eyeless

DEVELOPMENTAL BIOLOGY

Embryo

eyeless is expressed both early and late in Bolwig's organ cells, which serve as the larval photoreceptor. The ey expression in Bolwig's organ occurs during embryonic development at the end of stage 12. Krüppel expression can also be detected in all 12 Bolwig's organ precursors, but whether ey and Krüppel are coexpressed in all precursors is unknown. ey is down-regulated and absence during most phases of Bolwig's organ development, which includes morphogenetic movement and axonal growth, elongation and projection (Sheng, 1997).

The mushroom body (MB) is a uniquely identifiable brain structure present in most arthropods. Functional studies have established its role in learning and memory. The early embryonic origin of the four neuroblasts that give rise to the mushroom body has been described and its morphogenesis through later embryonic stages has been followed. In the late embryo, axons of MB neurons lay down a characteristic pattern of pathways. eyeless and dachshund are expressed in the progenitor cells and neurons of the MB in the embryo and larva. In the larval brains of the hypomorphic ey^R strain, beside an overall reduction of MB neurons, one MB pathway, the medial lobe, has been found to be malformed or missing. Overexpression of eyeless in MBs under the control of an MB-specific promoter results in a converse type of axon pathway abnormality, i.e. malformation or loss of the dorsal lobe. In contrast, loss of dachshund results in deformation of the dorsal lobe, whereas no lobe abnormalities can be detected following dachshund overexpression. These results indicate that ey and dachshund may have a role in axon pathway selection during embryogenesis (Noveen, 2000).

MB neurons are formed by four neuroblasts (MBNBs) that occupy a characteristic position on the vertex of the late embryonic and larval brain hemispheres. The origin and early embryonic development of the MBNBs have not been studied before. Using the early embryonic expression of ey, dac and other markers (e.g. seven-up) that have been identified as being expressed in the larval MB, it can be shown that MBNBs segregate from the central protocerebral neurectoderm as part of the Pc3 group of neuroblasts during early embryogenesis. ey and dac are expressed in a cluster of approximately 10-12 cells at stage 9, shortly before delamination of brain neuroblasts commences, which will be called here the MB neurectoderm. It is possible that the MB neurectoderm, defined by the early ey expression, corresponds to a proneural cluster, i.e. the equivalence group of cells that are competent to become MBNBs. Proneural genes of the AS-C, whose expression defines proneural clusters in the ventral neurectoderm, are expressed in wider domains in the head and include the MB neurectoderm (Noveen, 2000 and references therein).

As they delaminate, the four MBNBs keep expressing ey, whereas expression in the ectodermal cells that stay at the surface diminishes. In contrast, dac remains expressed in the MB ectoderm as well as the MBNBs throughout embryogenesis. Ectodermal expression of dac expands to include a cluster of cells laterally adjacent to the MB neurectoderm, referred to here as 'para-MB neurectoderm'. It should be noted that this designation is not meant to imply any other than a purely topological relationship between MB and para-MB neurectoderm. The para-MB neurectoderm does not contribute to the formation of the MB in any way (Noveen, 2000).

ey and dac are also expressed in other embryonic neuroblasts. ey is expressed in a small group of neuroblasts in the deuterocerebrum and tritocerebrum and in segmentally reiterated groups of three SII neuroblasts in the ventral nerve cord. Expression of dac at an early stage (stage 9-10) is restricted to the MB and para-MB neurectoderm and the MBNBs. Later, scattered groups of neurons in both ventral nerve cord and brain, as well as other embryonic tissues, turn on this gene (Noveen, 2000).

Once the MBNBs have delaminated, the MB and para-MB neurectoderm does not seem to give rise to any more neuroblasts. However, the fate of this part of the head ectoderm is an unusual one: all cells of the MB and para-MB ectoderm are incorporated at mid-embryogenesis into the cortex of the brain. Neurons and glial cells in Drosophila are typically produced by neuroblasts that delaminate at an early stage (stage 9-11) and proliferate inside the embryo. Cells that remain at the surface after neuroblast delamination typically form the epidermis of the larva. Portions of the brain do not stem from neuroblasts, but form small 'placode'-like groups of ectoderm cells that invaginate during stage 13. The MB and para-MB ectoderm form a subset of these placodes. Following their invagination from the surface, these cells are positioned at the surface of the lateral part of the brain hemisphere. Abundant cell death removes part of the cells, particularly in the case of the MB neurectoderm. The remaining cells spread out over the lateral aspect of the brain hemisphere (Noveen, 2000).

The time at which the MB neuroblasts can be first distinguished from other neuroblasts, and at which most MB-specific markers start being expressed is in the late embryo (stage 15). Surprisingly, however, MB neuroblasts are among the earliest neuroblasts delaminating from the head ectoderm. To confirm this observation the phenocritical period at which the activation of the hsp70-Notch(intra) construct is able to affect the MB neuroblasts was determined. N(intra) activation abolishes neuroblast delamination, resulting in the loss of the structures normally produced by these neuroblasts. Heat pulses applied to embryos during stages 9 and 10 strongly reduce the number of ey and dac positive MB neurons in the late embryo, whereas the later pulse (stage 11) has no such effect. This finding supports the notion that the MBNBs are born at an early stage (Noveen 2000).

Similar to most other neuroblasts of the brain and ventral nerve cord, the MBNBs start to proliferate as soon as they have delaminated, each producing lineages of 15-20 neurons, starting at embryonic stage 9. Neurons keep expressing both ey and dac, although the level of expression of both genes declines towards mid-embryogenesis. MBNBs and their early embryonic progeny form a coherent wedge-shaped cluster that is called here the early embryonic MB primordium (eMBp). During this early phase of MBNB proliferation (between embryonic stage 9-14), about 60-80 Kenyon cells are produced per hemisphere. Around stage 14, when all the other neuroblasts of the brain and ventral nerve cord have ended their proliferation, the MBNBs keep proliferating. This later phase of proliferation continues uninterruptedly into the larval period and gives rise to the large, circular plate of MB neurons characteristic of the larval brain (late embryonic/larval MB primordium, lMBp). The total number of the Kenyon cells at the end of embryogenesis has been estimated to be between 100 to 300 (Noveen, 2000).

It is believed that early neurons (born between stages 9 and 14) and late neurons (after stage 14) form two different populations. Thus, numerous molecular markers of the larval MB, among them Protein kinase A and Leonardo, are first produced at embryonic stage 14 in a small number of cells attached to the MB neuroblasts. The large population of earlier produced neurons (eMBp) does not show expression of these markers. Beside expressing different genes, early embryonic and larval MB primordium are strikingly different in their growth pattern. The early formed MB neurons form columns of cells that grow from the neuroblast towards the center of the brain, similar to the typical insect neuroblast lineage. In contrast, the later born neurons expand tangentially over the brain surface and form the typical appearance of the Kenyon cells, resembling the head of a mushroom. It appears from these observations that the neurons born early from the MB neuroblasts do not contribute to the Kenyon cells, since they are located centrally, close to the neuropile, as opposed to superficially where one can find the Kenyon cell bodies (Noveen, 2000).

Axons of MB neurons can be detected with antibody against Fasciclin II (FasII) from late stage 17 onward. At this stage, the typical, orthogonally arranged peduncle, dorsal lobe and medial lobe can already be recognized. During earlier stages, MB axons are FasII negative. These axons were labeled by applying DiI through a micropipette to the MB neurons located right underneath the easily recognizable quartet of MB neuroblasts. MB axons extend as a short bundle, the forerunner of the peduncle, during stage 14. They grow along one of the brain neuropile founder cells, P4l. During stage 16, a conspicuous 90° turn can be seen at the tip of the peduncle; this gives rise to the medial lobe. The dorsal lobe is formed last by collaterals of the MB axons. The above indicates that the medial and dorsal lobes are formed at different times (Noveen, 2000).

The expression and function of eyeless and twin of eyeless in the embryonic central nervous system of Drosophila were examined. Both genes are differentially expressed in specific neuronal subsets (but not in glia) in every CNS neuromere, and in the brain, specific cell populations co-expressing both proteins define a longitudinal domain that is intercalated between broad exclusive expression domains of ey and toy. Studies of genetic null alleles and dsRNA interference did not reveal any gross neuroanatomical effects of ey, toy, or ey/toy elimination in the embryonic CNS. In contrast, targeted misexpression of ey, but not of toy, resulted in profound axonal abnormalities in the embryonic ventral nerve cord and brain (Kammermeier, 2001).

For an initial characterization of ey and toy gene expression during embryogenesis, whole-mount in situ hybridization was carried out. The spatiotemporal expression patterns of the two Drosophila Pax6 genes in the embryonic CNS are clearly different. The embryonic expression of toy transcripts begins during stage 5 and precedes expression of ey which only starts during stage 9. This initial expression of toy is observed in a discrete anterior dorsal region of the embryo that will give rise to part of the procephalic neuroectoderm. During subsequent embryonic development, this anterior expression domain remains prominent and gives rise to a subset of cephalic neuroblasts. At stage 12, toy expression first becomes visible in the neuromeres of the embryonic VNC; this segmentally reiterated expression pattern persists throughout the remaining embryogenesis. Initial expression of ey transcripts commences at stage 9 and, in contrast to toy expression, is detectable from the onset in both the brain and the VNC. From stage 14 onwards throughout embryogenesis, prominent ey expression domains are visible in the brain as well as in all neuromeres of the VNC. Thus, in terms of their relative spatiotemporal expression patterns, toy transcripts are observed earlier than ey transcripts in the developing embryonic brain, but ey transcripts are detected earlier than toy transcripts in the VNC. The initiation of toy expression in cephalic neuroectoderm regions followed by a later appearance throughout the more posterior CNS is comparable to the spatiotemporal expression pattern of vertebrate Pax6. The mouse Pax6 gene is first expressed at embryonic day 8 in the prospective forebrain and only later appears in the more posterior regions of the embryonic CNS excluding the midbrain (Kammermeier, 2001).

To visualize the spatial distribution pattern of Ey and Toy proteins in the embryonic CNS, immunocytochemical experiments with polyclonal antibodies against the Ey and Toy proteins were carried out in combination with an antiHRP antibody. Anti-HRP immunoreactivity reveals the entire neural lineage of the developing CNS excluding the glial lineage. In the embryonic supraesophageal ganglion, Ey and Toy are expressed in specific clustered subsets of neurons in all three neuromeres (protocerebrum, deuterocerebrum and tritocerebrum) as well as in several neurons located along the preoral brain commissure. In addition, Ey and Toy are also expressed in a small subset of cells in the developing optic lobe of the embryo as well as in the mushroom body neuroblasts. Both proteins are also expressed in specific clustered subsets of neurons in the three brain neuromeres of the subesophageal ganglion. Similarly, in the VNC both Ey and Toy are expressed in characteristic segmentally reiterated patterns; expression of both proteins occurs in each neuromere (neuromere boundaries were visualized with anti-Engrailed immunocytochemistry), however, the two metameric patterns in the VNC are different for Ey versus Toy. Given that both proteins are expressed in complex spatial patterns in the embryonic brain, it was of interest to determine whether cells co-express both proteins. For this, double-immunostaining experiments were performed with antibodies against Ey and Toy on stage 11-15 embryos. In the embryonic brain of embryos at all of these stages, Ey is mainly expressed in the ventral, and Toy in the dorsal part of the embryonic brain (spatial coordinates according to neuraxis). Co-expression of both proteins is only observed in a small set of cells that are intercalated between the two broad dorsoventral regions of non-overlapping expression of Ey and Toy. Thus, the co-expressing cells define a longitudinal (according to neuraxis) domain which is bordered by toy-expressing and ey-expressing regions. This restricted spatial extent of Ey/Toy co-expressing cells in the embryonic brain was confirmed by optical sections through immunostained brains. In the embryonic mouse brain, a role of Pax6 in the establishment of longitudinal expression boundaries in the forebrain has been demonstrated, and similar functions may underlie ey and toy co-expression in the embryonic fly brain (Kammermeier, 2001).

To determine whether the cells that express either of the two Drosophila Pax6 genes are neuronal or glial in nature, a double-immunocytochemical experiment was carried out in which antibodies against Ey or Toy were used together with an anti-RK2 antibody that is a marker for glial cells. In these experiments, neither Ey nor Toy were co-expressed in cells that were labeled with the glial marker. This indicates that both Drosophila Pax6 genes are expressed in a neuron-specific manner in the embryonic CNS (Kammermeier, 2001).

In contrast to the lack of embryonic CNS phenotypes, pronounced defects occur postembryonically in ey mutants. Similarly, dramatic defects were observed postembryonically in toy RNAi mutants. In these mutants, which are characterized by pupal lethality, pharate adults lack either half of the head or the entire head structure. This defect, which is also observed in ey/toy RNAi double loss-of-function mutants, correlates with phenotypes observed in toy mutants. However, since pupal development was not the focus of this investigation these postembryonic defects were not studied further (Kammermeier, 2001)

Eye specification in Drosophila is thought be controlled by a set of seven nuclear factors that includes the Pax6 homolog, Eyeless. This group of genes is conserved throughout evolution and has been repeatedly recruited for eye specification. Several of these genes are expressed within the developing eyes of vertebrates and mutations in several mouse and human orthologs are the underlying causes of retinal disease syndromes. Ectopic expression in Drosophila of any one of these genes is capable of inducing retinal development, while loss-of-function mutations delete the developing eye. These nuclear factors comprise a complex regulatory network and it is thought that their combined activities are required for the formation of the eye. The expression patterns of four eye specification genes [eyeless (ey), sine oculis (so), eyes absent (eya), and dachshund (dac)] were examined throughout all time points of embryogenesis; only eyeless is expressed within the embryonic eye anlagen. This is consistent with a recently proposed model in which the eye primordium acquires its competence to become retinal tissue over several time points of development. The expression of Ey was compared with that of a putative antennal specifying gene, Distal-less (Dll). The expression patterns described here are quite intriguing and raise the possibility that these genes have even earlier and wide ranging roles in establishing the head and visual field (Kumar, 2001b).

Clonal analysis and fate mapping data have suggested that the antenna and compound eyes are specified during embryogenesis. Morphological analysis has further indicated that the antennal and eye disc primordia fuse to form a single complex near the end of embryogenesis. The expression of an ey-lacZ transgene, which is known to faithfully reflect ey expression within the developing imaginal disc, was examined in batches of embryos that were separated by 1 h and this was compared with the expression of Dll, an antennal specifying gene. Dll precedes ey-lacZ expression and is first seen in anterior regions of the head at approximately 5 h AED. Dll expression can be found in the maxillary and mandibular segments along with the leg precursors at approximately 6 h AED. ey-lacZ expression is first detectable at approximately 11 h AED in two symmetrical regions on the dorso-lateral surface of the embryo. At this time point the ey-lacZ domains are adjacent to two domains of Dll expression. Interestingly, Dll expression is not seen in the presumptive antennal discs. However, as development proceeds, the eye imaginal disc adopts a more dorso-medial position and the domains of Dll expression become further separated from that of ey-lacZ. This separation continues through to the end of embryonic development. While ey expression is seen throughout the eye-antennal disc complex at this time point, Dll expression is not detected in the eye-antennal imaginal disc complex until the second instar larval time point, and is not restricted to just the antennal portion of the disc until the beginning of the third larval instar (Kumar, 2001b).

Genetic experiments have established ey as residing near the top of the eye specification hierarchy and dac as the most-downstream member of this signaling cascade. Nevertheless, genetic and molecular epistasy experiments have shown a complex reciprocal interaction between the two genes. Both are able to induce the transcription of the other in ectopic expression experiments, and both require the function of the other for ectopic eye development. Although it is still unclear if these connections are through direct binding of these proteins to each other's promoters, it suggests that both genes should be expressed within the same cells during eye imaginal disc development. It is expected that dac expression is induced in all places where ey-lacZ is expressed and visa versa. The expression of ey-lacZ was compared with that of dac; it was surprising to find that dac is expressed in an ey-independent manner during embryogenesis. dac expression is first detected in two anterior-dorsal domains at approximately 4 h AED and increases in complexity throughout the embryonic head by approximately 5 h and 6 h AED. Subsets of cells within the brain hemispheres express dac beginning at approximately 7 h AED, while cells within each segment of the central nervous system (CNS) express dac at approximately 8 h AED. At approximately 11 h AED ey-lacZ expression, which demarcates the eye imaginal disc, is not co-localized with dac, which is predominant in the optic lobes, brain and CNS. As the imaginal disc continues to develop through embryogenesis, dac and ey-lacZ expression are never co-localized to the same groups of cells. This is further surprising, since ectopic dac expression in imaginal discs is sufficient on its own to induce ey transcription, and regions of ey-lacZ and dac colocalization are expected to be seen (Kumar, 2001b).

Ey directs the transcription of eya by binding to regulatory regions within the eya promoter. Ectopic expression of eya was not however observed to induce eye transcription. It is therefore possible to see cells that are Eya positive and Ey negative, but any cell that is Ey positive should also be Eya positive. To determine if this is indeed the case, the expression of ey-lacZ was compared with that of eya, with the expectation that all cells (especially the eye imaginal disc primordia) that express ey-lacZ would also express eya. Surprisingly, eya expression begins even earlier than that of dac and is obviously also independent of ey regulation (Kumar, 2001b).

Eya protein is first seen at approximately 2 h AED at which time the embryo is still in the synctial blastoderm time point and can be seen as a band of cells that runs along the dorsal surface of the embryo. By approximately 4 h AED this band is transformed into a crown that extends more laterally. As is the case with dac, eya begins to be expressed in subsets of cells within the embryonic brain by approximately 7 h AED, but these cells are distinct from those that express dac. Unlike dac, eya is not expressed within the ventral nerve cord, but rather is found in a small clustering of cells within the segmental grooves of the embryo. From the onset of ey-lacZ expression at approximately 11 h AED through the end of embryogenesis, eya is not expressed within the eye imaginal disc. Eya protein is first detected in the eye imaginal disc during the first larval instar (Kumar, 2001b).

These results have several implications for current thinking on how the Drosophila eye is specified. It has been shown that the ectopic expression of each of the eye specification genes (with the exception of so) is sufficient to induce the formation of ectopic eyes. What prevents the induction of retinal tissue throughout the embryo? It is argued here that expression of all eye specification genes are required for eye determination. Within the embryo no region is found in which all these factors are present. It is not until the second larval instar that all genes are expressed within the same tissue. Within the embryo, positive or repressive mechanisms must be in place to prevent the eye specification genes from being co-expressed. For example, ey is capable of directly inducing the transcription of both so and eya within the mature eye imaginal disc, but within the eye anlagen these genes are not expressed, although Ey protein is present. The nature of this regulatory mechanism is unknown (Kumar, 2001b).

It is still unclear as to why only three eye specification genes (toy, ey, and eyg) are expressed within the embryonic eye primordium. Is the expression of these three genes within the eye primodium a priming step for the eventual specification of the eye or is it simply a step that distinguishes one disc from another? Since Ey protein has been shown to directly bind to the so and eya promoters, there must be an inhibitory signal within the eye disc that prevents the transcription of these genes from being induced. This repression is first released for eya transcription because it is localized to the first instar eye disc. The inhibition upon the remaining genes is released during the second larval instar. Unraveling this mystery will certainly require extensive molecular and biochemical analysis on embryonic and early larval eye discs (Kumar, 2001b).

Are the earliest expression patterns of these eye specification genes homologous between vertebrates and invertebrates? This is certainly a much more difficult question to answer. A decade ago this question would be easily answered with a resounding 'no'. Now as more molecular and physiological similarities between the visual systems of vertebrates and invertebrates are being discovered, the answer to this developmental question may not be as easily or as negatively answered. It would be truly remarkable if a common developmental history underlies the use of identical molecules to create the different types of eyes seen throughout the animal kingdom. The key to such questions may lie in the precise fate mapping of individual cells that express each of the genes responsible for eye specification (Kumar, 2001b).

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).

eyeless (ey), which encodes a member of the Pax6 family of transcription factors, is a crucial regulator for eye development. It is expressed in the embryonic ventral nerve cord and brain and has been shown to be involved in the development of the mushroom bodies. Although it has been suggested that ey is expressed in the progenitor cells of the mushroom bodies, ey expression in the evolving NB pattern of the ventral nerve cord and brain has not been described to date. Using an Ey antibody (which principally shows the same pattern as ey mRNA in situ hybridization), Ey in the trunk is found to be expressed in segmentally reiterated ectodermal stripes and, at stage 11, in six NBs per hemineuromere. In the posterior pregnathal head segments (intercalary and antennal), a segmental expression of Ey appears to be fundamentally conserved. Ey/En double labelling reveals that the Ey-positive spots (and emerging NBs) in both segments are localized just anterior to their En-positive counterparts. Ey protein is detected, by stage 9, in five NBs: Pcv6, Pcv7, Pcv9, Pcd2 (which derive from an ocular ectodermal Ey domain) and Dv6 (which develops from a small ectodermal Ey spot in the antennal segment). By stage 10, ey becomes expressed in an intercalary ectodermal spot, from which, by that stage, the Ey-positive Td2 and (slightly later) Td1 delaminate, and in a second ocular ectodermal spot from which Ppd12 develops. In contrast to the intercalary and antennal ectodermal spot, Ey protein becomes depleted during stage 11 in the ocular domain. At late stage 11, Ey is found in 15 brain NBs, including two intercalary, three deutocerebral, nine ocular and one labral NB (Pad7). During subsequent development of the brain, the Ey expression pattern becomes complex, especially in the preantennal segments, but it appears to be mainly confined to the progeny of the identified NBs (Urbach, 2003).

The second Drosophila Pax6 gene, twin of eyeless (toy), has been shown to be expressed in the blastoderm in an anterodorsal patch that represents the posterior region of the procephalon anlage, and later in the embryonic brain (including the mushroom body and visual system). At stage 9, Toy expression encompasses the dorsal ocular and the anterodorsal part of the antennal ectoderm. All NBs that delaminate from this part of the neuroectoderm express Toy. By stage 11, these include about 40 protocerebral NBs, all of which derive from the ocular ectoderm, except the putative labral Pav1, and two deutocerebral NBs. This pattern of expression in NBs closely matches the pattern that has been observed for otd. In some of these NBs expression of Toy protein is transient and ceases during stage 11 (e.g., Dd3). To determine whether both Pax6 genes, ey and toy, are co-expressed in identified NBs, toy in situ hybridization was performed combined with an Ey antibody staining. Interestingly, toy appears to be expressed only in those Ey-positive NBs deriving from the ocular segment; a co-expression was not seen in Ey-positive NBs of the trito- and deuto-cerebrum, or in Pad7. During later embryogenesis, Toy protein is expressed in cells of the proto- and deutocerebrum, as well as in the tritocerebrum, although no Toy-positive tritocerebral NBs were identified (Urbach, 2003).

In order to establish further molecular markers that are specifically expressed in subsets of brain NBs, the expression of castor, Fasciclin 2, klumpfuss, ladybird early, POU-domain 1 gene, runt and unplugged were investigated for details of the spatiotemporal expression pattern of these genes in the neuroectoderm and brain NBs (stages 9, 10 and 11) (Urbach, 2003).

The origin of islet-like cells in Drosophila identifies parallels to the vertebrate endocrine axis

Single-cell resolution lineage information is a critical key to understanding how the states of gene regulatory networks respond to cell interactions and thereby establish distinct cell fates. This study identified a single pair of neural stem cells (neuroblasts) as progenitors of the brain insulin-producing neurosecretory cells of Drosophila, which are homologous to islet β cells. Likewise, a second pair of neuroblasts was identified as progenitors of the neurosecretory Corpora cardiaca cells, which are homologous to the glucagon-secreting islet α cells. Both progenitors originate as neighboring cells from anterior neuroectoderm, which expresses genes orthologous to those expressed in the vertebrate adenohypophyseal placode, the source of endocrine anterior pituitary and neurosecretory hypothalamic cells. This ontogenic-molecular concordance suggests that a rudimentary brain endocrine axis was present in the common ancestor of humans and flies, where it orchestrated the islet-like endocrine functions of insulin and glucagon biology (Wang, 2007).

The principal insulin producing-cells (IPCs) in higher metazoans, such as flies and mammals, direct organismal growth, metabolism, aging, and reproduction via a conserved signal transduction pathway. Gut- or pancreas-based IPCs, with endodermal origin, emerged as the principal IPC locus with the evolution of lower vertebrates such as the jawless fish. In contrast, the principal IPCs of invertebrates are found in the nervous system and are likely of ectodermal origin. Despite this difference, the possibility that gene regulatory modules may be conserved for cell fate programming the principal IPCs of all higher animals, irrespective of germ layer origin, has led the development of islet-like cells to be addressed in Drosophila (Wang, 2007).

Brain IPCs in Drosophila were first recognized by their expression of insulin (Drosophila insulin-like peptide, Dilp2) at the end of embryonic development. The goal of this work was to understand the developmental origin of these cells. The absence of morphological and vital markers for identifying brain neuroblasts for dye-labeled lineage tracing necessitated the combined use of mosaic analysis to demonstrate lineage relationships and immunohistology to follow cell identities. In this study, 16 molecular lineage markers corresponding to conserved genes were used to follow cells in fixed embryos. To identify genes involved in early IPC lineage development, before the differentiation of IPCs, 650 transposable GAL4-transgene insertions, obtained from public collections, that reported gene enhancer activity (GAL4 enhancer traps) in the CNS, were screened. Enhancer-driven GAL4 activity was used to trigger heritable and irreversible lineage labeling, which was assayed for coexpression with Dilp2 in late larval brains, thereby identifying lineage markers and potential developmental determinants. It was found that enhancers near the genes dachshund (dac), eyeless (ey), optix, and tiptop (tio) each triggered IPC lineage labeling by the time of Dilp2 expression onset just before hatching (late-stage 17). tio enhancer-triggered labeling was highly specific to the IPCs within the pars intercerebrallis (PI), the dorsomedial brain region harboring the IPCs and other neurosecretory cells. Antibody staining of Dac, Ey, and Optix proteins recapitulated enhancer reporter labeling and revealed expression in the tio⁺ cell cluster in late-stage embryos just after IPC differentiation, and before IPC differentiation at early-stage 17. Thus, a bilateral cluster of 10-12 Dac⁺ Ey⁺ cells were identified, 6-8 of which expressed tio before continuing on to express insulin (Dilp2) slightly later in development (Wang, 2007).

The hypothesis was tested that the Dac⁺ Ey⁺ cluster is generated by the proliferation of a single neuroblast. The pre-Dilp2 Dac⁺ Ey⁺ cluster comprised 10-12 cells at stage 17, but only a single Dac⁺ cell at stage 12, suggesting that a lineage expanded from a single progenitor beginning at stage 12. The Dac⁺ cluster maintains a posterior and lateral position within the anterior PI, identified by dChx1 expression, which allows following it during the morphogenetic changes in the developing brain. To mark progenitors and their lineage descendants, stage 11-12 embryos harboring both a heat-shock promoter-flip recombinase (hsp70-flp) transgene and an FRT-mediated flip-out Actin promoter-LacZ reporter were heat-shocked to induce random clone marking events in cell lineages. After aging embryos for 6 h at 25°C to reach stage 16-17, marked clusters of clonally related cells were occasionally recovered that comprised the 10-12 cell Dac⁺ Ey⁺ cluster. Clones that partly labeled the Dac⁺ Ey⁺ cluster, which were posterior in the cluster, were interpreted as being labeled by a lineage marking event induced after the neuroblast had divided one or more times. It was unlikely that multiple marking events accounted for the apparent clonal labeling of IPCs because the frequency of marked clone induction was extremely low (tens per brain). Clones were also found that labeled neighboring cells, but do not label Dac⁺ Ey⁺ cells, suggesting there is a lineage restriction that defined the Dac⁺ Ey⁺ cluster. Thus, all data are consistent with a lineage model whereby one neuroblast produced 10-12 Dac⁺ Ey⁺ cells, 6-8 of which were IPCs (Wang, 2007).

Whether the single Dac⁺ cell progenitor of IPCs seen at stage 12 was indeed a neuroblast was further tested by using markers of neuroblast lineage development. Asymmetrically dividing neuroblasts can be identified by nuclear expression of the pan-neuroblast marker Deadpan (Dpn) and Prospero (Pros) localization to the plasma membrane. It was found that the single Dac⁺ cell expressed Dpn and also showed Pros localization at the plasma membrane, which indicated that it was a neuroblast. As the Dac⁺ cluster increased in cell number with age, it was found that Pros was present in the nucleus of Dac⁺ cells anterior to the Dac⁺ neuroblast, which indicated that these were the neuroblast daughter cells, or ganglion mother cells (GMCs) generated by asymmetric neuroblast divisions. By stage 14, the most anterior Dac⁺ cells in the cluster lacked Dpn and Pros, suggesting that they were early, undifferentiated neurons or neurosecretory cells generated by GMC cell divisions. It was also found that tio expression occurs in the most anterior Dac⁺ cells of the lineage group, furthest from the posterior-located Dac⁺ neuroblast, suggesting that the six to eight IPCs are the products of the first three to four GMCs to be generated by asymmetric neuroblast division. This observation confirmed the interpretation of the marked clone data that showed partial labeling by a clone occupies the posterior, more recently formed region of the Dac⁺ Ey⁺ cluster, near the IPC neuroblast. Thus, a histological pattern of cell identities and divisions within the Dac⁺ IPC lineage group was observed that was consistent with the generic lineage development of a single neuroblast, with the IPCs being produced from the first three to four GMCs formed (Wang, 2007).

Further attempts were made to identify the precise origin of the IPC neuroblast within the neuroectoderm epithelium and the blastoderm embryo to place this lineage in the context of early axial patterning. The IPC neuroblast was first recognized by Dac expression only after neuroblast formation, but before its first division. However, preceding the formation of the IPC neuroblast, the markers Castor (Cas) and dChx1 and the proneural factor Lethal of Scute (L'Sc) showed coexpression in eight nearby cells of the neuroectoderm epithelium. Cas and dChx1 were maintained in all neuroblast lineages that delaminated from this group, as indicated by coexpression of Dpn. The IPC neuroblast was the only neuroblast from this group to express Dac, and it was always the first Dpn⁺ neuroblast to delaminate, becoming the most posterior in a chain of delaminating Cas⁺ dChx1⁺ neuroblasts. The Cas⁺ dChx1⁺ L'Sc⁺ proneural group lies within a 'gap gene' head stripe corresponding to the Bicoid responsive giant head stripe 1 (gt1), which suggested that the IPC neuroblast, or its earliest progenitor, arose from this pattern element of the precellular blastoderm (Wang, 2007).

β Cell and α cell development in mammals shares a largely common pathway. Thus attempts were made to study the origin of the α-like cells in Drosophila and their development relative to the IPC lineage. Corpora cardiaca (CC) cells are analogous in function to islet α cells. These neuroendocrine cells reside in the endocrine ring gland, just dorsal to the brain. CC cells produce and secrete a glucagon-like peptide, adipokinetic hormone, in response to circulating glucose levels, via a conserved K_atp sensor. The gene glass (gl) is a marker of CC cells and their precursors that specifically labels the CC lineage beginning at stage 10. The Gl⁺ group of cells expands in number to form a bilateral pair of six to eight cell clusters, aligned at the border of the brain and the developing foregut (stage 13). The Gl⁺ clusters then migrated out of the protocerebrum (stage 14), and posterior along the roof of the pharynx, to ultimately coalesce at the midline within the prospective ring gland (stage 16). Remarkably, the first Gl⁺ cells appeared a single cell diameter apart from the dChx1⁺ cluster containing the IPC neuroblast, also within the gt1 stripe (Wang, 2007).

These results suggested that the CC cell lineage, like the IPC lineage, is also generated from a progenitor within the gt1⁺ dorsal neuroectoderm. Indeed, a neuroblast progenitor for CC cells was suggested by expression of a Kruppel reporter (Kr-GFP) found to specifically label the Gl⁺ cells and an adjacent cell that both was Dpn⁺ and showed membrane localized Pros, indicating that it was a neuroblast. As for IPCs, tests were made to see if CC cells are derived from a single progenitor, perhaps the Kr-GFP⁺ neuroblast. Gl⁺ β-gal⁺-marked clones were recovered that comprised all or part of a CC cell cluster, after their migration to the prospective ring gland at stage 16. Because labeled CC cells had moved from their point of origin in the developing PI, it could not be determine whether a progenitor also produced other cells besides the CC cells, which did not similarly migrate. Together, these observations suggest that the CC cells are related by lineage to a neuroblast progenitor (Wang, 2007).

Typically, neuroblasts inherit the expression of cell specification factors from their point of origin in the patterned neuroectoderm before the neuroblast forms. It was found that this was the case with the IPC neuroblast, which retains dChx1 and Cas expression from the neuroectoderm. It was therefore hypothesized that this may also be the case for the CC cell neuroblast. CC cell specification was shown to require the function of gt, sine oculis (so), twist (twi), and snail (sna). Indeed, it was found that all of these factors are expressed in the Gl⁺ CC cell lineage. Moreover, the Kr-GFP⁺ cell group, containing the neuroblast and CC cell precursors, also expressed Eyes absent (Eya), the cognate protein tyrosine phosphatase of So. It was subsequently found that at stage 10, the time that Gl⁺ cells are first detected, a region of gt1⁺ neurectoderm shows expression of So. It was also found that one to two So⁺ gt1⁺ neuroblasts can be detected by labeling with Dpn at this stage. Thus, it is proposed that the So⁺ Eya⁺ gt1⁺ neuroectoderm gives rise to the Kr-GFP⁺ So⁺ Eya⁺ gt1⁺ neuroblast, which is the single progenitor of the CC cells (Wang, 2007).

The model of a dorsal neurectoderm origin for CC cells is in disagreement with another extant model. The anterior ventral furrow (AVF) epithelium was suggested to be the CC cell origin based on gene expression and function studies implicating So, Gt, Twi, and Sna in CC cell formation. To distinguish between the AVF and dorsal neuroectoderm as possible origins of CC cells, two newly available gt promoter fragment reporters were used whose expression persists late enough in development, beyond endogenous protein and transcript expression, to serve as a coarse-grain lineage marker of CC cells. The AVF is marked by the gt23 reporter, whose expression is limited to the two gt head stripes posterior to gt1 at the blastoderm stage. This reporter does not label the Gl⁺ cells. However, as has been shown, the Gl⁺ cells arise in the context of the most anterior gt head stripe, gt1, which reaffirms the proposed origin from the gt1⁺ neuroectoderm (Wang, 2007).

The organization of this gt1⁺ segment-derived proendocrine neuroectoderm was investigated with respect to the conserved factors Optix, So, Eya, and dChx1. Optix and Eya expression aligned with the gt1 reporter expression domain. The D-six4 gene also shows expression specific to this domain. Labeling studies showed that this domain is subdivided into several small compartments of 2-12 cells with discrete gene expression profiles. The data indicate that the IPC neuroblast was derived from compartment B (Optix⁺, dChx1⁺, Cas⁺, So^-, low-level Eya) and the CC cell neuroblast arose from the adjacent compartment C (Optix⁺, So⁺, Eya⁺, dChx1^-). This somewhat surprising finding suggests that the largely common developmental pathway of β and α cells may be partly conserved in Drosophila, perhaps with respect to a domain of Sine oculis/Six family and Eya gene expression (Wang, 2007).

The early expression of the mouse ortholog of the Drosophila homeodomain gene optix, Six6, demarcates the hypophyseal placode and infundibular region, which give rise to the anterior pituitary and neurosecretory hypothalamus, respectively. Mutation of the Six6 gene leads to reduction of the pituitary in mice and humans. The hypophyseal placode and adjacent ectoderm also expresses the other so-called 'placode genes,' Six1, Six4, and Eya, and this coexpression pattern is conserved in amphibians, fish, and lower chordates such as ascidians. In mice, the anterior pituitary is reduced in size in the double mutant of Eya1 and Six1, and in zebrafish, Eya1 is essential for differentiation of all pituitary cell types except for prolactin-expressing cells. In Drosophila, So and Eya are essential for CC cell formation. Thus, there is a striking conservation of the molecular signature of tissues that give rise to elements of the brain endocrine axis in flies, mammals, lower vertebrates, and lower chordates (Wang, 2007).

There are also parallels between vertebrate and fly with respect to tissue morphogenesis within the developing brain endocrine system and adjacent oral ectoderm, although there appears to be considerable variation on a general theme. For example, in mouse, the progenitors of the anterior pituitary and neurosecretory hypothalamus appear to arise respectively from Rathke's pouch, an invagination of the oral ectoderm, and the neurectoderm, which do not start as neighboring regions, but come into direct contact only after neurulation. However, in the zebrafish, which does not form a Rathke's pouch, the progenitors of the anterior pituitary and neurosecretory hypothalamic cells (GnRH1⁺) arise from neighboring regions of the hypohyseal placode, which is situated directly dorsal to the stomodeal ectoderm. In Drosophila, the ventral cells of the gt1⁺ Optix⁺ Eya⁺ ectoderm invaginate to form the roof of the pharynx, the fly's oral ectoderm, whereas the dorsal cells contribute to the endocrine axis. Therefore, there is considerable evidence for evolutionarily conservation of the close relationship between the oral ectoderm and the developing compartments of the endocrine axis, all of which express the hypophyseal placode genes. The gene expression profile and specification of endocrine cell functions from the anterior ectoderm appears to be more 'fixed' across the bilateria, whereas the pattern of accompanying tissue morphogenesis and diversity of cell types is more variable, just as has been demonstrated for the specification of the bilaterian CNS, eye, gut, and heart (Wang, 2007).

The model proposed in this study contrasts with the prior suggestion, based on the proximity of developing CC cells to the posterior foregut in the moth, Manduca, that CC cells originate from neurogenic placodes of the foregut that engender the stomatogastric nervous system. Because CC cell progenitors were not identified in those studies, and subsequent mutational analysis in Drosophila demonstrated that the CC cells develop independently of the stomatogastric nervous system and posterior foregut, it is suggested that the current model of CC cell origin is the most strongly supported (Wang, 2007).

It is proposed that the brain endocrine systems of invertebrates and vertebrates are derived from a common ancestry because they both develop from a domain of Eya and sine oculis/Six family gene expression that comprises the anterior neuroectoderm and adjacent oral ectoderm. Indeed, these results extend prior observations that the neurosecretory cells of the PI and ring gland show other aspects of homology to the hypothalamic-pituitary axis. The specification of islet-like cells within a conserved brain endocrine axis raises the intriguing possibility that islet organogenesis, which is a derived feature of vertebrates, may have coopted brain endocrine cis-regulatory modules for specification of islet fates in endoderm. Indeed, the ectopic expression of the nominal rat insulin promoter reporter in anterior pituitary and hypothalamus underscores the similar gene regulatory state of these endocrine tissues. It is expected that further genetic analysis of endocrine cell fate specification within the gt1 domain of Drosophila will lead to insights into the patterning and organogenesis of endocrine compartments and provide the basis for identifying conserved pan-IPC regulatory modules with relevance to mammalian systems (Wang, 2007).

Larval

eyeless transcripts are confined to eye imaginal discs and are not detected in leg or wing discs. The transcripts accumulate at the anterior of the eye disc, and extend posteriorly to the morphogenetic furrow and a few cells beyond the furrow. The Eyeless transcripts also accumulate in parts of the brain and the ventral nervous system, as well as in the salivary glands (Quiring, 1994).

The Drosophila compound eye is specified by the concerted action of seven nuclear factors: Twin of eyeless (Toy), Eyeless (Ey), Eyes absent (Eya), Sine oculis (So), Dachshund (Dac), Eye gone (Eyg), and Optix (Opt). These factors have been called 'master control' proteins because loss-of-function mutants lack eyes and ectopic expression can direct ectopic eye development. However, inactivation of these genes does not cause the presumptive eye to change identity. Surprisingly, several of these eye specification genes are not coexpressed in the same embryonic cells -- or even in the presumptive eye. Surprisingly, the EGF Receptor and Notch signaling pathways have homeotic functions that are genetically upstream of the eye specification genes; specification occurs much later than previously thought -- not during embryonic development but in the second larval stage (Kumar, 2001a).

Do Egfr and Notch Act upstream of the eye specification genes? A molecular epistasy study was undertaken, examining the expression of some of the eye and antennal specification genes in the transforming conditions during the third larval stage (before cell types differentiate). In eye specification gene mutants (such as ey), ommatidial development is blocked, but the eye disc remains in a reduced form. Conditions that produce eye to antenna transformations, whether through hyperactivation of Egfr or downregulation of Notch signaling, show a complete replacement of the eye disc with an antenna disc. Distal-less (Dll) and Spalt-Major are normally expressed within subdomains of the antenna disc and are required for antenna development. Dll and SalM are expressed in the correct locations in the transformed antenna disc suggesting that both endogenous and transformed antenna are also both morphologically and molecularly equivalent (Kumar, 2001a).

The transcription of five of the seven known eye specification genes (toy, ey, eya, so, and eyg) was examined. In transforming conditions, transcription levels of all five of the seven genes are below the levels of detection. This is consistent with both Egfr and Notch signaling acting genetically upstream to both the eye and antennal specification genes. The downregulation of ey suggests that the ey-GAL4 driver may also be downregulated via an autoregulatory mechanism. That the transformation occurs despite this may reflect a phenocritical period for the eye-antenna transformation; once the transformation has occurred the system is refractory to the loss of Egfr signaling (Kumar, 2001a).

When and where are the eye and antenna specified? The seven known eye specification genes are thought to act in a genetic and biochemical complex; by pairwise tests, their products have been shown to either directly regulate each other's transcription or to interact at the protein level, or both. From the few published reports of the early expression patterns of eye specification genes and from fate mapping experiments, it has been suggested that eye versus antennal fate specification occurs during the latter stages of embryogenesis. These concepts lead to a straightforward hypothesis: at some point in the developing embryo, the seven eye specification genes' products are coexpressed in the presumptive eye and act to specify its fate. A similar event (with the action of different genes) also specifies the antenna (Kumar, 2001a).

If this hypothesis is true, then three predictions should hold: (1) At some time during embryonic development, there should be two domains of expression of the eye specification genes that correspond to the future eyes, and anterior to these, should be two domains of antenna specification gene expression marking and acting to direct antenna fate. These gene products should be specific to the future structures they mark, and should not be found elsewhere. (2) The eye specification genes should be coexpressed in the same cells. This is known to be true of toy, ey, and eyg. (3) The phenocritical period for the eye to antenna transforming function of Egfr and Notch pathway signaling should be coincident with, or earlier than, the time at which the eye and antenna specification genes are first specifically coexpressed. All three of these predictions were tested (Kumar, 2001a).

To test the first prediction above, embryos were collected (at 1 hr intervals from 1 to 16 hr after egg deposition, AED) and analyzed for expression of the canonical eye specification gene ey (Pax6) and the antenna specification protein Dll. Dll is first detected at 7 hr in the leg imaginal disc primordia and in several segments in the embryonic head. ey transcription in the eye imaginal disc is first detectable at 11 hr while Dll is seen in an adjacent region as well as other sites. In latter stages of embryogenesis, the eye imaginal disc invaginates and assumes a more dorsal-medial position within the embryonic head, just above the developing embryonic brain. Regions of ey expression are observed that correspond to this. Furthermore, this ey expression corresponds to domains of Escargot expression (Esg), a general imaginal disc marker. However, Dll expression is more anterior and it is not clear if these sites correspond to the presumptive antennae. It thus appears that ey is expressed in both the presumptive eye and antenna by 13 hr and remains there through the last embryonic time point observed, and that Dll is not expressed in the future antenna at any embryonic time. It is also quite clear that ey is expressed in many sites in the embryo that will never form eye (such as the segmental grooves). In short, the position of the presumptive eye or antenna during embryonic development cannot be distinguish based on the specific expression of their respective 'master control' genes—neither ey nor Dll expression are sufficient to specify the eye or the antenna; therefore, prediction 1 (above) does not hold true (Kumar, 2001a).

Are the eye specification genes coexpressed during embryonic development? The expression pattern of Eya and Dac proteins and so transcription were examined at 1 hr time points (from 1 to 16 hr AED) and it was found that none of these three eye specification genes is coexpressed with ey within the presumptive eye. The fact that these genes are not expressed within the same cells during embryonic development precludes any possibility that their products act in a multiprotein complex critical for eye specification in the embryo and, thus, prediction 2 (above) does not hold true either. However, eye specification might occur later in development (Kumar, 2001a).

The eye specification genes are first coexpressed in the second larval stage. In second stage larva, the eye specification gene products are completely segregated into the eye portion of eye-antennal disc, but the antennal marker Dll is evenly expressed in both the eye and antennal segments. Interestingly, the expression patterns of the eye specification genes are still not completely overlapping. For instance, toy appears to be expressed throughout the entire eye field while both eya and dac are expressed just in the posterior portions of the eye disc. In the third larval stage, the eye specification genes remain within the eye portion and Dll is now segregated to just the antennal segments (Kumar, 2001a).

The phenocritical period for eye to antenna transformation is in the second larval stage. Use was made of the cold sensitivity of the GAL4 protein to determine the phenocritical period. GAL4 is a yeast protein and is fully functional at 25°C but is less active at 18°C. Flies of the ey-GAL4/UAS-Ser^DN genotype were raised at 18°C, shifted to 25°C for a consecutive series of 24 hr periods, and then returned to 18°C until late third instar imaginal discs could be examined. The use of the dominant negative Ser construct in this experiment effectively eliminates Notch pathway function in the developing eye. Indistinguishable transformations were observed in other experiments with constructs that either hyperactivate Egfr pathway signaling or inactivate Notch. The developing eye-antennal complex is completely normal if kept continuously at 18°C (negative control) while constant exposure to 25°C temperatures resulted in the eye to antenna transformation (positive control). These controls confirm that the cold sensitivity of GAL4 protein activity is sufficient to control the transformation (Kumar, 2001a).

Temperature shifts during the embryonic and first larval stages failed to induce any effects. The eye-antennal discs are completely normal. This is consistent with the expression data suggesting that the eye is not specified during embryogenesis. A temperature shift during the first half of the second larval stage results in a reduced eye field, but no transformation to antenna. Interestingly, this phenotype is similar to that seen in ey mutant homozygotes. The eye to antenna transformation is fully induced in all cases when the temperature shift occurs during the latter half of the second larval stage. The transformed antenna expresses Dll in an identical pattern as seen in the endogenous antenna. Subsequent temperature shifts during the earliest phase of the third larval stage do not result in a complete transformation. Interestingly, loss of Notch during the next day of the third larval stage results in defects in the regulation of the morphogenetic furrow. These results clearly indicate that the phenocritical period is chiefly within the latter half of the second larval stage. This phenocritical period does not predate the expression of any of the eye specification genes, but it is coincident with their first coexpression and, thus, prediction 3 (above) holds true (Kumar, 2001a).

Is the presumptive eye actually transformed into a second antenna under these conditions, or does the eye degenerate and get replaced by regrowth from elsewhere? The former interpretation (transformation) is favored because in hundreds of transformed L2 disc complexes dissected, a degenerating eye disc, or a small (presumably regrowing) antennal disc was never observed. In all cases, the transformed antenna is equal in size to the normal one. Indeed, both are somewhat larger than normal. This might suggest that a fixed number of cells that normally distribute preferentially to the eye are now equally allocated to both antennae (Kumar, 2001a).

The Decapentaplegic and Notch signaling pathways are thought to direct regional specification in the Drosophila eye-antennal epithelium by controlling the expression of selector genes for the eye (Eyeless/Pax6, Eyes absent) and/or antenna (Distal-less). The function of these signaling pathways in this process has been investigated. Organ primordia formation is indeed controlled at the level of Decapentaplegic expression but critical steps in regional specification occur earlier than previously proposed. Contrary to previous findings, Notch does not specify eye field identity by promoting Eyeless expression but it influences eye primordium formation through its control of proliferation. Analysis of Notch function reveals an important connection between proliferation, field size, and regional specification. It is proposed that field size modulates the interaction between the Decapentaplegic and Wingless pathways, thereby linking proliferation and patterning in eye primordium development (Kenyon, 2003).

This paper analyzes the role of Dpp and Notch in the regional specification of the eye-antennal disc. This study makes four observations: (1) domains of regional identity emerge in a complex pattern starting early in L2; (2) formation of eye and antenna primordia depend upon specific domains of dpp expression that emerge in early-L2 (eye) and mid-L2 (antenna); (3) neither Notch nor Dpp control the establishment of separate eye and antennal fields; (4) Notch can influence the establishment of an eye primordium through its control of proliferation in the eye field. Current models of regional specification have been evaluated based on these results and a new perspective on the emergence of regional identity in this tissue is presented (Kenyon, 2003).

It has been proposed that allocation of eye field and antennal field identity occurs in the latter half of L2 through the restriction of eye selectors, such as Ey, and antennal selectors, such as Dll, to distinct regions of the disc. However, two observations reported in this paper are not consistent with this interpretation: (1) Dll is not expressed ubiquitously at any time during disc development; (2) eye and antennal fields are clearly established by mid-L2 as evidenced by the restricted expression of Ey (eye field) and Cut (antennal field), and by distinct Dpp/Wg patterning centers within each field. These observations place the emergence of separate eye and antennal fields in the first half of L2 and not in the second half as previously proposed. Moreover, onset of Eya occurs in early-L2 and so is expressed by mid-L2. The beginning of eye primordium formation in early-L2, prior to the appearance of distinct fields, indicates that regional specification within this disc does not follow a two-step mechanism (i.e., establishment of separate fields followed by induction of organ primordia) but occurs in a more complex pattern. Further analysis of the transcription factors and signaling molecules active in the late-L1 and early-L2 disc is necessary to better understand how the establishment of eye field identity relates to eye primordium formation and the emergence of an antennal field (Kenyon, 2003).

Analyses of hypomorphic dpp alleles and tissue mutant for Mad implicate Dpp in the control of eya and Dll expression during late larval development. The onset of Eya and Dll expression correlate with specific changes in dpp expression during normal development. Using temperature shift experiments, it has also been established that Dpp signaling in L2 is required for the proper induction of both Eya and Dll in their respective fields. Gain-of-function analyses show that Dpp is also sufficient to induce Eya expression within the eye field and Dll expression within the antennal field. Clearly, though, Dpp must function in the context of selector factors such as Ey in order to produce two independent primordia within the eye-antennal epithelium. In the presence of Ey, Dpp signaling induces Eya expression as opposed to Dll. The absence of Ey in the antennal field at the time of Dpp signaling is of crucial importance to ensure the proper induction of Dll and subsequent formation of an antenna primordium. Indeed, as described in this study, the restriction of Ey to the eye field precedes the emergence of dpp and Dll in the antennal field during normal development (Kenyon, 2003).

In conclusion, Dpp, unlike Notch, functions as an inducer of tissue identity during specification of the eye-antennal disc, and the spatial and temporal aspect of organ primordia formation is controlled at the level of dpp transcription (Kenyon, 2003).

Notch is thought to function upstream of the Ey/Pax6 pathway in promoting eye field identity. Expression of Notch antagonists in the developing discs leads to adult flies lacking eyes and sometimes displaying double antennae; this latter phenotype has been interpreted as evidence for a transformation of the eye field into a second antennal field. However, eye and antennal selector gene expression is not perturbed in loss-of-function Notch mutant backgrounds (N^54/9, N^ts1, and N^ts2. Furthermore, expression of Ey and Dll is normally restricted to the eye field and the antennal field, respectively, in late-L2 Ser^DN or N^DN discs. These observations indicate that Notch does not control the specification of separate eye and antennal fields in L2 (Kenyon, 2003).

The occasional appearance of double antennae in flies expressing Ser^DN and N^DN results from a duplication of the antenna primordium rather than an eye-to-antenna transformation. Antenna duplications are frequently observed after surgical removal of eye tissue and have also been observed in mutant backgrounds that cause suppression of proliferation and/or cell death in the eye field. Thus, the formation of dual antennae in ey-Gal4 UAS-Ser^DN or ey-Gal4 UAS-N^DN discs is likely due to the extreme suppression of proliferation observed in these genetic backgrounds (Kenyon, 2003).

The proposal that Notch controls Ey in Drosophila has been extended to the control of Pax6 in vertebrates. Although it is concluded that Notch does not regulate the Ey/Pax6 pathway in the fly, Notch does influence regional specification through its control of proliferation. Hence, the interactions uncovered in vertebrates between Notch signaling and the Ey/Pax6 pathway may reflect a nonconserved aspect of eye development or alternatively an indirect relationship between Pax6 and Notch (Kenyon, 2003).

Although not a direct inducer of regional identity, Notch function is essential for the emergence of an eye primordium. In N^ts1and N^ts2 discs, onset of Eya expression is delayed. In ey-Gal4 UAS-Ser^DN discs, Eya expression is always severely affected and most often completely absent. Yet, in these discs, dpp is still expressed; hence lack of Eya expression is not due to the absence of this inducer. The observation that stimulating cell division is sufficient to rescue eye primordium formation in ey-Gal4 UAS-Ser^DN discs indicates that Eya induction depends upon cell proliferation or field size rather than Notch signaling per se. Since the normal onset of Eya expression in early-L2 does not correlate with the initiation of cell proliferation at mid-late-L1 and the stimulation of proliferation by constitutive Notch signaling (N^Intra) is not sufficient to directly induce Eya, the latter explanation, namely that Eya expression is influenced by the size of the morphogenetic field as opposed to cell division, is preferred (Kenyon, 2003).

In conclusion, this analysis of the effect of Notch on regional identity at the L2 stage reveals an important connection between proliferation, field size, and eye primordium formation. Through its control of proliferation in the eye field, Notch may indeed participate in generating the specific signaling environment that promotes the expression of Eya and the emergence of an eye primordium. Control of field size likely reflects a general mechanism through which cell proliferation can influence the patterning of a morphogenetic field and thereby contribute to the coordination of proliferation and patterning essential to the proper development of complex multicellular organisms (Kenyon, 2003).

eyg transcripts are distributed within several embryonic tissues as well as the leg, wing and eye-antennal imaginal discs. eyg and toe transcripts are first detected in stage 9 embryos within the salivary gland precursor (SGP) and a small cluster of cells within the dorsal head. The expression of toe transcripts in the SGP will persist through the rest of embryonic and larval development while eyg expression is terminated in late stage embryos and reinitiated later. By late stage 10 both transcripts are also found in identical patterns within the posterior spiracle (PS) and within a cluster of cells at the anterior edge of each thoracic and abdominal segment. Expression of eyg and toe expands to the larval antennal organ (AO) as well as the leg disc primordia by stage 12. During the latter stages of embryogenesis both eyg and toe transcripts accumulate in the presumptive eye-antennal imaginal disc. Only two other members of the eye specification cascade, ey and toy, share this expression pattern. The remaining members are added sequentially during the larval development. The only discernable difference between the expression patterns of either Pax6(5a) gene during embryogenesis is found within the SG: eyg expression is eliminated while toe transcriptional levels are maintained (Yao, 2008).

Within the developing larval eye-antennal discs both eyg and toe transcripts accumulate in identical patterns. Within the antennal segment both transcripts localize to the medial and distal segments while in the eye disc expression of both genes is found anterior to the morphogenetic furrow. Unlike the similarities found in the embryo, eyg and toe expression is somewhat different from that of ey and toy. The Pax6 transcripts are expressed broadly ahead of the advancing furrow. However, eyg and toe expression is restricted to a narrow domain of cells that straddle the dorsal-ventral compartment boundary and does not extend laterally. This difference in expression is likely due to the requirements of eyg (and possibly toe) in Notch mediated control of cell proliferation at the organizing center versus the role of ey and toy in tissue specification. Within the developing wing primordium both transcripts are expressed broadly within the notum and in two discrete regions within the presumptive wing. It is interesting that one of those areas is particularly susceptible to being transformed into retinal tissue in response to forced expression of ey. Both eyg and toe transcripts are also found within identical patterns of the leg primordium and the anterior duct cells of the salivary gland. The results from this and other studies of eyg and toe expression suggest at first glance that these genes may play redundant roles within several developing tissues including the compound eye. It is unlikely, however, that these genes play completely surplus roles (at least in the eye) as eyg loss-of-function mutants show near complete loss of retinal tissue and forced expression of toe is insufficient to restore eye development to these flies (Yao, 2008).

Combinatorial temporal patterning in progenitors expands neural diversity

Human outer subventricular zone (OSVZ) neural progenitors and Drosophila type II neuroblasts both generate intermediate neural progenitors (INPs) that populate the adult cerebral cortex or central complex, respectively. It is unknown whether INPs simply expand or also diversify neural cell types. This study shows that Drosophila INPs sequentially generate distinct neural subtypes, that INPs sequentially express Dichaete, Grainy head and Eyeless transcription factors, and that these transcription factors are required for the production of distinct neural subtypes. Moreover, parental type II neuroblasts also sequentially express transcription factors and generate different neuronal/glial progeny over time, providing a second temporal identity axis. It is concluded that neuroblast and INP temporal patterning axes act together to generate increased neural diversity within the adult central complex; OSVZ progenitors may use similar mechanisms to increase neural diversity in the human brain (Bayraktar, 2013).

Tests were carried out to determine whether D, Grh and Ey exhibit cross-regulation in INPs. wor-gal4, ase-gal80 was used to drive UAS-D^RNAi in a Dichaete heterozygous background (subsequently called D RNAi, in which RNAi denotes RNA interference), which removed detectable D from INP lineages. Compared to wild type, D RNAi resulted in a significant loss of early born Grh⁺ Ey⁻ INPs, without altering the number of later-born Grh⁺ Ey⁺ INPs. The same result was observed in D mutant clones. By contrast, misexpression of D did not lead to ectopic Grh expression. Thus, D is necessary for the timely activation of Grh in INP lineages, although D-independent inputs also exist (Bayraktar, 2013).

To test whether Grh regulates D or Ey, R9D11-gal4 was used to drive UAS-grh^RNAi in a grh heterozygous background (subsequently called grh RNAi), which significantly reduced Grh levels in middle-aged INPs. grh RNAi increased the number of D⁺ INPs at the expense of Ey⁺ INPs without altering the total number of INPs. As expected, grh RNAi did not change the numbers of D⁺ and Ey⁺ INPs in the DM1 lineage, which lacks Grh expression, nor did misexpression of Grh lead to ectopic Ey expression. It is concluded that Grh represses D and activates Ey within INP lineages (Bayraktar, 2013).

To determine whether Ey regulates D or Grh, R12E09^D-gal4 UAS-FLP actin-FRT-stop-FRT-gal4 was used to drive permanent expression UAS-ey^RNAi within INPs (subsequently called R12E09^D) act-gal4 or INP-specific ey RNAi. It was confirmed that INP-specific ey RNAi removed Ey expression from INPs, without affecting Ey in the mushroom body or optic lobes. ey RNAi resulted in a notable increase in the number of old D^-Grh⁺ INPs, without affecting the number of young D⁺ INPs. Conversely, Ey misexpression in INPs significantly reduced the number of Grh⁺ INPs without altering the total number of INPs. An increase was observed in D⁺ INPs, consistent with a regulatory hierarchy in which Ey represses Grh, which represses D. This effect was not due to ectopic Ey directly activating D because misexpression of Ey had no effect on D⁺ INP numbers in the DM1 lineage, which lacks Grh expression. It is concluded that Ey is necessary and sufficient to terminate the Grh expression window in INPs. A 'feedforward activation/feedback repression' model is proposed for D-to-Grh-to-Ey cross-regulation (Bayraktar, 2013).

Next, it was asked whether distinct neuronal or glial subtypes were generated during each transcription factor expression window. To determine the cell types produced by young D⁺ INPs or old Ey⁺ INPs, permanent lineage tracing was used. Cells labelled by R12E09^D but not OK107^ey are generated by young INPs, whereas cells labelled by OK107^ey are generated by old INPs. A collection of 60 transcription factor antibodies was screened and two were found that labelled subsets of young INP progeny, and two that labelled subsets of old INP progeny. The transcription factors D and Brain-specific homeobox (Bsh) labelled sparse, non-overlapping subsets of young INP progeny, but not old INP progeny. Thus, young INPs generate Bsh⁺ neurons, D⁺ neurons, and many neurons that express neither gene. By contrast, the glial transcription factor Reverse polarity (Repo) and the neuronal transcription factor Twin of eyeless (Toy) labelled sparse, non-overlapping subsets of old INP progeny, but not young INP progeny. Additional mechanisms must restrict each marker (D, Bsh, Repo and Toy) to small subsets of young or old INP progeny; for example, each population could arise from just early or late born INPs within a type II neuroblast lineage. It is concluded that INPs sequentially express the D, Grh and Ey transcription factors, and they generate distinct neuronal and glial cell types during successive transcription factor expression windows. These data provide the first evidence in any organism that INPs undergo temporal patterning (Bayraktar, 2013).

Experiments were designed to determine whether D, Grh and Ey act as temporal identity factors that specify the identity of INP progeny born during their window of expression. First, the role of Ey in the specification of late born INP progeny was investigated. INP-specific ey RNAi resulted in the complete loss of the late born Toy⁺ neurons and Repo⁺ neuropil glia, but did not alter the number of early born D⁺ and Bsh⁺ neurons. Removal of Toy⁺ neurons (using toy RNAi) does not alter the number of Repo⁺ glia, and conversely removal of Repo⁺ glia (using gcm RNAi) does not alter the number of Toy⁺ neurons; thus Ey is independently required for the formation of both classes of late INP progeny. Conversely, permanent misexpression of Ey in early INPs increased late born Toy⁺ neurons and decreased early born Bsh⁺ neurons, consistent with Ey specifying late INP temporal identity. Unexpectedly, ectopic Ey reduced the number of late born Repo⁺ glia. Itis concluded that Ey is an INP temporal identity factor that promotes the independent specification of late born Toy⁺ neurons and Repo⁺ glia (Bayraktar, 2013).

Next tests were performed to see whether D and Grh specify early and mid INP temporal identity. INP-specific D RNAi led to a small but significant reduction in the number of early born Bsh⁺ neurons, whereas INP-specific grh RNAi severely reduced the number of early born Bsh⁺ neurons without impairing INP proliferation or late INP progeny. This is consistent with the Bsh⁺ neurons deriving from the D⁺ Grh⁺ expression window. Interestingly, misexpression of D or Grh did not increase Bsh⁺ neuron numbers; perhaps D/Grh co-misexpression is required to generate Bsh⁺ neurons. It is concluded that both D and Grh are required, but not sufficient, for the production of Bsh⁺ early INP progeny (Bayraktar, 2013).

The function of early or late born INP progeny in adult brain development is unknown. This study determined the role of late born INP neurons and glia in the development and function of the adult central complex (CCX), an evolutionarily conserved insect brain structure containing many type II neuroblast progeny. The CCX consists of four interconnected compartments at the protocerebrum midline: the ellipsoid body, the fan-shaped body, the bilaterally paired noduli, and the protocerebral bridge; each of these compartments is formed by a highly diverse set of neurons. First, permanent lineage tracing was used to map the contribution of late born Ey⁺ INP progeny to the adult CCX. Cell bodies were detected in the dorsoposterior region of the CCX, and their axonal projections extensively innervated the entire ellipsoid body, fan-shaped body, and protocerebral bridge, with much weaker labelling of the paired noduli. It is concluded that old INPs contribute neurons primarily to the ellipsoid body, fan-shaped bod and protocerebral bridge regions of the CCX. Second, INP-specific ey RNAi was used to delete the late born Toy⁺ neurons and Repo⁺ glia. Loss of late born INP progeny generated major neuroanatomical defects throughout the adult CCX: the ellipsoid body and paired noduli were no longer discernible, the fan-shaped body was enlarged, and the protocerebral bridge was fragmented. Subsets of this phenotype were observed after removal of Toy⁺ neurons or Repo⁺ glia, showing that they contribute to distinct aspects of the CCX. Previous studies have described similar or weaker morphological CCX defects in ey hypomorphs, toy mutants, and after broad glia ablation during larval stages. In addition, ey RNAi adults were found to have relatively normal locomotion, but have a significant deficit in negative geotaxis. It is concluded that Ey is a temporal identity factor that specifies late born neuron and glial identity, and that these late born neural cell types are essential for assembly of the adult central complex (Bayraktar, 2013).

Bsh⁺ neurons and Repo⁺ glia were found to be sparse within the total population of young and old INP progeny, respectively, indicating that other mechanisms must help to restrict the formation of these neural subtypes. One mechanism could be temporal patterning within type II neuroblast lineages (Bayraktar, 2013).

To determine whether type II neuroblasts change their transcriptional profiles over time, known temporal transcription factors were examined for expression in type II neuroblasts at five time points in their lineage (24, 48, 72, 96 and 120 h ALH). No type II neuroblast expression for Hunchback, Kruppel, Pdm1/2 and Broad, and Grh was expressed in all type II neuroblasts at all time points. However, three transcription factors were identified with temporal expression in type II neuroblasts. D and Castor (Cas) were specifically detected in early type II neuroblasts: 3-4 neuroblasts at 24 h ALH, 0-1 neuroblast at 48 h ALH, and none later. Although D was never detected simultaneously in all type II neuroblasts at 24 h, permanent lineage tracing with R12E09^D labels all type II neuroblasts, indicating that all transiently express D. The third transcription factor, Seven up (Svp), showed a pulse of expression in a subset of type II neuroblasts at 48 h ALH, but was typically absent from younger or older type II neuroblasts. D, Cas and Svp are all detected in the anterior-most type II neuroblasts (probably corresponding to DM1-DM3), and thus at least these type II neuroblasts must sequentially express D or Cas, and Svp. It is concluded that type II neuroblasts can change gene expression over time (Bayraktar, 2013).

Next, tests were performed to determine whether type II neuroblasts produce different INPs over time. Permanently labelled clones were generated within the type II neuroblast lineages at progressively later time points. If type II neuroblasts change over time to make different INPs, early and late neuroblast clones should contain different neural subtypes. Clones were assayed for Repo⁺ glia and Bsh⁺ neurons, choosing these markers because Repo⁺ neuropil glia have been proposed to be born early in type II neuroblast lineages and Bsh⁺ neurons were positioned far from the Repo⁺ glia consistent with a different birth-order. Bsh⁺ neuron numbers began to decline only in clones induced at the latest time point, showing that they are generated late in the type II neuroblast lineage. By contrast, Repo⁺ glia were detected in clones induced early but not lat. This allows assigning of Repo⁺ glia to an 'early neuroblast, old INP' portion of the lineage, and Bsh⁺ neurons to a 'late neuroblast, young INP' portion of the lineage. It is concluded that type II neuroblasts undergo temporal patterning, and neuroblast temporal patterning was proposed to act together with INP temporal patterning to increase neural diversity in the adult brain (Bayraktar, 2013).

This study has shown that INPs sequentially express three transcription factors (D, Grh and then Ey), and that different neural subtypes are generated from successive transcription factor windows. It is likely that multiple GMCs are born from each of the four known INP gene expression windows; GMCs born from a particular gene expression window may have the same identity, or may be further distinguished by 'subtemporal genes' as in embryonic type I neuroblast lineages. This study also showed that each temporal factor is required for the production of a distinct temporal neural subtype. Loss of D or Grh leads to the loss of Bsh⁺ neurons; loss of Ey leads to loss of Toy⁺ neurons and Repo⁺ glia, although the fate of the missing cells is unknown. An unexpected finding was that Ey limits the lifespan of INPs. Mechanisms that prevent INP de-differentiation have been characterized -- loss of the translational repressor Brain tumour (Brat) or the transcription factor Earmuff (Erm) causes INPs to de-differentiate into tumorigenic type II neuroblasts, but factors that terminate normal INP proliferation have never before been identified (Bayraktar, 2013).

The D-to-Grh-to-Ey INP temporal identity factors are all used in other contexts during Drosophila development. Many embryonic neuroblasts sequentially express D and Grh. Ey is expressed in mushroom body neuroblasts, and is required for development of the adult brain mushroom body. Interestingly, mammalian orthologues of D and Ey (SOX2 and PAX6, respectively) are expressed in neural progenitors, including OSVZ progenitor, but have not been tested for a role in temporal patterning (Bayraktar, 2013).

This study has shown that there are two axes of temporal patterning within type II neuroblast lineages: both neuroblasts and INPs change over time to make different neurons and glia, thereby expanding neural diversity. It will be important to investigate whether INPs generated by OSVZ neural stem cells undergo similar temporal patterning (perhaps using SOX2 and PAX6), and whether combinatorial temporal patterning contributes to the neuronal complexity of the human neocortex (Bayraktar, 2013).

Temporal patterning of Drosophila medulla neuroblasts controls neural fates

In the Drosophila optic lobes, the medulla processes visual information coming from inner photoreceptors R7 and R8 and from lamina neurons. It contains approximately 40,000 neurons belonging to more than 70 different types. This study describes how precise temporal patterning of neural progenitors generates these different neural types. Five transcription factors - Homothorax, Eyeless, Sloppy paired, Dichaete and Tailless - are sequentially expressed in a temporal cascade in each of the medulla neuroblasts as they age. Loss of Eyeless, Sloppy paired or Dichaete blocks further progression of the temporal sequence. Evidence is provided that this temporal sequence in neuroblasts, together with Notch-dependent binary fate choice, controls the diversification of the neuronal progeny. Although a temporal sequence of transcription factors had been identified in Drosophila embryonic neuroblasts, this work illustrates the generality of this strategy, with different sequences of transcription factors being used in different contexts (Li, 2013).

In the developing medulla, the wave of conversion of neuroepithelium into neuroblasts makes it possible to visualize neuroblasts at different temporal stages in one snapshot, with newly generated neuroblasts on the lateral edge and the oldest neuroblasts on the medial edge of the expanding crescent shaped neuroblast region. An antibody screen was conducted for transcription factors expressed in the developing medulla and five transcription factors, Hth, Ey, Slp1, D and Tll, were identified that are expressed in five consecutive stripes in neuroblasts of increasing ages, with Hth expressed in newly differentiated neuroblasts, and Tll in the oldest neuroblasts. This suggests that these transcription factors are sequentially expressed in medulla neuroblasts as they age. Neighbouring transcription factor stripes show partial overlap in neuroblasts with the exception of the D and Tll stripes, which abut each other. Previous studies have reported that Hth and Ey< were expressed in medulla neuroblasts, but they had not been implicated in controlling neuroblast temporal identities. Hth and Tll also show expression in the neuroepithelium (Li, 2013).

To address whether each neuroblast sequentially expresses the five transcription factors, their expression was examined in the neuroblast progeny. Hth, Ey and Slp1 are expressed in three different layers of neurons that correlate with birth order, that is, Hth in the first-born neurons of each lineage in the deepest layers; Ey or Slp1 in correspondingly more superficial layers, closer to the neuroblasts. This suggests that they are born sequentially in each lineage. D is expressed in two distinct populations of neurons. The more superficial population inherit D from D⁺ neuroblasts. D⁺ neurons in deeper layers (corresponding to the Hth and Ey layers) turn on D expression independently and will be discussed later. Single neuroblast clones were generated, and the expression of the transcription factors was examined in the neuroblast and its progeny. Single neuroblast clones in which the neuroblast is at the Ey⁺ stage include Ey⁺ GMCs/neurons as well as Hth⁺ neurons. This indicates that Ey⁺ neuroblasts have transited through the Hth⁺ stage and generated Hth⁺ neurons. Clones in which the neuroblast is at the D⁺ stage contain Slp1⁺ GMCs and Ey⁺ neurons, suggesting that D⁺ neuroblasts have already transited through the Slp⁺ and Ey⁺ stages. This supports the model that each medulla neuroblast sequentially expresses Hth, Ey, Slp1 and D as it ages, and sequentially produces neurons that inherit and maintain expression of the transcription factor (Li, 2013).

slp1 and slp2 are two homologous genes arranged in tandem and function redundantly in embryonic and eye development. Slp2 is expressed in the same set of medulla neuroblasts as Slp1. Slp1 and Slp2 are referred to collectively as Slp (Li, 2013).

Tll is expressed in the oldest medulla neuroblasts. The oldest Tll⁺ neuroblasts show nuclear localization of Prospero (Pros), suggesting that they undergo Pros-dependent cell-cycle exit at the end of their life, as in larval nerve cord and central brain neuroblasts. Tll⁺ neuroblasts and their progeny express glial cells missing (gcm), and the progeny gradually turn off Tll and turn on Repo, a glial-specific marker. These cells migrate towards deeper neuronal layers and take their final position as glial cells around the medulla neuropil. Thus, Tll⁺ neuroblasts correspond to previously identified glioblasts between the optic lobe and central brain that express gcm and generate medulla neuropil glia. Clones in which the neuroblast is at the Tll⁺ stage contain Hth⁺ neurons and Ey⁺ neurons, among others, confirming that Tll⁺ neuroblasts represent the final temporal stage of medulla neuroblasts rather than a separate population of glioblasts. Therefore, these data clearly show that medulla neuroblasts sequentially express five transcription factors as they age. The four earlier temporal stages generate neurons that inherit and maintain the temporal transcription factor present at their birth, although a subset of neurons born during the Ey, Slp or D neuroblast stages lose expression of the neuroblast transcription factor. At the final temporal stage, neuroblasts switch to glioblasts and then exit the cell cycle (Li, 2013).

Whether cross-regulation among transcription factors of the neuroblast temporal sequence contributes to the transition from one transcription factor to the next was examined. Loss of hth or its cofactor, extradenticle (exd), does not affect the expression of Ey and subsequent progression of the neuroblast temporal sequence (Li, 2013).

ey-null mutant clones were generated using a bacterial artificial chromosome (BAC) rescue construct recombined on a chromosome containing a Flip recombinase target (FRT) site in an ey^J5.71 null background. ey^J5.71 homozygous mutant larvae were also tested. In both cases, Slp expression is lost in neuroblasts, along with neuronal progeny produced by Slp⁺ neuroblasts, marked by the transcription factor Twin of eyeless (Toy, see below). However, neuroblast division is not affected, and Hth remains expressed in only the youngest neuroblasts and first-born neurons. Targeted ey RNA interference (RNAi) using a Vsx-Gal4 driver that is expressed in the central region of the neuroepithelium and neuroblasts gives the same phenotype. This suggests that Ey is required to turn on the next transcription factor, Slp, but is not required to repress Hth (Li, 2013).

In clones of a deficiency mutation, slp^S37A, that deletes both slp1 and slp2, neuroblasts normally transit from Hth⁺ to Ey⁺, but older neuroblasts maintain the expression of Ey and do not progress to express D or Tll, suggesting that Slp is required to repress ey and activate D (Li, 2013).

Similarly, in D mutant clones, neuroblasts are also blocked at the Slp⁺ stage, and do not turn on Tll, indicating that D is required to repress slp and activate tll. Finally, in tll mutant clones, D expression is not expanded into oldest neuroblasts, suggesting that tll is not required for neuroblasts to turn off D. Thus, in the medulla neuroblast temporal sequence, ey, slp and D are each required for turning on the next transcription factor. slp and D are also required for turning off the preceding transcription factor (Li, 2013).

Gain-of-function phenotypes of each gene were studied. However, misexpression of Hth, Ey, Slp1 or Slp2, or D in all neuroblasts or in large neuroblast clones is not sufficient to activate the next transcription factor or repress the previous transcription factor in neuroblasts. Only misexpressing tll in all neuroblasts is sufficient to repress D expression (Li, 2013).

In summary, cross-regulation among transcription factors is required for at least some of the transitions. No cross-regulation was observed between hth and ey. Because ey is already expressed at low levels in the neuroepithelium and in Hth⁺ neuroblasts, an as yet unidentified factor might gradually upregulate ey and repress hth to achieve the first transition. As tll is sufficient but not required to repress D expression, additional factors must act redundantly with Tll to repress D (Li, 2013).

The temporal sequence of neuroblasts described above could specify at least four neuron types plus glia (in fact more than ten neuron types plus glia considering that neuroblasts divide several times at each stage with overlaps between neighbouring temporal transcription factors). As this is not sufficient to generate the 70 medulla neuron types, it was asked whether another process increases diversity in the progeny neurons born from a neuroblast at a specific temporal stage. Apterous (Ap) is known to mark about half of the 70 medulla neuron types. In the larval medulla, Ap is expressed in a salt-and-pepper manner in subsets of neurons born from all temporal stages. In the progeny from Hth⁺ neuroblasts, all neurons seem to maintain Hth, with a subset also expressing Ap. However, only half of the neurons born from neuroblasts at other transcription factor stages maintain expression of the neuroblast transcription factor. For instance, in the progeny of Ey⁺ neuroblasts, Ey⁺ neurons are intermingled with about an equal number of Ey⁻ neurons that instead express Ap. Neuroblast clones contain intermingled Ey⁺ and Ap⁺ neurons. This is also true for the progeny of Slp⁺ neuroblasts: Slp1⁺ neurons are intermingled with Slp1⁻ Ap⁺ neurons. In the progeny of D⁺ neuroblasts, D and Ap are co-expressed in the same neurons, and they are intermingled with neurons that express neither D nor Ap. Neurons in deeper neuronal layers (corresponding to the Ey⁺ and Hth⁺ neuron layers) also express D independently, and these neurons are Ap⁻. The expression of Ap is stable from larval to adult stages (Li, 2013).

The intermingling of Ap⁺ and Ap⁻ neurons raised the possibility that asymmetric division of GMCs gives rise to one Ap⁺ and one Ap⁻ neuron. Two-cell clones were generated to visualize the two daughters of a GMC. In every case, one neuron is Ap⁺ and the other is Ap^-, suggesting that asymmetric division of GMCs diversifies medulla neuron fates by controlling Ap expression (Li, 2013).

Asymmetric division of GMCs in Drosophila involves Notch (N)-dependent binary fate choice. In the developing medulla, the N pathway is involved in the transition from neuroepithelium to neuroblast, and loss of Su(H), the transcriptional effector of N signalling, leads to faster progression of neurogenesis and neuroblast formation. However, Su(H) mutant neuroblasts still follow the same transcription factor sequence and generate GMCs and neuronal progeny, allowing analysis of the effect of loss of N function on GMC progeny diversification. Notably, neurons completely lose Ap expression in Su(H) mutant clones. All mutant neurons born during the Hth⁺ stage still express Hth, but not Ap, suggesting that the N^ON daughters of Hth⁺ GMCs are the neurons expressing both Ap and Hth. In contrast to wild-type clones, all Su(H) mutant neurons born during the Ey⁺ neuroblast stage express Ey and none express Ap. Similarly, all mutant neurons born during the Slp⁺ neuroblast stage express Slp1 but lose Ap. These data suggest that, for Ey⁺ or Slp⁺ GMCs, the N^OFF daughter maintains the neuroblast transcription factor expression, whereas the N^ON daughter loses this expression but expresses Ap. In the wild-type progeny born during the D⁺ neuroblast stage, Ap⁺ neurons co-express D. Both D and Ap are lost in Su(H) mutant clones in the D⁺ neuroblast progeny, confirming that D is transmitted to the Ap⁺ N^ON daughter of D⁺ GMCs. By contrast, the D⁺ Ap⁻ neurons in the deeper layers (corresponding to the N^OFF progeny born during the Ey⁺ and Hth⁺ neuroblast stages, see above) are expanded in Su(H) mutant clones at the expense of Ap⁺ neurons. Therefore, the deeper layer of D expression is turned on independently in the N^OFF daughters of Hth⁺ and Ey⁺ GMCs (Li, 2013).

Finally, in wild type, a considerable amount of apoptotic cells were observed dispersed among neurons, suggesting that one daughter of certain GMCs undergoes apoptosis in some of the lineages. Together these data suggest that Notch-dependent asymmetric division of GMCs further diversifies neuronal identities generated by the temporal sequence of transcription factors (Li, 2013).

How does the neuroblast transcription factor temporal sequence, together with the Notch-dependent binary fate choice, control neuronal identities in the medulla? Transcription factor markers specifically expressed in subsets of medulla neurons, but not in neuroblasts, were examined including Brain-specific homeobox (Bsh) and Drifter (Dfr), as well as other transcription factors identified in the antibody screen, for example, Lim3 and Toy. Bsh is required and sufficient for the Mi1 cell fate, and Dfr is required for the morphogenesis of nine types of medulla neurons, including Mi10, Tm3, TmY3, Tm27 and Tm27Y (Hasegawa, 2011). Investigation were carried out to identify at which neuroblast temporal stage these neurons were born by examining co-expression with the inherited neuroblast transcription factors. Then whether the neuroblast transcription factors regulate expression of these markers and neuron fates was investigated. The results for each neuroblast stage are described below (Li, 2013).

Bsh is expressed in a subset of Hth⁺ neurons, suggesting that Bsh is in the N^ON daughter of Hth⁺ GMCs. Indeed, Bsh expression is lost in both Su(H) and hth mutant clones. Thus, both Notch activity and Hth are required for specifying the Mi1 fate, consistent with the previous report that Hth is required for the Mi1 fate. Ectopic expression of Hth in older neuroblasts is also sufficient to generate ectopic Bsh⁺ neurons, although the phenotype becomes less pronounced in later parts of the lineage. These data suggest that Hth is necessary and sufficient to specify early born neurons, but the competence to do so in response to sustained expression of Hth decreases over time. This is similar to embryonic CNS neuroblasts, where ectopic Hb is only able to specify early born neurons during a specific time window (Li, 2013).

Lim3 is expressed in all Ap⁻ progeny of both Hth⁺ and Ey⁺ neuroblasts. Toy and Dfr are expressed in subsets of neurons born from Ey⁺ neuroblasts, as indicated by their expression in the Ey⁺ neuron progeny layer. The most superficial row of Ey⁺ Ap⁻ neurons express Toy (and Lim3), suggesting that they are the N^OFF progeny of the last-born Ey⁺ GMCs. Dfr is co-expressed with Ap in two or three rows of neurons that are intermingled with Ey⁺ neurons, suggesting that they are the N^ON progeny from Ey⁺ GMCs. In addition to these Ap⁺ Dfr⁺ neurons, Dfr is also expressed in some later-born neurons that are Ap⁻ but express another transcription factor: Dachshund (Dac), in specific sub-regions of the medulla crescent (Li, 2013).

Whether Ey in neuroblasts regulates Dfr expression in neurons was tested. As expected, Dfr-expressing neurons are lost in ey-null mutant clones, suggesting that they require Ey activity in neuroblasts, even though Ey is not maintained in Ap⁺ Dfr⁺ neurons. Furthermore, in slp mutant clones in which neuroblasts remain blocked in the Ey⁺ state, the Ap⁺ Dfr⁺ neuron population is expanded into later-born neurons, suggesting that the transition from Ey⁺ to Slp⁺ in neuroblasts is required for shutting off the production of Ap⁺ Dfr⁺ neurons. In addition, Ap⁺ Dfr⁺ neurons are lost in Su(H) mutant clones. Thus, Ey expression in neuroblasts and the Notch pathway together control the generation of Ap⁺ Dfr⁺ neurons (Li, 2013).

In addition to its expression with Ey in the N^OFF progeny of the last-born Ey⁺ GMCs, Toy is also expressed in Ap⁺ (N^ON) neurons in more superficial layers generated by Slp⁺ and D⁺ neuroblasts. Consistently, in Su(H) mutant clones, an expansion of Toy⁺ Ey⁺ neurons is seen in the Ey progeny layer, followed by loss of Toy in the Slp and D progeny layer (Li, 2013).

Tests were performed to see whether Slp is required for the neuroblasts to switch from generating Toy⁺ Ap⁻ neurons, progeny of Ey⁺ neuroblasts, to generating Toy⁺ Ap⁺ neurons. Indeed, in slp mutant clones, the Toy⁺ Ap⁺ neurons largely disappear, whereas Toy⁺ Ap⁻ neurons expand (Li, 2013).

WAp and Toy expression was examined in specific adult neurons. OrtC1-gal4 primarily labels Tm20 and Tm5 plus a few TmY10 neurons, and these neurons express both Ap and Toy. To examine whether Slp is required for the specification of these neuron types, wild-type or slp mutant clones were generated using the mosaic analysis with a repressible cell marker (MARCM) technique by heat-shocking for 1 h at early larval stage, and the number of OrtC1-gal4-marked neurons in the adult medulla was examined. In wild-type clones, OrtC1-gal4 marks ~100 neurons per medulla. By contrast, very few neurons are marked by OrtC1-gal4 in slp mutant clones. Slp is unlikely to directly regulate the Ort promoter because Slp expression is not maintained in Ap⁺ Toy⁺ neurons. Furthermore, the expression level of OrtC1-gal4 in lamina L3 neurons is not affected by slp mutation. These data suggest that loss of Slp expression in neuroblasts strongly affects the generation of Tm20 and Tm5 neurons (Li, 2013).

In summary, these data show that the sequential expression of transcription factors in medulla neuroblasts controls the birth-order-dependent expression of different neuronal transcription factor markers, and thus the sequential generation of different neuron types (Li, 2013).

Although a temporal transcription factor sequence that patterns Drosophila nerve cord neuroblasts was reported more than a decade ago, it was not clear whether the same or a similar transcription factor sequence patterns neural progenitors in other contexts. The current identification of a novel temporal transcription factor sequence patterning the Drosophila medulla suggests that temporal patterning of neural progenitors is a common theme for generating neuronal diversity, and that different transcription factor sequences might be recruited in different contexts (Li, 2013).

There are both similarities and differences between the two neuroblast temporal sequences. In the Hb-Kr-Pdm-Cas-Grh sequence, ectopically expressing one gene is sufficient to activate the next gene, and repress the previous gene, but these cross-regulations are not necessary for the transitions, with the exception of Castor. In the Hth-Ey-Slp-D-Tll sequence, removal of Ey, Slp or D does disrupt cross-regulations necessary for temporal transitions (except the Hth-Ey transition). However, in most cases these cross-regulations are not sufficient to ensure temporal transitions, suggesting that additional timing mechanisms or factors are required (Li, 2013).

For simplicity, the medulla neuroblasts are represented as transiting through five transcription factor stages, whereas in fact the number of stages is clearly larger than five. First, neuroblasts divide more than once while expressing a given temporal transcription factor, and each GMC can have different sub-temporal identities. Furthermore, there is considerable overlap between subsequent temporal neuroblast transcription factors: neuroblasts expressing two transcription factors are likely to generate different neuron types from neuroblasts expressing either one alone (Li, 2013).

Although the complete lineage of medulla neuroblasts is still being investigated, this study shows how a novel temporal sequence of transcription factors is required to generate sequentially the diverse neurons that compose the medulla. The requirement for transcription factor sequences in the medulla and in embryonic neuroblasts suggests that this is a general mechanism for the generation of neuronal diversity. Interestingly, the mammalian orthologue of Slp1, FOXG1, acts in cortical progenitors to suppress early born cortical cell fates. Thus, transcription-factor-dependent temporal patterning of neural progenitors might be a common theme in both vertebrate and invertebrate systems (Li, 2013).

A temporal mechanism that produces neuronal diversity in the Drosophila visual center

The brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf/Vvl), correspond to types of medulla neurons. This study examined the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, transcription factors were sought that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates [Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)]. The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. The results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequential ly expressed in NBs (Suzuki, 2013).

In the embryonic central nervous system, the heterochronic transcription factors suchas Hb, Kr, Pdm, Cas and Grh are expressed in NBs to regulate the temporal specification of neuronal identity. They regulate each other to achieve sequential changes in their expression in NBs without cell-extrinsic factors. However, expression of the embryonic heterochronic genes was not detected in the medulla NBs.Instead this study found that Hth, Klu, Ey, Slp and D are transiently and sequentially expressed in medulla NBs. The expression of Hth and Klu was observed in lateral NBs, while that of Ey/Slp and D was observed in intermediate and medial NBs, respectively. These observations suggest that the expression of heterochronic transcription factors changes sequentially as each NB ages, as observed in the development of the embryonic central nervous system (Suzuki, 2013).

This study demonstrates that at least three of the temporal factors Ey, Slp and D regulate each other to form a genetic cascade that ensures the transition from Ey expression to D expression in the medulla NBs. Ey expression in NBs activates Slp, while Slp inactivates Ey expression. Similarly, Slp expression in NBs activates D expression, while D inactivates Slp expression. In fact, the expression of Slp is not strong in newer NBs in which Ey is strongly expressed, but is up regulated in older NBs in which Ey is weakly expressed in the wildtype medulla. A similar relationship is found between Slp and D, supporting the idea that Ey, Slp and D regulate each other's expression to control the transition from Ey-expression to D-expression. In the embryonic central nervous system, similar interaction is mainly observed between adjacent genes of the cascade hb-Kr-pdm-cas-grh, and this concept may also be applied to the medulla primordium. The expression pattern and function of Ey, Slp and D suggest that they are adjacent to each other in the cascade of transcription factor expression in medulla NBs (Suzuki, 2013).

However, no such relationship was found between Hth, Klu and the other temporal factors.The sequential expression of Hth and Klu could be regulated by an unidentified mechanism that is totally different from the genetic cascade that controls the transition through Ey-Slp-D. Or, there might be unidentified temporal factors that are expressed in lateral NBs which act upstream of Hth and Klu to regulate their expression. It is necessary to identify additional transcription factors that are transiently expressed in medulla NBs (Suzuki, 2013).

The expression of concentric transcription factors in the medulla neurons correlates with the temporal sequence of neuron production from the medulla NBs (Hasegawa, 2011). In the larval medulla primordium, the neurons are located in the order of Hth/Bsh-, Run- and Drf-positive cells from inside to outside, and these domains are adjacent to each other (Hasegawa, 2011). Given that NBs generate neurons toward the center of the developing medulla, Hth/Bsh-positive neurons are produced at first, and then Run-positive and Drf-positive neurons. Thus Hth/Bsh, Run and Drf were used as markers to examine roles of Hth, Klu, Ey, Slp and D expressed in NBs in specifying types of medulla neurons. The continuous expression of Hth and Ey from NBs to neurons and the results of clonal analyses that visualize the progeny of NBs expressing each one of the temporal transcription factors suggest that the temporal windows of NBs expressing Hth, Klu and Ey approximately correspond to the production of Hth/Bsh-, Run- and Drf- positive neurons, respectively. Indeed, the results of the genetic study suggest that Hth and Ey are necessary and sufficient to induce the production of Hth/Bsh- and Drf-positive neurons,respectively (Hasegawa, 2011, 2013). Ectopic Klu expression at least induces the produc tion of Run-positive neurons (Suzuki, 2013).

Slp and D expression in NBs may correspond to the temporal windows that produce medulla neurons in the outer domains of the concentric zones, which are most likely produced after the production of Drf-positive neurons. The results at least suggest that Slp is necessary and sufficient and D is sufficient to repress the production of Drf-positive neurons. Identification of additional markers that are expressed in the outer concentric zones compared to the Drf-positive domain would be needed to elucidate the roles of Slp and D in specification of medulla neuron types (Suzuki, 2013).

D mutant clones did not produce any significant phenotype except for derepression of Slp expression in NBs. Drf expression in neurons was not affected either. Since D is a Sox family transcription factor, SoxN, another Sox family transcription factor, is a potential candidate molecule that acts together with D in the medulla NBs. However, its expression was found in neuroepithelia cells and lateral NBs that overlap with Hth-positive cells but not with D-positive cells. All the potential heterochronic transcription factors examined in this study are expressed in three to five cell rows of NBs. Nevertheless, one NB has been observed to produce one Bsh- positive and one Run-positive neuron (Hasegawa, 2011). Therefore, the expression pattern of the heterochronic transcription factors is not sufficient to explain the stable production of one Bsh-positive and one Run-positive neuron from a single NB.The combinatorial action of multiple temporal factors expressed in NBs may play important roles in the specification of Bsh- and Run- positive neurons (Suzuki, 2013).

Another possible mechanism that guarantees the production of a limited number of the same neuronal type from multiple rows of NBs expressing a temporal transcription factor could be a mutual repression between concentric transcription factors expressed in medulla neurons. For example, Hth/Bsh, Run and Drf may repress each other to restrict the number of neurons that express either of these transcription factors. However, expression of Run and Drf was not essentially affected in hth mutant clones and in clones expressing Hth (Hasegawa, 2011). Similarly, expression of Hth and Drf was not essentially affected in clones expressing run RNAi under the control of AyGal4, in which Run expression is eliminated. Hth and Run expression was not affected in drf mutant clones (Hasegawa, 2011). These results suggest that Hth/Bsh, Run and Drf do not essentially regulate each other during the formation of concentric zones in the medulla (Suzuki, 2013).

During embryonic development, the heterochronic genes that are expressed in NBs (hb-Kr-pdm-cas-grh) are maintained and act in GMCs to specify neuronal type. Similarly, Hth and Ey are continuously expressed from NBs to neurons, suggesting that their expression may also be inherited through GMCs (Hasegawa, 2011). However, this type of regulatory mechanism may be somewhat modified in the case of Klu, Slp and D (Suzuki, 2013).

Klu is expressed in NBs and GMCs, but not in neurons. Slp and D are predominantly detected in NBs and neurons visualized by Dpn and Elav, respectively. Occasionally, however, expression of D was found in putative GMCs, which are situated between NBs and neurons. Additionally, both D-positive and D-negative cells were found among Miranda-positive GMCs. Slp expression was not found in Miranda-positive GMCs. Finally, D is expressed in medulla neurons forming a concentric zone in addition to its expression in medial NBs. However, D expression was abolished in slp mutant NBs but remained in the mutant neurons, suggesting that D expression in medulla neurons is not inherited from the NBs. These results suggest that Slp and D expression are not maintained from NBs to neurons and that not all the temporal transcription factors expressed in NBs are inherited through GMCs. However, it is possible to speculate that Klu, Slp and D regulate expression of unidentified transcription factors in NBs that are inherited from NBs to neurons through GMCs (Suzuki, 2013).

Effects of Mutation and Ectopic Expression

The eyeless mutation was first described in 1915 by Hoge. The characteristic phenotype is partial or complete absence of compound eyes. Weak recessive alleles lead to a reduction of compound eyes, or their complete absence, but affects neither the ocelli nor simple eyes found in the center of the fly's head (Quiring, 1994).

The Drosophila homeobox gene optix is capable of inducing ectopic eyes by an eyeless-independent mechanism.

Since ectopic expression of eyes absent, dachshund, eya plus sine oculis and eya plus dac requires eyeless to form ectopic eyes, an examination was performed to see whether ey expression is also induced during ectopic eye formation by Optix. In the eye discs of UAS-Optix dpp-GAL4 flies, no ectopic ey expression was detected. Therefore attempts were made to induce ectopic eye formation with Optix in an ey² mutant background. Targeted expression of the Optix gene in an ey² background results in ectopic eye formation. The efficiency of occurrence of ectopic eyes does not change from the wild-type background situation, but extra ocelli are induced more often than in a wild-type background. From these results, it is concluded that Optix does not require ey expression for the induction of ectopic eyes. Since ey is expressed much earlier in the eye anlagen than Optix, the fact that Optix can induce ectopic eyes only in the eye disc while ey can induce ectopic eyes in other discs as well suggests that ey induces a larger set of target genes than Optix, and that the activity of some of those genes are required for eye induction by Optix. This interpretation is supported by the observation that Optix cannot induce ectopic eyes in a so or eya mutant background. Furthermore, the ectopic expression of ey is sufficient to induce ectopic Optix expression, although in normal eye development Optix transcription is not regulated by ey. Since all these results come from an ectopic situation it will be necessary to analyze the relationship of Optix and ey in an Optix mutant background (Seimiya, 2000).

Genetic control of development of the mushroom bodies, the associative learning centers in the Drosophila brain, by the eyeless, twin of eyeless, and dachshund genes

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the Drosophila brain. By high-resolution neuroanatomy, it has been shown that eyeless, twin of eyeless, and dachshund, which are implicated in eye development, also are expressed in the developing MBs. Mutations of ey completely disrupt the MB neuropils, and a null mutation of dac results in marked disruption and aberrant axonal projections. Genetic analyses demonstrate that, whereas ey and dac synergistically control the structural development of the MBs, the two genes are regulated independently in the course of MB development. These data argue for a distinct combinatorial code of regulatory genes for MBs as compared with eye development and suggest conserved roles of Pax6 homologs in the genetic programs of the olfactory learning centers of complex brains (Kurusu, 2000).

Mushroom bodies (MBs) are a pair of prominent neuropil structures in the insect brain that are implicated as centers for higher-order behaviors including olfactory associative learning and elementary cognitive functions. Anatomically, each MB comprises a large number of densely packed parallel fibers organized into distinct neuronal structures in the brain. The MB cell bodies, Kenyon cells, are located at the dorsal cortex, extending their dendrites into the calyx and their axonal projections through the peduncles, which split dorsally into two lobes, alpha and alpha', and medially into three lobes, beta, beta', and gamma. The calyces of MBs receive olfactory information from the antennal lobes via the prominent antennoglomerular tracts. The peduncles and lobes send neural commands through their connections to the major brain regions including the lateral protocerebrum. These anatomical structures are consistent with the putative MB function: that MBs integrate various sensory information to compute behavioral outputs (Kurusu, 2000).

A Gal4 MB marker, 238Y, identifies the MB primordia in the embryonic brain. Neuroanatomical examination of the developing brains double-stained for 238Y and the Ey protein reveals that Ey is expressed in the embryonic MB primordia. High-resolution imaging shows that Ey is expressed in the MB neuroblasts, ganglion mother cells, and their progenies, suggesting pivotal functions of Ey in various stages of cell differentiation in MB development. In addition to ey, studies on Drosophila eye development have revealed a cascade of regulatory genes that function synergistically in the early specification of eye primordia. Among such regulatory genes involved in eye development, toy also is expressed in the embryonic MBs. Moreover, dac, another gene involved in eye development, also is expressed in the embryonic MBs. However, the expression of the Dac protein is rather confined to ganglion mother cells and embryonic Kenyon cells. Yet, in contrast to the eye development cascade, neither sine oculis (so) nor eyes absent (eya) is expressed in the embryonic MBs, though Eya is detected in nearby cell clusters in the anterior region of the embryonic brain (Kurusu, 2000).

The characteristic expression of ey, toy, and dac in the developing MBs is maintained in the larval brain. Ey is expressed in all of the larval MB cells at a significant level whereas expression of a Gal4 MB marker, 201Y, is absent in the central cells. Expression of toy is also evident in the Kenyon cells. As with ey, toy is expressed in all of the MB cells. On the other hand, Dac is not expressed in the central cells, including neuroblasts and ganglion mother cells, whereas it is clearly detected in distantly located cells. Double staining for Dac and Gal4 MB markers, 201Y, c831 and 238Y, demonstrated that the Gal4 MB markers are expressed in outer cells, which are located several cells diameters away from the central cells. Neither so nor eya is expressed in the larval MBs though they are expressed in nearby cells (Kurusu, 2000).

The distinctive expression profiles of ey, toy, and dac in the embryonic and larval MBs suggest combinatorial regulatory mechanisms in the initial formation and structural development of the MBs. To examine functional significance of these genes in the MBs, the neural structures of the developing MBs were examined in mutant backgrounds of either ey or dac. The larval MBs are topologically similar to the adult MBs but have only two orthogonal lobes, alpha^L and beta^L. Internally, the peduncles and lobes have simple concentric organization, in which the FAS II proteins are expressed homogeneously except for the central, unstained core. Mutational inactivation of ey results in moderate defects in the larval MBs in all the cases examined, with weak but consistent suppression of FAS II in the peduncles and lobes. The distribution of FAS II also is affected: the globular end of the alpha^L-lobe is often devoid of FAS II. In contrast, a null mutation of dac (dac⁴) barely affects the larval MBs. However, 50% reduction of dac activity in heterozygous larvae enhances the structural defects of ey mutants, suggesting synergistic regulatory functions of the two genes in the development of the MB structures. In the double mutant for ey and dac, most parts of the peduncles and lobes showed clear symptoms of neural degeneration including significant degeneration of the alpha^L-lobe in many cases (10%-20%). Furthermore, FAS II expression is markedly suppressed, leaving uneven residual expression in the peduncles and remaining lobes (Kurusu, 2000).

The significance of ey and dac in MB development was examined further in the early pupal stage, in which MBs undergo massive degeneration and reorganization to form the complex adult MB structures. Fifty hours after puparium formation, most of the MB structures are reorganized into the adult architecture, in which FAS II is strongly expressed in the alpha/beta-lobes and peduncles and moderately in the gamma-lobe. In addition, it is heavily expressed in the ellipsoid body, which belongs to the central complex. On the other hand, DIF is strongly expressed in the gamma-lobe and weakly in the other lobes and the peduncle (Kurusu, 2000).

Mutations of ey abolished all the neuropil structures of the pupal MBs in all the cases examined, whereas Kenyon cells expressing Dac are retained. Notably, the ellipsoid body also is disrupted in the mutant. The dac⁴ mutation disrupts most of the neuropil structures of the pupal MBs, leaving Kenyon cells expressing Ey protein intact. Occasionally dac⁴ causes ectopic projections of peduncles. In these cases, the structural profile of the FAS II expression resembles that of the larval MB structures, with homogeneous concentric patterns suggesting failure of reorganization of the MB structures at the onset of pupation. Thus, these results clearly demonstrate the functional importance of ey and dac in the structural formation of the adult MBs in the course of the massive neural reorganization in the early pupal stage. Studies of eye development have revealed a combinatorial network of key regulatory genes, in which toy acts upstream of ey, which initiates the regulatory feedback loop that additionally includes so, eya, and dac. These nuclear regulatory genes then synergistically control the subsequent stages of eye development. To dissect the regulatory network of MB development, an examination was made of the expression of ey, toy, and dac in various mutant backgrounds (Kurusu, 2000).

Whereas Ey and Dac are clearly coexpressed in the embryonic primordia, ey expression is not affected by the loss of dac activity and vice versa. Likewise, Ey and Dac expression is independent of one another's activity in the larval MBs. Ey and Dac are coexpressed in most of the Kenyon cells at the pupal stage except for the central cells, which express only Ey. Again, mutation of ey does not alter the Dac expression though the number of the Kenyon cells is slightly reduced. Mutation of dac does not alter Ey expression at all with the normal number of Kenyon cells (Kurusu, 2000).

Expression of toy is initiated from the cellular blastoderm stage earlier than the onset of ey and dac in both the eye and brain. Consistent with this temporal order of gene expression, neither ey nor dac mutation affects the expression of toy in the developing MBs. Moreover, Dac expression was examined in nullo 4 embryos, which lack both ey and toy genes because of the loss of the fourth chromosome. Despite the fact that the brain is largely deformed in nullo 4 embryos, characteristic MB neuroblasts expressing a nuclear marker are found at a dorsolateral position of each brain hemisphere with Dac-expressing progenies. Taken together, in contrast to the intricate feedback cascade in eye development, these results argue for distinct parallel cascades for the regulation of ey and dac in the developing MBs (Kurusu, 2000).

In vertebrates, Pax6 is expressed in various regions of forebrain, including the anlagen of the olfactory bulb, piriform cortex, and amygdala, which are important to olfactory information processing and emotional learning. Mutations of Pax6 result in profound defects in these forebrain structures as well as other telencephalon regions. Intriguingly, a mouse dac homolog also is expressed in the developing telencephalon in overlapping regions with the Pax6 gene. The findings that, in both Drosophila and mouse, homologs of Pax6 genes are expressed in and required for the development of the neural structures that are important to the olfactory perception and learning raises the possibility that these structures arose very early in brain evolution (Kurusu, 2000).

Early development of the Drosophila mushroom body: the roles of eyeless and dachshund

Both hypomorphic alleles of eyeless, ey^R and ey², are produced as a result of insertion of transposable elements in a regulatory element in the intron at the 5' terminus of ey. This results in reduced expression of ey transcripts in the embryonic and larval primordia of the eye imaginal discs. To find a function for ey in MB development, these hypomorphic alleles of ey were used, since at the present time a null allele of ey is not available. Neither of the two hypomorphic alleles reveal abnormalities in the embryonic pattern of MBNBs or the eMBp. These ey alleles were tested for defects in the larval MB morphology. Using anti-FasII antibody, 38% of ey^R flies have defects in MBs that are easily detected. The affected areas are the medial lobes, which are either fused with each other, drastically reduced in diameter, or one or both lobes are completely absent. In ey² flies, fusion of the medial lobes were found in about 35% of flies, very similar to the medial lobe fusion in ey^R; however, other abnormalities were not detected. Overexpression of ey in MBs results in reduced dorsal lobes and fused medial lobes. The number of MB Kenyon cells is reduced in both ey hypomorph and ey overexpression (Noveen, 2000).

dac4, a null allele of dac was used to see if any defects could be detected in the larval MB structure. Staining the larval brains with anti-FasII antibody, about 10% of the larvae were found to have dorsal lobes with extremely reduced diameter. The defect is only unilateral, as is usually observed with ey^R and ey-overexpression. Although the dorsal lobe defect is most noticeable, there are still other defects such as slanting of the dorsal lobe and widening of various regions of MB neuropile (Noveen, 2000).

A model for embryonic MB development is presented. The abnormalities caused by the hypomorphic ey allele and ey overexpression has to be interpreted in light of the temporal sequence in which medial and dorsal lobes are formed in the embryo. Thus the medial lobe is formed first, when the growth cones of the extending MB axons make a sharp medial turn. The dorsal lobe originates as a collateral at a later stage. Taken together, these findings lead to the following model. Signals localized near the dorsal midline of the brain act upon the outgrowing MB axons to form the medial lobe (signal 'm'), followed by a dorsal signal ('d') that induces the outgrowth of the dorsal lobe collaterals. Ey may be specifically involved in balancing the reception of the d- and the m-signals, such that it renders growth cones more sensitive to the m-signal and less sensitive to the d-signal. According to this hypothesis, reception of the m-signal is reduced, and reception of the d-signal increases in ey hypomorphs. As a result growth cones fail to grow medially and extend dorsally instead, resulting in the absence of medial lobes and either normal or thickened dorsal lobes. By overexpressing ey, the MB axons get more sensitive to the m-signal, so that the collaterals that should grow dorsally are diverted medially. A thickening of the medial lobe is often observed in brains where the dorsal lobe is absent. In contrast to ey, dac may function only in the regulation of the d-signal, since its null allele results in the absence of the dorsal lobe while its overexpression has little effect (Noveen, 2000).

ey could regulate the m and d signals by regulating the expression of various genes. In the vertebrate CNS, cell adhesion molecules, N-CAM and L1 appear to be under direct control of ey/Pax6. Studies in chimeric mice made from aggregation of wild-type and Pax6-/- embryos show that the wild-type cells have adhesive properties different from those that have mutant Pax6. Drosophila FasciclinII, a homolog of vertebrate N-CAM, may also be regulated by ey, since in the ey-null mutants it appears to be downregulated. Additionally, in Drosophila, the tyrosine kinase receptor, Derailed/Linotte (Drl/Lio), may be the receptor for the d-signal, since its absence causes loss of the dorsal lobes. ey could act directly or indirectly to downregulate Drl/lin expression, which would explain why ey overexpression causes the absence of the dorsal lobes. In vertebrates, both the loss of function and overexpression of Pax6 lead to defects in the eye and nervous system (Noveen, 2000 and references therein).

There is some evidence indicating that the embryonic Kenyon cells are made up of gamma-type neurons. This is based on hydroxyurea ablation of MB neuroblast in the newly hatched 1st instar larvae. Using various gal4 driver lines, it has been demonstrated that when the early 1st instar larvae are fed hydroxyurea, all the MB lobes are absent in the adult except the gamma lobe. Since no MB Kenyon cells are generated after the hydroxyurea treatment, the Kenyon cells that remain are those that are generated during the embryonic period. In the adult, the gamma lobe is generated by the gamma-type neurons. Thus the above implies that the MB neurons generated during the embryonic period are the gamma neurons; however, after embryogenesis, additional gamma neurons are added to the pool of previously existing gamma neurons. In the present experiments the formation of both the dorsal and medial lobes were observed after hydroxyurea treatment of the early 1st instar larvae. This is due to the fact that, during the 1st and 2nd larval stages, when the larvae are usually observed, the axons of the gamma neurons are branched and give rise to both the medial and dorsal lobes. It is interesting to note that when ey or dac are lacking, the most severe defects are found during the pupal stage, when the axons of the gamma neurons are degenerated and reformed along with the formation of the alpha/beta axons (Noveen, 2000 and references therein).

The eyeless homeodomain is dispensable for eye development in Drosophila

Pax-6 genes, known to be essential for eye development, encode an evolutionarily conserved transcription factor with two DNA-binding domains. To corroborate the contribution of each DNA-binding domain to eye formation, truncated forms of the Drosophila Pax-6 gene eyeless were generated and their capacity to rescue the ey² mutant was examined. Surprisingly, Ey deleted of the homeodomain is able to rescue the ey² mutant and can trigger ectopic eyes morphogenesis. In other words, Ey protein that lacks the homeodomain, is fully functional in promoting eye morphogenesis. In contrast, Ey lacking the paired domain fails to rescue the ey² mutant, but leads to truncation of appendages, and represses Distal-less when misexpressed. This result suggests distinct functions mediated differentially by the two DNA-binding domains of Eyeless (Punzo, 2001).

The data predict that ey downstream target genes required for eye development are activated by the paired domain. Therefore, the expression of so as a direct target gene of ey was tested. The various ey constructs were misexpressed by dpp^blink-Gal4. Induction of so expression is detected by lac-Z staining of an enhancer trap line. so is ectopically activated by the full-length as well as the EYdeltaHD protein. It is not ectopically activated when the paired domain is missing, showing that the PD within the Ey protein is sufficient to induce its direct target so (Punzo, 2001).

dac is an indirect target gene of ey and misexpression of dac has been shown to be in part responsible for appendage truncation and to be able to induce ectopic eye development. The misexpression experiments done for so was repeated and antibodies were used to detect both Ey and Dac. Like so, Dac is only ectopically activated if the PD in Ey is present, as expected for an essential gene in eye development. This suggests that, truncated appendages are not due to Dac misexpression, because it does not get ectopically activated by eydeltaPD (Punzo, 2001).

It was then asked whether the ectopic eyes generated by eydeltaHD also express late marker genes of eye development. rhodopsin-1 has been proposed to be directly regulated by the homeodomain of ey. The presence of rhodopsin-1 was examined in ectopic eyes generated by eydeltaHD in ey² mutants, in which neither EY-HD nor TOY-HD are required for ectopic eye development. Immunostainings on cryosections by use of an anti-Rhodopsin-1 antibody have revealed that Rhodopsin-1 is expressed in the retina of ectopic eyes generated by ey and eydeltaHD in ey² mutants. Rhodopsin-1 expression was also detected in the eyes of both ey² and ey^J5.71 mutants, but not in the eyes of the rhodopsin-1 mutant ninaE. This indicates that the expression of Rhodopsin-1 is independent of the homeodomain of ey and does not require the presence of Ey in the adult eye. It strengthens the hypothesis that rhodopsin-1 is likely to be activated by another paired type HD, containing a gene other than ey (Punzo, 2001).

Because the homeodomain of the Pax-6 proteins is highly conserved, its function during development was investigated. ey is able to repress Dll in an ectopic situation. So far, experiments have suggested that the HD may confer gene repression. Therefore, whether Dll repression by ey is mediated by the homeodomain was examined. ey, eydeltaPD, eydeltaHD, and eydeltaPDPMHD were ectopically expressed on all appendages with dpp^blink-Gal4 and Ey and Dll expression were monitored by antibody detection. Ectopic expression of ey and eydeltaPD are able to repress Dll expression in the respective areas of overlap, in contrast to ectopic expression of eydeltaHD and eydeltaPDPMHD. This result shows that the HD of ey mediates Dll repression by DNA binding. Therefore, it is concluded that the truncated appendages are due in part to the repression of Dll (Punzo, 2001).

Headless flies generated by developmental pathway interference

Ectopic expression of transcription factors in eye-antennal discs of Drosophila strongly interferes with the disc's developmental program. Early ectopic expression in embryonic discs interferes with the developmental pathway primed by Eyeless and generates headless flies, which suggests that Eyeless is necessary for initiating cell proliferation and development of both the eye and antennal disc. Interference occurs through a block in the cell cycle that for some ectopic transcription factors is overcome by D-CycE or D-Myc. Late ectopic expression in cone cell precursors interferes with their differentiation. It is proposed that this developmental pathway interference is a general surveillance mechanism that eliminates most aberrations in the genetic program during development and evolution, and thus seriously restricts the pathways that evolution may take (Jiao, 2001).

To investigate the effect of ectopic transcription factors on early eye development, Pax proteins were expressed under the control of the eye-specific enhancer of the eyeless (ey) gene, a Pax6 homolog active in eye-antennal disc precursor cells. Thus, D-Pax2/Shaven was ectopically expressed in the developing eye disc under the indirect control of the eye-specific enhancer of ey, by the use of ey-Gal4 and UAS-D-Pax2 transgenes. This resulted in a dramatic interference with eye development, and no flies eclosed. Surprisingly, however, when the ey-Gal4/+; UAS-D-Pax2/+ pharate adults were examined, they not only lacked eyes, like the strongest known ey mutants, but frequently had no head except for the proboscis, while thorax and abdomen were wild type. Very similar phenotypes were observed when Poxn, Pox meso (Poxm), Gooseberry (Gsb) or Paired (Prd), i.e. Pax proteins whose paired domains belong to a class different from that of Ey or Pax6, were ectopically expressed under the control of ey-Gal4 (Jiao, 2001).

These results suggest that Pax proteins that do not belong to the Ey class are able to interfere with the functions of ey in the eye-antennal disc to generate headless flies. If true, it might be possible to rescue the headless phenotype by elevating the levels of the Ey protein. Indeed, one copy of UAS-Ey is able to rescue the headless phenotype of ey-Gal4/UAS-Gsb flies partially to produce small-eyed flies, characteristic for hypomorphic ey alleles. Although only about a quarter of the rescued flies eclose, almost all pharate adults exhibit a small-eye phenotype and only a few (<5%) exhibit a more severe phenotype (Jiao, 2001).

It is concluded that the headless phenotype results from an interference with ey functions during development of the eye-antennal disc. Hence, it was anticipated that complete absence of these functions might generate headless flies, i.e. a much more severe phenotype than that of previously analyzed ey alleles. This prediction has been confirmed by analysis of strong ey mutants. Because ey is activated by the product of its paralog twin of eyeless (toy), attempts were made to rescue the headless phenotype of ey-Gal4/UAS-Gsb flies by a UAS-Toy instead of a UAS-Ey transgene. However, these experiments showed no rescue, which suggests that the activity of the ey gene is close to its maximum level and hence higher Toy levels are unable to raise the concentration of Ey sufficiently (Jiao, 2001).

The eye-antennal discs of ey-GAL4/+; UAS-Gsb/+ third instar larvae are absent or strongly reduced in size. Evidently, developmental pathway interference induced by the ectopic expression of transcription factors eventually results in the inhibition of cell proliferation and/or apoptosis in these discs. To investigate which of these two processes is responsible for the generation of headless flies, attempts were made to inhibit apoptosis or to stimulate cell proliferation in eye-antennal discs. While inhibition of apoptosis by the expression of the baculovirus P35 protein is unable to suppress the headless phenotype, stimulation of cell proliferation by the expression of D-Myc suppresses it in spontaneously eclosing adults (5%-20%), producing adults of variable eye size, from eyeless adults to adults whose eye size is only slightly reduced. The headless phenotype is rescued even more dramatically by D-CycE, which restores a wild-type phenotype in up to 50% of the adults and only rarely generates small-eyed flies. Rescue of the headless phenotype by CycE is not restricted to ey-GAL4/+; UAS-Gsb/+ flies, but is achieved for all Pax proteins and transcription factors whose potency to interfere with ey function in the eye-antennal disc was tested. However, in contrast to headless flies produced by Gsb, Prd, Poxm, D-Pax2 or Dac, many of which were rescued by CycE to adults that eclosed spontaneously, those generated by Mef2, Sim or Poxn were incompletely rescued. D-Myc is not as efficient in its rescue ability, except in the case of D-Pax2, in which nearly all flies were rescued to wild-type adults. It is concluded that developmental pathway interference through ectopic expression of transcription factors results in the inhibition of cell proliferation that is at least partially overcome by co-expression of D-Myc or D-CycE (Jiao, 2001).

It has been shown that ectopic expression of Antennapedia in the eye disc inhibits eye development and generates eyeless flies. On the basis of in vitro binding studies, it has been proposed that Antp as well as other homeodomain proteins exert this effect by binding through their homeodomain to the paired domain and homeodomain of the Ey protein, thus inhibiting the activation of Ey target genes in a dominant negative manner. Several results strongly suggest that the mechanism inhibiting eye and head development by the ectopic expression of a transcription factor does not crucially depend on the dominant negative interaction of an ectopic homeodomain factor with the Ey protein, but is of a more general nature. (1) When tested in vivo for its ability to generate headless or eyeless flies, the Gsb protein strictly depends on its paired domain without which it does not affect eye development, while in the absence of its homeodomain it is still able to produce eyeless flies. (2) A truncated Gsb protein, which consists of both DNA-binding domains, the paired domain and the homeodomain, but lacks its transactivation domains, has no effect on eye or head development. (3) If a missense mutation is introduced that abolishes the DNA-binding activity of its paired domain but does not affect its homeodomain, Gsb is unable to interfere with eye development. (4) Similarly, if the DNA-binding specificity of the paired domain of the ectopic Gsb is altered to that of Ey, its interference with head and eye development is abolished or reduced to that of ectopic Ey. (5) The two homeodomain proteins tested that have no paired domain, Ubx and En, inhibit eye development relatively weakly and with low penetrance. In fact, they exhibit the weakest phenotype of all transcription factors examined. (6) Many non-homeodomain transcription factors inhibit eye and head development very efficiently. (7) While elevating Ey levels may overcome the inhibition of some ectopic transcription factors, this is not the case for Sim and perhaps for several of the other factors tested. (8) By contrast, the inhibition of eye and head development by ectopic transcription factors can be reduced or entirely removed by elevating the concentrations of CycE or Myc. (9) Interference with eye and head development is limited to a critical short period in the embryonal eye-antennal primordium, long before the Ey targets so, eya and dac are activated in the larva (Jiao, 2001).

Taken together, these results demonstrate that the observed inhibition of eye and head development by an ectopic transcription factor cannot be explained by its interaction with Ey protein, but rather is caused by a block in the execution of the developmental program primed by Toy and Ey. In addition, they raise the possibility that the decisive inhibition by Antp does not occur through its binding to Ey, but through this mechanism of developmental pathway interference (Jiao, 2001).

It is important to note that complete interference with eye-antennal development primed by Toy and Ey produces headless flies that lack all structures derived from the eye-antennal disc, a phenotype that is much stronger than that reported for ey loss-of-function alleles. Its primary cause is a block during the G1 phase of the cell cycle, because in some cases it can be completely removed by overexpression of CycE. This block can occur only at a very early stage of eye-antennal disc development, which suggests that Toy and Ey prime eye-antennal development in the corresponding embryonic disc primordium, long before the fates of eye and antenna are specified during the second instar. In agreement with such an early role for Ey in the development of both eye and antenna, Ey is expressed throughout the eye-antennal disc of the embryo and first instar larva. If Toy and Ey prime the genetic program that activates the network regulating development not only of the eye, but also of the antenna, one would expect that ey mutants that lack any function in the eye-antennal disc would also display a headless phenotype. Indeed, strong ey mutants show a phenotype indistinguishable from the headless phenotype produced by interference with eye-antennal development through ectopic transcription factors. Therefore, one of the earliest functions of ey is the activation of the cell division cycle with which ectopic transcription factors interfere. Since interference is restricted to a short phenocritical period during the second half of embryogenesis, it is concluded that Ey primes cell division in eye-antennal development about 24 hours before eye-antennal disc cells divide in first instar larvae (Jiao, 2001).

It is expected that many transcription factors will be restricted to their realms and thus give rise to sharp boundaries between their domains of expression. Indeed, such boundaries are abundant during development and may result from the necessity of the factors to avoid interference with the developmental program of the adjacent domain. Examples of early developmental pathway boundaries established at the blastoderm stage are those between transcription factors encoded by pair-rule genes. A classical example is the ubiquitous expression of fushi tarazu (ftz) in Hs-ftz embryos at this stage, which results in the loss of those epidermal structures in which ftz is normally not expressed. Consistent with the cuticular phenotype of Hs-ftz embryos, it is assumed that interference results in a block of cell division followed by apoptosis. Similar to the observations presented in this study, interference by ftz is restricted to a very short time interval around cellular blastoderm. It should be noted that a complementary situation arises in ftz^- mutants in which absence of Ftz protein results in developmental pathway interference in those regions where it is normally required. Hence, absence of a transcription factor may also lead to developmental pathway interference if it results in an undefined developmental program. This is not the case, for example, in homeotic loss-of-function mutants. Other examples of sharp boundaries are observed in mouse embryos between different types of paired domain transcription factors, such as between Pax2 and Pax6 in the developing eye, or between Pax3 and Pax6 and between Pax2 and Pax6 in the developing neural tube (Jiao, 2001).

Temporal control of glial cell migration in the Drosophila eye requires gilgamesh, hedgehog, and eye specification genes

In the Drosophila visual system, photoreceptor neurons (R cells) extend axons towards glial cells located at the posterior edge of the eye disc. In gilgamesh (gish) mutants, glial cells invade anterior regions of the eye disc prior to R cell differentiation and R cell axons extend anteriorly along these cells. gish encodes casein kinase Igamma. gish, sine oculis, eyeless, and hedgehog (hh) act in the posterior region of the eye disc to prevent precocious glial cell migration. Targeted expression of Hh in this region rescues the gish phenotype, though the glial cells do not require the canonical Hh signaling pathway to respond. It is proposed that the spatiotemporal control of glial cell migration plays a critical role in determining the directionality of R cell axon outgrowth (Hummel, 2002).

A set of genes encoding nuclear proteins [e.g., eyeless (ey), eyes absent (eya), sine oculis (so) and secreted factors such as hedgehog (hh)] regulates the initiation of neuronal differentiation in the posterior region of the eye disc. The effect of loss-of-function mutations in these genes on glial cell migration was tested. As in gish mutants, glial cells migrate precociously out of the optic stalk in a hh temperature-sensitive mutation incubated at the nonpermissive temperature during first and second instar. This is an early function of hh, since ectopic glial cells are not observed in hh¹; in this allele, the posterior eye field develops normally, but anterior progression of the MF is inhibited. A similar early onset glial cell migration defect is observed in eye-specific alleles of so and ey. In contrast, glial cells did not migrate out from the optic stalk in an eye-specific allele of eya, raising the possibility that eya is required to activate glial cell migration. Since glial cells migrate out of the stalk precociously in eya/gish double mutants, the production of an eya-dependent signal is not necessary to promote anterior migration. Hence, in contrast to their role in R cell development, eye specification genes ey and so seem to function independent of eya to control the onset of glial cell migration (Hummel, 2002).

These observations raise the possibility that gish also contributes to the genetic circuitry regulating eye specification. Indeed, while ey-FLP-induced clones of gish lead to only minor defects in MF progression during third instar stage, gish mutant adult eyes are smaller and frequently contain a reduced number of ommatidia in the anterior region. These phenotypes are frequently seen in weak alleles of eye specification genes. Furthermore, double mutants of ey¹ and gish¹ , as well as so¹ and gish¹, reveal strong synergistic effects in R cell development. The glial cell migration phenotypes in double mutants is similar in severity to the single mutants. In summary, these data argue that gish acts in conjunction with eye specification genes to coordinate neuronal development and glial cell migration in the eye disc (Hummel, 2002).

Headless flies produced by mutations in the paralogous Pax6 genes eyeless and twin of eyeless

The two Pax6 gene homologs, eyeless and twin of eyeless, play decisive early roles in Drosophila eye development. Strong mutants of either twin of eyeless or eyeless are headless, which suggests that they are required for the development of all structures derived from eye-antennal discs. The activity of these genes is crucial at the very beginning of eye-antennal development in the primordia of eye-antennal discs when eyeless is first activated by the twin of eyeless gene product. This activation does not strictly depend on the Twin of eyeless protein, but is temperature-dependent in its absence. Twin of eyeless also acts in parallel to the eyeless gene and exerts functions that are partially redundant with those of Eyeless, while Eyeless is mainly required to prevent early cell death and promote eye development in eye-antennal discs (Kronhamn, 2002).

An old mutant, l(4)8, induced by X-ray in 1957, displays a headless phenotype. This toy^hdl allele turns out to be the first mutant allele of the Pax gene toy, previously isolated as a paralog of the ey gene and proposed to activate ey to initiate eye development. The dramatic phenotype of toy^hdl mutants, however, implies that toy is not only necessary for proper development of the eye, but for that of all structures derived from the eye-antennal disc. Moreover, the same is true for the ey gene; ey^D, an allele of ey and much stronger than any ey alleles previously characterized, exhibits the same phenotype. The headless phenotype of ey^D pharates (newly hatched adults) results from the induction of apoptosis in the eye-antennal disc, as evident from its rescue by the expression of the baculovirus P35 protein in eye-antennal primordia and discs. These findings are consistent with an old conclusion that 'at no time in development (except at the very end) is there a group of identified and committed cells which will give rise to the eye and to nothing else' (Kronhamn, 2002 and references therein).

Temperature shift experiments with toy^hdl mutants show that the headless phenotype critically depends on the absence of Toy protein activity during stages 12-16 of embryogenesis. In toy^hdl mutants, up to 80% of the pharate adults are headless at 28°C, whereas this phenotype is nearly completely suppressed at 18°C. Shifting the temperature down to the permissive temperature at the end of stage 16 demonstrates that after this stage Toy^hdl is unable to provide any function that would be able to rescue the headless phenotype, while shifting the temperature up to the non-permissive temperature at this time shows that Toy^hdl can provide all the functions necessary, if any, to rescue the headless phenotype at all temperatures after stage 16. It follows that the temperature-sensitive function of Toy^hdl with regard to the headless phenotype is restricted to the phenocritical period during stages 12 to 16 (Kronhamn, 2002).

Homozygous ey^D pharates exhibit the same headless phenotype as toy^hdl mutants. Since ey^D is a strong allele of the ey gene, it is concluded that the truncated Ey^D protein, if translated from the ey^D mRNA, is unable to provide the functions necessary for eye-antennal disc development. It was therefore important to know if ey transcription depends on Toy during the phenocritical period, as had been shown previously for ectopic eye formation. ey transcription indeed depends on toy activity. Surprisingly, however, even in the absence of Toy protein ey transcription (1) remains temperature-dependent during the phenocritical period, and (2) is not completely eliminated. Hence, the observed temperature-sensitivity of the headless phenotype of toy^hdl mutants is not a property of the truncated Toy^hdl protein, but rather of the transcriptional activation of the ey gene in the absence of a functional Toy protein (Kronhamn, 2002).

Does Toy serve to stabilize a temperature-dependent activation complex on the eye-antennal enhancer of the ey gene? It is proposed that during the phenocritical period, in addition to Toy, other transcription factors bind to the eye-antennal enhancer of the ey gene to activate its transcription in the eye-antennal primordia. In the absence of Toy, these factors are able to activate ey transcription sufficiently at low (but not at high) temperatures to ensure normal eye-antennal development. The simplest explanation for this temperature-sensitive activation of ey is that formation of the transcription factor complex bound to the eye-antennal enhancer or its activation of the basal transcription machinery becomes temperature-dependent in the absence of Toy protein. Thus, the main function of Toy is to stabilize this transcriptional activator complex on the eye-antennal enhancer of ey. It is interesting to note that three Toy binding sites have been found in the eye-antennal enhancer of ey, two of which are immediate neighbors. Truncated Toy^hdl probably binds with similar affinity to these sites as wild-type Toy protein. However, its lack of C-terminal activation domains may fail to stabilize its own binding and that of cofactors as a result of which the basal transcription machinery is not efficiently recruited and activated. The fact that a third of the toy^hdl mutants die during larval stages and that toy^hdl mutants that show normal eye-antennal development still die as pharate adults shows that Toy is more strictly required for the activation of other enhancers necessary for the development of viable adults (Kronhamn, 2002).

Interestingly, ey transcription is not abolished, but reduced levels of ey transcripts remain detectable in eye-antennal primordia of toy^hdl embryos, even at temperatures at which most of them develop to headless pharates. This finding implies that there is a delicate balance of ey activation for inducing eye-antennal development. If transcript levels do not exceed a relatively high threshold value, the program for eye-antennal disc development cannot proceed. This delicate balance is particularly evident when it is temperature-dependent in the absence of a functional Toy protein, for example, in toy^hdl mutants. In many instances, unequal ey transcript levels have been observed in the left and right eye-antennal primordia of toy^hdl embryos. Accordingly, ey transcripts may surpass the threshold in only one of the two eye-antennal primordia and thus give rise to pharates with only one half of the head developing normally. Even within the eye-antennal primordium, ey transcript levels may vary in toy^hdl mutants and thus explain phenotypes like the cleft-head (Kronhamn, 2002).

In toy^hdl mutants, reduced yet detectable (moderate) levels of ey transcripts in eye-antennal primordia are unable to rescue the headless phenotype. This is evident from the reciprocal correlation between ey transcript levels in eye-antennal primordia during the phenocritical period and the fraction of headless pharates at different temperatures of development. In contrast, ey² mutants, in which no ey transcripts have been detected in eye-antennal primordia, never display a headless phenotype and mostly have eyes of only slightly reduced size, whereas ey^D mutants show a high penetrance of headless pharates. The following considerations illustrate that this apparent contradiction is resolved by the assumptions that (1) Toy and Ey share partial functional redundancy in eye-antennal disc development, and (2) ey² expresses very low levels of wild-type Ey protein whose mRNA escapes detection. In the absence of functional Ey protein, as in ey^D mutants, normal levels of functional Toy protein rescue the headless phenotype at low efficiency. In ey² mutants, however, very low levels of functional Ey protein are sufficient to rescue the headless phenotype completely in the presence of normal Toy levels and are even able to promote nearly normal eye development. In contrast, in the absence of functional Toy protein, as in toy^hdl mutants, much higher levels of ey transcripts are necessary to rescue the headless phenotype, which is achieved more efficiently at lower temperatures. Consequently, the headless phenotype is observed only in the complete absence of functional Ey protein or in the absence of functional Toy if Ey does not exceed a moderate level (Kronhamn, 2002).

This partial redundancy of Toy and Ey functions implies that Toy does not act exclusively through the activation of ey, but also in a pathway parallel to ey, to promote eye-antennal disc development. Such an ey-independent pathway downstream of toy may include the optix gene, a paralog of the sine oculis gene, which is in turn a target of ey, and the eyg gene (Kronhamn, 2002).

The most crucial function of ey and most sensitive to the level of Ey during eye-antennal development is the inhibition of cell death. This became evident from a successful attempt to rescue the ey^D headless phenotype by the expression in eye-antennal discs of the baculovirus P35 protein, an inhibitor of apoptosis. Astonishingly, P35 is able to rescue more than half of the ey^D embryos to viable adults with normal head structures, with the exception of the eyes that still do not develop (Kronhamn, 2002).

The fact that inhibition of apoptosis in ey^D eye-antennal discs is unable to rescue eye development argues that additional functions of ey during eye-antennal development are required and restricted to the eye disc, in agreement with its expression pattern in eye-antennal discs. It has been shown that interference with the program of eye-antennal development by ectopic expression of other transcription factors also generates headless pharates if interference is restricted to exactly the same phenocritical period observed here. However, in this case interference cannot be antagonized by P35 expression, but only by overexpression of Ey, CycE or Myc. One of the earliest functions of ey must be the activation of the cell cycle. Combining this conclusion with the results reported here, it is proposed that in the presence of developmental pathway interference it is impossible to obtain rescue by the inhibition of apoptosis in the absence of cell cycle activation, whereas inhibition of apoptosis appears to suffice in ey^D mutants, possibly because the truncated Ey^D and/or the Toy protein are able to activate the cell cycle (Kronhamn, 2002 and references therein).

Eyeless collaborates with Hedgehog and Decapentaplegic signaling in Drosophila eye induction

eyeless (ey) is a key regulator of the eye development pathway in Drosophila. Ectopic expression of ey can induce the expression of several eye-specification genes (eya, so, and dac) and induce eye formation in multiple locations on the body. However, ey does not induce eye formation everywhere where it is ectopically expressed, suggesting that Ey needs to collaborate with additional factors for eye induction. Ectopic eye induction by Ey has been examined in the wing disc; eye induction was spatially restricted to the posterior compartment and the anterior-posterior (A/P) compartmental border, suggesting a requirement for both Hh and Dpp signaling. Although Ey in the anterior compartment induces dpp and dac, these are not sufficient for eye induction. Coexpression experiments show that Ey needs to collaborate with high level of Hh and Dpp to induce ectopic eye formation. Ectopic eye formation also requires the activation of an eye-specific enhancer of the endogenous hh gene (Kango-Singh, 2003).

These results indicated that Ey needs to collaborate with high levels of Hh and Dpp for eye induction. Since Hh and Dpp are secreted molecules and can act over long range, the requirement for their high levels restricts the site of eye development. At the time of MF initiation, hh and dpp are expressed at the posterior margin of the eye disc, and ey is expressed throughout the eye disc. So the coexistence of Ey, high Hh, and high Dpp occurs only at the posterior margin to induce MF initiation. After MF initiation, ey is downregulated in the developing photoreceptor cells posterior to the MF, where hh is expressed (Kango-Singh, 2003).

dpp is expressed only at the MF. Thus, the only location where Ey, high Hh, and high Dpp levels coexist is just anterior to the MF, allowing the MF to progress anteriorly. These results clearly show that even when ey, hh, and dpp are all provided, eye induction still does not occur everywhere, suggesting that additional factors are required. It is proposed that the coexpression of the eye-specification genes, eya, so, dac, ey, toy, and eyg, occurring first in the second instar eye disc, specifies the eye fate (Kango-Singh, 2003).

Two Pax genes, eye gone and eyeless, act cooperatively in promoting Drosophila eye development

eyegone (eyg) is required for eye development. Loss-of-function eyg mutations cause reduction or absence of the eye. Similar to the Pax6 eyeless (ey) gene, ectopic expression of eyg induces extra eye formation, but at sites different from those induced by ey. Several lines of evidence suggest that eyg and ey act cooperatively: (1) eyg expression is not regulated by ey, nor does it regulate ey expression; (2) eyg-induced ectopic morphogenetic furrow formation does not require ey, nor does ey-induced ectopic eye production require eyg; (3) eyg and ey can partially substitute for the function of the other, and (4) coexpression of eyg and ey has a synergistic enhancement of ectopic eye formation. These results also show that eyg has two major functions: to promote cell proliferation in the eye disc and to promote eye development through suppression of wg transcription (Jang, 2003).

Since eyg is a Pax gene that shares sequence similarity with ey and toy in the PD and HD domains, its relationship with ey is of particular interest. The results indicate that eyg and ey are transcriptionally and functionally independent for two reasons. (1) Except for a small amount of eyg expression ventral to the equator of the eye disc, eyg and ey do not regulate each other's expression. In this respect, eyg is different from dac, so and eya, whose expression is strongly regulated by ey (and can induce ey expression in some cases). Thus eyg transcription is neither downstream of ey, nor does eyg participate in the ey/eya/so/dac positive feedback loop. This transcriptional independence is similar to that of optix. (2) eyg and ey can each function (to induce ectopic eyes) in the absence of the other. Again, this is similar to optix, which can induce ectopic eyes in ey² mutant. Whether optix is required for ey function has not been tested, because of the lack of optix mutants (Jang, 2003).

However, other evidence indicates that the functions of eyg and ey must converge at some point in the pathway leading to eye development: (1) eyg; ey double hypomorphic mutants show a much stronger eye-loss phenotype; (2) coexpression of ey and eyg causes synergistic enhancement of the ectopic eye phenotype; (3) eyg and ey are able to substitute functionally for each other. Overall, the results suggest that these two Pax genes may act cooperatively. This genetic cooperativity might mean that eyg and ey interact and cooperate as proteins in the same pathway or that they act in parallel pathways. eyg and ey are coexpressed in the eye disc primordium in the embryo. Their expression domains also overlap in the eye disc, especially in the early eye disc when eyg function is critically required. So it is possible that the two Pax proteins act within the same cell, although the possibility they act in different cells to achieve a functional cooperativity has not been ruled out (Jang, 2003).

If eyg and ey are both required for eye development, how could ectopic expression of either one be sufficient for ectopic eye development? One possibility is that the two Pax proteins form heterodimers, directly or indirectly via other proteins, to activate target genes. When the level of either one is low, the target genes that lead to eye formation cannot be induced. However, when either one is strongly expressed ectopically, the high level of homodimer can partially substitute for the heterodimer. Since both genes are required for normal eye formation, this model predicts that the Eyg-Ey heterodimer is more effective than either homodimer in inducing eye formation. As expected by this model, coexpression of eyg and ey causes enhanced ectopic eye formation (Jang, 2003).

As predicted by the eyg and ey interaction, ey, like eyg, suppresses wg expression. Suppression of wg expression by eyg (and ey) is also seen in the wing disc. However, suppression does not occur in all cells expressing eyg, suggesting that additional factors are required for the wg suppression. The relationship of eyg/ey and wg may be mutually antagonistic, since ectopic ey cannot induce eya and so expression in regions of high wg expression (Jang, 2003).

Control of growth determines the size and shape of organs. Localized signals known as 'organizers' and members of the Pax family of proto-oncogenes are both elements in this control. Pax proteins have a conserved DNA-binding paired domain, which is presumed to be essential for their oncogenic activity. Evidence is presented that the organizing signal Notch does not promote growth in eyes of D. melanogaster through either Eyeless (Ey) or Twin of eyeless (Toy), the two Pax6 transcription factors. Instead, it acts through Eyegone (Eyg), which has a truncated paired domain, consisting of only the C-terminal subregion. In humans and mice, the sole PAX6 gene produces the isoform PAX6(5a) by alternative splicing; like Eyegone, this isoform binds DNA though the C terminus of the paired domain. Overexpression of human PAX6(5a) induces strong overgrowth in vivo, whereas the canonical PAX6 variant hardly effects growth. These results show that growth and eye specification are subject to independent control and explain hyperplasia in a new way (Dominguez, 2004).

Functional divergence between eyeless and twin of eyeless

Pax6 genes encode transcription factors with two DNA-binding domains that are highly conserved during evolution. In Drosophila, two Pax6 genes function in a pathway in which twin of eyeless (toy) directly regulates eyeless (ey), which is necessary for initiating the eye developmental pathway. To investigate the gene duplication of Pax6 that occurred in holometabolous insects like Drosophila and silkworm, different truncated forms of toy and small eyes (sey) were used, and their capacity to induce ectopic eye development was tested in an ey-independent manner. Even though the Paired domains of Toy and Sey have DNA-binding properties that differ from those of the Paired domain of Ey, they all are capable of inducing ectopic eye development in an ey mutant background. One of the main functional differences between toy and ey lies in the C-terminal region of their protein products, implying differences in their transactivation potential. Furthermore, only the homeodomain (HD) of Ey is able to downregulate the expression of Distal-less (Dll), a feature that is required during endogenous eye development. These results suggest distinct functions of the two DNA-binding domains of Toy and Ey, and significant evolutionary divergence between the two Drosophila Pax6 genes (Punzo, 2004).

The existence of two Pax6 genes in Drosophila raises the question of whether they have a redundant function or whether they have diverged to control different sets of target genes. A recent characterization of new alleles of ey and toy mutants by Kronham (2002) suggests a functional divergence, with a partial redundancy remaining. Epistasis studies show that toy lies upstream of ey, because ectopic toy is capable of inducing ectopic ey but not vice versa. Additionally, toy cannot induce ectopic eyes in an ey² mutant background whereas ey can. The regulation of ey by toy is due to a direct binding of the Toy-PD to the ey-enhancer, which is located in the second intron of the ey gene. The Ey-PD contains a glycine at position 14, whereas the Toy-PD has an asparagine at that same position. This difference allows Toy to regulate ey through the ey-enhancer, whereas Ey cannot regulate itself. Complementary experiments showed that ectopic expression of a HD-deleted version of the Ey protein did not induce the endogenous full-length gene, and therefore confirmed the lack of an auto-regulatory feedback loop for ey. Endogenous ey can only be induced by misexpression of the three downstream genes eya, so and dac, or by toy. Interestingly, the mouse Pax6 gene sey also has an asparagine at position 14 of the PD and has, therefore, the same DNA-binding properties as toy. Moreover, sey and toy have multiple stretches of conserved amino acids in their C termini, whereas the C terminus of ey diverged. Thus, ectopic sey is able to induce ectopic ey but it does not induce ectopic toy. This suggests that the auto-regulatory feedback loop found in the vertebrate Pax6 gene evolved into a hetero-regulatory interaction in Drosophila with toy regulating ey expression. Overall these data indicate that not only the PDs, but also the cis-regulatory sequences of the two Drosophila Pax6 genes, have diverged to control different sets of target genes. This hypothesis is further supported by the fact that only toy is expressed in the ocelli territory of the eye disc but both toy and ey regulate the eye-specific enhancer of the so gene, by binding partly to the same and partly to different binding sites, and by discriminating between eye and ocelli development during larval stages (Punzo, 2004 and references therein).

The PD and the HD are the most conserved regions within the Pax6 proteins, indicating evolutionary constraints imposed to maintain specific binding to target genes. Therefore, an investigation was carried out to what extent the PD of Tot and Sey, which diverged in their DNA binding properties from the PD of Ey, were able to induce the eye developmental pathway independently of ey. Moreover, whether only the HD of ey was able to downregulate Dll expression was investigated, and whether this function is required during endogenous eye development to specify the eye territory. These hypotheses were tested by generating deletion constructs of sey similar to those described for ey and toy, as well as Ey-Toy chimeric proteins, and ectopic eye formation was scored. Furthermore, the ey null mutant ey^J5.71 was rescued by transferring the genomic region of the ey gene onto the third chromosome. This allowed an analysis of mutant ey clones in a wild-type background. Both sey and toy were found to be able to activate eye development in an ey-independent manner, and one of the main differences between toy and ey, besides their DNA-binding properties, was found to lie in their C-terminal region, and therefore mainly in their transactivation potential. This suggests that most of the differences reside in their capacity to interact with different sets of proteins. Only the HD of ey is able to downregulate Dll expression in an ectopic situation, and this downregulation is required during endogenous eye development (Punzo, 2004).

In mammals Pax6 mutations are haploinsufficient and in heterozygotes eye development is critically affected. However, analysis of ey and toy mutants (ey^15.71 and toy^G3.39) and deletions indicate that ey and toy are completely recessive. Nevertheless, overexpression of ey⁺ in the eye disc leads to a reduced eye phenotype, indicating that expression levels are important (Punzo, 2004).

Because of its variable expressivity, the ey² mutant has been considered to be a hypomorph. However, neither ey mRNA nor Ey protein can be detected in ey² eye discs and the embryonic eye-anlagen. These findings strongly suggest that ey² is a null mutation with respect to eye development. Thus, the variable eye size observed in ey² flies may be due to redundant functions of ey and toy. This interpretation is supported by recent analyses of ey and toy mutants, which indicates partial complementation between the two genes. It has been shown that toy cannot induce ectopic eyes in a strongly selected ey² background with a high penetrance and expressivity of the eyeless phenotype. However, the data presented in this study show that higher expression levels of Toy protein are capable of inducing ectopic eyes in an ey² background. This is in line with the finding that both ey and toy directly activate sine oculis (so) by binding to its so10 enhancer, so that the prior activation of ey by toy is not absolutely required for initiation of the genetic cascade leading to eye development (Punzo, 2004).

To determine more precisely how the two Drosophila Pax6 proteins achieve functional specificity, the PDs and the C termini of both proteins were swapped. The C-terminal domains of Ey and Toy differ considerably, implying functional differences between the two proteins. This is suggested by the fact that it was possible to induce ectopic eyes on the antenna only when the C terminus of ey was present within the Pax6 protein, suggesting that the PD does not play a decisive role in this respect. Thus, the Ey-CT may interact with a different subset of transcription factors and co-factors to increase DNA-binding specificity, functional activity and transactivation potential. Interestingly, on the Western blot of the chimeric constructs, all of the proteins harboring the Ey-CT show a more diffuse band than the proteins harboring the Toy-CT. This is typically seen for phosphorylated proteins and suggests that ey function may also be regulated post-transcriptionally through multiple phosphorylation sites. At the CT of Pax6, there are two highly conserved domains that are present in SEy and Toy but absent from Ey, which may account for the observed differences in function (Punzo, 2004).

The results obtained on the C terminus complement previous findings on ey and toy, where it has been shown that the same binding site (e.g. so10 enhancer) can be bound by both, but depending either on the cellular context, the presence of co-factors, protein kinases or phospatases, the activity of ey and/or toy may be modulated in order to obtain the correct cellular response (Punzo, 2004).

eyDeltaPD can downregulate Dll expression at the transcriptional level in an ectopic situation leading to leg truncation, whereas toyDeltaPD and seyDeltaPD do not, even though all three HDs have the same amino acids at positions conferring DNA-binding specificity. These functional differences between Ey and Toy most likely reside in the CT of Ey, which differs significantly from that of Toy and SEy. Although previous findings have shown that DNA binding of the HD is required for the downregulation of Dll, the C terminus of Ey appears to confer the functional specificity of the Dll repression (Punzo, 2004).

Several lines of evidence point to the fact that the induction of Dll is not directly controlled by ey but rather by a secondary late event of postmitotic differentiation. (1) In ey² mutants Dll is normally not expressed. Only in very rare cases do those mutants show a transdifferentiation from eye to antenna. (2) Over expression of P35 in ey² mutants does not lead to Dll induction until the third larval stage when differentiation sets in. Those Dll-expressing cells reside at the posterior tip of the eye disc, where differentiation starts with the onset of MF movement. (3) Rescuing the ey² mutant by eyDeltaHD leads to normal eye development and not to uniform up-regulation of Dll. Only in rare cases was Dll found to be expressed in those eye discs and in even fewer cases showed antenna like outgrowth. These results are in line with the clonal analysis, where only a small percentage of clones show induction of Dll, but no clone displays an adult eye phenotype. Thus, only rarely might the size of Dll-expressing clones be big enough the lead to a transdifferentiation. Additionally, the ability of toy to function redundantly to ey may account for those observations. (4) The co-expression experiment of the various ey constructs with P35 in the ey² mutant strongly suggests that only the repression of Dll is ey dependent, not the induction, since P35 in conjunction with eyDeltaPD, which does not initiate eye development, completely abolishes antenna duplication. Antenna duplications are observed only in those rare cases where P35, in conjuction with eyDeltaHD, does not rescue eye development and thus fails to instruct the cells to enter the eye developmental pathway (Punzo, 2004).

Taken together, these findings suggest that expressing a PD-containing Pax6 protein is sufficient to prevent Dll activation. By contrast, the Ey-HD clearly confers downregulation of Dll. A more profound study with double mutant clones of ey and toy, preventing the presence of any Pax6-PD containing protein, may be more conclusive. The downregulation of Dll by ey may be direct or indirect, but the activation is ey independent. Other studies have shown that dpp is required for the activation of Dll in the antenna primodium. This may explain why in the absence of ey, Dll is activated only in cells located in or behind the MF that fail to differentiate to photoreceptors, cells that have already seen dpp and reside therefore normally in the posterior part of the eye disc or within the range of dpp signaling (Punzo, 2004).

Dll repression is shown to be required in the normal eye disc to prevent antennal development and to install the eye development program. The failure to repress Dll in the eye primordia leads to a transdetermination from eye to antennal structures, and the formation of an additional antenna in the eye field. The downregulation of Dll in the eye region of the eye-antennal discs depends on the Ey-HD and the Ey-CT, whereas the Ey-PD (and the PD of toy) are required to install the eye development program, mainly by activation of the subordinate target genes (Punzo, 2004).

The results strongly suggest that the functional differences between ey and toy are not only due to their different DNA-binding specificities and changes in the cis-regulatory sequences of their PDs, but also to interactions with different co-factors through their C termini. Recent studies have shown that the transcriptional activator Pax5 is converted into a repressor by interaction with the groucho protein through its C terminus and its octapeptide. Similarly, the Ey-CT, which differs strongly from that of Toy, is likely to interact with a different set of co-factors to confer specific activation or repression of target genes. This hypothesis is supported by the analysis of the CT. Only the Ey-CT, and not that of Toy, is capable of inducing ectopic eyes on the antenna, and only the Ey-HD with an Ey-CT is able to confer DLL repression, which is required for normal eye development. Thus, these experiments provide new insights into the evolutionary divergence of the two Pax6 genes in Drosophila, and their role in eye and head development (Punzo, 2004).

A direct functional antagonism of proboscipedia and eyeless in Drosophila head development

Diversification of Drosophila segmental and cellular identities requires the combinatorial function of homeodomain-containing transcription factors. Ectopic expression of the mouthparts selector proboscipedia (pb) directs a homeotic antenna-to-maxillary palp transformation. It also induces a dosage-sensitive eye loss that was used to screen for dominant Enhancer mutations. Four such Enhancer mutations were alleles of the eyeless (ey) gene that encode truncated Ey proteins. Apart from eye loss, these new eyeless alleles led to defects in the adult olfactory appendages -- the maxillary palps and antennae. In support of these observations, both ey and pb were seen to be expressed in cell subsets of the prepupal maxillary primordium of the antennal imaginal disc, beginning early in pupal development. Transient co-expression is detected early after this onset, but is apparently resolved to yield exclusive groups of cells expressing either Pb or Ey proteins. A combination of in vivo and in vitro approaches indicates that Pb suppresses Ey transactivation activity via protein-protein contacts of the Pb homeodomain and Ey Paired domain. The direct functional antagonism between Pb and Ey proteins suggests a novel crosstalk mechanism integrating known selector functions in Drosophila head morphogenesis (Benassayag, 2003).

To better understand the relationship between Pb and Ey in normal development, the phenotypic effects of ey mutations were studied in the sensitized HSPb^sy genetic context (ectopic expression of pb). Two copies of the HSPb^sy transgene (the sensitizing condition) showed no marked effect. In contrast, pharate adult females with 2x HSPb^sy and homozygous for ey^JD showed strong maxillary palp and antennal defects. In some cases, the maxillary palp, whose identity is indicated by the distinctive distal bristles, remains adjoined to the antennal appendage. However, differentiation of the proboscis (which likewise depends on pb function) is not affected. Thus in the sensitized context, ey mutations can provoke strong defects of the maxillary and antennal appendages. The phenotype suggests that ey⁺ may participate in partitioning imaginal disc cells into antenna and maxillary palp during morphogenesis (Benassayag, 2003).

To confirm a role of eyeless in this process, HSPb^sy was removed from the genetic background to examine the effects of the new eyeless mutations alone. All four ey alleles appear recessive in a non-sensitized background as shown for ey^JD, and can be interpreted as loss-of-function mutations in accord with their molecular lesions. All give homozygous escapers with visible defects, allowing for the composition of an allelic series, from weakest to strongest: ey¹¹>ey^D1Da>ey^EH>ey^JD. Analysis of the phenotypes of hemizygotes with Df(4)BA led to the same conclusion (Benassayag, 2003).

ey^JD homozygotes display eye reduction or loss, low viability and strong brain defects associated with abnormal behavior. Furthermore, a minority of surviving ey^JD homozygotes (10%-20%, after outcrossing) show alterations in the size and/or shape of maxillary palps and antennae. The altered maxillary palps of ey^JD homozygotes still harbor the two characteristic sensilla trichodea, suggesting that maxillary identity per se is not affected. Reduced, malformed maxillary palps are often accompanied by enlarged, misshapen antennae. Similar although weaker defects are likewise detected for ey^EH homozygotes, as well as for certain trans-heterozygous combinations with other Enhancer alleles in the sensitized background. The reciprocal effect of ey^JD on appendages that derive from the same antennal imaginal disc constitutes evidence of a potential role for ey in apportioning the maxillary portion of this disc. The defects observed in ey^JD homozygotes appeared stronger than in the hemizygous combination, ey^JD/Df(4)BA. Thus, the truncated protein may have a limited antimorphic character not detected in the presence of wild-type protein (Benassayag, 2003).

Although mutant phenotypes implicated both pb and now ey in maxillary development, no gene expression had been detected in the maxillary portion of the antennal disc in the third instar larvae. Pb and Ey expression were examined later, during the prepupal stage when maxillary and antennal structures evaginate from the eye-antenna imaginal disc. Using a rabbit anti-Pb serum directed against the C-terminal region (anti-E9), Pb accumulation was detected in the central part of the maxillary primordium beginning approximately eight hours after puparium formation, during evagination of the antennal and maxillary appendages from the composite disc. Ey protein as visualized by a rabbit anti-Ey serum accumulates in the same primordium and, within discrimination, at the same time. This expression appears to be limited to the borders of the primordium, rather than the center as for Pb. Results of tests for co-expression of the two proteins were mitigated: in situ hybridization or immunostaining experiments were inconclusive, whereas available antibodies that gave acceptable signals in this tissue were both rabbit polyclonal antisera. To address whether endogenous pb and ey patterns in the maxillary palps may overlap, a pb-GAL4 mini-gene was used based on descriptions of the pb-promoter region. Using a pb-GAL4 driver insertion to direct ß-galactosidase expression (pb-GAL4>UAS-lacZ), the patterns of pb>lacZ and ey expression were examined by double-immunofluorescence labeling and confocal microscopy. Early Pb expression is limited to a small number of cells in the distal maxillary primordium. At this stage, Ey expression can likewise be detected in a small group of cells partially overlapping those expressing Pb. Co-expression appears to be very limited in the progression of a dynamic pattern. In later prepupae, the expression patterns of pb>lacZ and ey in the maxillary primordium are adjacent but exclusive. Taken together, these data show a previously undisclosed co-temporal expression of both pb and ey in the maxillary primordium of prepupae, and support an ephemeral co-expression of these genes in a small number of cells. This is in agreement with the known function of pb in maxillary determination, and with the newly established function of ey in this tissue (Benassayag, 2003).

Thus, ctopically expressed homeotic Pb protein, even a form bereft of DNA-binding capacity, can suppress eye development in a dose-sensitive manner. Genetic and molecular results indicate a central role for direct contacts between conserved domains of the Hox selector protein Pb and the eye selector Ey. One physiological situation where the interaction between pb and ey is likely to be relevant was identified, based on their genetic interaction, mutant phenotypes and expression patterns in forming the adult antennae and maxillary palps (Benassayag, 2003).

This work has identified a previously unrecognized role for ey in the development of the maxillary palps and antennae. The mutation employed for most of these experiments, ey^JD, behaves as a strong allele affecting viability, formation of the adult eyes and brain mushroom bodies, but also of antennal and maxillary differentiation. Consistent with a late requirement for ey in the antennal disc, ey expression in the maxillary primordium appears in early stages of metamorphosis when both eye-antennal discs have fused, and the antennal and maxillary appendages start to evaginate. After evagination, the maxillary primordium migrates to join the labial disc in forming the adult mouthparts, whereas the antennal primordium remains near the eye. When a contiguous epidermal cell layer has been completed, the head sac is abruptly evaginated under the internal pressure. Maxillary ey expression in early stages of prepupal metamorphosis is limited to the boundary between the maxillary primordium and the antenna, and ey mutant phenotypes often involved simultaneously reduced palps and enlarged antennae. These reciprocal effects are consistent with communicating cell populations, suggesting that ey may contribute to a partitioning of the antennal disc permitting the establishment of two separate appendages. Further analysis of this process will require new maxillary-specific markers permitting the fates of these cells to be followed (Benassayag, 2003).

Starting from a dose-sensitive eye loss provoked by ectopic Pb, new ey alleles isolated as eye loss Enhancers were identified. These mutations reveal a role for ey, and a potential biological relevance for this Hox-PAX6 interaction, in the development of the antennal and maxillary sensory palps. ey loss-of-function defects in the sensory palps are exacerbated by Pb^sy. The most direct interpretation of the enhanced ey loss-of-function phenotype with HSPb^sy is that the newly isolated alleles retain a partial function that can be negated by adequate Pb levels. The molecular characterization of these alleles is consistent with this hypothesis, because all four alleles should encode truncated proteins that contain most or all of the interacting PD (Benassayag, 2003).

To better understand the in vivo relationship between these two selector genes, attempts were made to examine the effects of double mutants for pb and ey. Although homozygotes for pb^- or for the new ey mutants showed viabilities of up to 50% compared with heterozygotes, a double mutant adult was never obtained for any of the four ey alleles. This result, although suggesting that the double mutant is synthetic lethal, does not offer insight into the tissue(s) implicated in this lethality (Benassayag, 2003).

One tissue in which an interaction is clearly indicated from this analysis is the maxillary palp primordium, where a dynamic expression was detected of Ey and of Pb (directly or via the pbGAL4 driver) during pre-pupal development. Transient early co-expression of Pb and Ey in pre-pupae is limited to a small number of cells, whereas later expression appears exclusive. This result can be rationalized in two ways: first, co-expressing cells might be rapidly eliminated by apoptosis, through a coordinate gene-activation process triggered by a Hox-Pax dimer; second, co-expression of the Ey and Pb transcription factors could induce a developmental pathway interference resulting in a G1 cell-cycle arrest. These possibilities are not fully exclusive. Indeed, one or both mechanisms could serve to refine the boundaries between antennal and maxillary cell populations within the antennal disc (Benassayag, 2003).

In vertebrates, Pax6 has multiple known or inferred roles in eye, brain and nasal development. Apart from the fly eye, several groups have identified an eyeless function required for development of the mushroom bodies, neural structures important for olfactory perception and learning. This study describes a specific role for Ey in concert with Pb in the maxillary and antennal appendages; both of these are derived from the antennal disc and constitute the adult olfactory system. An analysis of mutations producing headless flies has revealed a role for Drosophila Pax6 in head morphogenesis and thereby suggests a requirement of ey for the development of all structures derived from eye-antennal discs. These studies involve mutations truncating the Ey protein, which induces head defects. Interestingly, because these truncated Ey proteins still contain the PD, the fact that the phenotypes obtained reflect an allele-specific antimorphic effect of the PD cannot be excluded. Taken together, these results strongly suggest that eyeless, apart from its known role in eye morphogenesis, may also play multiple other roles in head formation (notably for brain and olfactory sensory systems) (Benassayag, 2003).

The development of olfactory and visual systems has several common features in Drosophila. Both systems are derived from the composite eye-antennal imaginal disc. Moreover, both have similar signal transduction pathways and appear to share regulatory networks. However, when the expression of ey, so, eya and dac was examined in the pre-pupal maxillary primordium, only ey expression was detected. This observation suggests that ey acts there via a distinct combinatorial code of regulatory genes compared with eye development. One possibility proposed in this study is that ey activity is modulated by other co-factors or transcription factors whose activity is likely to be sensitive to Pb. In this light, it is worth noting that other Enhancer mutations isolated also similarly affect maxillary palps, either singly or in combination with ey. It will be of fundamental interest to better understand the molecular basis for how a single protein might function in multiple, distinct networks (Benassayag, 2003).

The ey mutants studied here were identified as dominant enhancers of pb-induced eye reduction. Consistent with the antagonism observed in vivo, Ey and Pb proteins interact directly in vitro, via the Ey Paired domain and the Pb homeodomain. This interaction with Pb that renders Ey unable to activate its downstream target genes can be extended to other homeotic genes because Antp, Scr, Ubx, abdA and AbdB repress eye development while increasing apoptosis in the eye disc, and their protein products likewise interact in vitro with Ey protein. This suggests a combinatorial interaction of homeodomain-containing proteins (Hox and Pax) to specify a given body segment. An inhibition through physical association has been proposed between Pax6 and En-1 during eye development in quail, and between Pax3 and Msx1 for muscle development in chicken. Moreover, a similar inhibitory mechanism involving a Hox protein HD has been reported in vertebrates; in contrast, physical interaction with Hox-B1 protein leads to increased Pax6 activity in Hela cells, raising the possibility that additional context-dependent partners modulate the action of Hox-Pax combinations to generate functional diversity. Based upon genetic and molecular data, it is proposed that variations on a PD-HD interface can serve to mediate combinatorial or hierarchical functional relationships among Hox and Pax genes in normal development (Benassayag, 2003).

The results presented here appear to favor a specific role for discrete protein-protein interactions rather than an indirect interference mechanism. Indeed, (1) by analysing the residues of Pb protein involved in its homeotic function, a Pb^sy protein was identified with diminished DNA binding but still able to inhibit eye development; (2) using this mutant in a genetic screen to isolate Pb functional partners, four independent eyeless mutations were isolated, all of them leading to a shortened Ey protein; (3) genetic interaction tests showed that Pb^sy-induced eye loss is highly sensitive to levels of ey function but independent of several other eye-determining genes including eyg, eya or so, and (4) ectopically expressed Pb interferes with ey activity in the eye imaginal disc by inhibiting so and eya activation without affecting ey transcription or Ey accumulation (Benassayag, 2003).

In conclusion, these results suggest that a specific Hox/Pax interaction between Pb and Ey is involved in a normal developmental process defining the boundary between the antenna and maxillary palp. More generally, the formation of such protein couples could afford a sensitive and delicate measure of the balance of Pax6 level, permitting a finely tuned integration to generate distinct transcriptional outputs during development (Benassayag, 2003).

Differential requirements for the Pax6(5a) genes eyegone and twin of eyegone during eye development in Drosophila

In eye development the tasks of tissue specification and cell proliferation are regulated, in part, by the Pax6 and Pax6(5a) proteins respectively. In vertebrates, Pax6(5a) is generated as an alternately spliced isoform of Pax6. This stands in contrast to the fruit fly which has two Pax6(5a) homologs that are encoded by the eyegone and twin of eyegone genes. This study set out to determine the respective contributions that each gene makes to the development of the fly retina. Both eyg and toe are shown to encode transcriptional repressors that are expressed in identical patterns but at significantly different levels. A molecular dissection of both proteins shows that Eyg makes differential use of several domains when compared to Toe and that the number of repressor domains also differs between the two Pax6(5a) homologs. It is predicted that these results will have implications for elucidating the functional differences between closely related members of other Pax subclasses (Yao, 2008).

An initial analysis of transcriptional patterns indicates that both Pax6(5a) genes are expressed in identical patterns within the retina. However, eyg is expressed at a much higher level than toe. Not surprisingly, while mutations in eyg nearly delete the eye, a reduction in toe via miRNA treatments has no effects on its own. Simultaneous reductions in both genes, in contrast, result in a 'headless' phenotype. Using a set of mini genetic screens and activator/repressor fusion assays, both proteins are demonstrated function as transcriptional repressors. In total, these characteristics suggest that eyg and toe might play redundant roles in during development (Yao, 2008).

However, the high level of sequence divergence within the non-DNA binding domains hints that their functions may only be partially redundant. This study set out to molecularly dissect both Pax6(5a) proteins and determine what, if any, differences exist between the activities of each protein. In two experimental contexts no such differences between eyg and toe exist were found. First, a comparison of eyg and toe loss-of-function phenotypes indicated that toe played a greater role in the development of the thorax than the eye. Second, forced expression of both full-length proteins throughout the developing fly identified 43 different instances in which expression of one Pax6(5a) gene induced a different phenotype than the other. Taken together, these results hint that the roles of eyg and toe may be not be completely redundant (Yao, 2008).

Studies were carried out to discover which domain(s) might account for the differences seen in loss-of-function mutants and forced expression assays. A set of deletion and chimeric proteins were generated to dissect the requirement for each domain as well as the level of functional conservation. Attempts were made to rescue eyg¹ mutants as well as generate extra eye fields with these protein variants. The results indicate that Eyg and Toe make differential use of several domains. Many of these differences map to the non-DNA binding domains. One possible mechanism for this is that Toe has only one repressor domain, while Eyg has two. The prediction is that the differences in the non-DNA binding domains are the primary determinants of how each Pax6(5a) protein will influence development. It is less likely that the two DNA binding domains functionally distinguish one protein from another, since there is an extremely high level of sequence conservation within these motifs. Thus the model for how Eyg and Toe function is that both transcription factors bind to similar target genes but can differentially influence transcription through differing levels of repressor activity and/or interactions with disparate binding partners (Yao, 2008).

These results may have broad implications for the activities of other Pax genes in both Drosophila and vertebrates. The fly genome contains two Pax6 genes, eyeless (ey) and twin of eyeless (toy), both of which also arose through a relatively recent duplication. Both share high degrees of homology within the DNA binding domains while having significantly lower levels of sequence conservation in the non-DNA binding regions. Functionally, Ey and Toy have differing abilities to induce eye formation when expressed in non-retinal tissues. Some of these differences have been attributed to the C-terminal tail section of each protein (Yao, 2008).

Mammalian Pax genes are grouped, in part, according to their structure. Individual classes are defined by the presence or absence of the octapeptide and the two DNA recognition (Paired and Homeobox) motifs. Like the fly genes, members within each Pax subclass share a very high degree of sequence conservation within the DNA binding domains thus they are likely to bind to very similar targets. The current results, if extended to these other Pax genes, would suggest that their activity could be distinguished by examining the localization of activation and repressor domains as well as the use of different binding partners (Yao, 2008).

Drosophila nemo promotes eye specification directed by the retinal determination gene network

Drosophila nemo (nmo) is the founding member of the Nemo-like kinase (Nlk) family of serine-threonine kinases. Previous work has characterized nmo's role in planar cell polarity during ommatidial patterning. This study examined an earlier role for nmo in eye formation through interactions with the retinal determination gene network (RDGN). nmo is dynamically expressed in second and third instar eye imaginal discs, suggesting additional roles in patterning of the eyes, ocelli, and antennae. Genetic approaches were used to investigate Nmo's role in determining eye fate. nmo genetically interacts with the retinal determination factors Eyeless (Ey), Eyes Absent (Eya), and Dachshund (Dac). Loss of nmo rescues ey and eya mutant phenotypes, and heterozygosity for eya modifies the nmo eye phenotype. Reducing nmo also rescues small-eye defects induced by misexpression of ey and eya in early eye development. nmo can potentiate RDGN-mediated eye formation in ectopic eye induction assays. Moreover, elevated Nmo alone can respecify presumptive head cells to an eye fate by inducing ectopic expression of dac and eya. Together, these genetic analyses reveal that nmo promotes normal and ectopic eye development directed by the RDGN (Braid, 2008).

This study describes novel roles for nmo in early eye patterning that are distinct from its known role in planar polarity during late larval development. The RDGN is composed of a highly complex cascade of positive feedback loops. The fundamental refinement of this delicate system is apparent from the dramatic defects resulting from reducing or ectopically expressing even a single component. Through loss-of-function and misexpression analyses, genetic evidence is provided that nmo contributes to patterning events orchestrated by the RDGN during eye development (Braid, 2008).

Co-expression of the RD genes is spatially and temporally regulated and confers cellular identity through the consequential formation of selector complexes. For example, So and Eya complex to activate dac expression. Subsequently, Dac can complex with So or Eya to direct expression of complex-specific gene targets. In addition, Ey and So complex to activate ato in cells entering the MF. Repression of ey in, and posterior to, the MF limits this interaction to the pro-neural cells. Spatio-temporal regulation of the RD genes is imperative for normal eye and head development, given the deleterious effects of their misexpression on normal eye development. It has been proposed that the availability and relative concentrations of these cofactors affect which protein-protein complexes form. As such, misexpression of the RD genes alters the pool of available cofactors, resulting in mis-specification of cell fate (Braid, 2008).

Interestingly, reducing any of the eye-specification factors also results in patterning defects, culminating in cell death and loss of tissue. Thus, reducing an RD factor may be analogous to its misexpression since the relative levels of RD factors are similarly perturbed, leading to abnormal development and hyperactivation of apoptosis. The data support such a model, since loss of nmo restores eye- and head-patterning defects associated with loss of ey and eya, as it does with early misexpression of these genes. The ey and eya alleles used in this study are not nulls and therefore may retain some level of activity. These interactions imply that reducing nmo can modulate the transcriptional output of RD complexes, restoring developmental integrity. Moreover, inhibiting apoptosis with co-expression of the caspase-inhibitor p35 did not phenocopy this rescue, further supporting the hypothesis that Nmo may contribute to eye development by affecting the activity of RD selector complexes rather than by generally promoting cell death (Braid, 2008).

Although driving nmo throughout the eye disc in all stages of development with ey-Gal4 has minimal effects on its own, and misexpression of ey or eya causes only small eyes, the combined presence of Nmo and Ey or Nmo and Eya is not compatible with eye and head development. This dramatic synergy, together with the rescue mediated by reducing nmo, is consistent with a model in which Nmo affects the function of one or more of the RD cofactors, thereby affecting the balance of selector factors. This study established that Nmo does not regulate Ey, so, Eya, or Dac levels in somatic clones, supporting the hypothesis that the observed genetic interactions occur at the protein level. Whether nmo is itself regulated by the RDGN is yet to be determined (Braid, 2008).

The context-specific nature of Nmo's role in mediating RD activity was revealed in the ectopic eye induction assay. Misexpression of ey using dpp-Gal4 not only induced ectopic eyes in the antennal, wing, and leg discs, but also interfered with endogenous eye development. Ectopic nmo rescued the dorso-ventral reduction in dpp>ey compound eyes, suggesting that Nmo promotes eye development. It further implies that Nmo may differentially affect Ey activity through cell-specific factors, since early co-expression of nmo with ey>ey had the converse effect, resulting in ablation of the eye and head. Spatial restriction of cofactors to achieve different outcomes is a common developmental strategy. nmo's dynamic pattern of co-expression with Ey, and their complementary expression in the third instar eye and head fields, respectively, supports the hypothesis that Nmo may promote early Ey activity to specify the eye field, while later contributing to patterning of the eye field by antagonizing Ey (Braid, 2008).

Using ectopic eye induction assays, Nmo's contribution to eye development was investigated in cells expressing exogenous Ey, Eya, and Dac. Endogenous nmo potentiates the induction of ectopic eyes in the antennal disc, as well as in the leg and wing. Interestingly, it was found that loss of nmo restricts the ability of Ey, more than Eya or Dac, to induce ectopic eyes. Ey is most potent inducer of ectopic eyes as it can effectively activate transcription of the downstream RD targets. Eya, Dac, and So are much less effective in ectopic eye assays because their transactivating potential is limited by the number of available RD cofactors. Thus, it is expected that misexpressed ey would have the least requirement for nmo in the dpp>ey assay. This finding suggests that Nmo may contribute to deployment of the RDGN by Ey, since cells with exogenous Eya or Dac more readily compensate for loss of endogenous nmo than Ey in the induction of ectopic eyes (Braid, 2008).

The most convincing evidence for Nmo's role in early eye specification is Nmo's ability to respecify a specific set of head cells as retinal cells when misexpressed alone. Importantly, these are the same subsets of cells able to be transformed by ectopic expression of RD genes and Tsh, which induces ey expression. Ectopic eyes induced by other factors such as Optix or Eyegone (Eyg), which promote eye specification through Ey-independent mechanisms, occur in different subsets of cells. This study determined that dac and eya are inappropriately activated in cells transformed by misexpressed nmo. It is tempting to speculate that ectopic Nmo perturbs the basal protein-protein interactions that normally repress them, resulting in deployment of the RDGN in the head primordia. Consistent with this model, loss of Hth was observed in cells ectopically expressing dac. (Braid, 2008).

The ectopic eye induction assay has been utilized to determine epistasis among the RD factors. Although loss of Hth was observed in dpp>3xnmo wing discs, this repression does not culminate in activation of any of the retinal genes. This is consistent with clonal analyses that demonstrate that nmo is not required for expression of the RD genes in the eye disc. Moreover, Nmo antagonizes Dpp and Wg signaling in the wing disc, both of which contribute to regulation of hth expression in the wing hinge. Thus, the observed loss of Hth in dpp>3xnmo eye and wing discs may be the result of different mechanisms. For example, elevated Nmo may promote Eya function to repress hth in the antennal disc. Repression of Hth is not sufficient to deploy the RDGN; therefore Nmo requires the presence of an unidentified factor in the antennal disc to activate eye development (Braid, 2008).

This study showed that nmo is not required for expression of Ey, so, Eya, or Dac or the secreted morphogen dpp. In the eye disc, Wg actively represses eya, so, and dac to antagonize progression of the eye field and promote head development. It has been previously showed that nmo is an inducible feedback inhibitor of Wg signaling in the wing imaginal disc. Although nmo expression is not coincident with wg in the ME during eye development, it was important to verify that the observed genetic interactions between Nmo and the RDGN are not due to repression of Wg signaling. Using mutant clonal analysis, it was confirmed that, as in the wing, Wg levels are unchanged in both somatic and flp-out nmo clones. Furthermore, no change was observed in Wg activity as assayed by stabilization of cytoplasmic Arm. These observations are consistent with a previous study indicating that nmo does not modulate Arm stability in the eye imaginal disc. It will be interesting to determine what unidentified factors are affected by loss of nmo, and how they contribute to patterning of the eye field (Braid, 2008).

Novel targets and modes of regulating RDGN activity are rapidly emerging. Recent studies have expanded the repertoire of transcriptional targets regulated by specific RD complexes beyond the scope of the RDGN itself. Moreover, additional proteins have been identified that modify activity of the canonical retinal factors by various mechanisms. For example, Ey acts as a transcriptional activator when bound to So. However, Ey represses the very same target genes when complexed to Tsh and Hth. Alternatively, the So-Eya interaction is physically inhibited when So is in complex with the transcriptional corepressor Groucho (Gro). In addition, Distal antenna (Dan) and Distal antenna related (Danr) were recently identified as retinal factors that complex with Ey and Dac to promote retinal specification through activation of ato. Whether Nmo directly modulates RDGN output through protein-protein interactions that alter the stoichiometry of available RD cofactors (through post-translational modification of their activity by phosphorylation or indirectly by interactions with noncanonical RDGN regulators) is being investigated. Further characterization of the molecular interactions between Nmo and the RD factors will aid in understanding how cells integrate multiple signals to achieve a specific outcome (Braid, 2008).

Cell migration in Drosophila optic lobe neurons is controlled by eyeless/Pax6

In the developing Drosophila optic lobe, eyeless, apterous and distal-less, three genes that encode transcription factors with important functions during development, are expressed in broad subsets of medulla neurons. Medulla cortex cells follow two patterns of cell movements to acquire their final position: first, neurons are arranged in columns below each neuroblast. Then, during pupation, they migrate laterally, intermingling with each other to reach their retinotopic position in the adult optic lobe. eyeless, which encodes a Pax6 transcription factor, is expressed early in progenitors and controls aspects of this cell migration. Its loss in medulla neurons leads to overgrowth and a failure of lateral migration during pupation. These defects in cell migration among medulla cortex cells can be rescued by removing DE-Cadherin. Thus, eyeless links neurogenesis and neuronal migration (Morante, 2011).

Medulla cortex cells indeed originate from the OPC. In adults, there are ~800 'columns' in the medulla, each formed by more than 60 different cell types that repetitively contact every R7-R8 fascicle (Morante, 2008). The spatial segregation of ey-, ap- and dll-positive cells observed early in larval brain lobes is lost during pupal development through two modes of cell movements happening during larval (radial) and pupal life (mostly lateral). As a result, each adult 'column' in wild-type medulla is composed of the precise complement of neurons necessary to process visual information. Each R7/R8 termination pair is surrounded by at least 47 different cell types, while 13 other cell types do not appear to contact PRs (Morante, 2008). Further experiments will determine whether the cell movements shown by medulla cortex cells during optic lobe organogenesis involve the dispersion of the entire neuron, or only the cell body (Morante, 2011).

ey-, dll- and ap-positive neurons originating from the OPC-derived neuroblasts show a radial organization, while the number of medulla neurons expands. This organization resembles the radial units present in the embryonic mammalian neocortex where cells reach their appropriate layer via radial migration along glial processes, expanding the size of the cerebral cortex . However, it is unlikely that there is active radial migration in the medulla, as neurons appear to be simply displaced by newly formed neurons above them (Morante, 2011).

The lack of eyeless causes an increase in neuroblast proliferation and impedes cells from reaching their final position in the medulla. It should be noted that some ey^DN cells still manage to disperse in the medulla cortex. They might represent cells that express ey-Gal4 but do not express or require ey for their migration, or cells that express lower levels of ey-Gal4 in which ey^DN is not sufficient to affect function (Morante, 2011).

The effect of ey^DN was examined on eye imaginal discs, and the same phenotype was observed as that reported in a previous study; expression of ey^DN resulted in underproliferation of eye imaginal disc cells and a partial or complete loss of eye structure, which is similar to what is seen in eyeless mutants. Thus, the effects of ey^DN in the eye disc are, surprisingly, the opposite of what was observed in medulla neuroblasts (Morante, 2011).

Furthermore, to address a possible role of eyeless in postmitotic cells, ey^DN was mis-expressed using an act-FRT-stop-FRT-Gal4 construct both in larvae and in postmitotic cells (after P25). This mis-expression resulted in lethality, even with very short heat-shocks, probably owing to the strength of the act-Gal4 driver; this precludes analysis in adults. Thus, at this point, a possible role of eyeless in postmitotic cells cannot be eliminated (Morante, 2011).

Interestingly, Sey mice embryos, which are mutant for the mouse ortholog of ey (Pax6), exhibit an increase in the number of Cajal-Retzius cells, which migrate tangentially. Simultaneously, late-born cortical precursors show abnormalities in their patterns of radial migration, forming heterotopic clusters in the path towards their final position in the embryonic cerebral cortex. Although this defective neuronal radial migration has been proposed to be due to an altered radial glia morphology, other studies have shown that these clusters exhibit an increase in adhesion molecules in neurons. Biochemical studies have demonstrated the presence of binding sites for the Pax6 transcription factor in the NCAM and L1 promoter. Thus, increased proliferation or adhesion among neurons in the clusters could prevent Sey mutant cells from detaching from each other to reach their final position in the cerebral cortex (Morante, 2011).

Therefore, development of medulla neurons of the optic lobe resembles the neurogenesis and migration mechanisms observed during the development of the embryonic mammalian brain (Morante, 2011).

Analysis of the transcriptomes downstream of Eyeless and the Hedgehog, Decapentaplegic and Notch signaling pathways in Drosophila melanogaster

Tissue-specific transcription factors are thought to cooperate with signaling pathways to promote patterned tissue specification, in part by co-regulating transcription. The Drosophila melanogaster Pax6 homolog Eyeless forms a complex, incompletely understood regulatory network with the Hedgehog, Decapentaplegic and Notch signaling pathways to control eye-specific gene expression. This study reports a combinatorial approach, including mRNAseq and microarray analyses, to identify targets co-regulated by Eyeless and Hedgehog, Decapentaplegic or Notch. Multiple analyses suggest that the transcriptomes resulting from co-misexpression of Eyeless+signaling factors provide a more complete picture of eye development compared to previous efforts involving Eyeless alone: (1) Principal components analysis and two-way hierarchical clustering revealed that í Eyeless+signaling factor transcriptomes are closer to the eye control transcriptome than when Eyeless is misexpressed alone; (2) more genes are upregulated at least three-fold in response to Eyeless+signaling factors compared to Eyeless alone; (3) based on gene ontology analysis, the genes upregulated in response to Eyeless+signaling factors had a greater diversity of functions compared to Eyeless alone. Through a secondary screen that utilized RNA interference, it was shown that the predicted gene CG4721 has a role in eye development. CG4721 encodes a neprilysin family metalloprotease (see Neprilysin4) that is highly up-regulated in response to Eyeless+Notch, confirming the validity of the approach. Given the similarity between D. melanogaster and vertebrate eye development, the large number of novel genes identified as potential targets of Ey+signaling factors will provide novel insights to understanding of eye development in D. melanogaster and humans (Nfonsam, 2012; full text of article).

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