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).

On the origin and evolution of the tripartite brain

The many different nervous systems found in bilaterally symmetric animals may indicate that the tripartite brain appeared several times during the course of bilaterian evolution (see Bilaterian evolutionary tree). However, comparative developmental genetic evidence in arthropods, annelids, urochordates, and vertebrates suggests that the development of a tripartite brain is orchestrated by conserved molecular mechanisms. Similarities in the underlying genetic programs do not necessarily reflect a common origin of structures. Nevertheless, 3 lines of evidence support a monophyletic origin of the tripartite brain and possibly also an elongated central nervous system (CNS): structural homology, character identity networks, and the functional equivalence of character identity genes. Monophyly of the brain also implies that the brain was secondarily reduced and lost multiple times during the course of evolution, leading to extant brainless bilaterians. The likelihood of secondary loss can be estimated by metazoan divergence times and through reconstructed cases such as limb loss in tetrapods or eye loss in fish. When scaled to molecular clock dates, monophyly of the tripartite brain indicates that existing brainless Bilateria had several hundred million years’ time for the secondary modification and eventual loss of a primitive/ancestral brain and CNS. To corroborate this conjecture, ancestral character identity genes of living brainless Bilateria can be tested for their potential to substitute Drosophila or Mus homologs in tripartite brain development (Hirth, 2010).

Comparative developmental genetic analyses in arthropods, annelids, urochordates, and vertebrates provide experimental evidence suggesting that brain and CNS development in these taxa is orchestrated by conserved molecular genetic mechanisms. Similarities in the underlying developmental genetic programs do not necessarily reflect a common origin of structures. However, monophyly of the tripartite brain is supported by 3 lines of evidence, namely (1) structural homology, (2) character identity networks (ChINs), and (3) functional equivalence of character identity genes. (Hirth, 2010).

Homology signifies common descent and can be defined as a relationship between traits of organisms that are shared as a result of common ancestry. Structural homology refers to a morphological character that is (a) derived from a common ancestor possessing this character, (b) built on the same basic plan, and (c) consists of comparable elements. The latter was already exemplified by Darwin, who referred to 'the relative position or connection in homologous parts; they may differ to almost any extent in form and size, and yet remain connected together in the same invariable order'. Textbook examples are the different forelimbs of tetrapods where similar bones are connected in the 'same invariable order', irrespective of the different functions they serve. The same principle of 'relative position or connection in homologous parts' applies to 'midbrain' structures in Drosophila and Mus, although function seems to be conserved as well; in both cases, GABAergic, serotonergic, and dopaminergic neural circuit elements involved in the control of locomotor behavior are located in the same relative position to other neural processing centers, namely posteriorly to light-sensing organs and anteriorly to gustatory and 'facial' innervation (Hirth, 2010).

The principle 'connected together in the same invariable order' of comparable elements may also apply to ventral/spinal cord motor neurons in Drosophila, Platynereis (Annelida), and Mus; they are located ventrally to nonneural tissue and dorsally to the midline. As a stand-alone criterion, however, 'relative position or connection in homologous parts' would be insufficient to signify homology because similar structures with similar positions can have multiple causes, including parallel or convergent evolution. However, related to brain evolution, this criterion is supported by developmental genetic evidence suggesting that insect and mammalian brains are built on the same basic plan, which is executed by the spatiotemporal activities of pleiotropic genes that constitute character identity networks (ChINs) (Hirth, 2010).

ChINs refer to genetic regulatory networks that control the developmental program and, hence, the specification of character identities, such as forelimbs. ChINs for brain and CNS development include Otx-Pax-Hox modules acting along the anterior-posterior axis, BMP-Msx-Nkx modules acting along a DV axis, and bHLH-Par-Numb modules acting along an apico-basal axis. Together, these modules are necessary for the correct formation of an orthogonal, species-specific CNS. Character identity genes are central to ChINs; their knockdown/mutational inactivation does obstruct the formation of a character identity. The majority of character identity genes are transcription factors, with textbook examples such as Pax6 genes, which are essential for the formation of light-sensing organs, or otd/Otx genes, which are essential for anterior brain formation. As stated earlier, similarities in developmental genetic programs do not necessarily reflect a common origin of character identities, which is exemplified, for example, by the role of distal-less(Dll/Dlx) genes in the formation of non-homologous appendages. However, the situation is different if taxa-specific ChINs, which comprise homologous genes, specify taxa-specific character identities to which structural homology applies, namely their construction depends on a conserved genetic program (basic plan) and consists of comparable elements that are arranged 'in the same invariable order'. This principle applies to the above-mentioned midbrain-derived neural circuit elements that are required for the control of locomotor behavior: in both flies and mice, FGF8, Engrailed, and Pax2/5/8 genes are essential for the formation and specification of these structures (Hirth, 2010).

ChINs of different taxa can comprise homologous genes that control the developmental program and, hence, the specification of character identities sharing a common descent. It has therefore been predicted that the phenotype caused by knockdown of a character identity gene (i.e. transcription factor) 'can be reversed with a gene from the clade that shares this character, but not by genes from species that diverged before the origin of this character'. Experimental evidence conforming to this prediction is available. Human Otx2 can rescue defective 'forebrain' formation in Drosophila orthodenticle (otd) mutants, and otd can replace mouse Otx2 in fore- and midbrain formation, but only if otd is accompanied by the Otx2 regulatory sequences required for epiblast-specific translational control. Drosophila engrailed can substitute for mouse Engrailed1 function in mid-hindbrain formation, but not for limb development. Mouse Emx1 can rescue brain defects in Drosophila empty spiracles(ems) mutants, but Acropora [Anthozoa, Cnidaria (radially symmetric organisms including corals, anemones and jelly fish)] Emx is not able to replace ems in fly brain development (Hirth, 2010).

Based on these results, and in line with Wagner's prediction of the existence of equivalent functions of character identity genes (Wagner, 2007), it can be concluded that: (1) The character of a 'fore-, mid-, and hindbrain' develops under the control of ChINs comprising otd/Otx, ems/Emx, and en/EN1 genes that are shared in a clade including flies and mice. (2) The failure of Drosophilaengrailed to rescue limb formation in MusEN1 mutants indicates that the appendages of mice and flies are not homologous. (3) Most likely, the epiblast did not exist in the last common ancestor of Drosophila and Mus and is an evolutionary novelty of the lineage leading to Mus (Boyl, 2001). (4) Drosophila and Acroporaems/Emx genes diverged before the origin of the character 'tripartite brain'. The last conclusion has major implications for the evolutionary origin of the tripartite brain and depends on the position of anthozoans/cnidarians relative to bilaterally symmetric animals in a phylogenetic tree (Hirth, 2010).

A phylogenetic tree is based on evolutionary relationships and can be reconstructed using cladistics. The cladistic concept is relative; it scores characters for their presence and absence and, if present, for their state in each of the taxa of interest. Current cladistics scores morphological as well as molecular characters (i.e. genes, genomes, and developmental pathways) against each other, and the resulting phylogenetic tree is based on sister and out groups in order to be rooted. For example sister groups like arthropods and onychophorans share a common panarthropod ancestor, and together they are a sister group to cycloneuralians (including nematodes), which together belong to the ecdysozoans, which are a sister group to lophotrochozoans. Out groups provide necessary additional information about the origin of a character in sister groups; they are used for character polarity which enables the application of the parsimony criterion in order to infer whether the character is primitive/ancestral or derived (Hirth, 2010).

Monophyly of bilaterally symmetric animals and subsequent interpretations about the origin and evolution of the brain and CNS hinge on the identification of genuine sister and out groups and, thus, on how deep a phylogenetic tree is rooted. Current cladistics is work in progress which is exemplified by the allocation of Cnidaria (but also Acoela) as either a sister or out group to bilaterians. The ambiguity is caused by mounting evidence suggesting that cnidarians possess genetic toolkits similar to those active in bilaterian axis and cell type specification, including neurogenesis (Gaillot, 2009). Depending on additional characters and genuine out groups to Cnidaria, this can be interpreted to mean that: (i) cnidarians are de facto bilaterians and a genuine sister group to the rest of bilaterally symmetric animals, namely P+D, or (ii) cnidarians are a genuine out group to P+D. It follows that the positioning of Cnidaria has an impact on the positioning of Urbilateria and the origin of a brain and CNS. Cnidarians possess nerve nets and nerve rings but, so far, no evidence of a centralized nervous system has been found [Gaillot, 2009]. Several extant Protostomia and Deuterostomia (P+D) also possess a net-like nervous system. In the first scenario, Urbilateria would be the last common ancestor of both Cnidaria and P+D, suggesting that a nerve net is a primitive/ancestral character. In the second scenario, Urbilateria would be the last common ancestor of P+D, and unrelated to Cnidaria, suggesting that the cnidarian nerve net is unrelated to the nerve net of extant bilaterally symmetric animals (the position of Acoela would require further considerations) (Hirth, 2010 and references therein).

As mentioned, the presence of genetic toolkits does not necessarily reflect a common origin of characters. The existence of equivalent functions of character identity genes, however, allows inferences as to whether genes diverged before the origin of a character or not (Wagner, 2007). Murine, but not the cnidarian AcroporaEmx gene, can rescue brain defects in Drosophila ems mutants, suggesting that Drosophila and Acroporaems/Emx genes diverged before the origin of the character 'tripartite brain', which is a primitive/ancestral character to mice and Drosophila. These data suggest that Acropora and the last common ancestor of mice and Drosophila did not share the character 'tripartite brain'; it may also indicate that the last common ancestor of Cnidaria and P+D possessed a nerve net-like nervous system. The latter notion is supported by the functional equivalence of another character identity gene. The cnidarian Hydra achaete scute homolog has proneural activity in Drosophila, can heterodimerize with daughterless, and is able to form ectopic sensory organs in the peripheral nervous system of Drosophila; in addition, it is also able to partially rescue adult external sensory organ formation in viable achaete scutecomplex mutations. Unfortunately, whether Hydra achaete scute is able to rescue defects in Drosophila achaete scute mutant brain and CNS development was not tested (Hirth, 2010).

Together, these data suggest that the morphological character 'tripartite brain' evolved after the Cnidaria-P+D split. It depends on the placement of cnidarians in- or outside the clade Bilateria, whether it is reasonable to propose that Urbilateria already possessed a tripartite brain and probably also a complex CNS, or whether Urbilateria was likely to be 'brainless'. If Cnidaria and P+D are true sister groups of a clade Bilateria, it follows that Urbilateria was brainless. Independent of the position of Urbilateria, the above-mentioned data corroborate monophyly of the tripartite brain, as well as its evolutionary origin after the cnidarian-P+D split but before the Protostomia-Deuterostomia (P/D) split. This concept, though, is challenged by the fact that several proto- and deuterostomians do not possess a brain and complex CNS. The conundrum is most obvious when the complex CNS of arthropods is compared to that of cycloneuralians, which are supposed to be sister groups. Comparative data for cycloneuralians are scant, except for Caenorhabditis elegans. The nematode does not possess a complex brain and CNS, yet monophyly of the brain is corroborated by functional equivalence of a character identity gene: the ems homolog ceh-2 of C. elegans is able to rescue ems mutant brain defects in Drosophila. These data suggest that C. elegans secondarily lost the character 'tripartite brain' and most likely also a complex CNS, as should be postulated for other brainless proto- and deuterostomians that are monophyletic to Drosophila and mice. (Hirth, 2010).

Monophyly of the tripartite brain and its evolutionary origin after the cnidarian-P+D split and before the P/D split implies that the brain and CNS were secondarily reduced and eventually lost multiple times and independently during the course of protostomian and deuterostomian evolution. For the quality and consistency of this conjecture, it is necessary to consider (1) metazoan divergence times, (2) the likelihood of secondary loss, as illustrated by reconstructed cases, and (3) the proposal of an experimental paradigm that can test ancestral character identity genes of brainless bilaterians for their potential to control brain development in Drosophila or mice. (Hirth, 2010).

Molecular clock dates suggest that the cnidarian-P+D split occurred somewhere around 630 million years ago (Mya), the P/D split around 555 Mya, and the Arthropoda-Priapulida split around 540 Mya. These estimates suggest that a primitive/ancestral tripartite brain likely evolved within 75 million years between 630 and 555 Mya and then continued to evolve into taxon- and species-specific characters such as the extant Drosophila and mouse brain. Monophyly implies that ancestral priapulids shared with arthropods the character of a tripartite brain and possibly a segmented CNS. Fossil evidence supports ancestral segmentation in priapulids (e.g. Markuelia) even though extant priapulids are nonsegmented and their CNS is 'only' composed of a nerve ring and a single ventral cord running the length of the body. Scaled against molecular clock dates, monophyly of the tripartite brain suggests that the derived character of the priapulid brain and CNS would have had several hundred million years' time for its secondary modification. Such a scale of divergence time can be extrapolated to other taxa as well, and implies that also other extant brainless P+D would have had several hundred million years' time for the secondary, independent modification and eventual loss of a tripartite brain and CNS (Hirth, 2010).

Comparative developmental genetics and phylogenomics reveal that morphological evolution is most likely driven by gene duplication and gene loss, together with changes in differential gene regulation, including mutations in cis-regulatory elements of pleiotropic developmental regulatory genes. These genetic modifications can account not only for the acquisition of novel morphological characters but also for the modification and eventual loss of a morphological character. The latter is illustrated by limbless tetrapods, such as whales, snakes, and flightless birds. Limbless tetrapods are descended from limbed ancestors, and limblessness has been shown to be polygenic, involving pleiotropic regulatory genes that act as modifiers to suppress limb development. In snakes, for example, differential regulation of HoxC genes accounts for the failure to activate the signaling pathways required for proper limb development, eventually leading to limbless snakes. Independent reduction and limb loss in tetrapods occurred repeatedly over several millions of years for lizards, and over 10-12 or up to 20 million years for whales (Hirth, 2010).

The secondary loss of morphological characters is also exemplified in fish. In different natural populations of threespined stickleback fish, the secondary loss of the pelvis occurred through regulatory mutations deleting a tissue-specific enhancer of the Pituitary homeobox transcription factor 1 (Pitx1) gene. The selective pressures causing secondary loss can be manifold, including energy limitation and environmental constraints which are most obvious for nervous system structures that are characterized by high energy consumption. For example, populations of cave fish have undergone convergent eye loss at least 3 times within the last 1 million years, whereas populations that continuously lived on the surface retained their eyes. These examples illustrate that the secondary loss of a morphological character can occur repeatedly during the course of evolution within a time frame of million years. In comparison, monophyly of the tripartite brain calibrated by metazoan divergence times suggests that extant brainless P+D would have had several hundred million years’ time, possibly from 555 Mya onwards, for the secondary modification and eventual loss of an ancestral/primitive tripartite brain and CNS, the mechanisms of which remain unknown (Hirth, 2010).

The secondary, independent loss of the brain and CNS multiple times during the course of protostomian and deuterostomian evolution is a conjecture that can be tested experimentally. Wagner's prediction states that the phenotype caused by knockdown of a character identity gene can be rescued with a gene from the clade that shares a particular character, but not by genes from species that diverged before the origin of this character (Wagner, 2007). Monophyly of the tripartite brain implies that extant brainless P+D species were once able to develop a primitive/ancestral tripartite brain; therefore, these brainless species should possess ChINs for the development and specification of a tripartite brain, unless they have secondarily lost the necessary genes during the course of evolution. The potential functional equivalence of character identity genes in brain development can be tested in those cases where brainless P+D species have retained the ChINs or at least a character identity gene. Thus, genes from species that have secondarily lost the character tripartite brain but have retained, for example, otd/Otx genes with an archetypical 'brain function' might be able to rescue, at least in part, brain phenotypes in Drosophila otd or Mus Otx2 mutants. These experiments are feasible and can be tested as homologs of character identity genes controlling tripartite brain development have been identified in brainless P+D species. It will be interesting to see whether Otx or Engrailed genes from brainless brachiopods or echinoderms like sea cucumber are able to substitute their Drosophila or murine homologs in the development and specification of a tripartite brain (Hirth, 2010).


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).

Opposite Feedbacks in the Hippo Pathway for Growth Control and Neural Fate

Signaling pathways are reused for multiple purposes in plant and animal development. The Hippo pathway in mammals and Drosophila coordinates proliferation and apoptosis via the coactivator and oncogene, YAP/Yorkie (Yki), which is homeostatically regulated through negative feedback. In the Drosophila eye, cross-repression between the Hippo pathway kinase, LATS/Warts (Wts), and growth regulator, Melted, generates mutually exclusive photoreceptor subtypes. This study shows that this all-or-nothing neuronal differentiation results from Hippo pathway positive feedback: Yki both represses its negative regulator, warts, and promotes its positive regulator, melted. This postmitotic Hippo network behavior relies on a tissue-restricted transcription factor network - including a conserved Otx/Orthodenticle-Nrl/Traffic Jam feedforward module - that allows Warts-Yki-Melted to operate as a bistable switch. Altering feedback architecture provides an efficient mechanism to co-opt conserved signaling networks for diverse purposes in development and evolution (Jukam, 2013).

A fundamental strategy in animal development is to re-purpose the same signaling pathways for a diversity of functions. This study identified a tissue-specific transcription factor network that enables the otherwise homeostatic Hippo growth control pathway to act as a bistable switch for terminal cell fate. This alteration in network level properties—such as positive versus negative feedback—within biochemically conserved pathways is an efficient means to re-use a signaling network in contexts as distinct as proliferation and terminal differentiation (Jukam, 2013).

How is the R8-specific Hippo regulatory circuit achieved? The two interlinked positive feedback loops (one with wts, one with melt) provide the R8 Hippo pathway with multiple points of potential regulation. Context-specific expression of wts and melt is defined by overlapping expression of four transcription factors: Otd, Tj, Pph13, and Sens. Otd and Pph13 are expressed in all photoreceptors and generate a permissive context that endows the initially equipotent R8s with the competence to become either subtype: Otd promotes melt/Rh5 whereas Pph13 promotes wts/Rh6 expression. This competence is further restricted by expression of Tj in R7 and R8, and Sens in R8s, which ensures that melt and wts cross-regulation is restricted to R8s. Importantly, it is the status of Yki activity and resulting feedback that assures the outcome of pR8/Rh5 vs. yR8/Rh6 (p vs. y) fate: in pR8s, Yki functions with Otd and Tj to promote melt and Rh5; in yR8s, wts inhibits Yki, preventing melt and Rh5 expression and allowing 'default' wts and Rh6 expression by Pph13 and Sens. Each of these four transcription factors regulates a partially overlapping subset of R8 subtype fate genes, and together, the network cooperates at multiple regulatory nodes to provide the specific context for repurposing the Hippo pathway (Jukam, 2013).

While other instances of pathways with both positive and negative feedback exist, these are conceptually different from R8 Hippo regulation. For example, in Sprouty (hSpry) regulation of Ras/MAPK-mediated EGFR signaling, EGFR induces hSpry2 expression but hSpry2 inhibits EGFR function (negative feedback); however, hSpry2 also promotes EGFR activity by preventing Cbl-dependent EGFR inhibition (positive feedback). hSpry2 positive feedback is likely coupled to its negative feedback to fine-tune the length and amplitude of receptor activation. In contrast, the opposite Hippo pathway feedbacks occur in vastly different cell types (mitotic epithelial cells versus post-mitotic neurons), and both forms of feedback cannot co-exist in R8 since Yki’s repression of wts expression (positive feedback) would make Yki up-regulation of Hippo regulators (negative feedback) inconsequential (Jukam, 2013).

Gaining positive feedback or losing negative feedback within Hippo signaling could permit oncogenesis. Indeed, the Yki ortholog, YAP, is an oncogene and is amplified in multiple tumors, and LATS1/2 (Wts) down-regulation is associated with non-small cell lung carcinomas, soft tissue sarcoma, metastatic prostate cancers, retinoblastoma, and acute lymphoblastic leukemia. Otx and MAF factors are also oncogenic in a number of tissues. Thus, understanding the regulatory networks identified here in other contexts will be crucial for deciphering how normal signaling pathways can go awry (Jukam, 2013).

The current findings also reveal that a Crx/Otd-Nrl/Tj feedforward module plays a conserved role in post-mitotic photoreceptor fate specification in both flies and mammals. Both induce one photoreceptor fate at the expense of another, and both regulate opsins with a feedforward loop wherein Crx/Otd activates Nrl/Tj expression and Crx-Nrl or Otd-Tj synergistically activate downstream targets (Hao, 2012). Given such deep evolutionary conservation, this module may be critical for generating photoreceptor diversity in other complex visual systems (Jukam, 2013).

This work has two main implications. First, although positive feedback is well documented in other switch-like, irreversible cell fate decisions such as in Xenopus oocyte maturation or cell cycle entry, this work suggests that positive feedback could have a broad role in terminal neuronal differentiation, which often requires permanent fate decisions to maintain a neuron’s functional identity. Second, the changes in network topology in R8 photoreceptors allows a finely tuned growth control pathway to be used as a switch in a permanent binary cell fate decision. Context-specific regulation allows the feedback architecture to change in an otherwise conserved signaling module. This may be a general mechanism to endow signaling networks with new systems properties and diversify cell fates in development and evolution (Jukam, 2013).

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-EGFRDN). The eyelet of these animals is not affected and three or four cells express Rh6 normally. This shows that larval Rh6 photoreceptors do not contribute to the eyelet (Sprecher, 2008).

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

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

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

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

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

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

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

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

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

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

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

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

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

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

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

OTX2 and CRX rescue overlapping and photoreceptor-specific functions in the Drosophila eye

Otd-related transcription factors are evolutionarily conserved to control anterior patterning and neurogenesis. In humans, two such factors, OTX2 and CRX, are expressed in all photoreceptors from early specification through adulthood and associate with several photoreceptor-specific retinopathies. It is not well understood how these factors function independently vs. redundantly, or how specific mutations lead to different disease outcomes. It is also unclear how OTX1 and OTX2 functionally overlap during other aspects of neurogenesis and ocular development. Drosophila encodes a single Otd factor that has multiple functions during eye development. Using the Drosophila eye as a model, tests were performed of the ability of the human OTX1, OTX2, and CRX genes, as well as several disease-associated CRX alleles, to rescue the different functions of Otd. The results indicate the following: OTX2 and CRX display overlapping, yet distinct subfunctions of Otd during photoreceptor differentiation; CRX disease alleles can be functionally distinguished based on their rescue properties; and all three factors are able to rescue rhabdomeric photoreceptor morphogenesis. These findings have important implications for understanding how Otx proteins have subfunctionalized during evolution, and cement Drosophila as an effective tool to unravel the molecular bases of photoreceptor pathogenesis (Terrell, 2012).

Previous cross- and intra-species experiments, as well as the experiments described above, are limited in their ability to define common vs. unique properties used by OTX factors to regulate development, largely because of a lack of known target genes. In contrast, the ability to quantify the rescue of specific gene targets (Rh3, Rh5, and Rh6) in distinct photoreceptor populations provided valuable tools to further explore these issues. Importantly, it was found that OTX1, OTX2, and CRX are each equally able to rescue Rh3-expressing photoreceptors, demonstrating that these factors are properly expressed and functional in the fly eye and can contribute to common processes. In contrast, only OTX2 can prevent Rh6 expansion into outer photoreceptors and only CRX significantly activates Rh5 in R8 cells, revealing that these factors can also mediate distinct functions (Terrell, 2012).

These data are in strong agreement with previous studies on Otx factors during mouse nervous system development, indicating that they exert similar and distinctive functions. Otx1 and Otx2, for example, are widely expressed and participate in overlapping processes during nervous system development and in the formation of ocular structures such as the lens, ciliary body, and retinal pigmented epithelia, while Otx2 and Crx have overlapping patterns of expression in bipolar cells and photoreceptors and regulate many of the same genes in the retina. This, together with data indicating that Otx1 and Otx2 can largely replace each other's functions in gene replacement studies, suggests that OTX factors can mediate common regulatory functions. However, it is also clear that Otx2 only partially restores many of Otx1-dependent functions, with no capacity to rescue Otx1-dependent regulation of inner ear development. Similarly, despite their similarities, Otx2 and Crx induce the formation of different cell types when overexpressed in Xenopus retinal explants, and photoreceptor-specific knockout studies of Otx2 do not phenocopy Crx mutants. Thus, OTX factors also appear have distinct functions. The ability to quantify the extent of rescue of specific target genes now allows the defining domains within the OTX proteins necessary to mediate their various functions and to dissect the molecular mechanisms underlying their overlapping and unique properties (Terrell, 2012).

These studies may also lend insight into previous studies suggesting that Otx1 and Otx2 function dose-dependently. For example, during ocular development, Otx1−/−; Otx2+/− mice exhibit strong defects in lens, retina, and retinal pigmented epithelium (RPE), which are not observed in Otx1−/− or Otx2+/− mice. This study found that OTX2 can strongly induce dve expression and subsequently repress Rh6 expression in outer photoreceptors, whereas OTX1 can only weakly activate defective proventriculus (dve) and this is not sufficient to repress Rh6. One could envision that similar events are under way on target genes in the mouse eye, with Otx2 primarily regulating the expression of a certain gene or set of genes required for some developmental events, and Otx1 contributing weakly to their expression. Hence, when both copies of Otx1 and a copy of Otx2 are removed, the target gene(s) are now no longer able to reach the appropriate threshold of expression necessary for function. Indeed, this may be the case with Otx2-dependent activation of the BEST-1 gene in the RPE. Thus, it is possible to begin to identify domains between OTX1 and OTX2 that would explain how they can equally activate some targets (e.g., Rh3) but differentially regulate other targets (e.g., dve) (Terrell, 2012).

Orthodenticle is necessary for survival of a cluster of clonally related dopaminergic neurons in the Drosophila larval and adult brain

The dopaminergic (DA) neurons present in the central brain of the Drosophila larva are spatially arranged in stereotyped groups that define clusters of bilaterally symmetrical neurons. These clusters have been classified according to anatomical criteria (position of the cell bodies within the cortex and/or projection pattern of the axonal tracts). However, information pertaining to the developmental biology, such as lineage relationship of clustered DA neurons and differential cell subtype-specific molecular markers and mechanisms of differentiation and/or survival, is currently not available. Using MARCM and twin-spot MARCM techniques together with anti-tyrosine hydroxylase immunoreactivity, this study analyzed the larval central brain DA neurons from a developmental point of view and determined their time of birth, their maturation into a DA neurotransmitter phenotype as well as their lineage relationships. In addition, it was found that the homeodomain containing transcription factor Orthodenticle (Otd) is present in a cluster of clonally related DA neurons in both the larval and adult brain. Taking advantage of the otd hypomorphic mutation ocelliless (oc) and the oc2-Gal4 reporter line, the involvement of orthodenticle in the survival and/or cell fate specification of these post-mitotic neurons was studied. The findings provide evidence of the presence of seven neuroblast lineages responsible for the generation of the larval central brain DA neurons during embryogenesis. otd is expressed in a defined group of clonally related DA neurons from first instar larvae to adulthood, making it possible to establish an identity relationship between the larval DL2a and the adult PPL2 DA clusters. This poses otd as a lineage-specific and differential marker of a subset of clonally related DA neurons. Finally, it was shown that otd is required in those DA neurons for their survival (Blanco, 2011).

The Drosophila larval central brain contains 21 DA neurons per hemisphere during L3, which express the cell type-specific marker gene TH. Different methods have been proposed to classify and annotate these neurons according to anatomical criteria (position of the cell bodies within the cortex and/or projection pattern of the axonal tracts). This paper has analyzed these neurons from a developmental point of view and classified them according to their lineage relationship. The MARCM technique is a powerful tool to study lineage progression and cellular pedigrees during Drosophila brain development. It allows the labeling of progenitor cells and their offspring at different times during development, depending on the timing of a heat-shock-induced flippase-mediated mitotic recombination event. Using this technique, it was shown that the larval central brain DA neurons are primary neurons born during early embryogenesis. However, when analyzing the lineage relationship among these neurons, two major problems were encountered. Firstly, implicit in the technique is the fact that a labeled NB clone is accompanied by a non-labeled twin clone (two post-mitotic cells derive from the first ganglion mother cell born just after the mitotic recombination event). The exclusion of two cells from the lineage analysis is negligible when larval lineages are analyzed (the average size of a standard larval lineage at L3 is 120 cells). However, embryonic lineages are small (on average between 10 and 20 cells at the end of embryogenesis and the exclusion of two cells can be significant. Secondly, MARCM-labeled NB clones induced during early embryogenesis can only be visualized with a considerable delay after their generation (from L2 onwards) due to the persistence of the Gal80 repressor protein. These two problems have recently been circumvented by the development of the twin-spot MARCM technique. This technique not only allows the visualization of cell clones earlier in development but also differentially labels the NB clone and the twin clone; thus, the study of the entire NB lineage is now possible (Blanco, 2011).

Using this technique, the lineage relationship among the DA neurons present in the Drosophila central brain was analyzed during larval development. It was found that seven NB lineages generate the 21 DA neurons present in the larval central brain: DM1a (one DA neuron), DM1b (three DA neurons), DM2 (four DA neurons), DL1a (six DA neurons), DL1b (one DA neuron), DL2a (four DA neurons) and DL2b (two neurons). At large, the lineage analyses agree with the clustering of DA neurons according to anatomical criteria, supporting the general assumption that cell bodies arrangement and axonal projection patterns are reliable ways to classify neurons in Drosophila. Just in the case of the DL1 cell cluster was a discrepancy found. The cell bodies of the seven DL1 DA neurons are compactly arranged in a cell cluster that occupies medial-lateral positions in the L3 central brain and their neurites display similar projection pattern. Yet, six DL1 DA neurons are clonally related (DL1a NB lineage) and the remaining DL1 DA neuron is generated by an additional NB (DL1b NB lineage). For future functional studies, it would be interesting to find molecular markers differentially labeling these two populations of DL1 DA neurons (Blanco, 2011).

Most studies involving the homeodomain transcription factor Otd in central nervous system development in Drosophila have dealt with its role in the specification and proliferation of progenitor cells during early neurogenesis, whereas a possible function in post-mitotic neurons has been largely overlooked. The observation that otd is expressed in the DL2a DA neurons during larval development prompted an investigation its role in the specification and/or survival of this DA cell cluster. According to anti-TH labeling, DL2a DA neurons mature mainly during early L1. Thus, null otd alleles, which are embryonic lethal, could not be used in this analysis. Therefore, the hypomorphic otd allele oc was studied. It was found that in oc mutant hemizygous larvae, otd expression in dorsolateral regions of the central brain was reduced and, as a consequence, only one of the four DL2a DA neurons showed anti-TH labeling during L3. The failure to detect three of the four DA neurons can be due to a defect in the regulation of TH expression or to the loss of DA neurons per se. Several lines of evidence support the latter hypothesis. Firstly, a general regulator of TH expression would be expected to be present in all or most of the central brain DA neurons; yet, otd expression during larval development is restricted to the DL2a DA cell cluster. Secondly, misexpression of otd in randomly induced cell clones in the central brain during larval development does not result in ectopic TH-expressing DA neurons. Thirdly, labeling of DL2a DA neurons with the oc2-gal4 driver shows that reporter gene expression is also abolished in oc mutant hemizygous larvae during L3. The oc2 enhancer has been shown to be positively regulated by otd during ocelli development and might not, therefore, be suitable to label DL2a DA neurons in an otd-independent way. However, a minimal version of this enhancer harboring the characterized Otd binding site (oc7) was active in the ocelli primordium, but did not show enhancer activity in DL2a DA neurons during larval development. This indicates that the oc2 enhancer is differentially regulated in the ocelli primordium and in DA neurons during development and, hence, the oc2. Taken together, the observations support the hypothesis that otd expression is required for survival of DL2a DA neurons during larval development.

The wild-type Drosophila adult brain is populated by about 200 DA neurons distributed in several bilaterally symmetric clusters. The PPL2 cluster contains seven cells that express otd and five of them also show oc2 enhancer activity in young adult brains. Similarly to the larval brain, otd expression in PPL2 DA neurons seems to be necessary for their survival, since neither anti-TH immunoreactivity nor transcriptional activity of the oc2 enhancer is detected in oc mutant adult brains. Moreover, the effects of targeted depletion of Otd in PPL2 DA neurons can be rescued by the simultaneous expression of the anti-apoptotic gene P35, pointing out a role in cell survival as the main function of otd in PPL2 DA neurons. Altogether, the simplest interpretation for these results would be that otd expression labels homologous DA neuron populations in both the larval (DL2a cell cluster) and adult (PPL2 cell cluster) brains and, hence, both clusters contain the same DA neurons. The discrepancy in cell number between both clusters of DA neurons can be interpreted by analyzing the NB lineage responsible for the generation of the DL2a DA neurons. At L3, this lineage contains seven otd expressing cells, four of them are primary neurons that have already undergone maturation and express TH. The other three cells might represent immature secondary neurons that differentiate during pupal stages to give rise to the additional three DA neurons present in the adult PPL2 cluster. The distinction between early-differentiating (four cells) and late-differentiating (three cells) PPL2 DA neurons finds support in the targeted depletion of Otd in DA neurons by RNAi. Expression of an otd-specific RNAi construct in DA neurons (using the TH-Gal4 driver) has no effect on the larval brain, but impairs the viability of four PPL2 DA neurons in the adult brain. Since these four cells differentiate during larval development, the RNAi machinery would have more time to completely deplete Otd than in the case of the late differentiating DA neurons. Further support for this interpretation also comes from the analysis in the adult brain of wild-type twin-spot MARCM cell clones induced during early L1. According to this analysis, at least two PPL2 DA neurons in the adult brain are secondary neurons, whereas the third DA neuron might represent an undifferentiated primary neuron that only matures during pupal development (Blanco, 2011).

Recently, the expression of Otx2, an otd ortholog, in DA neurons in the mouse adult brain has also been reported. It is selectively expressed in the central DA neurons of the ventral tegmental area, where it is cell autonomously required to antagonize identity features of the dorsal-lateral ventral tegmental area DA neurons. Thus, contrary to Drosophila, depletion of Otx2 in these DA neurons does not induce cell death, but it changes neuron subtype identity. Interestingly, otx2 expression in these DA neurons has been associated with their reduced vulnerability to Parkinsonian neurodegeneration [35]. Finally, in oc mutant adult flies most of the protocerebral bridge, a neuropile structure that is part of the central complex, is also missing. In several behavioral paradigms, these mutant flies walk slowly and show altered orientation behavior toward visual objects. It has been recently proposed that the protocerebral bridge is an essential part of a visual targeting network that transmits directional clues to the motor output. Thus, with regards to the data presented here, it would be interesting to analyze whether the lack of PPL2 DA neurons in oc mutant adult flies contributes to the behavioral phenotypes observed in these mutant flies (Blanco, 2011).

Using MARCM and twin-spot MARCM techniques together with anti-TH immunoreactivity, this study has classified the 21 DA neurons present in the Drosophila larval central brain into seven clusters of clonally related DA neurons. The homeobox gene otd is specifically expressed in DA neurons belonging to one of these clusters (DL2a cluster); thus, otd expression differentially labels a subset of DA neurons. Furthermore, by taking advantage of an otd hypomorphic mutation and the oc2-Gal4 reporter line, a cell lineage relationship was established between the larval DL2a and the adult PPL2 DA cell clusters. The role of otd in the survival and/or cell fate specification of these post-mitotic neurons was also studied. Contrary to mice, where Otx2 expression in DA neurons of the adult brain is necessary for neuron subtype identity, otd is required in the Drosophila larval and adult brain for survival of DL2a and PPL2 DA neurons. These findings suggest that otd acts as a post-mitotic selector gene whose differential expression among DA neurons might help to establish functional differences (Blanco, 2011).

Orthodenticle is required for the development of olfactory projection neurons and local interneurons in Drosophila

The accurate wiring of nervous systems involves precise control over cellular processes like cell division, cell fate specification, and targeting of neurons. The nervous system of Drosophila melanogaster is an excellent model to understand these processes. Drosophila neurons are generated by stem cell like precursors called neuroblasts that are formed and specified in a highly stereotypical manner along the neuroectoderm. This stereotypy has been attributed, in part, to the expression and function of transcription factors that act as intrinsic cell fate determinants in the neuroblasts and their progeny during embryogenesis. This study focuses on the lateral neuroblast lineage, ALl1, of the antennal lobe and shows that the transcription factor-encoding cephalic gap gene orthodenticle is required in this lineage during postembryonic brain development. Immunolabelling was used to demonstrate that Otd is expressed in the neuroblast of this lineage during postembryonic larval stages. Subsequently, MARCM clonal mutational methods were used to show that the majority of the postembryonic neuronal progeny in the ALl1 lineage undergoes apoptosis in the absence of orthodenticle. Moreover, it was demonstrated that the neurons that survive in the orthodenticle loss-of-function condition display severe targeting defects in both the proximal (dendritic) and distal (axonal) neurites. These findings indicate that the cephalic gap gene orthodenticle acts as an important intrinsic determinant in the ALl1 neuroblast lineage and, hence, could be a member of a putative combinatorial code involved in specifying the fate and identity of cells in this lineage (Sen, 2014a).

During early embryogenesis, the cephalic gap gene otd is expressed in a broad stripe in the anterior most domain of the cephalic region of the embryo where it is known to specify the entire segment, including the anterior brain that derives from this segment. Studies that have analysed the expression of otd in the later stages of embryonic brain development have shown that otd continues to be expressed in specific neuroblasts. For example, in the protocerebral part of the embryonic brain, otd is expressed in about 70% of the neuroblasts. Interestingly, 15% of the embryonic neuroblasts that express otd co-express the cephalic gap gene ems (Sen, 2014a).

This study reports that otd is also co-expressed with ems in a neuroblast lineage during postembryonic brain development. This study focused on the ALl1 neuroblast, which has been shown to express ems during larval development. While these findings indicate that the expression of otd is relatively low compared to the level of ems expression in the ALl1 neuroblast, mutant analysis indicates that otd is essential for the development of the neurons in this lineage. It will be interesting to see if otd might be similarly involved in the development of the other neuroblast lineages in the brain (Sen, 2014a).

Mutant analysis of the function of otd in the ALl1 lineage revealed several distinct requirements for this gene. The first, most evident defect observed in clonal loss-of-function experiments was the reduction in cell number of the ALl1 lineage; only 20% of the cells present in the wild-type adult brain were seen in the mutant condition. This phenotype is reminiscent of, but not exactly like, the phenotypes observed in this lineage due to the loss of function of three other genes, empty spiracles (ems), homothorax (hth) and extradenticle (exd). Upon the loss of function of any of these genes, the entire lineage is eliminated. In contrast, upon the loss of function of otd, 20% of the neural cells (~40 cells) survive and are present in the adult brain. This suggests that the mechanism of action of these genes might be different. In this respect, it is interesting to note that accompanied with the loss of function of ems, hth or exd a severe reduction in the size of the antennal lobe results, whereas following otd loss of function, the lobe size and its general glomerular organization remains largely unaffected (Sen, 2014a).

A different requirement for otd in the ALl1 lineage, determined by mutational analysis, was in the targeting of the dendrites and the arborization of the axons of the 20% of the cells that do survive to adulthood. Upon the loss of function of otd, ALl1 PNs displayed a variety of targeting defects including diffuse and disorganised dendritic arbours, innervations in non-antennal neuropiles, as well as extensive, premature defasciculation and misprojections of the axonal terminals. This suggests that patterning of the PNs at both the proximal and the distal terminals might be coupled. Such coupling of PN pattering has been uncovered for other genes as well, including other transcription factors like acj6, drifter, hth, exd and lola (Sen, 2014a).

It has been postulated that the identity of a NB and its lineage depends upon a certain constellation of transcription factors that acts as a code of identity. Expression analysis of NBs in the embryo has revealed that there do exist unique combinations of transcription factors in specific NBs. Moreover, recent studies, which are largely limited to a few well-described lineages in the brain, are beginning to identify the elements of putative 'combinatorial codes' of NB specification. Results from this study imply that the two cephalic gap genes otd and ems are included among the set of intrinsic cell fate determinants for the ALl1 lineage. As most postembryonic lineages have now been identified in both the larval and adult brains, such molecular genetic analyses can now be extended to other brain lineages. It is noteworthy that although analyses such as these have uncovered genes that are required in NB lineages for their survival or local targeting, none, so far, have identified genes that can actually switch the identity of one NB lineage into that of other. It will be interesting to see if future studies uncover such important factors that determine the identities of lineages (Sen, 2014a).

Genetic transformation of structural and functional circuitry rewires the Drosophila brain

Acquisition of distinct neuronal identities during development is critical for the assembly of diverse functional neural circuits in the brain. In both vertebrates and invertebrates, intrinsic determinants are thought to act in neural progenitors to specify their identity and the identity of their neuronal progeny. However, the extent to which individual factors can contribute to this is poorly understood. This study investigates the role of orthodenticle in the specification of an identified neuroblast (neuronal progenitor) lineage in the Drosophila brain. Loss of orthodenticle from this neuroblast affected molecular properties, neuroanatomical features and functional inputs of progeny neurons, such that an entire central complex lineage transformed into a functional olfactory projection neuron lineage. This ability to change functional macrocircuitry of the brain through changes in gene expression in a single neuroblast revealed a surprising capacity for novel circuit formation in the brain and provided a paradigm (Sen, 2014b; PubMed).

This study focused on two identified neuroblast lineages in the Drosophila brain, LALv1 and ALad1, which develop in close spatial proximity to each other in the larval brain but become spatially segregated in the adult brain. While the ALad1 neuroblast generates olfactory projection interneurons that innervate the antennal lobe, the LALv1 neuroblast generates wide-field interneurons that innervate the central complex. orthodenticle is expressed during development in the LALv1 neuroblast lineage but not in the ALad1 neuroblast lineage. Remarkably, loss of otd from the LALv1 neuroblast results in a complete transformation in the identity of the neurons that derive from this lineage. The otd null LALv1 neurons transform into antennal lobe projection interneurons similar to the ALad1 lineage, and this transformation includes a complete change in the neuroanatomy of the neurons, a change in their molecular properties as well as in their functional connectivity. This remarkably complete respecification of a neuroblast lineage upon the mutation of a single gene in the brain demonstrates that intrinsic determinants acting in the neuroblast during development specify the identity of its neural progeny and the macrocircuitry that these progeny establish. This large-scale modification of functional circuits in the brain by a single transcription factor in a single stem cell is unprecedented and reveals a surprising capacity for novel neural circuit formation in the developing brain, which may provide a paradigm for large-scale evolutionary modification of brain connectivity (Sen, 2014b).

orthodenticle: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | References

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