dachshund
See the embryonic expression pattern of dac at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site.
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 eyR 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).
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 Katp 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).
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).
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).
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).
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 ëratchetí 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).
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).
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).
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).
In the Drosophila wing, distal cells signal to proximal cells to induce the expression of Wingless, but the basis for this distal-to-proximal signaling is unknown. Three genes that act together during the establishment of tissue polarity, fat, four-jointed and dachsous, also influence the expression of Wingless in the proximal wing. fat is required cell autonomously by proximal wing cells to repress Wingless expression, and misexpression of Wingless contributes to proximal wing overgrowth in fat mutant discs. Four-jointed and Dachsous can influence Wingless expression and Fat localization non-autonomously, consistent with the suggestion that they influence signaling to Fat-expressing cells. dachs is identified as a gene that is genetically required downstream of fat, both for its effects on imaginal disc growth and for the expression of Wingless in the proximal wing. These observations provide important support for the emerging view that Four-jointed, Dachsous and Fat function in an intercellular signaling pathway, identify a normal role for these proteins in signaling interactions that regulate growth and patterning of the proximal wing, and identify Dachs as a candidate downstream effector of a Fat signaling pathway (Cho, 2004).
There is a progressive elaboration of patterning along the PD axis over the course of wing development. During the second larval instar, interactions among the Epidermal Growth Factor Receptor, Dpp and Wg signaling pathways divide the wing disc into a dorsal region, which will give rise to notum, and a ventral region, from which the wing will arise. An initial PD subdivision of the wing is then effected by signaling from the AP and DV compartment boundaries, which promotes the expression of two genes, scalloped and vestigial, that encode subunits of a heterodimeric transcription factor (Sd-Vg) in the center of the wing. This subdivides the wing into distal cells, which give rise to the wing blade, and surrounding cells, which give rise to proximal wing and wing hinge structures. The proximal wing is further subdivided into a series of molecularly distinct domains. Studies of Sd-Vg function in the wing led to the realization that the elaboration of this finer pattern depends in part upon signaling from the distal, Sd-Vg-expressing cells, to more proximal cells. Thus, mutation of vg leads to elimination, not only of the wing blade, where Vg is expressed, but also of more proximal tissue. Conversely, ectopic expression of Vg in the proximal wing reorganizes the patterning of surrounding cells (Cho, 2004 and references therein).
A key target of the distal signal is Wg, which during early third instar is expressed in a ring of cells that surround the SD-VG-expressing cells, and which later becomes expressed in a second, more proximal ring. Wg expression in the inner, distal ring within the proximal wing is regulated by an enhancer called spade-flag (spd-fg), after an allele of wg in which this enhancer is deleted (Neumann, 1996). Studies of this allele, together with ectopic expression experiments, have revealed that Wg is necessary and sufficient to promote growth of the proximal wing. Wg also plays a role in proximal wing patterning; it acts in a positive-feedback loop to maintain expression of Homothorax (Hth). The rotund (rn) gene has been identified as an additional target of distal signaling (Cho, 2004 and references therein).
This work identified Four-jointed (Fj), Dachsous (Ds), Fat and Dachs as proteins that influence signaling to proximal wing cells to regulate Wg and rn expression. Fj is a type II transmembrane protein, which is largely restricted to the Golgi. Null mutations in fj do not cause any obvious defects in the proximal wing. However, fj plays a role in the regulation of tissue polarity, yet acts redundantly with some other factor(s) in this process. Mutations in fat or ds can also influence tissue polarity. Although the molecular relationships among these proteins are not well understood, genetic studies suggest that fj and ds act via effects on fat, and both fj and ds can influence Fat localization in genetic mosaics (Cho, 2004 and references therein).
Interestingly, alleles of fj, ds and fat, as well as alleles of another gene, dachs, can result in similar defects in wing blade and leg growth. The similar requirements for these genes during both appendage growth and tissue polarity, together with the expression patterns of fj and ds in the developing wing, led to this investigation of their requirements for proximal wing development. All four genes influence the expression of Wg in the proximal wing, and genetic experiments suggest a pathway in which Fj and Ds act to modulate the activity of Fat, which then regulates transcription via a pathway that includes Dachs. These observations lend strong support to the hypothesis that Fj, Ds and Fat function as components of an intercellular signal transduction pathway, implicate Dachs as a key downstream component of this pathway, and identify a normal role for these genes in proximodistal patterning during Drosophila wing development (Cho, 2004).
The common feature of all of the manipulations of FJ and DS expression carried out in this study is that Wg expression, and by inference, Fat activity, can be altered when cells with different levels of Fj or Ds are juxtaposed. In the case of Fj, its normal expression pattern, and effects of mutant and ectopic expression clones are all consistent with the interpretation that juxtaposition of cells with different levels of Fj is associated with inhibition of Fat in the cells with less Fj and activation of Fat in the cells with more Fj. The influence of Ds, however, is more variable. Studies of tissue polarity in the eye suggest that Ds inhibits Fat activity in Ds-expressing cells, and/or promotes Fat activity in neighboring cells. The predominant effect of Ds during early wing development is consistent with this, but its effects in late discs are not. Studies of tissue polarity in the abdomen suggest that the Ds gradient might be interpreted differently by anterior versus posterior cells, and it is possible that a similar phenomena causes the effects of Ds to vary during wing development (Cho, 2004).
The influence of ds mutation on gene expression and growth in the wing is much weaker than that of fat. It has been suggested that Fj might influence Fat via effects on Ds, and fj mutant clones have been observed to influence Ds protein staining. The observations are consistent with the inference that both Ds and Fj can regulate Fat activity, but they do not directly address the question of whether Fj acts through Ds. They do, however, indicate that even the combined effects of Fj and Ds cannot account for FAT regulation, and, assuming that the strongest available alleles are null, other regulators of Fat activity must exist. It is presumably because of the counteracting influence of these other regulators that alterations in Fj and Ds expression have relatively weak effects. In addition, according to the hypothesis that Fat activity is influenced by relative rather than absolute levels of its regulators, the effects of Fj or Ds could be expected to vary depending upon their temporal and spatial profiles of expression, as well as on the precise shape and location of clones (Cho, 2004).
The observations imply the existence of at least two intracellular branches of the Fat signaling pathway. One branch involves the transcriptional repressor Grunge, influences tissue polarity, certain aspects of cell affinity, and fj expression, but does not influence growth or wg expression. An alternative branch does not require Grunge, but does require Dachs. Dachs is implicated as a downstream component of the Fat pathway, based on its cell autonomous influence on Fat-dependent processes, and by genetic epistasis. The determination that it encodes an unconventional myosin, and hence presumably a cytoplasmic protein, is consistent with this possibility. It also suggests that Dachs does not itself function as a transcription factor, and hence implies the existence of other components of this branch of the Fat pathway. This Grunge-independent branch influences Wg expression in the proximal wing and imaginal disc growth. However, further studies will be required to determine whether Dachs functions solely in Grunge-independent Fat signaling, or whether instead Dachs is required for all Fat signaling (Cho, 2004).
The observations that fj expression is regulated by Sd-Vg, and that fj is both necessary and sufficient to modulate the distal ring of Wg expression in the proximal wing, suggest that Fj influences the activity of a distal signal, which then acts to influence Fat activity. However, the relatively weak effects of fj indicate that other factors must also contribute to distal signaling, just as fj functions redundantly with other factors to influence tissue polarity. Since Ds expression is downregulated in a domain that is broader than the Vg expression domain, a direct influence of Vg on the Ds gradient is unlikely, and the essentially normal appearance of Wg expression in the proximal wing in fj ds double mutants implies that Ds is not a good candidate for the hypothetic factor Signal X. Rather, it is suggested that Ds acts in parallel to signaling from Vg-expressing cells to modulate Fat activity. This Vg-independent effect would account for the remnant of the distal ring that sometimes appears in vg null mutants. Importantly though, the observation that the phenotypes of hypomorphic dachs mutant clones on Wg expression are more severe than fj and ds suggests that the hypothesized additional factors also act via the Fat pathway. It is also noted that the limitation of Wg expression to the proximal wing even in fat mutant clones implies that Wg expression both requires Nubbin, and is actively repressed by distally-expressed genes (Cho, 2004).
The recovery of normal Wg expression by later stages in both fj and dachs mutant clones implies that the maintenance of Wg occurs by a distinct mechanism. Prior studies have identified a positive-feedback loop between Wg and Hth that is required to maintain their expression. It is suggested that once this feedback loop is initiated, Fat signaling is no longer required for Wg expression. Moreover, the recovery of normal levels of Wg at late stages suggests that this positive-feedback loop can amplify reduced levels of wg to near normal levels (Cho, 2004).
The distinct consequences of Vg expression and Fj expression in clones in the proximal wing suggest that another signal or signals, which are qualitatively distinct from the Fj-dependent signal, is also released from VG-expressing cells. When Vg is ectopically expressed, Wg is often induced in a ring of expression that completely encircles it. However, this is not the case for Fj-expressing clones. Both Vg- and Fj-expressing clones can activate rn and wg only within NUB-expressing cells, but Vg expression can result in non-autonomous expansion of the Nub domain, and this expansion presumably facilitates the expression of Wg by surrounding cells. Another striking difference between Vg- and Fj-expressing clones is that in the case of ectopic Fj, enhanced Wg expression is only in adjacent cells. By contrast, in the case of Vg, Wg expression initiates in neighboring cells, but often moves several cells away as the disc grows, resulting in a gap between Vg and Wg expression. This gap suggests that a repressor of Wg expression becomes expressed there, and recent studies have identified Defective proventriculus (Dve) as such a repressor (Cho, 2004).
In strong fat mutants, the wing discs become enlarged and have extra folds and outgrowths in the proximal wing. The disproportionate overgrowth of the proximal wing is due to upregulation of Wg in this region, as demonstrated by its suppression by wgspd-fg. At the same time, clones of cells mutant for fat overgrow in other imaginal cells, and fat wgspd-fg discs are still enlarged compared with wild-type discs. Thus, Fat appears to act both by regulating the expression of other signaling pathways (e.g. Wg), and via its own, novel growth pathway. The identification of additional components of this pathway will offer new approaches for investigating its profound influence on disc growth (Cho, 2004).
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).
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 eyR 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).
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).
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