orthodenticle

DEVELOPMENTAL BIOLOGY

Embryonic

Earliest OTD mRNA is found in the anterior region of the syncytial blastoderm. Expression then disappears from the anterior terminus, such that otd is expressed in a stripe in the cellular blastoderm extending from approximately 75 to 92% of the way to the anterior. Expression also retracts from the ventral region, such that the stripe is no longer circumferential (Gao, 1996).

empty spiracles helps define both antennal and intercalary segments, while buttonhead defines antennal, intercalary and mandibular segments [Images] (Finkelstein, 1991). Mutation of otd eliminates the first (procerebral) brain neuromere. Mutation of empty spiracles eliminates the second (deuterocerebral) and third (tritocerebral) neuromeres. otd is also necessary for the development of the dorsal protocerebrum of the adult brain (Hirth, 1995).

Early tailless expression (blastoderm stage) covers the anlage of the entire brain. Beginning approximately with the onset of gastrulation, an anterior-dorsal region with a high expression level (called HL domain) can be distinguished from a posterior-ventral domain expressing tll at a somewhat lower level. The HL domain coincides with part of the central and anterior protocerebral neurectoderm. The low expression level LL domain covers the remaining part of the protocerebral neuroectoderm. orthodenticle is expressed in a circumferential domain of the cellular blastoderm but during gastrulation becomes restricted to a domain that encompasses most of the protocerebral neurectoderm and an adjacent part of the deuterocerebral neurectoderm. All neurobasts segregating from this domain transiently express otd during stages 10 and 11. buttonhead is initially expressed in a wide domain including the anlagen of the antennal, intercalary and mandibular segments, as well as the acron. With the beginning of gastrulation, expression disappears from most of the procephalon, except for small domains of the posterior part of the deuterocerebral and tritocerebral neurectoderm and a dorsoanterior patch that partially overlaps with the dorsoanterior protocerebrum. Both the late deutocerebral and tritocerebral btd domains contain few, if any neuroblasts. empty spiracles is in an asymmetric circumferential domain of the cellular blastoderm. During gastrulation, this pattern resolves into two stripes that occupy anterior portions of the deuterocerebral neuroectoderm and the mandibular metamere, respectively. In addition, a small circular domain corresponding to the tritocerebral neurectoderm appears ventral to the deuterocerebral stripe (Younossi-Hartenstein, 1997).

Loss of tll function results in the absence of all protocerebral neuroblasts and loss of all four coherent domains of Fas II expression in the protocerebrum. Also missing is the optic lobe. orthodenticle functions in a domain that includes a large part of the protocerebrum and a smaller part of the adjacent deuterocerebrum. Loss of otd results in loss of protocerebral P1, P2 and P4 coherent domains of Fas II expression. Also missing is a nerve that carries axons from the antennal organ. In buttonhead mutation the D/T cluster is missing; consequently a cervical connection is missing that normally sends nerves to the labral sensory organ, the hypopharyngeal sensory organ and the stomatogastric nervous system (Younossi-Hartenstein, 1997).

otd is also expressed in the ventral midline where it affects survival of specific cells and axonal guidance (Klambt, 1991).

The orthodenticle-unplugged interface is positioned at the 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 (DTB) 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 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 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).

The Drosophila brain develops from the procephalic neurogenic region of the ectoderm. About 100 neural precursor cells (neuroblasts) delaminate from this region on either side in a reproducible spatiotemporal pattern. Neuroblast maps have been prepared from different stages of the early embryo (stages 9, 10 and 11, when the entire population of neuroblasts has formed), in which about 40 molecular markers representing the expression patterns of 34 different genes are linked to individual neuroblasts. In particular, a detailed description is presented of the spatiotemporal patterns of expression in the procephalic neuroectoderm and in the neuroblast layer of the gap genes empty spiracles, hunchback, huckebein, sloppy paired 1 and tailless; the homeotic gene labial; the early eye genes dachshund, eyeless and twin of eyeless; and several other marker genes (including castor, pdm1, fasciclin 2, klumpfuss, ladybird, runt and unplugged). Based on the combination of genes expressed, each brain neuroblast acquires a unique identity, and it is possible to follow the fate of individual neuroblasts through early neurogenesis. Furthermore, despite the highly derived patterns of expression in the procephalic segments, the co-expression of specific molecular markers discloses the existence of serially homologous neuroblasts in neuromeres of the ventral nerve cord and the brain. Taking into consideration that all brain neuroblasts are now assigned to particular neuromeres and individually identified by their unique gene expression, and that the genes found to be expressed are likely candidates for controlling the development of the respective neuroblasts, these data provide a basic framework for studying the mechanisms leading to pattern and cell diversity in the Drosophila brain, and for addressing those mechanisms that make the brain different from the truncal CNS (Urbach, 2003).

The cephalic gap genes are expressed in large domains of the procephalon and play a crucial role not only in the patterning of the peripheral ectoderm, but also in regionalizing the brain primordium. The segmental organization of the Drosophila brain is based on the expression pattern of segment polarity and DV patterning genes. To see whether the cephalic gap genes respect the neuromeric boundaries segment polarity and DV patterning genes, and to provide a basis for studying their potential role in the formation or specification of brain precursor cells, the expression was studied of orthodenticle, empty spiracles, sloppy paired 1, tailless, huckebein, and hunchback in the developing head ectoderm, as well as in the entire population of identified NBs during stages 9-11 (Urbach, 2003).

In the cellular blastoderm orthodenticle (otd) is expressed in an anterior, circumferential stripe and subsequently fades in the ventral region to become restricted to the procephalic ectoderm after gastrulation. In Otd/Engrailed (En) double labelling between stage 9 and 11, Otd expression in the pregnathal head is found to be confined to a large domain covering most of the antennal (the third neuromere) and preantennal (the second neuromere, termed 'ocular') neuroectoderm. Furthermore, Otd is detectable in all NBs delaminating from this domain (about 50 ocular and six antennal. NBs in the dorsal and most anterior region of the protocerebrum are Otd-negative, including most NBs of the labral neuromere (the most anterior neuromere). Thus Otd covers the NBs of the anterodorsal part of the antennal segment and most of the acron (which is equivalent to the ocular segment). Otd expression is also observed in cells along the dorsal midline of the head, as well as faint expression in neuroectodermal cells in the ventral part of the intercalary segment (the fourth neuromere - posterior to the antennal neuromere), from which the weakly Otd-positive Tv1 emerges (Urbach, 2003).

Larval

Both the mediolateral and medial domains of the dorsal head require otd activity. The medial domain is most sensitive to reduction in otd activity, particularly ocelli and associated bristles. In the eye antennal disc, otd is expressed behind the morphogenetic furrow and specifically in precursors of photoreceptor cells, ocelli (an alternative visual system in the fly) and bristles (Hirth, 1995, Royet, 1995 and Vandendries, 1996).

The eye-antennal imaginal discs of Drosophila melanogaster form the head capsule, the eyes and the antenna of the adult fly. Unlike the limb primordia, each eye-antennal disc gives rise to morphologically and functionally distinct structures. As a result, these discs provide an excellent model system for determining how the fates of primordia are specified during development. An investigation has been carried out of how the adjacent primordia of the compound eye and dorsal head vertex are specified. Subdivision of the eye-antennal disc is not based on compartmentalization: this is in contrast to the basis for subdivision in the wing and leg discs. Therefore, selector gene-mediated division of the disc into compartments, mediated by engrailed and invected, as in the wing disc for example, is not likely to be the basis for regionalization within the antennal primordium. Instead, in this region, the genes wingless and orthodenticle are expressed throughout the entire second instar eye-antennal disc, conferring a default fate of dorsal vertex cuticle. Mutations that decrease dpp expression in the eye primordia lead to the formation of severely reduced eyes. Similarly, the loss of otd or wg function in the vertex primordia causes the elimination of dorsal head structures (Royet, 1997).

Transplantation experiments show that the eye primordium occupies most of the posterior half of the eye-antennal disc (the so-called 'eye disc'). The head vertex forms from the dorsomedial region of the disc, while the antenna develops from the anterior half of the disc (the so-called 'antennal disc'). During the early third instar stage (70-80 hours after egg laying), dpp is expressed in a horseshoe-shaped domain along the ventral, posterior and dorsal periphery of the eye disc. Dorsal dpp expression does not extend as far anteriorly as ventral expression, but instead ends at the vertex primordium. At this stage, otd expression covers the vertex primordium and extends along the edge of the antennal disc. The posterior boundary of otd expression in the vertex anlage coincides, approximately, with the anterior boundary of the dpp domain. At the same stage of disc development, wg is expressed in two regions of the eye disc. One region corresponds to the future gena (the lateral part of the head capsule bounded above by the eye) and the other to the head vertex (Royet, 1997).

dpp expression prevents dorsal head development in the eye primordium. Flies homozygous for the dppd-blk allele that reduces dpp activity in the eye primordium greatly reduces the compound eye giving rise to an eye with only a few residual ommatidia. In these mutants the eyes are largely replaced by frons cuticle, which normally appears only on the dorsal areas of the head. This ectopic frons lies between the orbital cuticle and the remaining ommatidia, and to the anterior, between the shingle cuticle and the ommatidia. In other eye loss mutants, such as sine oculis or eyes absent, the eyes are completely lost but are not replaced by ectopic frons. This suggests that dorsal head cuticle does not result simply from loss of the eyes, but is caused instead by loss of dpp function. Clones of Mothers against dpp, coding for a protein involved in transmission of the Dpp signal, likewise transform ommatidia into frons (Royet, 1997).

Activation of decapentaplegic expression in the posterior eye disc eliminates wg and otd expression, thereby permitting eye differentiation. In dppd-blk mutants, the otd domain expands toward the anlagen of the shingle cuticle and the compound eyes, consistent with the location of ectopic frons cuticle on dppd-blk mutant heads. wg expression also expands in these mutant discs. Ectopic activation of the wingless pathway (the result of the generation of clones mutant for shaggy/zeste-white 3) in the eye primordium induces otd expression and vertex formation. Loss of shaggy function results in constitutively activated wg signaling and ectopic otd expression. This suggests that otd expression in the vertex primordium is normally activated or maintained by wingless. Early activation of dpp depends on hedgehog expression in the eye anlage prior to morphogenetic furrow formation. Loss of hh activity during the second instar larval stage eliminates dpp expression along the posterior and lateral margins of the eye disc and in the antennal primordium. This loss of dpp expression is associated with a dramatic expansion of the otd expression domain. wg expression also expands into the eye primordium (Royet, 1997).

Unlike the limb discs, which derive from single trunk segments, each eye-antennal disc arises from multiple embryonic head segments. Divisions between segment primordia within the disc could contribute to certain aspects of regional specification. It is proposed that wg and otd expression in the eye-antennal discs are inherited from the embryo, where the two genes are expressed in segments from which these discs are derived. The almost ubiquitous expression of these two genes serves to program the early disc for a vertex fate. Later, hh expression in the posterior region of the future eye disc induces dpp expression along the margins of the eye primordium. dpp represses wg, permitting the formation of the eye primordium (Royet, 1997).

Effects of Mutation or Deletion

orthodenticle mutants display anterior denticle belts pointing posteriorly, defects at the ventral midline and head defects (Wieschaus, 1984). There is a graded response of loss of head structure with orthodenticle mutants of different strength, the most severe being complete loss of medial and mediolateral head structure. Ventral midline defects include cell death restricted to identified neurons of the midline of the CNS and defects in axon pathway choice (Klambt, 1991).

One orthodenticle mutation results in altered photoreceptor cell development in the eye. This mutation is in a regulatory region of otd in the third intron. otdUV-insensitive (otduvi) is a hypomorphic allele of otd that only affects R-cell development. The R-cell rhabdomeres are disorganized in otduvi, and there is a disruption of proximal-distal development in the eye. Sequences encompassing this deletion are able to direct expression of otd at all stages of the developing visual system, including the photosensitive cells of Bolwig's organ, the ocelli, and the adult eye. The third intron enhancer is the primary regulatory element controlling otd in the R cells (Vandendries, 1996).

The CNS midline of Drosophila should not be considered as an isolated autonomous entity but as an organizing center for the rest of the CNS. Cells located at the midline of the developing central nervous system perform a number of conserved functions during the establishment of the lateral CNS (the rest of the CNS as distinguished from the midline). The midline cells of the Drosophila CNS are required for correct pattern formation in the ventral ectoderm (which gives rise to the rest of the CNS) and for induction of specific mesodermal cells. The midline cells are also required for the correct development of lateral CNS cells. Embryos that lack midline cells through genetic ablation show a 15% reduction in the number of cortical CNS cells. A similar thinning of the ventral nerve cord can be observed following mechanical ablation of the midline cells. A number of specific neuronal and glial cell markers have been identifed that are reduced in CNS midline-less embryos, as for example in single-minded embryos, in early heat-shocked Notch(ts1) embryos or in embryos where the midline cells have been mechanically ablated. Genetic data suggest that both neuronal and glial midline cell lineages are required for differentiation of lateral CNS cells. One marker, the rR226 enhancer trap insertion, reveals a reduction in the number of marker positive cells in midline ablated embryos. Loss of orthodenticle, a gene expressed specifically in midline neurons, results in the degeneration of many midline neurons. Compared to wild type, the number of rR226-positive cells is reduced in otd mutant embryos. It is thus concluded that the CNS midline plays an important role in the differentiation or maintenance of the lateral CNS cortex (Menne, 1997).

The molecular mechanisms of head development are a central question in vertebrate and invertebrate developmental biology. The anteriorly expressed Drosophila homeobox gene otd and its murine homolog Otx are required for the early development of the most anterior part of the body, suggesting that a fundamental genetic program of cephalic development might be conserved between vertebrates and invertebrates. This hypothesis has been examined by introducing the human Otx genes into flies. By inducing expression of the human Otx homologs with a heat shock promoter, it was found that both Otx1 and Otx2 functionally complement the cephalic defects of a fly otd mutant through specific activation and inactivation of downstream genes. Expression of transformant hsp-Otx1 and hsp-Otx2 in flies was induced by heat pulses in the oc1 mutant background. Both human Otx1 and Otx2 homologs complement the oc1 defect, generating either ocellar lenses or associated ocellar pigments. In some cases, both lenses and pigments are formed. Formation of the vertex bristles is also enhanced by the human Otx genes. Whereas human Otx homologs tended to produce more bristles on the median vertex than the otd gene, the fly gene is more potent in making postvertical-like bristles at the normal position. In wild-type flies, the postvertical bristles are located at the posterior edge of the medial vertex (Nagao, 1998).

The primordium of the vertex is situated near the dorsomedial edge of the eye-antennal disc. A network of cross-regulatory segment polarity gene interactions is involved in the development of the vertex primordium: en and hh are expressed and wg is suppressed in a medial patch of cells in the late third instar stage. These genetic interactions are unique to the vertex primordium and are very different from those in trunk development, where wg acts to maintain en and hh, and en and hh act to maintain wg. The oc1 mutation causes specific loss of expression of en and hh in this region whereas expression of wg is maintained in a continuous crescent-like pattern at the dorsomedial region of the eye-antennal disc. To determine whether the morphological complementation by the human Otx genes is a result of similar genetic interactions, the expression of en, hh, and wg were examined in the vertex primordium cells after heat induction in oc1 background. Induction of the fly otd gene stimulates en and hh and represses wg in the vertex primordium, confirming previous observations. Despite the ubiquitous induction of the otd gene by the hsp promoter over the eye-antennal disc, the regulatory effects on these downstream genes are restricted to the vertex primordium, suggesting that specific cofactor(s) in this region are present. Consistent with these morphological results, induction of the human Otx1 and Otx2 genes results in activation of en and hh and repression of wg in the vertex primordium. The specific regulatory effects on these downstream genes are somewhat more variable than those induced by the fly otd gene. However, similar cell type specificity is observed, despite the ubiquitous induction of the human Otx homologs, suggesting that the induced human OTX proteins might be able to interact with the vertex cofactor(s). Heat induction of the ems gene fails to affect expression of these segmentation genes, indicating that the downstream controls are specific to the otd/Otx homologs. Combined with previous morphological studies, these results are consistent with the view that a common molecular ground plan of cephalization was invented before the diversification of the protostome and the deuterostome in the course of metazoan evolution (Nagao, 1998).

In Drosophila, mutational inactivation of the orthodenticle gene results in deletions in anterior parts of the embryonic brain and in defects in the ventral nerve cord. In the mouse, targeted elimination of the homologous Otx2 or Otx1 genes causes defects in forebrain and/or midbrain development. To determine the morphogenetic properties and the extent of evolutionary conservation of the orthodenticle gene family in embryonic brain development, genetic rescue experiments were carried out in Drosophila. Ubiquitous overexpression of the orthodenticle gene rescues both the brain defects and the ventral nerve cord defects in orthodenticle mutant embryos; morphology and nervous system-specific gene expression are restored. Two different time windows exist for the rescue of the brain versus the ventral nerve cord. Ubiquitous overexpression of the human OTX1 or OTX2 genes also rescues the brain and ventral nerve cord phenotypes in orthodenticle mutant embryos; in the brain, the efficiency of morphological rescue is lower than that obtained with overexpression of orthodenticle. Overexpression of either orthodenticle or the human OTX gene homologs in the wild-type embryo results in ectopic neural structures. The rescue of highly complex brain structures in Drosophila by either fly or human orthodenticle gene homologs indicates that these genes are interchangeable between vertebrates and invertebrates and provides further evidence for an evolutionarily conserved role of the orthodenticle gene family in brain development (Leuzinger, 1998).

orthodenticle gene has been classified as a head gap gene for two reasons. (1) Its expression at the early cellular blastoderm stage is under the control of maternal positional information in a manner similar to that of (non-cephalic) gap genes. (2) Mutation of otd leads to a gap-like phenotype in the anterior head, which includes deletions in cuticular structures, the absence of the antennal and preantennal expression of engrailed and wingless, the loss of several cephalic sensory structures, and the deletion of the protocerebral anlage. Fate map studies relate the regionalized cephalic defects seen in otd mutants to the broad anterior region of otd expression in the early cellular blastoderm stage. It is, therefore, conceivable that the gap-like otd mutant phenotype is due to the absence of a functional otd gene at the cellular blastoderm stage. While this may apply to some of the non-CNS defects, the experiments described in this paper indicate that this is not the case for the embryonic brain defects observed in otd mutants. Genetic rescue experiments through ubiquitous overexpression of an otd transgene in otd mutant embryos indicate that the existence of a functional Otd gene product before embryonic stage 7 is not required for proper development of the anterior embryonic brain. This is because the gap-like brain defects in the otd mutant can be restored by overexpressing otd at stages 7-8. This, in turn, implies that the cells of the blastoderm embryo, which express otd in the wild type and are fated to give rise to the protocerebrum, are not deleted in the otd mutant at least up to stage 7-8. It is possible that other head gap genes with partially redundant function can compensate for the loss of otd in the cellular blastoderm embryo (Leuzinger, 1998).

Switch of rhodopsin expression in terminally differentiated Drosophila sensory neurons

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

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

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

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

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

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

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

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

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

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

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

Although EcR could directly control the switch of rhodopsin expression through binding to the promoters of rh5 and rh6, these promoters contain no potential EcR binding sites. Moreover, as no EcR expression is detectable when Rh6 starts to be expressed, this would make it unlikely for EcR to control directly the switch to Rh6. Finally, only allowing expression of the dominant negative form of EcR starting at mid-pupation (GMR-Gal4/Tub-Gal80^ts,UAS-EcR^DN), after rh5 is switched off, does not prevent activation of Rh6 in the eyelet. Thus EcR most likely acts in an indirect manner in regulating rhodopsins, likely through the activation of transcription factors that bind to rh5 and rh6 promoters (Sprecher, 2008).

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

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

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

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

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

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

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

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

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

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