SAL is first detected at the end of the syncytial blastoderm stage (late stage 4), forming a circumferential ring around the embryo at 60-70% egg length towards the anterior. During the blastoderm stage [Image], two additional expression domains appear. The first is a circumferential ring in the posterior 12-20% egg length, spanning parasegments 14 and 15 and the primordia of the hindgut up to the Malpighian tubule anlagen. The second is a"horseshoe-shaped domain" in the anterior region 80-86% egg length, covering the anlagen of the maxillary and labial segments [Image] in this region. labial and sal are expressed in adjacent domains, flanked but not overlapping the cap'n'collar expression domain. sal expression overlaps deformed and Sex combs reduced domains (Mohler 1995). Later SAL accumulates in the neuroectoderm giving rise to a repeated pattern in the CNS. Still later, SAL is found in the tracheal system (Kühnlein, 1994).

During formation af tracheal placodes spalt expression overlaps with the dorsal parts of all tracheal placodes, and spalt expression decreases in non-tracheal cells, becoming restricted to tracheal cells. Later spalt is found in the dorsal parts of all tracheal metameres, in the outgrowing dorsal trunk anterior and dorsal trunk posterior as well as in the dorsal branch, while no spalt expression is detectable in central and ventral tracheal structures (Kühnlein, 1996).

Decapentaplegic controls tracheal cell migration along the dorsoventral body axis of the Drosophila embryo. Dpp signaling determines localized gene expression patterns in the developing tracheal placode, and is also required for the dorsal expression of the recently identified Branchless (Bnl) guidance molecule, the ligand of the Breathless (Btl) receptor. spalt (sal) is strongly expressed in dorsal trunk cells in stage 14 embryos and is necessary for the directed anterior migration of these cells. sal is expressed in the dorsal trunk in punt and tkv mutant embryos, indicating that Dpp does not regulate sal expression. However, embryos in which the Dpp signaling pathway has been activated in all tracheal cells at the placode stage fail to accumulate Sal. This lack of Sal expression correlates with the absence of the dorsal trunk upon ectopic Dpp signaling (Vincent, 1997).

Morphogenesis of the Drosophila tracheal system relies on different signalling pathways that have distinct roles in specifying both the migration of the tracheal cells and the particular morphological features of the primary branches. The current view is that the tracheal cells are initially specified as an equivalent group of cells whose diversification depends on signals from the surrounding cells. The tracheal primordia are already specified as distinct dorsal and ventral cell populations. This subdivision depends on the activity of the spalt (sal) gene and occurs prior to the activity of the signalling pathways that dictate the development of the primary branches. The specification of these two distinct cell populations, which are not defined by cell lineage, are critical for proper tracheal patterning. These results indicate that tracheal patterning depends not only on signalling from surrounding cells but also in the different response of the tracheal cells depending on their allocation to the dorsal or ventral domains (Franch-Marro, 2002).

The tracheal placodes are first specified as clusters of ectodermal cells at each side of 10 embryonic segments. Thereafter, formation of the tracheal branches begins with the protrusion of small buds from the tracheal placodes; cells from each bud migrate in a stereotyped direction and give rise to a specific branch with a distinctive morphology. Different studies have identified some of the mechanisms that control the migration and specification of each branch, suggesting that each branch was specified independently by signals from surrounding cells. The results of this study, instead, indicate that there is a subdivision between two cell populations within the tracheal placode independent of branch specification. In particular, expression of the sal gene in the dorsal half of the tracheal placode establishes a distinction between the cells that will adopt a dorsal identity and the cells that will adopt a ventral identity. This role for sal is in agreement with its pattern of expression. By stage 10, sal is found in the dorsal part of the tracheal metameres, the precursors of the dorsal branch and the dorsal trunk, but later on it remains only in the dorsal trunk cells and declines in the dorsal branch cells. While the requirement of sal activity in the dorsal trunk cells is well established, the early role of sal expression in the whole dorsal region has not been clear. Indeed, it seemed that the unique role of the dorsal sal expression was to ensure its subsequent expression in the dorsal trunk cells. Now, a role for the dorsal expression of sal has been identified, since these results indicate that sal endows a dorsal identity to these tracheal cells (Franch-Marro, 2002).

At stages 10 and 11, the tracheal cells undergo the two last rounds of mitosis. However, cell clonal analysis has shown that cell lineage does not play an important role in the specification of the distinct identity of the different branches. For instance, the two descendants of a single cell can belong to a dorsal and a ventral branch, respectively. Thus, as opposed to the classic compartments, specification of a dorsal subdomain in the tracheal placode appears not to be linked to a restriction in cell lineage. This is reminiscent of what happens in the generation of medial and lateral dorsal body domains by pannier or in the specification of dorsal and ventral domains in the leg discs. In this case, again, it appears that the important event is the generation of groups of cells with distinct genetic addresses rather than the fact that these subdomains are inherited by cell lineage or not (Franch-Marro, 2002).

It has to be noted that sal confers a dorsal identity to these cells while they are specified as tracheal cells as indicated by the expression of the tracheal-inducing genes. Thus, they do not seem to be first specified as tracheal cells and thereafter as dorsal tracheal cells. Indeed, the broad ectodermal domains of sal expression in each metamere encompass both tracheal cells and nontracheal cells. Thus, as is the case for the specification of dorsoventral subdomains in the leg imaginal disc, it appears that the dorsal tracheal cells inherit their dorsal identity from its position in the ectoderm. Thereafter, the dorsal identity is reinforced by the specific expression of sal in the dorsal tracheal branches (Franch-Marro, 2002).

The identification of two subdomains in the tracheal placodes allows the establishment of a correlation between the branches in the dorsal region and those in the ventral region; the dorsal branch appears to be an analogue of the lateral trunk and the dorsal trunk to the visceral branch. Similarly, the tracheal branches can also be grouped according to the signalling pathway that dictates their features. Thus, both the dorsal branch and the lateral trunk are specified by the Dpp pathway; however, they differ in the expression of sal: dorsal branch cells initially express sal, while lateral trunk cells are devoid of sal activity. The expression of sal in the dorsal branch is transient and restricted to an early period around stages 10-13 and is subsequently repressed by Dpp signalling through the activation of knirps (kni). Indeed, the repression of sal by kni is necessary for the proper morphology of the dorsal branch. The data indicate that the transient expression of sal is also critical for dorsal branch development. Thus, for instance, sal might distinguish between dorsal branch identity and lateral branch identity by modulating the response of the dorsal branch cells to Dpp signalling. Such a role for sal as a switch between two cell fates has also been recently described in the induction of oenocytes vs chordotonal precursors by EGFR signalling. However, others factors are probably involved since unp appears not to be a direct target of the Dpp pathway since it is still expressed in embryos lacking the Dpp receptor Tkv (Franch-Marro, 2002).

There is a similar situation concerning the dorsal trunk and the visceral branch. In this case, again, expression of sal in the dorsal trunk is responsible for the choice between a dorsal trunk cell identity and a visceral branch cell identity. However, some differences are also observed. (1) sal expression in the dorsal ectodermal domains is not sufficient to pattern the dorsal trunk cells; instead, they have to maintain the expression of sal to acquire their proper differentiation. (2) The wg/wnt pathway is active in the dorsal trunk cells precisely to keep sal activated in those cells but not in the visceral branch, as opposed to the Dpp pathway that is required in both the dorsal branch and the lateral trunk. This will explain the observation that constitutive activation of the wg/wnt pathway can lead to the transformation of visceral branch into dorsal trunk (since this causes ectopic expression of sal) but the converse transformation of dorsal trunk into visceral branch does not occur in the absence of wg/wnt signalling (since the wg/wnt-independent expression of sal in the dorsal ectodermal domains is sufficient to endow a dorsal identity to the cells of the dorsal trunk) (Franch-Marro, 2002).

In summary, the following steps can be considered in the morphogenesis of the tracheal tree. First, the tracheal cells are determined by the coordinate activity of tracheal inducer genes, basically, trachealess (trh) and ventral veinless (vvl). At the same time, sal expression endows a dorsal identity to the cells located dorsally in the tracheal metameres. Accordingly, tracheal cells are specified as two distinct populations of dorsal and ventral cells. As a consequence of the tracheal inducer genes, the tracheal cells activate downstream genes that make them competent to the signalling from surrounding cells. Thus, the EGFR pathway plays a role in tracheal cell invagination, the Bnl/Btl pathway stimulates and guides cell migration, and the Dpp and wg pathway induces different sets of cells to adopt the specific features of distinct branches. In this regard, the role of the Dpp and wg pathways in determining a specific migratory direction can be depicted as a part of its role in branch specification; their activity differentiates tracheal cells to enable them to recognise specific migratory substrates. However, the response to the branch-specific pathways differs according to whether they act upon the cells of the dorsal or ventral populations in the placodes (Franch-Marro, 2002).

Finally, it is worth noting that the visceral branch is the sole branch whose cells do not require either the Dpp or the wg/wnt pathway for its morphogenesis. This is also the case for the transverse connective. Therefore, both structures could be considered as a basal state for the tracheal tree. Reinforcing this view, it is worth noting that in many apterygotes the rudimentary tracheal system is a discrete entity in each half-segment that lacks longitudinal trunks connecting adjacent segments and connections with the opposite segmental side (Franch-Marro, 2002).

Identity, origin, and migration of peripheral glial cells in the Drosophila embryo

Glial cells are crucial for the proper development and function of the nervous system. In the Drosophila embryo, the glial cells of the peripheral nervous system are generated both by central neuroblasts and sensory organ precursors. Most peripheral glial cells need to migrate along axonal projections of motor and sensory neurons to reach their final positions in the periphery. This paper studied the spatial and temporal pattern, the identity, the migration, and the origin of all peripheral glial cells in the truncal segments of wildtype embryos. The establishment of individual identities among these cells is reflected by the expression of a combinatorial code of molecular markers. This allows the identification of individual cells in various genetic backgrounds. Furthermore, mutant analysis of two of these marker genes, spalt major and castor, reveal their implication in peripheral glial development. Using confocal 4D microscopy to monitor and follow peripheral glia migration in living embryos, it was shown that the positioning of most of these cells is predetermined with minor variations, and that the order in which cells migrate into the periphery is almost fixed. By studying their lineages, the origin of each of the peripheral glial cells was uncovered and they were linked to identified central and peripheral neural stem cells (von Hilchen, 2008).

This study has characterized the expression of a collection of cell-specific molecular markers, which allows to identify and distinguish all glial cells in the embryonic peripheral nervous system. The reproducibility with which enhancer-trap lines and marker genes are expressed in the individual peripheral glial cells, indicates that these cells display unique identities. Furthermore, the spatial and temporal pattern of migration and the final arrangement of these cells are relatively stereotypic. This suggests that the specification of the unique identity of each cell does not only define a specific combination of genes to be expressed, but also includes the information about the timing of migration, the nerve tract it is associated with, and to some degree the final position to be occupied along the respective nerve. How the cell receives this information is still unknown. The individual characteristics could be determined (1) by lineage or (2) during migration by cell-cell interactions (between the glial cells or between the glia and other closely associated cells, e.g. neurons, tracheae), or (3) by a combination of both (von Hilchen, 2008).

The master regulatory gene glial cells missing (gcm) is required to induce the glial cell fate. Gcm as a transcription factor switches on downstream target genes, of which the gene encoding for the homeobox transcription factor Reversed polarity (Repo) is well described. As this cascade of gene activation is required for all glial cells in the Drosophila embryo (except the midline glia), it is unlikely to contribute to cell fate diversification among the glia. Whereas central glial cells migrate over rather short distances, in literally any possible direction, to finally occupy stereotypic positions within the CNS, the peripheral glial cells behave differently as they have to migrate over remarkable distances into the periphery. It has been recently shown that the migration of PGs depends on Notch signalling. In Notch mutants or in mutants where Notch signalling is altered in PGs, the migration is impaired or even completely blocked. However, this signalling does not appear to supply the cells with characteristics of their fate apart from the onset and/or maintenance of the migration itself. Sepp (2000) described the developmental dynamics and morphology of a subset of peripheral glial cells and could show that a signalling cascade mediated by the small GTPases RhoA and Rac1 influences the actin cytoskeleton of migrating PGs. Sepp further showed, that, within the analysed population of cells, a 'leading glia’ expresses filopodia-like structures whereas the ‘follower’ cells do not. Similar results were reported by Aigouy (2004). Aigouy established a 4D microscopy technique to record and analyse the developmental dynamics and migratory behaviour of PNS glia during pupal stages in the developing fly wing. In this system, differences between 'leading' and 'follower' glia cells were also observed. The glial cells in the wing PNS migrate along wing veins in a chain with one 'leading' cell in front. If this chain is interrupted by laser ablation of either the leading or intermediate cells, a new 'leading’ cell starts to form filopodia and explores the surrounding. Once this new 'leading' cell catches up with the previous chain or reaches its target area, the filopodia disappear and the cells' morphology changes again. Hence, these differences in glial cell morphology and behaviour in the wing PNS are based on interactions of the glial cells with each other rather than on a predetermined intrinsic cell fate (von Hilchen, 2008).

Findings for the embryonic PNS glia suggest that these cells are predetermined at least to a certain extent. The 4D microscopy approach allowed tracing of the migration of individually identified PGs in living embryos from the moment they leave the CNS until they reach their final position. Apart from the dorsal SOP-derived cells, which never change their position or behaviour, it is always the ePG9 that leaves the CNS first and 'leads' the track. This cell expresses filopodia-like structures, while the following cells do not, although it remains to be experimentally shown whether they can take over the leading function in the absence of ePG9. It is worth mentioning that the SOP-derived ePG12 migrates along trajectories of the ISN prior to ePG9. It is not clear whether ePG12 has any leading function for ePG migration or functions as a guidepost cell for axonal projections. It is the only cell, though, that swaps nerve tracts and finally associates with the TN. Most likely, cell-cell communication between ePG12 and axonal projections and/or neighbouring cells is required for proper pathfinding and positioning. It is always the ePG4 that migrates along and finally enwraps the segmental nerve. As this cell is the only cell associated with the distal part of the segmental nerve, it functions as 'leading' glia for this nerve and expresses filopodia-like structures at least in later stages when it enwraps the SN. This enwrapment occurs in a bidirectional fashion, i.e. the filopodia occur at both ends of the glial cell (von Hilchen, 2008).

Lineage analysis revealed that the PGs mentioned above originate from the CNS neuroblast NB 1-3 and a ventrally located SOP. Interestingly, the two NB 2-5 derived PGs (ePG6 and ePG8) differ from these cells with respect to both identity and behaviour. They express fewer of the analysed PG-specific markers (cas-Gal4 and mirr-lacZ) and it is not possible to distinguish between these two cells so far. Whether the lack of identifying markers is a consequence of or a prerequisite for their different identity and behaviour is not yet clear. The cells migrate along the ISN independently of the NB 1-3- and SOP-derived PGs and frequently overtake them (and occasionally even one another). The correlation of such characteristics with the different origin of these three subpopulations of PGs lends support to the hypothesis that some aspects of cell fate diversification among the PGs may be predetermined by lineage. It is likely, that such predetermined characteristics include the competence to respond to specific external signals that guide the respective cell along the correct nerve to its target position (von Hilchen, 2008).

One incidence for lineage-dependent cell fate determination comes from the analysis of the ladybird homeobox genes. The ladybird genes are expressed in the developing CNS in only few NBs including NB 5-6. The NB 5-6 lineage produces one of the proximal PGs (ePG2) which expresses the Ladybird early (Lbe) protein. It has been shown that a loss of ladybird gene function results in a loss of the ePG2 in a third of all analysed hemisegments, accompanied with a higher number of medially located glial cells in the CNS. An opposite phenotype with excessive cells in the transition zone was observed by ectopic expression of the ladybird genes throughout the CNS. Using an anti-Repo antibody as well as a subset specific reporter transgene (K-lacZ), De Graeve (2004) provided evidence suggesting that the ladybird genes play a role in glial subtype specification in particular NB lineages. Another factor shown to be required for the specification of a lineage-specific set of glial cells (NB1-1-derived subperineurial glia) is Huckebein, which interacts with Gcm to amplify its expression specifically in these cells (von Hilchen, 2008).

Furthermore, in cas mutants, it was shown that the two NB 2-5-derived glia (ePG6 and ePG8) do not migrate into the periphery but most likely stay at their place of birth, although they acquire glial cell fate (as can be deduced from Repo stainings). Thus, similar to Ladybird and Huckebein, Cas seems to be involved in lineage-dependent glial subtype specification rather than determination of glial fate in general. In contrast to ladybird (De Graeve, 2004), though, Cas is not sufficient to ectopically induce glial cell fate or PG subtype specification (von Hilchen, 2008).

This study shows that salm is a likely candidate participating in the control of glial development. Embryos homozygous for salm445 show a pleiotropic and variable phenotype affecting not only glial cells but also PNS neurons, sensory organs, and other tissues. Yet, nearly all ventrally derived PGs stall in the transition zone between CNS and PNS and do not migrate properly into the periphery. In about 50% of the analysed hemisegments, a variable number of one to three PGs are missing, even though these cells could remain in the CNS. salm-lacZ is expressed in the two ventral SOP-derived ePG4 and ePG5, as well as in the dorsal SOP-derived ePG11 along the DLN, and in some of the ligament cells of the lateral chordotonal organ. In salm445 mutants the ePG4 cell can sometimes be detected at its wildtypic position along the SN. If ePG4 is missing along the SN, it could well be a secondary effect, as the SN itself is affected with the SNc shortened or occasionally missing. The ePG5 however, cannot be unambiguously identified in Repo-staining within the group of cells stalling in the transition zone (von Hilchen, 2008).

It needs to be further shown whether the differences between the PGs derived from certain progenitor cells result in functional differences in the larva. The peripheral nerves of the larva are ensheathed by two distinct types of glial cells, the perineurial and the subperineurial glial cells. The subperineurial glia build septate junctions with each other (or themselves) and thereby form the blood-nerve barrier, whereas the perineurial glia form an outer layer and secrete the neural lemma. In order to allow proper electrical conductance, the peripheral nerves must be enwrapped and insulated at the end of embryogenesis when hatching of the larva requires coordinated muscle contractions. It is not known to date which of the embryonic PGs will become perineurial or subperineurial glia, or what other functions they might fulfill (von Hilchen, 2008).

The comprehensive description of the ancestry, identity and dynamics of the developing embryonic peripheral glia, and the molecular markers at hand, provide a crucial basis for further clarification of the mechanisms controlling development, migration, and function of peripheral glia on a single cell level (von Hilchen, 2008).

The ETS domain transcriptional repressor Anterior open inhibits MAP kinase and Wingless signaling to couple tracheal cell fate with branch identity

Cells at the tips of budding branches in the Drosophila tracheal system generate two morphologically different types of seamless tubes. Terminal cells (TCs) form branched lumenized extensions that mediate gas exchange at target tissues, whereas fusion cells (FCs) form ring-like connections between adjacent tracheal metameres. Each tracheal branch contains a specific set of TCs, FCs, or both, but the mechanisms that select between the two tip cell types in a branch-specific fashion are not clear. This study shows that the ETS domain transcriptional repressor anterior open (aop) is dispensable for directed tracheal cell migration, but plays a key role in tracheal tip cell fate specification. Whereas aop globally inhibits TC and FC specification, MAPK signaling overcomes this inhibition by triggering degradation of Aop in tip cells. Loss of aop function causes excessive FC and TC specification, indicating that without Aop-mediated inhibition, all tracheal cells are competent to adopt a specialized fate. Aop plays a dual role by inhibiting both MAPK and Wingless signaling, which induce TC and FC fate, respectively. In addition, the branch-specific choice between the two seamless tube types depends on the tracheal branch identity gene spalt major, which is sufficient to inhibit TC specification. Thus, a single repressor, Aop, integrates two different signals to couple tip cell fate selection with branch identity. The switch from a branching towards an anastomosing tip cell type may have evolved with the acquisition of a main tube that connects separate tracheal primordia to generate a tubular network (Caviglia, 2013).

This work has investigated how the choice between the two types of specialized tip cells in the tracheal system is controlled. The transcriptional repressor Aop plays a key role in linking tracheal tip cell fate selection with branch identity. First, a novel tube morphogenesis phenotype is described in aop mutants, which is due to the massive mis-specification of regular epithelial cells into specialized tracheal tip cells. aop is specifically required for controlling tracheal cell fate, whereas aop, like pnt, is dispensable for primary tracheal branching, thus uncoupling roles of RTK signaling in cell fate specification and cell motility. The finding that tracheal branching morphogenesis proceeds normally in the presence of excess tip cell-like cells suggests that collective cell migration is surprisingly robust and that mis-specified cells apparently do not impede the guided migration of the tracheal primordium. Second, it was demonstrated that in the absence of inhibitors of MAPK signaling (aop and sty), all tracheal cells are competent to assume either TC or FC fate. The transcriptional repressor Aop globally blocks both TC and FC differentiation, but high-levels of MAPK signaling in tip cells relieve Aop-mediated inhibition, thus permitting differentiation. Third, the results suggest that in the DT region Aop limits FC induction through a distinct mechanism by antagonizing Wg signaling in addition to MAPK signaling. Conversely, in the other branches, Aop limits TC differentiation by blocking MAPK-dependent activation of Pnt. Fourth, it was shown that the region-specific choice between the two cell fates in the DT is determined by Wg signaling and by the selector gene salm. Based on these results, a model is proposed in which a single repressor, Aop, integrates MAPK and Wg signals to couple tip cell fate selection with branch identity. High levels of Bnl signaling trigger Pnt activation and Aop degradation in tracheal tip cells. It is proposed that in the DT, unlike in other tracheal cells, MAPK-induced degradation of Aop releases inhibition of Wg signaling. This is consistent with recent work showing an inhibitory effect of Aop on Wg signaling, possibly through direct interaction of Aop and Arm, or through Aop-mediated transcriptional repression of Wg pathway component. The current work extends the evidence for this unexpected intersection between two major conserved signaling pathways, suggesting that this function of Aop is likely to be more widespread than previously appreciated. The findings also provide an explanation for the puzzling observation that, in pnt mutants, TCs are lost, while FCs become ectopically specified. As pnt is required for expression of the feedback inhibitor sty, loss of pnt is expected to lead to MAPK pathway activation and consequently to increased Aop degradation. This would release Aop-mediated repression of Wg signaling, resulting in extra FCs, whereas TCs are absent because of the lack of pnt-dependent induction. This suggests that excessive FC specification in the DT of aop and sty mutants is mainly due to deregulated Wg signaling, rather than to de-repression of pnt-dependent MAPK target genes. Consistent with this notion, it was shown that pnt is not required for Delta and Dys expression in tracheal cells, although constitutively active AopACT represses their expression (Caviglia, 2013).

The results further show that salm function constrains the fate that is chosen by cells when released from the Aop inhibitory block. MAPK signaling triggers Aop degradation in all tip cells, but only in the absence of salm does this signal lead to TC induction. In salm-expressing cells, degradation of Aop releases Wg signaling, resulting in FC specification. Thus, salm biases the choice between two morphologically different types of seamless tubes. This is reminiscent of the role of salm in switching between different cell types in the peripheral nervous system and in muscles. salm expression is sufficient to repress TC formation. The genetic results, consistent with biochemical data showing that Salm acts as a transcriptional repressor, suggest that salm promotes FC fate by repressing genes involved in TC development. However, salm is not sufficient to overcome the requirement for Wg signaling in FC induction, indicating that Wg does not act solely via salm to induce FC fate. Indeed, FC induction requires genes whose expression is independent of salm (esg, dys). In addition, it is proposed that a feedback loop between Wg signaling and salm expression maintains levels of Wg signaling in the DT sufficiently high to induce FC fate. Taken together, these results suggest that the default specialized tip cell fate, and possibly an ancestral tracheal cell state, is TC fate. Although FCs and TCs differ in their morphology, they share a unique topology as seamless unicellular tubes. FCs and TCs might therefore represent variations of a prototypical seamless tube cell type. Salm might modify cellular morphology by repressing TC genes, including DSRF, which mediates cell elongation and shape change. Intriguingly, Wg-dependent salm expression in the DT of dipterans correlates with a shift towards FC as the specialized fate adopted by the tip cells of this branch. This study has shown that salm expression inhibits TC fate, while promoting the formation of a multicellular main tracheal tube by inhibiting cell intercalation. It is therefore tempting to speculate that the salm-dependent switch from a branching towards an anastomosing tip cell type in the DT may have evolved with the acquisition in higher insects of a main tube that connects separate tracheal primordia to generate a tubular network. It will be of great interest to identify the relevant target genes that mediate the effect of Salm on tube morphology and tip cell fate (Caviglia, 2013).

The mechanisms of tip cell selection during angiogenesis in vertebrates are beginning to be understood at the molecular level. However, the signals that control the formation of vascular anastomoses by a particular set of tip cells are not known. Intriguingly, the development of secondary lumina in aop mutants is reminiscent of transluminal pillar formation during intussusceptive angiogenesis, which is thought to subdivide an existing vessel without sprouting. Although the cellular basis for this process is not understood, it is conceivable that specialized endothelial cells are involved in transluminal pillar formation. This work provides a paradigm for deciphering how two major signaling pathways crosstalk and are integrated to control cell fate in a developing tubular organ. It will be interesting to see whether similar principles govern tip cell fate choice during tube morphogenesis in vertebrates and invertebrates (Caviglia, 2013).


Patterning of the developing limbs by the secreted signaling proteins Wingless, Hedgehog and Dpp takes place while the imaginal discs are growing rapidly. Cells born in regions of high ligand concentration may be displaced through growth to regions of lower ligand concentration. A novel lineage-tagging method was used to address the reversibility of cell fate specification by morphogen gradients. Responses to Hedgehog and Dpp in the wing disc are readily reversible. In the leg, cells readily adopt more distal fates, but do not normally shift from distal to proximal fate. However, they can do so if given a growth advantage. These results indicate that cell fate specification by morphogen gradients remains largely reversible so long as the imaginal discs are growing. In other systems, where growth and patterning are uncoupled, nonreversible specification events or ‘ratchet’ effects may be of functional significance (Weigmann, 1999).

Hh induces dpp expression in anterior cells adjacent to the anteroposterior (AP) boundary of the wing disc. In mature third instar discs, a dpp-lacZ reporter gene is expressed in a narrow stripe of cells in the center of the disc, whereas in young third instar discs, the dpp-lacZ stripe occupies the central third of the disc. This comparison illustrates that the proportion of the disc occupied by Hh-responsive cells is relatively larger in small discs and decreases as the disc grows. Further, it suggests that Hh-responsive cells must be able to lose expression of Hh target genes as the cells are displaced out of range of the Hh signal by growth of the disc. To verify that this is indeed the case, cells born in the Hh-responsive region were lineage-tagged using dppGal4 to direct expression of FLP recombinase. In larvae carrying dppGal4, UAS-Flp and act5c>stop>lacZ, FLP recombinase is expressed in cells expressing dppGal4 and mediates excision of the flip-out ‘stop’ cassette from the inactive reporter construct to generate an active act5c>lacZ transgene. After excision of the cassette, reporter gene expression is regulated by the actin promoter and is clonally inherited in all the progeny of dppGal4-expressing cells in which the recombination event took place. Cells expressing lacZ fill most of the anterior compartment of the wing pouch, hinge and the notum. By comparison, the dppGal4 domain is much narrower. This indicates that cells born in the dppGal4 domain contribute to most of the A compartment of the wing and that they change their pattern of gene expression as they are displaced out of range of Hh (Weigmann, 1999).

Dpp signaling induces Spalt expression in the wing pouch. Clones of cells unable to transduce the Dpp signal lose Spalt expression, suggesting that expression of Spalt depends on continuous input of the Dpp signal. Spalt is first induced in early third instar discs. To ask whether cells that initiate Spalt expression at this stage revert to a more lateral identity as the disc grows in the course of normal development, cells born in the Spalt domain were lineage-tagged in larvae carrying spaltGal4, UAS-Flp and act5c>stop>lacZ. betaGal-expressing cells are found lateral to the endogenous Spalt domain in both the A and P compartments, indicating that cells can alter their pattern of target gene expression when displaced out of range of the Dpp signal. Taken together, these results suggest that, in general, cells are not committed to maintain a particular threshold response to the Hh or Dpp morphogens. Rather, cells in the wing disc appear to be able to revert to lower threshold responses when morphogen levels decrease (Weigmann, 1999).

Cell death-induced regeneration in wing imaginal discs requires JNK signalling

Regeneration and tissue repair allow damaged or lost body parts to be replaced. After injury or fragmentation of Drosophila imaginal discs, regeneration leads to the development of normal adult structures. This process is likely to involve a combination of cell rearrangement and compensatory proliferation. However, the detailed mechanisms underlying these processes are poorly understood. A system was established to allow temporally restricted induction of cell death in situ. Using Gal4/Gal80 and UAS-rpr constructs, targeted ablation of a region of the disc could be performed and regeneration monitored without the requirement for microsurgical manipulation. Using a ptc-Gal4 construct to drive rpr expression in the wing disc resulted in a stripe of dead cells in the anterior compartment flanking the anteroposterior boundary, whereas a sal-Gal4 driver generated a dead domain that includes both anterior and posterior cells. Under these conditions, regenerated tissues were derived from the damaged compartment, suggesting that compartment restrictions are preserved during regeneration. These studies reveal that during regeneration the live cells bordering the domain in which cell death was induced first display cytoskeletal reorganisation and apical-to-basal closure of the epithelium. Then, proliferation begins locally in the vicinity of the wound and later more extensively in the affected compartment. Finally, regeneration of genetically ablated tissue was shown to require JNK activity. During cell death-induced regeneration, the JNK pathway is activated at the leading edges of healing tissue and not in the apoptotic cells, and is required for the regulation of healing and regenerative growth (Bergantiños, 2010).

Two main conclusions can be drawn from this work: (1) that genetically induced regeneration entails compartment-specific proliferation; and (2), that this type of regeneration requires JNK signalling for early regeneration events (Bergantiños, 2010).

This study established that the proliferation response to ptc>rpr induction is concentrated in the A compartment and consists of two activities: a local and a compartment-associated response. The local proliferation response resembles the activity of blastemas, a feature found in discs after fragmentation and implantation. The late compartment-restricted proliferation could be indicative of a reutilisation of developmental programs. The entire A compartment responds to the lack of the original ptc region by reactivating proliferation in order to achieve the final organ size. Thus, it is concluded that genetically induced regenerating discs restore the overall organ size by activation of proliferation, not only near the wound, as in fragmented and implanted discs, but also in the whole affected compartment. Thus, it is believed that the local proliferation is a fast and early response to the lost structures and that the later compartment-associated proliferation is a response to adjust the size of the tissue (Bergantiños, 2010).

ptc and sal were selected because of the precise removal of cells and also because they enabled testing whether both A and P compartments are involved in regeneration. The results suggest that when the A compartment is damaged (ptc>rpr), the P compartment only responds to the injury by sealing the gap that separates it from the A compartment through the generation of F-actin-rich cell extensions. These are projected to anchor the extensions from the cells at the edge of the A compartment as they proceed towards recovery of the intact cell sheet. In this situation, the regenerated tissue is derived exclusively from the A compartment. By contrast, when cells from both the A and P compartments are killed (sal>rpr), proliferation increases in both compartments. The boundaries between compartments are rapidly re-established after injury and prevent cells from crossing into adjacent compartments. Thus, boundaries are respected and compartments act as units of growth during regeneration (Bergantiños, 2010).

Following genetic ablation driven by either the ptc or sal drivers, healing starts at the DV boundary and spreads laterally towards the proximal regions, which are the last to close the wound. Cells at the DV boundary are arrested in G1-S, through a mechanism based on Notch and wg signalling. These arrested cells are the first to respond to healing and drive the cytoskeletal machinery for tissue reorganisation. This is consistent with the idea that the requirements for cell proliferation and for cell shape changes that occur during normal fly and vertebrate development and wound repair place incompatible demands on the cytoskeletal machinery of the cell. Another issue to be considered is that the DV boundary is the first zone of closure for F-actin extensions. This is reminiscent of Drosophila embryonic dorsal closure and wound repair, in which matching filopodia on both sides of the opening are recognised by the code of segment polarity genes in each parasegment. In addition, mechanical forces may be involved in tissue reorganisation. Stretching forces could be altered upon the induction of cell death, and they could have an important role in mounting a quick healing response. For example, mechanical forces, which have been proposed to act in the developing wing disc and compress the tissue through the central region, could stretch it towards the DV border after ablation of the ptc domain. Thus, either by matching affinities or by stretching forces, wound repair spreads from the apical DV border to basal and proximal domains (Bergantiños, 2010).

It has been shown in the Drosophila wing disc that massive loss of cells after irradiation gives rise to apparently normal adult wings as a result of compensatory proliferation driven by surviving cells. Experiments involving irradiation or induction of apoptosis in a p35 background have suggested that this compensatory proliferation is controlled by signals, including JNK, emerging from cells that have entered apoptosis, and that cell-death regulators, such as p53 and the caspase Dronc (Nedd2-like caspase), function as regulators of compensatory proliferation and blastema formation in the surviving cells. By contrast, the results show that proliferation is compartment specific and occurs independently of the dead tissue following targeted ablation. Two observations strongly support this interpretation. First, puc expression, as a marker of JNK activity, is concentrated in a narrow strip of apical cells, suggesting that JNK signalling is activated in the leading edges during wound closure. This again resembles other repair mechanisms described not only in imaginal discs, but also in other healing tissues, and reiterates epithelial fusion events observed in embryogenesis. Second, perturbation of the JNK pathway within the dying domain has no effect on either healing or regeneration. Even the early peak of localised mitosis near the wound and the later A-compartment-associated mitoses are present when UAS-bskDN and UAS-puc are driven in the dying domain. Effects on healing and regeneration are found only in hep mutant backgrounds, when JNK is impaired in the whole epithelium and not only in the dead domain. This requirement for the JNK pathway at the edges of the wound has also been found in studies of microsurgically induced regeneration. Cell lineage analysis of puc-expressing cells near the wound has shown that puc sets the limits of a blastema and that puc derivatives are able to reconstitute most of the missing tissue (Bergantiños, 2010).

Finally, whether JNK is required for healing alone or also functions as a signal for proliferation remains an open issue. Rapid local proliferation is affected in unhealed hep heterozygotes. Also, salPE>rpr wing regeneration cannot be achieved after 10 hours induction in a hepr75 background. Reduced proliferation could be due to a lack of healing or to loss of JNK activity. The possibility canot be ruled out that the JNK cascade, through the active AP-1 (Kayak and Jun-related antigen -- FlyBase) transcription factor complex, targets not only genes required for healing and epithelial fusion, but also those required for regenerative growth. In mammals, inhibition of the JNK pathway or lack of c-Jun results in eyelid-closure defects and also impairs proliferation by targeting Egfr transcription. Reconstruction of normal pattern and size might also require multiple signals. It has recently been found that regenerative growth induced by cell death requires Wnt/Wg signalling to increase dMyc stability, suggesting the involvement of other signalling pathways and also cell competition. It is very likely that an integrated network of signals and cell behaviours is necessary to reconstitute the damaged tissue (Bergantiños, 2010).

Taken together, these results suggest a model for cell-induced regeneration that includes two phases. The first, which occurs near the wound edges, involves JNK activity and is important for healing and rapid local proliferation. The second involves proliferation to compensate for the lost tissue and is extended throughout the damaged compartment. As in normal development, the regenerative growth that occurs in this second phase requires the reconstitution of morphogenetic signals that drive proliferation (Bergantiños, 2010).

Sumoylation modulates the activity of Spalt-like proteins during wing development in Drosophila

The Spalt-like family of zinc finger transcription factors is conserved throughout evolution and is involved in fundamental processes during development and during embryonic stem cell maintenance. Although human SALL1 is modified by SUMO-1 in vitro, it is not known whether this post-translational modification plays a role in regulating the activity of this family of transcription factors. This study shows that the Drosophila Spalt transcription factors are modified by sumoylation. This modification influences their nuclear localization and capacity to induce vein formation through the regulation of target genes during wing development. Furthermore, spalt genes interact genetically with the sumoylation machinery to repress vein formation in intervein regions and to attain the wing final size. These results suggest a new level of regulation of Sall activity in vivo during animal development through post-translational modification by sumoylation. The evolutionary conservation of this family of transcription factors suggests a functional role for sumoylation in vertebrate Sall members (Sánchez, 2010).

The activity and the location of some transcription factors can be influenced by their post-translational modifications. For instance, murine Sall1 phosphorylation by protein kinase C disrupts its interaction with the histone deacetylase complex and its transcriptional ability (Lauberth, 2007). This study investigated how sumoylation might affect the activity of Drosophila Sall proteins in vivo and their localization in cultured cells. The results show that both Sal and Salr are sumoylated and that their sumoylation status might influence their subnuclear localization and their activity in vivo (Sánchez, 2010).

The results indicate that localization of Sall proteins depends on their sumoylation status. On one hand, it was shown that the subnuclear localization of Sal changes in wing imaginal discs when sumoylation is compromised. On the other hand, Sal localization changes when the Drosophila SUMO homolog Smt3 is overexpressed in cultured cells. Although HEK-293FT cells express SUMO, only a proportion of the overexpressed Sall factors might be sumoylated. This proportion might be augmented when Smt3 is overexpressed, making the change in localization visible. Therefore, the results in cultured cells are in accordance with the changes in Sal localization seen in imaginal discs: in WT discs, where Smt3 is expressed throughout the wing blade (Talamillo, 2008), Sal shows a more diffuse localization than in discs deficient for sumoylation. Interestingly, analysis in imaginal discs also showed that Sal expression is not abolished when sumoylation is impeded. In addition, Sal is still expressed when smt3 is down-regulated by RNA interference in imaginal discs, despite that the function of signaling of Vg and Decapentaplegic, two known positive regulators of sal in the wing, could be affected by the lack of sumoylation (Sánchez, 2010).

The role of Sal and Salr in the formation of vein LII is not fully understood. Whereas the absence of both genes inhibits vein formation, the absence of only Sal promotes the formation of ectopic vein LII, suggesting a possible role for Salr as an vein activator. However, the overexpression of either protein using Gal4 lines that drive expression throughout the wing blade, such as 638-Gal4 and Nubbin-Gal4, inhibits vein LII formation through the inhibition of kni. Using SalEPv-Gal4, expression of sall constructs in the central part of the wing was increased, with this effect being more acute in the presumptive LII region. It was hypothesized that by overexpressing either the WT or sumoylation mutant forms of the proteins, the proportion of sumoylated versus non-sumoylated forms might be alteredin vivo, with this affecting the regulation of kni. In the case of vein LIII, analysis of trans-heterozygous flies suggests that Sall proteins and Smt3 collaborate in the repression of vein LIII in the LII-LIII intervein region. In addition, the overexpression of the sumoylation mutant Salr-IKEA-IKVA promoted the formation of ectopic vein LIII, suggesting that sumoylated Salr is necessary for LIII repression in the LII-LIII intervein region (Sánchez, 2010).

One conclusion that emerges from these results is that sumoylation seems to affect the localization and vein repressor capacities of Sal and Salr in opposite ways: whereas sumoylation modifies the subnuclear localization of Sal toward a more diffuse pattern in the nucleus, Salr sumoylation promotes the formation of large subnuclear aggregates. Sal and Salr exhibit transcriptional repressor capacity in cultured cells, and it could be possible that these dramatic changes in the localization of Sall proteins could alter their transcriptional repressor capacity in vivo. In the adult wing, overexpression of the sumoylation mutant of Sal produces a milder phenotype than the overexpression of WT Sal, whereas the sumoylation mutant of Salr increases the formation of ectopic veins in comparison with WT Salr. In addition, the results on the capacity to regulate the expression of kni fit well with the phenotypic consequences observed in adult wings: the overexpression of the mutant form of Sal affects kni expression in a milder way than the overexpression of the WT form. In contrast, the overexpression of mutant Salr increases the kni expression territory, suggesting that the sumoylated form of Salr regulates the repression of kni in the intervein region more effectively compared with the non-sumoylated form. Thus, sumoylation seems to affect Sal and Salr in opposite ways. As both proteins are in many cases coexpressed and might participate in the same biological processes, their regulation by sumoylation opens an interesting venue in which the same modification produces opposite effects, contributing to the fine-tuning of the role of Sall proteins in gene regulation (Sánchez, 2010).

In summary, the sumoylation of Sall proteins affects their nuclear localization. Sumoylation is necessary for repressing vein LIII by Sall in the LII-LIII intervein region and for attaining the wing final size, suggesting that these two functions are sensitive to the doses of sumoylated Sall proteins (Sánchez, 2010).

Effects of Mutation or Deletion

Mutation in the sal gene leads to incomplete transformation of pattern elements of the posterior head and interior tail towards trunk. Specifically, the labial segment is partially transformed to anterior prothorax, without altering Scr expression in this segment (Casanova, 1989). Abdominal-B/sal double mutants develop thoracic structures in place of ectopic head structures found in the tail region of Abd-B single mutant embryos (Jürgens, 1988).

spalt activity suppresses the molecular pathway that establishes tracheal development. spalt function is also necessary for the directed migration of dorsal trunk cells, a distinct subset of tracheal cell. This process is a prerequisite for the dorsal trunk generated by fusion of adjacent tracheal metameres into a common tubular structure (Kühnlein, 1996).

The formation of the tracheal network in Drosophila is driven by stereotyped migration of cells from the tracheal pits. No cell divisions take place during tracheal migration and the number of cells in each branch is fixed. This work examines the basis for the determination of tracheal branch fates, prior to the onset of migration. The EGF receptor pathway is activated by localized processing of the ligand Spitz in the tracheal placodes and is responsible for the capacity to form the dorsal trunk and visceral branch. Prominent double phosphorylation of Erk (Rolled) is detected in the tracheal placodes at stage 10-11. This pattern is Efgr-dependent and is abolished in rhomboid mutants. The double phosphorylated Erk domain is broader than the region of rhomboid expression. Since Rhomboid is known to regulate Spitz processing, this pattern probably reflects the diffusion of the secreted form of Spitz originating within the rhomboid-expressing cells, in the central part of the placode. In mutants for Egfr, tracheal pits appear normal, although certain tracheal branches fail to develop: specifically, the dorsal trunk and visceral branch are missing or incomplete. spalt mutants show specific defects in the migration of dorsal trunk cells, pointing to an important role for spalt in subdivision of tracheal fates. The Dpp pathway is induced in the tracheal pit by local presentation of Dpp from the adjacent dorsal and ventral ectodermal cells. This pathway patterns the dorsal and lateral branches. Elimination of both Dpp and Egfr pathways blocks migration of all tracheal branches. Antagonistic interactions between the two pathways are demonstrated. The opposing activities of two pathways may refine the final determination of tracheal branch fates. Egfr-dependent activation of Erk (Rolled) in the tracheal placode precedes the activation of the same pathway by Breathless. Only after Egfr induction is diminished, does a new double phosphorylated Erk pattern appear, induced by Breathless. It is proposed that two opposing gradients of Dpp and Spitz are operating within the placode. the cells in the center of the tracheal placode encounter high concentrations of secreted Spitz, and low or negligable levels of Dpp. Conversely, the cells located at the dorsal and ventral domains of the placode encounter high concentrations of Dpp and low levels of secreted Spitz. Therefore, induction by the Egfr and Dpp pathways creates three subsets of cells: dorsal, central and ventral (Wappner, 1997).

Spalt function during eye development

The Drosophila ommatidia contain two classes of photoreceptor cells (PRs), R1-R6 and R7 and R8, the outer and the inner PRs respectively. An enhancer trap screen was carried out in order to target genes specifically expressed in PRs. Using the UAS/GAL4 method with enhanced green fluorescent protein (eGFP) as a vital marker, 180,000 flies were screened. Out of 2730 lines exhibiting new eGFP patterns, a focus was placed on 16 lines expressing eGFP in particular subsets of PRs. In particular, three lines are described with inserts near the spalt major, m-spondin and furrowed (fw) genes, whose respective expression patterns resemble those genes. These genes had not been reported to be expressed in the adult eye. These examples clearly show the ability of this screen to target genes expressed in the adult Drosophila eye (Mollereau, 2000).

salm has a very dynamic expression pattern in several organs and has been implicated in multiple developmental processes. Interestingly it has been shown to be expressed in R3, R4 and cone cells in the eye imaginal disc. It is expressed in R7 and R8 PR's in the adult eye, suggesting multiple roles at different stages of eye development. However, no morphological defects have been observed in eye mosaic clones for salm suggesting a very subtle phenotype or possible redundancy with the related gene salr. Indeed salm and salr have very similar expression patterns due to the organization of their regulatory region in a common DNA fragment located 3' of both transcription units. The detection of a primary pigment cell staining by X-Gal in the enhancer trap line R7T3.8 but not in the salm antibody staining, suggests that GAL4 may be under the control of salr enhancer. As a transcription factor expressed in R7 and R8, Salm could play a role in the transcription of rh3-6 in R7 and R8; alternatively Salm could mediate repression of rh1 in inner PR cells. This hypothesis is currently being tested in salm mosaic clones generated in the eye (Mollereau, 2000).

The mspo insertion line shows X-Gal staining in R7 cells, together with strong lamina and medulla staining. No phenotype has been observed at embryo muscle attachment sites in mspo null mutants. Similarly no morphological defects were found in the adult eye of the mspo null mutant. This could be due to a possible redundancy between Mspo and two other related genes found in Drosophila (Mollereau, 2000). furrowed (fw) mutants have been reported to exhibit a strong morphological phenotype in the adult eye as well as defects at the level of the mechanosensory bristles. A possible explanation for the eye phenotype has been derived from the fact that fw is expressed in the eye imaginal disc, suggesting a role for fw in early eye development. However, it is possible that the mutant phenotype is due to the loss of the strong adult expression that has been observed in the primary pigment cells (Mollereau, 2000).

The formation of photoreceptor cells (PRCs) in Drosophila serves as a paradigm for understanding neuronal determination and differentiation. During larval stages, a precise series of sequential inductive processes leads to the recruitment of eight distinct PRCs (R1-R8)1. But, final photoreceptor differentiation, including rhabdomere morphogenesis and opsin expression, is completed four days later, during pupal development. It is thought that photoreceptor cell fate is irreversibly established during larval development, when each photoreceptor expresses a particular set of transcriptional regulators and sends its projection to different layers of the optic lobes. The spalt (sal) gene complex encodes two transcription factors that are required late in pupation for photoreceptor differentiation. In the absence of the sal complex, rhabdomere morphology and expression of opsin genes in the inner PRCs R7 and R8 are changed to become identical to those of outer R1-R6 PRCs. However, these cells maintain their normal projections to the medulla part of the optic lobe, and not to the lamina where outer PRCs project. These data indicate that photoreceptor differentiation occurs as a two-step process: (1) during larval development, the photoreceptor neurons become committed and send their axonal projections to their targets in the brain; (2) terminal differentiation is executed during pupal development and the photoreceptors adopt their final cellular properties (Mollereau, 2001).

The two zinc finger proteins of the sal gene complex are expressed in distinct subsets of PRCs throughout eye development. sal major (salm) and sal related (salr) have almost identical expression patterns in most tissues, including the imaginal disc, and thus are likely to have similar or overlapping roles. In eye imaginal discs, the sal genes are expressed in a very dynamic pattern including R3 and R4 PRCs and cone cells. However, no obvious defects have been reported in salm mutant eye discs. In the adult, salm is no longer expressed in outer PRCs but is restricted to the inner PRCs, R7 and R8. To determine when this transition happens, salm expression was analysed during pupal life. After 24 h of pupation, salm is expressed in R3, R4 and cone cells. After 48 h, salm expression is strongly diminished in the cone cells with only weak labelling detected at 72 h in these cells. Between 48 h and 60 h of pupation, salm expression is turned off in R3 and R4 but is activated in R7 and R8 where it is maintained throughout adult life. This activation of salm expression in inner PRCs occurs at about the same time as the onset of rhabdomere morphogenesis and rhodopsin expression, suggesting a role for these genes in the differentiation of R7 and R8. To examine the role of the sal complex in eye morphogenesis, tissue that was mutant for both salm and salr was generated using a small chromosomal deficiency [Df(2L)32FP5] uncovering only these genes. No large phenotypic changes were detected in the PRCs in imaginal discs and the projections to the optic lobes appeared to be normal. However, the mutant ommatidia in the adult eye are greatly altered: the small central rhabdomeres of inner R7 and R8 PRCs are absent, and extra PRCs with large rhabdomeres are observed. Mosaic analysis indicates that the individual mutant R7 and R8 cells exhibit features of outer R1-R6 PCRs in a cell-autonomous manner. Sections through mutant ommatidia showed that most of the transformed PRCs have rhabdomeres that extend throughout the thickness of the retina, and hence ommatidia with eight outer PRCs are found in apical sections. Together these data suggest that both R7 and R8 are transformed into outer PRCs. In addition, a small proportion of ommatidia is also observed with as many as nine or ten outer photoreceptor rhabdomeres, which appear to arise both from rhabdomere duplication and from recruitment of additional PRCs, on the basis of the presence of extra photoreceptor cell bodies in some ommatidia. Because of the position of this extra rhabdomere, between R3 and R4 cells in most cases, the idea is favored that there is a transformation of 'mystery cells' toward outer photoreceptor cell fate. Finally, in a few cases, six or less PRCs were present. This could be accounted for by photoreceptor loss: older flies exhibit dramatic pathology of some PRCs reminiscent of degeneration (Mollereau, 2001).

In wild-type flies, the six outer PRCs, R1-R6, express rhodopsin1 (rh1) and mediate image formation and dim light vision. The inner PRCs R7 and R8 express distinct rhodopsins (rh3 or rh4 in R7; rh5 or rh6 in R8) that mediate color perception. To define more precisely the cell fates adopted by sal mutant ommatidia, rhodopsin expression was examined in eyes that were completely mutant for salm/salr. All rhabdomeres contained Rh1 but none of the inner rhodopsins (Rh3, Rh4, Rh5 and Rh6) were detected at significant levels, strongly supporting the model proposed above that both R7 and R8 are transformed into outer R1-R6 PRCs. A similar, but weaker and less penetrant phenotype was observed with a single mutant for salm, probably owing to a partially redundant function for this family of related transcription factors. Because salm is normally only expressed in non-Rh1-positive PRCs, and because rh1 expression is expanded in salm/salr mutants, it is possible that rh1 is repressed by salm/salr. However, the extent to which Sal proteins regulate rhodopsin promoters is as yet unknown (Mollereau, 2001).

The fairly late timing of salm expression in inner PRCs during pupal life (that is, much later than the time PRCs send out their axons in third instar larvae) suggests that its role in photoreceptor differentiation is not related to early photoreceptor specification or axon pathfinding. Consistent with this hypothesis, it was found that, in the salm/salr mutants, the transformed R7 and R8 cells project their axons to the medulla, which is their normal site of projection. Therefore, it appears that the determination of R7 and R8 is correctly initiated, but that these cells later adopt features typical of outer R1-R6 PRCs. Furthermore, the expression of prospero (pros), an early cell-marker for R7 neurons whose expression is maintained in the adult and controls aspects of R7 differentiation, is normal in adult clones of salm/salr. These results demonstrate that although the mutant R7 and R8 cells have the morphology of outer PRCs and express rh1, they express early R7-specific markers. There are examples of transformation of inner into outer PRCs, for instance in mis-expression experiments with rough or seven-up. However, in these cases, the transformation occurs much earlier in the disc and concerns only R7 cells. Although this has not been addressed, it is predicted that, in this case, the projections of transformed R7 cells are to the lamina and no longer to the medulla (Mollereau, 2001).

Together, these data show that the sal complex is essential for the terminal differentiation of inner R7 and R8 PRCs. In its absence, these PRCs exhibit characteristics of both inner and outer PRCs. Although initial work had predicted that eye development occurs in different stages marked by the successive expression of various molecules in PRCs, most recent studies largely assume that PRCs are fully determined in the third instar imaginal disc. This study demonstrates that photoreceptor development is a two-step process and that each step is under different genetic regulation. In the first step, the cells adopt their fate as neurons, become committed, and send specific axonal projections. During this recruitment stage, the PRCs are predetermined but their fate is not fully and irreversibly established. In a second step these neurons become mature photoreceptors. They execute their differentiation program and acquire their final properties with rhodopsin gene expression and rhabdomere morphogenesis. Atypical terminal differentiation of inner PRCs occurs naturally in specific parts of the retina. For instance, two rows of ommatidia at the dorsal margin of the eye display normal R7 and R8 specification but later acquire different terminal fates, with much larger rhabdomeres and cells that express only rh3 in both R7 and R8. These cells still project normally to the medulla. It is noted that in Crx-deficient mice, a model for human cone-rod dystrophy, the photoreceptors are specified but fail to undergo terminal differentiation, with no outer segment morphogenesis and loss of cone and rod opsins (Mollereau, 2001).

Effects of Mutation: Spalt function in the peripheral nervous system

Spalt works as a cell fate switch between two EGFR-induced cell types, the oenocytes and the precursors of the pentascolopodial organ in the embryonic peripheral nervous system. Removal of spalt increases the number of scolopodia, as a result of extra secondary recruitment of precursor cells at the expense of the oenocytes. In addition, the absence of spalt causes defects in the normal migration of the pentascolopodial organ. The dual function of spalt in the development of this organ, recruitment of precursors and migration, is reminiscent of its role in tracheal formation and of the role of a spalt homolog, sem-4, in the C. elegans nervous system (Rusten, 2001).

In order to understand the role of sal and salr during PNS development, a detailed analysis of their expression pattern in the trunk region was carried out during embryonic stages. Double immunostaining was performed using anti-Sal antibodies together with different markers for the developing PNS in stage 16 embryos. To identify neuronal cells, the monoclonal antibody 22C10 that labels all PNS neurons was used. Antibodies specific for Elav, an RNA binding protein located in the nuclei of all neuronal cells, was also used. The A18 and A37 lacZ insertion lines, as well as anti-Cpo antibodies (which are all markers for most if not all PNS cells) were also used. Sal-positive cells are located in the three abdominal PNS clusters: ventral, lateral and dorsal. In the lateral cluster sal is expressed in the sheath cell of the single chordotonal organ, v'ch1 as well as all the pentascolopodial support cells, but not the pentascolopodial neuron. Moreover, sal is also expressed in the two accessory cells associated with lch5. In the ventral cluster neurons v'esA and v'esB are sal positive, as are two unidentified cells in close proximity, as well as the sheath cells of the vchA and vchB chordotonal organs. In the dorsal cluster sal is expressed in the dorsal bipolar neuron (dbp) and its associated glia (PG3), as well as another unidentified neuron (Rusten, 2001).

In addition to the PNS, other cells in the region stain prominently with anti-sal antibodies. These cells are the oenocytes (oe), which are situated between the epidermis and lch5 in late embryos. Little is known about the development of these putative nephrocytes, except that they are located exclusively in abdominal segments and are of ectodermal origin. The analysis of a number of lacZ lines shows that oenocytes originate in the epidermis of stage 11 embryos (Rusten, 2001 and reference therein).

Using the expression pattern of sal as reference, the expression of salr was analyzed by in situ hybridization and double immunostaining using anti-Sal and anti-Salr specific antisera. Salr is first detected at stage 13 in the oenocytes, where it colocalizes with Sal and at stage 14 in some ventral cells. These are likely to be v'esA and v'esB since they are positive for sal and salr later in development. At stage 16, Salr is expressed in the oenocytes, the dbp neuron, v'esA and v'esB, but it is absent from other PNS organs in the abdomen. In summary, sal and salr are expressed in a partially overlapping pattern in the PNS. However, sal and not Salr is expressed in distinct support cells of lch5, indicating that salr may not play an important role in the development of this organ (Rusten, 2001).

Given the possibility that the Sal-positive cells surrounding the most dorsal lch5 precursor, C1, are developing oenocytes, it was hypothesised that the extra scolopodia observed in sal mutants would develop at the expense of these cells. To test this, the drifter-lacZ insertion line was used as a marker for oenocytes. Double staining using anti-Spalt and anti-beta-Gal antibodies confirmed the colocalization of the two proteins in the cells surrounding the C1 precursor. Later in development these cells migrate ventrally in close association with the lch5 organ and are finally located in the lateral position between the lch5 and the epidermis. This strongly suggests that the C1-surrounding cells are indeed the oenocytes. In concordance with this hypothesis, the cells surrounding the C1 precursors disappear in the sal mutants. At later stages of development, while wild-type embryos have 5.9 oenocytes per hemisegment, sal mutant embryos have an average of 0.4 oenocytes per hemisegment, respectively (Rusten, 2001).

By analogy with the developing lch5, it was hypothesized that the oenocytes require Egfr signalling for proper development. Embryos mutants for Star and spitz were examined at different stages of development. Interestingly, in stage 11 embryos the sal pattern of expression remains unaltered in the cells surrounding the C1 precursor, as well as in the epidermis. However, later on, the development of the oenocytes is inhibited. These results indicate that sal regulation is independent of the Egfr pathway and that the oenocytes development depends on both sal and Egfr signaling activity. Furthermore, if the signaling arises from the precursor C1, the formation of oenocytes would be restrained in the absence of SOPs. Indeed, in ato mutant embryos oenocytes originate only in the segments where remnant SOPs develop (Rusten, 2001).

In conclusion, the results are consistent with a model where sal restricts the ability of C1-surrounding cells, receiving Egfr signaling, to adopt sensory organ precursor cell fate; these cells then develop as oenocytes rather than chordotonal organs (Rusten, 2001).

The Egfr pathway is implicated in the development of the chordotonal organs in Drosophila. The pathway is necessary for the second step of recruitment of SOPs from ectodermal precursors, and for the consequent increase of number of scolopodia in the lch5 and in the vchA/B organs. Thus, during development of the lch5 organ, where two secondary SOPs are recruited, removal of positive Egfr pathway components like rho, S, spi, pnt, sos, Drk, or Egfr itself, reduces the number of scolopodia in the lch5 from five to three. Conversely, mutations in negative regulators of Egfr signaling like argos, gap1 or spry result in an increase of secondary recruited SOPs in the thorax as well as in the abdominal segments (Rusten, 2001 and references therein).

Sal plays a role in the formation of the lch5 in parallel with the Egfr signaling pathway: the absence of sal generates supernumerary scolopodia, while the overexpression of Sal reduces the number of scolopodia from five to three. These results are consistent with the idea that under wild-type conditions, sal modifies the Egfr signaling output in the cells surrounding the primary precursor C1, which instead of becoming secondary SOPs adopt the oenocytes cell fate. Five lines of evidence support this idea. (1) Supernumerary support cells accompany the supernumerary neurons observed in sal mutants. Thus, the phenotype is not caused by cell fate transformation within the SOP lineage. (2) The C1-surrounding cells receive the Egfr signal (shown by the antibody staining for activated Rolled/MAPK) and, therefore, are capable of becoming secondary precursors. These cells are sal positive while the other potential secondary precursors, also showing activated Rolled and overlying the more ventrally located C2-C5, are not. Given that the number of cells receiving the Egfr signal is larger than the number of cells that become secondary SOPs (two for lch5 and one for vchA/B), the output of the Egfr pathway must be modified in the rest of the cells receiving the signal. (3) The analysis of allelic combinations between sal and Egfr pathway mutants reveals that the supernumerary neuronal phenotype observed in the absence of sal is Egfr dependent. (4) The oenocyte precursors depend on sal and Egfr signaling to develop, and (5) in the absence of primary precursors, oenocytes do not develop, as shown in ato mutants (Rusten, 2001).

The effects of sal loss- and gain-of-function are similar, but not identical, to the ones exhibited by corresponding changes in negative regulators of Egfr signaling. There are at least two important differences between the role of these regulators and sal. (1) aos, pnt and spry are expressed in all the cells receiving the Egfr signal from the primary SOPs, while sal is expressed only in a subset of them. Consistent with this, the loss of function of these regulators affects the secondary recruitment of SOPs to other chordotonal organs, like vchA/B and v'ch1, while sal seems to modify only lch5. (2) The increase of scolopodia numbers in lch5 is moderate in the spry and aos mutants, while in sal mutants, up to eight scolopodia are observed. In conclusion, sal is involved specifically in the formation of lch5 in a manner different from that of the Egfr pathway regulators that are involved in the development of all the chordotonal organs (Rusten, 2001).

The cells surrounding C1 migrate along the dorsoventral axis, closely associated with the pentascolopodial organ. These cells are easy to recognize by the elongated shape of their nuclei and the strong sal expression that they display. These cells occupy the location of oenocytes in late embryonic stages. It is then likely that sal plays a role in deciding the fate of the Egfr responding cells surrounding the C1 precursor. In the presence of sal these cells will become oenocytes while in the absence of sal (as is true for the presumptive secondary precursors overlying C2, C3, C4 and C5), the cells will become sensory organ precursors. Since the putative precursors of the oenocyte cells need Egfr signaling to accomplish some aspects of their development, sal is thought to act as a selector gene being necessary to direct them to their correct fate (Rusten, 2001).

In addition to the extra recruitment phenotype, sal mutants have aberrantly located lch5 along the dorsoventral axis. In the wild type, lch5 precursors are recruited in a dorsal position and then migrate ventrally. In the mutant, the ventral migration does not seem to take place. The phenotype is similar, but not identical to that of Homothorax, Abdominal-A or extradenticle mutants, where the lch5 organ remains in a dorsal position and scolopodial numbers are reduced to three. The involvement of sal in the migration process has been reported for tracheal development. There, in cells of the dorsal tracheal trunk, sal is required for anteroposterior migration and morphogenesis. Furthermore, it has been shown that sal is necessary for the correct location of some neurons in the CNS. The molecular mechanisms involved in the specification of migration are largely unknown, and whether the same mechanism applies in the three cases mentioned remains unexplored (Rusten, 2001).

The pleiotropic functions that Spalt proteins exert during development are remarkable. In C. elegans, sem-4 phenotypes include cell fate changes, cell death, defects in axonal morphologies, extra cell divisions or migration. The same is true in Drosophila, where sal genes play a role in establishing homeotic identities in the blastoderm, positioning the wing veins, localizing sensory organ clusters and affecting the migration of the dorsal tracheal trunk. It therefore appears that the Spalt proteins can function with different signaling pathways and act in combination with other transcription factors to serve diverse roles during development (Rusten, 2001).

spalt-dependent switching between two cell fates that are induced by the Drosophila EGF receptor

Signaling from the EGF receptor can trigger the differentiation of a wide variety of cell types in many animal species. The mechanisms that generate this diversity have been explored using the Drosophila peripheral nervous system. In this context, Spitz ligand can induce two alternative cell fates from the dorsolateral ectoderm: chordotonal sensory organs and non-neural oenocytes. The overall number of both cell types that are induced is controlled by the degree of Egfr signaling. In addition, the spalt gene is identified as a critical component of the oenocyte/chordotonal fate switch. Genetic and expression analyses indicate that the Sal zinc-finger protein promotes oenocyte formation and supresses chordotonal organ induction by acting both downstream of and in parallel to the Egfr. To explain these findings, a prime-and-respond model is proposed. Here, sal functions prior to signaling as a necessary but not sufficient component of the oenocyte prepattern that also serves to raise the apparent threshold for induction by Spi. Subsequently, sal-dependent Sal upregulation is triggered as part of the oenocyte-specific Egfr response. Thus, a combination of Sal in the responding nucleus and increased Spi ligand production sets the binary cell-fate switch in favour of oenocytes. Together, these studies help to explain how one generic signaling pathway can trigger the differentiation of two distinct cell types (Elstob, 2001).

The larval oenocytes of Drosophila are conspicuous secretory cells of ectodermal origin. They are arranged in clusters of, on average, 6 cells per abdominal hemisegment, occupying a characteristic lateral and subepidermal location. In contrast to the invariant peripheral nervous system, the number of cells in each larval oenocyte cluster can vary between 4 and 9. Using many different molecular markers, the development of larval oenocytes has been followed from the third larval instar back to the extended germ band stage of embryogenesis. Developing oenocytes express four genes from very early stages, all of which encode DNA-binding proteins. These are seven up, pointed, spalt and ventral veins lacking which produce proteins of the nuclear receptor, ETS-domain, zinc-finger and POU-homeodomain class, respectively (Elstob, 2001).

In the late embryo, immunolabelling experiments were carried out with two independent oenocyte markers: svp-lacZ, an enhancer trap into the svp gene and BO-lacZ, a regulatory construct containing an oenocyte-specific enhancer from the sal complex. Using these markers, in conjunction with the sensory neuronal marker anti-Futsch/ 22C10, it can be seen that each cluster of oenocytes is closely associated with an array of five lateral chordotonal organs, termed an Lch5. In each abdominal hemisegment, there are eight chordotonal organs that are partitioned into arrays consisting of one dorsolateral (V'ch1), five lateral (Lch5) and two ventral (VchAB) organs. The close apposition of mature oenocyte clusters and Lch5 arrays in late embryos suggests that their formation might be linked in some way. In order to investigate this possibility, the spatial relationship between the precursors of both cell types were examined in early embryos. Each chordotonal organ is formed by a single chordotonal organ precursor (COP) that divides asymmetrically to produce four cells, a sensory neuron, scolopale, ligament and cap cell. The progeny of the most dorsal COP (C1) constitute the most anterior chordotonal organ of the lateral cluster (Lch5a). A rho-lacZ insertion that is specific for Lch5a and its precursor COP was used, together with anti-Sal, to follow the development of C1 and oenocytes simultaneously. By stage 10, C1 has delaminated and does not express Sal, despite lying directly underneath a dorsal domain of Sal-positive ectoderm (termed the dorsal Sal domain). By stage 11, C1 has already divided and its progeny are surrounded by a whorl of sickle-shaped nuclei expressing higher levels of Sal than surrounding cells. The whorl structure always appears in a dorsal and posterior segmental position, close to the ventral limit of the Sal domain, and corresponds to oenocyte precursors in the process of delamination. In addition to high levels of Sal, the oenocyte precursor whorl also expresses svp-lacZ and vvl. Since only one oenocyte precursor whorl per hemisegment is seen, and this surrounds C1, it is concluded that more ventral COPs are not associated with the formation of oenocytes (Elstob, 2001).

Oenocytes are induced from the ectoderm by an inductive signal that is generated in the developing PNS. The production of active Spi by the C1 precursor cell, under the control of ato and rho, triggers Egfr activation and thus oenocyte induction in adjacent ectoderm. Oenocyte induction by the PNS appears to be a short-range event with only the cells immediately surrounding C1 switching on oenocyte markers. In argos mutants, however, the range of the response is increased from one to two concentric rings of cells. Hence, as in photoreceptor recruitment, Spi ligand is not intrinsically limited to immediate neighbors but the response is nevertheless kept short-range by argos-mediated feedback inhibition of the receptor (Elstob, 2001).

In wild-type embryos and in all of the mutant backgrounds examined, the number of cells in the whorl at any one time is less than the final number of mature oenocytes. For example, a wild-type whorl contains 3-4 cells with sickle-shaped nuclei but the final oenocyte cluster contains on average 6 cells. The basis for this difference is not yet understood but it might be explained by cell division or by sequential delamination of oenocyte precursors (Elstob, 2001).

The specification of secondary COP and oenocyte fates requires the Egf pathway. In ato, rho, spi and EgfrDN backgrounds, where signaling is compromised, the induction of both cell types is blocked. Conversely, when the Egfr is hyperactivated, both cell types become more numerous. These results indicate that the number of recruited cells is controlled by the amount of Egf pathway signal. It is important to realize, however, that the level, duration and spatial extent of ligand production are all being altered in these experiments. More sophisticated methods would be needed to clearly distinguish which of these three signaling parameters is critical for controlling cell number (Elstob, 2001).

Surprisingly, there is no parity between the numbers of excess oenocytes and lateral chordotonal organs that are produced by Egfr hyperactivation. Thus for a given increase in ligand, more oenocyte precursors than COPs are recruited. This implies the existence of an additional tier of control that restricts neural but not oenocyte induction. Such a selective inhibition process would ensure that the number of chordotonal organs is more tightly controlled than that of oenocytes, as is observed in wild-type embryos (Elstob, 2001).

The expression pattern and mutant phenotype of sal can account for the restriction of oenocyte induction to a single whorl surrounding C1, the most dorsal primary COP. It has been suggested that C1 and C3 each induce one secondary COP. However, the results presented here argue that the presence of Sal is incompatible with chordotonal recruitment. Therefore, the idea is favored that C3 recruits both of the secondary COPs that contribute to the Lch5 array (Elstob, 2001).

The sal gene plays opposite roles in oenocyte and chordotonal induction. It is both necessary and sufficient for repressing secondary COP induction and it is necessary but not sufficient for promoting oenocyte formation. The lack of sufficiency for oenocyte induction is revealed when sal is misexpressed using the en-GAL4 driver. Oenocytes are not ectopically induced in ventral regions, even in the presence of excess Spitz. It is likely that other factors are required, together with Sal, to promote the oenocyte induction process (Elstob, 2001).

Using epistasis tests and gene expression analysis the regulatory relationship between sal and the Egf pathway in oenocyte and COP formation has been elucidated. These data allow the exclusion of the possibility that sal acts upstream of spi in the signaling cell. Importantly, the results indicate that sal functions in the responding ectoderm, either downstream of the Egfr or in a parallel pathway leading to oenocyte induction and secondary COP repression (Elstob, 2001).

In fact, it is probable that sal plays a dual role that is downstream and also in parallel to the Egfr. In rho and spi mutants, the normal upregulation of Sal in the vicinity of C1 is abolished. Conversely, Spitz misexpression produces ectopic Sal upregulation in dorsal locations. Both results indicate that sal lies downstream of the Egfr and that Sal protein levels are controlled by receptor activation. However, Sal is also expressed at moderate levels in presumptive oenocyte precursors prior to Egf pathway activation and this expression remains normal in rho and spi mutants. For these reasons, it is likely that at least part of the function of sal lies in a parallel pathway that, in conjunction with the Egf signal, promotes oenocyte induction and inhibits COP recruitment (Elstob, 2001).

A prime-and-respond model is presented to integrate the dual roles of sal downstream and also in parallel to the Egfr. In this model, sal functions in the parallel pathway as a competence switch. Thus, Sal prepatterns the dorsal ectoderm so that, on receipt of the Egf signal, oenocytes rather than COPs are induced. One consequence of the Sal oenocyte prepattern is to increase the apparent induction threshold in responding cells. This makes the prediction that the signaling cell inducing oenocytes needs to express more ligand than those that recruit secondary COPs, and this is indeed the case. C1 is known to express high levels of rho for longer than any of the other primary COPs. Thus, the Egf pathway does contribute to the cell-type specificity of the induction event in the sense that more Spi ligand is required to induce oenocytes than to recruit chordotonal organs (Elstob, 2001).

One of the early oenocyte-specific responses to the Sal prepattern is the subsequent upregulation of Sal itself. This, in turn, stimulates the expression of the sal target gene svp, one member of the repertoire of oenocyte early differentiation genes. A key feature of the prime-and-respond model is that moderate levels of sal expression serve to prime the responding cells to further upregulate Sal when they receive Spi ligand. In support of this priming mechanism, it has been demonstrated that upregulation in response to constitutive Spitz expression is restricted to those cells that have already expressed sal. Hence, Sal proteins provide a molecular link between the prepattern and the Egfr response (Elstob, 2001).

In the prime-and-respond model, it is implicit that the early and late phases of sal expression produce distinct effects on the responding cell. As the levels of Sal are different in the two phases, it may be that there are at least two different concentration-dependent effects for this transcription factor. In agreement with this, it has been shown that strong expression of the sal target gene, svp, correlates with the domain of sal upregulation and not with the lower-level prepattern. In another system, wing vein development, there is a very extreme example of a concentration difference, with low and high levels of Sal producing completely opposite transcriptional effects on the knirps target gene (Elstob, 2001 and references therein).

Mutations in spalt cause a severe but reversible neurodegenerative phenotype in the embryonic central nervous system of Drosophila

The gene spalt is expressed in the embryonic central nervous system of Drosophila but its function in this tissue is still unknown. To investigate this question, a combination of techniques was used to analyse spalt mutant embryos. Electron microscopy shows that in the absence of Spalt, the central nervous system cells are separated by enlarged extracellular spaces populated by membranous material at 60% of embryonic development. Surprisingly, the central nervous system from slightly older embryos (80% of development) exhibited almost wild-type morphology. An extensive survey by laser confocal microscopy has revealed that the spalt mutant central nervous system has abnormal levels of particular cell adhesion and cytoskeletal proteins. Time-lapse analysis of neuronal differentiation in vitro, lineage analysis and transplantation experiments have each confirmed that the mutation causes cytoskeletal and adhesion defects. The data indicate that in the central nervous system, spalt operates within a regulatory pathway that influences the expression of the ß-catenin Armadillo, its binding partner N-Cadherin, Notch, and the cell adhesion molecules Neuroglian, Fasciclin 2 and Fasciclin 3. Effects on the expression of these genes are persistent but many morphological aspects of the phenotype are transient, leading to the concept of sequential redundancy for stable organization of the central nervous system (Cantera, 2002).

Sal is expressed by ~60 postmitotic neurons per hemisegment in the embryonic nerve cord of the CNS. The Sal-positive neurons include motorneurons RP2 and aCC and interneurons dMP2, vMP2, pCC and serotonergic neurons. Sal is not expressed by Repo positive CNS glia, although it is expressed by glial cells of the peripheral nervous system. Initial analysis has revealed that mutations in the sal locus do not interfere with the development of the overall organization of the CNS. Staining with the axonal markers Futsch/22C10 and BP102 reveals that the major axonal tracts are formed with only small (although variable and frequent) departures from the wild-type plan. The precise location of RP2 cell bodies is somewhat variable while the cell bodies of aCC, pCC and CQ neurons are in their correct locations. The mutant CNS appears to be wider and less compact, and exhibits abnormal fragility during dissection. These initial data suggest that the mutation has a widespread effect, perhaps on the integrity of the tissue. Therefore, the ultrastructure of the mutant CNS was examined (Cantera, 2002).

A strong and fully penetrant phenotype was detected in the CNS of Df(2L)32FP-5;sal445 mutant embryos by TEM at early stage 16 (~60% of embryonic development). Neuronal and glial cells in the brain and nerve cord were seen to be loosely attached or widely separated by a dramatic enlargement of the extracellular space. At this stage neuronal cell bodies are tightly packed in wild-type tissue. Membrane 'whorls', autophagosomes, and other membranous profiles typical of neurodegenerative processes were observed in the cytoplasm of neuronal and glial cells. The lacunar spaces between cell bodies and neuronal fibers in the neuropil contain large amounts of extracellular membranous material, mostly in the form of vacuoles of a wide size range, the largest approaching the size of whole cell bodies. These vacuoles either seem empty or contain smaller vacuoles, but not organelles or cytoplasmic remnants characteristic of cellular debris resulting from cell death. No increase in apoptotic profiles wasobserved as compared with wild-type tissue. Similar membranous formations result from mutations in spongecake. Axonal caliber is reduced in sal null mutants and filopodia emanating from growth cones are often clumped. The phenotype is observed in all sal mutant embryos, but not in heterozygous or wild-type embryos examined as controls. The phenotype is not observed in peripheral nerves (Cantera, 2002).

Surprisingly, the CNS appeared to recover rapidly from the degenerative process, since embryos fixed a few hours later (by late stage 16 or stage 17 -- between 80% and 90% of embryonic development) exhibit an almost normal organization for cell bodies and for neuropil. At this stage, most of the extracellular membranous material has disappeared and the neuronal cell bodies in the mutant are almost as tightly packed as in the wild type (Cantera, 2002).

If the recovery depends on the activity of another protein, with the capacity to compensate for the loss of Sal, simultaneous deletion of this protein should substantially diminish the capacity of the tissue to recover and perhaps make the phenotype irreversible. A potential candidate for this hypothetical redundant function could be the paralogous protein Salr. To test this hypothesis, embryos lacking Sal and Salr due to a small deficiency were examined. However, these homozygous Df(2L)32FP-5; Df(2L)32FP-5 mutants exhibit the same phenotype caused by the lack of Sal alone and the phenotype reverses within the same developmental interval (Cantera, 2002).

A possible interpretation of the phenotype defined above would be that components of cell adhesion are seriously compromised in the CNS of sal embryos during early stage 16. To test this hypothesis specific antibodies and laser confocal microscopy were used to survey the expression of molecules known to be important for cell adhesion in embryonic CNS at early stage 16. All the markers are detectably expressed in Df(2L)32FP-5;sal445 mutant embryos at both stages, and their spatial patterns of expression in the CNS are normal, showing that sal is not essential for any of these proteins to be expressed. However, the quantification of fluorescence intensity revealed that most markers were present in abnormally high or low levels. In transheterozygous Df(2L)32FP-5;sal445 mutants at early stage 16, when the strong transmission electron microscopy TEM phenotype is manifest, lower fluorescence levels were measured for Armadillo, N-Cadherin, Neuroglian, Fasciclin 2 and Fasciclin 3; higher fluorescence levels were measured for Notch; and levels similar to wild type for Neurotactin, Neurexin IV and Faint Sausage. Comparison between wild-type, heterozygous and null sal mutant embryos revealed a stepwise decrease in the fluorescence levels for Armadillo and N-Cadherin, indicating that the effect of the mutation is dominant (Cantera, 2002).

Fluorescence levels were measured at the stage when the TEM phenotype is reverted (stage 17). The wild-type fluorescence for the three markers studied in this regard (Armadillo, Fasciclin 2, Neuroglian) changes between early stage 16 and stage 17, indicating that during this short developmental interval the levels of cell adhesion proteins are regulated. Relative to these new wild-type levels, the three proteins that are not affected during the expression of the TEM phenotype (Neurotactin, Neurexin IV and Faint Sausage) remain normal in the mutant. The levels of Notch switch from abnormally high to slightly lower than normal. All other markers still exhibit lower-than-normal fluorescence levels, with the exception of N-Cadherin, which exhibits a partial recovery. Taken together, these data led to the conclusions that the expression of sal is necessary to maintain correct dynamic levels of several adhesion molecules in the CNS and that sal exerts this function in a persistent and dominant fashion (Cantera, 2002).

To gain a more detailed understanding of the dynamics of the sal phenotype, cell cultures were used derived from single neuronal precursors isolated either from the neuroectoderm or the midline region of mutant embryos. Unlike the wild type, mutant cells were extremely fragile and sometimes disintegrated upon suction into the microcapillary. Moreover, the cells had a rounded morphology and showed obvious difficulties in establishing and maintaining a normal attachment to the substrate. Upon inspection of time-lapse recordings, examples of cells were found that attached and lost contact with the substrate repeatedly. Wild-type neuroectodermal precursors, however, strongly adhered to the bottom of the culture chamber and adopted a more flattened morphology. Proliferation of the sal mutant-derived cells did not appear to be affected. However, their progenies exhibited a clearly slower rate of branch growth and differentiation. Even after several days of culture, most mutant clones still had a poor branching when compared with wild-type clones. The fibers growing from sal-derived cells were often very thin, confirming the original TEM observations, and sometimes displayed abnormal fasciculation. Mutant-derived clones were often surrounded by debris, probably representing material shed from living cells (Cantera, 2002).

Time-lapse recordings suggested that the cells derived from sal mutant CNS have a deficient cytoskeleton. To investigate this possibility staining was performed in vitro for tubulin and two major differences with wild-type neurons were found. In mutant neurons, tubulin did not reach into the growth cone and patches of poor fluorescence were also detected along the axon, suggesting the existence of interruptions along the core of axonal microtubules. In wild-type neurons, anti-tubulin fluorescence extends homogeneously along the entire axon length and reaches almost the distal border of the growth cone. Laser confocal microscopy of embryos stained with three cytoskeletal markers (F-actin, tubulin and the tubulin-associated protein Futsch) revealed additional differences. The three markers were correctly expressed across brain and nerve cord, with the typical accumulation along major axonal tracts, but the fluorescence levels were abnormally higher for F-actin, and lower for tubulin and its associated protein Futsch (Cantera, 2002).

On the single cell level, the data gathered from these cell cultures show that neurons derived from sal mutant neuroectodermal precursors differentiate poorly in vitro. To what extent the lack of Sal affects the differentiation of individual cell lineages was tested in the developing CNS tissue. In Drosophila each neural precursor (neuroblast) produces a stereotyped combination of cells identifiable by cell body position and the pattern of axonal projections. Extensive data obtained from lineage analysis in wild type make it possible to identify each neuroblast lineage on the basis of its neuroanatomy. This knowledge was exploited to investigate the capacity of sal mutant neuroblasts to produce normal cell clones in situ. Single neuroectodermal and midline precursors were labelled with DiI and cell lineages derived from these precursors were analysed at late stage 17 (Cantera, 2002).

The clones obtained in sal null embryos can be classified into three categories according to their degree of neuroanatomical abnormality. Some clones differentiated into morphologies showing no obvious similarities to identified wild-type lineages; others exhibited abnormally projecting axons but were as a whole identifiable as particular wild-type lineages, and finally, others were almost indistinguishable from their wild-type counterparts. Interestingly, some of the clones derived from labelled midline precursors also developed abnormalities, although sal expression has not been detected in these precursors. At high magnification, spherical thickenings were found along the axons that resembled the blistering observed in the time-lapse studies (Cantera, 2002).

The rapid recovery of sal CNS during the course of stage 16 could be explained by the robustness inherent to a system in which adhesion is mediated by a combination of proteins and the possible compensatory effect mediated by upregulation of other members of the system. However, an alternative view is proposed. The ultrastructural recovery may as well reflect the normal dynamics of combinations of adhesion proteins expressed successively along embryonic development. From this point of view, the rapid recovery from the adhesion phenotype will reflect the normal transition between two particular combinations of adhesion proteins expressed at early or late stage 16. For this to be valid, the expression levels of several adhesion proteins must change along this interval during normal development. Interestingly, the data do support this possibility, since the fluorescence levels for Armadillo, Fasciclin 2 and Neuroglian change between stages 16 and 17 in wild-type CNS. Whether sal is required for the regulation of a combination of cell adhesion and cytoskeletal proteins at a particular developmental stage could be tested by deleting the expression of Sal exclusively in CNS tissue within short developmental intervals. This approach could now be possible using techniques based on combinations of the GAL4-UAS system and RNA interference (Cantera, 2002).

Drosophila spalt/spalt-related mutants exhibit Townes-Brocks' syndrome phenotypes

Mutations in SALL1, the human homolog of the Drosophila spalt gene, result in Townes-Brocks' syndrome, which is characterized by hand/foot, anogenital, renal, and ear anomalies, including sensorineural deafness. spalt genes encode zinc finger transcription factors that are found in animals as diverse as worms, insects, and vertebrates. This study examined the effect of losing both of the spalt genes, spalt and spalt-related, in the fruit fly, and reports defects similar to those in humans with Townes-Brocks' syndrome. Loss of both spalt and spalt-related function in flies yields morphological defects in the testes, genitalia, and the antenna. Furthermore, spalt/spalt-related mutant antennae show severe reductions in Johnston's organ, the major auditory organ in Drosophila. Electrophysiological analyses confirm that spalt/spalt-related mutant flies are deaf. These commonalities suggest that there is functional conservation for spalt genes between vertebrates and insects (Dong, 2003; full text of article).

In light the observation that Drosophila sal and salr mutants exhibit eye defects, it was intriguing that a patient of age 44 with sudden optic neuropathy was subsequently diagnosed with TBS because of both physical deformities typical of the syndrome and a heterozygous mutation in SALL1 (Blanck, 2000). Together with the reported expression of sal in the mouse eye, this leads to the thought that sal is involved in vertebrate eye development. It is suggested that Drosophila sal and salr mutant phenotypes may provide clues to other overlooked or less penetrant defects associated with TBS. That sal functions in both Drosophila and human limb development, in conjunction with its putative function in vertebrate eye development, and the finding that the auditory organ, genitalia, and testes are affected by loss of sal function in both humans and flies indicates that sal functions may be evolutionarily conserved between insects and vertebrates. Studying sal in Drosophila therefore may lead to insights into the roles of sal genes in vertebrate development (Dong, 2003).

Although human SALL1 and Drosophila sal mutants exhibit some strikingly similar phenotypes, there are fundamental differences in the nature of the mutations leading to the defects. The Drosophila phenotypes result from being null for at least one sal gene, whereas TBS is caused by a heterozygous mutation in a single human sal, SALL1, that leads to production of a truncated protein. The two Drosophila genes are adjacent, similarly expressed, share enhancers, exhibit significant redundancy, and probably result from a relatively recent duplication event. The four known human genes reside on separate chromosomes and their mouse and chick homologs exhibit overlapping, but still distinct, expression patterns. Therefore, they are less likely to be as functionally redundant. However, it remains unclear whether the effects of SALL1 mutations in TBS patients result from haploinsufficiency or whether the truncated SALL1 proteins act dominantly and negatively to interfere with normal SALL1 function (and possibly the functions of other SALL genes). Sall1 heterozygous null mice are viable and appear normal, whereas homozygous null mice die perinatally with only severe kidney defects. These results lead to the idea that the mutations in TBS are dominant negative. However, the expression patterns of mouse Sall1 and human SALL1 are not identical, so they may not serve identical functions, and the effects of Sall1 mutations might not be comparable between mice and humans (Dong, 2003).

The observations that humans and Drosophila with sal mutations exhibit both conductive and sensorineural deafness has implications for thinking about the evolution of auditory systems. sal and ato are activated by Distal-less (Dll) during antennal development. The data presented in this study, when integrated with existing information, allow building of a model in which Dll regulates both ato and sal and all three are required for the normal development of a functional Drosophila auditory system. Interestingly, the homologs of Dll and ato as well as sal are required for the development of a normal vertebrate auditory system. A second human syndrome that includes limb and auditory defects, split hand/split foot malformation has been linked to vertebrate homologs of Dll, the Dlx genes, and mice null for the ato homolog Math1 lack inner ear hair cells. Thus, despite the fact that sal/salr/SALL1, ato/Math1, and Dll/Dlx have pleiotropic effects in both Drosophila and vertebrates, their expression in the developing ear appears to represent a unique and tissue-specific constellation. If the regulatory interactions among Dll, ato, and sal homologs prove to be the same in vertebrates as in Drosophila, it would provide strong support for a view that these genes were used in the development of an ancestral auditory system and that the existence of such a system predated the arthropod/vertebrate divergence. One prediction of this model is that the developing auditory organs of other animals also would require this genetic cascade (Dong, 2003).

Spalt transcription factors are required for R3/R4 specification and establishment of planar cell polarity in the Drosophila eye

The establishment of planar cell polarity in the Drosophila eye requires correct specification of the R3/R4 pair of photoreceptor cells. In response to a polarizing factor, Frizzled signaling specifies R3 and induces Delta, which activates Notch in the neighboring cell, specifying it as R4. The spalt zinc-finger transcription factors (spalt major and spalt-related) are part of the molecular mechanisms regulating R3/R4 specification and planar cell polarity establishment. In mosaic analysis, spalt genes have been shown to be specifically required in R3 for the establishment of correct ommatidial polarity. In addition, spalt genes are required for proper localization of Flamingo in the equatorial side of R3 and R4, and for the upregulation of Delta in R3. These requirements are very similar to those of frizzled during R3/R4 specification. spalt genes are required cell-autonomously for the expression of seven-up in R3 and R4, and seven-up is downstream of spalt genes in the genetic hierarchy of R3/R4 specification. Thus, spalt and seven-up are necessary for the correct interpretation of the Frizzled-mediated polarity signal in R3. Finally, it has been shown that, posterior to row seven, seven-up represses spalt in R3/R4 in order to maintain the R3/R4 identity and to inhibit the transformation of these cells to the R7 cell fate (Domingos, 2004a).

Therefore, the results suggest that sal is required upstream or in parallel to the Fz/PCP pathway for R3/R4 specification. Also, in support of this model, sal expression is not affected in R3/R4, either in gain- or loss-of-function experiments with members of the Fz/PCP and Notch signaling pathways. sal is required cell-autonomously in R4 for normal levels of mdelta0.5-lacZ expression. This requirement of sal in R4 could be due to a defect in the activation of Notch signaling [e.g. sal may be required for the expression of Notch or Su(H)]. Alternatively, sal may be required for transcriptional activation of E(spl)mdelta, in parallel to Notch signaling. The latter possibility is favored, since the expression of a transgenic line, where lacZ is under the regulation of 12Suppressor of Hairless multimerized-binding sites [12Su(H)-lacZ], is not affected when R4 is sal. The 12Su(H)-lacZ transgenic line is a reporter for Su(H)-dependent Notch signaling, and thus, sal is not required for the expression or activation of Notch, Su(H) or other components required for signaling. In addition, exogenous expression of a constitutively activated Notch (sev-Nact) can rescue mdelta0.5-lacZ expression in sal clones. Altogether, these results suggest that sal acts in parallel to Notch signaling for the transcriptional activation of E(spl)mdelta. Finally, although there is a reduction of E(spl)mdelta expression when R4 is sal, this does not correspond to chirality defects in mature ommatidia. This suggests that other genes may be redundant to sal in R4 for PCP establishment (Domingos, 2004a).

Several pieces of evidence demonstrate that sal is required upstream of svp for R3/R4 specification: (1) sal is required for svp expression in R3/R4; (2) both sal and svp are required in R3 for the establishment of proper ommatidial chirality; (3) in both sal and svp mutants Fmi is not asymmetrically localized in R3/R4 and mdelta0.5-lacZ expression is lost in R4, and (4) exogenous expression of svp in R3/R4 (sev-svp) can rescue the expression of mdelta0.5-lacZ in sal clones (Domingos, 2004a).

In addition, posterior to row seven, svp is required to repress sal expression in R3/R4 , and sal is responsible for the transformation of R3/R4 into R7 in svp mutants. Based on these results, which demonstrate that sal is both necessary and sufficient for R7 differentiation posterior to row seven, a model for the action of sal and svp during R3/R4 specification: from rows three to seven, sal is required for svp expression in R3/R4 and for R3/R4 specification: posterior to rows seven to nine, repression of sal by svp in R3/R4 is necessary for the maintenance of R3/R4 identity and the inhibition of R7 fate. This dual regulation between sal and svp helps to understand the complex sal- phenotype in R3/R4. Strikingly, although svp expression is lost in sal- R3/R4, these cells do not get transformed into R7, but keep an outer PR identity. Thus, in the absence of sal, the presumptive R3/R4 remain as outer PRs with an unspecified subtype identity (Domingos, 2004a).

In conclusion, these results demonstrate that sal is required in R3 to allow normal Fz/PCP signaling to specify the R3 and R4 cell fates. Ommatidia mutant for sal show defects that are very similar to those observed in fz and dsh mutants, as judged by the loss of asymmetric Fmi localization at the equatorial side of the R3/R4 precursors, and by the lack of Dl and E(spl)mdelta upregulation within the R3/R4 pair. In addition, sal is required upstream of svp for normal R3/R4 specification. Finally, these results show that, posterior to row seven, svp represses sal in R3/R4 in order to maintain R3/R4 identity and to inhibit transformation of these cells to the R7 cell fate (Domingos, 2004a).

Regulation of R7 and R8 differentiation by the spalt genes

Photoreceptor development begins in the larval eye imaginal disc, where eight distinct photoreceptor cells (R1-R8) are sequentially recruited into each of the developing ommatidial clusters. Final photoreceptor differentiation, including rhabdomere formation and rhodopsin expression, is completed during pupal life. During pupation, spalt has been proposed to promote R7 and R8 terminal differentiation. spalt is shown to be required for proper R7 differentiation during the third instar larval stage since the expression of several R7 larval markers (prospero, enhancer of split mdelta, and runt) is lost in spalt mutant clones. In R8, spalt is not required for cell specification or differentiation in the larval disc but promotes terminal differentiation during pupation. spalt is necessary for senseless expression in R8 and sufficient to induce ectopic senseless in R1-R6 during pupation. Moreover, misexpression of spalt or senseless is sufficient to induce ectopic rhodopsin 6 expression and partial suppression of rhodopsin 1. spalt and senseless are part of a genetic network that regulates rhodopsin 6 and rhodopsin 1. Taken together, these results suggest that while spalt is required for R7 differentiation during larval stages, spalt and senseless promote terminal R8 differentiation during pupal stages, including the regulation of rhodopsin expression (Domingos, 2004b).

Photoreceptor cell (PRC) development has been used as a paradigm to understand neuronal specification and differentiation. In the absence of the sal genes, inner PRCs R7 and R8 are transformed into the outer PRC subtype, and this phenotype has been interpreted as a result of the role of sal in R7 and R8 terminal differentiation during pupal stages. As a consequence, a model has been proposed in which PRC differentiation occurs as a two-step process. In the first step, during larval stages, the cells adopt their fate as neurons, become committed and send specific axonal projections. In the second step, during pupal stages, these neurons execute their terminal differentiation program and become mature photoreceptors. In this model, sal is required for the second step of differentiation in R7 and R8. This study shows that sal has distinct roles during R7 and R8 differentiation. In R7, sal is necessary for the expression of the larval markers pros, E(spl)mdelta, and runt. In addition, misexpression of sal during larval stages is sufficient to induce ectopic expression of Pros (R7 marker) and suppress BarH1 (R1/R6 marker). These results demonstrate that sal is required for R7 differentiation during larval stages. However, the majority of sal mutant presumptive R7 cells do not get transformed into the outer PRC subtype during larval stages since the expression of outer PRC markers (Svp, Ro, and BarH1) is not induced. Moreover, R7 specification is not disrupted in sal mutants since R7 still acquires a neuronal fate (expresses Elav), expresses detectable levels of the R7 marker H214-klg, and projects to the medulla. Therefore, it is concluded that the requirement for sal during R7 differentiation occurs soon after R7 specification in a continuum rather than in temporally distinct steps (Domingos, 2004b).

In R8, sal is not required for specification or early differentiation in the larval imaginal disc but is necessary for its terminal differentiation during pupation. During pupal stages, sal is necessary for sens expression in R8 and is sufficient to induce ectopic sens in R1-R6. Misexpression of salm, salr, or sens is sufficient to induce ectopic expression of Rh6 and partial suppression of Rh1 in the outer PRCs. Furthermore, the results place sens genetically downstream of sal during R8 pupal development and show that the regulation of Rh1 and Rh6 by sal can occur both via sens-dependent and -independent mechanisms. These findings raise a number of interesting issues with respect to the differentiation of R7 during larval stages, the terminal differentiation of R8 at pupation, and the role of sal and sens in these processes (Domingos, 2004b).

Current models account for three developmental stimuli in R7 specification and differentiation during larval stages: EGFR pathway activation, which is required for neuronal differentiation; Sevenless (Sev) receptor signaling, which is required for R7 fate assumption since in Sev mutants the presumptive R7 is transformed into a nonneural cone cell, and Notch signaling, which is also required for R7 fate assumption since loss of Notch function causes the presumptive R7 to be transformed into the R1/R6 subtype (Domingos, 2004b).

In salm/salr mutant tissue, the presumptive R7 becomes a neuron since it expresses Elav. This result implies that activation of EGFR and Sev signaling is not significantly affected by the loss of salm/salr function, placing sal downstream of EGFR and Sev activation during R7 differentiation. sal is required for activation of the Notch signaling pathway in R7 since expression of E(spl)mdelta is lost in salm/salr mutants. However, since expression of H214-klg is only partially suppressed in salm/salr mutants and BarH1 is ectopically expressed in only 4.8% of the mutant ommatidia, it is possible that some residual Notch signaling is present in salm/salr mutant R7 cells. Following Notch loss of function, all presumptive R7 cells that are transformed into the R1/R6 subtype show ectopic BarH1 and complete loss of H214-klg expression in larvae. Thus, in salm/salr loss of function, the expression of E(spl)mdelta is lost in R7 but this is not sufficient to respecify the presumptive R7 into R1/R6 subtype as is observed in Notch loss of function mutants. Only later, during pupal development, does the presumptive R7 mutant for salm/salr acquire features of outer PRCs, including large rhabdomere size and expression of rh1 (Domingos, 2004b).

Previous studies led to a model for R7 and R8 rhodopsin regulation in the 'yellow' and 'pale' ommatidial subtypes where the 'yellow' subtype (Rh4 in R7 and Rh6 in R8) corresponds to the default state and the 'pale' subtype (Rh3 in R7 and Rh5 in R8) corresponds to the acquired state. This model was based on the observation that, in sev mutants where R7 is absent, all R8 cells express Rh6, suggesting that communication between an R7 expressing Rh3 and the underlying R8 is responsible for the repression of Rh6 and the induction of Rh5 in R8. This study shows that although salm and sens are expressed in all R8 cells, misexpression of these genes in outer PRCs under the control of the rh1 promoter induces ectopic expression of Rh6 but not Rh5. These results suggest that sal and sens regulate the default state of rhodopsin expression in R8 ('yellow' subtype) and that additional factors may be required to repress Rh6 and activate Rh5 expression in the R8 'pale' subtype. The results suggest a model for the regulation of rhodopsin by sal and sens in R8 during pupal stages. In this model, sal regulates sens expression, which in turn suppresses Rh1 and induces Rh6 expression. In addition, sal can also regulate Rh1 and Rh6 independently of sens, in a direct manner or in conjunction with other target genes (Domingos, 2004b).

sal is normally expressed in both R7 and R8, which raises the question as to why sal does not also induce Rh6 expression in R7. A possible explanation for the absence of Rh6 in R7 could be the presence of an Rh6 repressor in R7. In accordance with this hypothesis, it has recently been shown that in pros mutant adult retinae, Rh5 and Rh6 expression expands to R7 and that pros is a direct repressor of rh5 and rh6. In pros mutants, salm but not sens is expressed in R7. These results indicate that in the absence of pros, induction of Rh6 expression in R7 occurs independently of sens, and that sal may be involved in this process. Moreover, in R7 cells mutant for pros, since sal is not sufficient to induce sens, factors other than pros should repress sens expression in R7. Alternatively, cofactor(s) required for sens induction by sal in R1-R6 may be absent in R7. Further investigations are necessary to validate these hypotheses and to determine if the regulation of rh1 and rh6 by sal and sens occurs in a direct or indirect manner (Domingos, 2004b).

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).

Asymmetric distribution of Spalt in Drosophila wing squamous and columnar epithelia ensures correct cell morphogenesis

The Drosophila wing imaginal disc is a sac-like structure that is composed of two opposing cell layers: peripodial epithelium (PE, also known as squamous epithelia) and disc proper (DP, also known as pseudostratified columnar epithelia). The molecular mechanism of cell morphogenesis has been well studied in the DP but not in the PE. Although proper Dpp signalling activity is required for proper PE formation, the detailed regulation mechanism is poorly understood. This study found that the Dpp target gene spalt (sal) is only expressed in DP cells, not in PE cells, although pMad is present in the PE. Increasing Dpp signalling activity cannot activate Sal in PE cells. The absence of Sal in the PE is essential for PE formation. The ectopic expression of sal in PE cells is sufficient to increase the PE cell height. Down-regulation of sal in the DP reduced DP cell height. It was further demonstrated that the known PE cell height regulator Lines, which can convert PE into a DP cell fate, is mediated by sal mis-activation in PE because sal-RNAi and lines co-expression largely restores PE cell morphology. By revealing the microtubule distribution, it was demonstrated that Lines- and Sal-heightened PE cells are morphologically similar to the intermediate cell with cuboidal morphology (Tang, 2016).

The wing disc is a sac-like structure composed of PE, DP, and intermediate cells linking the PE and DP. To investigate the potential role of Dpp signalling in PE morphogenesis, the distribution of Dpp signalling activity in late 3rd instar (L3) wing imaginal discs was revealed in both the x-y and x-z views. Using an antibody of phospho-Mothers against dpp (pMad) to reveal Dpp signal transduction activity, it was found that Dpp signal transduction was ubiquitously present in both the PE and DP. The pMad level was relatively reduced in the central PE compared with the central DP. Dpp target gene expression patterns were detected in the PE. The main Dpp target genes are brinker (brk), omb, and sal in the L3 wing discs. brk was transcribed in both the PE and DP, with a relatively weaker level in the PE, as indicated by a brk-lacZ reporter. However, both omb and sal were transcribed only in the DP, not in the PE. These data indicate that the Dpp target genes omb and sal are asymmetrically expressed in the PE and DP. Brk is also a repressor of other Dpp target genes, including sal and omb, and thereby restricts their expression domains to the medial DP region. The presence of Brk in the PE might be a direct cause of the absence of Omb and Sal in the PE. To assess this possibility, brk-RNAi was expressed in the PE. Sal expression was not detectable in the central PE. The efficiency of brk-RNAi was demonstrated by the elevation of Brk targets Omb and Sal in lateral wing discs of C765>brk-RNAi. To further confirm that Dpp signalling cannot induce sal expression in the PE, a constitutive active form of the Dpp receptor tkvQD was expressed in the PE. Sal was not induced in central PE. When tkvQD clones were generated, Sal was induced only in clones within the DP and not in clones located in the PE). Similarly, Omb was not induced in clones located in the PE. Ubiquitous expression of tkvQD failed to induce Omb in the PE. These data demonstrate that Dpp signalling cannot activate omb and sal in PE (Tang, 2016).

The expression patterns of Dpp target genes are well studied in wing DP. Dpp controls target genes (sal and omb) expression indirectly through repression of the transcriptional repressor Brk. Dpp target gene expression patterns have not been studied in wing PE to date. The results revealed that pMad is ubiquitously present in both DP and PE. However, brk-lacZ was still present in PE. Either suppressing brk or elevating Dpp signalling by expressing tkvQD cannot induce Sal and Omb in the PE. Except for Lin, other factors, such as Bowl, Wg, and EGFR, cannot induce Sal in the PE. The factors that suppress sal in the PE require further investigation (Tang, 2016).

As omb and sal are expressed only in the DP, not the PE, it was therefore asked whether this asymmetric transcription of omb and sal is essential for correct PE formation. To test this possibility, sal was mis-expressed in the PE using the Gal4-UAS system. dpp-Gal4 line is expressed in narrow stripes in the lateral PE and in the middle DP. When sal was ectopically expressed in the dpp-Gal4 domain, the height of lateral PE was notably elongated to a height similar to that of intermediate cells. Then, sal was expressed driven by C765-Gal4, which is ubiquitously expressed in both DP and PE. A similar elongation phenotype was observed in the PE. The cell height of the central PE was elongated to a height similar to that of cuboidal cells. The extent of elongation as a result of dpp-Gal4 was stronger than that of C765-Gal4. This difference might be due to the differences in Gal4 activity because dpp-Gal4 is stronger than C765-Gal4. The quantification of cell height, using the ratio between PE and DP cells within one wing disc, revealed a significant increase in sal mis-expression discs. Consistently, the PE height ratio between sal mis-expression and control also revealed a significant increase. To confirm this result, sal over-expression clones were generated in PE. From the x-z cross view, the clonal height was apparently elongated to a height similar to that for cuboidal cells. Therefore, it is concluded that sal is sufficient to elongate PE height (Tang, 2016).

Unlike the effect of sal mis-expression, the elongation of PE height in case of omb ectopic expression was not apparent, however, the differences in the PE/DP ratio and normalized PE height for C765>omb wing discs are statistically significant. Strong overexpression of omb induces severe extrusion and basal delamination, and cell motility can thicken the wing disc. Although a relatively weaker UAS-omb line was used, the side effect from cell movement may remain, thus leading to the statistic difference in the PE measurement. A previous report demonstrates that if Dpp signalling is suppressed in the PE by Ubx-Gal4 driven dad, a portion of the PE cells are elongated to a cuboidal shape. Therefore, suppressing Dpp signalling in the PE and expressing sal in the PE exhibit similar effects. When carefully assessing the Ubx-Gal4 expression domain, a portion of the expressing cells were, surprisingly, located in the DP. Thus, a non-autonomous effect from a loss of Dpp signalling in the DP in the Ubx>dad wing disc is reasonable. Because when dad-expressing clones were generated within PE, the height of PE did not increase. When Omb-RNAi was driven by hh-Gal4 which is expressed in PE and the posterior compartment of DP, the posterior DP height was reduced. Interestingly, the height of opposite PE was increased. Thus, it is possible that there is a connection between DP and PE during cell morphogenesis. To directly confirm the non-autonomous effect on PE elongation, the DP height was shortened by expression of either dad or brk within the DP specific nub-Gal4 domain. Consistently, the PE height was apparently increased (Tang, 2016).

Previous studies have revealed that mis-expressing lin induces ectopic sal expression in the PE. Thus, sal may mediate lin’s role in PE elongation. First, the experiment of lin mis-expression in the PE was repeated and the elongation phenotype was consistently observed in the PE. Then, the transcription state of sal was revealed using a sal-lacZ reporter. sal was apparently transcribed in the PE. The sal gene complex is composed of two functionally redundant genes: spalt major (salm) and spalt-related (salr). A rescue experiment was performed by co-expressing lin and salm-RNAi. The morphology of the wing imaginal discs was rescued to an approximate normal state, and PE height was no longer elongated. The cell heights of the corresponding genotypes were also measured. sal down-regulation largely rescued the abnormal cell height induced by lin mis-expression. These results indicate that sal mediates the role of lin in promoting PE elongation. However, Lin elongated PE to a greater extent than Sal did, according to the statistic measurements. Other mediators may be involved downstream of Lin. Since that Ubx-Gal4 line is also expressed in part of the DP cells, potential non-autonomous effects between DP and PE can not be ruled out (Tang, 2016).

Given that the mis-expression of sal in the PE elongates cell height, whether down-regulating Dpp-Sal signalling in DP is sufficient to shorten the DP was assessed. nub-Gal4 is only expressed in the wing pouch region in the DP. When Dpp signalling was mildly inhibited by expressing a dominant negative form of the Dpp receptor, tkvDN, in the nub-Gal4 domain, DP cell height was reduced. Unlike the strong inhibition of Dpp signalling by expressing dad, the non-autonomous effect on PE height was not apparent in nub>tkvDN. The Dpp signalling activities in discs of nub>tkvDNand nub>dad were revealed by anti-Sal staining. The DP height was slightly reduced when sal was down-regulated either by salm-RNAi or salr-RNAi; however, the extent of this reduction was weaker than that of tkvDN. Then, sal mutant clones were generated, marked by the loss of GFP in the DP. The intensity of F-actin labelled by Phalloidin was much stronger in the clone regions. The x-z cross-section showed that the apical side of sal mutant clones in the DP was retracted toward the basal side. These data suggested that Dpp-Sal signalling is required to maintain DP elongation. Interestingly, a similar retraction phenotype was also observed in sal-overexpressing clones. Therefore, both sal loss- and gain-of-function clones induce an apical retraction phenotype in the DP. This phenotype is observed in both omb loss- and gain-of-function clones in the DP. Omb exhibits a graded distribution in the DP along the A/P axis and specifies unknown apically distributed adhesion molecules. A continuous Omb level is essential for maintaining the epithelial integrity of the wing disc. Therefore, sharp discontinuity in either Omb or Sal levels in the DP induces apical retraction of cells. To confirm this conclusion, a sharp discontinuity of Sal was generated in the DP using dpp-Gal4 driven UAS-sal. Sal continuity was disrupted at the A/P boundary and where deep apical folds were formed. The expression domain of sal in the DP is narrower than that of omb and vg. Beyond the sal domain, omb and vg can ensure the correct cell morphogenesis in the DP. Clones lacking Vg function are also extruded from the DP layer (Tang, 2016).

Microtubule cytoskeleton is polarized during cell morphogenesis in the wing imaginal disc. To reveal microtubule-based cytoskeleton changes induced by either lin or sal mis-expression and the microtubule dynamics during normal development, the microtubule level was monitored via antibody staining. In the 2nd instar, all cells were cuboidal shape, and microtubules were uniformly distributed. During the early 3rd instar, the cell shape begins to differentiate. PE cells were largely shortened, whereas DP cells were remarkably elongated. Correlating with DP elongation, the microtubule network was asymmetrically enriched to the apical side of the DP. When lin was mis-expressed in the PE by Ubx-Gal4, sal was activated in the PE. Both direct and indirect sal expression induced PE height elongation with an even microtubule distribution. The microtubule levels of PE were increased compared with the wild type PE. The microtubule distribution in Sal-elongated PE was similar to that in very lateral PE and intermediate cells in the L3 stage or undifferentiated cells in earlier larval stages. In the rescue experiment, lin and salm-RNAi were co-expressed. PE height was restored were similar to that in wild type PE. Therefore, based on the cell height and microtubule distribution, Sal mis-expression converts the PE into a cuboidal cell shape (Tang, 2016).

During tissue morphogenesis, cell-shape changes always accompany microtubule cytoskeleton rearrangement. Dpp signalling activity has been proposed to play a basic function in microtubule organization. Dpp signalling is graded in the DP along the A/P axis, with higher levels in the medial DP region, which is enriched in apical microtubules. Thus, a correlation is noted between Dpp signalling activity and microtubule levels in the DP. Clones with loss-of-function of Dpp receptors in the DP appear extruded (cell height is severely shortened) and exhibit reduced apical microtubule levels. Consistently, clones with both loss- and gain-of-function sal and omb in the DP consistently exhibit severe apical retraction with shortened cell height and loss of apical microtubule enrichment. Therefore, the data support the hypothesis that Dpp-Omb/Sal signalling activity plays a more general function in microtubule-based cell morphogenesis. Other transcription factors that induce reductions in DP cell height also correlate with the loss of apical microtubule enrichment. The Tbx6 subfamily gene cluster Dorsocross (Doc) initiates wing hinge/blade fold formation. In the Doc expression domain in the DP, cells are shortened from the apical side with severe loss of apical microtubules (Tang, 2016).


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

date revised: 20 November 2016

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