GMR-Gal4/Tub-Gal80ts,UAS-EcRDN), after rh5 is switched off, does not prevent activation of Rh6 in the eyelet. Thus EcR most likely acts in an indirect manner in regulating rhodopsins, likely through the activation of transcription factors that bind to rh5 and rh6 promoters (Sprecher, 2008).

The differential response to ecdysone of Rh6 photoreceptors (which die) and of Rh5 photoreceptors (which switch to Rh6) must be due to intrinsic differences between the two subtypes before EcR signalling. Likely candidates are Sal and Svp. However, late misexpression of Svp in Rh5 photoreceptors (rh5-Gal4/UAS-H2B::YFP;UAS-svp) or of Sal in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-sal) neither affects rhodopsin expression or cell number in the eyelet nor alter the expression of rhodopsins in the Bolwig organ (which is only affected by very early expression of these transcription factors, through so-Gal4. Thus neither Sal nor Svp are sufficient to alter the response of larval photoreceptors to EcR (Sprecher, 2008).

An additional factor, independent from svp and sal, must therefore allow survival of Rh5 photoreceptors, or promote Rh6 photoreceptor death. It was found that the transcription factor Sens is specifically expressed in larval Rh5 photoreceptors and remains expressed in all cells in the eyelet where it might act to promote cell survival. To test this, sens was misexpressed in Rh6 photoreceptors (rh6-Gal4/UAS-H2B::YFP;UAS-sens). This results in an eyelet that consists of 12 photoreceptors, all expressing Rh6. Thus, expression of Sens in Rh6 photoreceptors is sufficient to rescue them from death, without affecting Sal and Svp expression and subtype specification of larval photoreceptors (Sprecher, 2008).

Ecdysone hormonal signalling thus acts in two independent ways during the formation of the adult eyelet. First, it induces the degeneration of the Rh6 subtype, thereby assuring the correct number of eyelet photoreceptors. This apoptotic death requires the absence of Sens, whose expression is restricted to Rh5 photoreceptors that survive. Second, ecdysone signalling is also required to trigger the switch of spectral sensitivity of blue-sensitive (Rh5) larval photoreceptors to green-sensitive (Rh6) eyelet photoreceptors (Sprecher, 2008).

Thus terminally differentiated sensory neurons switch specificity by turning off one Rhodopsin and replacing it with another. Although examples of such switches in sensory specificity of terminally differentiated, functional, sensory receptors are extremely rare, this strategy might be more common than currently anticipated. In the Pacific pink salmon and rainbow trout, newly hatched fish express an ultraviolet opsin that changes to a blue opsin as the fish ages. As in flies, this switch might reflect an adaptation of vision to the changing lifestyle. The maturing salmon, born in shallow water, later migrates deeper in the ocean where ultraviolet does not penetrate. The rhodopsin switch in the eyelet may similarly be an adaptation to the deeper location of the eyelet within the head, as light with longer wavelengths (detected by Rh6) penetrates deeper into tissue than light with shorter wavelengths (detected by Rh5) (Sprecher, 2008).

The eyelet functions with retinal photoreceptors and Cryptochrome to entrain the molecular clock in response to light. The larval eye, on the other hand, functions in two distinct processes: for the entrainment of the clock and for the larva to avoid light. Interestingly, the Rh5 subtype appears to support both functions whereas Rh6 photoreceptors only contribute to clock entrainment. Thus, the photoreceptor subtype that supports both functions of the larval eye is the one that is maintained into the adult and becomes the eyelet. Why are Rh6-sensitive photoreceptors not maintained? As these photoreceptors are recruited to the larval eye secondarily, the ancestral Bolwig organ might have had only Rh5 photoreceptors and had to undergo a switch in specificity. Larval Rh5 photoreceptors appear to maintain their overall connectivity to the central pacemaker neurons. However, they are also profoundly restructured and exhibit widely increased connectivity during metamorphosis. This might be due to the increase in number of their target neurons, and the switch of Rh might be part of more extensive plasticity during formation of the eyelet, including increased connectivity and possibly the innervation of novel target neurons (Sprecher, 2008).

The general model that sensory neurons express only a single sensory receptor gene does not hold true for salmon and the fruitfly. Interestingly, reports from several other species, including amphibians, rodents and humans, show co-expression of opsins. In humans, for instance, it has been proposed that cones first express S opsin and later switch to L/M opsin. However, this likely reflects a developmental process rather than a functional adaptation (Sprecher, 2008).

This study identified two major players in the genetic programme for the transformation of the larval eye to the eyelet. (1) EcR acts as a trigger for both rhodopsin switch and apoptosis. Surprisingly, the upstream regulators specifying larval photoreceptor-subtype identity, Sal, Svp and Otd, do not contribute to the genetic programme of sensory plasticity of the rhodopsin switch. Therefore a novel genetic programme is required for regulating rhodopsin expression in the eyelet, which likely depends on downstream effectors of EcR (Sprecher, 2008).

(2) Larval Rh5 and Rh6 photoreceptors respond differently to ecdysone, either switching rhodopsin expression or undergoing apoptosis. This appears to depend on Sens, which is likely to be required for the survival of Rh5 photoreceptors. The role of Sens in inhibiting apoptosis is not unique to this situation: Sens is essential to promote survival of salivary-gland precursors during embryogenesis. The vertebrate homologue of sens, Gfi-1, acts to inhibit apoptosis of T-cell precursors in haematopoiesis and cochlear hair cells of the inner ear. Thus the anti-apoptotic function of Sens/Gfi-1 may be a general property of this molecule (Sprecher, 2008).

Ecdysone acts in remodelling neurons during metamorphosis. In γ-neurons of the mushroom body, a structure involved in learning and memory, ecdysone is required for the pruning of larval processes. Similarly, dendrites of C4da sensory neurons undergo large-scale remodelling that depends on ecdysone signalling. Interestingly, in the moth Manduca, 'lateral neurosecretory cells' express cardio-acceleratory peptide 2, which is switched off in response to ecdysone before expression of the neuropeptide bursicon is initiated in the adult (Sprecher, 2008).

The transformation of larval blue-sensitive photoreceptors to green-sensitive photoreceptors of the eyelet reveals an unexpected example of sensory plasticity by switching rhodopsin gene expression in functional, terminally differentiated sensory neurons (Sprecher, 2008).

Binary cell fate decisions and fate transformation in the Drosophila larval eye

The functionality of sensory neurons is defined by the expression of specific sensory receptor genes. During the development of the Drosophila larval eye, photoreceptor neurons (PRs) make a binary choice to express either the blue-sensitive Rhodopsin 5 (Rh5) or the green-sensitive Rhodopsin 6 (Rh6). Later during metamorphosis, ecdysone signaling induces a cell fate and sensory receptor switch: Rh5-PRs are re-programmed to express Rh6 and become the eyelet, a small group of extraretinal PRs involved in circadian entrainment. However, the genetic and molecular mechanisms of how the binary cell fate decisions are made and switched remain poorly understood. This study shows that interplay of two transcription factors Senseless (Sens) and homeodomain transcription factor Hazy [PvuII-PstI homology 13, Pph13] control cell fate decisions, terminal differentiation of the larval eye and its transformation into eyelet. During initial differentiation, a pulse of Sens expression in primary precursors regulates their differentiation into Rh5-PRs and repression of an alternative Rh6-cell fate. Later, during the transformation of the larval eye into the adult eyelet, Sens serves as an anti-apoptotic factor in Rh5-PRs, which helps in promoting survival of Rh5-PRs during metamorphosis and is subsequently required for Rh6 expression. Comparably, during PR differentiation Hazy functions in initiation and maintenance of rhodopsin expression. Hazy represses Sens specifically in the Rh6-PRs, allowing them to die during metamorphosis. These findings show that the same transcription factors regulate diverse aspects of larval and adult PR development at different stages and in a context-dependent manner (Mishra, 2013).

In the larval eye, determination of primary or secondary precursors to acquire either Rh5-PR or Rh6-PR identity depends on the transcription factors Sal, Svp and Otd. Primary as well as secondary precursors have the developmental potential to express Rh5 or Rh6. During differentiation, a pulsed expression of Sens acts as a trigger to initiate a distinct developmental program: Sens acts genetically in a feedforward loop to inhibit the Rh6-PR cell-fate determinant Svp and to promote the Rh5-PR cell-fate determinant Sal. Similarly, in the adult retina, differentiation of 'inner' PRs R7 and R8 requires sens and sal. Sal is necessary for Sens expression in R8-PRs and misexpression of Sal is sufficient to induce Sens expression in the 'outer' PRs R1-R6 (Mishra, 2013).

Svp is exclusively expressed in R3/R4 and R1/R6 pairs of the outer PRs in early retina development. Initially, Sal is expressed in the R3/R4 PRs in order to promote Svp expression. Later, Svp represses Sal in R3/R4 PRs in order to prevent the transformation of R3/R4 into R7. Similarly in larval PRs Svp is repressing Sal in secondary precursors (Mishra, 2013).

Intriguingly, in R8 development in the adult retina Sens also provides two temporally separable functions: First, during R8 specification, lack of Sens in precursors results in a transformation of the cell into R2/R5 fate; second, during differentiation, Sens counteracts Pros to inhibit R7 cell fate and promotes R8 cell fate. Thus, Sens is an early genetic switch in R8-PRs and larval Rh5-PRs that represses an alternate cell fate (Mishra, 2013).

The lack of Sens results in a larval eye composed of only Rh6-PRs. Thus, the default state for both primary and secondary precursors is to differentiate into Rh6-expressing PRs. Rh6 is also the default state in adult R8 PRs: In the absence of R7 PRs (e.g. sevenless mutants) that send a signal to a subset of underlying R8 PRs, the majority of R8 PRs express Rh6. Thus, the genetic pathway initiated by the Sens pulse ensures that primary precursors choose a distinct developmental pathway by repressing the Rh6 ground state. The mechanisms that initiate and control this pulse of Sens remain to be discovered (Mishra, 2013).

In larval PRs as well as in the formation of sensory organ precursors (SOP) in the wing, Sens functions as a binary switch between two alternative cell fates. In the larval eye, this switch occurs when Sens is expressed in one cell type and not in the other. However, during wing disc development the cell fate choice in SOP formation is controlled by the levels, and not the presence or absence of Sens expression: high levels of Sens act synergistically with proneural genes to promote a neuronal fate, while in neighboring cells, low levels of Sens repress proneural gene expression, thereby promoting a non-SOP fate. Thus, Sens uses distinct molecular mechanisms to act as a switch between Rh5 versus Rh6-PR cell fate and SOP versus non-SOP cell fate (Mishra, 2013).

Transcription factors regulate developmental programs in a context- dependent fashion. An example is Sens, which has distinct functions in BO and eyelet development. First, during embryonic development, Sens acts as a key cell fate determinant by regulating transcription factors controlling PR-subtype specification. Second, during metamorphosis Sens inhibits ecdysone-induced apoptotic cell death. Third, in the adult eyelet Sens promotes Rh6 expression. Interestingly, the pro-survival function of Sens appears to be a conserved feature of Sens in other tissues and also in other animal species. In the salivary gland of Drosophila, Sens acts also as a survival factor of the salivary gland cells under the control of the bHLH transcription factor Sage. pag-3, a C.elegans homolog of Sens is involved in touch neuron gene expression and coordinated movement (Jia, 1996; Jia, 1997). Pag-3 was shown to act as a cell-survival factor in the ventral nerve cord and involved in the neuroblast cell fate and may affect neuronal differentiation of certain interneurons and motorneurons. In mice, Gfi1 is expressed in many neuronal precursors and differentiating neurons during embryonic development and is required for proper differentiation and maintenance of inner ear hair cells. Gfi1 mutant mice lose all cochlear hair cells through apoptosis, suggesting that its loss causes programmed cell death (Wallis, 2003). Taken together, these findings support that Sens and its orthologs function in cell fate determination and cell differentiation both in nervous system formation, but also play an essential role in the suppression of apoptosis (Mishra, 2013).

Hazy plays distinct roles in larval PRs and during metamorphosis. First, Hazy is essential during embryogenesis for proper PR differentiation. This early function of Hazy is essential for PRs to differentiate properly during embryogenesis, to express Rhodopsins and to subsequently maintain Rhodopsin expression during larval stages. This function of Hazy is similar to its role in rhabdomere formation in adult PRs and subsequent promotion of Rh6 expression, although it is not required for Rh5 in the adult retina. It is likely that Hazy exerts this function by binding to the RCSI site of the rhodopsin promoters, as has been suggested for the adult retin. Second, during metamorphosis Hazy is required in Rh6-PRs to repress sens, thus allowing these cells to undergo apoptosis. This highlights the reuse of a small number of TFs for distinct functions in the same cell type at distinct time points of PR development. How these temporally distinct developmental programs are controlled on a molecular level remains unresolved. It seems likely that the competence of the cell to respond to a specific transcription factor changes during development (Mishra, 2013).

rh5 and rh6 are expressed in different PRs at different developmental stages: rh5 is expressed in the larval eye and in the adult retina, whereas rh6 is expressed in the larval eye, the adult eyelet and the adult retina. However, the gene regulatory networks controlling rhodopsin expression are distinct in these organs. In the adult retina, a bi-stable feedback loop of the growth regulator melted and the tumor suppressor warts acts to specify Rh5 versus Rh6 cell fate, respectively, while in the larva, Sens, Sal, Svp and Otd control Rh5 versus Rh6 identity whereas Hazy has been shown to maintain Rhodopsin expression. A third genetic program acts downstream of EcR during metamorphosis in Rh5-PRs to switch to Rh6, which requires Sens (Mishra, 2013).

An intriguing question is how the developmental pathways to specify Rh5- or Rh6-cell fates converge on the regulatory sequences of these two genes. It seems likely that parts of the regulatory machinery acting on the rh5 and rh6 promoters are shared between the larval eye, adult retina and eyelet, especially as short minimal promoters are functional in all three different contexts. Future experiments will show how the activity of the identified trans-acting factors is integrated on these promoters to yield context-specific outcomes (Mishra, 2013).


REFERENCES

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. PubMed Citation: 9465099

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

Alfano, C., et al. (2011). COUP-TFI promotes radial migration and proper morphology of callosal projection neurons by repressing Rnd2 expression. Development 138(21): 4685-97. PubMed Citation: 21965613

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

Baek, M. and Mann, R. S. (2010). Lineage and birth date specify motor neuron targeting and dendritic architecture in adult Drosophila. J. Neurosci. 29(21): 6904-16. PubMed Citation: 19474317

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. PubMed Citation: 7867504

Benito-Sipos, J., et al. (2011). Seven up acts as a temporal factor during two different stages of neuroblast 5-6 development. Development 138(24): 5311-20. PubMed Citation: 22071101

Bovetti, S., Bonzano, S., Garzotto, D., Giannelli, S. G., Iannielli, A., Armentano, M., Studer, M. and De Marchis, S. (2013). COUP-TFI controls activity-dependent tyrosine hydroxylase expression in adult dopaminergic olfactory bulb interneurons. Development 140: 4850-4859. PubMed ID: 24227652

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. PubMed Citation: 8575299

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. PubMed Citation: 11144353

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 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. PubMed Citation: 8675007

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

Erclik, T., Li, X., Courgeon, M., Bertet, C., Chen, Z., Baumert, R., Ng, J., Koo, C., Arain, U., Behnia, R., Rodriguez, A. D., Senderowicz, L., Negre, N., White, K. P. and Desplan, C. (2017). Integration of temporal and spatial patterning generates neural diversity. Nature [Epub ahead of print]. PubMed ID: 28077877

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. PubMed Citation: 9651479

Fjose, A., et al. (1993). Functional conservation of vertebrate seven-up related genes in neurogenesis and eye development. EMBO J 12: 1403-14. PubMed Citation: 8467797

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. PubMed Citation: 8541212

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. PubMed Citation: 8681384

Hiromi, Y., et al. (1993). Ectopic expression of seven-up causes cell fate changes during ommatidial assembly. Development 118: 1123-35. PubMed Citation: 8269843

Hoshizaki, D. K., et al. (1994). Embryonic fat-cell lineage in Drosophila melanogaster. Development 120: 2489-99. PubMed Citation: 7956826

Jia, Y., Xie, G. and Aamodt, E. (1996). pag-3, a Caenorhabditis elegans gene involved in touch neuron gene expression and coordinated movement. Genetics 142: 141-147. PubMed ID: 8770591

Jia, Y., Xie, G., McDermott, J. B. and Aamodt, E. (1997). The C. elegans gene pag-3 is homologous to the zinc finger proto-oncogene gfi-1. Development 124: 2063-2073. PubMed ID: 9169852

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. PubMed Citation: 9637680

Kohwi, M., Hiebert, L. S. and Doe, C. Q. (2011). The pipsqueak-domain proteins Distal antenna and Distal antenna-related restrict Hunchback neuroblast expression and early-born neuronal identity. Development 138(9): 1727-35. PubMed Citation: 21429984

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. PubMed Citation: 7789267

Krishnan, V., et al. (1997). Mediation of Sonic hedgehog-induced expression of COUP-TFII by a protein phosphatase. Science 278(5345): 1947-1950. PubMed Citation: 9395397

Laudet, V. (1997). Evolution of the nuclear receptor superfamily: early diversification from an ancestral orphan receptor. J. Mol. Endocrinol. 19(3): 207-26. PubMed Citation: 9460643

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. PubMed Citation: 8119130

Ma, T., Wang, C., Wang, L., Zhou, X., Tian, M., Zhang, Q., Zhang, Y., Li, J., Liu, Z., Cai, Y., Liu, F., You, Y., Chen, C., Campbell, K., Song, H., et al. (2013) Subcortical origins of human and monkey neocortical interneurons. Nat Neurosci 16: 1588-1597. PubMed ID: 24097041

Maurange, C., Cheng, L. and Gould, A. P. (2008). Temporal transcription factors and their targets schedule the end of neural proliferation in Drosophila. Cell 133(5): 891-902. PubMed Citation: 18510932

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. PubMed Citation: 10354476

Mishra, A. K., Tsachaki, M., Rister, J., Ng, J., Celik, A. and Sprecher, S. G. (2013). Binary cell fate decisions and fate transformation in the Drosophila larval eye. PLoS Genet 9: e1004027. PubMed ID: 24385925

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. PubMed Citation: 2105166

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

Naka, H., Nakamura, S., Shimazaki, T. and Okano, H. (2008). Requirement for COUP-TFI and II in the temporal specification of neural stem cells in CNS development. Nat. Neurosci. 11: 1014-1023. PubMed Citation: 19160499

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. PubMed Citation: 10215630

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. PubMed Citation: 10336677

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. PubMed Citation: 9271116

Rehorn, K.-P., et al. (1996). A molecular aspect of hematopoiesis and endoderm development common to vertebrates and Drosophila. Development 122: 4023-4031. PubMed Citation: 9012522

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

Sieglitz, F., Matzat, T., Yuva-Adyemir, Y., Neuert, H., Altenhein, B. and Klambt, C. (2013). Antagonistic feedback loops involving rau and sprouty in the Drosophila eye control neuronal and glial differentiation. Sci Signal 6: ra96. PubMed ID: 24194583

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. PubMed Citation: 9133437

Sprecher, S. G. and Desplan, C. (2008). Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons. Nature 454(7203): 533-7. PubMed Citation: 18594514

Srinivasan, R. S., et al. (2010). The nuclear hormone receptor Coup-TFII is required for the initiation and early maintenance of Prox1 expression in lymphatic endothelial cells. Genes Dev. 24(7): 696-707. PubMed Citation: 20360386

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

Syed, M. H., Mark, B. and Doe, C. Q. (2017). Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity. Elife 6 [Epub ahead of print]. PubMed ID: 28394252

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

Tang, K., et al. (2010). COUP-TFs regulate eye development by controlling factors essential for optic vesicle morphogenesis. Development 137(5): 725-34. PubMed Citation: 20147377

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. PubMed Citation: 8020091

Tomassy, G. S., et al. (2010). Area-specific temporal control of corticospinal motor neuron differentiation by COUP-TFI. Proc. Natl. Acad. Sci. 107: 3576-358. PubMed Citation: 20133588

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. PubMed Citation: 10656770

Wallis, D., Hamblen, M., Zhou, Y., Venken, K. J., Schumacher, A., Grimes, H. L., Zoghbi, H. Y., Orkin, S. H. and Bellen, H. J. (2003). The zinc finger transcription factor Gfi1, implicated in lymphomagenesis, is required for inner ear hair cell differentiation and survival. Development 130: 221-232. PubMed ID: 12441305

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. PubMed Citation: 8557044

Younossi-Hartenstein, A., et al. (1996). Early neurogenesis of the Drosophila brain. J. Comp. Neur. 370: 313-329. PubMed Citation: 8799858

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. PubMed Citation: 9070327

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. PubMed Citation: 9658405

Zelhof, A. C., et al. (1995a). Seven-up inhibits ultraspiracle-based signaling pathways in vitro and in vivo. Mol Cell Biol 15: 6736-6745. PubMed Citation: 8524239

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. PubMed Citation: 7479823

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. PubMed Citation: 9852581


seven up: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 23 August 2017

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

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