sparkling

DEVELOPMENTAL BIOLOGY

Embryonic

During development, transcripts of the sparkling gene are first detected in a segmentally reiterated pattern after completion of germ-band eleongation (stage 10). The expression is initiated in one or two cells per hemisegment, and then rapidly expands to a cluster of a few cells; subsequently, expression becomes more complex (early stage-ll embryos). Expression persists in each hemisegment during germ-band retraction until stage 14; thereafter, it declines. This dynamic expression pattern is typical of the developing peripheral nervous system which consists mainly of external and internal sensory organs. The expression pattern of sparkling was compared with that of cut and couch potato. sparkling is expressed in a similar manner to cut in the thoracic and abdominal segments of the embryo, while its expression differs from that of cpo. These comparative expression patterns suggest that sparkling is prodominantly transcribed in the external sensory organs but not the chordotonal organs. Transcripts are also observed in the sensory organs of the head region, including antennal, maxillary, ventral and labial sense organs. At stage 15, the expression of spa is initiated in the CNS of the ventral cord and head region, where it persists throughout later embryogenesis (Czerny, 1997).

Pax2 and Poxn define the presumptive deutocerebral-tritocerebral boundary in Drosophila

Studies on expression and function of key developmental control genes suggest that the embryonic vertebrate brain has a tripartite ground plan that consists of a forebrain/midbrain, a hindbrain and an intervening midbrain/hindbrain boundary region, each of which are characterized by the specific expression of the Otx, Hox and Pax2/5/8 genes, respectively. The embryonic brain of Drosophila expresses all three sets of homologous genes in a similar tripartite pattern. Thus, a Pax2/5/8 expression domain is located at the interface of brain-specific otd/Otx2 and unpg/Gbx2 expression domains anterior to Hox expression regions. This territory is identified as the deutocerebral/tritocerebral boundary region in the embryonic Drosophila brain. Mutational inactivation of otd/Otx2 and unpg/Gbx2 result in the loss or misplacement of the brain-specific expression domains of Pax2/5/8 and Hox genes. In addition, otd/Otx2 and unpg/Gbx2 appear to negatively regulate each other at the interface of their brain-specific expression domains. These studies demonstrate that the deutocerebral/tritocerebral boundary region in the embryonic Drosophila brain displays developmental genetic features similar to those observed for the midbrain/hindbrain boundary region in vertebrate brain development. This suggests that a tripartite organization of the embryonic brain was already established in the last common urbilaterian ancestor of protostomes and deuterostomes (Hirth, 2003).

In the embryonic CNS of vertebrates, the Pax2, Pax5 and Pax8 genes are expressed in specific domains that overlap in the presumptive MHB region. Drosophila has two Pax2/5/8 orthologs, Pox neuro (Poxn) and Pax2/Sparkling (Hirth, 2003).

The embryonic brain of Drosophila can be subdivided into the protocerebrum (PC or b1), deutocerebrum (DC or b2) and tritocerebrum (TC or b3) of the supra-esophageal ganglion and the mandibular (S1), maxillary (S2) and labial (S3) neuromeres of the sub-oesophageal ganglion. Expression of engrailed (en) delimits these subdivisions by marking their most posterior neurons. Because of morphogenetic processes, such as the beginning of head involution, the neuraxis of the embryonic brain curves dorsoposteriorly within the embryo. Accordingly, anteroposterior coordinates will here henceforth refer to the neuraxis rather than the embryonic body axis (Hirth, 2003).

It was first determined whether Pax2 is expressed in specific domains of the Drosophila brain, by analyzing its expression pattern using in situ hybridization, immunolabelling and lacZ reporter gene expression. Pax2 transcripts initially appear during gastrulation and at stage 9/10 are observed in a segmentally reiterated pattern of the developing procephalic and ventral neuroectoderm, with its anteriormost expression domain located at the future deutocerebral-tritocerebral boundary. Expression of Pax2 transcripts in the developing brain begins at stage 10/11 and is most prominent in a longitudinal stripe at the medial part of the protocerebrum and in a transversal stripe at the posterior border of the deutocerebrum. Immunolabelling with a Pax2-specific polyclonal antibody reveals that Pax2 protein distribution resembles that of Pax2 transcripts, as does a Pax2-lacZ reporter gene expressing ß-galactosidase. In addition to its expression in the developing anterior brain, Pax2 expression is also seen in six to eight cells located at the lateral margin of each hemisegment throughout the more posterior CNS regions of the sub-oesophageal ganglion and ventral nerve cord (Hirth, 2003).

To determine the expression of the second Drosophila Pax2/5/8 ortholog, Poxn expression was characterized using immunolabelling and lacZ reporter genes. Poxn protein is first detected in the developing brain at the end of germband extension (stage 10/11) in two stripes of the procephalic neuroectoderm, that subsequently become restricted to the posterior protocerebrum and the posterior deutocerebrum. Poxn expression in more posterior regions of the CNS also occurs in segmentally reiterated patterns. A comparison between Pax2 and Poxn expression domains reveals, that Pax2 and Poxn are never co-expressed in the same cells of the CNS. Moreover, and with one exception, expression of Pax2 and Poxn does not occur at a comparable anteroposterior position along the neuraxis. The exception is in the posterior deutocerebrum where adjacent Pax2 and Poxn expression domains define a transversal domain immediately anterior to the tritocerebral brain neuromere. This transversal domain of adjacent Pax2 and Poxn expression is distinguishable from segmentally reiterated expression in more posterior regions by the fact that it is the only position along the neuraxis where expression of both genes coincides with a neuromere boundary. This transversal domain of adjacent Pax2/5/8 ortholog expression is referred to as the deutocerebral-tritocerebral boundary (DTB) region (Hirth, 2003).

It is important to note that the DTB is located anterior to the expression domain of the Drosophila Hox1 ortholog labial (lab), which is expressed in the posterior tritocerebrum. Moreover, the DTB is located posterior to the expression domain of the Drosophila Otx orthologue otd in the protocerebrum and anterior deutocerebrum. Thus, in Drosophila as in vertebrates, a Pax2/Poxn (Pax2/5/8) expression domain is located between the anterior otd/Otx2 and the posterior Hox-expressing regions. This raises the question of whether the DTB in the embryonic Drosophila brain might have developmental genetic features similar to those observed for the MHB in vertebrate brain development (Hirth, 2003).

In the embryonic vertebrate brain, Otx2 is expressed anterior to and abutting Gbx2. The future MHB as well as the overlapping domains of Pax2, Pax5 and Pax8 expression are positioned at this Otx2-Gbx2 interface. To investigate if comparable expression patterns are found in the embryonic fly brain, the brain-specific expression of the Drosophila Gbx2 ortholog unplugged (unpg) was determined in relation to that of otd, using immunolabelling and an unpg-lacZ reporter gene that expresses ß-galactosidase like endogenous unpg. The otd gene is expressed in the protocerebrum and anterior deutocerebrum of the embryonic brain, as well as in midline cells in more posterior regions of the CNS. Expression of unpg-lacZ in the embryonic CNS is first detected at stage 8 in neuroectodermal and mesectodermal cells at the ventral midline, with an anterior limit of expression at the cephalic furrow. Subsequently, the unpg expression domains in the CNS widen and have their most anterior border in the posterior deutocerebrum. Double immunolabelling of Otd and ß-galactosidase reveal that the posterior border of the brain-specific otd expression domain coincides with the anteriormost border of the unpg expression domains along the anteroposterior neuraxis. There is no overlap of otd and unpg expression in the brain or in more posterior regions of the CNS (Hirth, 2003).

These findings indicate that the otd-unpg interface is positioned at the anterior border of the DTB. This was confirmed by additional immunolabelling studies examining unpg-lacZ, otd, Poxn and en expression in the protocerebral/deutocerebral region of the embryonic brain. Thus, double immunolabelling of Otd and En confirms that the posterior border of otd expression extends beyond the protocerebral en-b1 stripe into the anterior deutocerebral domain. Labelling Otd and Poxn confirms that the Poxn expression domain of the DTB is posterior to this deutocerebral otd expression boundary. Labelling En and ß-galactosidase (indicative of unpg expression), confirms that the anteriormost unpg expression domain overlaps with the en-b2 stripe. Finally, labelling ß-galactosidase and Poxn confirms that this anteriormost unpg expression domain overlaps with the Poxn expression domain of the DTB. Therefore, in terms of overall gene expression patterns, it is found that a transversal domain of adjacent Pax2/Poxn expression defines the DTB region of the embryonic Drosophila brain. Furthermore, this region is located between an anterior otd expression domain and a posterior Hox expression domain. Moreover, it is located abutting and posterior to the interface of otd and unpg expression along the anteroposterior neuraxis (Hirth, 2003).

In mammalian brain development, homozygous Otx2-null mutant embryos lack the rostral brain, including the MHB-specific Pax2/5/8 expression domain, whereas Gbx2 null mutants misexpress Otx2 and Hoxb1 in the brain. Moreover, Otx2 and Gbx2 negatively regulate each other at the interface of their expression domains. To test if similar regulatory interactions occur in the embryonic brain of Drosophila, the expression of the corresponding orthologs was analyzed in otd and unpg mutant embryos. In otd-null mutant embryos, the protocerebrum is absent because protocerebral neuroblasts are not specified. Analysis of unpg, en and Poxn expression in otd-null mutant embryos reveals that the anteriormost border of unpg expression shifts anteriorly into the anterior deutocerebrum, while Poxn fails to be expressed in the deutocerebrum. In contrast to inactivation of otd, inactivation of unpg does not result in a loss of cells in the mutant domain of the embryonic brain, as is evident from the expression of an unpg-lacZ reporter construct in unpg-null mutant embryos. Analysis of otd expression in unpg-null mutants shows that the posterior limit of brain-specific otd expression shifts posteriorly into the posterior deutocerebrum, thus extending into the DTB. This was confirmed by additional immunolabelling studies examining otd, Poxn and en expression in the protocerebral/deutocerebral region of the embryonic brain in unpg-null mutants. Double immunolabelling of Otd and En in unpg-null mutants confirms that the posterior border of brain-specific otd expression extends posteriorly to the deutocerebral en-b2 stripe into the posterior deutocerebrum. In addition, double immunolabelling of Otd and Poxn in unpg-null mutants confirms that the posterior border of brain-specific otd expression extends posteriorly into the Poxn expression domain of the DTB. Moreover, analysis of lab expression in unpg-null mutants shows that brain-specific lab expression shifts anteriorly into the anterior tritocerebrum. Thus, in both Drosophila and mammals, mutational inactivation of otd/Otx2 and unpg/Gbx2 results in the loss or misplacement of the brain-specific expression domains of orthologous Pax and Hox genes. Moreover, otd and unpg appear to negatively regulate each other at the interface of their expression domains (Hirth, 2003).

In vertebrate brain development, the Pax2 gene, and subsequently the Pax5 and Pax8 genes, are among the first genes expressed at the Otx2/Gbx2 interface, followed by the overlapping expression of En1 and Fgf8 genes. Inactivation of Pax2, Pax5, En1 or Fgf8 results in the loss of the midbrain and cerebellum because of a failure to maintain development of this brain region. In Drosophila, no obvious brain phenotypes were seen after mutational inactivation of Pax2, Poxn, en/inv or the Drosophila Fgf homolog branchless (bnl). The absence of brain phenotypes in these mutants contrasts with those observed in the vertebrate brain following mutational inactivation of the orthologous Pax2, Pax5, En1 and Fgf8 genes (Hirth, 2003).

Comparative developmental studies in urochordates and vertebrates have led to the notion that the basic ground plan for the chordate brain consists of a forebrain/midbrain region characterized by Otx gene expression, a hindbrain region characterized by Hox gene expression, and an intervening boundary region characterized by expression of Pax2/5/8 genes. This suggests that a corresponding, evolutionarily conserved, tripartite organization also characterized the brain of the last common ancestor of insects and chordates. A comparison of the brain-specific topology of gene expression patterns that define this tripartite organization in Drosophila and in mouse suggests that the vertebrate midbrain/hindbrain boundary (MHB) region corresponds to the insect deutocerebral-tritocerebral boundary (DTB) region. If this is the case, one might expect that other patterning genes that characterize the MHB region are also expressed at the insect DTB. Although this expectation is fulfilled for the segment-polarity genes en and wingless (wg) in Drosophila, these two genes are expressed at the borders of all CNS neuromeres, as well as at parasegmental boundaries in the epidermis; hence, their expression may not be indicative of brain-specific requirements (Hirth, 2003).

In addition to remarkable similarities in orthologous gene expression between insects and chordates, this study also shows that several functional interactions among key developmental control genes involved in establishing the Pax2/5/8-expressing MHB region of the vertebrate brain are also conserved in insects. Thus, in the embryonic brains of both fly and mouse, the intermediate boundary regions, DTB and MHB, are positioned at the interface of otd/Otx2 and unpg/Gbx2 expression domains. These boundary regions are deleted in otd/Otx2-null mutants and mispositioned in unpg/Gbx2-null mutants. Moreover, otd/Otx2 and unpg/Gbx2 genes engage in crossregulatory interactions, and appear to act as mutual repressors at the interface of their brain-specific expression domains. However, not all of the functional interactions among genes involved in MHB formation in the mouse appear to be conserved at the Drosophila DTB. Thus, in the embryonic Drosophila brain, no patterning defects are observed in null mutants of Pax2, Poxn, en or bnl. It remains to be seen if these genes play a role in the postembryonic development of the Drosophila brain (Hirth, 2003).

It is conceivable that the similarities of orthologous gene expression patterns and functional interactions in brain development evolved independently in insects and vertebrates. However, a more reasonable explanation is that an evolutionary conserved genetic program underlies brain development in all bilaterians. This would imply that the generation of structural diversity in the embryonic brain is based on positional information that has been invented only once during evolution and is provided by genes such as otd/Otx2, unpg/Gbx2, Pax2/5/8 and Hox, conferring on all bilaterians a common basic principle of brain development. If this is the case, comparable orthologous gene expression and function should also characterize embryonic brain development in other invertebrate lineages such as the lophotrochozoans. This prediction can now be tested in lophotrochozoan model systems such as Platynereis or Dugesia (Hirth, 2003).

Taken together, these results indicate that the tripartite ground plan that characterizes the developing chordate brain is also present in the developing insect brain. This implies that a corresponding tripartite organization already existed in the brain of the last common urbilaterian ancestor of insects and chordates. Therefore, an urbilaterian origin of the tripartite brain is proposed (Hirth, 2003).

Larval and Pupal

sparkling (spa) is expressed in the embryonic nervous system, and in cone, primary pigment, and bristle cells of larval and pupal eye discs. Transcripts are expressed in the posterior portion of the eye disc, with the anterior boundary of expression lagging clearly behind the morphogenetic furrow. Isolated cells of the antennal and wing discs exhibit strong expression. In the CNS, Spa expression is observed mainly in the thoracic ventral ganglion and in the brain (Fu, 1997).

During embryogenesis, Spa is expressed in the central nervous system (CNS) and in the peripheral nervous system (PNS) where it is observed in developing external sensory organs (Czerny, 1997) and chordotonal organs of the larva (Fu, 1998). Later, during late third instar larval and early pupal stages, Spa is not only detected in cone and primary pigment cells but also in all four precursor cells of the mechanosensory bristles of the developing pupal retina. At subsequent pupal stages, Spa expression is lost from socket cells and neurons whereas shaft and glial cells continue to express it. In addition, Spa protein is expressed in what appear to be SOPs of antennal, leg and wing discs. These cells include SOPs of chordotonal organs, such as those of Johnston's organ in the second antennal segment, as well as of external sensory organs. Particularly, these last observations point to an additional role for the Spa protein in bristle development (Fu, 1998).

In the developing eye, spa transcripts are first detectable in eye discs of late third instar larvae. The anterior margin of Spa expression lags behind the morphogenetic furrow by six to seven rows of ommatidial precursors, in which the assembly of ommitidia begin. Five rows behind the furrow, the assembly of the eight photoreceptor precursors is complete; the accretion of the future anterior and posterior cone cells first becomes apparent in row 6, whereas a few rows more posterior, the polar and equatorial cells are added to the ommatidia. At this stage, the Spa protein is localized exclusively in the nuclei of all four cone cell precursors. Thus, expression of Spa in cone cell precursors begins very shortly after their accretion to the growing ommatidial clusters. During early pupal stages, Spa is also found in the nuclei of primary pigment cell precursors, which are recruited to the developing ommatidia at this time. In addition, Spa appears in the basal nuclei of mechanosensory bristle cells, which in contrast to all other ommatidial cells are derived clonally from a bristle mother cell at this stage. At no other time is Spa expressed in immature or differentiated photoreceptor cells or in secondary and tertiary pigment cells (Fu, 1997).

Hamlet modulates Pax2 fate-determining signals in external sensory organs

The Drosophila external sensory organ forms in a lineage elaborating from a single precursor cell via a stereotypical series of asymmetric divisions. Hamlet transcription factor expression demarcates the lineage branch that generates two internal cell types, the external sensory neuron and thecogen. In Hamlet mutant organs, these internal cells are converted to external cells via an unprecedented cousin-cousin cell-fate respecification event. Conversely, ectopic Hamlet expression in the external cell branch leads to internal cell production. The fate-determining signals Notch and Pax2 act at multiple stages of lineage elaboration and Hamlet acts to modulate their activity in a branch-specific manner (Moore, 2004).

To investigate the fate of the 'thecogen' in a ham mutant, wild-type and ham mutant embryos were stained with antibodies to detect expression of the transcription factors Cut, Pros, and Pax2, and the placW enhancer trap A1-2-29. Cut marks all cells descended from the ESOP. Upon terminal differentiation of the ESO, Pax2 marks the thecogen and trichogen, Pros marks only the thecogen, and A1-2-29 drives ß-galactosidase expression in the trichogen and tormagen. In the dorsal external sensory (des) and ventral pore (vp) organs of ham mutant embryos, the total number of cells remains unchanged, and the thecogen cell (expressing Cut, Pax2, and Pros) is replaced by a cell expressing Cut, Pax2, and A1-2-29; this combination of markers normally defines trichogen fate. Given that this staining pattern implies that this cell is a trichogen, the ESO structures on the cuticle surface of ham mutant second instar larvae were examined. Sixty-five percent of organs showed a wild-type one-trichogen and one-tormagen phenotype, and 34% a two-trichogen and one-tormagen phenotype (Moore, 2004).

The expression of I-branch specific markers was investigated in ham mutant ESO lineages during elaboration. ham mutant MARCM clones positively marked with mCD8GFP fusion protein expression were made in all ESOP-derived cells. ham mutant clones at all stages of ESO lineage elaboration were stained with antibodies that detected the following markers: Pros, Pax2, Elav (Embryonic lethal abnormal vision), which labels all differentiated neurons, and Suppressor of Hairless [Su(H)], which labels differentiated tormagen. In both wild-type and ham mutant lineages, the IIB, IIIB, and IIIBsib cells expressed Pros, thus confirming the live imaging findings that showed no differences between the elaborating I-branch in wild-type and ham mutant lineages (Moore, 2004).

The IIIB progeny in ham mutant clusters, however, shifted their patterns of differentiation over time. Shortly after division of the IIIB cell (22-24 h APF), one daughter in ham mutant clones continued to express Pros, similar to a wild-type thecogen, and the other began to express ELAV, a marker of neuron fate. By 28-30 h APF, the ham mutant ESO cell clearly differed from the wild-type control: expression of the I-branch-specific maker Pros was lost. Moreover, Elav-positive neurons were no longer present, but several ham mutant ESO showed small Elav-positive apoptotic cell fragments, indicative of the ES neuron undergoing cell death. Live confocal imaging of ham mutant clones showed the frequency of this event to be 15%. By 36-40 h, wild-type ESO clearly contained one trichogen and one thecogen (both Pax2-positive), one tormagen [Su(H)-positive], and one ES neuron (Elav-positive). In contrast, ham mutant ESOs contained no ES neurons or thecogen, two trichogen (Pax2-positive), and either one or two tormagen [Su(H)-positive]. Whereas mCD8GFP activity and Pax2 staining revealed the two-trichogen phenotype with 100% frequency, the appearance of two trichogens on the adult cuticle was less frequent, indicating that one supernumerary trichogen must have failed to grow out onto the cuticle surface. Costaining of ham mutant embryos with antibodies to detect Cut, Pros, and ELAV revealed that Pros is also transiently expressed in the thecogen cell before it undergoes conversion to a trichogen. Therefore, in both the adult and embryonic ham mutant ESO lineage, the daughters of the IIIB cell first express markers specific to internal cells, but expression of such markers ceases as these cells undergo respecification to an external cell fate (Moore, 2004).

If the cell-fate conversions that are proposed take place, then it would mean that the thecogen (high N) becomes a trichogen (low N) and the ES neuron (low N) can become a tormagen (high N). To confirm this, whether the high-N or low-N IIIB daughter cell becomes the supernumerary trichogen was investigated in a ham mutant ESO. ham MARCM clones were made in an N^ts (temperature sensitive) background and N was inactivated in these clones at the stage where N-mediated signaling is generating asymmetry between the IIIB daughter cells. The resulting nota were stained with antibodies to detect mCD8, Su(H), and Pax2 and the fate of the IIIB daughter cells was examined. In the ham¹, N^ts ESOs 28/28 had a one-trichogen/multiple-tormagen phenotype, whereas in the ham¹, N⁺ control 37/37 ESOs had a two-trichogen/multiple-tormagen phenotype (Moore, 2004).

This experiment clearly demonstrates that it is the high-N IIIB cell daughter that becomes the supernumerary trichogen, since reducing N activity results in a reduction in the number of trichogen to one in the ham mutant organ. The conclusion to be drawn from this experiment is, therefore, that whereas N is acting to determine the difference between daughters of an asymmetric division, of either IIA or IIIB, a second signal is acting that makes the trichogen similar to the thecogen and the tormagen similar to the ES neuron (Moore, 2004).

This study shows that in both the embryo and adult, ESO Ham is expressed solely in the IIIB cell and its daughters, the ES neuron and thecogen. Loss of Ham causes the conversion of the internal-cell-branch thecogen into an external-cell-branch trichogen by cell-fate respecification. In addition, loss of Ham in the ES neuron leads either to its conversion to an internal-cell-branch IIIBsib cell or an external-cell-branch tormagen. Therefore, Ham appears to act to determine the fate of the IIIB daughters with respect to all other terminally differentiated cell types in the ESO lineage. In other words, it acts as an intrinsic transcription factor determinant of IIIB cell-derived identity (Moore, 2004).

It was of interest to test whether Ham expression alone determines the difference between IIIB-derived and non-IIIB-derived fate. Ectopic expression of Ham in the embryonic IIB and IIIBsib cell (MD neuron) converts the IIIBsib into an ES neuron. This analysis was extended to the E-branch by using gal4^109-68 to drive UAS-ham and UAS-mCD8GFP in all cells of the adult lineage. Ham expression in all ESO lineage cells led to the loss of both the E-branch-derived trichogen and tormagen from the surface of the cuticle. Antibody staining of the ESOs showed that I-branch-specific cell types had replaced these external cells. When Ham was ectopically expressed in the entire ESO lineage, five or six cells were seen, all of which were expressing Pros or Elav or (rarely) both Pros and Elav. In contrast, in a wild-type cluster, there are four cells including only one thecogen (Pros-positive) and one ES neuron (Elav-positive). These ectopic expression experiments confirm that Ham determines IIIB-derived versus non-IIIB-derived fate (Moore, 2004).

These ectopic expression experiments confirm that Ham determines IIIB-derived versus non-IIIB-derived fate. However, why in ham mutant ESOs are the transformations that occur thecogen (high N) to tricogen (low N) and ES neuron (low N) to tormagen (high N)? Pax2 expression in the ESO highlights a connection between these pairs of cell types; the thecogen and trichogen both express Pax2, whereas the tormagen and ES neuron do not. Moreover, Pax2 itself is required for hair-shaft differentiation in the trichogen, and ectopic Pax2 expression in the tormagen leads to ectopic hair-shaft development. In the thecogen, where Pax2 and Ham are coexpressed, Pax2 expression does not lead to hair-shaft development; however, in the absence of Ham, this cell now takes on a trichogen fate including the development of a hair shaft. Therefore one role of Ham in the thecgoen cell may be to suppress the ability of Pax2 to promote hair-shaft formation (Moore, 2004).

To investigate whether Ham can repress the hair-shaft-promoting activity of Pax2, Ham or Pax2 were expressed or Ham and Pax2 were coexpressed at high levels in all cells of the ESO lineage. neu-gal4 was used to drive expression of UAS-ham and/or UAS-Pax2; however, this causes embryonic lethality. To get around this problem, ectopic gene expression was driven only in notum clones. Ectopic expression of Pax2 led to organs with multiple hair shafts, some organs with misshapen external cells, and in some cases loss of external cells. In contrast, ectopic Ham or Ham and Pax2 caused the loss of external cells in almost 100% of ESO clones and never the formation of multiple hair shafts. These experiments demonstrate that Ham has the ability to modulate a Pax2-driven differentiation program, in this case, hair-shaft growth. Therefore, loss of Ham from the "thecogen" could lead to the depression of a program at least in part controlled by Pax2, which drives this cell to a trichogen fate (Moore, 2004).

The Pax2 cell-fate-determining signal is used at multiple points during the development of the ESO lineage, as is N. The presence or absence of Ham provides a branch-specific background state against which differentiating cells of the organ can interpret these signals. It is likely that a similar modulation of iterated signals by branch-specific factors occurs in vertebrate systems. For example, N signaling occurs at multiple points during the development of the hematopoietic lineage and could be modulated by the presence of branch-specific transcription factors. It is suggested that the analysis of Ham function in ESO lineage elaboration presented in this study provides useful insight into how cell fate is determined in many invertebrate and vertebrate lineages in which a single stem/precursor cell gives rise to multiple cell types via iterative cell divisions (Moore, 2004).

Effects of Mutation or Deletion

sparkling is named for its eye phenotype, where it is required for the proper specification of cone and primary pigment cells in late larval and pupal eye discs. All sparkling mutants have rough eyes. The strongest spa alleles also produce unpigmented tarsal claws, whose precursor cells are located in the center of leg discs (Fu, 1997).

Clearly, the abnormal development of cone and primary pigment cells seen in strong spa mutants exerts secondary effects on the recruitment and/or development of interommatidial secondary and tertiary pigment cells. The fact that many ommatidial bristle cells are misplaced is a secondary effect, resulting from the disturbed development or absence of primary, secondary, and tertiary pigment cells, which are probably required to constrain and assign proper positions to the bristle cells and to support their differentiation by intercellular signals (Fu, 1997).

According to the model proposed by Freeman (1996), cone cells are recruited by the Ras signaling pathway activated by the release from neighboring photoreceptor cells of the epidermal growth factor-like Spitz signal, which overcomes the inhibitory Argos signal. Cone cell precursors are also competent to become R7 cells in response to the Ras signaling pathway. Because Spa is not expressed in R7 cells, its expression in newly recruited cone cells distinguishes their fate from that of R7 cells. spa is thus a direct or indirect target of a transcription factor actived by selective phosphorylation through the Ras signaling pathway. Such a factor, whose synthesis would have to precede that of Spa, might be Lozenge, which is required in the R1/R6 photoreceptor pair and R7, as well as in cone cells and which helps define the R7 equivalence group by repressing seven-up. The observed eye phenotype of strong spa mutants clearly shows that Spa protein is required for the proper development and assembly of both cone and primary pigment cells rather than for their recruitment. After the accretion of all four cone cells to the developing ommatidia, primary pigment cells are recruited, presumably by the release of Spitz from neighboring anterior and posterior cone cells. As a result, Spa is again expressed in primary pigment cells, where it is required for the activation of the two Bar genes. Bar expression in primary pigment cells depends completely on Spa and Spa also plays an indirect role in the recruitment or specification of all pigment cells (Fu, 1997).

The eyes of spa1 mutants reveal many ommatidia with defective corneal lenses and pseudocones, occasionally fused facets, and frequently mispositioned bristles or two bristles protruding from the same vertex, an effect that is most pronounced dorsally and ventrally (see The Drosophila Adult Ommatidium: Illustration and explanation with Quicktime animation). The strongest phenotype is exhibited by spapol flies whose eye size is somewhat reduced. Their corneal lenses and pseudocones, which are secreted by the cone cells and the primary pigment cells, are blurred and irregular, and many of them are fused. In addition, numerous necrotic pits are apparent between an irregular array of ommatidia of variable size. In about 15% of spa1 ommatidia the number of photoreceptors is reduced or their orientation and types are abnormal. Although many ommatidia in young adults have retained eight photoreceptors, their orientation and positions are disturbed, most rhabdomeres are malformed, and frequently the ommatidia appear fused to each other. Some ommatidia have also lost two to five photoreceptor cells. At the early pupal stage, the four cone cells frequently fail to assemble in the typical rhomboid-like configuration, whereas after completion of ommatidial assembly one or two cone cells or one of the primary pigment cells may be missing. In the latter case, a single primary pigment cell expands beyond its normal dorsoventral boundaries and attempts to encircle all four cone cells (Fu, 1997).

Genomic DNAs of spa1 and spapol flies exhibit rearrangements characteristic of spontaneously induced mutations. Thus, the spapol mutation is a 1.58-kb deficiency that removes exons 3 and 4 and flanking sequences, whereas spa1 consists of a 7.5-kb insertion into the same region of intron 4 (Fu, 1997).

Drosophila Pax2 also encodes the shaven (sv) function, which is crucial during bristle development. Both sv and spa alleles, previously thought to represent different genes, are mutations in two widely separated enhancers of Drosophila Pax2. The sv function of Drosophila Pax2 acts in at least two distinct steps of mechanosensory bristle development: the specification of the alternative fate of shaft cell, as opposed to socket cell, and the later differentiation of the shaft cell (Fu, 1997).

spa expression is lost in sv mutants. While Spa protein is not detectable in the cone and primary pigment cells of spa^pol mutants, its expression in bristle cells appears unaffected. The situation is reversed in svⁿ mutants: Spa protein is absent from nearly all shaft and glial cells; both cell types continue to express Spa in wild type mid-pupal eye discs, but its expression in cone and primary pigment cells is the same as in wild type. At earlier pupal stages, Spa protein is still expressed in all four bristle cells of svⁿ eye discs, in agreement with the observation that svⁿ flies display a hypomorphic sv phenotype. That sv and spa are alleles of the same gene was also consistent with the result of a molecular analysis of mutant sv alleles. When the three available sv alleles, svⁿ, sv35a and svde, were examined for molecular lesions in, or close to, the spa transcription unit, all were found to carry insertions of transposable elements within a 0.25 kb EcoRI fragment about 1.6 kb upstream from the transcriptional start site of spa. While svⁿ and sv35a turned out to be identical insertions of the 5.0 kb blastopia retrotransposon, a 5.1 kb Tirant element is inserted in svde. Although this evidence strongly suggests that sv and spa are alleles of the same transcription unit, it is conceivable that sv and spa are adjacent or very closely linked genes and that Spa expression in bristle cells depends on the product of sv (Fu, 1998).

To decide whether sv and spa are different genes, or alleles of the same gene, attempts were made to rescue both spa and sv mutant phenotypes with transgenes in which the coding and promoter regions of spa were combined with the spa enhancer or a putative sv enhancer. Two different upstream fragments were analyzed in combination with a spa-cDNA for rescue of svⁿ mutants. Indeed, when expression of the spa transgene is controlled by a 6.7 kb upstream fragment of spa, the bristle phenotype of homozygous svⁿ mutants is rescued completely. In contrast, a shorter 2.1 kb upstream region is able to rescue the bristle phenotype of svⁿ mutants entirely on the notum and partially in the eye but not on the abdomen. Since these results do not exclude the possibility that the partial rescue of svⁿ is attributable to a gene located largely within the 2.1 kb upstream region of, but different from, spa, an attempt was made to rescue both the spa^pol and the svⁿ phenotypes by a Hs-spa transgene in which expression of a spa-cDNA is under the control of the hsp70 heat-shock promoter. Such a transgene is able to rescue the eye phenotype of spa^pol mutants to a considerable extent, as well as the complete rescue of the bristle phenotype of svⁿ mutants on the notum, and partially in the eye (Fu, 1998).

Nevertheless, these results are still consistent with the formal possibility that the 2.1 kb upstream region of spa includes the entire coding region of sv, whose product acted mainly, if not entirely, through the transcriptional activation of spa. If true, it should be possible to rescue sv mutants with a transgene that does not include the spa coding region. Therefore, the rescue of svⁿ mutants was attempted with a transgene that consists only of the 6.7 kb upstream region of spa. Since none of 22 such independent transgenic lines shows any rescue of svⁿ, it follows that this region does not encode a transactivator but rather a cis-regulatory region of spa and that sv and spa are alleles of the same gene that have been mutated in different tissue-specific enhancers (Fu, 1998).

What is the function of Spa in the differentiation of bristles? All bristles or mechanosensory organs of the adult fly arise in a simple, stereotyped manner by three consecutive asymmetric divisions during which a single SOP gives rise to a neuron and three support cells. The first division generates different siblings, one of which by subsequent division produces the cells that form the socket (tormogen) and shaft (trichogen); the other sibling produces the neuron and glial cell (thecogen). Sparkling appears to be required at various stages during bristle development. Initially, it is observed in SOPs and all four bristle precursor cells (Fu, 1997). However, by mid-pupal stages it is no longer expressed in the tormogen and neuron, but continues to be expressed in the thecogen and is strongly elevated in the trichogen. If thecogen and trichogen, in response to opposite Notch (N) signaling, arise from their precursors by analogous asymmetric divisions, Sparkling expression must also react to N signaling in these cells in opposite ways. For example, while N signaling may activate sparkling through Su(H) in glial cells, it may repress sparkling in socket cells. Comparison of Spa protein levels in the developing eye bristles of svⁿ mutants with those of wild type suggests that its expression needs to be maintained at least to mid-pupal stages for proper differentiation of trichogens and the formation of a shaft of normal size. However, trichogen cells are able to differentiate to some extent in hypomorphic svⁿ mutants that express Spa during early pupal stages. If the levels of Spa are further reduced, the shafts are also shortened or completely missing. In that case, the shaft is replaced by a second socket, which indicates that the trichogen is no longer able to differentiate properly but becomes a tormogen. It appears therefore that Sparkling protein is not only required during differentiation of a shaft cell, but also serves at an earlier time to specify the fate of shaft versus socket cell. This would be consistent with Sparkling being a target of the N signaling pathway. The roles that Sparkling plays in developing cone and primary pigment cells or in bristle cells, which manifest themselves in the various enhancer mutant alleles of spa and sv, are probably not the only functions of Sparkling as evident from several additional locations where Spa protein can be detected during embryonic and larval development. The expression of Sparkling in the embryonic PNS may indicate that Sparkling plays a function in the development of larval sense organs analogous to that of sv in the development of the mechanosensory bristles of the adult (Fu, 1998).

Expression of Sparkling in the antennal disc is particularly interesting because it might reflect the conservation of another gene network in which Drosophila and vertebrate Pax2 participate and thus lends further support to the proposed conservation of gene networks. The expression patterns of Sparkling protein in the antennal disc, as well as in the leg and wing disc, include expression in precursors of chordotonal organs, as judged by their striking resemblance to those of atonal (ato) and, more convincingly, by their absence in ato mutant discs. The chordotonal organs of the second antennal segment form the fly's auditory device, the so-called Johnston's organ. Similar to Sparking in the development of Johnston's organ, Pax2 expression begins very early during development of the mouse inner ear, in the otic placode; differentiation of the auditory portion of the inner ear, the cochlea and cochlear ganglion, is completely blocked in Pax2 null mutants (Torres, 1996). A hearing defect observed in human patients heterozygous for a gene encoding a truncated PAX2 protein suggests a very similar role for PAX2 in man. Although it remains to be shown if Sparkling plays a similarly decisive role in the development of Johnston's organ, the ear of the fly, it is an attractive hypothesis that the functions of vertebrate and invertebrate Pax2 have been conserved in the development of both the eye and the ear (Fu, 1998).

The adult peripheral nervous system of Drosophila includes a complex array of mechanosensory organs (bristles) that cover much of the body surface of the fly. The four cells (shaft, socket, sheath, and neuron) that compose each of these organs adopt distinct fates as a result of cell-cell signaling via the Notch (N) pathway. However, the specific mechanisms by which these cells execute their conferred fates are not well understood. shaven, called in this paper D-Pax2, the Drosophila homolog of the vertebrate Pax2 gene, has an essential role in the differentiation of the shaft cell. In flies bearing strong loss-of-function mutations in shaven, shaft structures specifically fail to develop. Consistent with this, Shaven protein is expressed in all cells of the bristle lineage during the mitotic (cell fate specification) phase of bristle development, but becomes sharply restricted to the shaft and sheath (glial) cells in the post-mitotic (differentiative) phase. Three cell types, early on the pIIA secondary precursor and later the tormogen, and the thecogen, are responsive to the N-mediated signals sent by their sister cells, but pIIB, the trichogen, and the neuron are made resistant to the reciprocal signal by N pathway antagonists such as Numb and Hairless. Interestingly, while the tormogen/socket and thecogen/glial/sheath cells are recipients of Notch signals, it is the trichogen/shaft and thecogen that express Shaven (Kavaler, 1999).

An anti-Shaven antiserum was used to examine the temporal and spatial pattern of Shaven accumulation in the pupal notum during microchaete development. Shaven expression is first apparent in the SOP nucleus before division (14 hours after puparium formation (APF). After SOP division (16 hours APF), Shaven is present at similar levels in the nuclei of the two daughter cells, the secondary precursors pIIA and pIIB. Following the completion of the pIIA and pIIB divisions (18 hours APF), all four cells of the microchaete lineage express Shaven at fairly comparable levels, though one cell (the presumptive trichogen/shaft) is regularly distinguishable at this stage by its slightly elevated accumulation of the protein. Subsequently, the pattern of Shaven expression is refined so that by 32 hours APF, Shaven protein is present in only two cells, one containing a large polyploid nucleus (either the trichogen or the tormogen/socket) and one containing a small nucleus (either the neuron or thecogen/glia). By double-labeling nota with anti-Shaven antibody and the neuron/shaft marker mAb 22C10, the two cells that express Shaven at 32 hours APF could be identified. Microchaete neurons (clearly identifiable by their 22C10-positive cell bodies and axons) are Shaven-negative, but are positioned adjacent to small, Shaven-positive nuclei in 22C10-negative cells, which by inference are thecogens. This interpretation is confirmed by a second double-labeling experiment using anti-Elav (a specific nuclear marker for post-mitotic neurons) and anti-D-Shaven antibodies at 32 hours APF. In both macrochaetes and microchaetes, the small nucleus that labels with anti-Shaven is clearly distinct from the small nucleus that labels with anti-Elav. Thus, the two bristle cells that exhibit specific nuclear expression of Shaven at 32 hours APF are the thecogen and trichogen. These results distinguish two phases of Shaven expression in the bristle lineage: an early stage, in which the protein appears at similar levels in the SOP and all of its progeny as cell division proceeds, and a late stage, in which Shaven expression is restricted to the postmitotic trichogen (shaft) and thecogen (sheath) cells (Kavaler, 1999).

Two lines of evidence described here indicate that shaven expression and function is at least in part downstream of cell fate specification mechanisms such as N signaling. (1) The lack of late shaven expression in the socket cell (the sister of the shaft cell) is controlled by N pathway activity; (2) loss of shaven function is epistatic to the socket-to-shaft cell fate transformation caused by reduced N signaling. Overexpression of H after the completion of the microchaete cell divisions results in the expression of the shaft cell fate by both the normal trichogen and the transformed tormogen; that is, reduction of N pathway function promotes the expression of the shaft differentiation program. Loss of shaven function has the opposite effect: the stronger shaven mutant genotypes cause a broad failure of shaft development while permitting the normal or nearly normal expression of the other bristle cell fates. The epistatic relationship between excess H activity and loss of shaven function was examined by combining these two conditions and observing the effect on shaft differentiation. Homozygosity for even the comparatively weak shaven allele effectively suppresses the double shaft phenotype of heat-treated Hs-H transgenic flies. Thus, in the combined genotype large territories devoid of external bristle structures are observed. This effect may be explained as follows: excess H activity alone causes both the normal trichogen and the transformed tormogen to adopt the shaft fate, but reduction of shaven function prevents the expression of this fate by either cell, so no external cuticular structures are produced by the double mutant bristles. In other words, loss of shaven activity is epistatic to the effects of reduced N pathway function on the expression of the shaft differentiation program. This result strongly suggests that shaven acts at least in part downstream of the N-dependent trichogen/tormogen cell fate decision in the bristle lineage, and plays a major role in the differentiation phase of shaft cell development. Additional experiments show that misexpression of shaven is sufficient to induce the production of ectopic shaft structures. From these results, it is proposed that Shaven is a high-level transcriptional regulator of the shaft cell differentiation program, and acts downstream of the N signaling pathway as a specific link between cell fate determination and cell differentiation in the bristle lineage (Kavaler, 1999).

In the developing Drosophila eye, cell fate determination and pattern formation are directed by cell-cell interactions mediated by signal transduction cascades. Mutations at the rugose locus (rg) result in a rough eye phenotype due to a disorganized retina and aberrant cone cell differentiation, which leads to reduction or complete loss of cone cells. The cone cell phenotype is sensitive to the level of rugose gene function. Molecular analyses show that rugose encodes a Drosophila A kinase anchor protein (DAKAP 550). Genetic interaction studies show that rugose interacts with the components of the Egfr- and Notch-mediated signaling pathways. These results suggest that rg is required for correct retinal pattern formation and may function in cell fate determination through its interactions with the Egfr and Notch signaling pathways. rugose interacts with Egfr and N signaling pathways. Sparkling is a Drosophila homolog of the vertebrate pax-2 gene and is involved in cone cell specification. The runt family transcription factor, Lozenge, has been shown to directly regulate spa and Lozenge is a key downstream mediator of the Notch and Egfr pathways. A single copy of the spa mutation acts as dominant enhancer of the rg eye phenotype (Shamloula, 2002).

Determination of cell fates in the R7 equivalence group of the Drosophila eye by the concerted regulation of D-Pax2 and TTK88

In the developing Drosophila eye, the precursors of the neuronal photoreceptor cells R1/R6/R7 and non-neuronal cone cells share the same developmental potential and constitute the R7 equivalence group. It is not clear how cells of this group elaborate their distinct fates. This study shows that both TTK88 and D-Pax2 play decisive roles in cone cell development and act in concert to transform developing R1/R6/R7 into cone cells: while TTK88 blocks neuronal development, D-Pax2 promotes cone cell specification. In addition, ectopic TTK88 in R cells induces apoptosis, which is suppressed by ectopic D-Pax2. It was further demonstrated that Phyllopod (Phyl), previously shown to promote the neuronal fate in R1/R6/R7 by targeting TTK for degradation, also inhibits D-Pax2 transcription to prevent cone cell specification. Thus, the fates of R1/R6/R7 and cone cells are determined by a dual mechanism that coordinately activates one fate while inhibiting the other (Shi, 2009).

Members of the R7 equivalence group have the developmental potential to become a neuronal R1/R6/R7 or a non-neuronal cone cell. TTK88 and D-Pax2 are both specifically expressed in developing cone cells. The absence of cone cells in ttk¹; spa^pol double mutants and their presence in ttk¹ and spa^pol single mutants strongly suggest that both D-Pax2 and TTK88 function in cone cell development. This study has shown that (1) blocking the neuronal fate by TTK88 and (2) promoting the non-neuronal fate by D-Pax2 are simultaneous and coordinated steps in the transformation of R cells into cone cells. The TTK88 function of blocking the neuronal fate is largely redundant with that of TTK69 because ectopic Phyl, which down-regulates D-Pax2 and TTK88 as well as TTK69, is sufficient to transform cone cells into R7 cells, whereas removal of only TTK88 and D-Pax2 in ttk¹; spa^pol double mutants results in the loss of cone cells but not their conversion into R cells. In contrast, during R1/R6/R7 development, Phyl promotes the neuronal fate not only (1) through targeting TTK (both TTK88 and TTK69) for degradation, thereby releasing the inhibition of R cell specification, but also (2) by down-regulating D-Pax2 to block the cone cell fate. Therefore, it is proposed that the cell fates of the R7 equivalence group are determined by a dual mechanism that coordinately promotes the fate of one cell type and blocks that of the other. For cone cell development, it is not sufficient to provide D-Pax2 that activates the cone cell fate, the alternative neuronal fate has to be blocked as well by TTK protein. Conversely, for R cell development, it is not sufficient to specify this fate by removing its TTK block, but inhibition of the alternative cone cell fate by preventing D-Pax2 activation is equally important (Shi, 2009).

In third instar larval and early pupal eye discs, TTK88 protein is initially detected in all undifferentiated basal cells, but later restricted to cone cells. TTK88 blocks neuronal R cell differentiation, but is unable to promote non-neuronal cone cell specification. Thus, TTK88 serves as a safeguard in basal cells that maintains them in an undifferentiated state. The binary switch between R1/R6/R7 and cone cell fates is regulated by Phyl, which is present in the former but absent from the latter. Activation of phyl depends on Svp in R1/R6, or on high levels of Ras signaling in R7 where svp is repressed by Lz. This follows from the observation that Phyl is absent from cone cells, while ectopic Svp or high levels of Ras signaling in cone cells transforms these into R7 cells in a Phyl-dependent manner. Moreover, it has been shown that phyl is activated by Sev-induced Ras signaling in R7 precursor cells. Since Svp is absent from cone cells and Ras signaling too low because Sev is not activated, phyl is inactive in and its product absent from cone cells (Shi, 2009).

In R1/R6/R7 precursors, Phyl forms a complex with Ebi and Sina, activating a ubiquitin-proteasome machinery that targets TTK for degradation and hence releases the block of the neuronal fate in these cells. In addition to TTK, D-Pax2 must be absent from R cells because ectopic D-Pax2 in R cells of GMR-D-Pax2 flies causes frequent loss of R cells or their strong deformation, even though early R cell development seems normal as judged by staining for Elav. It is proposed that the same ubiquitin-proteasome machinery also targets an activator X of D-Pax2 transcription for degradation and thus indirectly down-regulates D-Pax2 in R1/R6/R7 precursors. Therefore, the cone cell fate is blocked in these cells while R1/R6/R7 specification begins (Shi, 2009).

In developing cone cells, TTK is not degraded because Phyl is absent. Strong N signaling in cone cell precursors is activated by high concentrations of the N ligand, Dl, on neighboring R cells. As a consequence, D-Pax2 transcription is activated in cone cells by the combinatorial effect of Lz, N-activated Su(H), and the concomitant activation of PntP2 and inhibition of Yan by EGFR-activated Ras signaling. Thus, as TTK and D-Pax2 are both present, the R cell fate is blocked and cone cell specification is initiated (Shi, 2009).

Absence of TTK88 in ttk¹ or of D-Pax2 in spa^pol mutants does not result in the transformation of cone cells into R7 cells. According to the model see A dual mechanism determines cell fates in the R7 equivalence group, efficient transformation depends on the absence of both TTK (TTK88/TTK69) and D-Pax2. This is achieved by ectopic expression of Phyl in cone cells under control of the sev enhancer or by combining the homozygous ttk¹ mutation with a heterozygous null allele of yan, which results in the derepression of phyl in cone cells. Similarly, homozygous hypomorphic yan^P mutations combined with heterozygous strong ttk alleles results in a large fraction of ommatidia with supernumerary R7 cells. In this case, Phyl levels induced in cone cells suffice to unblock the neuronal fate by further reducing TTK levels and to down-regulate D-Pax2 levels to an extent that cone cells are transformed efficiently into R7 cells. Ectopic Phyl transforms cone cells into R7 rather than R1/R6 photoreceptor cells because Svp, which specifies the R1/R6 versus the R7 cell fate, is not expressed in cone cells. Thus, the model is in good agreement with earlier observations that ttk and yan mutations act synergistically to alter the fate of cone cell precursors to that of R7 cells. It further confirms an earlier suggestion that TTK88 functions in a pathway distinct from and parallel to Ras signaling (Shi, 2009).

In addition, the reverse situation was shown to be true as well. Ectopic expression of both D-Pax2 and TTK88 in R cell precursors transforms R1/R6/R7 with a much higher efficiency into cone cells than R2-R5. For when D-Pax2 and TTK88 are expressed under the control of the sev enhancer, which results in their ectopic expression in R3/R4/R7 precursors, only one additional cone cell appears with high efficiency. By contrast, when ectopic expression occurs in all R cell precursors under the control of the GMR enhancer, only three supernumerary cone cells appear with high efficiency although up to 7 additional cone cells were observed. Moreover, ectopic co-expression in R1/R6/R7 of TTK88 and D-Pax2 in phyl mutants efficiently transforms these R cell precursors into supernumerary cone cells. The evidence, therefore, suggests the existence of a dual mechanism regulated by a binary switch between the non-neuronal cone cell and neuronal R cell fate in the R7 equivalence group. The state of this switch depends on the presence or absence of Phyl that coordinately regulates TTK and D-Pax2 levels through ubiquitin-directed proteolysis (Shi, 2009).

The model might suggest Sina as an alternative switch to Phyl. However, this possibility is excluded because Sina is expressed in photoreceptors as well as cone cells. Moreover, in sina mutants only R7 is transformed into a cone cell even though Sina is expressed in all photoreceptors. This observation has been explained by a redundancy of sina function with that of musashi in the down-regulation of TTK in R1 and R6. It is not known how TTK is down-regulated in photoreceptor precursors different from R1/R6 and R7. However, it appears that this mechanism is not only independent of Sina but also independent of Phyl, as Phyl is not expressed in photoreceptors different from R1/R6/R7 (Shi, 2009).

Degradation of TTK is mediated through an E3 ubiquitin ligase complex, including Phyl/Sina/Ebi, which targets TTK to the proteasome. The results suggest that the same complex also functions to down-regulate D-Pax2 transcription. It is therefore conceivable that this complex targets one or several of the activators of D-Pax2 for degradation (X in the model). It is improbable that TTK is this activator because TTK is only known to act as repressor (Shi, 2009).

Transcription of D-Pax2 in cone cells is regulated by the combinatorial action of Lz, N-activated Su(H), and the EGFR-regulated effectors PntP2 and Yan. EGFR signaling and Lz are both active in R1/R6 and R7 precursors. Ectopic expression in R7 of the constitutively active intracellular domain of N, N^IC, activates D-Pax2. It follows that the Phyl/Sina/Ebi-dependent proteasome machinery down-regulates D-Pax2 transcription in R7 by antagonizing N signaling. Consistent with this conclusion, it has been suggested that the E3 ubiquitin ligase complex Phyl/Sina/Ebi, targeting TTK for degradation, may inhibit the transcription-activating activity of Su(H). It is thus attractive to speculate that Su(H) is the target of this complex that may not include all of its components and thus may not degrade Su(H) but only inhibit its activity required in a complex with N^IC to activate D-Pax2. Such an interpretation is consistent with the observation that Su(H) levels in R1/R6/R7 are indistinguishable from those in cone cells. However, it cannot be excluded that other targets like N^IC might be modified rather than degraded by the Phyl/Sina/Ebi complex, since the concentration of N^IC, when expressed under the control of the sev enhancer, is independent of co-expression with Phyl (Shi, 2009).

The mechanism by which TTK blocks neuronal development is unclear. However, since TTK encodes a transcriptional repressor, it may repress one or several genes (Y in the model) that are required for neuronal development. Down-regulation of TTK would then derepress the Y genes, which act to specify neuronal development. One of the factors encoded by Y is the transcription factor Prospero (Pros), which is required for proper development of R7 since ectopic expression of TTK88 in R7 precursors abolishes the elevation of Pros. Since loss-of-function alleles of pros do not affect other R cells, other Y factors must exist (Shi, 2009).