spalt

DEVELOPMENTAL BIOLOGY

Embryonic

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).

Larval

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 ëratchetí 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 ëstopí 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).

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 Egfr^DN 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-N^act) 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).

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date revised: 10 April 2008 

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