InteractiveFly: GeneBrief

dachshund: Biological Overview | Regulation | Developmental Biology | Effects of Mutation | Evolutionary Homologs | References

BIOLOGICAL OVERVIEW

Visual motion detection in sighted animals is essential to guide behavioral actions ensuring their survival. In Drosophila, motion direction is first detected by T4/T5 neurons. Their axons innervate one of the four lobula plate layers. How T4/T5 neurons with layer-specific representation of motion-direction preferences are specified during development is unknown. This study shows that diffusible Wingless (Wg) between adjacent neuroepithelia induces its own expression to form secondary signaling centers. These activate Decapentaplegic (Dpp) signaling in adjacent lateral tertiary neuroepithelial domains dedicated to producing layer 3/4-specific T4/T5 neurons. T4/T5 neurons derived from the core domain devoid of Dpp signaling adopt the default layer 1/2 fate. Dpp signaling induces the expression of the T-box transcription factor Optomotor-blind (Omb), serving as a relay to postmitotic neurons. Omb-mediated repression of Dachshund transforms layer 1/2- into layer 3/4-specific neurons. Hence, spatio-temporal relay mechanisms, bridging the distances between neuroepithelial domains and their postmitotic progeny, implement T4/T5 neuron-subtype identity (Apitz, 2018).

Visual signals received by the retina are generally not stationary because objects in the environment and/or the bodies of animals move. To detect motion, visual circuits perform complex spatio-temporal comparisons that convert luminance changes collected by photoreceptors into signals containing information about direction or speed. Despite the seemingly divergent anatomy of vertebrate and insect visual systems, they display remarkable parallels in the computations underlying motion vision and the neuronal elements performing them. In most sighted animals, this involves neurons that respond to motion signals in specific directions. Direction-selectivity emerges from differences in the connectivity of their dendrites. Motion-direction preferences by their axons are represented by layer-specific innervation. Thus, anatomical characteristics such as layer-specificity seem to be intricately linked with motion-directionality. However, how these are implemented during circuit development is poorly understood (Apitz, 2018).

The Drosophila visual system has emerged as a powerful model for elucidating the neural circuits and computations underlying motion detection. Photoreceptors (R-cells) in the retina extend axons into the optic lobe consisting of the lamina, medulla, lobula plate, and lobula. Neuronal projections in these ganglia are organized into retinotopically arranged columnar units. The medulla, lobula plate, and lobula are additionally subdivided into synaptic layers. They are innervated by more than a 100 neuronal subtypes that extract different visual features in parallel pathways. T4 and T5 lobula plate neurons are the first direction-selective circuit elements. Each optic lobe hemisphere contains ~5300 T4/T5 neurons. T4 dendrites arborize within medulla layer 10, and T5 dendrites in lobula layer Lo1. Their axons project to one of the four lobula plate layers, thereby defining four different neuron subtypes each. Axons segregate according to their motion-direction preferences. Thus, front-to-back, back-to-front, upward, and downward cardinal motion directions are represented in lobula plate layers. T4 neurons are part of the ON motion detection pathway reporting brightness increments, while T5 neurons are part of the OFF pathway reporting brightness decrements. Distinct neuron sets in the lamina and medulla relay ON and OFF information to T4 and T5 neurons. Direction-selectivity emerges within T4/T5 dendrites and involves the non-linear integration of input from these upstream neurons for enhancement in the preferred direction and suppression in the null-direction. Dendritic arbors of the four T4 neuron subtypes have characteristic orientations, that correlate with the direction preferences of lobula plate layers innervated by their axons. Thus, direction-selectivity involves the establishment of neuron subtypes, each with distinct spatial connectivities. This study addresses when and how T4 and T5 neuron subtypes with different layer identities are specified during development (Apitz, 2018).

Optic lobe neurons originate from two horseshoe-shaped neuroepithelia, called the outer and inner proliferation centers (OPC and IPC). These are derived from the embryonic optic lobe placode and expand by symmetric cell divisions during early larval development. At the late 2nd instar larval stage, neuroepithelial (NE) cells from the medial OPC edge begin to transform into medulla neural stem cells, called neuroblasts (Nbs). These undergo asymmetric divisions to self-renew and give rise to ganglion mother cells (GMCs), which divide to generate two neurons or glia. Apposing the OPC, two dorsal and ventral NE domains, called the glial precursor cell (GPC) areas, produce neuron subtypes associated with all ganglia. At the mid 3rd instar larval stage, the lateral OPC begins to generate lamina neurons (Apitz, 2018).

The IPC generates lobula and lobula plate neurons, including T4/T5 neurons from the early 3rd instar larval stage onward. Recent studies showed that NE cells in one domain, the proximal (p-)IPC, convert into progenitors in an epithelial-mesenchymal transition (EMT)-like process. Progenitors migrate to a second proliferative zone, the distal (d-)IPC, where they mature into Nbs. These transition through two competence windows to first produce C and T neurons, corresponding to C2 and C3 ascending neurons connecting the medulla and lamina, as well as T2/T2a and T3 neurons connecting the medulla and lobula, and then T4/T5 lobula plate neurons. Cross-regulatory interactions between Dichaete (D) and Tailless (Tll) control the switch in Nb competence defined by the sequential expression of the proneural bHLH transcription factors Asense (Ase) and Atonal (Ato). The latter is co-expressed with the retinal determination protein Dachshund (Dac). The molecular mechanisms that control layer-specific T4/T5 neuron subtype identities within this sequence of developmental events occurring at different locations have remained elusive (Apitz, 2018).

T4/T5 neuron diversity resulting in differential layer-specificity could be achieved by postmitotic combinatorial transcription factor codes upstream of distinct guidance molecules. Although not mutually exclusive, layer-specificity of T4/T5 neurons could also be determined by temporal differences in the expression of common postmitotic determinants, similar to the birth-order dependent R-cell growth cone segregation strategy described in the medulla. This study provides evidence for another mechanism, whereby layer-specific T4/T5 neuron subtype identity is determined early in the p-IPC neuroepithelium. Their specification depends on two relay mechanisms involving Wnt and Bone morphogenetic protein (Bmp) signaling and transcription factor interactions. These establish and translate the spatial patterning of NE cells into postmitotic neuronal subtype identities to bridge distances inherent to this particular neurogenesis mode (Apitz, 2018).

The spread of Wg is dispensable for patterning of many tissues. However, this study uncovered a distinct requirement for diffusible Wg in the nervous system, where it orchestrates the formation of T4/T5 neurons innervating lobula plate layers 3/4. Their generation depends on inductive mechanisms that are relayed in space and time. The spatial relay consists of a multistep-signaling cascade across several NE domains: Wg from the GPC areas induces wg expression in the s-IPC and Nb lineage adjacent to ventral and dorsal p-IPC subdomains; this secondary Wg source activates dpp expression. Dpp signaling mediates EMT of migratory progenitors from these subdomains. The p-IPC core produces Dac-positive layer 1/2 specific T4/T5 neurons. Dpp signaling in p-IPC NE subdomains triggers a temporal relay across intermediate cellular states by inducing omb. Omb in turn suppresses Dac, conferring layer 3/4 identity to postmitotic T4/T5 neurons (Apitz, 2018).

When Wg is membrane-tethered, the first step of this cascade is disrupted. This defect is not caused by decreased signaling activity of NRT-Wg protein in wg{KO;NRT-wg} flies. First, wild-type Wg signaling activity inside the GPC areas and the adjacent OPC was not affected. Second, in allele switching experiments, ectopic expression of a highly active UAS-NRT-wg transgene in the GPC areas was unable to rescue. By contrast, restoring wild-type wg function in the GPC areas was able to rescue, supporting the notion that Wg release and spread from the GPC areas are required to induce its own expression in the s-IPC and the Nb clone (Apitz, 2018).

Although Wg release is essential, the range of action is likely limited. Wg expression in the s-IPC commences in early 3rd instar larvae, when it is still in close proximity with the GPC. Half of the wg{KO;NRT-wg} flies showed residual dpp expression in one progenitor stream at the 3rd instar larval stage and a 25% reduction of T4/T5 neurons, correlating with three lobula plate layers in adults. The other half lacked dpp-lacZ expression and showed a 50% reduction of T4/T5 neurons correlating with two remaining layers. While this partial phenotypic penetrance is not fully understood, NRT-Wg likely partially substituted for Wg because of the initial close proximity of the GPC areas and the s-IPC and Nb clone. Occasional residual NRT-Wg expression in the s-IPC argues against an all-or-nothing inductive event and suggests a model, whereby cell-intrinsic signaling thresholds have to be reached. Theoretically, the dpp expression defect in the p-IPC of wg{KO;NRT-wg} flies could reflect the dependence on long-range Wg from the GPC areas. However, as this study has shown, IPC-specific wg knockdown leads to dpp loss in the p-IPC. Propagation of sequential Wnt signaling could explain long-range activities. Moreover, sequentially acting primary and secondary sources of Wg have been described in the developing Drosophila eye, suggesting that the regulatory mechanism observed in the optic lobe might be employed in several contexts. The different outcomes of early and late allele wg to NRT-wg allele switching indicate that Wg secretion is required for the induction but not long-term maintenance of wg expression in the s-IPC. The GPC areas become rapidly separated from the s-IPC and Nb clone by compact rows of newly generated neurons. As part of a relay system, diffusible Wg may therefore be required to bridge distances over a few cell diameters during the initial phase of neurogenesis. The s-IPC in wg{KO;NRT-wg} flies expressed Hth and generated two neuron clusters as in wild-type. Thus, the sole function of wg in the s-IPC is to relay the GPC-derived Wg signal to induce dpp expression in the p-IPC. Since Wg release is not required in the GPC areas to induce dpp in the adjacent OPC, this secondary wg function in the s-IPC is most likely juxtacrine (Apitz, 2018).

Compared to approximately 80 medulla neuron subtypes derived from the OPC, the specification of 13 distinct subtypes originating from the p-IPC appears simple. However, the distinct mechanisms employed are surprisingly complex. Previous work has shown that cross-regulatory interactions between D and tll regulate a Nb competence switch from generating early-born C2, C3, T2, T2a, and T3 neurons to eight distinct layer-specific T4/T5 subtypes. Ato and Dac are expressed in the second Nb competence window and depend on tll. Functional studies showed that dac mutant T4/T5 neurons adopted early-born T2/T3 neuron-like morphologies. Similarly, ato mutant T4/T5 neurons displayed neurite connectivity defects. Notably, simultaneous knockdown of dac and ato resulted in the absence of T4/T5 neurons, demonstrating that both are required together for the ability of d-IPC Nbs to produce new neuron subtypes in the second competence window (Apitz, 2018).

Dac is initially expressed in all T4/T5 neurons but only maintained in layer 1/2 innervating subtypes. This suggests that an essential step for the specification of layer 3/4 innervating neurons is the downregulation of Dac and the suppression of the T4/T5 default neuron fate, i.e., layer 1/2 identity. Although the mode of this inhibitory mechanism depends on the outcome of the Nb-specific switching mechanism in the d-IPC, it is already primed in p-IPC NE cells. Thus, layer-specificity and therefore motion-directionality are determined early in the NE precursors of T4/T5 neurons. Molecularly, it involves the Omb-mediated relay of Dpp-signaling-dependent NE cell patterning information across intermediate cell states to postmitotic T4/T5 neurons resulting in the repression of Dac. In contrast to the OPC, this study found no link between NE patterning in the p-IPC and Notch-dependent differential apoptosis of region-specific T4/T5 subtypes. Instead, Notch controls the choice between T4 and T5 identity, likely during the second competence window, indicating that the distinction between layer 1/2 and 3/4 fates precedes T4 and T5 neuron specification (Apitz, 2018).

The mechanisms controlling the maintenance of omb expression, and Omb-mediated downregulation of Dac are unclear. Hypotheses regarding the latter have to be reconciled with the fact that dac, together with ato, is required for the formation of all T4/T5 neurons and hence is expressed in all d-IPC Nbs during the second competence window. Omb and Dac are initially co-expressed in Nbs and young T4/T5 neurons, suggesting that Omb does not directly repress dac transcription. Yet, expression of the dacp7d23 enhancer trap Gal4 line showed that dac is only transcribed in layer 1/2 neurons in adults. A possible scenario is that Omb could break Dac autoregulation by triggering degradation of Dac. Since T-box genes can act as transcriptional activators and repressors and their effects are influenced by various co-factors, future studies will need to explore the molecular details underlying Omb-mediated repression of Dac. It will also be important to determine whether layer 3/4 specification is mediated solely by Dac downregulation, or whether omb has additional instructive roles (Apitz, 2018).

Consistent with the observation that C2 and C3 neurons have distinct developmental origins, this study found that Nbs derived from the Dpp-expression domain produce C2 and possibly T2a neurons during the first Nb competence window, while the core p-IPC generates C3, T2, and T3 neurons. dac mutant T4/T5 neurons adopt T2/T3-like morphologies suggesting that this is the default neuron fate in this neuron group. While Omb is maintained in C&T neurons derived from the Dpp-expression domain, Dac is not expressed, suggesting that Omb interacts with other molecular determinants in these neurons. While this study did not explore how layer 1 and 2 neurons or layer 3 and 4 neurons become distinct from each other because of the lack of specific markers, the data suggest a possible contribution of Ato/Dac and Notch signaling, as these are active within the d-IPC. Findings in a concurrent study of Pinto-Teixeira (2018) align with the current data concerning the role of Dpp and Notch signaling. Furthermore, a second study of Mora (2018) reported an additional role for Ato in controlling the transient amplification of d-IPC Nbs by symmetric cell division to ensure that the correct number of T4/T5 neurons is produced. It will be fascinating to identify the transcriptional targets of Notch, Ato/Dac, and Omb that mediate ganglion- and layer-specific targeting of T4/T5 dendrites and axons, respectively. Finally, future behavioral studies of layer 3/4-deficient flies will address to what extent direction selectivity is affected or compensatory mechanisms are in place (Apitz, 2018).

Signaling centers, also called organizers, pattern tissues in a non-autonomous fashion. The vertebrate roof plate and the cortical hem, for instance, both release Wnts and Bmps to pattern NE cells in the developing dorsal spinal cord and in the surrounding forebrain, respectively. In the Drosophila visual system, the GPC areas express wg and pattern the OPC by inducing dpp expression in adjacent dorsal and ventral OPC subdomains. Together with the current insights into the function of GPC-derived wg in IPC patterning and neurogenesis, this firmly establishes the GPC areas as local organizers of optic lobe development. At the onset of neurogenesis, wg is first expressed in the GPC areas followed by the s-IPC, explaining the well-established delay in neurogenesis between the IPC and OPC. Wg release from the GPC areas could coordinate the timely onset of neurogenesis in the OPC and IPC to safeguard the alignment of matching partner neurons across several retinotopically organized neuropils. The intercalation of new-born neurons between both neuroepithelia may have driven the need for a relay system using primary and secondary sources of Wg. Wg induces Dpp to subdivide the adjacent OPC and p-IPC NE into distinct regions as basis for generating neuronal diversity. The temporal relay mediated by Omb represents an efficient strategy to pass the memory of spatial NE patterning information by Dpp signaling on to postmitotic neurons generated at a distance. It is thus intricately tuned to the distinct neurogenesis mode of the p-IPC essential for spatially matching birth-order-dependent neurogenesis between the OPC and IPC. Interestingly, the progressive refinement of NE patterning by the induction of secondary signaling centers plays a central role in vertebrate brain development. Furthermore, similar signaling cascades have been recently identified in mammalian optic tissue cultures where sequential Wnt and Bmp signaling induces the expression of the Omb-related T-box transcription factor Tbx5 to specify dorsal retinal NE cells. Hence, such cascades could represent conserved regulatory modules that are employed repeatedly during invertebrate and vertebrate nervous system development (Apitz, 2018).

Widespread activation of developmental gene expression characterized by PRC1-dependent chromatin looping

Polycomb repressive complexes 1 and 2 have been historically described as transcriptional repressors, but recent reports suggest that PRC1 might also support activation, although the underlying mechanisms remain elusive. This study shows that stage-specific PRC1 binding at a subset of active promoters and enhancers during Drosophila development coincides with the formation of three-dimensional (3D) loops, an increase in expression during development and repression in PRC1 mutants. Dissection of the dachshund locus indicates that PRC1-anchored loops are versatile architectural platforms that persist when surrounding genes are transcriptionally active and fine-tune their expression. The analysis of mammalian RING1B binding profiles and 3D contacts during neural differentiation in mice suggests that this role is conserved in mammals (Loubiere, 2020).

Polycomb group proteins (PcG) assemble into two main epigenetic complexes called Polycomb repressive complexes 1 and 2 (PRC1 and PRC2), which are highly conserved across metazoans and collaborate at multiple levels to maintain their target genes in a repressed state. PRC1 also binds a subset of active promoters and enhancers devoid of the PRC2-mediated H3K27me3 repressive mark in both Drosophila and mammals. Loss-of-function experiments suggest that PRC1 might contribute to the transcriptional activation of a subset of genes. However, since PRC1 binds to a large number of sites, disentangling direct from indirect regulatory effects has been proven difficult, and the molecular mechanisms that might support transcriptional activation by PRC1 are obscure. This study tested whether PRC1 might mediate gene activation by forming enhancer-promoter loops, in addition to the repressive chromatin loops that were previously described (Loubiere, 2020).

PRC1 plays important roles during normal physiology and in cancer, but how it might play a dual silencing and an activating role is a matter of great interest. The data provided in this study suggest that, rather than behaving as transcriptional repressors, PRC1-mediated loops establish versatile architectural platforms that can induce repression and activation. In the absence of transcription factors, PRC1 might cooperate with PRC2 to establish repressive loops and to form silent Polycomb domains, whereas the binding of developmental transcription factors might exploit PRC1-dependent enhancer-promoter contacts to coordinate the timely induction of cognate genes during development. The net effect of looping appears to be gene specific since expression of the CG5888 gene is more sensitive to disruption of a PRC1-dependent loop than the neighboring dac gene. At dac, one of the PRC1 binding sites is within a few hundred base pairs (bp) from the TSS, whereas the PRC1-binding site closest to the two alternative CG5888 alternative promoters is located, respectively, around 16 and 25 kb away. One possibility is therefore that the relative location of regulatory elements might modulate the effects of PRC1-dependent loops. In addition to the role in 3D looping, recent work has shown that PRC1 might assist transcription by modulating occupancy and phosphorylation of RNA polymerase II, as well as association of the pausing-elongation factor Spt5 to enhancers and promoters. In future studies, it will be important to analyze the relative role and the interplay of these mechanisms in individual cells at the onset of silencing, as well as during transcriptional activation to understand the chain of molecular events that triggers this dual function for Polycomb-bound regions (Loubiere, 2020).

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).

Earlier studies of Dachshund

The mutant phenotype of dachshund inspired its gene name: the legs of mutant flies, in comparison to wild-type and in relation to body length (as in the breed of dog), are extremely short. The wild-type leg is composed of ten discrete segments. Moving from proximal (nearest the body) to distal, they are: the coxa, trochanter, femur, tibia, plus five tarsal segments and the claw. While the proximal and distal morphology of these segments in dac mutant legs appears to be normal, the intermediate segments are fused and condensed. Upon eclosion from the pupal cases, these mutants are unable to locomote normally; if allowed, they will quickly fall into their food and die. However, if kept away from wet medium, dac homozygotes can remain, unable to move about, for several days before dying, presumably from dehydration. These helpless homozygotes are able to flail their misshapen legs, albeit to no avail, indicating that at least a portion of the leg neuromusculature develops normally and is functional (Mardon, 1994).

The expression pattern of dachshund during larval leg disc development is consistent with the mutant phenotype of the adult leg. The leg imaginal disc is composed of concentric folds of epithelia such that the outermost portions of the disc give rise to the most proximal segments (i.e. the coxa) in the adult leg while the most distal structures are derived from the central portion of the disc. dachshund is expressed specifically in the presumptive epithelium that is fated to give rise to the femur, tibia and proximal tarsal segments, the same structures most severely affected in dac mutant legs. Although the imaginal discs from dac mutants appear morphologically normal, a significant increase in cell death in dac mutant leg discs is apparent. Elongation, the outgrowth of the leg disc that forms the extended structure of the adult leg, does not occur in mutant flies. Thus there is a failure of morphogenesis in dac mutant leg discs during larval and early pupal development (Mardon, 1994).

dachshund was originally isolated as a dominant suppressor of the Ellipse mutation of the Epidermal growth factor receptor. Ellipse is a hypermorphic allele of Egfr, a dominant hyperactive receptor function that causes a rough eye phenotype. Mutations of the Egfr gene prevent normal spacing and differentiation of photoreceptor cells in the developing eye. These results suggest that loss-of-function mutations in dac, which produce the defective leg phenotype, reduce the eye specific activity of the hyperactive Elp allele of Egfr. The eyes of dac mutant homozygotes are reduced and roughened. In contrast to the compound eye, the external morphology of the adult ocelli appears normal in all dac mutants (Mardon, 1994).

Photoreceptor development is prevented in dachshund mutant eye discs, suggesting a role for dac in ommatidial assembly. Since dac is a nuclear protein, it may be a candidate for regulating the Egfr gene, which is required for normal photoreceptor determination. A highly variable, but reduced number of photoreceptors is present in mutant eyes and just a few Elav positive clusters are formed. Elav is normally expressed in all neurons, and its expression is indicative of the degree of neural maturation of photorecepter precursors. dachshund does not appear to be required specifically for neural differentiation, instead dac function is required for normal movement of the morphogenetic furrow. In the absence of furrow movement, cells in dac mutants fail to adopt a neural fate, remain in an undifferentiated state and eventually die (Mardon, 1994).

The morphogenetic furrow, is a self propagating line of shortened cells that coordinates gene expression and initiation of neural differentiation in the developing eye. dachshund is required for the initiation of the furrow and for the normal movement of the furrow across the eye disc. Clonal analysis reveals that dac is cell autonomously required for initiation of movement of the away from the posterior margin of the eye disc where the furrow originates. This may reflect a direct requirement for dac in furrow initiation (Mardon, 1994).

Dac may have an even more fundamental role in eye development (see Specification of the eye disc primordium and establishment of dorsal/ventral asymmetry). Ectopic expression of dac is sufficient to induce ectopic retinal development in a variety of tissues, including the adult head, thorax and legs. This result is similar to that observed with Drosophila's pax6 homolog, eyeless. The external morphology of dac-induced ectopic eyes closely resembles that of normal adult eyes. Three independent results suggest that dac functions downstream of eyeless:

1. Misexpression of eyeless in imaginal discs is sufficient to induce ectopic dac expression.
2. Targeted eyeless expression is unable to induce ectopic eye formation in a dac null mutant, again suggesting that eyeless cannot compensate for the absence of dac. If eyeless functions downstream of dac ectopic eyeless should be able to direct eye differentiation in the absence of dac.
3. eyeless is still expressed in dac mutant eye discs, demonstrating that dac is not essential for eyeless expression.

These results suggest that dac functions downstream of eyeless and, considering that dac can induce ectopic retinal development, are constent with the idea thad dac may be a direct target of eyeless. If this is true, then dac can also function in eyeless independent pathways, as in the leg, for example. This result points to the likelihood that dac functions in independent combinatorial pathways and highlights the complexity of interactions that may characterize some gene action in development (Shen, 1997).

Since dac is able to induce ectopic eyeless, dac can function as a positive regulator of eyeless. However, neither gene is able to induce photoreceptor in all cells in which it is expressed. For example, ectopic dac expression is unable to efficiently induce retinal development along any part of the A-P compartment boundary of the wing disc. Both dac and eyeless are unable to act alone in the control of gene expression or retinal cell-fate specification. Instead, these genes are likely to require other factors that are expressed in a spatially or temporally restricted pattern during development. Genes acting early in retinal development are potential candidates for such factors, including sine oculis, coding for a homeodomain protein, and clift/eyes absent, coding for a novel protein that like Dachshund, acts in multiple tissues (Shen, 1997).

REGULATION

Promoter

Regulation of the retinal determination gene dachshund in the embryonic head and developing eye of Drosophila

Drosophila eye development is controlled by a conserved network of retinal determination (RD) genes. The RD genes encode nuclear proteins that form complexes and function in concert with extracellular signal-regulated transcription factors. Identification of the genomic regulatory elements that govern the eye-specific expression of the RD genes will allow a better understanding of how spatial and temporal control of gene expression occurs during early eye development. Conserved non-coding sequences (CNCSs) between five Drosophilids were compared along the ~40 kb genomic locus of the RD gene dachshund (dac). This analysis uncovers two separate eye enhancers, in intron eight and the 3' non-coding regions of the dac locus, defined by clusters of highly conserved sequences. Loss- and gain-of-function analyses suggest that the 3' eye enhancer is synergistically activated by a combination of eya, so and dpp signaling, and only indirectly activated by ey, whereas the 5' eye enhancer is primarily regulated by ey, acting in concert with eya and so. Disrupting conserved So-binding sites in the 3' eye enhancer prevents reporter expression in vivo. These results suggest that the two eye enhancers act redundantly and in concert with each other to integrate distinct upstream inputs and direct the eye-specific expression of dac (Anderson, 2006).

The smallest fragment in the 3' dac eye enhancer that can respond to dpp, eya and so is 3EE^{194 bp}, which is centered around two CNCS blocks of ~40 bp and 20 bp. These two CNCS blocks are also common to all active fragments of the 3' eye enhancer. These two evolutionarily conserved stretches were scanned for known, genetically upstream transcription factor binding sites. The 40 bp conserved stretch contains two putative consensus So-binding sites, S1-5'-CGATAT and S2-5'-CGATAC, compared with the consensus 5'-(C/T)GATA(C/T) described previously. Each of these putative So-binding sites in 3EE were mutated individually and in combination to test their requirement for normal enhancer activity in vivo. Mutation of individual So-binding sites causes a severe reduction, but not complete elimination, of enhancer activity in vivo. However, simultaneous mutation of both So binding sites completely abolishes enhancer activity in vivo. These results, coupled with loss-and gain-of-function analyses with dpp, eya and so, suggest that So binds to the 3' eye enhancer directly and nucleates a protein complex that includes Eya to regulate 3EE. However, despite much effort using a wide variety of binding conditions, it was not possible to demonstrate specific, direct binding of So protein to oligos that contain these So-binding sites. The 5' eye enhancer, which has four CNCS blocks, were scanned for potential upstream transcription factor binding sites and no strong candidate binding sites were found within the CNCS blocks (Anderson, 2006).

Loss- and gain-of-function analyses with the two eye enhancers suggest that each enhancer is regulated by a distinct set of protein complexes. The 5' eye enhancer is activated by a combination of ey, eya and so, but is not activated by Dpp signaling. 5EE is activated by ectopic ey expression even in eya and so mutants, suggesting that it is regulated exclusively by ey. However, somewhat paradoxically, expression of 5EE, the intron 8 enhancer, is lost in eya and so mutants even though ectopic expression of a combination of dpp, eya and so does not activate this enhancer. Furthermore, driving high levels of ey in so¹ mutant eye discs restores 5EE-lacZ expression. Coupled together, these results suggest that 5EE is primarily regulated by ey but that the regulation of 5EE by ey also requires eya and so (Anderson, 2006).

By contrast, the 3' dac eye enhancer is regulated by a combination of eya, so and dpp signaling, but is not directly dependent on ey. 3EE-GFP expression is lost in eya² and so¹ mutant eye discs, and in posterior margin mad^1-2 mutant clones. Furthermore, ey cannot bypass the requirement for eya and so to activate 3EE. Conversely, 3EE is strongly induced by co-expression of eya and so. Moreover, dpp signaling via the tkv receptor can synergize with eya and so to induce 3EE in ectopic expression assays. Furthermore, neither Mad nor Medea, the intracellular transducers of Dpp signaling, is sufficient to bypass the requirement for activation of the Dpp receptor Tkv in these assays. Thus, it is concluded that events downstream of Dpp-Tkv signaling, such as the phosphorylation of Mad, are essential for the synergistic activation of the 3' dac eye enhancer by eya and so. Taken together, these results suggest that there are distinct requirements for the activation of the 5' and 3' dac eye enhancers. However, the exact nature of the protein complexes that regulate 5EE and 3EE remain to be determined (Anderson, 2006).

Morphogenetic furrow (MF) initiation is completely blocked in posterior margin dac³-null mutant clones. However, dac³ clones that do not include any part of the posterior margin develop and do not prevent MF progression, but do cause defects in ommatidial cell number and organization. This dichotomy in dac function is reflected in the two eye enhancers characterized in this study. Analysis of dac⁷ homozygotes demonstrates that the 3' eye enhancer is dispensable for MF initiation and progression. It is proposed that in dac⁷ mutants, the intact 5EE enhancer is sufficiently activated by ey to drive high enough levels of dac expression to initiate and complete retinal morphogenesis. However, dac⁷ mutants have readily observable defects in ommatidial organization. Thus, it is further proposed that this lack of normal patterning in dac⁷ mutants is most likely due to the loss of 3EE, which normally acts in concert with 5EE after MF initiation, to integrate patterning inputs from extracellular signaling molecules such as Dpp with tissue-specific upstream regulators such as ey, eya and so. However, it is not known if the 3' eye enhancer is sufficient to initiate dac expression in the absence of the 5' eye enhancer (Anderson, 2006).

Based on the results, a two-step model is proposed for the regulation of dac expression in the eye. First, the initiation of dac expression in the eye disc is dependent on Ey binding to 5EE. However, Ey is fully functional only when So and Eya are present. It is possible that Ey recruits So and Eya to 5EE, but a model is favored in which Ey bound to 5EE cooperates with an So/Eya complex bound to 3EE to initiate dac expression in the eye. After initiation of the MF, dac expression is maintained by an Eya and So complex bound to 3EE. In addition, 3EE can integrate patterning information received via dpp signaling, thereby allowing the precise spatial and temporal expression of dac in the eye. This two part retinal enhancer ensures that dac expression is initiated only after ey activates eya and so expression. Thus, the dac eye enhancers provide a unique model with which the sequential activation of RD proteins allows the progressive formation of specialized protein complexes that can activate retinal specific genes (Anderson, 2006).

The redundancy in dac enhancer activity also explains the inability to isolate eye-specific alleles of dac, despite multiple genetic screens. The modular nature of the two enhancers and their potential ability to act independently or in concert suggest that both enhancers must be disrupted to block high levels of transcription of dac. Thus, two independent hits in the same generation, a phenomenon that occurs infrequently in genetic screens, would be required to obtain an eye-specific allele in dac (Anderson, 2006).

Despite much investigation, very few direct targets of RD proteins, especially for Eya and So, have been identified. One study suggests that So can bind to and regulate an eye-specific enhancer of the lz gene. However, lz is not expressed early during eye development and is required only for differentiation of individual cell types. The results suggest that regulation of dac expression occurs via the interaction of two independent eye enhancers that are likely to be bound by Ey, Eya and So, and respond to dpp signaling. This analysis of the 3' eye enhancer suggests that two putative conserved So-binding sites are essential for 3EE activity in vivo. Mutation of individual So-binding sites dramatically reduces, but does not completely eliminate, reporter expression in the eye. Mutating both predicted So-binding sites completely blocks enhancer activity in vivo. Thus, it is concluded that So binds to 3EE via these conserved binding sites. However, it has not been possible to demonstrate a direct specific interaction of either So alone or a combination of Eya and So with oligos that contain these putative So-binding sites in vitro. It is possible that other unidentified proteins are required for stabilizing the Eya and So complex. Furthermore, the 194 bp fragment that responds to ectopic expression of dpp, eya, and so contains no conserved or predicted Mad-binding sites. This raises the intriguing possibility that dpp signaling activates other genes, which then directly act with eya and so to regulate the 3' eye enhancer. Alternatively, a large complex that includes Eya, So and the intracellular transducers of dpp signaling, such as Mad and Medea, may be responsible for activation of 3EE. Similarly, the results suggest that the 5' eye enhancer is regulated primarily by ey. However, it is unclear whether Ey directly binds 5EE. Furthermore, Ey is fully functional only in the presence of Eya and So. Thus, Ey either independently recruits Eya and So into a 5' complex or is activated by virtue of its proximity to the So/Eya complex bound to the 3' enhancer or both (Anderson, 2006).

The exact order and dynamics of protein complex assembly at 5EE and 3EE requires further investigation. However, the two dac eye enhancers are extremely useful tools with which to investigate fundamental issues about the mechanism of RD protein action. One significant issue concerns the mechanism of Eya function during eye development. Eya consists of two major conserved domains, an N-terminal domain that has phosphatase activity in vitro and a C-terminal domain that can function as a transactivator in cell culture assays. So contains a conserved Six domain and a DNA binding homeodomain. However, it is unclear if Eya provides phosphatase activity, transactivator function, or both, in this complex. Characterization of the components of the protein complexes that regulates dac expression may uncover the targets of Eya phosphatase activity during eye development. Thus, the isolation of two eye enhancers with distinct regulation provides very useful tools with which to study protein complex formation and function during Drosophila retinal specification and determination (Anderson, 2006).

Transcriptional Regulation

Dachshund and the eye/antennal disc

Three independent results suggest that dachshund functions downstream of eyeless: (1) Misexpression of ey in the antennal, leg and wing imaginal discs is sufficient to induce ectopic dac expression in all discs. These results suggest that EY positively regulates dac expression. (2) Targeted expression of ey is unable to induce ectopic eye formation in a dac mutant background. (3) ey is expressed in a mutant dac background, indicating that dac is not required for ey expression (Shen, 1997).

Dachshund and eyes absent proteins form a complex and function synergistically to induce ectopic eye development in Drosophila

The eyeless, dachshund, and eyes absent genes encode conserved, nuclear proteins that are essential for eye development in Drosophila. Misexpression of eyeless or dachshund is also sufficient to induce the formation of ectopic compound eyes. Like ey and dac, targeted expression of eya alone is sufficient to induce ectopic eye formation. However, in contrast to ey, the penetrance of the ectopic eye phenotype induced by either dac or eya alone is incomplete and, when induced, such eyes are small. When dac expression was strongly induced in all imaginal discs, ectopic eye development was observed only on the anterior surface of the fly head ventral to the antenna, in just 56% (61/109) of animals examined. In contrast to the low penetrance of ectopic eye formation induced by dac or eya expressed alone, coexpression of dac and eya induces substantial ectopic eyes on the head, legs, wings, and dorsal thorax of 100% of animals examined. On the head, the cuticle between the normal eye field and antennae is transformed into retinal cells such that the normal retinal field is expanded. Large patches of pigment are induced on the dorsal surface of the femur and tibia of all legs, which are severely truncated. Ectopic eya alone can induce small patches of glass expression in the pouch area of the wing disc with 25% penetrance. In no case has ectopic Glass staining been observed in leg discs with either dac or eya alone. However, when dac and eya are coexpressed, ectopic Glass staining is induced with 100% penetrance along the ventral margin of the eye-antennal disc, the dorsal half of the leg disc along the anterior-posterior compartment (A/P) boundary, and along the A/P boundary of the dorsal wing disc. In each case, the sites of ectopic glass expression in discs correspond to the positions of ectopic retinal development observed in adults. Taken together, these data demonstrate that dac and eya show strong genetic synergy to induce ectopic retinal development in Drosophila (Chen, 1997).

Ectopic Elav-positive cells are induced in the antennal, leg, and wing discs, in response to dac and eya coexpression, suggesting ectopic neural differentiation. These ectopic neurons must be photoreceptor cells, since the visual system-specific Glass protein is also induced in the same pattern. Ectopic eyes observed in adults corresponding to these positions contain all of the normal cell types associated with the wild-type eye, including pigment cells, lens-secreting cone cells, and interommatidial bristles. The ectopic neurons induced by dac and eya misexpression send out axonal projections. The axons of ectopic photoreceptors in the eye-antennal disc form a bundle that extends posteriorly into the eye imaginal disc. These axons appear to fuse with the axon tracts sent out by photoreceptors of the normal retinal field that exit through the optic stalk to synapse in the brain. It is likely, therefore, that the fly can perceive light through ectopic photoreceptors formed in the eye-antennal disc as a result of dac and eya coexpression. Coexpression induces ectopic dpp expression in the eye-antennal disc adjacent to the field of ectopic photoreceptors. In the leg disc, dpp expression is split and forms a ring around the ectopic photoreceptors, again suggesting that an ectopic MF is initiated and propagates (Chen, 1997).

While eya expression in the eye disc does not depend on dac function, dac expression is greatly reduced in an eya2 mutant background, demonstrating that dac expression requires eya activity. Similarly, eyeless (ey) induction of ectopic dac expression is greatly reduced in an eya2 mutant background. These results suggest that dac may function downstream of eya. Consistent with this interpretation, eya is unable to induce ectopic eye formation in a dac mutant background. eyeless misexpression is sufficient to induce eya, suggesting that eya may be required for ey function. Indeed, ectopic retinal development driven by targeted ey expression fails to occur in an eya2 mutant background. Induction of eya expression by ey does not depend on dac activity, consistent with the idea that eya functions downstream of ey but upstream of dac. However, these genes do not act in a simple, linear pathway; targeted expression of dac and eya strongly induce the expression of one another, and eya is required for ectopic eye induction by dac. Misexpression of dac or eya is also sufficient to induce ectopic ey expression in the antennal disc. These results suggest that multiple positive-feedback loops exist among these genes during normal eye development and raises the possibility that ey may be required for ectopic retinal induction by eya and dac. Indeed, ectopic eye formation driven by coexpression of dac and eya is completely blocked in an eyeless2 mutant background, indicating that induction of ey is essential. It is proposed that a conserved regulatory network, rather than a linear hierarchy, controls retinal specification and involves multiple protein complexes that function during distinct steps of eye development (Chen, 1997).

Signaling by the TGF-beta homolog decapentaplegic functions reiteratively within the network of genes controlling retinal cell fate determination in Drosophila

Retinal cell fate determination in Drosophila is controlled by an interactive network of retinal determination (RD) genes, including eyeless, eyes absent, sine oculis and dachshund. The role of decapentaplegic in this pathway was investigated. During eye development, while eyeless transcription does not depend on dpp activity, the expression of eyes absent, sine oculis and dachshund are greatly reduced in a dpp mutant background. dpp signaling acts synergistically with, and at multiple levels within, the retinal determination network to induce eyes absent, sine oculis and dachshund expression and ectopic eye formation. These results suggest a mechanism by which a general patterning signal such as Decapentaplegic cooperates reiteratively with tissue-specific factors to determine distinct cell fates during development (Chen, 1999).

During ectopic photoreceptor determination there is a tight correlation between the location of ectopic eyes and the endogenous pattern of dpp expression. In particular, the dpp-GAL4 driver is the most efficient means of retinal induction by any of the RD genes: ubiquitous eyeless (ey) expression induces downstream genes only in the vicinity of the anteroposterior (AP) compartment boundary of discs where dpp is normally expressed. These results suggested that dpp signaling may be essential for the RD genes to specify retinal cell fates. dpp is normally expressed along the AP boundary of the larval wing disc. The GAL4 line 30A drives gene expression in a ring that surrounds the wing pouch, which will become the wing blade in the adult. The 30A ring pattern corresponds to tissue that will form the hinge of the adult wing and overlaps endogenous dpp at only two spots. When ey is misexpressed using 30A-GAL4, ectopic eye formation is induced only at two positions: dorsal and ventral to the pouch at the AP boundary. One explanation for this phenomenon is that dpp activity is essential for ey to induce ectopic eye development. Coexpression of dpp and ey is sufficient to expand the domain of ectopic retinal development induced by ey alone. To test whether dpp and ey act synergistically to induce RD genes, mRNA levels of ey, eya, so and dac were measured in a dpp loss-of-function background. ey is normally expressed throughout the entire eye disc prior to MF initiation and anterior to the furrow during MF progression. In dpp mutants, the eye-antennal disc is much smaller than in wild-type due to a proliferation defect, and MF initiation and photoreceptor development does not occur. Nevertheless, EY mRNA is still detectable in dpp mutant eye discs throughout second and third instar larval development. In contrast, although eya is still expressed in the ocellar region, almost no EYA, SO or DAC mRNA is detected in dpp mutant eye discs prepared from second or third instar larvae. These data indicate that dpp is not essential for ey expression but is required upstream of eya, so and dac in the eye disc (Chen, 1999).

If eya and dac are the primary downstream targets of dpp during eye development, then it should be possible to bypass the requirement for dpp and induce ectopic eye formation by overexpressing ey with eya or dac. While targeted expression of either eya or dac alone driven by 30A-GAL4 is unable to induce photoreceptor development, strong synergistic induction of ectopic eye formation is observed when ey is coexpressed with either dac or eya. Although there is clear synergy between ey and dac or eya, ectopic photoreceptor induction in both imaginal discs and adults is still limited to the vicinity of the AP boundary and the source of dpp signaling. Moreover, photoreceptor differentiation is still restricted to the vicinity of the AP boundary when ey, dac, eya and so are simultaneously induced by 30A-GAL4, indicating that dpp and ey must regulate other essential targets in this process (Chen, 1999).

It is possible that dpp signaling might cooperate directly and exclusively with ey. Alternatively, dpp could interact at multiple levels within this pathway. To distinguish these two models, a test was performed to see whether dpp functions synergistically with eya and so to regulate the expression of dac. No ectopic dac expression is induced by so alone: targeted expression of eya induces ectopic dac expression only at a single ventral spot on the AP boundary of the wing disc when driven by 30A-GAL4. Consistent with the idea that the Eya and So proteins function cooperatively as a complex, strong synergistic induction of dac is observed when eya and so are coexpressed. However, dac expression is still restricted mainly to places where endogenous dpp is present. In contrast, when dpp is coexpressed with eya, strong dac expression is induced all along the ventral-posterior pouch margin Moreover, ectopic Dac is detected around the entire circumference of the wing pouch as a result of dpp, eya and so coexpression. Since coexpression of dpp, eya and so is sufficient to induce dac expression in places where dpp and ey cannot, it is concluded that dpp interacts with the network at multiple levels to control the expression of retinal determination genes. Consistent with this interpretation, no induction of ey transcription could be detected in response to misexpression of dpp, eya and so with 30A-GAL4 (Chen, 1999).

Thus dpp signaling is reiteratively used to regulate gene expression within the retinal cell fate determination pathway in Drosophila. Specifically, dpp signaling enables ey to induce strong eya, so and dac expression in the posterior, but not anterior, wing disc compartment. In contrast, dpp functions synergistically with eya and so to activate the expression of dac in both compartments. This activation of dac expression by dpp, eya and so is unlikely to result from feedback induction of ey for two reasons: (1) targeted expression of ey and dpp is unable to induce dac in the anterior wing disc compartment, and (2) ectopic ey transcription is not detected in response to misexpression of dpp, eya and so driven by 30A-GAL4 in the wing disc. Thus, these data suggest that dpp signaling interacts with the retinal determination pathway at (at least) two levels to regulate RD gene expression. Interestingly, while targeted expression of dpp, eya and so with 30A-GAL4 is unable to induce ey expression or ectopic photoreceptor development in the wing disc, coexpression of eya and so using dpp-GAL4 is sufficient to induce ey expression and photoreceptor development in the antennal disc. These differences most likely reflect the unique transcriptional environments present in the specific portions of each imaginal disc tested in these assays (Chen, 1999).

Targeted expression of teashirt induces ectopic eyes in Drosophila

teashirt was initially identified as a gene required for the specification of the trunk segments in Drosophila embryogenesis and encodes a transcription factor with zinc finger motifs. Targeted expression of teashirt in imaginal discs is sufficient to induce ectopic eye formation in non-eye tissues, a phenotype similar to that produced from targeted expression of eyeless, dachshund, and eyes absent. The expression of so and dac are induced in the antennal disc by the ectopic expression of tsh, suggesting that tsh may act upstream of these genes in eye development. Furthermore, teashirt and eyeless induce the expression of one another, suggesting that teashirt is part of the gene network that functions to specify eye identity (Pan, 1998).

However, these results do not prove that tsh does play a role in specifying the eye identity during normal development. To address this issue, an examination was carried out to see if tsh is expressed at the right time and the right place to have a role in specifying the eye identity. Indeed, TSH mRNA is expressed in the eye disc, with the strongest expression anterior to the morphogenetic furrow. This pattern of expression is similar to that of ey, a gene that is known to play an essential role in specifying eye identity. An examination was carried out to see if loss-of-function mutations of tsh affect eye development. Several weak loss-of-function tsh alleles were examined and no eye defects were found. X-ray-induced mitotic recombination was used to generate mutant clones of a null tsh allele. tsh mutant clones were recovered at a frequency similar to the wild-type control, and sections through the mutant clones revealed a normal ommatidial organization. These data suggest that tsh may play a redundant role during normal eye development, and the requirement for tsh may be masked by other factor(s) that play a role similar to tsh (Pan, 1998).

Morphogenetic furrow initiation and progression during eye development in Drosophila: the roles of decapentaplegic, hedgehog and eyes absent

decapentaplegic mediates the effects of hedgehog in tissue patterning by regulating the expression of tissue-specific genes. In the eye disc, the transcription factors eyeless, eyes absent, sine oculis and dachshund participate with these signaling molecules in a complex regulatory network that results in the initiation of eye development. Analysis of functional relationships in the early eye disc indicates that hh and dpp play no role in regulating ey, but are required for eya, so and dac expression. Ey is expressed throughout the eye portion of the wild-type eye disc during early larval stages, prior to MF initiation. Eya and Dac are expressed throughout the posterior half of the eye imaginal disc, with stronger expression at the posterior margin. Ey is expressed normally in homozygous Mad^1-2 clones that touch the posterior margin and in clones that are positioned internally in the disc, indicating that Dpp signaling is not required for Ey expression prior to MF initiation. In contrast, neither Eya nor Dac is expressed in homozygous Mad^1-2 clones that touch the margin of the eye disc. In addition, Eya and Dac are not expressed, or are expressed weakly, in internal clones that lie well anterior of the posterior margin. However, strong Eya and Dac expression is observed in internal clones that lie within a few cell diameters of the posterior margin. Like Eya and Dac protein, SO mRNA is expressed in the posterior region of the eye disc prior to MF initiation. Mad^1-2 posterior margin clones fail to express so. These results suggest that dpp function is required to induce or maintain Eya, SO and Dac expression, but not Ey expression, at the posterior margin prior to MF initiation. This function is consistent with the pattern of DPP mRNA expression along the posterior and lateral margins at this stage of eye disc development. Whereas dpp is not necessary for Eya and Dac expression in internal, posterior regions of the early eye disc, it does play a role in regulating Eya and Dac expression in internal, anterior regions of the disc. Although DPP mRNA expression does not extend to the very center of the eye disc, it is expressed in a significant proportion of the interior of the disc. The possibility that dpp may regulate gene expression in more central regions may be attributed to the fact that it encodes a diffusible molecule (Curtiss, 2000).

Restoring expression of eya in loss-of-function dpp mutant backgrounds is sufficient to induce so and dac expression and to rescue eye development. Thus, once expressed, eya can carry out its functions in the absence of dpp. These experiments indicate that dpp functions downstream of or in parallel with ey, but upstream of eya, so and dac. Additional control is provided by a feedback loop that maintains expression of eya and so and includes dpp. The fact that exogenous overexpression of ey, eya, so and dac interferes with wild-type eye development demonstrates the importance of such a complicated mechanism for maintaining proper levels of these factors during early eye development. Whereas initiation of eye development fails in either Hh or Dpp signaling mutants, the subsequent progression of the morphogenetic furrow is only slowed down. However, clones that are simultaneously mutant for Hh and Dpp signaling components completely block furrow progression and eye differentiation, suggesting that Hh and Dpp serve partially redundant functions in this process. Interestingly, furrow-associated expression of eya, so and dac is not affected by double mutant tissue, suggesting that some other factor(s) regulates their expression during furrow progression (Curtiss, 2000).

The lack of eya, so and dac expression in Mad^1-2 clones that lie at the margins of the eye disc prior to MF initiation reflects a role for dpp in controlling early eye gene expression at these stages of eye development. Evidence from several studies suggests that ey acts together with dpp at or near the top of the hierarchy: (1) ey expression is not regulated by dpp; (2) ey and dpp are both required for eya, so and dac expression prior to MF initiation; (3) ey is not capable of rescuing dpp^blk eye development or of inducing ectopic eyes in regions of imaginal discs in which dpp is not already expressed. These observations suggest that ey functions upstream of or in parallel with dpp. The possibility that ey is responsible for dpp expression, leading indirectly to eya, so and dac expression, is unlikely. Since ey cannot induce ectopic eyes without a source of dpp, it probably cannot induce dpp expression, at least not in the absence of factors that are specific to the eye disc. Moreover, Ey protein binds to the regulatory region of so, suggesting it is directly involved in so regulation. Thus, it is likely that ey and dpp cooperate to induce expression of the other early eye genes (Curtiss, 2000).

Such cooperation could achieve two ends. (1) ey is expressed throughout the eye disc and from embryonic stages of development through MF initiation. However, induction of eya, so and dac expression and MF initiation occurs approximately 48 hours later, around the time of the transition between second and third instars. Moreover, eya, so and dac are not expressed throughout the eye disc as ey is, but have stronger levels of expression around the margins than in other regions. The initiation of dpp expression at the posterior margin at approximately the same time suggests that it could be the spatiotemporal signal that sets the MF in motion. (2) dpp induces expression of tissue-specific genes as part of its role in patterning many diverse structures in Drosophila. An interaction with ey could be essential to ensuring that in the eye imaginal disc dpp initiates factors that are appropriate to eye development, such as eya, so and dac (Curtiss, 2000).

Distal-less and homothorax regulate multiple targets to pattern the Drosophila antenna

The Drosophila antenna is a highly derived appendage required for a variety of sensory functions including olfaction and audition. To investigate how this complex structure is patterned, the specific functions of genes required for antenna development were examined. The nuclear factors, Homothorax, Distal-less and Spineless, are each required for particular aspects of antennal fate. Coexpression of Homothorax, necessary for nuclear localization of its ubiquitously expressed partner Extradenticle with Distal-less is required to establish antenna fate. This study tests which antenna patterning genes are targets of Homothorax, Distal-less and/or Spineless. Antennal expression of dachshund, atonal, spalt, and cut requires Homothorax and/or Distal-less, but not Spineless. It is concluded that Distal-less and Homothorax specify antenna fates via regulation of multiple genes. Phenotypic consequences of losing either dachshund or spalt and spalt-related from the antenna are reported. dachshund and spalt/spalt-related are essential for proper joint formation between particular antennal segments. Furthermore, the spalt/spalt-related null antennae are defective in hearing. Hearing defects are also associated with the human diseases Split Hand/Split Foot Malformation and Townes-Brocks Syndrome, which are linked to human homologs of Distal-less and spalt, respectively. It is therefore proposed that there are significant genetic similarities between the auditory organs of humans and flies (Dong, 2002).

In contrast, there are other genes expressed in both antenna and leg precursors that have distinct patterns in the two appendages. Among these are dac, ato, ct and ss. The domain of dac expression in the antenna (a3) is much smaller than in the leg where it is expressed in multiple segments. The function of dac in antennal development has not been described previously (Dong, 2002).

In contrast to the leg, in the antenna dac expression is restricted primarily to a single segment (a3). Trace levels of Dac can be detected in areas of the antennal disc immediately distal and proximal to a3. Because no antennal phenotypes have been reported for loss-of-function dac mutants, it is unclear whether dac plays a role in patterning this appendage. In transheterozygous dac null mutants, a fusion of the a5 segment with the arista occurs, accompanied by a reduction in the width of the a5 segment. This fusion phenotype is similar to what is observed in dac hypomorphic and null legs. However, unlike the leg phenotype, no obvious reductions in length or loss of segments is found in the dac mutant antenna. In addition, this antennal phenotype is observed in dac null animals but not in strong hypomorphic combinations such as dac^lacZ/dac⁴. Therefore, high levels of Dac are probably not necessary for dac function in the antenna (Dong, 2002).

If Dac levels are elevated in the antenna, expression of Dll and hth is repressed and medial leg structures are induced. Therefore if Dac levels are too high, antenna development is compromised. Because bab mutants exhibit phenotypes similar to those of dac, and dac regulates bab expression in the antenna, it is likely that antennal dac function is mediated via its regulation of bab (Dong, 2002).

The antennal dac expression domain expands in Dll hypomorphs and in hth null clones. This expansion of dac expression in Dll and hth mutant antennae resembles the leg pattern of dac expression. In contrast, in the ss null antenna, there appears to be neither expansion nor reduction of dac expression. The only detectable difference in the ss null antennal disc is overgrowth in the central (distal) area such that the ring of dac expression has a larger radius. This correlates with the transformation phenotype of the ss null arista into a tarsus, which is a larger structure. Since the expression of dac relative to other genes appears normal in ss null antennae, ss is not thought to regulate dac (Dong, 2002).

Homeotic genes, Dll and hth, regulate multiple targets during antennal development. These targets function in specifying antenna structures and/or in repressing leg development. For example, the ss mutant phenotype suggests that it represses leg tarsal differentiation. But ss is also required for the formation of olfactory sensory sensilla normally found in a3. Although Dll and hth repress distal leg development via activation of ss, their repression of medial leg development appears to be, at least in part, independent of ss. Instead, this is achieved via their regulation of the medial leg gene, dac, to a narrower domain of expression with lower levels in the antenna as compared to the leg. sal/salr and ato are required for proper differentiation of a2. However, no transformation phenotypes are associated with the sal/salr and ato null antenna. This indicates that while sal/salr and ato are required to make particular antenna-specific structures, they do not appear to repress leg fates. Therefore homeotic genes such as Dll and hth repress the elaboration of other tissue fates in addition to activating genes required for the differentiation of particular tissues (Dong, 2002).

Combinatorial control of Drosophila eye development by Eyeless, Homothorax, and Teashirt

In Drosophila, the development of the compound eye depends on the movement of a morphogenetic furrow (MF) from the posterior (P) to the anterior (A) of the eye imaginal disc. Several subdomains along the A-P axis of the eye disc have been described that express distinct combinations of transcription factors. One subdomain, anterior to the MF, expresses two homeobox genes, eyeless (ey) and homothorax (hth), and the zinc-finger gene teashirt (tsh). Evidence suggests that this combination of transcription factors may function as a complex and that their combination plays at least two roles in eye development: it blocks the expression of later-acting transcription factors in the eye development cascade, and it promotes cell proliferation. A key step in the transition from an immature proliferative state to a committed state in eye development is the repression of hth by the BMP-4 homolog Dpp (Bessa, 2002).

Anterior to the MF, at least three cell types can be distinguished by the patterns of Hth, Ey, and Tsh expression. The most anterior domain in the eye field, which is next to the antennal portion of the eye-antennal imaginal disc, expresses Hth, but not Tsh or Ey. In a slightly more posterior domain, all three of these factors are coexpressed (region II). In a more posterior domain, Tsh and Ey, but not Hth, are coexpressed. This domain, which also expresses hairy, is equivalent to the pre-proneural (PPN) domain. The MF, marked by the expression of Dpp, is immediately posterior to the PPN domain, and therefore abuts Tsh + Ey-expressing cells (Bessa, 2002).

Domain II is the only region of the eye-antennal imaginal disc that strongly expresses all three of these transcription factors. Posterior to the MF, Hth, but not Tsh or Ey, is expressed in cells committed to become pigment cells. Hth and Ey, but not Tsh, are coexpressed in a narrow row of margin cells that frame the eye field and separate the main epithelium of the eye disc from the peripodial membrane. Finally, Hth is also strongly expressed in peripodial cells, whereas Ey and Tsh are weakly expressed in a subset of these cells (Bessa, 2002).

The expression patterns of So, Dac, and Eya were also examined in wild-type eye discs. All three of these transcription factors are expressed in the PPN domain but not in domain II. Their expression domains have the same anterior limit but different posterior limits. Furthermore, the anterior limits of their expression domains are not sharp, but instead decrease gradually as Hth levels increase. Thus, cells in the PPN domain express So, Dac, and Eya as well as Tsh, Ey, and Hairy. Anterior to the PPN domain there is a gradual transition into domain II, where cells express Hth, Ey, and Tsh, but not So, Eya, Dac, or Hairy (Bessa, 2002).

The complementary patterns of Hth versus So, Eya, and Dac at the transition between domain II and the PPN domain suggested that these factors may also be playing a role in hth repression. To test this idea, clones of cells mutant for eya were examined. eya^- clones de-repress hth. Part of this de-repression is probably due to the fact that dpp expression requires eya. However, the de-repression of hth is observed in all eya^- cells, even in cells that are next to wild-type, dpp-expressing cells. Thus, Dpp expressed in wild-type neighboring cells is not able to repress hth in adjacent eya^- cells. These data suggest that eya is required for Dpp to repress hth in the PPN domain. hth was also de-repressed in dac^- clones, suggesting that dac also plays a role in hth repression (Bessa, 2002).

Because Hth is coexpressed and can interact in vitro with Tsh and Ey, the possibility was considered that combinations of these transcription factors might be required to repress eya and dac. Consistent with this idea, it was found that the simultaneous expression of Tsh and Hth efficiently represses eya and dac expression. Importantly, the dual expression of Tsh and Hth is maintained by Ey expression; consequently, these clones expressed all three of these transcription factors. Other pairs of these transcription factors (Hth + Ey and Tsh + Ey) were also tested, and it was found that they can also partially repress eya (Bessa, 2002).

The above results suggest that the combination of Hth + Ey + Tsh, which is normally present in domain II, is able to repress the expression of eya. To test if hth normally plays a role in the repression of these genes, hth^- clones were examined. Although hth^- clones anterior to the MF are rare, it was found that both dac and eya are de-repressed in anterior hth^- clones (Bessa, 2002).

In summary, these data suggest that the combination of the factors expressed in domain II is necessary and sufficient to repress eya and dac. In contrast, Hth is sufficient to repress the pre-proneural gene hairy. Conversely, eya and dac, together with Dpp, repress hth as the MF advances. It is suggested that one function for this reciprocal antagonism may be to prevent premature and uncoordinated differentiation anterior to the MF. However, as the MF advances, hth must be repressed to allow differentiation to occur (Bessa, 2002).

These experiments suggest that one of the functions mediated by Ey-Hth-Tsh is to repress eya and dac. This proposal stems from both ectopic expression experiments, showing that the coexpression of Ey, Hth, and Tsh represses these genes, and from loss-of-function experiments, showing that hth^- clones anterior to the MF de-repress these genes. Similarly, hth is de-repressed in both eya^- and dac^- clones, suggesting that this antagonism exists in both directions. Interestingly, the antagonism between these two sets of genes is analogous to that observed in other appendages. In the leg, hth and tsh are required for the development of proximal fates, and have been shown to be mutually antagonistic with dac and Distal-less (Dll), two genes required for intermediate and distal leg fates, respectively. Similarly, in the wing, hth and tsh are required for proximal wing fates, and oppose the activity of vestigial (vg), which is required for more distal wing fates (Bessa, 2002).

Control of Drosophila eye specification by Wingless signaling

The Wingless protein plays an important part in regional specification of imaginal structures in Drosophila, including defining the region of the eye-antennal disc that will become retina. Wingless signaling establishes the border between the retina and adjacent head structures by inhibiting the expression of the eye specification genes eyes absent, sine oculis and dachshund. Ectopic Wingless signaling leads to the repression of these genes and the loss of eyes, whereas loss of Wingless signaling has the opposite effects. Wingless expression in the anterior of wild-type discs is complementary to that of these eye specification genes. Contrary to previous reports, it has been found that under conditions of excess Wingless signaling, eye tissue is transformed not only into head cuticle but also into a variety of inappropriate structures (Baonza, 2002).

In order to analyse the effect of ectopic activation of the Wingless pathway during the development of the eye-antennal imaginal disc, clones either mutant for the negative regulator of Wingless signaling, Axin, or expressing an activated form of Armadillo (Arm*) were induced. The loss of eye identity caused by the ectopic activation of Wingless, suggests a possible function for Wingless in the regulation of the eye selector genes. The top of the genetic hierarchy involved in eye specification appears to be the Pax6 homolog, Eyeless. In the third instar eye disc the expression of Eyeless is restricted to the region anterior to the furrow and, despite the Wingless-induced inhibition of eye development, the expression of Eyeless in this region is not affected by axin^- clones. This lack of an effect anterior to the furrow, despite the overgrowth and abnormal Distal-less expression in the same region, implies that misregulation of Eyeless is not the primary cause of the transformations caused by ectopic Wingless activity (Baonza, 2002).

Downstream of Eyeless (although feedback relationships makes the epistatic relationship complex) are other transcription factors required for eye specification, including Eyes absent, Sine oculis and Dachshund. A phenotype similar to axin^- clones of excess proliferation and consequent overgrowth is caused by loss of Eyes absent and Sine oculis. Moreover, as in axin^- clones, clones mutant for sine oculis ectopically express Eyeless in the region posterior to the furrow. The similar mutant phenotypes shown by the loss of function of these genes and the ectopic activation of Wingless signaling make them good candidates to be regulated by the Wingless pathway (Baonza, 2002).

The expression patterns of Eyes absent, Sine oculis and Dachshund, in axin^- and/or arm^* mutant clones, were examined in third instar eye discs. At this stage, Dachshund is expressed at high levels on either side of the morphogenetic furrow, whereas Eyes absent and Sine oculis are expressed in all the cells of the eye primordium. In order to produce large patches of mutant tissue, the Minute technique was used. In axin^- M⁺ clones the expression of Eyes absent in front of the furrow is always autonomously eliminated. This effect is not only seen in large clones that touch the eye margin but also in small internal clones. Identical results were obtained with Sine oculis and Dachshund: their expression was autonomously lost from anterior axin^- M⁺ clones. Consistent with these results, in arm^*-expressing clones Eyes absent, Dachshund and sine oculis (detected with a lacZ reporter construct) are similarly autonomously eliminated. It is therefore concluded that Wingless signaling represses the expression of the eye selector genes eyes absent, dachshund and sine oculis anterior to the morphogenetic furrow. Posterior to the furrow, however, some clones express high levels of Eyes absent, and Dachshund. This effect is always associated with overgrowth, and this expression is restricted to only some cells in these clones (Baonza, 2002).

The conclusion that Wingless signaling negatively regulates the expression of Eyes absent, Dachshund and Sine oculis anterior to the furrow leads to the prediction that in normal development, domains of high Wingless activity in the anterior region of the eye disc will be associated with low expression of these genes. Previous work indicates that their expression is broadly non-overlapping, but to analyse this precisely, discs were double-labelled to detect the expression of Wingless and Eyes absent or Sine oculis throughout the third instar larval stage. The expression of these eye specification genes is precisely complementary to that of Wingless in the anterior lateral margins of the eye throughout the third instar. This is consistent with a role for Wingless signaling in initiating the borders between eye and other head structures. Note that in posterior lateral regions slight overlap is observed between the expression of Wingless and these genes; this is presumably analagous to the expression of eye specification genes seen in some posterior axin^- clones, and confirms that in posterior regions of the eye disc, Wingless signaling is not incompatible with the expression of these genes (Baonza, 2002).

These results indicate that Wingless regulates the final size of the eye field of cells by controlling the expression of eyes absent, sine oculis and dachshund. The expression pattern of these genes in the anterior eye margin is complementary to the expression of Wingless throughout the third instar, indicating that in anterior regions, high activity of Wingless signaling corresponds to absence of these gene products. Moreover, ectopic activation of Wingless signaling represses their expression anterior to the furrow (where they act to specify the eye field) throughout eye development. Finally, the loss of Wingless signaling causes ectopic expression of Eyes absent and Dachshund (Baonza, 2002).

It is proposed that the initial expression of Eyes absent, Sine oculis and Dachshund is negatively regulated by Wingless signaling in the eye disc, and that this regulation initiates the border between the eye field and adjacent head cuticle. Attempts were made to define whether Wingless represses the eye specification genes independently or whether eyes absent is the primary target but the data confirms earlier reports of the complexity of the regulatory relationships between eyes absent, sine oculis and dachshund. The observation that Eyes absent is able partially to restore the expression of the other two genes but cannot rescue the overgrowth and differentiation phenotype of axin^- clones has two possible explanations. Either Wingless represses eye development through at least one additional gene, or high level Wingless signaling blocks eye development later in the developmental program -- e.g., it is known to inhibit morphogenetic furrow initiation, even after its earlier effects are rescued by eyes absent expression (Baonza, 2002).

Dfrizzled-3, a new Drosophila Wnt receptor, acting as an attenuator of Wingless signaling in wingless hypomorphic mutants

The absence of Drosophia Frizzled-3 produces no apparent phenotype. Binding studies reveal that Wg can interact with Dfz3 in cultured cells. In order to reveal a role for Dfz3 in development, the possiblity of a genetic interaction of Dfz3 with wingless has been investigated. Dfz3 may be involved in Wg signaling required for adult appendage formation. For example, Dfz3 may serve as an attenuator of Wg signaling, at least in a wg hypomorphic mutant background; the absence of Dfz3 may increase Wg signaling and stimulate wing formation. For analysis of this possiblility, a study was made to find possible interaction between Dfz3 and Wg signaling in various wg mutant backgrounds. Wing blades are frequently absent from flies mutant for wg ¹. Thus, the first question to be examined was is the wg ¹ phenotype affected by the absence of Dfz3? The absence of wing blades is partially rescued through the elimination of Dfz3 activity. On a wg ¹/wg ^CX4 background, fractions of flies with two wings increased from 46% to 87%, while those flies with one wing and wing-less flies, respectively, reduced from 44% and 10% to 13% and 0.5%. The wing-less phenotype of wg ¹ is enhanced in a heterozygous apterous (ap) mutant background: no wing blade is generated at approx. 90% of the presumptive wing-blade-forming sites in wg ¹ homozygous flies heterozygous for ap. Wing blade formation increases 3-fold in the absence of Dfz3 activity. Since wg ^CX4 and wg ¹ are null and regulatory mutant alleles, respectively, these effects are not due to possible change in Wg protein conformation. Thus, wild-type Dfz3 may serve as an attenuator of Wg signaling at least in a wg hypomorphic mutant background; the absence of Dfz3 may increase Wg signaling and stimulate wing formation (Sato, 1999).

To confirm that Dfz3 attenuates Wg signaling, an examination was made of the effects of Dfz3 absence in a different developmental context. Nearly all wg^11en/wg^CX4 flies lack antennal structures. This antenna-less phenotype is significantly rescued by removing Dfz3 activity; complete antennal structures, as well as incomplete ones, areregenerated at more than 70% of putative antennal sites. Distal antennal segment formation requires the circular expression of Bar homeobox genes. Dachshund (Dac) is required for the formation of proximal leg structures and expressed circularly in leg and antenna discs. Thus, wg ^11en/wg^CX4 fly discs with or without Dfz3 activity were stained for Wg, BarH1 and Dac. When there is Dfz3 activity, antennal discs are small and no or little expression of BarH1 and Dac is detected. In the absence of Dfz3 activity, about 10% of the discs, probably corresponding to the completely rescued type, exhibit circular BarH1 and Dac expression similar to that of wild-type discs. In about 50% of discs, presumably corresponding to the partially rescued type, Dac expression is partially restored without recovery of BarH1 expression. In contrast to BarH1 and Dac, no Wg expression is detected in the rescued mutant discs, indicating that wg expression is not enhanced by the absence of Dfz3. That wg^CX4 and wg ^11en are regulatory mutant alleles of wg suggests again that the genetic interactions found here would not be due to possible change in Wg protein structure, but simply to reduction in transcription products of wg. Thus it follows that in wg hypomorphic mutants, Dfz3 reduces Wg signaling activity required for antennal formation without changing wg expression; accordingly, Dfz3 would appear to function as a negative factor or attenuator of Wg signaling at least on a wg hypomorphic mutant background (Sato, 1999).

Homeotic proboscipedia function modulates hedgehog-mediated organizer activity to pattern adult Drosophila mouthparts

Drosophila proboscipedia (HoxA2/B2 homolog) mutants develop distal legs in place of their adult labial mouthparts. How pb homeotic function distinguishes the developmental programs of labium and leg has been examined. The labial-to-leg transformation in pb mutants occurs progressively over a 2-day period in mid-development, as viewed with identity markers such as dachshund (dac). This transformation requires hedgehog activity, and involves a morphogenetic reorganization of the labial imaginal disc. These results implicate pb function in modulating global axial organization. Pb protein acts in at least two ways. (1) Pb cell autonomously regulates the expression of target genes such as dac; (2) Pb acts in opposition to the organizing action of hedgehog. This latter action is cell-autonomous, but has a nonautonomous effect on labial structure, via the negative regulation of wingless and decapentaplegic. This opposition of Pb to hedgehog target expression appears to occur at the level of the conserved transcription factor cubitus interruptus/Gli that mediates hedgehog signaling activity. These results extend selector function to primary steps of tissue patterning, and leads to the notion of a homeotic organizer (Joulia, 2005).

The labial palps, the drinking and taste apparatus of the adult fly head, are highly refined ventral appendages homologous to legs and antennae. As for most adult structures, these mouthparts are derived from larval imaginal discs, the labial discs. Wild-type pb selector function acts together with a second Hox locus, Scr, to direct the development of the labial discs giving rise to the adult proboscis. In the absence of pb activity, the adult labium is transformed to distal prothoracic (T1) legs, reflecting the ongoing expression and function of Scr in the same disc. Though the pb locus shows prominent segmental embryonic expression, as for the other Drosophila homeotic genes of the Bithorax and Antennapedia complexes, it is unique in that it has no detected embryonic function and null pb mutants eclose as adults that are unable to feed. Thus, normal pb selector function is required relatively late, in the labial imaginal discs that proliferate and differentiate during larval/pupal development to yield the adult labial palps. Though the genetic pathway guiding development of the ventral labial imaginal discs to adult mouthparts remains relatively unexplored both in flies and elsewhere, study of P-D patterning has identified several genes subject to pb regulation in the labial discs (notably Dll, dac, and hth) and a distinct organization of normal labial discs has been indicated compared to other imaginal discs (Joulia, 2005).

This study pursued an investigation of how pb homeotic function distinguishes between labial and leg developmental programs. The results implicate pb function at the level of global axial organization. Employing identity markers such as dachshund (dac), a 2-day period late in larval development has been identified when normal pb function is required for labial development. The labial-to-leg transformation occurs during the third larval instar stage, involves a progressive morphogenetic reorganization of the labial imaginal disc, and is hedgehog-dependent. This analysis of the transformation indicates that normal pb action is required at least at two distinct levels. One is in the cell-autonomous regulation of target genes such as dac likely to be implicated in cell identity. A second level involves an autonomous action with a nonautonomous effect on labial structure, through the negative regulation of wingless and decapentaplegic downstream of hh signaling. This opposition to hh targets is likely to occur at the level of the transcription factor cubitus interruptus/Gli, a crucial and conserved mediator of hh signaling activity. These results led to a proposal that homeotic function may exist in intimate functional contact with the hedgehog organizer signaling system: the 'homeotic organizer' (Joulia, 2005).

Segmental organization in the imaginal discs involves the reiterated deployment of segment polarity genes that organize the fundamental segmental form. This involves a cascade proceeding from posteriorly expressed Engrailed protein through a short-range Hh morphogen gradient in anterior cells favoring the activator form of Ci transcription factor, which in turn activates wg and dpp to establish two concurrent, instructive concentration gradients that structure gene expression along the proximo-distal axis. In contrast with this elaborate choreography of the segment polarity genes, the homeodomain proteins encoded by Hox genes are expressed in a segmental register, which obscures how they can direct the differentiation of distinct cell types within the segment. The present investigation of homeotic proboscipedia function during labial palp formation indicates a multipronged action for pb in the labial disc. Pb acts cell-autonomously in the negative regulation of target genes including dac, which is normally extinguished in Pb-expressing cells of labial or leg imaginal discs but is activated in labial discs in the absence of pb activity. This activation of dac in mutant labial cells is hh-dependent and is likely a response to wg and dpp morphogen signals as for leg discs. The data further indicate that pb acts cell autonomously to regulate the level of both wg and dpp expression in response to hh. Thus, pb appears to negatively regulate dac expression directly, but also by withholding positive instructions from Wg and Dpp morphogens. The interweaving of homeotic selector proteins with strategic target genes including morphogens (wg, dpp) and targets of signaling activity (dac, Dll) may influence segment patterning from global size and shape to specific local pattern and cell identity. This positioning offers a powerful yet economical mode of selector function that helps to better understand how a single selector gene can integrate global patterning with cellular identity (Joulia, 2005).

pleiohomeotic gene is required for maintaining expression of genes functioning in ventral appendage formation in Drosophila

Polycomb group (PcG) proteins are negative regulators that maintain the expression of homeotic genes and affect cell proliferation. Pleiohomeotic (Pho) is a unique PcG member with a DNA-binding zinc finger motif and has been proposed to recruit other PcG proteins to form a complex. The pho null mutants exhibits several mutant phenotypes such as the transformation of antennae to mesothoracic legs. This study examined the effects of pho on the identification of ventral appendages and proximo-distal axis formation during postembryogenesis. In the antennal disc of the pho mutant, Antennapedia (Antp), which is a selector gene in determining leg identity, is ectopically expressed. The homothorax (hth), dachshund (dac) and Distal-less (Dll) genes involved in proximo-distal axis formation are also abnormally expressed in both the antennal and leg discs of the pho mutant. The engrailed (en) gene, which affects the formation of the anterior-posterior axis, is also misexpressed in the anterior compartment of antennal and leg discs. These mutant phenotypes are enhanced in the mutant background of Posterior sex combs (Psc) and pleiohomeotic-like (phol), which are also PcG genes. These results suggest that pho functions in maintaining expression of genes involved in the formation of ventral appendages and the proximo-distal axis (Kim, 2008).

Many PcG genes act as zygotic as well as maternal effect genes during whole Drosophila development, but it is not well known when and how they function. Pho is known to work with its redundant DNA-binding protein, Phol and recruits other PcG complexes by binding its binding sites on PREs. pho functions as a maternal effect gene. Its maternal effect mutant embryos show several segment defects and weak homeotic transformation. When pho functions as a zygotic gene, its zygotic mutant adults show homeotic transformation of antennae and legs. In accord to these results, pho functions in identification of ventral appendage were investigated (Kim, 2008).

Mutations in a few PcG genes result in the transformation of antennae to legs. Mutation in esc induces the ectopic expression of Antp and Ubx in the antennal disc, thus transforming antennae to legs. This indicates that esc represses Antp and Ubx expression in the antennal disc during antennal development. Therefore, the possibility was investigated that pho mutation, like esc mutation, would affect the expression of the selector genes that determine the identity of antenna or leg. In the wild type antennal disc, Antp is not expressed, but hth is expressed in almost all cells except for the presumptive arista, allowing for the development of antenna. However, in the leg disc, Antp is expressed and restricts hth expression to the proximal cells, which permits leg development (Kim, 2008).

Antp is ectopically expressed in the antennal disc of the pho mutant, and its expression subsequently but partially represses hth expression in the presumptive a2 or a3. Moreover, in the pho mutant, dac, which is expressed in the presumptive a3 of wild type antennal discs, is overexpressed in the presumptive a2 or a3 where hth expression is reduced. Ectopic expression of Antp in the presumptive a2 represses hth expression, which subsequently results in the transformation from antenna to leg. Ectopic Antp expression in the presumptive a1 permits expression of hth. In addition, when dac is ectopically expressed in a3 using the UAS/GAL4 system, leg-like bristles are newly formed in a3, indicating transformation of a3 to femur. However, the antennal disc of pho mutant shows that hth expression does not completely disappear in all regions of the presumptive a2 and a3 where Antp is ectopically expressed. These indicate that a pho single mutation partially affects expression of Antp, which leads to the incomplete repression of hth. Moreover, as the increased dosage of PcG mutants causes stronger mutant phenotypes than each single mutant, double mutation of pho and Psc strongly affects the expression of Antp, which leads to the complete repression of hth. Therefore, these results indicate that a pho mutation results in the ectopic expression of Antp, which directly represses hth expression in antennal disc and indirectly regulates dac expression through hth expression, which consequently transforms antennae to legs (Kim, 2008).

In the wing imaginal disc, Polycomb (Pc) and Suppressor of zeste (Su(z)) regulate the expression of teashirt (tsh), which specifies the proximal domain with hth. The polyhomeotic (ph) gene regulates the expression of en and the hedgehog (hh) signaling pathway in the wing imaginal disc. Pc also regulates eye specification genes such as tsh and eyeless (ey). PcG genes have recently been found to regulate organ specification genes in addition to homeotic genes, segmentation genes and cell cycle genes (Kim, 2008).

Therefore, it was proposed that pho might regulate the expression of organ specification genes for several reasons. First, Dll is ectopically expressed in the proximal region of the posterior compartment in the antennal disc of the pho mutant. Additionally, Dll is ectopically expressed in the more proximal region of the leg disc in the pho mutant, while dac is ectopically expressed in both the proximal and distal regions. These ectopic expressions do not antagonize each other in their normal region of expression, and result in duplication of distal tibia. Finally, en expression extends to the anterior compartment of both the antennal and leg discs of the pho mutant (Kim, 2008).

According to these reasons the following is proposed; first, pho regulates the expression of Antp in the antennal disc, which in turn might activate Dll. It has been shown that Dll is activated in Antp^NS discs, which is similar in younger and older pho discs. Second, pho regulates the expression of en, which affects the expression of Dll. As a gene determining the A/P axis during antenna and leg development, en affects expression of wg and dpp, which determine the D/V axis via Hh signaling. Wg and Dpp act as morphogens, restricting the expression domain of hth, dac and Dll. This study has demonstrated that en is misexpressed in the anterior compartment in the antennal and leg discs of the pho mutant, which leads to misexpression of wg in the anterior-dorsal compartment. Although it has been shown that in the pho zygotic mutant embryos en is hardly derepressed, the current study showed that it is depressed in the pho zygotic mutant adults, suggesting that pho is involved in regulation of en expression and indirect regulation of Dll expression. Finally, pho might directly regulate expression of Dll, because recent studies using X-ChIP analysis have shown that PcG proteins bind PREs of appendage genes including Dll and hth. Hence, pho may directly or indirectly maintain the expression of Antp and en and regulates P/D patterning genes during ventral appendage formation (Kim, 2008).

Pho and Phol are the only PcG proteins that have a zinc finger domain. A mutation in pho results in weaker phenotypes than other PcG mutations despite the functioning of Pho as a DNA-binding protein. Therefore, Pho may interact with other corepressors and repress the homeotic selector genes. In fact, Pho binds to PRE, which is facilitated by GAGA. PRE-bound Pho and Phol directly recruit PRC2, which leads to the anchoring of PRC1. Pho interacts with PRC1 as well as with the BRM complex. Pho has recently been used to construct a novel complex, called the Pho-repressive complex (PhoRC), which has selective methyl-lysine-binding activity. It is currently known that pho interacts with two other PcG genes, Pc and Pcl, in vivo (Kim, 2008 and references therein).

Pho binds to approximately 100 sites on the polytene chromosome and colocalizes with PSC in about 65% of these binding sites. PSC is a component of PRC1 and inhibits chromatin remodeling. In the third instar larvae, PSC is found in the nuclei in all regions of all imaginal discs. Therefore, it is possible that pho and Psc interact with each other during the adult structure formation from the imaginal discs. pho and Psc interact in ventral appendage formation. While the Psc heterozygote was normal, it enhanced the adult mutant phenotypes exhibited by the pho homozygous mutant. Antp is more widely expressed in the antennal disc of the double mutant of pho and Psc than in that of the pho single mutant, while Psc mutant clones induced by FRT/FLP system showed normal expression of Antp, which indicated that Psc does not directly act by itself in regulating expression of Antp, but it certainly interacts with pho (Kim, 2008 and references therein).

hth is expressed in the distal region regardless of Antp expression so that dac was expressed not only in presumptive a3 but also in other segments, which results in the formation of a new P/D axis. According to recent study showing that hth may have a PRE, these results suggest that pho and Psc might interact to maintain hth expression during antennal development. Moreover, Dll expression in the antennal disc might be repressed by an unknown factor that was affected by the double mutation of pho and Psc, suggesting that the factor might be regulated by pho interaction with Psc during antennal development. In addition, legs of the double mutant had fused segments and weakly jointed tarsi, which may be because extension of Hh signal lead to the abnormal expression of the P/D patterning genes. In sum, pho functions as a regulator of selector genes for the identification of ventral appendages and axis formation by interaction with Psc during postembryogenesis (Kim, 2008).

In addition, Pho interacts with Phol in ventral appendage formation. Adults of double mutants showed more severe defects in appendage formation than those of single mutant. The stronger ectopic expression of Antp in the antennal disc of phol; pho double mutant seems to be one of reasons for severe defects. While Antp is not expressed in phol mutant clones of the wild type antennal discs, it is more strongly ectopically expressed in phol mutant clones of the pho mutant antennal discs than in their surrounding phol/+; pho/pho cells, indicating that Phol may not regulate the expression of Antp alone, but it may do that by interaction with Pho, suggesting that this may lead to recruit PRC1 including PSC to PRE sites of Antp and other appendage genes (Kim, 2008).

Sex determination genes control the development of the Drosophila genital disc, modulating the response to Hedgehog, Wingless and Decapentaplegic signals

In both sexes, the Drosophila genital disc contains the female and male genital primordia. The sex determination gene doublesex controls which of these primordia will develop and which will be repressed. In females, the presence of Doublesex^F product results in the development of the female genital primordium and repression of the male primordium. In males, the presence of Doublesex^M product results in the development and repression of the male and female genital primordia, respectively. This report shows that Doublesex^F prevents the induction of decapentaplegic by Hedgehog in the repressed male primordium of female genital discs, whereas Doublesex^M blocks the Wingless pathway in the repressed female primordium of male genital discs. It is also shown that Doublesex^F is continuously required during female larval development to prevent activation of decapentaplegic in the repressed male primordium, and during pupation for female genital cytodifferentiation. In males, however, it seems that Doublesex^M is not continuously required during larval development for blocking the Wingless signaling pathway in the female genital primordium. Furthermore, Doublesex^M does not appear to be needed during pupation for male genital cytodifferentiation. Using dachshund as a gene target for Decapentaplegic and Wingless signals, it was also found that Doublesex^M and Doublesex^F both positively and negatively control the response to these signals in male and female genitalia, respectively. A model is presented for the dimorphic sexual development of the genital primordium in which both Doublesex^M and Doublesex^F products play positive and negative roles (Sanchez, 2001).

The gene dachsund (dac) is also a target of the Hh pathway in the leg and antenna. In the present study, it was found that dac is differentially expressed in female and male genital discs. In the female genital discs, which have Dsx^F product, dac expression mostly coincides with that of wg in both the growing female primordium and the RMP. In contrast, in male genital discs, which have Dsx^M product, dac is not similarly expressed to wg but its expression partially overlaps that of dpp and no expression is observed in the RFP. In pkA minus clones, which autonomously activate Wg and Dpp signals in a complementary pattern, dac was ectopically expressed only in mutant pkA minus cells at or close to the normal dac expression domains in male and female genital discs. In pkA minus;dpp minus double clones, which express wg, dac is not ectopically induced in the male primordium of the male genital disc, but is still ectopically induced in both the growing female genital primordium and the RMP of female genital disc. Conversely, in pkA minus wg minus double clones, which express dpp, dac is not ectopically induced in the growing female or in the RMP of female genital discs, but is ectopically induced in the growing male primordium of the male genital disc. These results indicate that dac responds differently to Wg and Dpp signals in both sexes (Sanchez, 2001).

In dsx^Mas/+ intersexual genital discs, which have both Dsx^M and Dsx^F products, and in dsx¹ intersexual genital discs, which have neither Dsx^M nor Dsx^F products, dac is expressed in Wg and Dpp domains although at lower levels than in normal male and female genital discs. These results suggest that Dsx^M plays opposing, positive and negative roles in dac expression in male and female genital discs, respectively; and that Dsx^F plays opposing, positive and negative roles in dac expression in female and male genital discs, respectively. To test this hypothesis, tra2 clones (which express only Dsx^M ) were induced in female genital discs. The expression of dac is repressed in tra2 clones located in Wg territory. Therefore, Dsx^F positively regulates dac expression in the Wg domain, and Dsx^M negatively regulates dac expression in this domain, otherwise dac would be expressed in tra2 clones at the low levels found in dsx intersexual genital discs. However, when the tra2 clones are induced in the RMP, in the territory competent to activate dpp, they show ectopic expression of dac (Sanchez, 2001).

Therefore, Dsx^M positively regulates dac expression in the Dpp domain, whereas Dsx^F negatively regulates dac expression in this domain, since in normal female genital discs with Dsx^F dac is not expressed in Dpp territory. This is further supported by the induction of dac in the Wg domain and repression of dac in the Dpp domain by ectopic expression of Dsx^F in the male genital primordium of male genital discs. It is concluded that in male genital discs, Dsx^M positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, Dsx^F positively and negatively regulates dac expression in Wg and Dpp domains, respectively (Sanchez, 2001).

Homozygous tra2^ts larvae with two X-chromosomes develop into female or male adults if reared at 18°C or 29°C, respectively, because at 18°C they produce Dsx^F and at 29°C they produce Dsx^M. A shift in the temperature of the culture is accompanied by a change in the sexual pathway of tra2_ts larvae. Analysis of the growth of genital primordia and their capacity to differentiate adult structures of tra2_ts flies was performed using pulses between the male- and the female-determining temperatures in both directions during development (Sanchez, 2001).

Regardless of the stage in development at which the female-determining temperature pulse was given (transitory presence of functional Tra2_ts product; i.e. transitory presence of Dsx^F product and absence of Dsx^M product), the male genital disc develops normal male adult genital structures and not female ones. This occurs even if the pulse is applied during pupation. Pulses of 24 hours at the male-determining temperature (temporal absence of functional Tra2 ts product; i.e. transitory absence of Dsx^F product and presence of Dsx^M product) before the end of first larval stage produces female and not male genital structures. However, later pulses always give rise to male genital structures, except when close to pupation. Further, the capacity of the female genital disc to differentiate adult genital structures is also reduced when the temperature pulse is applied during metamorphosis (Sanchez, 2001).

When the effect of the male-determining temperature pulses was analyzed in the genital disc, it was found that overgrowth of the RMP is always associated with the activation of dpp in this primordium. However, this activation and the associated overgrowth only occurs when the temperature pulse is given after the end of first larval instar. This suggests that there is a time requirement for induction of dpp (Sanchez, 2001).

The activation of this gene in the RMP and the cell proliferation resumed by this primordium, as well as its capacity to differentiate adult structures is irreversible, because they are maintained when the larvae are returned to the female-determining temperature, which is when functional Tra2^ts product is again available (i.e. the presence of Dsx^F product and absence of Dsx^M product). This time requirement for induction of dpp is also supported by the fact that dsx¹¹ clones (which lack Dsx^M) induce differentiated normal male adult genital structures in the developing male genital primordium of XY; dsx¹¹/+ male genital discs (which express only Dsx^M ) after 24 hours of development. However, when the dsx¹¹ clones are induced in the time period between 0 and 24 hours of development, they do not differentiate normally and give rise to incomplete adult male genital structures. This different developmental capacity shown by the dsx¹¹ clones depending on their induction time is explained as follows. When the clones are induced after 24 hours of development, dpp is already activated. Indeed, these clones show no change in the expression pattern of dpp or their targets. Accordingly, these clones display normal proliferation and capacity to differentiate male adult genital structures. However, when the clones are induced early in development, dpp is not yet activated, since this gene is not expressed in the male genital primordium of male genital discs early in development. Therefore, when the male genital disc reaches the state in development when dpp is induced, the cells that form the clones activate this gene as in dsx mutant intersexual flies because the clones have neither Dsx^M nor Dsx^F products. Consequently, these clones do not achieve a normal proliferation rate, and then do not differentiate normal adult male genital structures (Sanchez, 2001).

As described above, it has been shown that dsx regulates the expression of gene dac. Recall that in male genital discs, Dsx^M positively and negatively regulates dac expression in Dpp and Wg domains, respectively; and in female genital discs, Dsx^F positively and negatively regulates dac expression in Wg and Dpp domains, respectively. The expression of the gene dac was analyzed in genital discs of tra2^ts flies using pulses between the male- and the female-determining temperatures in both directions. It was found that the dac expression pattern switches from a 'female type' to a 'male type' when male-determining temperature pulses were applied to tra2^ts larvae after first larval instar. Note that dac expression is reduced in the Wg domain of the RMP and is progressively activated in the Dpp domain. It should be remembered that these pulses lead to the transient presence of Dsx^M instead of Dsx^F product. Thus, these results are consistent with the previously proposed suggestion that Dsx^M activates dac in the Dpp domain and represses it in the Wg domain (again the converse is true for Dsx^F). When the pulse is given during first larval instar, dac is not activated in the Dpp domain of RMP, in spite of the fact that there is also a transient presence of Dsx^M instead of Dsx^F. This is explained by the lack of competence of cells to express Dpp, which is acquired after first larval instar. When the tra2^ts larvae reach such a developmental stage, these cells now produce Dsx^F because they have returned to the female-determining temperature (Sanchez, 2001).

Dsx^F prevents activation of dpp in the RMP, and consequently no induction of dac expression occurs. In the female genital primordium, dac expression is strongly reduced in the Wg domain and absent in the Dpp domain. Taken together, these results suggest that the development of male and female genital primordia have different time requirements for Dsx^M and Dsx^F products (Sanchez, 2001).

The Drosophila sex determination hierarchy modulates wingless and decapentaplegic signaling to deploy dachshund sex-specifically in the genital imaginal disc

The integration of multiple developmental cues is crucial to the combinatorial strategies for cell specification that underlie metazoan development. In the Drosophila genital imaginal disc, which gives rise to the sexually dimorphic genitalia and analia, sexual identity must be integrated with positional cues, in order to direct the appropriate sexually dimorphic developmental program. Sex determination in Drosophila is controlled by a hierarchy of regulatory genes. The last known gene in the somatic branch of this hierarchy is the transcription factor doublesex (dsx); however, targets of the hierarchy that play a role in sexually dimorphic development have remained elusive. The gene dachshund (dac) is differentially expressed in the male and female genital discs, and plays sex-specific roles in the development of the genitalia. Furthermore, the sex determination hierarchy mediates this sex-specific deployment of dac by modulating the regulation of dac by the pattern formation genes wingless (wg) and decapentaplegic (dpp). The sex determination pathway acts cell-autonomously to determine whether dac is activated by wg signaling, as in females, or by dpp signaling, as in males (Keisman, 2001).

A number of obstacles make it difficult to demonstrate that the sex determination pathway is responsible for the sex-specific regulation of a gene in the genital disc. These obstacles stem from the fact that the male and female primordia, which are the primary constituents of their respective discs, differ in their segmental origin. This raises the possibility that 'sex-specific' gene regulation is really just segment-specific gene regulation, made to look sex specific by the fact that only one primordium develops in each sex. Attempts were made to address this concern by creating clones of the opposite genetic sex in chromosomally male and female genital discs. Thus, for example, dac regulation could be examined in the male (A9) primordium, in both male and female cells. By varying the genetic sex of cells in a context where segmental identity is uniform, it was hoped that the contributions of sex and segmental identity to dac regulation could be disentangled (Keisman, 2001).

In the male primordium of both male and female discs, the regulation of dac varies according to the genetic sex of the cell. Genetically female clones in the male (A9 derived) primordium of the male genital disc are unable to express dac in the lateral male (dpp-dependent) domain, but are able to express dac when they extended medially, towards the source of Wg. Conversely, in the female genital disc, genetically male clones in the repressed male primordium (A9) lose their ability to express dac in the medial, wg-dependent domain, and begin to express dac laterally, presumably in response to Dpp. Finally, dac expression is abnormal in intersexual genital discs from dsx mutant larvae: the male primordium of dsx genital discs expresses dac in both the endogenous, lateral male domains, and in a slightly weaker medial domain that corresponds roughly to the region where tra + clones are able to activate dac. Thus, it is concluded that in the male primordium, the sex determination pathway determines how a cell will regulate dac (Keisman, 2001).

In the female primordium the results fail to show a role for the sex determination pathway in dac regulation. If such a role exists, it would be expected that genetically male clones in the female primordia of a female genital disc would activate dac laterally, like their counterparts in the male primordia. They do not, even when they take up much of the presumptive dpp-expressing domain. It would also be expected that such clones would repress dac medially. Only a few clones were observed to extend into the medial wg-expressing domain, and as expected these appear to repress dac. Interpretation of these results is complicated by the fact that changing the genetic sex of a cell in the genital disc can cause it to enter the 'repressed' state. Thus, for example, if a genetically male clone represses dac when it intersects the medial dac domain in the female primordia, it can be concluded either that the sex determination pathway regulates dac expression or that the cells, which are now male, have adopted a repressed state and are generally unresponsive. A similar caveat prevents interpreting the failure of tra2IR clones to activate dac ectopically in the female primordium. That tra + clones in the male primordium of male genital discs enter such a generally non-responsive state was not of concern, because these clones both repress and activate dac expression. The expression pattern of dac in the female primordium of a dsx mutant genital disc is also difficult to interpret. dac is not activated ectopically in the lateral domains of the dsx female primordium, which is consistent with the failure of tra2IR clones to cause such activation. However, even the medial, wg-dependent dac domain is frequently absent or severely reduced in the dsx female primordium, and thus the authors are reluctant to draw any conclusions from the absence of ectopic dac laterally (Keisman, 2001).

A model is proposed for dac regulation in the male primordium, in which the different isoforms of Dsx protein modulate dac regulation by wg and dpp. In the absence of dsx, both wg and dpp can activate dac, producing the two domains of dac expression observed in the male primordium of a dsx disc. In the female, Dsx^f modulates dpp activity so that dpp becomes a repressor of dac; Dsx^f may also potentiate the activation of dac by wg. In the male, Dsx^m modulates wg activity so that it becomes a repressor of dac, leaving dpp alone to activate dac. In support of this model, it is noted that the Dsx proteins act in a similar manner to positively or negatively modulate the effect of tissue-specific regulators on the yp genes (Keisman, 2001 and references therein).

The behavior of tra + and tra2IR clones provides insight into the mechanism of repression in the undeveloped genital primordium. It was anticipated that such clones would be difficult to recover when they occurred in the male and female primordium, respectively, because they should adopt the repressed state. Instead, large tra + (female) clones were recovered in the male primordium of a male disc, and large tra2IR (male) clones were recovered in the female primordium of a female disc. Some of these clones constitute a substantial fraction of the primordium in question. Though tra + or tra2IR clones were not scored in adults, previous studies strongly suggest that such clones would fail to differentiate adult genital structures (Keisman, 2001).

It has been shown that tra - (male) clones cause large deletions in the female genitalia, indicating that genetically male cells like those in a tra2IR clone divide but cannot differentiate female genital structures. Further, it has also been shown that male structures are deleted when the mosaic border passes through the male genitalia, suggesting that female tissue cannot differentiate male structures. To reconcile these data, it is proposed that repression of the inappropriate genital primordium involves two separable processes: repression of growth and the prevention of differentiation. Thus, clones of cells of the inappropriate genetic sex cannot differentiate, but they can grow and contribute to a morphologically normal genital primordium. This poses yet another question. Cells in a tra + clone in the male primordium of a male genital disc are analogous to the cells in the repressed male primordium of a wild-type female genital disc: both are genetically female, and both have A9 segmental identity. Why do tra + clones in the male primordium grow, while the repressed male primordium in a female disc does not? One possibility is that the decision of the male primordium to grow in a male disc is made before tra + clones were induced and cannot be over-ridden by a later switch of genetic sex. However, temperature-shift experiments with tra-2 ts alleles suggest that the decision of a genital primordium to develop can be reversed later in development. Furthermore, occasional, large tra + clones can cause severe reductions in male genital discs. This observation leads to the suggestion of a model in which growth in the genital disc is regulated from within organizing zones, such as the domains of wg and dpp expression. According to this model, the sex of the cells in the organizing regions would determine how the disc grows, while cells in other regions would respond accordingly, regardless of their sex. The tra + clones that cause reduction could result when such a clone intersects with one of the postulated organizing centers within the disc. The implication is that the sex determination pathway acts in yet undiscovered ways to modulate the function of the genes that establish pattern in the genital disc. One such interaction was found in the regulation of dac; further study is needed to determine if others exist, and what role they play in producing the sexual dimorphism of the genital disc and its derivatives (Keisman, 2001).

The female-specific doublesex isoform regulates pleiotropic transcription factors to pattern genital development in Drosophila.

Regulatory networks driving morphogenesis of animal genitalia must integrate sexual identity and positional information. Although the genetic hierarchy that controls somatic sexual identity in Drosophila is well understood, there are very few cases in which the mechanism by which it controls tissue-specific gene activity is known. In flies, the sex-determination hierarchy terminates in the doublesex (dsx) gene, which produces sex-specific transcription factors via alternative splicing of its transcripts. To identify sex-specifically expressed genes downstream of dsx that drive the sexually dimorphic development of the genitalia, genome-wide transcriptional profiling was performed of dissected genital imaginal discs of each sex at three time points during early morphogenesis. Using a stringent statistical threshold, 23 genes that have sex-differential transcript levels at all three time points were identified, of which 13 encode transcription factors, a significant enrichment. This study focused on three sex-specifically expressed transcription factors encoded by lozenge (lz), Drop (Dr) and AP-2. In female genital discs, Dsx activates lz and represses Dr and AP-2. It was further shown that the regulation of Dr by Dsx mediates the previously identified expression of the fibroblast growth factor Branchless in male genital discs. The phenotypes observed upon loss of lz or Dr function in genital discs explain the presence or absence of particular structures in dsx mutant flies and thereby clarify previously puzzling observations. This time course of expression data also lays the foundation for elucidating the regulatory networks downstream of the sex-specifically deployed transcription factors (Chatterjee, 2011).

A common theme in the evolution of development is that a limited 'toolkit' of regulatory factors is deployed for different purposes during morphogenesis. It is therefore not surprising that the key regulators of genital morphogenesis that this study identified are pleiotropic factors with roles in other developmental processes (Chatterjee, 2011).

Two genes that are expressed sex-differentially in the genital disc, branchless (bnl) and dachshund (dac), provide the best picture of how dsx controls genital morphogenesis. Bnl, which is the fly fibroblast growth factor (FGF), is expressed in two bowl-like sets of cells in the A9 primordium in male discs; there is no expression in female discs because DsxF cell-autonomously represses bnl. Bnl recruits mesodermal cells expressing the FGF receptor Breathless (Btl) to fill the bowls; these Btl-expressing cells develop into the vas deferens and accessory glands (Chatterjee, 2011 and references therein).

Dac, a transcription factor, is expressed in male discs in lateral domains of the A9 primordium and in female discs in a medial domain of the A8 primordium. These lateral and medial domains correspond to regions exposed to high levels of the morphogens Decapentaplegic (Dpp) and Wingless (Wg), respectively. Dsx determines whether these signals activate or repress dac. Male dac mutants have small claspers with fewer bristles and lack the single, long mechanosensory bristle. Female dac mutants have fused spermathecal ducts (Chatterjee, 2011 and references therein).

As with bnl and dac, it remains to be determined whether these downstream genes are direct Dsx targets. Each contains at least one match within an intron to the consensus Dsx binding sequence ACAATGT. Future work will determine whether these matches are indeed contained within Dsx-regulated genital disc enhancers. Moreover, efforts are underway to define Dsx binding locations genome-wide through experiments rather than bioinformatics (B. Baker and D. Luo, personal communication to Chatterjee, 2011); combined with the current expression data, these binding data could speed the discovery of a large number of sex-regulated genital disc enhancers (Chatterjee, 2011).

An important future direction will be to determine how spatial and temporal cues are integrated with dsx to regulate downstream genes. Because lz is expressed in the anterior medial region of the female disc, it is hypothesized that, like dac, it is activated by Wg and repressed by Dpp. Such combinatorial regulation could explain the spatially restricted competence of cells in the male disc to activate lz in response to DsxF. Although Dr, AP-2 and lz are expressed at L3, P6 and P20, many other genes are differentially expressed at only one or two of these time points. How these timing differences are regulated is an important unanswered question, especially for genes such as ac, which shifts from highly female biased at P6 to highly male biased at P20. The finding that Dsx binding sites are most enriched in genes with sex-biased expression at L3 suggests that indirect regulation through a cascade of interactions might contribute to expression timing differences (Chatterjee, 2011).

It has already been shown that DsxF indirectly represses bnl by repressing Dr. To date, Dr has been shown to repress, but not activate, transcription. Therefore, activation of bnl by Dr might itself be indirect, via repression of a repressor. The regulation of bnl by Dr is sufficient to explain the sex-specific expression of bnl. However, upstream of bnl are two sequence clusters that match the consensus binding motif of Dsx. Thus, bnl might be repressed both directly and indirectly by Dsx, in a coherent feed-forward loop (FFL). FFLs attenuate noisy input signals. An FFL emanating from Dsx could provide a mechanism of robustly preventing bnl activation in female discs, despite potential fluctuations in DsxF levels (Chatterjee, 2011).

Understanding how Dr controls the morphogenesis of external structures is also important. The posterior lobe will be of particular interest because it is the most rapidly evolving morphological feature between D. melanogaster and its sibling species. Mutations in Poxn and sal also impair posterior lobe development. Understanding how these two regulators work with Dr to specify and pattern the developing posterior lobe could substantially advance efforts to understand its morphological divergence. Likewise, understanding how lz governs spermathecal development could advance evolutionary studies, as this organ also shows rapid evolution (Chatterjee, 2011).

The extent to which the regulators that were identified play deeply conserved roles in genital development remains to be determined. Although sex-determination mechanisms evolve rapidly, some features are shared by divergent animal lineages. The observation that FGF signaling is crucial to male differentiation in mammals, or that mutations in a human sal homolog cause anogenital defects, could reflect ancient roles in genital development or convergent draws from the toolkit (Chatterjee, 2011).

Whether AP-2, Dr and lz play conserved roles in vertebrate sexual development is similarly uncertain. In mice, an AP-2 homolog is expressed in the urogenital epithelium (albeit in both sexes) and at least one AP-2 homolog shows sexually dimorphic expression (albeit in the brain). The mouse Dr homolog Msx1 is expressed in the genital ridge and Msx2 functions in female reproductive tract development. In chick embryos, Msx1 and Msx2 are expressed male specifically in the Müllerian ducts. The mouse lz homolog Aml1 (Runx1) is expressed in the Müllerian ducts and genital tubercle. As more data accumulate on the genetic mechanisms controlling genital development in other taxa, the question of how deeply these mechanisms are conserved might be resolved (Chatterjee, 2011).

Proximal-distal axis formation in the Drosophila leg

Limb development requires the formation of a proximal-distal axis perpendicular to the main anterior-posterior and dorsal-ventral body axes. The secreted signaling proteins Decapentaplegic and Wingless act in a concentration-dependent manner to organize the proximal-distal axis. Discrete domains of proximal-distal gene expression are defined by different thresholds of Decapentaplegic and Wingless activities. distal-less is expressed in a central domain that corresponds to the presumptive tarsal segments and the distal tibia. The dachshund gene is required for development of the femur and tibia. Dac is expressed in a ring corresponding to the presumptive femur, tibia and first tarsal segment, but is absent from the more distal tarsal segments of the leg disc. Although there is little or no overlap between Dll and Dac domains at early stages, by mid third instar the combination of Dac and Dll expression defines three regions along the P-D axis. Dll and Dac are expressed in circular domains centered on the point at which the ventral Wg domain and the dorsal Dpp domain meet. Dll expression in the center of the disc depends on the combined activities of wg and dpp. Wg and Dpp act directly to induce Dll, as analysis of constitutively active Thick-veins clones has shown (Tkv is the receptor for Dpp); analysis of shaggy/zeste white 3 clones (Sgg is required for transduction of the Wingless signal) reveals that both Wg and Dpp transduction pathways are activated cell autonomously. Continuous signalling is not required to maintain Dll or Dac expression. The spatial domains of Dac and Dll expression are defined by different threshold levels of both Wg and Dpp activities. Both Dpp and Wg act to directly repress Dac in the center of the disc. Dac repression is actively maintained by Wg and Dpp signaling long after Dac and Dll have been induced and are stably expressed in the absence of further signaling. Subsequent modulation of the relative sizes of these domains by growth of the leg is required to form the mature pattern (Lecuit, 1997).

Generation of multiple antagonistic domains along the proximodistal axis during Drosophila leg development

Homothorax is shown to limit Dpp and Wg expression. Expression of the Dpp and Wg targets omb and H15 is restricted to those cells that do not express Hth. To determine if hth inhibits target gene activation by Dpp and Wg, hth was either removed from its endogenous domain or either a GFP-Hth fusion protein or the murine hth homolog MEIS-1B was misexpressed in the distal portion of the leg disc. Removing hth function results in the expansion of wg and dpp target gene expression. Dorsally situated hth- clones result in the expansion of omb expression, as marked by the omb-lacZ reporter gene. Does hth repress Distal-less and dachshund? Similar to removing exd function, when hth loss-of-function clones were examined, dac was found to be only partially derepressed, and derepression was found to be more likely to occur in clones that arise near endogenous dac expression. hth- clones have no effect on Dll expression, regardless of where they are situated. However, when clones of GFP-Hth- or MYC-MEIS-expressing cells are generated, both Dll and dac can be repressed. These results suggest that the expression of Dll and dac requires two conditions: (1) the absence of Hth and (2) sufficient activity in the Dpp and Wg pathways. High levels of Wg and Dpp signaling are shown to repress the nuclear localization of Exd by repressing hth transcription. The direct action of both the Wg- and Dpp-signaling pathways is required to specify cell fates along the P/D axis. High levels of Wg and Dpp signaling are required to activate Dll, a determinant of distal cell fates, and to repress expression of dac, a determinant of intermediate fates along the P/D axis. At intermediate levels of Wg and Dpp signaling, dac, but not Dll, is activated. The distal edge of hth expression coincides with the proximal edge of dac expression, suggesting that the threshold of Dpp and Wg signaling required to activate dac is similar to that required to repress hth. To test this idea, either Wg or Dpp signaling was elevated in the hth expression domain by generating clones of cells that express either a membrane-tethered form of Wg or an activated Dpp receptor, Thickveins QD (TKV QD). When Wg-expressing clones were generated dorsally, where endogenous Wg levels are low but where Dpp is present at high concentrations, there was a loss of Hth protein and a shift of Exd protein to the cytoplasm. This suggests that sufficient levels of both Wg and Dpp signaling are required to repress Hth (Abu-Shaar, 1998).

High levels of Wg and Dpp signaling are shown to affect Hth and Exd, at least in part, by repressing hth transcription. The ability of Wg and Dpp to repress hth appears to be indirectly mediated by Dll and dac. Like Dll, Dac appears to have the capacity to repress hth. The expression patterns of Hth and Dll were examined in leg discs that were entirely devoid of dac function. In these discs, the hth domain appears expanded distally and the Dll domain appears to be expanded proximally, consistent with the idea that dac normally represses both hth and Dll (Abu-Shaar, 1998).

The domains of gene expression for Hth, Dac and Dll, as well the regulatory interactions between them, suggest that the leg is functionally divided into two major domains. The first is a proximal domain, which expresses hth, has nuclear Exd and does not express at least some of the potential target genes of the Wg- or Dpp-signaling pathways. The second is a distal domain, which does not express hth, has Exd localized to the cytoplasm, and expresses the targets of Wg, Dpp and Wg+Dpp signaling. These data suggest that the proximal domain is what has been referred to as the coxopodite, or an extension of the body wall, and is distinct from the distal domain, the telopodite. hth expression and nuclear Exd in the coxopodite would restrict the ability of the Wg and Dpp signals to activate their target genes. This idea is consistent with the observation that these two domains differ with respect to their requirement for Hh signaling: unlike the telopodite, which exhibits severe truncations upon the reduction of hh function, the coxopodite is less severely affected. These two domains also appear to have different cell surface properties; cells from one domain prefer not to mix with cells from the other domain. For example, Dll mutant clones almost always relocalize to the hth-expressing domain and hth mutant clones frequently sort into distal regions of the leg disc. This phenomenon is not observed in the wing disc, where hth and Dll are restricted to outside and within the wing pouch, respectively: hth or Dll mutant clones are positioned randomly in this tissue. The mutant phenotypes displayed by the loss of coxopodite gene function are qualitatively different from those displayed by the loss of telopodite gene function. Removal of coxopodite genes such as exd results in either ‘nonsense’ or proximal to distal cell fate transformations, whereas removal of telopodite gene functions such as Dll and dac results in deletions of the appendage. In summary, the data support the idea that the proximal and distal regions of the leg have independent origins and differ from each other primarily due to the expression of hth, which limits or alters the ability of proximal cells to respond to Wg and Dpp signaling (Abu-Shaar, 1998 and references).

dac and Dll are shown to mediate Wg and Dpp mediated repression of hth. The demonstration that Wg and Dpp signaling repressed hth transcription and Exd’s nuclear localization was surprising, because these two signaling molecules induce Exd’s nuclear localization in the endoderm of the embryonic midgut. An investigation was carried out into the possibility that the repression of hth by Wg and Dpp is indirect and perhaps mediated by dac and Dll, which are not expressed in the midgut. TKV QD-expressing clones were generated and Hth, Dll and Dac were examined. Loss of function clones of Dll and dac were generated. When Dll- clones were generated before ~72 hours of development, hth was found to be derepressed and Exd was nuclear. However, clones generated after ~72 hours have no effect on hth or Exd, suggesting that there is an alternative mechanism for maintaining hth repression. Like Dll, Dac appears to have the capacity to repress hth. The ability of Dac to repress hth expression was confirmed by generating dac- clones. These dac- clones suggest that there might be other regulators of hth in addition to dac and Dll. Completely removing dac function results in viable animals that have deletions along the P/D axes of their legs. The expression patterns of Hth and Dll were examined in leg discs that were entirely devoid of dac function. In these discs, the hth domain appears expanded distally and the Dll domain appears to be expanded proximally, consistent with the idea that dac normally represses both hth and Dll. It is an apparent paradox that Wg and Dpp repress hth in the leg disc while these same signals activate hth expression and nuclear Exd in the midgut endoderm. This may be explained because in the leg, Wg and Dpp repress hth indirectly, by activating the hth repressors Dll and dac. In the absence of Dll or dac, hth is derepressed in the leg disc, even in cells that receive high levels of the Wg and Dpp signals. In contrast, in the embryonic endoderm, dac and Dll are not activated by Wg and Dpp, nor are any other known hth repressors, allowing hth to be activated in these cells (Abu-Shaar, 1998).

Proximal to distal cell communication in the Drosophila leg provides a basis for an intercalary mechanism of limb patterning

Patterning in insect legs is organized along anteroposterior (AP), dorsoventral (DV) and proximodistal (PD) axes. In the case of Drosophila, AP and DV axes of the leg imaginal discs are established along the embryonic AP and DV axes, which are set up based on maternal positional information. The PD axis, however, is zygotically specified by cellular interactions involving the secreted signaling molecules Wingless and Decapentaplegic (Goto, 1999 and references).

PD axis formation in the leg disc first becomes evident when cells expressing either Escargot (Esg) or Distal-less (Dll) are arranged in a circular pattern. Dll expression defines the central, distal domain. Esg-expressing cells become the proximal domain, which surrounds the distal domain. The Meis family homeodomain protein Homothorax (Hth) is expressed in the proximal domain as well as in the surrounding body wall. Hth regulates nuclear localization of another homeodomain protein, Extradenticle (Exd). Exd is active in the nucleus but inactive in the cytoplasm. The genetic requirements for Dll, Exd and Hth suggest that the distal domain gives rise to the majority of the adult leg including tarsus, tibia, femur and trochanter and that the proximal domain gives rise to the coxa and the ventral thoracic body wall. Initial PD subdivision in the embryonic leg disc becomes elaborated during larval stages by activation of additional genes, such as dachshund (dac), in a circular intermediate domain between the distal and proximal domains. dac is required for specification of the intermediate fate (Goto, 1999 and references).

The leg imaginal disc is also divided into a posterior compartment, which expresses the secreted molecule Hedgehog (Hh) and an anterior compartment, which responds to Hh by expressing Wg and Dpp along the AP compartment boundary. Mutual repression between Wg and Dpp limits Wg expression to the ventral side and Dpp expression to the dorsal side. This spatial restriction of Wg and Dpp expression is essential for DV patterning of the leg. In addition, graded activities of Wg and Dpp are required for the expression of Dll and dac and repression of hth in the distal domain. In the proximal domain, target gene activation by Dpp and Wg is inhibited by Hth and Exd, suggesting that the distal and proximal domains have distinct characters to respond to Dpp and Wg (Goto, 1999 and references).

Based on the above observations, it was proposed that the circular patterns of gene expression along the PD axis in the distal domain are organized by the gradient of the combined activity of Dpp and Wg. In the central, distal region, where combined activity of Dpp and Wg would be high, Dll is activated and dac is repressed. An intermediate level of Wg and Dpp activities would allow dac expression in the intermediate domain. Ectopic expression of Dll in the dorsal-proximal region induces wg, which is thought to interact with dpp to specify a new PD axis. These results suggest that the combination of Wg and Dpp constitute a 'distalizing' signal for the PD axis (Goto, 1999 and references).

Although these results suggest that the combination of Wg and Dpp activities centered at the distal tip is essential for PD patterning, it is not known whether Wg and Dpp are sufficient to account for all aspects of PD positional information. In fact, the grafting and regeneration experiments using larval cockroach legs suggest that the reciprocal communication between distal and proximal parts of a leg segment promotes regeneration of the intermediate part. Thus it can be speculated that a proximal to distal cell communication may also be used in PD patterning of the leg during development. Esg is expressed in the proximal domain throughout leg development. Ectopic expression of Esg and its activator Hth in the distal domain induces the intermediate fate in surrounding cells by inducing dac expression. Esg and Hth-expressing cells in the distal domain undergo a change in their adhesive property to sort out from surrounding cells. The proximal to distal inductive communication is unexpected from the model based on the graded activity of Dpp and Wg. Thus an intercalary mechanism that elaborates the PD axis pattern of the leg has been proposed. During the transition from the second to third instar, dac expression in the intermediate domain is induced by (1) a combination of a signal from proximal cells, and (2) Wg and Dpp signaling from the AP compartment boundary. The range of each signaling limits dac expression to the intermediate domain. The proximal to distal signaling dependent on Esg and Hth may provide a molecular basis for the intercalary expression of dac (Goto, 1999 and references).

Thus, it has not been clear whether Wingless and Decapentaplegic are sufficient for the circular pattern of gene expression in the Drosophila leg. A proximal gene escargot and its activator homothorax have been shown to regulate proximodistal patterning in the distal domain. Clones of cells expressing either escargot or homothorax placed in the distal domain induce intercalary expression of dachshund in surrounding cells and reorient the planar cell polarity of those cells. escargot and homothorax-expressing cells also sort out from other cells in the distal domain. Thus, inductive cell communication between the proximodistal domains is the cellular basis for an intercalary mechanism, involving expression of dachshund, during proximodistal axis patterning of the limb (Goto, 1999).

The first sign of proximodistal axis formation in the leg imaginal disc was a circular arrangement of cells expressing either Esg or Dll during embryogenesis. As the disc grows in size and evolves circular folds that separated tarsus, tibia, femur, trochanter and coxa, the pattern of esg expression is maintained. At the late stage of the third instar, more Esg protein is detected in the proximal region corresponding to the coxa and trochanter. The distal most part of the esg-expressing domain partially overlaps with the Dac-expressing domain in the trochanter. The esg expression in the overlapping domain is weaker than that in the more proximal domain, where only esg is detected. The domain of Esg expression appears to overlap with the proximal domain defined by expression of homothorax and teashirt, and nuclear localization of Extradenticle (Goto, 1999 and references).

Dll induces distal leg development when expressed ectopically in the proximal domain. To determine if any of the proximal genes have an organizing activity analogous to that of Dll, esg was induced ectopically using the flip-out technique. In the adult, Esg-positive clones marked by GFP are found as vesicles inside the leg cuticle and are often associated with malformation. In the region proximal to the clones, the bristles and epidermal hairs, which normally point distally, are often reversed. These bristles and hairs are genetically wild type, suggesting that the polarizing activity of Esg is non-cell-autonomous (Goto, 1999).

In the third instar leg disc, dac is expressed in a partially overlapping manner with the expression of Dll and esg in an intermediate ring that corresponds to the proximal tarsus, tibia, femur and trochanter. When esg expression is induced during the second instar, clones in the distal tarsal region show compact morphology; and many of them are associated with ectopic dac expression in cells within and surrounding the clone. The ectopic dac expression results in a local reversion of the proximal-distal order of the gene expression, which prefigures the change in the cell polarity in the adult leg. The esg-positive clones in the coxa spread normally and do not show induced dac. The non-cell-autonomy of the Esg function could be due to a modulation of known secreted molecules controlling anteroposterior and dorsoventral patterning. However, the expression patterns of the Hh target genes wg and dpp, and optomotor-blind (omb, 1996), a target gene of Dpp, are unaffected by misexpression of Esg (Goto, 1999).

esg^G66B null mutant clones were used to assess the requirement of Esg for dac expression. esg^G66B is a derivative of an enhancer trap and lacks the coding region of esg but retains the lacZ gene that reproduces the expression pattern of esg. esg mutant cells are marked by the loss of Myc antigen or by the high expression of beta-gal produced from the two copies of the lacZ gene. dac expression is frequently lost in clones induced at the late second instar larval stage. The partial loss of dac expression in large clones may have been due to a non-cell-autonomous rescue by esg+ cells next to the clones. The clones are sometimes associated with ectopic fold formation. Taken together with the gain-of-function analysis, these data suggest that Esg is necessary and sufficient for dac induction (Goto, 1999).

Proximal cell identity is, at least in part, controlled by the homeodomain protein Hth, which regulates nuclear localization of Exd. When expressed ectopically in the tarsal region, Hth causes non-cell-autonomous induction of dac expression and reversal of bristle and cell polarity. These phenotypes are very similar to those caused by Esg. Unlike esg-expressing clones, which secrete a smooth cuticle, hth-expressing clones in the distal part of the leg sometimes form thick socketed bristles without bracts, a characteristic of the bristles in the proximal part of the leg. Hth strongly activates a reporter gene under the control of the esg enhancer in the distal domain, but it does so weakly, if at all, in the proximal domain. This effect is cell-autonomous, suggesting that Hth may directly regulate transcription of esg. In contrast, neither a loss nor a gain of esg expression affects the activity of Hth/Exd as assessed by the expression of Hth and nuclear localization of Exd, nor is esg expression affected by the expression of another proximal gene, teashirt. These results suggest that Esg acts downstream of Hth/Exd to regulate proximodistal patterning (Goto, 1999).

The esg- or hth-expressing clones in the distal region are round in shape with smooth borders and often invaginated basally to form vesicles in the adult legs and in the larval discs. In contrast, control clones expressing non-functional esg, which lacks the zinc-finger domain, and esg-expressing clones located in the coxa and trochanter, have ragged borders. The epithelial-type homophilic cell adhesion molecule DE-cadherin is expressed throughout the leg discs and its apical localization is maintained normally in esg-expressing clones, suggesting that these cells keep their epithelial character. These results of ectopic expression studies, together with the loss of function studies on hth, indicate that Hth and Esg regulate a cell surface property that distinguishes the proximal and distal domains. It is suggested that inductive cell communication between the proximodistal domains, which is maintained in part by a cell-sorting mechanism, is the cellular basis for an intercalary mechanism of the proximodistal axis patterning of the limb (Goto, 1999).

Formation and specification of distal leg segments in Drosophila by dual Bar homeobox genes, BarH1 and BarH2

BarH1 and BarH2 play essential roles in the formation and specification of the distal leg segments of Drosophila. In early third instar, juxtaposition of Bar-positive and Bar-negative tissues causes central folding that may separate future tarsal segments 2 from 3, while juxtaposition of tissues differentially expressing Bar homeobox genes at later stages gives rise to segmental boundaries of distal tarsi including the tarsus/pretarsus boundary. Tarsus/pretarsus boundary formation requires at least two different Bar functions: early antagonistic interactions with a pretarsus-specific homeobox gene, aristaless, and the subsequent induction of Fas II expression in pretarsus cells abutting tarsal segment 5. Bar homeobox genes are also required for specification of distal tarsi. Bar expression requires Distal-less but not dachshund, while early circular dachshund expression is delimited interiorly by BarH1 and BarH2 (Kojima, 2000).

Circular Dac expression appears in second-instar leg discs before Bar ring appearance. This early Dac-ring is associated interiorly with Bar-positive Keilin's organ cells, which are situated along the interior circumference of or within the early Bar ring. Although they are separated from each other by a Bar-negative, Dac-negative region just before the onset of central fold formation Dac and Bar rings are immediate neighbors at earlier stages. Dac expression is derepressed in Bar minus clones observed in early third instar, while repressed by Bar misexpression, indicating that Bar is essential for distal restriction of Dac expression. Since early Bar expression normally occurs in dac minus clones, dac appears dispensable for proximal restriction of the early Bar ring. Interestingly, in dac minus mutants, Bar misexpression occurs in regions fated to become trochanter, indicating that Dac represses Bar in future trochanter (Kojima, 2000)

Proximal distal axis formation in the Drosophila leg: distinct functions of Teashirt and Homothorax in the proximal leg

The proximal distal axis of the Drosophila leg is patterned by expression of a number of transcription factors in discrete domains along the axis. The homeodomain protein Homothorax and the zinc-finger protein Teashirt are broadly coexpressed in the presumptive body wall and proximal leg segments. Homothorax has been implicated in forming a boundary between proximal and distal segments of the leg. Evidence is presented that Teashirt is required for the formation of proximal leg segments, but Tsh has no role in boundary formation (Wu, 2000).

The leg disc consists of a single epithelial sheet in which the presumptive distal segments are specified in the center and the presumptive proximal segments are specified in the periphery. Cross-sections show that proximal segments, which express Hth and Tsh, fold back over the distal segments, which express Dll and Dac. Hth and Tsh expression is limited to the proximal region of the disc through repression by the combined activities of Wg and Dpp. Although the Hth and Tsh expression domains overlap through much of the proximal region, Hth expression extends more distally than Tsh. This is visible as a band of Hth expression that does not overlap Tsh in a basal optical section. This band coincides with the outer ring of Dll expression. The Tsh domain overlaps the proximal edge of the Dll ring by one or two cells. Tsh expressing cells are also found beneath the disc epithelium. Their location suggests that these may be adepithelial cells. Hth functions as a repressor to modulate Tsh expression. More distally located hth mutant clones lose Tsh expression. Loss of Tsh expression in hth correlates with ectopic expression of Dachshund. hth mutant clones cause ectopic expression of Dac close to the endogenous Dac domain, but do not do so in more proximal regions. The differential effect on Dac expression of hth clones located at different positions along the PD axis has been attributed to a role of Hth as a repressor of Wg and Dpp signaling. Thus the paradoxical loss of Tsh in more distal hth clones can be explained as an indirect effect of Hth on Dac expression. Dac can repress both Tsh and Hth when overexpressed. Thus the different distal limits of the Hth and Tsh expression domains presumably reflect a difference in their sensitivity to repression by Dac. The observation that Tsh levels increase in proximal hth clones suggests that Hth serves as a repressor of Tsh. Thus Hth modulates Tsh expression levels in the proximal leg in two ways. Hth may act directly to reduce Tsh expression levels in the proximal leg, and indirectly via repression of Dac to define the distal limit of Tsh expression (Wu, 2000).

The establishment of segmentation in the Drosophila leg. Development 128: 4511-4521

Segmentation is a developmental mechanism that subdivides a tissue into repeating functional units, which can then be further elaborated upon during development. In contrast to embryonic segmentation, Drosophila leg segmentation occurs in a tissue that is rapidly growing in size and thus segmentation must be coordinated with tissue growth. Segmentation of the Drosophila leg, as assayed by expression of the key regulators of segmentation, the Notch ligands and fringe, occurs progressively and this study defines the sequence in which the initial segmental subdivisions arise. The proximal-distal patterning genes homothorax and dachshund are positively required, while Distal-less is unexpectedly negatively required, to establish the segmental pattern of Notch ligand and fringe expression. Two Serrate enhancers that respond to regulation by dachshund are also identified. Together, these studies provide evidence that distinct combinations of the proximal-distal patterning genes independently regulate each segmental ring of Notch ligand and fringe expression and that this regulation occurs through distinct enhancers. These studies thus provide a molecular framework for understanding how segmentation during tissue growth is accomplished (Rauskolb, 2001).

A general theme in patterning during development is the subdivision of tissues initially by genes expressed in broad, partially overlapping domains, which through combinatorial control, subsequently regulate the expression of downstream genes to generate a repeating pattern. The studies presented here demonstrate that leg segmentation follows this same theme. The 'leg gap genes' Hth, Dac, and Distal-less are expressed in broad domains in the leg disc that encompass more than a single segment. Initially expression of these genes is largely nonoverlapping, but as the leg disc grows, the expression patterns of the leg gap genes change such that five different domains of gene expression are established. The analysis of the regulation of Notch ligand and fringe expression during leg development reveals two fundamental aspects of leg development. (1) These leg gap genes are key components in regulating the expression of the molecules controlling segmentation. Indeed, the effect of these leg gap genes on leg segmentation and growth can be accounted for by their regulation of Serrate, Delta and fringe expression. (2) The expression of each ring of Serrate, Delta and fringe is controlled by its own unique combination of regulators, apparently acting through independent enhancers (Rauskolb, 2001).

How do these three transcription factors regulate the formation of nine segments? Since the requirements for and the expression of the leg gap genes encompasses all leg segments, it is unlikely that there are additional leg gap genes yet to be identified. Rather, a collection of distinct combinatorial approaches is used to establish a segmental pattern of Serrate, Delta and fringe expression (Rauskolb, 2001).

In early third instar leg discs, there are two domains of gene expression: proximal cells express Hth and distal cells express Distal-less. Hth autonomously promotes the expression of Serrate, while Distal-less may prevent expression more distally, giving rise to a ring of expression in the coxa. Additionally, Distal-less-expressing cells may signal to the Hth-expressing cells to restrict Serrate expression to the distal edge of the Hth domain. As the leg disc grows, cells in an intermediate position, lying between the Hth and Distal-less domains, begin to express Dac. Dac, as shown in this study, is both necessary and sufficient to induce the expression of Serrate, Delta and fringe within the femur. Since they are not expressed in all Dac-expressing cells, other factors appear to be required to promote their expression in the proximal femur. The nonautonomous induction of Serrate expression by Hth suggests that this may be accomplished by a signal (X) emanating from the neighboring Hth-expressing cells. By mid third instar stages, expression of Serrate, Delta and fringe is also observed in tarsal segments 2 and 5, within cells expressing Distal-less but not Dac. Given that Distal-less is necessary and sufficient to repress their expression, Serrate, Delta and fringe expression within the tarsus appears to be induced by a mechanism that overrides the repressive effects of Distal-less. Subsequently, expression of Serrate, Delta and fringe is observed within the tibia, in cells expressing both Dac and Distal-less. Dac is necessary for expression of Serrate within the tibia, and its role here may be to overcome the repressive effects of Distal-less. It is also worth noting that the tibia ring of expression is not established at the time when cells first express both Dac and Distal-less. This may be because Dac levels may not be sufficiently high enough to overcome the repression by Distal-less. Clearly levels of Dac expression are critical because simply increasing Dac levels is sufficient to promote Serrate expression in cells already expressing endogenous levels of Dac. This observation can be explained if high levels of Dac expression in cells already expressing Dac override the function of inhibitory regulators of Serrate expression, such as Distal-less, where the expression of these genes overlap. Although late stages of leg segmentation were not investigated in this study, it has been noted that Hth, Dac and Distal-less are co-expressed in the presumptive trochanter late in leg development. It is thus hypothesized that Serrate, Delta and fringe expression is established by the combined activities of the three leg gap genes in the trochanter (Rauskolb, 2001).

Although these here have focused on the regulation of Serrate expression, it is thought that not only Serrate, but also Delta and fringe, receive primary regulatory input from the leg gap genes. Delta and fringe expression, like Serrate, is positively regulated by Dac. Moreover, Dl and fringe mutants have stronger leg segmentation phenotypes than Ser mutants, and thus Delta and fringe expression cannot simply be regulated downstream of Ser. The identification of two separate Ser enhancers, directing expression in the proximal versus distal leg, argues against Serrate being regulated downstream of Dl and fringe. Thus, the simplest model is that expression of all three genes is regulated directly by the leg gap genes. The regulation of Serrate, Delta and fringe expression in each segment appears to occur through independent and separable enhancer elements, supported by the analysis of the Ser reporter genes. This is reminiscent of what occurs during Drosophila embryonic segmentation, where separable enhancer elements direct different stripes of pair-rule gene expression (Rauskolb, 2001).

Most of the tarsus of the Drosophila leg derives from cells expressing Distal-less, but not Dac or Hth. Surprisingly, the studies presented here have shown that Distal-less actually represses Notch ligand expression. This negative regulatory role for Distal-less contrasts with the positive promoting role of Dac and Hth, and further indicates that a distinct molecular mechanism must promote segmentation within the tarsus. One key gene is spineless-aristapedia (ss), since simple, unsegmented tarsi develop in ss mutant flies. Moreover, ss regulates the expression of bric-à-brac (bab), which is also required for the subdivision of the tarsus into individual segments. Together, ss and bab must, in some way, ultimately overcome the repression of Notch ligand and fringe expression by Distal-less. If the sole function of ss and bab is to overcome the inhibitory effects of Distal-less, then in the absence of ss and/or bab, Serrate expression is expected to remain repressed (Rauskolb, 2001).

Intriguingly, the only notable variation between insect species is in the number of tarsal segments, with an unsegmented tarsus believed to be the ancestral state. Thus, the combinatorial regulation of segmentation by the leg gap genes may represent an ancient mechanism common to all insect species, a hypothesis supported by the conserved expression of Hth, Dac and Distal-less in the developing legs of many insect species (Rauskolb, 2001 and references therein).

Distalization of the Drosophila leg by graded EGF-receptor activity

Arthropods and higher vertebrates both possess appendages, but these are morphologically distinct and the molecular mechanisms regulating patterning along their proximodistal axis (base to tip) are thought to be quite different. In Drosophila, gene expression along this axis is thought to be controlled primarily by a combination of transforming growth factor-ß and Wnt signalling from sources of ligands, Decapentaplegic (Dpp) and Wingless (Wg), in dorsal and ventral stripes, respectively. In vertebrates, however, proximodistal patterning is regulated by receptor tyrosine kinase (RTK) activity from a source of ligands, fibroblast growth factors (FGFs), at the tip of the limb bud. This study revises understanding of limb development in flies and shows that the distal region is actually patterned by a distal-to-proximal gradient of RTK activity, established by a source of epidermal growth factor (EGF)-related ligands at the presumptive tip. This similarity between proximodistal patterning in vertebrates and flies supports previous suggestions of an evolutionary relationship between appendages/body-wall outgrowths in animals (Campbell, 2002).

In addition to activating genes, EGFR signalling is required to repress genes in distal regions, and again different genes appear to be differentially sensitive, with some, such as B and rn, possibly being both activated and repressed above different thresholds. B, rn and dac are repressed in the center of wild-type discs, with dac being repressed over a wider region than B and rn. Lowering EGFR activity in Egfr^ts discs to a level sufficient only for loss of al, results in expression of B and rn in the center, but not dac. Raising the temperature still further results in extension of the dac domain to fill the center. Clonal analysis shows that Egfr acts autonomously to repress dac. Ectopic EGFR activity can also repress B, dac and rn but again predominantly in ventral regions (B is repressed mainly at later stages). Previous studies have shown that repression of dac in distal regions requires high levels of Wg and Dpp signalling, so all three pathways appear to be required to achieve this (Campbell, 2002).

The role of buttonhead and Sp1 in the development of the ventral imaginal discs of Drosophila

The related genes buttonhead (btd) and Drosophila Sp1 (the Drosophila homolog of the human SP1 gene) encode zinc-finger transcription factors known to play a developmental role in the formation of the Drosophila head segments and the mechanosensory larval organs. A novel function of btd and Sp1 is reported: they induce the formation and are required for the growth of the ventral imaginal discs. They act as activators of the headcase (hdc) and Distal-less (Dll) genes, which allocate the cells of the disc primordia. The requirement for btd and Sp1 persists during the development of ventral discs: inactivation by RNA interference results in a strong reduction of the size of legs and antennae. Ectopic expression of btd in the dorsal imaginal discs (eyes, wings and halteres) results in the formation of the corresponding ventral structures (antennae and legs). However, these structures are not patterned by the morphogenetic signals present in the dorsal discs; the cells expressing btd generate their own signalling system, including the establishment of a sharp boundary of engrailed expression, and the local activation of the wingless and decapentaplegic genes. Thus, the Btd product has the capacity to induce the activity of the entire genetic network necessary for ventral imaginal discs development. It is proposed that this property is a reflection of the initial function of the btd/Sp1 genes that consists of establishing the fate of the ventral disc primordia and determining their pattern and growth (Estella, 2003).

In a search for genes with restricted expression in the adult cuticle, the MD808 Gal4 line was found to direct expression in the ventral derivatives of the adult body; proboscis, antennae, legs and genitalia. In the abdomen and analia no clear expression was discerned. It was also noticed that the insertion was located in the first chromosome and associated with a lethal mutation. The mutant larvae showed a head phenotype resembling that described for mutants at the btd gene: loss of antennal organ and the ventral arms of the cephalopharyngeal skeleton, and complementation analysis indicated that the chromosome carrying the insert contained a mutation at btd. The expression pattern found in MD808/UAS-lacZ embryos was also similar to that reported for btd, suggesting that the Gal4 insertion was located at this gene. In addition, the imaginal expression of MD808 and of btd was largely coincident (Estella, 2003).

Further to the genetic analysis and the expression data, DNA fragments at the insertion site were cloned to map the position of the P-element on the genome. It is located 753 bp 5' of the btd gene. The related gene Sp1 is immediately adjacent. It is likely that btd and Sp1 have originated by a tandem duplication of a primordial btd-like gene (Estella, 2003).

One particularly significant result about the mode of action of btd comes from the analysis of the ectopic leg patterns observed with ectopic btb expression in the wing and halteres. The clones of cells ectopically expressing btd tend to recapitulate the complete development of leg and antennal discs. For example, the whole genetic network necessary to make a leg appears to be activated. btd induces the activity of hth, dac and Dll, the domains of which account for the entire disc. Furthermore, hth, dac and Dll are activated in a spatially discriminated manner. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds. In one clone, for example hth is expressed only in the peripheral region, resembling the normal expression in the leg disc; in another clone the discriminate expressions of dac and Dll define three distinct regions. The formation of the dac and Dll domains is dependent on signalling from Wg and Dpp, although they require different signal thresholds, but the hth domain is independent from Wg and Dpp (Estella, 2003).

The generation of distinct hth, dac and Dll domains within the clones suggested that btd-expressing cells in the wing and haltere generate their own signalling process. Indeed, within these clones there is local activation of en, the transcription factor that initiates Hh/Wg/Dpp signalling in imaginal discs. btd-expressing clones also acquire wg and dpp activity in subsets of cells. It is probably in the boundary of en-expressing with non expressing cells where the Wg and Dpp signals are generated de novo; subsequently, their diffusion initiates the same patterning mechanism which operates during normal leg development. The result of this process is that the hth, dac and Dll genes are expressed in different domains contributing to form leg patterns containing DV and PD axes. One question for which there is no clear answer is how the initial asymmetry is generated, so that a few cells within the group gain (or lose) en activity. The cells expressing en within the clones are those closer to the posterior compartment cells. It is conceivable that there might be an external signal, perhaps mediated by Hh, which triggers the initial asymmetry (Estella, 2003).

Homeotic proboscipedia function modulates hedgehog-mediated organizer activity to pattern adult Drosophila mouthparts

Drosophila proboscipedia (HoxA2/B2 homolog) mutants develop distal legs in place of their adult labial mouthparts. How pb homeotic function distinguishes the developmental programs of labium and leg has been examined. The labial-to-leg transformation in pb mutants occurs progressively over a 2-day period in mid-development, as viewed with identity markers such as dachshund (dac). This transformation requires hedgehog activity, and involves a morphogenetic reorganization of the labial imaginal disc. These results implicate pb function in modulating global axial organization. Pb protein acts in at least two ways. (1) Pb cell autonomously regulates the expression of target genes such as dac; (2) Pb acts in opposition to the organizing action of hedgehog. This latter action is cell-autonomous, but has a nonautonomous effect on labial structure, via the negative regulation of wingless and decapentaplegic. This opposition of Pb to hedgehog target expression appears to occur at the level of the conserved transcription factor cubitus interruptus/Gli that mediates hedgehog signaling activity. These results extend selector function to primary steps of tissue patterning, and leads to the notion of a homeotic organizer (Joulia, 2005).

The labial palps, the drinking and taste apparatus of the adult fly head, are highly refined ventral appendages homologous to legs and antennae. As for most adult structures, these mouthparts are derived from larval imaginal discs, the labial discs. Wild-type pb selector function acts together with a second Hox locus, Scr, to direct the development of the labial discs giving rise to the adult proboscis. In the absence of pb activity, the adult labium is transformed to distal prothoracic (T1) legs, reflecting the ongoing expression and function of Scr in the same disc. Though the pb locus shows prominent segmental embryonic expression, as for the other Drosophila homeotic genes of the Bithorax and Antennapedia complexes, it is unique in that it has no detected embryonic function and null pb mutants eclose as adults that are unable to feed. Thus, normal pb selector function is required relatively late, in the labial imaginal discs that proliferate and differentiate during larval/pupal development to yield the adult labial palps. Though the genetic pathway guiding development of the ventral labial imaginal discs to adult mouthparts remains relatively unexplored both in flies and elsewhere, study of P-D patterning has identified several genes subject to pb regulation in the labial discs (notably Dll, dac, and hth) and a distinct organization of normal labial discs has been indicated compared to other imaginal discs (Joulia, 2005).

This study pursued an investigation of how pb homeotic function distinguishes between labial and leg developmental programs. The results implicate pb function at the level of global axial organization. Employing identity markers such as dachshund (dac), a 2-day period late in larval development has been identified when normal pb function is required for labial development. The labial-to-leg transformation occurs during the third larval instar stage, involves a progressive morphogenetic reorganization of the labial imaginal disc, and is hedgehog-dependent. This analysis of the transformation indicates that normal pb action is required at least at two distinct levels. One is in the cell-autonomous regulation of target genes such as dac likely to be implicated in cell identity. A second level involves an autonomous action with a nonautonomous effect on labial structure, through the negative regulation of wingless and decapentaplegic downstream of hh signaling. This opposition to hh targets is likely to occur at the level of the transcription factor cubitus interruptus/Gli, a crucial and conserved mediator of hh signaling activity. These results led to a proposal that homeotic function may exist in intimate functional contact with the hedgehog organizer signaling system: the 'homeotic organizer' (Joulia, 2005).

Segmental organization in the imaginal discs involves the reiterated deployment of segment polarity genes that organize the fundamental segmental form. This involves a cascade proceeding from posteriorly expressed Engrailed protein through a short-range Hh morphogen gradient in anterior cells favoring the activator form of Ci transcription factor, which in turn activates wg and dpp to establish two concurrent, instructive concentration gradients that structure gene expression along the proximo-distal axis. In contrast with this elaborate choreography of the segment polarity genes, the homeodomain proteins encoded by Hox genes are expressed in a segmental register, which obscures how they can direct the differentiation of distinct cell types within the segment. The present investigation of homeotic proboscipedia function during labial palp formation indicates a multipronged action for pb in the labial disc. Pb acts cell-autonomously in the negative regulation of target genes including dac, which is normally extinguished in Pb-expressing cells of labial or leg imaginal discs but is activated in labial discs in the absence of pb activity. This activation of dac in mutant labial cells is hh-dependent and is likely a response to wg and dpp morphogen signals as for leg discs. The data further indicate that pb acts cell autonomously to regulate the level of both wg and dpp expression in response to hh. Thus, pb appears to negatively regulate dac expression directly, but also by withholding positive instructions from Wg and Dpp morphogens. The interweaving of homeotic selector proteins with strategic target genes including morphogens (wg, dpp) and targets of signaling activity (dac, Dll) may influence segment patterning from global size and shape to specific local pattern and cell identity. This positioning offers a powerful yet economical mode of selector function that helps to better understand how a single selector gene can integrate global patterning with cellular identity (Joulia, 2005).

The role of the histone H2A ubiquitinase Sce in Polycomb repression

Polycomb group (PcG) proteins exist in multiprotein complexes that modify chromatin to repress transcription. Drosophila PcG proteins Sex combs extra (Sce; dRING) and Posterior sex combs (Psc) are core subunits of PRC1-type complexes. The Sce:Psc module acts as an E3 ligase for monoubiquitylation of histone H2A, an activity thought to be crucial for repression by PRC1-type complexes. This study created an Sce knockout allele and showed that depletion of Sce results in loss of H2A monoubiquitylation in developing Drosophila. Genome-wide profiling identified a set of target genes co-bound by Sce and all other PRC1 subunits. Analyses in mutants lacking individual PRC1 subunits reveals that these target genes comprise two distinct classes. Class I genes are misexpressed in mutants lacking any of the PRC1 subunits. Class II genes are only misexpressed in animals lacking the Psc-Su(z)2 and Polyhomeotic (Ph) subunits but remain stably repressed in the absence of the Sce and Polycomb (Pc) subunits. Repression of class II target genes therefore does not require Sce and H2A monoubiquitylation but might rely on the ability of Psc-Su(z)2 and Ph to inhibit nucleosome remodeling or to compact chromatin. Similarly, Sce does not provide tumor suppressor activity in larval tissues under conditions in which Psc-Su(z)2, Ph and Pc show such activity. Sce and H2A monoubiquitylation are therefore only crucial for repression of a subset of genes and processes regulated by PRC1-type complexes. Sce synergizes with the Polycomb repressive deubiquitinase (PR-DUB) complex to repress transcription at class I genes, suggesting that H2A monoubiquitylation must be appropriately balanced for their transcriptional repression (Gutiérrez, 2012).

This study analyzed how PRC1 regulates target genes in Drosophila to investigate how the distinct chromatin-modifying activities of this complex repress transcription in vivo. Because H2A monoubiquitylation is thought to be central to the repression mechanism of PRC1-type complexes, focus was placed on the role of Sce. The following main conclusions can be drawn from the work reported in this study. First, in the absence of Sce, bulk levels of H2A-K118ub1 are drastically reduced but the levels of the PRC1 subunits Psc and Ph are undiminished. Sce is therefore the major E3 ligase for H2A monoubiquitylation in developing Drosophila but is not required for the stability of other PRC1 subunits. Second, PRC1-bound genes fall into two classes. Class I target genes are misexpressed if any of the PRC1 subunits is removed. Class II target genes are misexpressed in the absence of Ph or Psc-Su(z)2 but remain stably repressed in the absence of Sce or Pc. At class II target genes, Ph and the Psc-Su(z)2 proteins work together to repress transcription by a mechanism that does not require Sce and Pc and is therefore independent of H2A monoubiquitylation. Third, removal of the Ph, Psc-Su(z)2 or Pc proteins results in imaginal disc tumors that are characterized by unrestricted cell proliferation. However, removal of Sce does not cause this phenotype, suggesting that this tumor suppressor activity by the PcG system does not require H2A monoubiquitylation. Finally, these analyses reveal that PRC1 subunits are essential for repressing the elB, noc, dac and pros genes outside of their normal expression domains in developing Drosophila. This expands the inventory of developmental regulator genes in Drosophila for which PcG repression has been demonstrated in a functional assay (Gutiérrez, 2012).

Targets of Activity

Molecular analysis of Drosophila eyes absent mutants reveals features of the conserved Eya domain

Genes involved in eye development are highly conserved between vertebrates and Drosophila. Given the complex genetic network controlling early eye development, identification of regulatory sequences controlling gene expression will provide valuable insights toward understanding central events of early eye specification. The focus of this study is the defining of regulatory elements critical for Drosophila eyes absent expression. Although eya has a complex expression pattern during development, analysis of eye-specific mutations in the gene reveal a region selectively deleted in the eye-specific alleles. Detailed analysis has been performed of a small 322 bp region immediately upstream of transcriptional start that is deleted in the eye-specific eya² allele. This analysis shows that this region can direct early eya gene expression in a pattern consistent with that of normal eya in eye progenitor cells. Functional studies indicate that this element will restore appropriate eya transcript expression to rescue the eye-specific allele. Regulation of this element during eye specification has been examined, both in normal eye development and in ectopic eye formation. These studies demonstrate that the element is activated upon ectopic expression of the eye specification genes eyeless and dachshund, but does not respond to ectopic expression of eya or sine oculis. The differential regulation of this element by genes involved during early retinal formation reveals new aspects of the genetic hierarchy of eye development (Bui, 2000).

The eya enhancer is expressed in ey, so, and dac mutant eye discs in a pattern consistent with previous studies of Eya protein expression during normal eye development. Normally, eya expression is dependent upon ey activity, partially dependent upon so activity, and independent of dac activity. Regulation during ectopic eye formation was addressed in order to define genes that control the expression of this eya enhancer region and to observe differential activation of the eya enhancer. Activity of the enhancer was detected upon ey- and dac-induced eye formation, as anticipated by previous studies. However, enhancer activation is not apparent upon ectopic eya or so gene expression or the combination of eya and so together. Thus, this eya enhancer appears to be selectively activated during ectopic eye formation, indicating a molecular distinction in how ey and dac genes induce ectopic retinal tissue compared to induction by the eya and so genes, at least with respect to regulation revealed by this element (Bui, 2000).

The regulation of this defined eye enhancer for eya suggests that eya and so function distinctively, at least in part, from dac and ey in ectopic eye formation. Whereas ey and dac either directly activate or feedback to activate eya expression, eya and so do not participate in regulatory loops to the level of activation of eya gene expression as defined by the eya eye enhancer (Bui, 2000).

eya can also synergize with dac in ectopic eye development, and physically interact with the Dac protein. However, the loss-of-function phenotype of dac in the eye is not identical to that of eya and so. These studies also suggest that dac is not acting the same way as eya with respect the eya enhancer: dac strongly activates expression, but eya does not. Based on observations from expression studies, dac has previously been placed downstream of eya. However, Dac is reduced, but not missing from eya mutant eye discs. The reduced expression may reflect massive loss of eye progenitor cells in eya mutant eye discs; alternatively, or in addition, there may be a partial dependence of Dac expression upon eya gene function. Thus, Dac may indeed be involved normally in aspects of eya gene expression. Previous studies showing Eya expression on ectopic eye formation are confounded by the fact that Eya is expressed both prior to and after the appearance of the furrow, but this expression is likely to be under the control of different regulatory elements. The element defined here presents a probe for at least some aspects of the early regulation of eya gene expression. The functional requirement by eya for ey and dac activity (and vice versa) in ectopic eye formation may reflect concurrent roles or other, later roles of these genes in eye formation. ey clearly has multiple roles at distinct times in eye development, such as regulation of genes important for late events of photoreceptor cell differentiation, in addition to the early function stressed here (Bui, 2000).

With respect to eya enhancer activation, ey and dac may directly bind to the eya eye enhancer or the regulation may be indirect through additional, yet-to-be defined genes. It is suggested the regulation may not be direct, at least for Ey, as Ey binding sites are not clearly apparent within the element. Whether Dac protein directly binds to DNA has yet to be determined, but it likely interacts with known transcriptional regulators in addition to interacting with Eya. Yeast one-hybrid experiments have also failed to support direct activation of the eya enhancer by Dac or Ey (as well as confirmed lack of activation by Eya and So). These studies provide a framework from which to define additional molecular genetic controls on early retinal specification. Recent studies showing that the fundamentals of ey/Pax-6 regulation can cross species boundaries suggests that not only are elements of the genetic pathway controlling eye development conserved in vertebrates, but fundamental aspects of the regulatory mechanisms may also be conserved. Given that vertebrate Eya homologs display functional rescue of Drosophila eya mutants, key regulatory aspects of eya gene expression, in addition to the function of the protein, may also be conserved. Eya is a critical gene of eye formation, with complex regulation of expression as shown here, as well as complex protein interactions, and multiple downstream targets. This eye enhancer controlling early eya expression provides a molecular genetic tool to help dissect additional regulatory events of eye specification that are involved in the conserved pathways of eye formation (Bui, 2000).

Diversification of cell types in the Drosophila eye by differential expression of prepattern genes

According to the recruitment theory of eye development, reiterative use of Spitz signals emanating from already differentiated ommatidial cells triggers the differentiation of around ten different types of cells. Evidence is presented that the choice of cell fate by newly recruited ommatidial cells strictly depends on their developmental potential. Using forced expression of a constitutively active form of Ras1, three developmental potentials (rough, seven-up, and prospero expression) were visualized as relatively narrow bands corresponding to regions where rough-, seven-up or prospero-expressing ommatidial cells would normally form. Ras1-dependent expression of ommatidial marker genes is regulated by a combinatorial expression of eye prepattern genes such as lozenge, dachshund, eyes absent, and cubitus interruptus, indicating that developmental potential formation is governed by region-specific prepattern gene expression (Hayashi, 2001).

In contrast to ato broad expression just anterior to the furrow, which disappears within 2 h after Ras1 activation, the misexpression of ro, svp, and pros becomes evident only 5-6 h after Ras1 activation. A similar delayed response to Ras1 signal activation is evidenced by the observation that Sev needs to be continuously required at least for 6 h to commit R7 precursors to the neuronal fate. Thus several hours' exposure to Ras1 signals might be essential for uncommitted cells to acquire ommatidial cell fate or the ability to express ommatidial marker genes. Consistent with this, weak, uniform dually phosphorylated ERK (dpERK) expression persists at least for 3 h in the eye developing field after Ras1 activation. This prolonged MAPK activation may be responsible for the marker gene misexpression (Hayashi, 2001).

This study suggests that ommatidial marker gene expression or developmental potential is regulated by a combinatorial expression of eye prepattern genes, according to distance from the morphogenetic furrow. Uncommitted cells just posterior to the morphogenetic furrow are presumed to acquire ro expression potential at the earliest stage of the model (stage 1). In stage 2, R3/R4 precursors expressing ro acquire svp expression potential. svp expression in wild type R3/R4 precursors along with Ras1 activation-dependent svp misexpression in uncommitted cells is assumed to be not only positively regulated by the concerted action of Ras1 signaling and Dac and Eya but also negatively regulated by the protein product of the prepattern gene, lz. R1/R6 photoreceptors are recruited into ommatidia between stages 2 and 3. R1/R6 fate is previously shown specified by dual Bar homeobox genes, BarH1 and BarH2, whose expression is positively regulated by the cell-autonomous function of lz and svp. Consistent with this, in the putative R1/R6 arising area (around row 6), considerable svp expression occurs even in the presence of Lz. svp expression is regulated by Dac and Eya, so that normal Bar expression or R1/R6 fate eventually comes under the control of putative eye prepattern genes Lz, Dac, and Eya (Hayashi, 2001).

In stage 3, which may correspond to R7 and cone cell formation stages, pros is positively regulated through the concerted action of Ras1 signaling and prepattern gene lz (Hayashi, 2001).

In the developing Drosophila eye, differentiation of undetermined cells is triggered by Ras1 activation but their ultimate fate is determined by individual developmental potential. Presently available data suggest that developmental potential is important in the neurogenesis of vertebrates and invertebrates. In the developing ventral spinal cord of vertebrates, neural progenitors exhibit differential expression of transcription factors along the dorso-ventral axis in response to graded Sonic Hedgehog signals and this presages their future fates. Subdivision of originally equivalent neural progenitors through the action of prepattern genes may accordingly be a general strategy by which diversified cell types are produced through neurogenesis (Hayashi, 2001).

Direct control of the proneural gene atonal by retinal determination factors during Drosophila eye development

The determination of neuronal identity in Drosophila cells depends on the accurate expression of proneural genes. The proneural gene atonal (ato) encodes a basic-HLH protein required for photoreceptor and chordotonal organ formation. The initial expression of ato in imaginal discs is regulated by sequences that lie 3' to its open reading frame. This report shows that the initial ato transcription in different imaginal discs is regulated by distinct 3' cis-regulatory sequences. The eye-specific ato 3' cis-regulatory sequence consists of two distinct elements termed 2.8PB and 3.6BP that regulate ato transcription during different stages of eye development. The 2.8PB enhancer contains a highly conserved consensus binding site for the retinal determination (RD) factor Sine oculis (So). Mutation of this So binding site abolishes 2.8PB enhancer activity. Furthermore the RD factors So and Eyes absent (Eya) are required for 2.8PB enhancer activity and can induce ectopic 2.8PB reporter expression. In contrast, ectopic Dpp signaling is not sufficient to induce ato 3' enhancer activation but can induce increased levels of RD factor Dachshund (Dac) and synergize with So and Eya to increase ato 3' enhancer activity. These results demonstrate a direct mechanism by which the RD factors regulate ato expression and suggest an important role of Dpp in the activation of ato 3' enhancer is to regulate the levels of RD factors (Tanaka-Matakatsu, 2008).

In addition to RD factors, Dpp signaling is also known to be involved in eye development although little is known about its role in the activation of the ato 3′ enhancer. This study found that induction of the ato 3′ enhancer by ectopic expression of So and Eya under the 30A-GAL4 driver was limited mainly to specific regions near the A/P compartment boundary where endogenous Dpp is expressed. In addition, co-expression of Dpp with So and Eya led to expansion of ectopic ato 3′ reporter expression, indicating that Dpp signaling can synergize with So and Eya to activate the 2.8PB enhancer. As the 2.8PB enhancer does not contain Mad binding sites, it is unlikely that Dpp signaling regulates 2.8PB expression directly through binding of Mad protein to 2.8PB. It is hypothesized that some of the downstream targets of Dpp signaling may mediate the ability of Dpp signaling to synergize with So and Eya in the activation of the ato 3′ eye enhancer. Interestingly, Dac, a RD factor regulated by Dpp signaling, can also synergize with So and Eya in activating the ato 3′ eye enhancer, raising the possibility that induction of Dac contributes to the ability of dpp to synergize with so and eya in the activation of ato 3′ enhancer. The level of Dac in the posterior of the wing disc is significantly lower than that in the anterior in the absence of Dpp co-expression, while similar levels of Dac in the anterior and the posterior are observed when Dpp is co-expressed. Therefore the difference in the subset of cells induced to activate the ato 3′ enhancer by dpp + so + eya and by dac^7c4 + so + eya expression could be in part due to differences in the level of Dac induced by Dpp expression and that reached with the 30A-GAL4 driver. Alternatively, it is possible that Dpp signaling has additional targets that contribute to its synergistic induction of the ato 3′ enhancer with So and Eya (Tanaka-Matakatsu, 2008).

During Drosophila sensory organ formation, transcriptional regulation of the proneural gene ato plays a key role to determine the position of proneural clusters. Tissue-specific expression of ato is governed by the flanking cis-regulatory regions immediately upstream (5′) and downstream (3′) of the ato transcription unit. ato 5′ transcription largely depends on the Ato-dependent autoregulatory mechanism, while the ato 3′ cis-regulatory region appears to encode tissue- and temporal-specific information. This analysis of the ato 3′ cis-regulatory region revealed a modular organization of tissue-specific enhancers, each of which determine the initial ato expression in sensory organ precursors of a specific tissue type for the formation of ch organs or photoreceptors. For example, the 1.7 kb BamHI–StuI fragment immediately downstream of the ato transcription unit controls ato expression specifically in the leg discs while the 1.9 kb StuI–PstI fragment located 1.7 kb downstream of the ato transcription unit regulates ato expression specifically in the antennal ch organ precursors. Similarly, the eye enhancer lies within the BglII–PstI–EcoRI fragment located 2.8 kb downstream of the ato transcription unit. Finally the 1.5 kb EcoRI–BamHI fragment located 4.8 kb downstream of the ato transcription unit regulates ato expression during embryonic development (Tanaka-Matakatsu, 2008).

Taken together, these results demonstrate that the modular organization of the ato 3′ cis-regulatory region determines the spatial control of ato expression in the ch organs and photoreceptors in different imaginal discs. A surgical experiment of eye disc fragments has revealed that cells immediately anterior to the MF have already acquired the potential to differentiate into retina. Cells ahead of the MF express RD genes and anti-proneural genes to precisely control retinal cell fate determination and proneural cell differentiation. This region is referred to as the pre-proneural (PPN) domain, based on competence for retinal differentiation. The observation that the 2.8PB but not the 6.4BB enhancer os activated precociously in the PPN region suggests the presence of repressor elements residing within the 3.6BP fragment that contribute to the timing of atonal activation during MF progression. Interestingly, gain of function experiments in the wing disc did not reflect significant differences between 2.8PB and 6.4BB. Both enhancers conferred reporter expression only in groups of cells near the A/P compartment boundary in response to So and Eya and co-expression of dpp with so and eya led to an expansion of GFP expression mostly in the posterior domain. It is possible that some positive and negative factors required for the proper regulation of the ato 3′ enhancer in eye discs were not present in the wing disc. Previous studies have identified a number of genes sufficient to induce retinal tissue development or precocious photoreceptor differentiation, and these genes are potential candidates that contribute to the precise expression of ato. For example, ectopic expression of eyegone (eyg) or Optix (Optx) induces retinal tissue development while induction of mutant clones for either extradenticle (exd) or homothorax (hth) lead to ectopic eye formation in the ventral head region. Additionally, ectopic activation of the Hh signaling pathway or removal of hairy (h)/extramacrochaetae (emc) is sufficient to induce precocious furrow advancement and photoreceptor differentiation. Furthermore, removal of the Notch effector Su(H) causes slight advancement of neural differentiation. This search of conserved non-coding DNA sequences did not find predicted Ci binding sites in the ato 3′ cis-regulatory region. In contrast, a highly conserved transcription factor binding site for Su(H) is observed in the ato 3′ cis-regulatory region. Further analysis of ato 3′ eye enhancer should help to define the mechanisms that contribute to the precise control of its expression (Tanaka-Matakatsu, 2008).

Dynamic rewiring of the Drosophila retinal determination network switches its function from selector to differentiation

Organ development is directed by selector gene networks. Eye development in Drosophila is driven by the highly conserved selector gene network referred to as the 'retinal determination (RD) gene network,' composed of approximately 20 factors, whose core comprises twin of eyeless (toy), eyeless (ey), sine oculis (so), dachshund (dac), and eyes absent (eya). These genes encode transcriptional regulators that are each necessary for normal eye development, and sufficient to direct ectopic eye development when misexpressed. While it is well documented that the downstream genes so, eya, and dac are necessary not only during early growth and determination stages but also during the differentiation phase of retinal development, it remains unknown how the retinal determination gene network terminates its functions in determination and begins to promote differentiation. This study identified a switch in the regulation of ey by the downstream retinal determination genes, which is essential for the transition from determination to differentiation. Central to the transition is a switch from positive regulation of ey transcription to negative regulation and that both types of regulation require so. These results suggest a model in which the retinal determination gene network is rewired to end the growth and determination stage of eye development and trigger terminal differentiation. It is concluded that changes in the regulatory relationships among members of the retinal determination gene network are a driving force for key transitions in retinal development (Atkins, 2013).

This work has found that a switch from high to low levels of Ey expression is required for normal differentiation during retinal development. A mechanism is presented of Ey regulation by the RD gene network members Eya, So, and Dac. Specifically, So switches from being an activator to a suppressor of ey expression, both depending on a So binding site within an ey eye-specific enhancer. It is additionally reported that the So cofactors Eya and Dac are required for ey repression posterior to the furrow but not for its maintenance ahead of the furrow, and are sufficient to cooperate with So to mediate Ey repression within the normal Ey expression domain (Atkins, 2013).

The results support a Gro-independent mechanism for the suppression of target gene expression by the transcription factor Sine oculis (So). An independent study has also shown that So can repress the selector gene cut in the antenna in a Gro-independent process though the mechanism was not determined (Anderson, 2012). It was observed that Ey is expressed at low levels posterior to the morphogenetic furrow. However, when so expression is lost in clones posterior to the furrow, Ey expression and ey-dGFP expression are strongly activated. This is not simply a default response of ey to So loss, as removing So from developmentally earlier anterior cells results in reduced ey expression. Knockdown of So specifically in differentiating cells using RNAi causes a similar phenotype, suggesting that an activator of Ey expression is expressed in differentiating photoreceptors. Mutation of a known So binding site in ey-dGFP results in activation of the reporter posterior to the furrow, supporting a model that binding of So to the enhancer prevents inappropriate activation of ey expression posterior to the furrow. Finally, in vitro it was observed that an excess of So is sufficient to prevent activation of the enhancer; in vivo overexpression of So can also suppress normal Ey expression. The observations are consistent with what in vitro studies have indicated about So function: when So binds DNA without Eya, it can only weakly activate transcription. However, the current work introduces a novel mechanism of regulation for So targets, in which So occupancy of an enhancer prevents other transcription factors from inducing high levels of target gene expression. The results also indicate that suppression of robust ey expression is an important developmental event. It is not yet clear if maintaining basal expression of ey, rather than completely repressing it, is developmentally important; however, it is possible that the ultimate outcome of a basal level of ey transcription may be necessary for the completion of retinal development (Atkins, 2013).

The results also show that eya is required for Ey suppression in vivo. However, consistent with its characterization as a transcriptional coactivator, in vitro analysis does not indicate a direct role for Eya in repression. Previous studies, and the current observations, indicate that Eya is required for the expression of So posterior to the furrow in the third instar. Additionally, reporter analysis shows that Eya regulation of ey requires the So binding site. It is proposed that the simplest model for Eya function in the suppression of ey is through its established function as a positive regulator of So expression, as it was observed that overexpression of So alone is sufficient to weakly repress Ey expression and to block reporter activation in vitro. This model could also account for the results reported regarding the inability of this UAS-so construct to induce ectopic eye formation. Briefly, the primary function of So in ectopic eye formation is to repress the non-eye program (Anderson, 2012). Overexpressing the So construct used in this study alone is not sufficient to induce this program, possibly because the transgene expression level is not sufficient; however, co-expression of the so positive regulator Eya is sufficient to induce robust ectopic eye formation. In light of the current findings, it is proposed that Eya co-expression is necessary to induce So expression to sufficient levels to block transcriptional activation of non-eye targets to permit the induction of the ectopic eye program; however other functions of Eya may play a role (Atkins, 2013).

It was further demonstrated that dac expression is required specifically near the furrow for Ey repression. In addition, this study showed that the So binding site is required for strong ey expression in dac clones near the furrow, suggesting that So activates ey in these clones. This suggests that repression by Dac occurs before the transition to repression by So, making Dac the first repressor of ey expression at the furrow, and identifying how the initiation of repression occurs before So levels increase. It was further shown that Eya and So are sufficient to repress ey expression in dac mutant clones anterior to the furrow, though not as completely as in cells that express Dac. This result indicates that Dac is not an obligate partner with Eya and So in ey repression, but is required for the full suppression of ey. One model would be that Dac and So can cooperate in a complex to modestly repress eyeless directly. This would be consistent with loss-of-function and reporter data as well as the observation that Dac and So misexpression can weakly cooperate to repress Ey anterior to the furrow. However, while a similar complex has been described in mammalian systems, previous studies have been unable to detect this physical interaction in Drosophila. An alternative model is that Dac suppresses ey expression indirectly and in parallel to Eya and So. A previous study has shown that dac expression is necessary and sufficient near the furrow to inhibit the expression of the zinc finger transcription factor Teashirt (Tsh). Tsh overlaps Ey expression anterior to the furrow, and can induce Ey expression when misexpressed. Furthermore, tsh repression is required for morphogenetic furrow progression and differentiation. In light of these previous findings, a simpler model is proposed based on current knowledge that Dac repression of tsh at the morphogenetic furrow reduces Ey expression indirectly. Future studies may distinguish between these mechanisms (Atkins, 2013).

In addition to the role of the RD gene network in ey modulation,signaling events within the morphogenetic furrow indirectly regulate the switch to low levels of ey expression. It has been shown that signaling pathways activated in the morphogenetic furrow increase levels of Eya, So and Dac; furthermore, it is proposed that this upregulation alters their targets, creating an embedded loop within the circuitry governing retinal development and allowing signaling events to indirectly regulate targets through the RD network. The identification of ey regulation by So posterior to the morphogenetic furrow represents a direct target consistent with this model (Atkins, 2013).

In conclusion, a model is presented that rewiring of the RD network activates different dominant sub-circuits to drive key transitions in development (see A model for dynamic RD gene network interactions during the third instar). To the interactions previously identified by others, this study adds that strong upregulation of So, dependent on Eya, results in minimal levels of ey transcription. It is proposed that the identification of this novel sub-circuit of the RD network provides a mechanism for terminating the self-perpetuating loop of determination associated with high levels of Ey, permitting the onset of differentiation and the completion of development. Together, these results give a new view into how temporal rewiring within the RD network directs distinct developmental events (Atkins, 2013).

Protein interactions

The Dachshund and Eyes Absent proteins can physically interact through conserved domains, suggesting a molecular basis for the genetic synergy observed; it has been shown that a similar complex may function in mammals. The C-terminal portion of Eya interacts with Dac while the amino-terminal portion does not, suggesting that the C-terminal conserved domain of the Eya protein is contacting a portion of the Dac protein that is also conserved (Chen, 1997).

Molecular analysis of Drosophila eyes absent mutants reveals features of the conserved Eya domain

The eyes absent gene is critical to eye formation in Drosophila; upon loss of eya function, eye progenitor cells die by programmed cell death. Moreover, ectopic eya expression directs eye formation, and eya functionally synergizes in vivo and physically interacts in vitro with two other proteins of eye development, Sine oculis and Dachshund. The Eya protein sequence, while highly conserved to vertebrates, is novel. To define amino acids critical to the function of the Eya protein, eya alleles have been sequenced. Loss of the entire Eya Domain is null for eya activity, but alleles with truncations within the Eya Domain display partial function. The molecular genetic analysis was extended to interactions within the Eya Domain. This analysis has revealed regions of special importance to interaction with Sine Oculis or Dachshund. Select eya missense mutations within the Eya Domain diminish the interactions with Sine Oculis or Dachshund. Taken together, these data suggest that the conserved Eya Domain is critical for eya activity and may have functional subregions within it (Bui, 2000).

This analysis of the mutations in the Eya Domain was extended to the situation in vivo by generating transgenics expressing the selective point mutants that disrupt interactions with So and Dac in the yeast two-hybrid system. Although this has failed to provide evidence in support of a special functional relevance of the Dac interaction (both mutant forms appeared to interact similarly in ectopic eye formation upon coexpression with Dac) evidence has been found supporting the importance of the So interaction. These data indicate that the Eya^E11 mutant form shows a diminished ability to synergize with So upon coexpression. This supports the hypothesis that the eya^E11 mutation within the Eya Domain disrupts interactions in vivo with so (and/or possibly with other Six homologs) that are critical for the function in eye formation. The Eya^E7 mutant form, which shows a disrupted Dac interaction, still supports ectopic eye formation, although at decreased penetrance compared to normal Eya. dac null mutations frequently show some degree of eye development, suggesting that dac may be partially redundant in eye formation. Therefore, even if interaction with Dac in vivo were disrupted by the eya^E7 mutation, eye formation might still occur due to compensation by such mechanisms. Nevertheless, this eya allele also shows a dominant reduced-eye phenotype when coexpressed with so -- this is a new property not observed with the wild-type Eya protein. The eya^E7 mutation may generate a protein with some dominant-negative property in eye formation. The data that So and Dac may interact, in part, differentially within the conserved domain of Eya supports the idea that the three proteins have the potential to interact in a single complex in vivo. Such a hypothesis, however, is complicated by other data indicating that the molecular activity of Eya-So coexpression in eye formation is at least in part distinct from that of Dac or Eya-Dac coexpression: whereas Dac, and Eya coexpression with Dac, activate an eya enhancer, Eya alone or Eya with So fails to activate enhancer activity, despite ectopic eye formation (Bui, 2000).

Inducible RNA interference uncovers the Drosophila protein Bx42 as an essential nuclear cofactor involved in Notch signal transduction

The UAS/GAL4 two component system was used to induce mRNA interference (mRNAi) during Drosophila development. In the adult eye the expression from white transgenes or the resident white locus is significantly repressed by the induction of UAS-wRNAi using different GAL4 expressing strains. By induced RNAi it was demonstrated that the conserved nuclear protein Bx42 is essential for the development of many tissues. Phenotypically the effects of Bx42 RNAi resemble those obtained for certain classes of Notch mutants, pointing to an involvement of Bx42 in the Notch signal transduction pathway. The wing phenotype following overexpression of Suppressor of Hairless is strongly enhanced by simultaneous Bx42 RNAi induction in the same tissue. Target genes of Notch signaling like cut and Enhancer of split m8 were suppressed by induction of Bx42 RNAi (Negeri, 2002).

Phenotypically, the consequences of Bx42RNAi often resemble effects obtained by interference with components of the Notch pathway. Studies of protein interaction in vitro suggest an involvement of Bx42 and its human homolog Skip in the Notch signal transduction. Both Skip and Bx42 were found to interact with Notch-IC, CBF1 and components of the CBF1 corepressor complex like SMRT, N-CoR, CIR, Sin3a and HDAC2 proteins (Zhou, 2000a; Zhou, 2000b; Zhang, 2001). By yeast two hybrid interaction and coimmunoprecipitation it was found that Bx42 physically interacts with the Drosophila CBF1-homolog Su(H) and with its antagonist Hairless, for which so far no vertebrate counterpart is known. The current study presents evidence that these interactions are biologically meaningful. Ubiquitous early induction of Bx42 RNAi results in embryos with dorsal cuticle only, a phenotype similar to Notch mutations. Induction of Bx42 RNAi in the eye disc results in an eye to antenna transformation as is observed following overexpression of dominant negative forms of Notch in the same tissue. Both effects could be interpreted that Bx42 normally functions as a coactivator of the Notch pathway. However, Other studies have demonstrated that overexpression of Notch-IC in the eye-antennal discs results in the formation of ectopic antennae too, but only if the eyeless function is reduced by a hypomorphic mutation. eyeless is one of the master regulators for eye development and functions in a cross-regulatory circuitry together with six other master regulatory genes. One of them is dachshund, whose human homolog Ski is a known interactor for the Bx42 human homolog Skip (Dahl, 1998). Thus, the observed eye antennal transformation by Bx42 RNAi could also be interpreted as a downregulation of eye master regulatory genes via diminished dachshund activity and a simultaneous derepression of the Notch pathway. Negative interference of Bx42 with Notch signaling is consistent with the results of ptc-GAL4 driven induction of Bx42 RNAi. A similar loss of scutellar bristles is observed on ptc-GAL4-driven overexpression of the Notch-ankyrin repeats, a part of Notch-IC involved in active signal transductio. However, this is not fully understood, since overexpression of Notch extracellular domains missing certain EGF repeats results in a similar antineurogenic phenotype. Moreover, Notch mutant clones result in a loss of bristles as well. A negative role of Bx42 in Notch signaling is suggested by the wing phenotype of dpp-GAL4/Bx42RNAi. Following reduction of Bx42 the veins are thinner or missing consistent with a Notch gain of function (Negeri, 2002).

Ectopic expression of Su(H) prevents sensory organ development in a similar manner to activated Notch. This may reflect an excess of lateral inhibition or it may be due to interference with the establishment of the correct fates in the progeny of the sensory organ precursor cells. The failure of sensory organ formation in the scutellum and the wing following local Bx42 RNAi may be related to a gain of Su(H) function. The enhancement of the Su(H) overexpression phenotype by Bx42 RNAi and the similar effects of Bx42 RNAi and Su(H) overexpression on Notch target genes strongly support this argument and suggest a functional relationship between both proteins (Negeri, 2002).

Although the data suggest a negative role, it is not believed that Bx42 protein acts as a repressor within the Notch pathway. Earlier work, which suggested an activation function for Su(H) was at odds with data demonstrating a repressive role for its mammalian homolog CBF1. More recent work provides evidence for Su(H) acting as a switch between repression and activation of Notch target genes. It is proposed that Bx42 contributes to the switch provided by the Su(H) protein. How this is accomplished can only be speculated at the moment. By its direct interaction with Su(H) Bx42 may stabilize a switching complex. By its direct interaction Bx42 could recruit Hairless into the complex contributing to its repressive function. Modification or removal of Bx42 protein (as by RNAi) would result in a destabilization of this repressive complex allowing to switch to the active state. Similarly, due to squelching by a large excess of overexpressed Su(H), only a reduced amount of Bx42 protein would be available to stabilize the repression complex. It is important to emphasize that Bx42 is able to physically interact with Notch-IC (Zhou, 2000a) and may act as a switching protein in the recruitment of activators as well (Negeri, 2002).

An important role in Notch signaling and functional relation between Su(H) and Bx42 were also suggested when the effects of reducing Bx42 on Notch target gene expression were studied. Both cut and E(spl)m8 were suppressed by Bx42 RNAi as was the vestigial quadrant element (vgQE) enhancer, indicating a Bx42 activating function for these genes in wild type. wingless expression was not affected under these conditions, excluding Bx42 RNAi induced cell death as an explanation for the observed effects. The effects on Notch target gene expression are consistent with the proposed role for Bx42 as part of a switch. Following Su(H) overexpression repression of cut, E(spl)m8 and the vgQE enhancer was demonstrated. wingless, on the other hand, was not affected. The similar effects of Su(H) overexpression and Bx42 RNAi on Notch target gene expression underscores the close relationship in the function of both proteins (Negeri, 2002).

Besides its involvement in Notch signaling the observed phenotypic effects of Bx42 RNAi suggest that the Bx42 protein is involved in other signaling pathways as well. Data from its vertebrate homolog, which indicate that Bx42 takes part in nuclear receptor pathways, are supported by observations on chromosomal binding to sites occupied by the ecdysone receptor complex. A possible interaction with Dachshund, one of the master regulators in eye development, which is widely expressed in the nervous system, has already been mentioned. It remains to be established how Bx42 is involved in these other pathways (Negeri, 2002).

Conserved role for the Dachshund protein with Drosophila Pax6 homolog Eyeless in insulin expression

Members of the insulin family peptides have conserved roles in the regulation of growth and metabolism in a wide variety of metazoans. The Drosophila genome encodes seven insulin-like peptide genes, dilp1-7, and the most prominent dilps (dilp2, dilp3, and dilp5) are expressed in brain neurosecretory cells known as 'insulin-producing cells' (IPCs). Although these dilps are expressed in the same cells, the expression of each dilp is regulated independently. However, the molecular mechanisms that regulate the expression of individual dilps in the IPCs remain largely unknown. This study shows that Dachshund (Dac), which is a highly conserved nuclear protein, is a critical transcription factor that specifically regulates dilp5 expression. Dac was strongly expressed in IPCs throughout development. dac loss-of-function analyses revealed a severely reduced dilp5 expression level in young larvae. Dac interacted physically with the Drosophila Pax6 homolog Eyeless (Ey), and these proteins synergistically promoted dilp5 expression. In addition, the mammalian homolog of Dac, Dach1/2, facilitated the promoting action of Pax6 on the expression of islet hormone genes in cultured mammalian cells. These observations indicate the conserved role of Dac/Dach in controlling insulin expression in conjunction with Ey/Pax6 (Okamoto, 2012).

DEVELOPMENTAL BIOLOGY

Embryonic

dachshund is expressed in the central nervous system of embryos (Mardon, 1994).

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 is described and its morphogenesis through later embryonic stages is 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, is 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).

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).

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).

Recently it has been shown that the patterning genes hedgehog (hh) and decapentaplegic (dpp) are required for the specification in the eye. In an interesting model it has been proposed that Hh signals to Eya which then in turn induces (directly or indirectly) the transcription of both so and dac. This would then suggest that during embryogenesis all three proteins should have overlapping expression patterns during the allocation of the eye disc. The expression of a so-lacZ transgene was compared with that of dac. Interestingly, while the onset of expression of both genes are first detected at approximately 4 h AED, their expression patterns abut each other and are not overlapping. While dac is expressed in two clusters of dorsal medially located cells, so-lacZ expression is seen in a broad swathe of cells that extends from one lateral surface to another. Its expression appears to be delimited by the more-anterior domain of dac expression and the cephalic furrow. By approximately 5 h AED there is a cluster of cells along the lateral margins just anterior to the cephalic furrow in which both so-lacZ and dac are co-expressed. However, the vast majority of so-lacZ and dac expression is non-overlapping. Not unlike eya, so-lacZ is expressed in a subset of cells within the developing brain but is not expressed in the ventral nerve cord. There is considerable overlap between the dac and so-lacZ expression patterns within the developing brain lobes. In the segmental grooves so-lacZ expression can be seen much like that of eya. At approximately 11-14 h AED there is no expression of so-lacZ within the developing eye imaginal disc (Kumar, 2001b).

A lingering question focuses on the fates of the cells that are derived from the initial expression of so, eya, and dac. All three of these genes are expressed very early; for instance eya is expressed in a cluster of cells at the cellular blastoderm time point. Do these cells contribute to the formation of the visual field? Are these three proteins committing cells to adopt an eye imaginal disc fate, an event that will occur much later in embryogenesis? Such questions can only be addressed by precise single cell fate mapping experiments. Only by labeling a single cell and tracing its progeny will it be possible to know if the earliest cells that express so, eya, and dac will later become cells of the eye imaginal disc. How the expression patterns described here correlate with the genetic, molecular, and biochemical interactions of the eye specification genes is an interesting problem that will undoubtedly require the identification of additional instructive and inhibitory signals (Kumar, 2001b).

Finally, 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).

dachshund (dac) is involved in the development of the eye and the mushroom bodies where it is expressed already in the progenitor cells. Using an antibody, Dac expression was found in the trunk CNS not before stage 12; it is expressed in only two or three cells (not NBs) per neuromer. In the procephalon, Dac is already detected by stage 9 in a small area of the dorsal ocular neuroectoderm from which four Dac-positive NBs (Pcd4, Pcd8, Pcd9, Pcv9) delaminate. It has been suggested that the NBs delaminating from this Dac domain represent the progenitors of the mushroom body and co-express eyeless (ey). In disagreement with this, it was found that, at that stage, the co-expression of both genes is confined only to a small region of the Dac-positive neuroectoderm and to only one of the four identified Dac-positive NBs. As evidenced by Dac/Ey antibody double labelling, this NB (Pcv9) is one of the five Ey-positive brain NBs identified at stage 9. Until stage 11, the Dac-expressing ocular domain expands into the antennal segment and into the optic lobe anlage (now encompassing also the ectodermal region called 'para-MB neuroectoderm'), and a further spot appears in the clypeolabral ectoderm. At this stage, Dac protein can be observed in 13 protocerebral NBs and in the tritocerebral Tv2, but in no deutocerebral NBs. From stage 12 onwards, Dac becomes expressed in an increasing number of scattered cell clusters in the brain and ventral nerve cord (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

Expression of dac is readily apparent prior to imaginal disc morphogenesis. Dac protein is detected at the posterior margin of the eye disc prior to furrow initiation. This expression pattern is similar to that of decapentaplegic at the same early stage of development. Strong dac expression is also detected in the unpatterned epithelium preceding the morphogenetic furrow as it moves anteriorly across the eye disc. Posterior to the furrow dac is expressed only in the part of the eye disc fated to become retina. Posterior to the furrow dac is expressed primarily in photoreceptors R1, R6 and R7, as well as the cone cells. The ring pattern of dac expression in the leg disc is established at an early stage of leg disc development, well before the characteristic epithelial folds of a mature leg disc are seen. Dac protein is found in the third antennal disc segment and the wing imaginal disc, as well as the larval brain (Mardon, 1994).

Signals transmitted along retinal axons in Drosophila: Hedgehog signal reception and the cell circuitry of lamina cartridge assembly

The arrival of retinal axons in the Drosophila brain triggers the assembly of glial and neuronal precursors into a neurocrystalline array of lamina synaptic cartridges. Retinal axons arriving from the eye imaginal disc trigger the assembly of neuronal and glial precursors into precartridge ensembles in the crescent-shaped lamina target field. In the eye disc, photoreceptor cells assemble into ommatidial clusters behind the morphogenetic furrow (mf) as it moves to the anterior. The ommatidial clusters project their axon fascicles into the crescent-shaped lamina. Neuronal precursor cells of the lamina (LPCs) are incorporated into the axon target field at its anterior margin, which is demarcated by a morphological depression known as the lamina furrow. Glia precursor cells (GPCs) are generated in two domains that lie at the dorsal and ventral anterior margins of the prospective lamina. These glial precursors migrate into the lamina along an axis perpendicular to that of LPC entry. Postmitotic LPCs within the lamina axon target field express the nuclear protein Dac, as revealed by anti-Dac antibody staining. Like the eye, lamina differentiation occurs in a temporal progression on the anterioposterior axis. Axon fascicles from new ommatidial R-cell clusters arrive at the anterior margin of the lamina (adjacent to the lamina furrow) and associate with neuronal and glia precursors in a vertical lamina column assembly. At the anterior of the lamina, at the trough of the lamina furrow, LPCs await a retinal axon-mediated signal in G1-phase and enter their terminal S-phase at the posterior margin of the furrow. Postmitotic (Dac-positive) LPCs assemble into columns at the posterior margin of the furrow. In older columns at the posterior of the lamina, a subset of postmitotic LPCs express definitive neuronal markers as they become specified as the lamina neurons L1-L5. Lamina neurons L1-L4 form a stack in a superficial layer, while L5 neurons reside in a medial layer near the R1-R6 axon termini. These neurons arise at cell-type specific positions along the column's vertical axis. Lamina glial cells take up cell-type positions in the precartridge assemblies. Epithelial (E-glia) and marginal (Ma-glia) glia are located above and below the R1-R6 termini, respectively. Satellite glia are interspersed among the neurons of the L1-L4 layer. The Ma-glia and E-glia layers, both located ventral to the neuronal precursor column, sandwich the R1-R6 axon termini. The medulla neuropil serves as the target for R7/8 axons and is separated from the lamina by the medulla glia, situated just below the Ma-glia (Huang, 1998 and references).

Hedgehog, a secreted protein, is an inductive signal delivered by retinal axons for the initial steps of lamina differentiation. In the development of many tissues, Hedgehog acts in a signal relay cascade via the induction of secondary secreted factors. Lamina neuronal precursors respond directly to Hedgehog signal reception by entering S-phase, a step that is controlled by the Hedgehog-dependent transcriptional regulator Cubitus interruptus. The terminal differentiation of neuronal precursors and the migration and differentiation of glia appear to be controlled by other retinal axon-mediated signals. Thus retinal axons impose a program of developmental events on their postsynaptic field utilizing distinct signals for different precursor populations (Huang, 1998).

A number of markers distinguish glial and neuronal precursor cells from the corresponding mature cell types. The expression of optomotor-blind (omb) labels both glial precursors in the dorsal and ventral anlagen and mature glia that have migrated into the lamina target field. The glia cell marker Repo and the enhancer-trap lacZ insertion 3-109 are expressed by glia once they have entered the lamina target field. Cubitus interruptus (Ci), a transcriptional mediator of Hh signaling is expressed by LPCs anterior of the lamina furrow and by the postmitotic neuronal precursors within the lamina. The nuclear protein Dachshund is expressed only by neuronal precursors that have begun terminal differentiation and lie posterior to the lamina furrow. Thus, Omb and Ci label the glial and neuronal precursors, respectively, while the mature cells, following their interaction with retinal axons, additionally express Repo and Dac. In the lamina target field of eyeless mutants (mutants that project no neurons toward the optic disc), such as eyes absent (eya) or sine oculis (so), Dac expression is not detected and Repo expression is greatly diminished (Huang, 1998).

The migration and early differentiation of lamina glia are independent of Hh. Enhanced transcription of the putative Hh receptor, patched (ptc) is a universal characteristic of Hh signal reception. All classes of glia in the lamina region upregulate ptc expression in an hh-dependent fashion. These cells are thus Hh-responsive. All three classes of lamina glia, as well as medulla glia, that express a ptc-lacZ reporter construct are in close proximity to Hh-bearing retinal axons. Glia cell ptc reporter gene expression is not observed in hh- animals. This raises the question of whether Hh signal reception is responsible for the migration and/or subsequent maturation of glia cells. To determine whether the migration of glial precursors into the lamina target field is Hh-dependent, the distribution of Omb-positive cells was examined in hh- animals. In the wild type, a trail of Omb-positive cells delineates a path of glia migration from the dorsal and vental anlagen. Is glia precursor migration Hh-dependent? This was investigated by examining the distribution of Omb-positive cells in hh¹ mutant animals. hh¹ is a regulatory mutation that specifically affects hh expression in the visual system. In hh¹ animals, approximately 12 columns of ommatidia initiate differentiation in the eye imaginal disc before the anterior progression of the morphogenetic furrow ceases. hh¹ retinal axons lack Hh immunoreactivity by the time they reach the lamina target field and thus the Hh-dependent steps of LPC maturation fail to occur in hh¹ animals. Omb staining reveals a relatively normal number of glia precursors in the lamina target field of hh¹ animals, despite the absence of Dac induction. The Omb-positive cells are distributed uniformly along the dorsoventral axis among the retinal axon fascicles, but appear more closely spaced than in the wild type. A likely explanation for this spacing defect is the absence of the neuronal precursors that would constitute the majority of lamina cells at this point in development. To determine whether the glial precursors that enter the lamina target field in hh- animals express a retinal innervation-dependent marker, their expression of Repo was examined. In hh¹ animals, the Omb-positive cells within the lamina also express Repo. Moreover, the Repo-positive cells occupy proper layers above and below the R1-R6 axon termini expected for satellite, marginal and epithelial glia, though the lack of markers specific for these three glia types precludes an unambiguous determination of glial cell type. The presence of marginal and epithelial glia is consistent with the observation that R1-R6 growth cones terminate in their proper positions between these layers in hh- animals. The ectopic expression of Hh in the brains of `eyeless' animals is sufficient to induce the initial steps of LPC maturation in the absence of retinal axons. However, neither Hh nor the Hh-mediated events of LPC maturation are sufficient for glia cell migration and maturation (Huang, 1998).

The activities of a number of Hh signal transduction pathway components are now well characterized. Mutations at these loci have been shown to either mimic or block Hh signal reception in a cell-autonomous fashion. Examining the cellular requirements for these genes in mosaic animals should help illuminate the cellular circuitry that mediates the Hh-dependent events of lamina development. The seven-pass transmembrane protein encoded by smoothened (smo) acts as a positive effector of Hh signal reception, downstream of the Hh receptor Patched. If Hh exerts its effects directly on LPCs, it would be expected that loss of smo function should block the entry of G1-phase LPCs into S-phase and/or prevent the expression of Hh-dependent markers of lamina differentiation such as Dac. Inducing smo mutant clones reveals that with respect to lamina differentiation, smo acts cell autonomously. smo clones that extended to the posterior of the lamina are rare. It is possible that LPCs that cannot respond to Hh are not readily incorporated into the lamina and displaced by smo+ LPCs. LPCs that are unable to respond to Hh might be eliminated by cell death (Huang, 1998).

Proximodistal axis formation in the Drosophila leg: subdivision into proximal and distal domains by Homothorax and Distal-less

The developing legs of Drosophila are subdivided into proximal and distal domains by the activity of the homeodomain proteins Homothorax (Hth) and Distal-less (Dll). The expression domains of Dll and Hth are initially reciprocal. In the mature third instar disc, Dll is expressed in a large central domain that corresponds to the presumptive tarsus and distal tibia. Dll is also expressed in a secondary ring. X-gal staining of adult legs carrying a Dll-lacZ reporter gene shows that this ring is located at the proximal edge of the femur, possibly extending slightly into the distal trochanter. The central domain of Dll expression is controlled by Wg and Dpp. The proximal ring arises in third instar and does not depend on Wg or Dpp activity. The leg disc is a continuous single-layered epithelial sheet that forms a series of folds as it grows. The peripheral region of the disc forms the proximal segments. This region is folded back over the central region where Dll is expressed. The domain of Hth expression extends from the peripodial membrane at the top, through the coxa and trochanter segment primordia. The distal-most portion of the Hth domain overlaps the proximal part of the dac-lacZ domain within the proximal ring of Dll expression in the femur. Dll is expressed alone in the central folds of the disc (which correspond to tarsal segment primordia). In proximal tarsus and tibia, Dll and Dac overlap. Dac is expressed alone in the presumptive femur. Because the disc is highly folded, horizontal optical sections make proximal and distal regions of the disc appear to be closely apposed, although they are actually far apart along the PD axis in the plane of the disc epithelium. Hth is expressed in the upper layer and around the lateral sides of the epithelial sac. Dll is expressed in the center of the lower layer. The two expression domains abut, but do not overlap. dac-lacZ is not detectably expressed at this stage, but can be reliably detected in slightly older discs at the transition from second to third instar. These observations suggest that the primary subdivision of the disc is into two domains: a central Dll-expressing domain and a proximal Hth-expressing domain. Wg and Dpp act together to induce Dll and Dac in the center of the leg disc. Wg and Dpp repress Hth and Teashirt, but not through activation of Dll (Wu, 1999).

The expression patterns of Dll and Hth/Exd reflect an early subdivision of the disc into proximal and distal domains. At early stages of disc development, Dll and Hth/Exd are expressed in reciprocal domains which account for all cells of the disc. At this stage, Dac is not yet expressed. What is the relationship between Dll and Hth/Exd expression in the early disc? The Dll domain is defined by Wg and Dpp signaling. The same signals repress nuclear localization of Exd and Hth expression. The reciprocity of Dll and Hth expression suggests a model in which Wg and Dpp act through Dll to repress Hth in the early disc. However, the analysis of marked Dll mutant clones reported here shows that this is not the case. Clones of Dll mutant cells located in the distal region of the leg do not express Hth. How is Hth repressed by Wg and Dpp? Dac is induced by Wg and Dpp toward the end of second instar. Hth expands distally, to some extent, in Dac mutant discs. These observations suggest that Dac contributes to Hth repression. However, Hth is repressed prior to the onset of Dac expression indicating that Dac cannot be the primary repressor. Whether Wg and Dpp act directly to repress Hth expression or act via another as unidentified repressor remains to be determined (Wu, 1999).

In conclusion, Hth and Dll expression appear to define alternative fates in the second instar disc. Under normal circumstances, there does not appear to be a cell lineage restriction between these populations (i.e. no compartment boundary). These results suggest that cells can cross between these territories if they are able to switch between Hth and Dll expression. This situation appears to be analogous to the DV subdivision of the leg disc (as opposed to the proximal distal subdivision reported here). DV subdivision is stable at the level of gene expression in a cell population, but is not a clonal lineage restriction boundary. Similarly, the separation of proximal and distal cell populations requires Hth function. These results suggest that cells at the interface between these two territories are specialized to allow integration of otherwise immiscible populations of cells (Wu, 1999 and references).

Lineage-tracing cells born in different domains along the PD axis of the developing Drosophila leg

Patterning of the developing limbs by the secreted signaling proteins Wingless, Hedgehog and Dpp takes place while the imaginal discs are growing rapidly. Cells born in regions of high ligand concentration may be displaced through growth to regions of lower ligand concentration. A novel lineage-tagging method was used to address the reversibility of cell fate specification by morphogen gradients. Responses to Hedgehog and Dpp in the wing disc are readily reversible. In the leg, cells readily adopt more distal fates, but do not normally shift from distal to proximal fate. However, they can do so if given a growth advantage. These results indicate that cell fate specification by morphogen gradients remains largely reversible so long as the imaginal discs are growing. In other systems, where growth and patterning are uncoupled, nonreversible specification events or ‘ratchet’ effects may be of functional significance (Weigmann, 1999).

In the developing leg disc, some responses to Wg and Dpp are readily reversible, while others are not. Lineage tracing of cells born in the TshGAL4 domain (proximal) suggests that cells readily lose Tsh (and Hth) expression and instead express Dll and Dac (distal markers). In young discs, a small proportion of cells expressing low levels of both Dll and Tsh are found at the edge between these domains. It is possible that these are cells in transition between the domains. These results suggest that cells born in the presumptive body wall readily contribute to formation of more distal leg regions. Under normal circumstances cells born in the Dll-expressing distal domain of the leg do not contribute to the body wall. However, they are not prohibited from doing so when given a strong growth advantage. The progeny of Dll-expressing cells in second instar are mostly fated to give rise to the tarsus and do not contribute to femur. In contrast, femur, tibia and tarsal segments (the distal segments) derive from cells that have expressed Dll in early third instar. The difference between these stages suggests that new cells must be induced to turn on Dll in order to provide the population of cells that contribute to the femur (the most proximal of the distal segments). These cells must derive from the Tsh domain in second instar and acquire Dll and Dac expression. At later stages, Dll is expressed at very low levels in the femur, where it may be repressed by Dac. Downregulation of Dll expression in the femur is unlikely to be a direct response to a lowering of Wg and Dpp signaling, because clones of cells unable to respond to these signals do not show abnormal Dll or Dac expression. This contrasts with the situation in the wing where removal of Dpp signaling leads to loss of Spalt expression. The low level of Dll expression in the femur is in part due to Dac activity, since dac mutant clones show elevated levels of Dll. The transient induction of Dll in the precursors of the femur is consistent with genetic analyses showing that formation of all leg segments except coxa depends on Dll activity in early development, whereas the low level of Dll expressed later is apparently not required for normal femur development (Weigmann, 1999).

When the distal part of a leg imaginal disc, or of an amphibian or a cockroach leg is removed, distal structures will regenerate from the cut edge. If the distal part of the leg disc is cultured in isolation, distal structures will regenerate from the cut edge, leading to a duplication of the fragment. The fact that distal structures regenerate but proximal structures do not has been termed distal transformation. These classical experiments have shown that cells have a general tendency to distalize, whereas their capability to proximalize is restricted. The current experiments show that distal transformation happens during normal development. Some proximal cells switch their pattern of gene expression as the disc grows and they acquire distal fate. Distal cells do not normally switch to proximal fate, but can do so if forced during early development. The classical regeneration studies suggest that the ability to shift from distal to proximal fate may be lost as development proceeds (Weigmann, 1999).

Proximodistal domain specification and interactions in developing Drosophila appendages

The morphological diversification of appendages represents a crucial aspect of animal body plan evolution. The arthropod antenna and leg are homologous appendages, thought to have arisen via duplication and divergence of an ancestral structure. To gain insight into how variations between the antenna and the leg may have arisen, the epistatic relationships among three major proximodistal patterning genes, Distal-less, dachshund and homothorax, have been compared in the antenna and leg of Drosophila. Drosophila appendages are subdivided into different proximodistal domains specified by specific genes, and limb-specific interactions between genes and the functions of these genes are crucial for antenna-leg differences. In particular, in the leg, but not in the antenna, mutually antagonistic interactions exist between the proximal and medial domains, as well as between medial and distal domains. The lack of such antagonism in the antenna leads to extensive coexpression of Distal-less and homothorax, which in turn is essential for differentiation of antennal morphology. Furthermore, a fundamental difference between the two appendages is the presence in the leg and absence in the antenna of a functional medial domain specified by dachshund. These results lead to a proposal that the acquisition of particular proximodistal subdomains and the evolution of their interactions has been essential for the diversification of limb morphology (Dong, 2001).

Each segment in the Drosophila leg is considered to be homologous to part or all of a segment in the antenna. The correspondences are based on reproducible homeotic transformations that can occur between parts of the two limbs. Such correlation enables a comparison of the expression domains of Dll, dac and hth between the antenna and the leg. The relative wild-type expression of these three important PD patterning genes of the leg differs in the antenna, indicating that their PD axes are differentially subdivided (Dong, 2001).

For example, at late third instar, Dll expression extends more proximally in the antenna into regions homologous to the leg trochanter. In addition, dac is expressed at lower levels and is expressed in fewer segments in the antenna than in the leg. The dac expression domain in the antenna lies completely within the Dll expression domain. In contrast, the dac and Dll domains in the leg are exclusive when dac expression is activated and remain largely non-overlapping at late third instar. hth is expressed only proximally in the leg, but is expressed throughout the antenna disc until early larval stages when it is lost from distal cells. Because Dpp and Wg, which regulate Dll, dac and hth in the leg, are similarly expressed in the antenna, it is thought unlikely that the differences in Dll, dac and hth expression could be accounted for by variations in Dpp and Wg expression. Instead, it is hypothesized that the differences are due to limb type-specific interactions between Dll, dac and hth. The results of experiments described here confirm that this is the case (Dong, 2001).

Gradients of the morphogens, Wg and Dpp, initiate the PD organization of the Drosophila leg by activating Dll and repressing dac distally and by repressing hth in the distal and medial leg. This creates three domains, distal, medial and proximal, that are specified by the expression Dll, dac and hth, respectively. The expression of dac is derepressed in clones of Dll-null cells in the presumptive distal region of the leg disc. The reciprocal is observed in dac null clones, where Dll expression expands into the medial domain. Mutually repressive interactions between the distal and medial domains therefore are required to keep these domains distinct from one another (Dong, 2001).

The interactions between proximal and medial domains were analyzed. dac is only rarely derepressed in hth-null clones, and ectopic expression of Hth is insufficient to downregulate dac expression in the medial leg. Thus, proximal-to-medial antagonism does not occur via hth. However, ectopic expression of a second proximal leg gene, tsh, can repress dac, and dac expression expands proximally in tsh hypomorphic leg discs. Proximal-to-medial antagonism therefore does occur in the Drosophila leg. Derepression of tsh expression in the dac-null clones has not been observed, but derepression of hth in dac-null clones has been observed. It is therefore concluded that mutually antagonistic interactions between the proximal and medial domains occur via the repression of dac by Tsh and repression of hth by Dac (Dong, 2001).

If the antenna is homologous to the leg, one might expect to find many genetic parallels, particularly with respect to the three major PD patterning genes of the leg, Dll, dac and hth. As in the leg, Dll and hth are required to specify the distal and proximal domains of the antenna. However, dac has a different function in the antenna. No deletions of antennal segments are observed in dac-null flies. In addition, the genetic relationships between Dll, dac and hth are different in the developing antenna. Specifically, the extensive overlap in expression of these three genes in the antenna indicates that domains are not kept separated as they are in the leg. The normal expression domain of dac in the antenna lies completely within an area of hth and Dll coexpression, making it unlikely that dac represses either gene. Nonetheless, because Dll and hth appear to have slightly lower levels of expression where dac is normally expressed, a test was performed to see whether either Hth or Dll levels would be elevated if dac were removed. No detectable changes in the levels of either Dll or Hth were observed in clones of cells that lack Dac. Therefore unlike the situation in the leg, Dac is insufficient to antagonize the expression of either Dll or hth in the antenna. Taken together, these data indicate that mutual antagonism is not a universal feature of appendage development (Dong, 2001).

dac has been shown to be essential for normal development of the medial leg, including trochanter, tibia, femur and first tarsal segment. Because dac also is expressed in the antenna, it has been thought that dac is not involved in specifying leg identity. Instead, the current view is that dac functions during leg development to specify medial addresses. However, lower levels of dac are reproducibly detected in antennal discs than in leg discs and the fact that only a single antennal segment (a3) expresses dac is intriguing. Therefore the consequences of increasing the domain and level of dac expression in the antenna were tested. This leads to the differentiation of medial leg structures in 100% of antennae examined, indicating that dac does play a role in the specification of leg fates (Dong, 2001).

The antennal regulation of dac by Dll also differs from that of the leg. The regulation of dac by Dll in the antenna varies depending on the proximodistal location. Dll can be a dac repressor or activator, or exert no effect on dac. Dac expression is not activated in Dll-null clones in the presumptive arista, whereas Dll-null clones in the presumptive base of the arista (segments a4 and a5) exhibit non-cell-autonomous dac activation, and Dll-null clones in the presumptive third antennal segment (a3), where dac is normally expressed, result in loss of dac. These data indicate that the regulation of dac by Dll in the antenna is different from that in the leg. They also indicate that the normal antennal expression of dac both requires Dll and has PD regional specificities. Because both Dll and Hth are required for antennal identity and are coexpressed with dac, Hth may also be required for the antennal expression of dac. Consistent with this view, ectopic expression of either Dll in antennal cells expressing Hth or of Hth in antennal cells expressing Dll can activate dac, as can ectopic coexpression of Dll and Hth in the wing disc. Furthermore, antennal dac expression, is not efficiently repressed by ectopic Hth (Dong, 2001).

Unlike Dll-null clones, both Dll hypomorphs and hth-null clones exhibit antenna-to-leg transformations. Examination of Dll hypomorphs and hth-null clones therefore reveals their homeotic functions. One such function may be the repression of leg dac. Leg expression of dac encompasses more segments and occurs at higher levels compared with the antenna. As in Dll hypomorphic leg discs, in Dll hypomorphic antenna discs, dac expression expands distally. hth-null clones exhibit derepression of dac in a1, a2 and a4 and elevation of Dac levels in a3. It is therefore proposed that the derepression of dac in Dll hypomorphs and in hth-null clones may represent leg-specific dac expression. Conclusive evidence for this awaits identification of dac enhancer elements and analysis of their regulatory inputs. Nonetheless, taken together, these data support the view that the regulation of leg and antennal dac expression occurs via distinct mechanisms and that the homeotic functions of Dll and hth are mediated not only through activation of antenna-specific genes such as spalt, but also through the active repression of leg development (Dong, 2001).

Appendages are subdivided by mutually antagonistic domains. Gradients of the morphogens Dpp and Wg initiate the PD organization of the Drosophila leg by activating Dll and repressing dac and hth distally, and by allowing the activation of dac while repressing hth medially. This creates three domains, distal, medial and proximal, that are specified respectively by expression of Dll, dac and hth. Further refinement and maintenance of the borders between domains requires mutually antagonistic interactions between proximal and medial domains as well as between medial and distal domains. Specifically, Dll and dac are mutually repressive. Also, mutually repressive interactions between the proximal and medial domains do exist via Tsh repression of dac and Dac repression of hth. Thus, pattern formation in the leg requires mutually antagonistic interactions among all three domains in order to refine and maintain borders that initially were set up by morphogens (Dong, 2001).

In contrast to the situation in the Drosophila leg, Dll, dac and hth are expressed in largely overlapping patterns in the antenna. This suggests that there is not mutual antagonism between Dll and hth in the antenna. Furthermore, that the entire antennal expression domain of dac lies within an area of Dll and hth coexpression indicates that Dac was unlikely to repress the antennal expression of either Dll or hth. Analysis of dac mutants confirms that Dac does not antagonize either proximal or distal development in the antenna but it does so in the leg. Therefore mutual antagonism is not a universal feature of appendage development (Dong, 2001).

It is noted that the absence of antagonism of any single PD domain towards another leads to overlap of otherwise exclusively expressed transcription factors. This, in turn, may permit the coexpressed factors to execute additional functions. Indeed, while Hth is required for proximal patterning of both antenna and leg, and Dll is required for distal patterning of both antenna and leg, their coexpression leads to the differentiation of antenna-specific cell fates. Thus, expression of distinct combinations of transcription factors such as Dll, Dac and n-Exd/Hth both in specific domains along the PD axis and between appendage types is likely to activate and repress particular suites of target genes, thereby contributing to differences in appendage morphologies (Dong, 2001).

The ability of Dll, Dac and n-Exd/Hth to repress the expression of one another undoubtedly is context-dependent. However, the only known factor involved in context specification is the Hox protein Antp. In the presence of Antp in the antenna, Dll and Hth are no longer coexpressed. Conversely, when Antp is removed from the leg, hth is derepressed in cells expressing Dll. Thus Antp appears to play a role in some aspects of domain antagonism. It remains unclear whether Antp directly modulates interactions among Dll, Dac and n-Exd/Hth or whether there are other molecules that intervene (Dong, 2001).

This comparison of the Drosophila antenna and leg leads to the conclusion that a fundamental difference between these homologous appendages is the presence of a functional medial domain in the leg, specified by dac. The antenna has fewer segments, with dac expressed at relatively low levels and in only one of the segments, whereas dac is expressed in at least four leg segments. Loss of dac results in medial deletions in the leg but not in the antenna. Repression of proximal and distal genes by dac is not observed in the antenna, as it is in the leg. Consequently, the antennal expression of n-Exd/hth and Dll are not separated in the antenna by a medial domain that expresses dac. For these reasons, it is proposed that the acquisition of a medial domain, possibly through the use of dac, may have been a distinct step in appendage evolution. Consistent with this, increasing the territory and levels of dac expression in the antenna leads to repression of hth and Dll and to the differentiation of medial leg structures (Dong, 2001).

Two scenarios by which the existing Drosophila domain organizations may have arisen can be envisioned, given primitive appendages that had only proximal and distal domains. One possibility is that the medial domains were initially acquired by both the antenna and leg, but lost from the antenna sometime prior to the evolution of Drosophila. A second possibility is that the medial domain is an innovation of only the leg and may never have existed in the antenna. The expression of dac in the legs and its absence in the antennae of other arthropods may provide support for the latter scenario. Comparison of the relative domains of expression and the functions of Dll, dac and hth in other organisms will undoubtedly lead to further insights into how distinct PD domains were acquired and became patterned during the course of appendage evolution (Dong, 2001).

EGF receptor and Notch signaling act upstream of Eyeless/Pax6 to control eye specification: Dac is an eye specification gene

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).

Embryonic and larval development of the Drosophila mushroom bodies: concentric layer subdivisions and the role of fasciclin II: Dac expression in Kenyon cells

Mushroom bodies (MBs) are the centers for olfactory associative learning and elementary cognitive functions in the arthropod brain. In order to understand the cellular and genetic processes that control the early development of MBs, high-resolution neuroanatomical studies of the embryonic and post-embryonic development of the Drosophila MBs have been performed. In the mid to late embryonic stages, the pioneer MB tracts extend along Fasciclin II (Fas II)-expressing cells to form the primordia for the peduncle and the medial lobe. As development proceeds, the axonal projections of the larval MBs are organized in layers surrounding a characteristic core, which harbors bundles of actin filaments. Mosaic analyses reveal sequential generation of the MB layers, in which newly produced Kenyon cells project into the core to shift to more distal layers as they undergo further differentiation. Whereas the initial extension of the embryonic MB tracts is intact, loss-of-function mutations of fas II causes abnormal formation of the larval lobes. Mosaic studies demonstrate that Fas II is intrinsically required for the formation of the coherent organization of the internal MB fascicles. Furthermore, ectopic expression of Fas II in the developing MBs results in severe lobe defects, in which internal layers also are disrupted. These results uncover unexpected internal complexity of the larval MBs and demonstrate unique aspects of neural generation and axonal sorting processes during the development of the complex brain centers in the fruit fly brain (Kurusu, 2002).

Studies of MB development with mosaic clones have shown that MB neurons in the adult brain are classified into three groups that project dorsally to the alpha and alpha' lobes and medially to the ß, ß' and gamma lobes. Based on this classification, all the Kenyon cells born before the mid-third larval instar belong to the gamma group. Only in the late third instar, the second group of neurons projecting into the alpha' and ß' lobes is produced. In this study, using various MB markers, it has been demonstrated that the larval Kenyon cells can be further subdivided into distoproximal concentric groups surrounding each of the neuroblasts. Furthermore, the axonal projections of the Kenyon cells are also organized into concentric layers in the peduncle and lobes. Axons of newly born Kenyon cells project into the core that is constituted of densely packed thin fibers rich in actin filaments (Kurusu, 2002).

Distoproximal expression patterns of nuclear regulatory genes in the larval MB cell clusters have been described. In particular, whereas ey is expressed in all the MB cells, including the neuroblasts and ganglion mother cells (GMCs), dac is expressed in differentiated Kenyon cells but not in the centrally located proliferating cells. GAL4 MB markers, such as 201Y and c739, are expressed in an outer group of the differentiated Kenyon cells that is located several cell diameters away from the proliferating neuroblasts (Kurusu, 2002).

While the four MB neuroblasts continue dividing up to the late pupal stage supplying increasing numbers of Kenyon cells, the newly formed larval MB axons follow the medial and the dorsal lobe projections that were pioneered at the embryonic stage with a concomitant increase in the sizes of the lobes. By contrast, a set of genes is turned on in the Kenyon cells after the hatching of the first instar larvae in slightly different patterns in both the cell bodies and their projections. As development proceeds, these differential gene expression patterns became more evident in the second instar larval stage. While the Dac protein is expressed in most of the Kenyon cells, dnc-lacZ is expressed in a small subset of cells peripherally positioned in each of the Kenyon cell clusters originated by the four MB neuroblasts. Expression of 201Y is detected in another subset of cells located more centrally in each of the Kenyon cell clusters, whereas c739 is widely expressed in most of the Kenyon cells (Kurusu, 2002).

Remarkably, these differential expression patterns observed in the Kenyon cells were topologically reflected in their axonal projections in the peduncle and lobes: dnc-lacZ is detected in the outermost surface layer of the peduncle and lobes; 201Y is detected in both the surface and middle layers; and c739 is detected in most axons, a pattern similar to that of FAS II (Kurusu, 2002).

As development proceeds further, further subdivisions emerge in the third instar larval stage with increasing numbers of Kenyon cells and their axons. Moreover the expression patterns of the 201Y and c739 markers change in both cell bodies and their projections; 201Y is then detected in many of the Kenyon cells and their projections, obscuring the 201Y peripheral pattern in the previous larval instar; c739 is then detected in a group of cells located near each of the neuroblasts. The axons of the c739-expressing cells project into an inner layers of the peduncle and lobes. By contrast, dnc-lacZ is maintained in the peripheral subdivisions both in the Kenyon cells and their projections. Double staining with anti-Fas II antibody confirms discrete internal organization of the peduncle and lobes, which are concentrically subdivided into at least three layers surrounding a core that is not labeled with the MB markers, including Fas II (Kurusu, 2002).

Interestingly, the reporter molecule for dnc-lacZ exhibits a characteristic patchy appearance in the calyx, peduncle and lobes, suggesting uneven distribution of the dnc-lacZ fibers. Indeed, higher magnification of the calyces double labeled with anti-ß-gal and anti-synaptotagmin antibodies reveals extensive arborization of the dnc-lacZ expressing neurons around the synaptic terminals, which are likely to represent the afferent terminals of axonal collaterals of the antennocerebral neurons (Kurusu, 2002).

Based on these expression profiles of nuclear regulatory genes and GAL4 markers in the cell bodies, it is suggested that the Kenyon cells that are labeled with both Dac and 201Y project their axons into the concentric layers that also are labeled with Fas II. However, the proximally located Kenyon cells that are labeled with DAC but not 201Y may correspond to the newly differentiated MB neurons that project thin fibers into the core of the peduncle and lobes. Recently described (using a DsRed variant) has been a similar concentric generation of Kenyon cell fibers in the surrounding layers of the peduncle and lobes, in which younger axons extend into the inner layer to shift older fibers into the outer layers. Clonal studies on the larval projection patterns support this temporal order of layer generation and further show that axons of the newly produced Kenyon cells first project into the core as actin-rich thin fibers to shift to the surrounding layers as they undergo further differentiation (Kurusu, 2002).

glial cells missing and gcm2 cell autonomously regulate both glial and neuronal development in the visual system of Drosophila: Dachshund acts dowstream of gcm

The transcription factors Glial cells missing (Gcm) and Gcm2 are known to play a crucial role in promoting glial-cell differentiation during Drosophila embryogenesis. A central function for gcm genes has been revealed in regulating neuronal development in the postembryonic visual system. Gcm and Gcm2 are expressed in both glial and neuronal precursors within the optic lobe. Removal of gcm and gcm2 function shows that the two genes act redundantly and are required for the formation of a subset of glial cells. They also cell-autonomously control the differentiation and proliferation of specific neurons. The transcriptional regulator Dachshund acts downstream of gcm genes and is required to make lamina precursor cells and lamina neurons competent for neuronal differentiation through regulation of epidermal growth factor receptor levels. These findings further suggest that gcm genes regulate neurogenesis through collaboration with the Hedgehog-signaling pathway (Chotard, 2005).

To explore the mechanisms by which gcm genes mediate neuronal development in the optic lobe, the role of Dac was examined because its expression depends on both the activation of the Hh pathway and on gcm and gcm2 function. Genetic analysis added two findings to an understanding as to how Hh and EGF signaling work in concert to regulate neurogenesis in the lamina. It was shown that (1) dac is not required for cell divisions of LPCs and (2) that expression of dac is necessary for the upregulation and maintenance of EGF receptor expression in lamina neurons to promote their further maturation. This is consistent with findings in the developing eye imaginal disc, demonstrating that Dac promotes early progression of the morphogenetic furrow and aspects of R-cell specification but is not required for cell proliferation. In the eye, genetic interaction assays have previously established a link between Dac and EGFR signaling because dac mutant alleles were identified as suppressors of the dominant-active EGFR allele Ellipse, although the precise mechanism underlying this interaction is unclear. The current findings present evidence for one possible mechanism by demonstrating that Dac controls EGF receptor levels in the optic lobe and, in this way, makes LPCs and their progeny competent for neuronal differentiation. In Drosophila, processing of EGF ligands by Rhomboids rather than the regulation of the receptor itself has been considered to be a limiting step in EGF receptor signaling. In the rodent retina, both ligand and receptor levels have been reported to mediate different cellular responses such as proliferation and cell-fate specification. Therefore, regulating receptor levels by Dac represents an additional mechanism to modulate activity of the EGF receptor pathway in the optic lobe of flies. gcm genes can contribute to neuronal differentiation through induction of Dac. Their role in promoting mitotic divisions of LPCs, however, must involve another mechanism. Indeed, genetic analysis suggests that gcm genes regulate both developmental processes through interaction with the Hh-signaling pathway (Chotard, 2005).

Sex-specific repression of dachshund is required for Drosophila sex comb development

The origin of new morphological structures requires the establishment of new genetic regulatory circuits to control their development, from initial specification to terminal differentiation. The upstream regulatory genes are usually the first to be identified, while the mechanisms that translate novel regulatory information into phenotypic diversity often remain obscure. In particular, elaborate sex-specific structures that have evolved in many animal lineages are inevitably controlled by sex-determining genes, but the genetic basis of sexually dimorphic cell differentiation is rarely understood. This report examines the role of dachshund (dac), a gene with a deeply conserved function in sensory organ and appendage development, in the sex comb, a recently evolved male-specific structure found in some Drosophila species. dac is shown to acts during metamorphosis to restrict sex comb development to the appropriate leg region. Localized repression of dac by the sex determination pathway is necessary for male-specific morphogenesis of sex comb bristles. This pupal function of dac is separate from its earlier role in leg patterning, and Dac at this stage is not dependent on the pupal expression of Distalless (Dll), the main regulator of dac during the larval period. Dll acts in the epithelial cells surrounding the sex comb during pupal development to promote sex comb rotation, a complex cellular process driven by coordinated cell rearrangement. These results show that genes with well-conserved developmental functions can be re-used at later stages in development to regulate more recently evolved traits. This mode of gene co-option may be an important driver of evolutionary innovations (Atallah, 2013).

EFFECTS OF MUTATION

In the absence of dachshund function, cells at the posterior margin of the eye disc, where the hedgehog driven morphogenic furrow initiates, fail to follow a retinal differentiation pathway and appear to adopt a cuticle fate instead. These cells are therefore unable to respond to pattern propagation signals such as hedgehog and furrow initiation does not occur. In contrast, cells in more anterior portions of the eye disc are able to differentiate as retinal cells in the absence of dachshund activity and respond normally to patterning signals. It is concluded that posterior margin cells are distinct from other cells of the eye imaginal disc by early stages of development (Mardon, 1994).

The furrow can propagate through mutant clones but the rate of movement is reduced. In addition photoreceptor maturation in mosaic clones at the border of mutant clones is defective both inside mutant clones and in adjacent wild-type cells. Thus dac-dependent activity from surrounding wild-type cells is unable to rescue the aberrant ommatidial structure of dac mutant clones (Mardon, 1994).

Genetic analysis reveals an interaction between dac and hedgehog. hh is required for the progression of the morphogenetic furrow. hh loss-of-function mutants dominantly enhance the recessive eye phenotype of weak dac alleles. These genetic results suggest that dac and hh act in the same or in parallel pathways during eye development. hh is not expressed in dac mutant eye discs, but hh expression in other imaginal discs, including the antennal, leg and wing discs, is not affected by loss of dac function. hh expression is normal in dac clones. Thus, dac function is not cell autonomously required for hh expression (Mardon, 1994).

Targeted expression of dachshund is sufficient to direct ectopic retinal development in a variety of tissues, including the adult head, thorax and legs. This result is similar to that observed with the highly conserved Drosophila gene eyeless (ey) that can induce ectopic eye formation on all major appendages. dachshund and eyeless induce the expression of one another. dachshund is required for ectopic retinal development driven by eyeless misexpression (Shen, 1997).

The dachshund gene encodes a putative transcriptional regulator required for eye and leg development. dachshund is also required for normal brain development. Dac is strongly expressed in mushroom body (MB) neurons that are 8-10 hours old, but not in neuroblast, GMC or newly born MB neurons. This is consistent with a role for Dac in mature MB neurons, rather than in the developmental events such as cell division that lead to their birth. The mushroom bodies of dachshund mutants exhibit a marked reduction in the number of a lobe axons, a disorganization of axons extending into horizontal lobes, and aberrant projections into brain areas normally unoccupied by mushroom body processes. The phenotypes become pronounced during pupariation, suggesting that dachshund function is required during this period. GAL4-mediated expression of dachshund in the mushroom bodies rescues the mushroom body phenotypes. Moreover, dachshund mutant mushroom body clones in an otherwise wild-type brain exhibit the phenotypes, indicating an autonomous role for dachshund. Dac is required autonomously within the MB for three major aspects of MB cell differentiation: (1) Dac is required for MB neurites to respect their normal neuropil borders; (2) it is required for axons to arrange themselves properly within the horizontal lobes; (3) and most dramatically, Dac is required for MB axons to be able to fill the alpha lobe neuropil properly. Although eyeless, like dachshund, is preferentially expressed in the mushroom body and is genetically upstream of dachshund for eye development, no interaction of these genes was detected for mushroom body development. Thus, dachshund functions in the developing mushroom body neurons to ensure their proper differentiation (Martini, 2000).

dac4, a null allele of dac, was used to see if any defects could be detected in the larval mushroom body (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).

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).

Independent roles of the dachshund and eyes absent genes in BMP signaling, axon pathfinding and neuronal specification

In the Drosophila nerve cord, a subset of neurons expresses the neuropeptide FMRFamide related (Fmrf). Fmrf expression is controlled by a combinatorial code of intrinsic factors and an extrinsic BMP signal. However, this previously identified code does not fully explain the regulation of Fmrf. The Dachshund (Dac) and Eyes Absent (Eya) transcription co-factors participate in this combinatorial code. Previous studies have revealed an intimate link between Dac and Eya during eye development. Here, by analyzing their function in neurons with multiple phenotypic markers, it is demonstrated that they play independent roles in neuronal specification, even within single cells. dac is required for high-level Fmrf expression, and acts potently, together with apterous and BMP signaling, to trigger Fmrf expression ectopically, even in motoneurons. By contrast, eya regulates Fmrf expression by controlling both axon pathfinding and BMP signaling, but cannot trigger Fmrf ectopically. Thus, dac and eya perform entirely different functions in a single cell type to ultimately regulate a single phenotypic outcome (Miguel-Aliaga, 2004).

Phenotypic and transcriptional synergy between So, Dac and Eya during development and in vitro has been well documented. By contrast, the current results indicate that these genes can act independently in the embryonic nervous system to specify neuronal identity. This is the case even when they are coexpressed in the same neuron; while no evidence of so expression was found in the ap-cluster, dac and eya functioned together with the previously identified ap/sqz/BMP combinatorial code to activate Fmrf expression in Tv neurons. However, eya controls additional aspects of Tv neuronal identity, such as axon pathfinding and the ability to respond to a BMP signal. Furthermore, the expression of Dac, but not Eya, So or Ap, in a large number of interneurons has suggested that Dac has additional, independent functions in postmitotic neurons (Miguel-Aliaga, 2004).

The molecular mechanisms underlying transcriptional synergy between So (Six), Eya and Dac (Dach) have proven to be quite complex. In most cases examined, So/Six binds DNA and Dac/Dach and Eya regulate its activity. These biochemical models would not appear to explain the current observations fully. In these studies, Dac appears to act as a potent activator of Fmrf expression but to rely on Eya for activating Fmrf expression only within ap-neurons; when dac and ap are co-misexpressed in all neurons there is widespread ectopic Fmrf expression without any ectopic Eya expression. Why Eya is required in the ap-neurons for both endogenous and ectopic Fmrf expression, but not for ectopic Fmrf expression outside ap-neurons, is currently unclear (Miguel-Aliaga, 2004).

The current findings illustrate the fact that regulators acting within a postmitotic neuron can act together in a combinatorial fashion to specify one aspect of neuronal identity (Fmrf expression, in this case). However, some of these regulators can simultaneously function in combinatorial sub-codes to control other aspects of neuronal identity; the additional roles of ap and eya in Tv axon pathfinding may be one such example. In abdominal hemisegments, Ap is expressed in the two vAp and the single dAp neurons. Normally, the axons of these neurons join a common ipsilateral longitudinal fascicle running the length of the VNC. Previous studies have revealed that ap is important for proper ap-axon fasciculation as well as for their avoidance of the midline. Eya is not expressed in vAp neurons, and the results indicate that it specifically controls dAp pathfinding. The eya mutant phenotype only partially phenocopies the ap phenotype, since eya affects midline crossing but not fasciculation; once dAp neurons have aberrantly crossed the midline they join the contralateral ap-fascicle. Neither the ap nor the eya mutant phenotypes are due to any apparent crossregulation between these two genes. Surprisingly, these findings indicated that different genetic mechanisms underlie the indistinguishable, ap-dependent axon pathfinding of dAp and vAp neurons; dAp axons crucially depend upon eya to avoid crossing the midline, whereas vAp axons neither express eya nor depend upon it (Miguel-Aliaga, 2004).

Together with previous findings these results indicate that Fmrf expression is triggered by the combinatorial action of ap, sqz, dimm, dac, eya and BMP signaling. However, with the exception of BMP signaling, none of these factors are absolutely necessary for endogenous Fmrf expression - in all mutants, expression of Fmrf is not lost from all Tv neurons. Similarly, although misexpression of a partial code can lead to ectopic Fmrf expression, its expression levels are consistently weaker than those seen in Tv neurons. Thus, it appears that a partial code is sufficient for some level of Fmrf expression: the ectopic expression of Fmrf in BMP-positive RP neurons (cells that do not express sqz, eya or dimm) in response to dac and ap is one such example. However, the complete code (ap/sqz/dimm/dac/eya/BMP) appears to be necessary for wild-type (high) levels of expression, as seen in the Tv neurons. It is possible that the simultaneous misexpression of all these factors would lead to robust ectopic Fmrf expression in all neurons. Due to obvious technical limitations, this idea has not been tested (Miguel-Aliaga, 2004).

Multiple signal transduction inputs/outputs appear to revolve around Eya: (1) phosphorylation of Eya by the Ras/MAPK pathway has been found to regulate Eya activity and synergy with So; (2) the transcriptional activity of Eya itself depends upon an intrinsic tyrosine phosphatase activity that is also required for ectopic eye induction in Drosophila. The target(s) of Eya phosphatase activity are currently unknown. (3) It is found that Eya regulates the BMP pathway in Tv neurons and pMad cannot be reactivated in eya mutants even by cell-autonomous introduction of the BMP ligand Gbb. A probable explanation for this result is that eya regulates the expression or activity of the BMP type receptors Wit, Tkv or Sax. When the BMP pathway is dominantly activated by the use of activated type I receptors, nuclear pMad is restored. However, this still does not reactivate Fmrf expression, indicating that Eya additionally plays important roles downstream of pMad activation. One interpretation of these findings is that Eya acts directly on the Fmrf gene. However, it is also tempting to speculate that Eya may act to modulate pMad activity directly. There are several reasons for this proposal. It is known that several other kinase pathways, such as MAPK, can phosphorylate Smad proteins on residues other than those phosphorylated by TGFß/BMP type I receptors. The in-vivo roles of such modifications are unclear, but in-vitro evidence points to both repression and activation of Smad activity. Nevertheless, given its nuclear localization and phosphatase activity, it is possible that Eya acts to de-phosphorylate inhibitory residues in pMad. A regulatory circuitry between MAPK (and other kinases), Eya and the TGFß/BMP pathway is an intriguing possibility. Moreover, recent studies reveal that vertebrate orthologs of Dac can physically interact with the Smad complex, thereby affecting TGF-ß signaling. Together with these previous findings, the current results point to a model wherein Eya and Dac play central roles in integrating input from, and controlling the activity of, multiple signal transduction networks. Determination of the precise mechanisms by which Eya and Dac orchestrate these events should enhance understanding of how both intrinsic and extrinsic signals intersect to affect cellular differentiation (Miguel-Aliaga, 2004).

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).

Developmental transcriptional networks are required to maintain neuronal subtype identity in the mature nervous system

During neurogenesis, transcription factors combinatorially specify neuronal fates and then differentiate subtype identities by inducing subtype-specific gene expression profiles. But how is neuronal subtype identity maintained in mature neurons? Modeling this question in two Drosophila neuronal subtypes (Tv1 and Tv4), tests were performed to see whether the subtype transcription factor networks that direct differentiation during development are required persistently for long-term maintenance of subtype identity. By conditional transcription factor knockdown in adult Tv neurons after normal development, it was found that most transcription factors within the Tv1/Tv4 subtype transcription networks are indeed required to maintain Tv1/Tv4 subtype-specific gene expression in adults. Thus, gene expression profiles are not simply 'locked-in,' but must be actively maintained by persistent developmental transcription factor networks. The cross-regulatory relationships were examined between all transcription factors that persisted in adult Tv1/Tv4 neurons. Certain critical cross-regulatory relationships that had existed between these transcription factors during development are no longer present in the mature adult neuron. This points to key differences between developmental and maintenance transcriptional regulatory networks in individual neurons. Together, these results provide novel insight showing that the maintenance of subtype identity is an active process underpinned by persistently active, combinatorially-acting, developmental transcription factors. These findings have implications for understanding the maintenance of all long-lived cell types and the functional degeneration of neurons in the aging brain (Eade, 2012).

The data provide novel insight supporting the view of Blau and Baltimore (1991) that cellular differentiation is a persistent process that requires active maintenance, rather than being passively 'locked-in' or unalterable. Two primary findings are made in this study regarding the long-term maintenance of neuronal identity. First, all known developmental transcription factors acting in postmitotic Tv1 and Tv4 neurons to initiate the expression of subtype terminal differentiation genes are then persistently required to maintain their expression. Second, it was found that key developmental cross-regulatory relationships that initiated the expression of certain transcription factors were no longer required for their maintained expression in adults. Notably, this was found to be the case even between transcription factors whose expression persists in adults (Eade, 2012).

In this study, all transcription factors implicated in the initiation of subtype-specific neuropeptide expression in Tv1 and Tv4 neurons were found to maintain subtype terminal differentiation gene expression in adults (see Summary of changes in subtype transcription network configuration between initiation and maintenance of subtype identity). In Tv1, col, eya, ap and dimm are required for Nplp1 initiation during development. In this study, knockdown of each transcription factor in adult Tv1 neurons was shown to dramatically downregulate Nplp1. In Tv4 neurons, FMRFa initiation during development requires eya, ap, sqz, dac, dimm and retrograde BMP signaling. Together with previous work showing that BMP signaling maintains FMRFa expression in adults (Eade, 2009), this study now demonstrates that all six regulatory inputs are required for FMRFa maintenance. Most transcription factors, except for dac, also retained their relative regulatory input for FMRFa and Nplp1 expression. In addition, individual transcription factors also retained their developmental subroutines. For example, as found during development, dimm was required in adults to maintain PHM (independently of other regulators) and FMRFa/Nplp1 expression (combinatorially with other regulators) (Eade, 2012).

The few genetic studies that test a persistent role for developmental transcription factors support their role in initiating and maintaining terminal differentiation gene expression. In C. elegans, where just one or two transcription factors initiate most neuronal subtype-specific terminal differentiation genes, they then also appear to maintain their target terminal differentiation genes. In ASE and dopaminergic neurons respectively, CHE-1 and AST-1 initiate and maintain expression of pertinent subtype-specific terminal differentiation genes. In vertebrate neurons, where there is increased complexity in the combinatorial activity of transcription factors in subtype-specific gene expression, certain transcription factors have been demonstrated to be required for maintenance of subtype identity. These are Hand2 that initiates and maintains tyrosine hydroxylase and dopa ß-hydroxylase expression in mouse sympathetic neurons, Pet-1, Gata3 and Lmx1b for serotonergic marker expression in mouse serotonergic neurons, and Nurr1 for dopaminergic marker expression in murine dopaminergic neurons (Eade, 2012).

However, while these studies confirm a role for certain developmental transcription factors in subtype maintenance, it had remained unclear whether the elaborate developmental subtype transcription networks, that mediate neuronal differentiation in Drosophila and vertebrates, are retained in their entirety for maintenance, or whether they become greatly simplified. This analysis of all known subtype transcription network factors in Tv1 and Tv4 neurons now indicates that the majority of a developmental subtype transcription network is indeed retained and required for maintenance. Why would an entire network of transcription factors be required to maintain subtype-specific gene expression? The combinatorial nature of subtype-specific gene expression entails cooperative transcription factor binding at clustered cognate DNA sequences and/or synergism in their activation of transcription. In such cases, the data would indicate that this is not dispensed with for maintaining terminal differentiation gene expression in mature neurons (Eade, 2012).

How the transcription factors of the subtype transcription networks are maintained is less well understood. An elegant model has emerged from studies in C. elegans, wherein transcription factors stably auto-maintain their own expression and can then maintain the expression of subtype terminal differentiation genes. The transcription factor CHE-1 is a key transcription factor that initiates and maintains subtype identity in ASE neurons. CHE-1 binds to a cognate DNA sequence motif (the ASE motif) in most terminal differentiation genes expressed in ASE neurons, as well as in its own cis-regulatory region. Notably, a promoter fusion of the che-1 transcription factor failed to express in che-1 mutants, indicative of CHE-1 autoregulation, and for the cooperatively-acting TTX-3 and CEH-10 transcription factors in AIY neurons. Thus, subtype maintenance in C. elegans is anchored by auto-maintenance of the transcription factors that initiate and maintain terminal differentiation gene expression (Eade, 2012).

In contrast, all available evidence in Tv1 and Tv4 neurons fails to support such an autoregulatory mechanism. An ap reporter (apC-t-lacZ) is expressed normally in ap mutants, and in this study ap^dsRNAi was not found to alter ap^GAL4 reporter activity. Moreover, col transcription was unaffected in col mutants that express a non-functional Col protein. This leaves unresolved the question of how the majority of the transcription factors are stably maintained. For transcription factors that are initiated by transiently expressed inputs, a shift to distinct maintenance mechanisms have been invoked and in certain cases shown. In this study, this was found for the loss of cas expression in Tv1 (required for col initiation) and the loss of cas, col and grh in Tv4 (required for eya, ap, dimm, sqz, dac initiation). However, it was surprising to find that the cross-regulatory relationships between persistently-expressed transcription factors were also significantly altered in adults. Notably, eya initiated but did not maintain dimm in Tv4. In Tv1, col initiated but did not maintain eya, ap or dimm. This was particularly unexpected as eya remained critical for FMRFa maintenance and col remained critical for Nplp1 maintenance. Indeed, although tests were performed for cross-regulatory interactions between all transcription factors in both the Tv1 and Tv4 subtype transcription networks, only Dimm was found to remain dependent upon its developmental input; Eya and Ap in Tv1 as well as Ap in Tv4. However, even in this case, the regulation of Dimm was changed; it no longer required eya in Tv4, and in Tv1 it no longer required col, in spite of the fact that both col and eya are retained in these neurons. It is anticipated that such changes in transcription factor cross-regulatory relationships will be found in other Drosophila and vertebrate neurons, which exhibit high complexity in their subtype transcription networks. Indeed, recent evidence has found that in murine serotonergic neurons, the initiation of Pet-1 requires Lmx-1b, but ablation of Lmx-1b in adults did not perturb the maintenance of Pet-1 expression (Eade, 2012).

The potential role of autoregulation for the other factors in the Tv1/Tv4 subtype transcription networks is being pursued. However, there are three additional, potentially overlapping, models for subtype transcription network maintenance. First, regulators may act increasingly redundantly upon one another. Second, unknown regulators may become increasingly sufficient for transcription factor maintenance. Third, transcription factors may be maintained by dedicated maintenance mechanisms, as has been shown for the role of trithorax group genes in the maintenance of Hox genes and Engrailed. Moreover, chromatin modification is undoubtedly involved and likely required to maintain high-level transcription of Tv transcription factors as well as FMRFa, Nplp1 and PHM. However, the extent to which these are instructive as opposed to permissive has yet to be established. In this light, it is intriguing that MYST-HAT complexes, in addition to the subtype transcription factors Che-1 and Die-1, are required for maintenance of ASE-Left subtype identity in C. elegans (Eade, 2012).

Taken together, these studies have identified two apparent types of maintenance mechanism that are operational in adult neurons. On one hand, there are sets of genes that are maintained by their initiating set of transcription factors. These include the terminal differentiation genes and the transcription factor dimm. On the other, most transcription factors appear to no longer require regulatory input from their initiating transcription factor(s). Further work will be required to better understand whether these differences represent truly distinct modes of gene maintenance or reflect the existence of yet unidentified regulatory inputs onto these transcription factors. One issue to consider here is that the expression of certain terminal differentiation genes in neurons, but perhaps not subtype transcription factors, can be plastic throughout life, with changes commonly occurring in response to a developmental switch or physiological stimulus. Thus, terminal differentiation genes may retain complex transcriptional control in order to remain responsive to change. It is notable, however, that FMRFa, Nplp1 and PHM appear to be stably expressed at high levels in Tv1/4 neurons, and no conditions were found that alter their expression throughout life. Thus, these are considered to be stable terminal differentiation genes akin to serotonergic or dopaminergic markers in their respective neurons that define those cells' functional identity and, where tested, are actively maintained by their developmental inputs. Tv1/4 neurons undoubtedly express a battery of terminal differentiation genes, and sets of unknown transcription factors are likely required for their subtype-specific expression. Subtype transcription networks are considered to encompass all regulators required for differentiating the expression of all subtype-specific terminal differentiation genes. Further, differentiation of subtype identity is viewed as the completion of a multitude of distinct gene regulatory events in which each gene is regulated by a subset of the overall subtype transcription network. As highly restricted terminal differentiation genes expressed in Tv1 and Tv4 neurons, it is believed that Nplp1, FMRFa and PHM provide a suitable model for the maintenance of overall identity, with the understanding that other unknown terminal differentiation genes expressed in Tv1 and Tv4 may not be perturbed by knockdown of the transcription factors tested in this study. In the future, it will be important to incorporate a more comprehensive list of regulators and terminal differentiation genes for each neuronal subtype. However, it is believed that the principles uncovered in this study for FMRFa, Nplp1 and PHM maintenance will hold for other terminal differentiation genes (Eade, 2012).

Finally, it is proposed that the active mechanisms utilized for maintenance of subtype differentiation represent an Achilles heel that renders long-lived neurons susceptible to degenerative disorders. Nurr1 ablation in adult mDA neurons reduced dopaminergic markers and promoted cell death. Notably, Nurr1 mutation is associated with Parkinson's disease, and its downregulation is observed in Parkinson's disease mDA neurons. Adult mDA are also susceptible to degeneration in foxa2 heterozygotes, another regulator of mDA neuron differentiation that is maintained in adult mDA neurons. Studies in other long-lived cell types draw similar conclusions. Adult conditional knockout of Pdx1 reduced insulin and ß-cell mass and, importantly, heterozygosity for Pdx1 leads to a rare monogenic form of non-immune diabetes, MODY4. Similarly, NeuroD1 haploinsufficiency is linked to MODY6 and adult ablation of NeuroD in β-islet cells results in β-cell dysfunction and diabetes. These data, together with current results, underscore the need to further explore the transcriptional networks that actively maintain subtype identity, and hence the function, of adult and aging cells (Eade, 2012).

EVOLUTIONARY HOMOLOGS

Dachshund homologs in other insects

The homolog of the Drosophila gene dachshund (dac) was isolated from the beetle Tribolium castaneum. Tc'dac is expressed in all appendages except urogomphi and pleuropodia. Tc'dac is also active in the head lobes, in the ventral nervous system, in the primordia of the Malpighian tubules and in bilateral stripes corresponding to the presumptive dorsal midline. Expression of Tc'dac in the labrum lends support to the interpretation that the insect labrum is derived from a metameric appendage. The legs of Tribolium accommodate two Tc'dac domains, of which the more distal corresponds to the single dac domain described for Drosophila leg discs. In contrast to Drosophila, where this domain is thought to intercalate between the homothorax (hth) and the Distal-less (Dll) domains, in Tribolium it arises from within the Dll domain. Both the distal Tc'dac domain in the legs as well as the expression in the labrum are deleted in embryos mutant for the Tc'Dll gene, while the proximal leg domain and the mandibular expression are unaffected. Based on Tc'dac expression in wild-type and mutant embryos, serial homology of the complete mandible with the coxa of the thoracic legs has been demonstrated. This homology affirms the gnathobasic nature of the insect mandible (Prpic, 2001).

dac expression in the leg primordia of Tribolium and the imaginal discs of Drosophila is similar. In both species, strong expression of dac appears relatively late during leg development, and at intermediate proximal-distal positions. In mature leg primordia, this dac domain is flanked proximally and distally by the 'ring' and the 'sock' of Dll expression, respectively. Also, a region of overlap between dac and the Dll 'sock' is present in both species. One difference between the Tribolium patterns and those in Drosophila is that distal regions appear to be underrepresented in the beetle leg primordia. While the region of Dll/dac overlap is large in Drosophila,including most of the primordia of tibia and tarsus, in Tribolium this overlap is restricted to a narrow ring. However, this correlates well with the fact that certain distal structures of the adult beetle leg are only formed during metamorphosis. The tibia and the tarsomeres of the imaginal leg are represented in the larva only as a single rather short podomere, termed tibiotarsus. The final number and size of adult podomeres arise in a second growth phase during metamorphosis (Prpic, 2001).

In addition to the difference just mentioned, there are three more discrepancies in dac expression in the embryonic thoracic leg primordia of Tribolium versus the postembryonic imaginal discs of Drosophila . (1) A surprising finding is the presence of an additional proximal domain in the thoracic appendages of Tribolium. In Drosophila, dac is reported to form a single domain in the leg discs. In crustaceans and chelicerates, too, only one dac domain has been observed. Despite its low abundance and although it is not yet known if a proximal dac domain is typical for insect leg primordia in general, it is believed this domain is relevant since its existence provides a rationale for understanding expression and regulation of Tc'dac in the gnathal segments (Prpic, 2001).

(2) A second apparent difference between the expression patterns of Tc'dac and Dm'dac in the leg concerns the position where the (distal) dac domain arises relative to the Dll domain. In Drosophila,it is believed that dac expression intercalates between the hth and Dll domains. The proximal 'ring' of Dll in mature discs is thought to arise as a secondary expression domain once the dac domain has already established itself. In Tribolium, however, it is evident that the distal Tc'dac domain arises within the still unbroken Dll domain, and that increasing abundance of Tc'dac coincides with loss of Tc'Dll expression in these cells, which results in the division of the initial Tc'Dll domain into the proximal 'ring' and distal 'sock' observed at later stages. In other words, the Dll ring in Tribolium is a remnant of the original Tc'Dll domain and not a newly formed, secondary domain. Future work will show if the two species really differ in this expression detail or if persistence of Dll expression proximal to the dac domain has been missed in Drosophila due to the folded architecture of leg imaginal discs. Emergence of the ring-and-sock pattern by downregulation of Dll in intermediate positions has also been described for Schistocerca, suggesting that this mode is ancestral in insects. Since antagonism between dac and Dll occurs in the Drosophila leg, dac may well be required for the generation of the ring-and-sock pattern (Prpic, 2001).

(3) The third difference between Tc'dac and Dm'dac expression in the leg provides an example of how limb patterning during dipteran evolution has adapted to the specific condition of imaginal disc development. In Drosophila,the dac domain is asymmetric along the dorso-ventral axis of the leg imaginal disc. This expression domain can be described as a ring which is much broader dorsally than ventrally. This dorsal expansion correlates with the way femur and tibia form in Drosophila,and how the leg disc evaginates during metamorphosis. Most leg podomeres are derived from ring-shaped primordia in the imaginal disc. During disc eversion, these rings are transformed into podomeres along the elongated leg in a process often compared to the extension of a telescope. However, the first podomeres that exit the disc cavity through its connection to the body wall epidermis are not the distal-most tarsus, but a dorsal lobe of the leg disc which comprises distal femur and proximal tibia -- which both express dac. Only after disc eversion do these two podomers become separated by longitudinal fission. The asymmetric expression of dac in Drosophila appears, therefore, to be an adaptation to the mechanical needs of disc eversion, while the symmetric pattern in Tribolium represents a more simple ancestral situation. It is intriguing to speculate that the need for an asymmetric dac domain may be the reason behind the altered expression of dpp in Drosophila, where the dpp expression domain is greatly expanded dorsally, while it is confined to the distal tip of the appendages in more ancestral arthropods like Tribolium, Schistocerca and Cupiennius (Prpic, 2001).

The labrum expresses Dll in all insects investigated; nevertheless, its metameric nature is still disputed since Dll can be seen as a general marker for structures growing out from the body wall rather than a specific marker for segmental limbs. Based on a homeotic phenotype in adult Tribolium, previous studies have concluded that the labrum was indeed of appendicular origin. Additional molecular evidence for this interpretation is provided by demonstrating that another gene with an essential function in Drosophila leg development, dac, is expressed in the Tribolium labrum. Even though the Tc'dac expression domain in the labrum is incomplete in the sense that it does not form a full ring, its intermediate proximal-distal position -- and its dependence on Dll function -- provides additional support for the labrum indeed being serially homologous to the other metameric arthropod appendages (Prpic, 2001).

It is generally believed that the maxillary and labial palps represent the telopodite of these appendages, while the maxillary stipes, cardo, galea and lacinia, and the labial mentum, prementum and glossae represent the coxopodite. In the labium, Tc'dac is expressed exclusively in the proximal part (which after metamorphosis will form mentum, prementum and glossae), while the labial palp remains free of expression. In Tc'Dll mutants, the labial palp is deleted, but Tc'dac expression remains unaffected. In this respect, the labial Tc'dac expression resembles the proximal expression domain in the thoracic legs. These data strongly suggest that the labial Tc'dac expression domain is actually homologous to the proximal rather than to the distal leg domain (Prpic, 2001).

Tc'dac expression in the maxilla is stronger than in the labium, but also for the maxilla the expression in Tc'Dll mutants makes it clear that most of the expression is in the coxopodite. However, in wild-type animals a small Tc'dac domain forms in the maxillary palp. This domain is missing in Tc'Dll mutant embryos, similar to the distal domain in the legs. This suggests that the palp domain is serially homologous to the distal leg domain, while the major maxillary Tc'dac expression again is homologous to the proximal leg domain. That no distal Tc'dac domain is present in the labial palps concurs with the fact that positional values present in the maxilla are missing in the labium: the maxillary palp in Tribolium larvae has three podomeres, while the labial palp has only two (Prpic, 2001).

It is argued that the expression pattern of dac in Tribolium actually provides evidence for the gnathobasic origin of the mandible, i. e. formed from the limb base. Like the coxopodite domains in leg, maxilla and labium, the Tc'dac expression in the mandible also is independent of Tc'Dll, and the similarity in size and expression of all three gnathal appendages in embryos mutant for Tc'Dll strongly suggests serial homology of the mandible to the coxopodite of maxilla and labium. This line of reasoning also makes it clear that the mandibular Tc'dac expression corresponds to the proximal leg domain, not to the distal domain -- even though it has been upregulated such that it is as strongly active as the distal leg domain. Since the proximal leg domain is located in the coxa, serial homology of coxa, mandible and the coxopodites of maxilla and labium can be inferred. This lends further support to the notion that the insect trochanter is part of the telopodite. Accordingly, the maxillary galea and the labial paraglossa are probably not serial homologs of the trochanter, but represent coxal endites (Prpic, 2001).

In summary, the expression of Tc'dac is strong confirmative evidence in favor of the gnathobasic origin of the mandibles. This interpretation of gnathal Tc'dac expression is based on the presence of a proximal dac domain in the developing Tribolium legs, a domain which apparently is missing in Drosophila. This shows once again the importance of comparative analyses for the correct interpretation of morphological structures and developmental processes. It remains to be seen how the proximal Tc'dac domains in the legs and in the gnathal appendages are regulated, whether they serve similar functions, and to which degree differential regulation of Tc'dac is responsible for the morphological differences among these appendages (Prpic, 2001).

The expression patterns of Gryllus (cricket) homothorax (Gbhth) and dachshund (Gbdac) are described, together with localization of Distal-less or Extradenticle protein during leg development. Their expression patterns have been correlated with the morphological segmentation of the leg bud. The boundary of Gbhth/GbDll subdivision is correlated with the segment boundary of the future trochanter/femur at early stages. Gbdac expression subdivides the leg bud into the presumptive femur and more distal region. During the leg proximodistal formation, although the early expression patterns of GbDll, Gbdac, and Gbhth significantly differ from those of Drosophila imaginal disc, their expression patterns in the fully segmented Gryllus leg are similar to those in the Drosophila late third instar disc (Inoue, 2002).

Although the legs of adult flies and crickets are very similar in their segmental compositions, the developmental processes producing their morphologies are quite distinct. In the Drosophila imaginal disc, leg formation occurs through the concentric folding and the subsequent segmentation of monolayered epithelia in the leg disc during later larval stages, although the early leg disc remains as a flattened two-dimensional structure. In contrast, the cricket leg bud is formed directly from the body wall and segmented during the subsequent outward growth in the early embryogenesis period. The leg segmentation in Gryllus occurs intercalatively step by step until stage 11 (6 days after EL). The changes have been classified into the five following stages: (1) formation of the leg bud at stages 6-7 (2.5 days after EL); (2) the first morphological segmentation at the future trochanter/femur boundary at stage 8 (3 days after EL); (3) the second segmentation in the femur/distal telopodite boundary at stage 9 (4 days after EL); (4) the third segmentation in the tibia/tarsus boundary at stage 10 (5 days after EL); (5) the fourth segmentation at the coxa/trochanter boundary. Then, the elongation of each segment takes place to form the legs of the nymph until hatching (14 days after EL) (Inoue, 2002).

Before the onset of the leg bud formation (stage 5), Gbhth is expressed uniformly throughout the embryos. Just before the onset of leg bud formation (stage 5, 40 h after egg laying), GbDll starts to be expressed in the presumptive thoracic leg region. Subsequently, Gbhth expression was downregulated in the same regions (stage 6, 48 h after egg laying). Thus, the Gbhth/GbDll antagonistic subdomains seem to be established in the early cricket embryos, as observed in Drosophila. In the early stages of leg bud formation (stages 7-9), Gbhth is expressed in the proximal region of the leg bud. In embryos at stages 8-10, when the leg segments are visible, the distal boundary of the Gbhth expression domain is localized at the proximal region of the femur segment (Inoue, 2002).

Expression patterns of Gbdac during leg development are dynamic, although Dmdac is expressed as a single ring in the Drosophila leg imaginal disc throughout its development. Gbdac expression is first detected in an anterior patch of cells in the leg bud at stage 7. At stage 8, the Gbdac expression domain becomes a narrow circumferential ring in the middle of the leg bud. At stage 9, the expression domain divides into two rings. Gbdac expression is not observed in the presumptive intersegmental border between the femur segment and more distal segments. Between stages 10 and 11, the proximal ring of expression corresponds to the distal part of the femur segment, while the distal ring of expression covers a region from the distal tibia to the proximal tarsus segment. In sagittal sections of the metathoracic femur, Gbdac expression is detected in the invaginating apodeme, as well as in the epithelial tissue of the distal femur (Inoue, 2002).

To compare expression patterns of Gbhth and Gbdac with localization of GbDll or GbExd, double staining was performed, using the corresponding RNA probes and an antibody against Dll or Exd, which are used as distal and proximal markers, respectively, in Drosophila, Acheta (Orthoptera, cricket), and Schistocerca (Orthoptera, grasshopper).Prior to the leg bud formation (stage 5), due to weak expression of GbDll, no double stainings could be observed. Prior to the first leg segmentation, Gbhth is expressed in the proximal region of the leg bud, while GbDll is detected in the distal region of the leg bud (stages 6-7). A merged panel reveals that the leg bud is stained with red and green without significant overlap, indicating that the bud is divided into two domains: a distal GbDll-localized domain and a proximal Gbhth-expressing domain. These domains are complementary at this stage, but by the end of stage 7 they partially overlap. At the same stage, GbExd accumulation, caused by nuclear localization of GbExd, is strongly detected in the proximal region of the leg bud, though low levels of GbExd expression can be seen in the distal region. The GbExd accumulation domain in the proximal region overlaps the Gbhth expression domain, as is observed in Drosophila (Inoue, 2002).

The subdivision of the leg bud into proximal and distal domains becomes morphologically discernible as a circumferential constriction, which is the boundary between the trochanter and femur (stage 8). The proximal limit of the GbDll domain corresponds to this boundary at this stage. However, the GbDll domain includes the distal region of the trochanter in later stages (Inoue, 2002).

At stages 8-9, Gbhth is expressed continuously in the proximal domain of the leg bud, overlapping the GbExd accumulation domain. By early stage 8, GbDll is localized throughout the distal tip of the leg bud, including the telopodite. Gbdac is expressed in the middle of the leg bud as a narrow ring, overlapping the GbDll expression domain. GbDll expression starts to become downregulated in the central region at the end of stage 8, in a region where Gbdac is expressed. The domain of GbDll and Gbhth co-expression continues to be observed at stage 9. The proximal ring of GbDll expression, corresponding to the proximal domain of the femur segment, remains unchanged from stage 9 to stage 11 (Inoue, 2002).

By stage 9, the Gbdac expression domain observed at stage 8 has divided into two domains: the proximal and distal domains. Meanwhile GbDll expression becomes undetectable in the middle region. The distal Gbdac domain therefore overlaps a proximal region of the distal GbDll domain, while no such co-expression is observed in the proximal domain. A narrow domain showing no expression of Gbhth, Gbdac, or GbDll can be observed between the two Gbdac domains. Consequently, six proximodistal expression domains appear in the Gryllus leg (Inoue, 2002).

At stage 10, the third morphological segmentation is observed in the distal leg bud, which divides into the tibia and tarsus. Subsequently, the fourth segmentation is discernible in the proximal leg bud, which divides into the coxa and trochanter. It has not been possible to find any of the boundaries of expression of the known appendage genes corresponding of the tibia/tarsus and coxa/trochanter boundaries (Inoue, 2002).

At stages 11-12, following the establishment of the leg segments, structures that connect adjacent segments such as articulates, muscle patterns, etc., are constructed at the segment boundaries. These stages are therefore designated here as the articulation phases. In Gryllus, since the major leg segments can be identified morphologically, the leg segments and the six domains determined by expression patterns of Gbhth, Gbdac, and GbDll can be easily correlated. The proximal-most domain in which only Gbhth is expressed, corresponds to the body wall, coxa segment, and proximal trochanter. The proximal boundary of the GbDll/Gbhth domain lies in the trochanter, and the overlapping GbDll/Gbhth domain extends into the proximal femur. The Gbdac domain corresponds to the middle of the femur segment, including the articulation between the femur and tibia. At stages 11-12, a narrow domain can also be found in which neither GbDll nor Gbdac is expressed, corresponding to the proximal tibia. The Gbdac/GbDll domain includes the articulate between the tibia and tarsus, and extends to the presumptive boundary between tarsal segments 1 and 2. The distal-most GbDll domain corresponds to presumptive tarsal segments 2 and 3 and the pretarsus. The intermediate three expression domains, i.e. the GbDll/Gbhth, Gbdac, and GbDll/Gbdac domains, include the segmental boundaries of the trochanter/femur, the femur/tibia, and the tibia/tarsus, respectively, but do not correspond to the segments (Inoue, 2002).

Expression patterns of Gryllus hth, dac, and Dll in the leg bud with those in the Drosophila leg imaginal disc were compared. The results reveal that these expression domains resolve into similar patterns. In contrast, some differences in the elaborating processes of the expression patterns of the three genes can be seen between Gryllus and Drosophila (Inoue, 2002).

The position of the proximal limit of the GbDll domain has a very sharp boundary between stages 6-8, corresponding to the morphological segmental boundary between the femur and trochanter. Thus, the most proximal segment of the telopodite, i.e. the trochanter, is not included in the GbDll domain in early stages. In contrast, in Drosophila, genetic evidence has demonstrated the subdivision of the leg disc by Dmexd and DmDll into the coxopodite and telopodite. In addition, it has been reported that Dll is detected throughout the telopodite during early development in Acheta (Orthoptera, cricket). Despite these discrepancies, the observations of the expression patterns of GbDll and Gbhth or GbExd in later stages are basically consistent with the results obtained for Acheta, Schistocerca (Orthoptera, grasshopper, and Drosophila (Inoue, 2002).

Following the primary subdivision by GbDll and Gbhth, their expression domains overlap at the boundary at later stages. The domain of Gbhth expression does not strictly correspond to the coxopodite, but expands distally into the femur. This is the same for Drosophila leg, in which the limit of expression of Dmhth expands into the proximal femur. In Drosophila, the first intercalated region between the Dmhth and DmDll domains is the intermediate region expressing Dmdac, and as a result, three discrete domains are established in the leg imaginal disc. In contrast, in Gryllus, three discrete expression domains of the three genes are not observed during leg development (Inoue, 2002).

The expression domain of Gbdac transiently overlaps with that of GbDll at early stages. In Drosophila, Dmdac expression is asymmetrically turned on in dorsal cells that still express DmDll, in the early third instar leg disc. Thus, in both insects, the overlapping Dll/dac domain is observed transiently and subsequently resolves into two domains; proximally Gbdac and distally Gbdac/GbDll in Gryllus, and proximally Dmdac and distally DmDll in Drosophila. At stages 8-9, the GbDll expression fades in the intermediate portion of the leg, and this leads to a new proximal boundary of GbDll. This boundary is correlated with the second segmentation of the femur/distal telopodite. No corresponding expression boundary has been reported in the Drosophila leg imaginal disc. In Drosophila, the expression boundary between the DmDll/Dmdac domain and distal DmDll domain corresponds to that between tarsal segments 1 and 2. Future tarsal segment 1 may be generated in the distal-most region of the Dmdac expression domain. In Gryllus, the distal limit of the Gbdac domain is likely to correspond to the boundary between tarsal segments 1 and 2 (Inoue, 2002).

From these results it was found that the expression patterns of the three genes are essentially conserved between Drosophila and Gryllus, although the time course of the pattern varies according to the developmental mode. The following conclusions were reached: (1) at early stages, expressions of Gbhth and GbDll do not correspond to the future coxopodite and telopodite, but rather to the presumptive trochanter/femur boundary; (2) Gbdac expression subdivides the leg bud into the presumptive femur and more distal region; (3) the expression patterns of GbDll, Gbdac, and Gbhth in the fully segmented Gryllus leg are similar to those in the Drosophila late third instar disc (Inoue, 2002).

The genes Distal-less, dachshund, extradenticle, and homothorax have been shown in Drosophila to be among the earliest genes that define positional values along the proximal-distal (PD) axis of the developing legs. In order to study PD axis formation in the appendages of the pill millipede Glomeris marginata, homologs of these four genes were isolated and their expression patterns examined. In the trunk legs, there are several differences from Drosophila, but the patterns are nevertheless compatible with a conserved role in defining positional values along the PD axis. However, their role in the head appendages is apparently more complex. Distal-less in the mandible and maxilla is expressed in the forming sensory organs and, thus, does not seem to be involved in PD axis patterning. No components of mouthparts could be identified that are homologous to the distal parts of the trunk legs and antennnae. Interestingly, there is also a transient premorphogenetic expression of Distal-less in the second antennal and second maxillary segment, although no appendages are eventually formed in these segments. The dachshund gene is apparently involved both in PD patterning as well as in sensory organ development in the antenna, maxilla, and mandible. Strong dachshund expression is specifically correlated with the tooth-like part of the mandible, a feature that is shared with other mandibulate arthropods. homothorax is expressed in the proximal and medial parts of the legs, while extradenticle RNA is only seen in the proximal region. This overlap of expression corresponds to the functional overlap between extradenticle and homothorax in Drosophila (Prpic, 2003).

In order to reconstruct the evolution of the gene expression pattern, the expression of Dll homologs has been studied in a great number of different arthropod species. This study is the first detailed account of Dll expression in a myriapod and the first report on expression of dac, hth, and exd in a myriapod. The data on Dll presented here show that, in contrast to the antenna and trunk legs, in the two gnathal appendages, mandible and maxilla, no Gm-Dll expression can be found that can be confidently correlated with PD axis formation. This implies that the Glomeris mouthparts lack elements serially homologous to the distal Gm-Dll-expressing parts of the antenna and legs. Thus, both mandible and maxilla are gnathobasic in nature and lack distal parts (telopodite, palp). A gnathobasic mandible traditionally is considered as a synapomorphy of crustaceans, insects, and myriapods, uniting them into a monophyletic Mandibulata. A gnathobasic maxilla, however, is found only in myriapods [apart from Glomeris (diplopod) also the maxilla of chilopods, symphylans, and pauropods apparently lacks a telopodite, to judge from external morphology] and adults of several crustacean species. The maxilla in insects and most crustaceans is not gnathobasic and has a palp (telopodite). This demonstrates that the gnathobasic condition of an appendage can evolve independently by convergence and thus questions the homology of the mandibular gnathobasy in crustaceans, insects, and myriapods. Indeed, recent molecular phylogenies corroborate two monophyletic groups comprising chelicerates and myriapods in one case and crustaceans and insects in the other. This would argue for an independent origin of the gnathobasic mandible in myriapods and the so-called Tetraconata (insects and crustaceans). However, the results with Gm-dac presented in this study support the homology of the gnathobasy in the mandibles of crustaceans, insects, and myriapods. It has been shown previously that expression of dac is very strong in the mandibles of crustaceans and insects. Similarly, strong expression of Gm-dac is seen in the mandible of Glomeris. The role of the strong dac expression in the development of the mandible is unclear, but it seems possible to correlate it with the specific tooth-like morphology of this appendage. Thus, a common genetic mechanism appears to underlie the gnathobasic nature of the mandibles in myriapods, crustaceans, and insects. In contrast to this, no comparable dac expression has been found in the first locomotory leg (L1) in chelicerates. The L1 segment in chelicerates corresponds to the mandibular segment in the other arthropods (Prpic, 2003).

In summary, the results presented here support earlier reports that the mandible in myriapods is indeed gnathobasic. Moreover, the results indicate that the maxilla is also gnathobasic, which is different from insects and most crustaceans and also from chelicerates, which do not possess a single gnathobasic appendage. Strong expression of dac in the mandible in crustaceans, insects, and myriapods suggests that the mandibular morphology is produced by a homologous mechanism and supports the homology of the mandible in all mandibulate arthropods. Although a homologous mandible in crustaceans, insects, and myriapods would support the monophyly of the Mandibulata, it has to be pointed out that a homologous mandible is also compatible with recent molecular phylogenies on the assumption that a true mandible already existed in the last common ancestor of all extant arthropod classes and has been lost secondarily in the chelicerates (Prpic, 2003a).

Leg development in Drosophila has been studied in much detail. However, Drosophila limbs form in the larva as imaginal discs and not during embryogenesis as in most other arthropods. Appendage genes have been analyzed in the spider Cupiennius salei and the beetle Tribolium castaneum. Differences in decapentaplegic expression suggest a different mode of distal morphogen signaling suitable for the specific geometry of growing limb buds. Also, expression of the proximal genes homothorax and extradenticle (exd) is significantly altered: in the spider, exd is restricted to the proximal leg and hth expression extends distally, while in insects, exd is expressed in the entire leg and hth is restricted to proximal parts. This reversal of spatial specificity demonstrates an evolutionary shift, which is nevertheless compatible with a conserved role for this gene pair as instructor of proximal fate. Different expression dynamics of dachshund and Distal-less point to modifications in the regulation of the leg gap gene system. The significance of this finding is discussed in terms of attempts to homologize leg segments in different arthropod classes. Comparison of the expression profiles of H15 and optomotor-blind to the Drosophila patterns suggests modifications also in the dorsal-ventral patterning system of the legs. Together, these results suggest alterations in many components of the leg developmental system, namely proximal-distal and dorsal-ventral patterning, and leg segmentation. Thus, the leg developmental system exhibits a propensity to evolutionary change, which probably forms the basis for the impressive diversity of arthropod leg morphologies (Prpic, 2003b).

Patterning of the adult mandibulate mouthparts in the red flour beetle, Tribolium castaneum

Specialized insect mouthparts, such as those of Drosophila, are derived from an ancestral mandibulate state, but little is known about the developmental genetics of mandibulate mouthparts. The metamorphic patterning of mandibulate mouthparts of the beetle Tribolium castaneum was studied RNA interference to deplete the expression of 13 genes involved in mouthpart patterning. These data were used to test three hypotheses related to mouthpart development and evolution. First, the prediction was tested that maxillary and labial palps are patterned using conserved components of the leg-patterning network. This hypothesis was strongly supported: depletion of Distal-less and dachshund led to distal and intermediate deletions of these structures while depletion of homothorax led to homeotic transformation of the proximal maxilla and labium, joint formation required the action of Notch signaling components and odd-skipped paralogs, and distal growth and patterning required epidermal growth factor (EGF) signaling. Additionally, depletion of abrupt or pdm/nubbin caused fusions of palp segments. Second, the hypotheses was tested for how adult endites, the inner branches of the maxillary and labial appendages, are formed at metamorphosis. The data reveal that Distal-less, Notch signaling components, and odd-skipped paralogs, but not dachshund, are required for metamorphosis of the maxillary endites. Endite development thus requires components of the limb proximal-distal axis patterning and joint segmentation networks. Finally, adult mandible development is considered in light of the gnathobasic hypothesis. Interestingly, while EGF activity is required for distal, but not proximal, patterning of other appendages, it is required for normal metamorphic growth of the mandibles (Angelini, 2012).

In D. melanogaster, Dll mutants lack maxillary structures and portions of the proboscis (i.e., labium), although Dll expression in the maxillary anlagen is weaker than in the leg or antennal discs. Paralleling the results for T. castaneum, in the horned beetle Onthophagus taurus distal regions of the adult mouthparts were deleted with larval Dll RNAi (Simonnet 2011). The embryonic and metamorphic functions of Dll in T. castaneum are also similar: the gene is required for the development of distal structures at both stages, and during embryogenesis Dll is expressed throughout the developing palps. Interestingly, removal of T. castaneum Dll expression earlier during larval life led to delayed metamorphosis, as well as changes in appendage morphology (Suzuki, 2009). Many insects delay molting after appendage loss to allow time for regeneration, and this dual role of Dll suggests a mechanism linking these processes (Angelini, 2012).

The data from T. castaneum provide evidence for a conserved gap gene role of dac during patterning of mouthparts and legs of this species. dachshund is not expressed in or required for development of the labial and maxillary anlagen of D. melanogaster. In T. castaneum embryos dac is expressed strongly in the proximal maxilla and part of the developing endite. Embryonic dac expression is weaker in the distal maxillary palp and the labium. The current data show a clear metamorphic requirement for dac in the intermediate regions of the maxillary and labial palps, as does a recent study of O. taurus (Simonnet, 2011). A function for dac in the development of an intermediate portion of the maxillary and labial appendages has so far only been observed in these two beetles, while data from two species with specialized mouthparts (the milkweed bug O. fasciatus and D. melanogaster) found that dac is not required for PD patterning of the mouthparts. Thus, comparative data from other species do not support the hypothesis that this mouthpart patterning role is ancestral. However, if mandibulate mouthparts evolved from leg-like structures similarities in the expression and function of genes patterning both legs and mouthparts are expected to be plesiomorphic. This hypothesis can be further tested by examining the role of dac in mouthpart development in additional insect orders, particularly those that retain mandibulate mouthparts, and in other arthropods (Angelini, 2012).

The effects of hth depletion are distinct in different species, but typically involve some degree of homeotic transformation. In D. melanogaster, hth is expressed in the labial discs, but without nuclear expression of its cofactor Extradenticle. Maxillary palps are retained in hth loss-of-function flies, but they may possess bristles typical of legs, indicating a partial proboscis-to-leg transformation. In the cricket Gryllus bimaculatus, which has mandibulate mouthparts, hth depletion causes transformation of proximal mouthpart structures towards antennal identity, with a loss of endites, while distal structures are transformed towards leg identity (Ronco, 2008). hth RNAi in T. castaneum transformed intermediate regions of the maxilla and labium towards distal mouthpart identity. Proximal regions also appeared transformed, but their identity could not be established, while distal regions appeared wild type. In the beetle O. taurus, proximal regions of the labium are transformed towards maxillary endite identity, but distal regions of the labium and the entire maxilla remain relatively unaffected (Angelini, 2012).

These results highlight the similarity between patterning of the maxilla, labium and legs in T. castaneum. Functional data from two species with highly derived mouthpart morphologies, D. melanogaster and the milkweed bug Oncopeltus fasciatus, suggest only limited similarity between mouthpart and leg patterning. One explanation for this low degree of conservation is that evolution of the ancestral patterning mechanism has occurred in concert with the functional and morphological diversification of these mouthparts. A correlation between generative mechanisms and structural morphology has been used as a common null hypothesis, although exceptions in which similar morphologies result from different developmental pathways are documented. Nevertheless, this hypothesis predicts that developmental patterning should be more highly conserved across appendage types in species that retain the ancestral mandibulate mouthpart morphology (Angelini, 2012).

The maxillary and labial palps are an interesting case of serial homology. Despite a difference in overall size, their shape and arrangement of sensillae are similar. The intermediate segments of each palp type are also similar, but differ in number, which suggests that segment number is regulated independently from other morphological traits. The RNAi depletion of pdm in T. castaneum caused the reduction and deletion of the third maxillary palp segment, producing a phenotype closely resembling the wildtype morphology of the labial palps. While a role for pdm in the labium cannot be excluded, the absence of observed labial phenotypes was significant compared to maxillary results. Therefore, it is hypothesized that the difference in the number of palp segments results from specific activation of pdm in the maxillary palp. Loss of function in the Hox gene Deformed during T. castaneum embryogenesis causes a transformation of the larval maxillae towards labial identity. Since Hox genes are the primary determinants of body segment identity, it is proposed that pdm is activated by Deformed, and repressed by the labial Hox gene Sex combs reduced. RNAi targeting pdm in another mandibulate insect, the cricket Acheta domesticus, generated defects in the antenna and legs, but no defects in the mouthparts, despite similar pdm expression in these appendages (Turchyn; 2011; Angelini, 2012).

Endites are a primitive component of arthropod appendages, and they are retained in insect mouthparts, as well as in the mouthparts and thoracic appendages of many crustaceans (Boxshall 2004). At least three hypotheses have been put forward for how endites are patterned, and these hypotheses are not mutually exclusive. The first hypothesis states that multiple PD axes result from redeployment of a PD axis patterning mechanism shared by palps and endites. A second hypothesis posits that endites and appendage segments form by the same mechanism, Notch-mediated in-folding of the cuticle. A third hypothesis states that dac expression initiates endite branching from the main appendage axis. The axis redeployment hypothesis predicts that depletion of genes involved in PD axis patterning will have similar effects on the development of palps and endites. Some support for this hypothesis comes from studies of endite morphogenesis and the expression and function of leg gap genes in the embryos of T. castaneum and the orthopteran Schistocerca americana, but not all data are consistent with it. The segmentation hypothesis predicts that endites will fail to differentiate if genes required for joints are depleted. This hypothesis was posed based on a comparative developmental study of segmented and phyllopodous crustacean limbs. Finally, the dac-mediated hypothesis predicts that depletion of dac will lead to reduced endites. This hypothesis emerged from the observation that dac expression is reiterated along the medial edges of larval endites in the crustacean Triops longicaudatus. Comparative expression data from the isopod Porcellio scaber are also consistent with the dac-mediated hypothesis (Angelini, 2012).

The current data are consistent with predictions of the axis redeployment and segmentation hypotheses but do not support a role for dac in endite metamorphosis. Adult endites were disrupted by depletion of Dll, Krn, the odd-related genes, and Notch signaling, and to a lesser degree hth. In the maxilla depletion of most of these genes led to the failure of the single larval endite to divide into two distinct branches, while in the labium, their depletion caused reduction of the ligula. Their requirement in the endites is consistent with the hypothesis that these structures are generated by redeploying appendage PD axis determinants. Depletion of Notch signaling components and the odd paralogs produced reductions and fusions between palp segments, between the palps and endites, and between the lacinia and galea. Thus, these data are compatible with both the hypothesis that a reiterated PD axis is used to pattern the endites and the hypothesis that endite formation is linked to joint formation. Normal endite development in dac-depleted specimens is inconsistent with the dac-mediated hypothesis (Angelini, 2012).

It is noteworthy that endite specification and the division of the single larval endite into the adult galea and lacinia appear to be separable functions. For example, Ser RNAi resulted in a single endite lobe with lacinia identity medially and galea identity laterally. In contrast, severe Dll RNAi individuals had a single endite that lacked also obvious lacinia identity (Angelini, 2012).

The mandibulate structure of Tribolium mouthparts is the pleisomorphic state for insects and is shared by a majority of insect orders. These mouthparts are characterized by robust mandibles, lacking segmentation. A classic debate in arthropod morphology concerns whether the mandibles of insects and myriapods are derived from a whole appendage or only from proximal appendage regions; the latter are called gnathobasic mandibles. Palps are retained on the mandibles of many crustaceans, making it clear that the biting regions of their mandibles are gnathobasic. Phylogenetic support for the gnathobasic hypothesis comes from phlyogenetic studies that place insects nested within crustaceans (Regier, 2010). The first developmental genetic support for the gnathobasic hypothesis came from the discovery that insect mandibles lack Dll expression. Furthermore, neither mutations in Dll nor its depletion through RNAi have been observed to alter mandible development in insects, including T. castaneum. This evidence has led to widespread acceptance of the gnathobasic hypothesis. Of the 13 genes depleted in this study, two (Krn and hth) produced results that would not be predicted by the most straightforward form of the gnathobasic hypothesis for mandible origins (Angelini, 2012).

Loss of EGF function in insects leads to distal appendage defects, including pretarsal or tarsal deletions. The role of EGF signaling in distal appendage regions is conserved in T. castaneum metamorphosis, since depletion of the EGF ligand Krn leads to reduction of the antennal flagellum, and maxillary and labial palps, as well as to deletion of the pretarsus and malformation of the tarsus. In light of the restriction of Krn’s role to distal appendage regions and regulation of distal EGF ligand expression by Dll in D. melanogaster, the gnathobasic hypothesis predicts that Krn should not be required for normal development of the mandible in T. castaneum. In contrast to this prediction, depletion of Krn produced a significant reduction in mandible length (Angelini, 2012).

The hypothesis of a gnathobasic mandible also predicts that hth depletion should produce effects in the mandible similar to those in the proximal regions of other appendage types. In T. castaneum, hth RNAi during metamorphosis caused homeotic transformation of proximal regions of the maxilla, labium and legs. However, the mandibles were not affected by hth depletion. In the beetle O. taurus, hth depletion slightly altered mandible shape, but also without apparent homeosis. In contrast, hth RNAi in embryos of the cricket G. bimaculatus transformed the mandible towards a leg-like structure distally and an antenna-like structure proximally, paralleling the transformation observed in other appendages. Because these results come from only two lineages and from different life stages, additional data are needed to determine whether a homeotic role for hth was present ancestrally in insect mandibles (Angelini, 2012).

These data must be weighed alongside other evidence bearing on the gnathobasic hypothesis. In T. castaneum, the lack of phenotypic effects on mandible metamorphosis of other genes in this study is consistent with the gnathobasic hypothesis. In particular, it was observed that mandible metamorphosis was normal following depletion of genes involved in distal growth and patterning or joint formation. Moreover, homology at one biological level, such as anatomy, does not preclude divergence at other levels, such as development. Nevertheless, since developmental genetic studies of Dll and other appendage-patterning genes have been used as strong support for the gnathobasic homology of the insect mandible, the findings of Krn function highlight the difficulties in establishing serial homology based solely on developmental data (Angelini, 2012).

This study provides a genetic model of adult mouthpart development in Tribolium castaneum based on 13 genes. While previous studies have examined patterning in species with derived mouthpart morphologies, T. castaneum retains the pleisomorphic, mandibulate state of insect mouthparts. These results demonstrate the conservation of many gene functions in the maxilla and labium, relative to the legs, thus supporting the interpretation of novel gene functions in groups with derived mouthpart morphology as indicative of their specialized morphogenetic roles in those species. Mandibulate mouthparts such as those of T. castaneum include medial maxillary and labial endites, and the current data are consistent with hypotheses of reiteration in the PD axis and specification by Notch signaling, but rule out a direct role for dac in branch generation or patterning at metamorphosis. Additionally these results demonstrate that a regulator of distal leg development, Krn, which encodes an EGF ligand, is required for normal mandible elongation. This finding underscores the complex relationship between homology at the levels of anatomy and developmental patterning (Angelini, 2012).

Dach1 regulates neural crest migration during embryonic development

Dachshund 1(Dach1) is a key component of the retinal determination gene network that plays significant roles in cell fate regulation. The vertebrate homolog of Drosophila dachshund has gained considerable importance as an essential regulator of development, but its functions during embryonic development remain elusive. This study investigated the functional significance of dach1 during Xenopus embryogenesis using loss-of-function studies. Reverse transcription-polymerase chain reaction demonstrated the maternal nature of dach1, showing enhanced expression at the neurula stage of development, and morpholino oligonucleotide injection of dach1 induced phenotypic anomalies of microcephaly and reduced body length. Animal cap assays followed by whole-mount in-situ hybridization indicated the perturbed expression of neural and neural crest (NC) markers. These data suggest the prerequisite functions of dach1 in NC migration during Xenopus embryogenesis. However, the developmental pathways regulated by dach1 during embryogenesis require further elucidation (Kim, 2020).

Chicken Dachshund homologs

Based on recent data, a new view is emerging that vertebrate Dachshund (Dach) proteins are components of Six1/6 transcription factor-dependent signaling cascades. Although Drosophila data strongly suggest a tight link between Dpp signaling and the Dachshund gene, a functional relationship between vertebrate Dach and BMP signaling remains undemonstrated. Chick Dach1 is shown to interact with the Smad complex and the corepressor mouse Sin3a (see Drosophila Sin3A), thereby acting as a repressor of BMP-mediated transcriptional control. In the limb, this antagonistic action regulates the formation of the apical ectodermal ridge (AER) in both the mesenchyme and the AER itself, and also controls pattern formation along the proximodistal axis of the limb. These data introduce a new paradigm of BMP antagonism during limb development mediated by Dach1, which is now proven to function in different signaling cascades with distinct interacting partners (Kida, 2004).

Mammalian Dachshund homologs

Mammalian homologs of the Drosophila dachshund gene have now been characterized. The genes have been designated DACH (human) and Dach (mouse). Two domains of high conservation show similarity to the Ski family of genes. The N-terminal domain is referred to as Dachbox-N and is 83 amino acids in length, with an overall similarity of 87% between the Drosophila and mammalian proteins. The C. elegans homolog of Drosophila dac also contains Dachbox-N and shows 73% identity with the mammalian proteins. The C-terminal domain, Dachbox-C, is 72 amino acids long and the degree of similarity to Drosophila dac is 63%. Dachbox-C possesses an alpha-helical, coiled-coil motif. This motif starts 20 amino acids from the N-terminal end of Dachbox-C and extends 28 amino acids beyond the box. It is proposed that Dachshund belongs to a superfamily including these genes. Dach shows a weak but significant identity with Ski, a proto-oncogene that normally functions during myogenesis and neurulation. The homology between Dach and Ski is based around the two Dachboxes. Dachbox-N has 28% identity with the consensus sequence of all vertebrate Ski and Sno proteins (see Drosophila snoN). Sno is a Ski-related protein of unknown function. Dachbox-C has very weak identity with the C-terminal region of Ski and Sno, confined to the occasional alignment of basic and hydrophobic amino acids. When the predicted alpha-helical domain of mammalian Dach is projected on a helical wheel, a striking motif is revealed: one face of the helix comprises alternating basic and hydrophobic residues while the adjacent face comprises alternating acid and hydrophobic residues. A similar motif is found in the Drosophila Dac protein (Hammond, 1998).

Mouse Dachshund is expressed in the eye and limb, structures affected by the Drosophila loss-of-function mutant, and in rib primordia, the CNS and the genital eminence. Dach is expressed in both the fore and hind limbs at all stages analysed, from E10.5 to E13.5. Dach limb expression becomes increasingly peripheral, extending around the entire handplate in the mesenchyme beneath the apical ectodermal ridge. Dach is localized to the mesenchyme at the distal tips of the digits. Dach is expressed in the mesenchyme surrounding the eye, which is predominantly neural-crest derived. Dach expression also occurs within the neural retina at these stages but not in the lens or the retinal pigmented epithelium. Pax6 and Dach show overlapping but non-identical expression patterns, with Pax6 expression excluded from the mesenchyme. Dach expression is unaffected in smalleye mouse brain, indicating that Pax6 is not directly activating Dach. In Drosophila eye development, dachshund is a component of an interacting network of proteins. Genes homologous to many of these exist in mammals, and Dach now joins this expanding group (Hammond, 1998).

A novel vertebrate homolog of the Drosophila gene dachshund, Dachshund2 has been identified. Dach2, is expressed in the developing somite prior to any myogenic genes, with an expression profile similar to Pax3, a gene previously shown to induce muscle differentiation. Pax3 and Dach2 participate in a positive regulatory feedback loop, analogous to a feedback loop that exists in Drosophila between the Pax gene eyeless (a Pax6 homolog) and the Drosophila dachshund gene. Although Dach2 alone is unable to induce myogenesis, Dach2 can synergize with Eya2 (a vertebrate homolog of the Drosophila gene eyes absent) to regulate myogenic differentiation. Moreover, Eya2 can also synergize with Six1 (a vertebrate homolog of the Drosophila gene sine oculis) to regulate myogenesis. This synergistic regulation of muscle development by Dach2 with Eya2 and Eya2 with Six1 parallels the synergistic regulation of Drosophila eye formation by dachshund with eyes absent and eyes absent with sine oculis. This synergistic regulation is explained by direct physical interactions between Dach2 and Eya2, and Eya2 and Six1 proteins, analogous to interactions observed between the Drosophila proteins. This study reveals a new layer of regulation in the process of myogenic specification in the somites. Moreover, the Pax, Dach, Eya, and Six genetic network has been conserved across species. However, this genetic network has been used in a novel developmental context -- myogenesis rather than eye development -- and has been expanded to include gene family members that are not directly homologous, for example Pax3 instead of Pax6 (Heanue, 1999).

A chick homolog of Drosophila dachshund (dac), termed Dach1, has been cloned. Dach1 is the ortholog of mouse and human Dac/Dach (hereafter referred to as Dach1). Chick Dach1 is expressed in a variety of sites during embryonic development, including the eye and ear. Previous work has demonstrated the existence of a functional network and genetic regulatory hierarchy in Drosophila in which eyeless, eyes absent, and dac operate together to regulate Drosophila eye development; ey regulates the expression of eya and dac. In the developing eye of both chick and mouse, expression domains of Dach1 overlap with those of Pax6, a gene required for normal eye development. Similarly, in the developing ear of both mouse and chick, Dach1 expression overlaps with the expression of another Pax gene, Pax2. In the mouse, Dach1 expression in the developing ear also overlaps with the expression of Eya1 (an eya homolog ). Both Pax2 and Eya1 are required for normal ear development. Expression studies suggest that the Drosophila Pax-eya-dac regulatory network may be evolutionarily conserved such that Pax genes, Eya1, and Dach1 may function together in vertebrates to regulate neural development. To address the further possibility that a regulatory hierarchy exists between Pax, Eya, and Dach genes, the expression of mouse Dach1 was examined in Pax6, Pax2 and Eya1 mutant backgrounds. Pax6, Pax2, and Eya1 do not regulate Dach1 expression through a simple linear hierarchy (Heanue, 2002).

Mouse Dach, a homolog of Drosophila dachshund, has been cloned and characterized. Sequence analysis reveals the presence of two motifs, DD1 and DD2, which may be involved in the function of Dach/Dachshund as gene regulatory factors. In addition, DD1 shares sequence similarity to N-terminal sequences of Ski and SnoN, which are involved in cellular transformation and differentiation. Mouse and human Dach/DACH were localized to chromosome 14E1 and 13q21.3-22, respectively, by fluorescence in situ hybridization. In situ hybridization analysis demonstrates that Dach is expressed in tissues similar to those observed in Drosophila, including the embryonic nervous system, sensory organs, and limbs. The finding of Dach expression in the eye completes the list of vertebrate homologs of eyeless, eyes absent, sine oculis, and dachshund that, as a group, may function to control cell-fate determination in the vertebrate eye (Davis, 2000).

The longest open reading frame encodes a protein of 699 amino acids. Although the predicted protein does not contain any recognizable functional protein motifs, comparative sequence analysis with Drosophila Dachshund reveals the presence of four conserved structural features. The first, Dachshund domain 1 (DD1), is located near the N-terminus and is 107 amino acids in length. The second conserved region, DD2, is located near the C-terminus and is 84 amino acids in length. The mouse and Drosophila proteins share 78% and 58% identity between DD1 and DD2, respectively. Interestingly, a BLAST database search also reveals similarities between DD1 and the proto-oncoprotein Ski and the Ski-related SnoN. These protein domains share 27% identity and 35% similarity. This domain in Ski/SnoN is required for transcriptional regulation and cellular transformation. A third sequence similarity, a trinucleotide repeat, was found N-terminal of DD1. This repeat encodes polyserine (AGC) in mouse Dach and polyglutamine (CAG) in Dachshund. Finally, a splice site at mouse Dach codon 276 precisely matches a splice site position in Dachshund, indicating the conservation of an exon-intron boundary. Comparison of available human EST and mouse Dach sequences demonstrates that DD1 and DD2 are 100% identical, with near-complete identity throughout the remaining sequences (Davis, 2000).

At E8.5 weak Dach expression is detected in the neural folds but not in the neuroepithelium of the anterior neural plate or groove. From E9.5 to E14.5, strong Dach expression is detected in the dorsal neural tube. In the developing brain Dach is first detected in the prosencephalon and hindbrain at E9.5. Subsequently, from E10.5 to E14.5, Dach continues to be expressed in the telencephalic vesicles, mesencephalon, and hindbrain. Within layers of the forebrain Dach is localized to the frontal cortex, subthalamus, dorsal thalamus, ganglionic eminence, and areas surrounding the optic recess. Within the midbrain and hindbrain, Dach transcripts are detected in the tectum, pons, cerebellum, and medulla. Dach transcripts are also detected in various nerve ganglia. In the cranium Dach expression is found in the trigeminal ganglia. In the trunk at E10.5 the long stripes of Dach-expressing cells parallel to the neural tube can be attributed to expression in the spinal ganglia. Dach expression in the dorsal neural tube and spinal ganglia suggests that it is expressed in neural crest cells before, during, and after their migration from the dorsal neural tube (Davis, 2000).

Dach transcripts are detected during the development of the eye. At E9.5 expression is observed in the optic vesicle and then at E10.5 in the optic cup. In cross-sections of whole-mount E10.5 embryos expression in the retina has been confirmed. Analysis of E16.5 eye sections was performed to define the Dach expression domains within the eye. Strong Dach expression is detected throughout all layers of the retina. Lower levels are detected in the epithelium of the anterior and equatorial regions of the lens. Expression in the developing ear is detected in the auditory vesicles, endolymphatic duct, and pinna (Davis, 2000).

Dach is expressed in the head mesenchyme and lateral mesoderm. At E8.5, cells expressing Dach near the anterior neural plate lie below the neuroepithelium and are therefore part of the head mesenchyme. From E8.5 to E9.5 Dach expression can be seen in the lateral plate mesoderm along the anterior-posterior axis. Dach is also expressed in a dynamic pattern in the myotome. In E9.5 and E10.5 embryos Dach transcripts are found along the posterior half of the somites. Dach expression in the myogenic cells migrating into the inter-limb bud region has also been observed (Davis, 2000).

In order to analyze the Pax6 pathway in vertebrates the cDNA and genomic clones corresponding to the human and mouse homologs of Drosophila dac have been isolated and characterized. A full-length human cDNA encoding dachshund (DACH) encodes the 706 amino acid protein with a predicted molecular weight of 73 kDa. A 109 amino acid domain located at the N-terminus of DACH shows significant sequence and secondary structure homologies to the ski/sno oncogene products. Northern blot analysis has found human DACH predominantly in adult kidney, heart, and placenta, with less expression detected in the brain, lung, skeletal muscle and pancreas. A panel of human cell lines was studied and most notably a large proportion of neuroblastomas expressed DACH mRNA. Mouse Dach encodes a protein of 751 amino acids with predicted molecular weight of 78 kDa that is 95% identical to the human DACH. RNase protection analysis shows the highest Dach mRNA expression in the adult mouse kidney and lung, whereas lower expression is detected in the brain and testis. RT/PCR analysis readily detects Dach mRNA in the adult mouse cornea and retina. Dach mRNA expression in the mouse E11.5 embryo is observed primarily in the fore and hind limbs, as well as in the somites (Kozmik, 1999).

Using a yeast two hybrid system and pull-down assays, mouse Dac (mDac) has been demonstrated to specifically bind mouse ubiquitin-conjugating enzyme mUbc9. In contrast to a direct interaction between Drosophila Dachshund and Eyes absent, mDac interaction with mEya2 could not be detected. mDac protein is found predominantly in the nucleus but translocates to the cytoplasm and condensates along the nuclear membrane in a cell-cycle dependent manner. Deletion analysis of mDac shows the intracellular localization and protein stability correlates with the binding to mUbc9. The C-terminal half of mDac, which associates with mUbc9, remains cytoplasmic and is degraded in proteasome whereas the non-interacting N-terminus is exclusively nuclear and more stable than the full-length mDac or its C-terminal portion. In situ hybridization on whole-mount embryos or tissue sections detects mUbc9 transcripts in complementary and overlapping areas with mDac expression, particularly in the proliferation zone of the limb buds, the spinal cord and forebrain. Mouse embryos stained with an anti-mDac antibody document that mDac is localized both in the nucleus and the cytoplasm with a cytoplasmic predominance in migrating neural crest cells. In the proliferation zone, visible nuclear envelopes are not formed and mDac is detected throughout the cells (Machon, 2000).

The Drosophila genes eyeless, eyes absent, sine oculis and dachshund cooperate as components of a network to control retinal determination. Vertebrate homologs of these genes have been identified and implicated in the control of cell fate. Mouse Dach2, a homolog of dachshund, has been cloned and characterized. In situ hybridization studies demonstrate Dach2 expression in embryonic nervous tissues, sensory organs and limbs. This pattern is similar to mouse Dach1, suggesting a partially redundant role for these genes during development. Dach2 expression in the forebrain of Pax6 mutants and dermamyotome of Pax3 mutants is not detectably altered. Genetic mapping experiments place mouse Dach2 on the X chromosome between Xist and Esx1. The identification of human DACH2 sequences at Xq21 suggests a possible role for this gene in Allan-Herndon syndrome, Miles-Carpenter syndrome, X-linked cleft palate and/or Megalocornea (Shen, 2001).

Mammalian organogenesis requires the expansion of pluripotent precursor cells before the subsequent determination of specific cell types, but the tissue-specific molecular mechanisms that regulate the initial expansion of primordial cells remain poorly defined. It has been genetically established that Six6 homeodomain factor, acting as a strong tissue-specific repressor, regulates early progenitor cell proliferation during mammalian retinogenesis and pituitary development. Six6, in association with Dach corepressors, regulates proliferation by directly repressing cyclin-dependent kinase inhibitors, including the p27Kip1 promoter. These data reveal a molecular mechanism by which a tissue-specific transcriptional repressor-corepressor complex can provide an organ-specific strategy for physiological expansion of precursor populations (Li, 2002).

Dac is a novel nuclear factor in mouse and humans that shares homology with Drosophila dachshund (dac). Alignment with available sequences defines a conserved box of 117 amino acids that shares weak homology with the proto-oncogene Ski and Sno. Dac expression is found in various neuroectodermal and mesenchymal tissues. At early developmental stages Dac is expressed in lateral mesoderm and in neural crest cells. In the neural plate/tube Dac expression is initially seen in the prosencephalon and gets gradually restricted to the presumptive neocortex and the distal portion of the outgrowing optic vesicle. Furthermore, Dac transcripts are detected in the mesenchyme underlying the Apical Ectodermal Ridge (AER) of the extending limb bud, the dorsal root ganglia and chain ganglia, and the mesenchyme of the growing genitalia. Dac expression in the Gli 3 mutant extra toes (Xt/Xt) shows little difference compared to the expression in wild-type limb buds. In contrast, a significant expansion of Dac expression is observed in the anterior mesenchyme of the limb buds of hemimelic extra toes (Hx/+) mice. FISH analysis reveals that human DAC maps to chromosome 13q22.3-23 and further fine-mapping defined a position of the DAC gene at 54cM or 13q21.1, a locus that associates with mental retardation and skeletal abnormalities (Caubit, 1999).

Drosophila sine oculis, eyes absent, and dachshund are essential for compound eye formation and form a gene network with direct protein interaction and genetic regulation. The vertebrate homologues of these genes, Six, Eya, and Dach, also form a similar genetic network during muscle formation. To elucidate the molecular mechanism underlying the network among Six, Eya, and Dach, the molecular interactions among the encoded proteins was examined. Eya interacts directly with Six but never with Dach. Dach transactivates a multimerized GAL4 reporter gene by coproduction of GAL4-Eya fusion proteins. Transactivation by Eya and Dach is repressed by overexpression of VP16 or E1A but not by E1A mutation, which is defective for CREB binding protein (CBP) binding. Recruitment of CBP to the immobilized chromatin DNA template is dependent on FLAG-Dach and GAL4-Eya3. These results indicate that CBP is a mediator of the interaction between Eya and Dach. Contrary to expectations, Dach binds to chromatin DNA by itself, not being tethered by GAL4-Eya3. Dach also binds to naked DNA with lower affinity. The conserved DD1 domain is responsible for binding to DNA. Transactivation was also observed by coproduction of GAL4-Six, Eya, and Dach, indicating that Eya and Dach synergy is relevant when Eya is tethered to DNA through Six protein. These results demonstrate that synergy is mediated through direct interaction of Six-Eya and through the interaction of Eya-Dach with CBP and explain the molecular basis for the genetic interactions among Six, Eya, and Dach. This work provides fundamental information on the role and the mechanism of action of this gene cassette in tissue differentiation and organogenesis (Ikeda, 2002).

DACH1 inhibits TGF-beta signaling through binding Smad4

The vertebrate homologues of Drosophila dachsund, DACH1 and DACH2, have been implicated as important regulatory genes in development. DACH1 plays a role in retinal and pituitary precursor cell proliferation and DACH2 plays a specific role in myogenesis. DACH proteins contain a domain (DS domain) that is conserved with the proto-oncogenes Ski and Sno. Since the Ski/Sno proto-oncogenes repress AP-1 and SMAD signaling, it is hypothesized that DACH1 might play a similar cellular function. DACH1 has been found to be expressed in breast cancer cell lines and to inhibit transforming growth factor-ß-induced apoptosis. DACH1 represses TGF-ß induction of AP-1 and Smad signaling in gene reporter assays and represses endogenous TGF-ß-responsive genes by microarray analyses. DACH1 binds to endogenous NCoR and Smad4 in cultured cells and DACH1 co-localizes with NCoR in nuclear dotlike structures. NCoR enhances DACH1 repression, and the repression of TGF-ß-induced AP-1 or Smad signaling by DACH1 required the DACH1 DS domain. The DS domain of DACH is sufficient for NCoR binding at a Smad4-binding site. Smad4 was required for DACH1 repression of Smad signaling. In Smad4 null HTB-134 cells, DACH1 inhibits the activation of SBE-4 reporter activity induced by Smad2 or Smad3 only in the presence of Smad4. DACH1 participates in the negative regulation of TGF-ß signaling by interacting with NCoR and Smad4 (Wu, 2003).

DACH1 functions as a transcriptional repressor of TGF-ß signaling. DACH1 represses TGF-ß-induced activity of both Smad/FAST1 Binding Element (SBE) and AP-1 activity and inhibits TGF-ß-induced apoptosis in MDA-MB-231 cells. NCoR enhances repression of TGF-ß signaling by DACH1. Repression by DACH1 requires Smad4, being abrogated in Smad4-deficient cells and restored by Smad4 coexpression. Repression by DACH1 requires a conserved DS domain that binds the transcriptional co-repressor NCoR. DACH1 and NCoR co-localize in a substantial proportion of subnuclear dotlike structures by confocal microscopy. Together, these findings suggest NCoR may participate in DACH1-mediated repression of gene expression (Wu, 2003).

DACH1 is detectable in MDA-MB-231 cells by Western blotting, and genome-wide analysis of DACH1-responsive genes in these cells indicates that 422 genes of 17,000 are regulated >2-fold by DACH1 expression. Consistent with the reporter gene analysis demonstrating DACH1 inhibition of AP-1 activity, several AP-1-responsive genes are repressed by DACH1 expression, including c-fos, Egr1, cyclin E2, neuregulin, tumor necrosis factor alpha-induced protein 3, cdc25A, FGF5, GRO3, MEF2C, ETR101, and BMP4. A comparison between genes regulated significantly by DACH1 with recent studies of TGF-ß signaling using a similar approach has demonstrated that genes induced by TGF-ß in other cell types are repressed by DACH1 (ATF3, interleukin-11, P2RY2) and several genes repressed by TGF-ß are induced by DACH1 (ID1 and interleukin-1-ß). Comparison between genome wide analysis 'fingerprints' must be considered with caution; however, it is of interest that of 70 genes regulated by TGF-ß, 22 of those genes are also significantly regulated by DACH1 expression; similarly, there is overlap with TGF-ß response genes in recent publications. The functions of these genes are diverse and include cell division, transcriptional regulation, cellular adhesion, extracellular matrix remodeling, and signal transduction. The use of genome-wide expression studies to identify clusters of genes representing a molecular signature of DACH1-regulated activity suggests a normal function for DACH1 in the inhibition of AP-1-regulated genes. The current studies suggest DACH1 may function to regulate aberrant TGF-ß signals that play important roles in human breast cancer progression. TGF-ß itself plays an important role in cancer progression by functioning both as an antiproliferative factor and as a tumor promoter. The numerous components of the signal conduction pathway are tumor suppressors that are functionally mutated in cancer (Wu, 2003).

DACH1 was found within a complex bound to a FAST1/SBE DNA binding site with Smad4. Immunopurified DACH1, however, does not bind DNA directly, suggesting that Smad4 serves as a DNA-bound platform to recruit DACH1. The DACH1 DS domain alone is insufficient for Smad4 binding, which requires the EYAD domain and is defective in SBE and AP-1 repression. DACH1 co-immunoprecipitates with Smad4 from cultured cells, and the association of DACH1 with Smad4 was observed in reciprocal immunoprecipitation. DACH1 associates with Smad4 in vitro using GST pull-down experiments, and, like Ski, multiple domains in DACH1 are required, including both the DS and EYA domains. Using saturating immunoprecipitation, the relative amount of co-precipitated Smad4 was greater for Ski than DACH1. In contrast, the relative abundance of NCoR coprecipitating with DACH1 is relatively greater than that associated with Ski. The finding that the DACH1DeltaDS domain mutant abrogates Ski-mediated repression of SBE activity suggests that DACH1 and Ski may function in a similar pathway (Wu, 2003).

DACH1, like Ski, represses Smad3-regulated transactivation of either SBE or AP-1 activity. These findings with Ski are similar to previous findings but contrast with the effect of Sno-N, which has little effect on Smad3 transactivation. Sno-N is degraded rapidly in response to Smad3 or TGF-ß, whereas Ski expression and DACH1 expression are not affected greatly by TGF-ß. These findings suggest distinct roles for Sno-N versus Ski-N and DACH1 in TGF-ß signaling (Wu, 2003).

DACH1 inhibits TGF-ß- and Smad-induced AP-1 activity. Inhibition of TGF-ß and Smad-induced AP-1 activity requires the DACH1 DS domain. TGF-ß induction of several genes, including PAI-1, clusterin, monocyte chemoattractant protein-1 (JE/MCP-1), type I collagen, and TGF-ß itself depends on AP-1 DNA-binding sites in the promoter region of these genes. Induction of AP-1 activity by TGF-ß involves interactions between Smads and AP-1 transcription factors. Smads bind directly to the Jun family, and both Smad3 and Smad4 can bind JunB, c-Jun, and JunD. Since the regions of DACH1 that bind Smads are required for repression of TGF-ß-induced AP-1 activity, it is likely that DACH1 mediates AP-1 repression through Smad4 association (Wu, 2003).

The identification of DACH1 as a new co-repressor of TGF-ß signaling extends understanding of this key pathway. The role of TGF-ß in cancer includes a complex function as both an antiproliferative activity and as a tumor promoter. DACH1, like Sno-N and v-Ski oncogenes, bind directly to NCoR/SMRT and mSin3. TGF-ß controls a plethora of cellular functions and regulates development and homeostasis. Since DACH1 and SKI have only partially overlapping expression patterns, with DACH1 expressed in neuroblastomas and in cell lines derived from pancreas and breast cancer cell lines, it is possible that DACH1 contributes in a cell type-specific manner to regulate TGF-ß signaling (Wu, 2003).

Attenuation of Forkhead signaling by the retinal determination factor DACH1

The Drosophila Dachshund (Dac) gene, cloned as a dominant inhibitor of the hyperactive growth factor mutant ellipse, encodes a key component of the retinal determination gene network that governs cell fate. In this study cyclic amplification and selection of targets identified a DACH1 DNA-binding sequence that resembles the FOX (Forkhead box-containing protein) binding site. Genome-wide in silico promoter analysis of DACH1 binding sites identified gene clusters populating cellular pathways associated with the cell cycle and growth factor signaling. ChIP coupled with high-throughput sequencing mapped DACH1 binding sites to corresponding gene clusters predicted in silico and identified as weight matrix resembling the cyclic amplification and selection of targets-defined sequence. DACH1 antagonized FOXM1 target gene expression, promoter occupancy in the context of local chromatin, and contact-independent growth. Attenuation of FOX function by the cell fate determination pathway has broad implications given the diverse role of FOX proteins in cellular biology and tumorigenesis (Zhou, 2010).

This study provides evidence that the RDGN and Forkhead pathways integrate to control the expression of genes that associate with contact-independent growth. The key RDGN protein, DACH1, directly binds DNA and competes in the context of local chromatin with FOXM1, thereby attenuating key Forkhead regulatory gene networks. The cell fate determination factor, DACH1, is thus a DNA sequence-specific inhibitor of Forkhead signaling (Zhou, 2010).

The FOX proteins are a family of evolutionarily conserved transcription regulators involved in diverse biological processes. FOX protein function can either promote or inhibit tumorigenesis, and deregulation of FOX protein function in human tumorigenesis may occur by alteration in upstream regulators or genetic events such as mutations of the DBD, or translocations, which often disrupt the DBD. FOXM1 is overexpressed in human tumors and promotes tumor growth. Inhibition of FOXM1 expression reduces growth of murine tumors in response to carcinogens, and diminishes DNA replication and mitosis of tumor cells. FOXC2, associated with aggressive basal-like breast cancer, enhances tumor metastasis and invasion (Zhou, 2010).

In this study DACH1 was shown to inhibit FOXM1-mediated contact-independent growth of U2OS cells, antagonize FOXM1-mediated gene expression, and reduce occupancy of FOXM1 at target genes known to regulate the G2/M phase progression. Through competitive inhibition of FOXM1 occupancy at target genes, DACH1 could be essential for transcription of F-Box protein S-phase kinase-associated protein 2 (Skp2) and the Cdk subunit 1 (Cks1), which are substrate-specific subunits of the Skp1-Cullin-F-box protein (SCF) ubiquitin-ligase complex, that regulate p21^CIP1 and p27^KIP1. FOXM1 protein activates gene transcription and DACH1 competes with FOXM1 in the context of local chromatin to repress gene expression. Loss of DACH1 expression and increased oncogenic FOX protein expression could lead to deregulation of a subset of genes required for tumorigenesis. It has been shown that loss of DACH1 expression in human breast and endometrial cancers predicts poor outcome. Future studies will address whether loss of DACH1 correlates with increased FOXM1 expression during the progression of breast and endometrial cancers as well as other type of cancers (Zhou, 2010).

Identification of DACH1 DNA binding sequences by CAST, together with genome-wide in silico screening for putative target genes and ChIP-Seq for genes whose promoters were engaged by DACH1 through direct DNA binding, provided an alternative approach to establish the regulatory networks or individual genes that are governed by DACH1. In silico screening identified 2,887 genes whose promoter regions contain a potential DACH1 binding site. Many FOXM1-targeted genes including Aurora A (STK6), Aurora B (AURKB), CDC25A, CDC25C, E-cadherin (CDH1), and CENPB have DACH1 binding sequences within their 2-kb promoter region. A subset of these genes, chosen based on gene expression change as a result of DACH1 induction into MDA-MB-231 cells, were also enriched in their promoter region for the Forkhead family of transcription factors, supporting the competition/cooperation model of DACH1 and Forkhead transcription factors in gene regulation. Genes with DACH1 binding sites in their promoters populate cellular pathways associated with cancers such as the cell cycle pathway and the glioma pathway. Taken together, these results provide a rationale for further investigations into DACH1-mediated gene regulation in tumorigenesis. Given the diverse roles of Forkhead family proteins in cellular differentiation, survival, and DNA repair, the finding that the RDGN network protein DACH1 intersects FOX signaling may have broad implications for the understanding of cellular biology and tumorigenesis (Zhou, 2010).

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Search PubMed for articles about Drosophila dachshund

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