The discovery of direct downstream targets of transcription factors (TFs) is necessary for understanding the genetic mechanisms underlying complex, highly regulated processes such as development. In this report, a combinatorial strategy was used to conduct a genome-wide search for novel direct targets of Eyeless (Ey), a key transcription factor controlling early eye development in Drosophila. To overcome the lack of high-quality consensus binding site sequences, phylogenetic shadowing of known Ey binding sites in sine oculis (so) was used to construct a position weight matrix (PWM) of the Ey protein. This PWM was then used for in silico prediction of potential binding sites in the Drosophila melanogaster genome. To reduce the false positive rate, conservation of these potential binding sites was assessed by comparing the genomic sequences from seven Drosophila species. In parallel, microarray analysis of wild-type versus ectopic ey-expressing tissue, followed by microarray-based epistasis experiments in an atonal (ato) mutant background, identified 188 genes induced by ey. Intersection of in silico predicted conserved Ey binding sites with the candidate gene list produced through expression profiling yielded a list of 20 putative ey-induced, eye-enriched, ato-independent, direct targets of Ey. The accuracy of this list of genes was confirmed using both in vitro and in vivo methods. Initial analysis reveals three genes, eyes absent, shifted, and Optix, as novel direct targets of Ey. These results suggest that the integrated strategy of computational biology, genomics, and genetics is a powerful approach to identify direct downstream targets for any transcription factor genome-wide (Ostrin, 2006).
Two members from the Six class of homeobox transcription factors, Sine oculis (SO) and Optix, function during development of the fly visual system. Differences in gain-of-function phenotypes and gene expression suggest that these related factors play distinct roles in the formation of the fly eye. However, the molecular nature of their functional differences remains unclear. This study reports the identification of two novel factors that participate in specific partnerships with Sine oculis and Optix during photoreceptor neurons formation and in eye progenitor cells. This work shows that different cofactors likely mediate unique functions of Sine oculis and Optix during the development of the fly eye and that the repeated requirement for SO function at multiple stages of eye development reflects the activity of different SO-cofactor complexes (Kenyon, 2005).
The fly SIX-HD transcription factors SO and Optix function during eye development and are expressed in different but overlapping patterns within the eye epithelium. Targeted expression of SO or Optix results in distinct phenotypes. Optix is able to induce eye development when ectopically expressed in the antenna and blocks eye morphogenesis when overexpressed within the eye epithelium. On the contrary, SO does not induce ectopic eye formation nor does it block neuronal differentiation within eye tissue. These observations strongly suggest that SO and Optix fulfill different roles during eye development. The identification of multiple unique partners for these Six factors clearly supports a model in which differences in cofactor recruitment contribute significantly to their specific function. This paper describes the identification of two novel potential partners of SO and Optix: one that is SO-specific by virtue of its expression pattern, and another likely to be Optix-specific by virtue of its protein binding profile (Kenyon, 2005).
The Sbp gene (Flybase ID FBgn0033654) encodes a novel factor of unknown function. It does, however, contain three highly conserved motifs and a proline-rich region. The high level of similarity from fly to vertebrates suggests that these sequences represent novel protein motifs or domains with specific functions. Since they do not appear related to previously characterized domains, little can be concluded about their potential function. However, Box 1 is not required for binding to the SD of SO, because the clone isolated through the yeast two-hybrid screen does not include this sequence. Its interaction with SO suggests that Sbp may function as a transcriptional regulator. The presence of a proline-rich region supports this hypothesis because proline-rich domains have been implicated in both transcriptional activation and repression. In a manner similar to Gro and unlike Eya, Sbp can interact in yeast with both SO and Optix. However, Sbp is specifically coexpressed with SO and not Optix within the eye epithelium. Hence, its interaction in vivo is restricted to SO by virtue of its expression posterior to the MF. This hypothesis is consistent with the observation that ectopic expression of Sbp in progenitor cells, where Optix is also expressed, interferes with normal eye development. However, alternative explanations are also possible. The effect of Sbp misexpression may be due to its abnormal association with SO itself, or other nuclear factors, in progenitor cells. Similarly, the disruption of eye development due to the misexpression of Optix in the developing neuronal field may result from abnormal interactions with other yet unidentified factors. Nonetheless, restricted expression of Sbp posterior to the MF and Optix anterior to the MF is obviously critical for normal eye development and effectively excludes binding between Optix and Sbp (Kenyon, 2005).
Sbp expression within the developing neuronal array, and not in progenitor cells, indicates that this gene is not required at an early stage but functions at the time of neuronal differentiation. On the contrary, SO function is required at multiple stages of eye development, including during specification of eye progenitor cells, at the time of patterning (in the MF) and during neuronal development. The Eya cofactor has also been implicated in these steps, and both proteins are continuously expressed in eye tissue through these developmental stages. These studies do not provide clues regarding the function of SO at each developmental stage. Based on these data, SO could function simply by driving expression of a few genes required at all times to maintain eye identity. The identification of an SO partner specifically expressed in the differentiating epithelium contradicts this hypothesis and shows that there are significant differences in at least some of the SO-based complexes that function at distinct developmental stages. Thus, posterior to the MF, Sbp may associate with the SO-Eya complex and modify its activity turning it from an activator into a repressor. Evidence of complex interactions between the mouse Six1, Dac, and Eya proteins has been described. Alternatively, Sbp may form a distinct SO-Sbp complex. In either case, however, SO function at various times during eye development would be modulated by its interaction with specific partners (Kenyon, 2005).
The Obp gene (Flybase ID FBgn0050443) encodes a protein characterized by nine putative zinc-finger domains of the C2H2 type. Although most members of this family are thought to regulate transcription through DNA binding, recent reports have also implicated C2H2-type zinc-fingers in protein-protein interactions. Because zinc-fingers 4 through 9 as well as the C-terminal sequence of Obp are present in the fragment isolated in the screen, it cannot be excluded that one or more of these zinc-finger domains mediate the interaction with the Optix transcription factor. Obp is coexpressed with both SO and Optix in eye progenitor cells. However, it behaves as an Optix-specific interactor in yeast two-hybrid tests and does not impair eye development when ectopically expressed in the differentiating neuronal field (where only SO is present). Hence, this factor is likely to be a specific partner of Optix in vivo (Kenyon, 2005).
This work has not identified clear homologues of this factor. This finding is not entirely unexpected, because zinc-finger domains display only low-level conservation at positions other than the C and H residues that bind zinc. In the related proteins from D. pseudoobscura and A. mellifera, conservation is restricted to the region encoding zinc-fingers 4 through 8. Hence, additional criteria (such as expression in eye progenitor cells, interaction with Optix, and/or mutant rescue) are required to assess whether they are indeed homologues. Obp is reported in Flybase to encode a product putatively involved in cell proliferation. In fact, the fourth and sizth zinc-fingers show similarity to zinc-fingers present in the Sfp1 transcription factor. In yeast, Sfp1 functions in cellular growth and proliferation by regulating ribosomal proteins expression. Obp expression, specifically anterior to the MF where undifferentiated progenitors proliferate and then arrest just before the start of eye morphogenesis, is consistent with a potential role in regulating cell proliferation. However, the alignment between Obp and Sfp1 is limited to less than 20% of what is currently defined as the Sfp1 domain. Given the limited conservation and the lack of functional data, a role for Obp in proliferation control is speculative at this time (Kenyon, 2005).
The expression pattern of Optix during embryonic and larval stages was examined by whole-mount in situ hybridization and compared to the pattern of sine oculis expression. Optix is first expressed in a ring at the anterior end of the blastoderm embryo. This expression is similar to so expression, but lies more anteriorly. During germ band extension, Optix is restricted to the anterior and in contrast to so, is not expressed in the optic lobe primordia. At stage 11, Optix expression is detected in the clypeolabrum and remains limited to the anterior region, whereas so expression is also detected bilaterally at the segmental boundaries in a set of unidentified epidermal cells, and in the optic lobe primordium. At stage 14 Optix is expressed in the ectoderm covering the supraesophageal ganglion, which will give rise to parts of the brain, but Optix is not expressed in Bolwig's organ (Seimiya, 2000).
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).
In wild-type eye imaginal discs, Optix RNA is detected in front of the morphogenetic furrow (MF), in the head region and just anterior to the vertex region. In late second instar larvae, before the MF forms, Optix expression covers the entire eye disc but later the expression becomes restricted to a region anterior to the MF. This expression pattern is similar to the pattern of eyeless and twin of eyeless (toy) (Quiring, 1994; Czerny, 1999) and it suggests that Optix may play an important role in early eye disc development as do ey and toy. In contrast, so starts to be expressed at early third instar just before MF initiation and later becomes restricted to a zone just anterior, inside and posterior to the MF. In addition, Optix is expressed in wing and haltere discs, but not in leg discs. This expression continues throughout the third instar larval stage, whereas so is not expressed in wing and haltere discs, but in the leg discs (Seimiya, 2000).
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date revised: 10 April 2009
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