org Interactive Fly, Drosophila

atonal


REGULATION

Promoter

atonal is a proneural gene for the development of Drosophila chordotonal organs and photoreceptor cells. atonal expression is controlled by modular enhancer elements located 5' or 3' to the atonal-coding sequences. During chordotonal organ development, the 3' enhancer directs expression in proneural clusters; whereas successive modular enhancers located in the 5' region drive tissue-specific expression in chordotonal organ precursors in the embryo and larval leg, wing and antennal imaginal discs. Similarly, in the eye disc, the 3' enhancer directs initial expression in a stripe anterior to the morphogenetic furrow. These atonal-expressing cells are then patterned through a Notch-dependent process into initial clusters, representing the earliest patterning event yet identified during eye morphogenesis. A distinct 5' enhancer drives expression in intermediate groups and R8 cells within and posterior to the morphogenetic furrow. Both enhancers are required for normal atonal function in the eye. The 5' enhancer, but not the 3' enhancer, depends on endogenous atonal function, suggesting a switch from regulation directed by other upstream genes to atonal autoregulation during the process of lateral inhibition. The regulatory regions identified in this study can thus account for atonal expression in every tissue and essentially in every stage of its expression during chordotonal organ and photoreceptor development (Sun, 1998).

Restriction of proneural gene expression from proneural clusters to SOPs is usually Notch (N) dependent. During eye development, N is known to function within and posterior to the MF in restricting ato expression to R8 cells within intermediate groups. Anterior to the morphogenetic furrow, N has been shown to promote ato expression. To test the function of N in the formation of the ato prepattern anterior to the MF and in regulating the 3' enhancer, lacZ expression was examined from the 3' enhancer-lacZ reporter gene in a temperature-sensitive N mutant background. When larvae carrying the temperature sensitive N allele and the 3' enhancer-lacZ fusion gene are shifted to the restrictive temperature for 2 hours, the 3' enhancer-directed lacZ expression anterior to the MF becomes continuous and appears broader and stronger than that in wild type, and the initial clusters normally seen within the initial stripe fail to form. The endogenous ato gene responds similarly to N inactivation in the initial stripe. It is concluded that N is involved in refining ato expression anterior to the MF from a continuous band to patterned initial clusters, which prefigure the future ommatidia (Sun, 1998).

Induction and autoregulation of the anti-proneural gene Bar during retinal neurogenesis in Drosophila: Bar regulates atonal

Atonal (Ato)/Math (Mammalian atonal homolog) family proneural proteins are key regulators of neurogenesis in both vertebrates and invertebrates. In the Drosophila eye, Ato is essential for the generation of photoreceptor neurons. Ato expression is initiated at the anterior ridge of the morphogenetic furrow but is repressed in the retinal precursor cells behind the furrow to prevent ectopic neurogenesis. Ato repression is mediated by the conserved homeobox proteins BarH1 and BarH2. Loss of Bar causes cell-autonomous ectopic Ato expression, resulting in excess photoreceptor clusters. The initial ommatidial spacing at the furrow occurs normally in the absence of Bar, suggesting that the ectopic neurogenesis within Bar mutant clones is not due to the lack of Notch (N)-dependent lateral inhibition. Targeted misexpression of Bar is sufficient to repress ato expression. Furthermore, evidence is provided that Bar represses ato expression at the level of transcription without affecting the expression of an ato activator, Cubitus interruptus (Ci). Thus, it is proposed that Bar is essential for transcriptional repression of ato and the prevention of ectopic neurogenesis behind the furrow (Lim, 2003).

Each ommatidium of the adult compound eye consists of eight photoreceptors that are generated by the proneural function of Ato expressed within and anterior to the furrow in the eye disc. The domain of Ato expression is juxtaposed to the Bar-expressing undifferentiated cells behind the furrow. Although Bar is also expressed in R1 and R6 photoreceptors, this study focuses specifically on the Bar expression in the undifferentiated cells and the Ato expression in adjacent anterior cells. These Bar-expressing undifferentiated cells will be referred as the 'basal cells' since their nuclei stay in the basal region while photoreceptor cell nuclei migrate apically, although cell bodies of both cell types are connected to the top and bottom of the eye disc epithelium. Nuclei of Ato-expressing cells are located basally during the stages 1 and 2, but migrate apically as they become R8 founder neurons posterior to the furrow (Lim, 2003).

Thus Ato expression is highly elevated in the absence of Bar behind the furrow, suggesting that Bar is necessary for downregulation of Ato expression. Furthermore, Bar represses ato expression at the transcriptional level through both 3'- and 5'-regulatory regions of ato. The 9.3 kb of ato 5' sequence (5'F:9.3) has been shown to be responsible for ato expression in the equivalence groups and the R8 founder cells in an Ato-dependent manner (stages 2-4). Sca and Egfr-mediated MAP kinase signaling may inhibit this enhancer function of 5'-regulatory element of ato within interommatidial regions to establish regularly spaced intermediate groups. By contrast, the 5.8 kb of ato 3' enhancer (3'F:5.8) is only activated anterior to the furrow to drive the initial stripe of ato expression (stage 1). How this enhancer activity is inhibited posterior to the stage 1 Ato domain of the eye disc is unknown. The results now indicate that the initial stripe (stage 1) of ato expression driven by 3'-regulatory element is strongly inhibited by Bar behind the furrow (Lim, 2003).

Ectopically elevated Ato expression within Bar loss-of-function clones is sufficient to induce the formation of mature ectopic photoreceptor clusters. This suggests that Bar mutations specifically eliminate the repression of initial ato expression with little effects on subsequent steps of photoreceptor recruitment. This is consistent with the observations that Sca, Egfr signaling and N-mediated lateral inhibitions function properly within Bar loss-of-function clones. Therefore, the major role of Bar during retinal neurogenesis appears to be the inhibition of initial stripe ato expression through 3'-regulatory elements of ato behind the furrow (Lim, 2003).

Bar is a DNA-binding homeodomain transcription factor. Mammalian homolog Barx2 was shown to bind directly to regulatory elements of several neural cell adhesion molecules, which contains target sites including the core sequence CCATTAGPyGA. Interestingly, the 5'F9.3 and 3'F5.8 regulatory regions of ato also have multiple potential Bar binding sites containing the same core sequence, suggesting that Bar may directly bind to these target sites of ato regulatory elements and repress ato transcription (Lim, 2003).

It is important to note that CiFL induced by Hh signaling can activate ato expression. Furthermore, Bar and CiFL are expressed complementarily to each other. These observations raise the possibility that ato repression by Bar may be mediated by Bar repression of CiFL. However, the results indicate that Bar function is independent of CiFL, supporting the idea that the primary cause of ato repression behind the furrow is a direct function of Bar as a repressor rather than indirect effects of the removal of the activator, CiFL. Furthermore, overexpression of CiFL by the lz-Gal4 driver in the presence of Bar does not activate ato expression, indicating that Bar-mediated ato repression is epistatic to an overexpression of CiFL activator (Lim, 2003).

Based on the findings, a model of Bar function in retinal neurogenesis is proposed. Ato is expressed within the furrow and is required for the generation of R8 founder neurons. Bar homeodomain proteins are expressed in the basal cells behind the furrow and represses ato expression, showing a complementary expression pattern to Ato. This function of Bar on ato-repression occurs independent of CiFL, a transcriptional activator of ato. Rather, Bar may directly repress ato transcription by binding to 3'- and 5'-regulatory regions of ato through its potential binding sites (Lim, 2003).

The finding that Bar inhibits the expression of the proneural gene ato as a transcriptional repressor in the eye disc raises the interesting issue of whether Bar can also repress the expression of other proneural genes in the eye or other tissues. Misexpression of BarH1 or BarH2 using a dpp-Gal4 driver shows increased expression of proneural gene scute in the eye and wing discs rather than repressing its expression, generating more sensory bristles. By contrast, the deficiency in Bar causes the loss of sensory interommatidial bristles in the eye. These results suggest that Bar can act as an activator for the expression of ASC proneural genes to generate bristle sensory neurons. Therefore, Bar can act as transcriptional activator as well as repressor for different proneural genes depending on developmental contexts in Drosophila. This dual function of Bar was also observed in mammalian Barx2. Barx2 has activator and repressor domains in the C- or N-terminal regions, respectively. Mbh1, another mammalian homolog of the Bar class genes, functions as either activator or repressor for the expression of neural bHLH genes in cell culture system. Therefore, the dual function of transcriptional activation and repression may be a general property of Bar family homeodomain proteins in the control of expression of neural target genes. These opposite actions of Bar may be dependent on the binding of their specific partners to the activator or repressor domain (Lim, 2003).

It has been shown that loss of groucho (gro) results in increased ato expression behind the furrow of the eye disc. Gro represses the expression of proneural genes during N-mediated lateral inhibition. It is interesting to note that Bar family homeodomain proteins have a conserved ~10 amino acid motif termed the FIL domain at the N-terminal region of the homeobox. This domain shows sequence similarity to the core region of the engrailed homology-1 (eh1) domain in Engrailed (En) repressor, which can directly interact with Gro co-repressor through its eh1 motif. Therefore, Bar may interact with Gro through its FIL domain for its repressor function (Lim, 2003).

Bar class homeodomain proteins are evolutionarily highly conserved from Drosophila to human. Vertebrate Bar homologs include Xenopus XBH1 and XBH2, mouse and human Barhl1 and Barhl2, rat Mbh1 [same gene as Barhl2], and murine and human Barx1 and Barx2 genes. Although in vivo function of Bar homologs has not been extensively analyzed, some members of the Bar class homeobox genes may be involved in the genesis and fate specification of neuronal cells. A mammalian homolog, Mbh1, is expressed in a complementary pattern to Mash1, a homolog of ASC, in the rat eye. Hence, Mbh1 may be involved in inhibition of Mash1 expression, similar to the ato repression by Drosophila Bar proteins (Lim, 2003).

In vertebrate eye development, a mammalian homolog of ato, Math5 (and/or Xath5), is crucial for the generation of retinal ganglion cells, which are the first neurons to arise and therefore may be analogous to the R8 founder cells in the Drosophila eye. The essential role of Math5 in the genesis of ganglion cells suggests that Math5 plays Ato-like proneural function in vertebrate eye development. It will be interesting to see whether a specific Bar homolog(s) may be involved in the repression of Math5, since the Drosophila Bar inhibits ato expression. In addition, Bar class genes are attractive candidates for many human genetic disorders, including Joubert syndrome and Rieger syndrome. The new function of Drosophila Bar in negative regulation of neurogenesis may provide insights into the function of Bar family genes in vertebrates and the molecular basis of human diseases associated with altered Bar function (Lim, 2003).

EGF receptor signaling triggers recruitment of Drosophila sense organ precursors by stimulating atonal autoregulation

In Drosophila, commitment of a cell to a sense organ precursor (SOP) fate requires bHLH proneural transcription factor upregulation, a process that depends in most cases on the interplay of proneural gene autoregulation and inhibitory Notch signaling. A subset of SOPs are selected by a recruitment pathway involving EGFR signaling to ectodermal cells expressing the proneural gene atonal. EGFR signaling drives recruitment by directly facilitating atonal autoregulation. Pointed, the transcription factor that mediates EGFR signaling, and Atonal protein itself bind cooperatively to adjacent conserved binding sites in an atonal enhancer. Recruitment is therefore contingent on the combined presence of Atonal protein (providing competence) and EGFR signaling (triggering recruitment). Thus, autoregulation is the nodal control point targeted by signaling. This exemplifies a simple and general mechanism for regulating the transition from competence to cell fate commitment whereby a cell signal directly targets the autoregulation of a selector gene (zur Lage, 2004).

Paracrine signaling is a widespread trigger of cell fate determination during development. However, it is well known that the information that such signals impart depends on the context. Thus, signaling allows or prevents a target cell from committing to a fate for which it is already predisposed or competent. Sense organ precursor (SOP) determination in the developing Drosophila PNS provides an important model system for understanding the mechanisms underlying competence and commitment, and particularly how the transition from competence to commitment is controlled. In this case, competence and commitment requires the function of the bHLH proneural genes achaete and scute (ac/sc), atonal (ato), and amos, which can be viewed as selector genes of SOP fate. Much progress has been made in understanding this process during 'classical' SOP selection. Proneural genes are initially expressed in groups of ectodermal cells known as proneural clusters (PNCs). This initial expression provides cells with neural competence but does not necessarily lead to commitment. The key event in SOP commitment is the upregulation of proneural protein expression in specific PNC cells. For ac/sc, a complex network of cell interactions and signaling feedback loops determines whether a cell upregulates or downregulates ac/sc expression, thereby ensuring that a single SOP is selected. In this process, Notch signaling within proneural clusters inhibits ac/sc autoregulation by directly interacting with autoregulatory enhancers (zur Lage, 2004 and references).

In addition to this classical mode, there is a second mode of SOP formation that has been characterized for ato during chordotonal stretch receptor development: chordotonal SOPs can be recruited by EGFR signaling. In each embryonic abdominal segment, five chordotonal SOPs are selected from two ato-expressing PNCs in a conventional manner, involving the interplay of ato with Notch signaling. These 'primary' precursors express rhomboid, (rho) which activates secretion of the EGFR ligand Spitz, and the subsequent signaling to adjacent ectodermal cells leads to their recruitment as 'secondary' chordotonal SOPs. As a result, in each abdominal segment five primary precursors recruit three secondary precursors, which together differentiate as the eight chordotonal organs. As in all cases of EGFR signaling, the recruited cell's immediate response is the activation of the ETS family transcription factor, Pointed (Pnt). The pnt gene encodes two isoforms, Pnt-P1 and P2 (the combined activity of the two isoforms is referred to as Pnt). Pnt-P2 is activated by ERK MAPK phosphorylation upon EGFR stimulation, whereas Pnt-P1 is regulated by EGFR signaling at the transcriptional level. The favored model is that ERK phosphorylates Pnt-P2, which then activates the transcription of Pnt-P1. Apart from their regulation, it is thought that Pnt-P1 and P2 function similarly as transcription factors by binding to the same sites via their common ETS domains. Interestingly, signaling from the dorsal-most primary SOP triggers the recruitment of oenocyte precursors rather than chordotonal SOPs. Clearly, specificity of cellular response must depend on factors other than Pnt (zur Lage, 2004).

A similar process of recruitment occurs for the adult femoral chordotonal organ (FCO), but with key differences. As in the embryo, chordotonal SOPs recruit further SOPs from the ectoderm, but in this case recruitment is reiterative: newly recruited SOPs themselves express rho and in turn recruit further SOPs from the ato-expressing PNC. Thus, unlike in the embryo, the recruitment cycle is repeated many times as new SOPs become new signaling sources. As a result, some 80 SOPs are recruited over time. In the leg disc, SOP recruitment correlates with ato upregulation. To understand the basis of this, ato regulation during recruitment has been investigated. Analysis of key target gene enhancers has greatly increased understanding of the logic of cell fate determination. An enhancer upstream of ato is active specifically during recruitment in both the leg disc and the embryo. This enhancer is regulated directly by Pnt and Ato binding cooperatively to adjacent sites. The consequence of this is that the enhancer responds only to the combined input of both Pnt and Ato. Thus, Ato ensures the specificity of EGFR signaling in this context. Importantly, SOP recruitment depends on the direct manipulation of Ato autoregulation: such autoregulation is contingent on EGFR signaling. Thus, to promote the transition from competence to cell fate commitment, a cell signal directly targets the autoregulation of a selector gene (zur Lage, 2004).

Sun (1998) described the approximate location of several regulatory elements up- and downstream of the ato ORF. An enhancer supporting reporter gene expression in FCO precursors was inferred indirectly to exist between SmaI and BamHI restriction sites 3.6-5.5 kb upstream of the ato ORF. When this 1.9 kb region was cloned into a Gal4 P element vector, it indeed supported Gal4 expression in the FCO precursors. The fragment was subdivided and each subfragment was inserted into the GFP reporter vector, pHStinger. Using these transgenes, the FCO enhancer could be localized to a 367 bp fragment. GFP expression driven by this fragment was observed in the chordotonal SOPs (marked by Ato and the SOP protein, Senseless [Sens] but not in the overlying PNC (marked by Ato), suggesting that it is active during SOP commitment (zur Lage, 2004).

The ato FCO enhancer is also active during embryonic chordotonal recruitment. The enhancer drives GFP expression in embryonic sensory cells that derive from a subset of Ato-dependent SOPs. Owing to the delayed acquisition of GFP fluorescence, the onset of GFP expression appears shortly after Ato is downregulated in these SOPs. Nevertheless, examination of perduring GFP in older embryos relative to a sensory neuron differentiation marker, 22C10, revealed expression in two chordotonal sensilla of the five that make up the lateral chordotonal array (lch5). This correlates with the two recruited SOPs that contribute to lch5. The GFP-expressing sensilla are usually the most posterior of the lch5 cluster. Significantly, the anterior-most sensillum never expresses GFP, which is consistent with it deriving from a primary chordotonal precursor. Similarly, GFP expression is observed weakly in one of the two ventral chordotonal organs (vchB), which derives from a recruited SOP. In the head, there is notable expression in cells that give rise to the larval eye (Bolwig's organ). This is also an ato-specified sense organ that requires EGFR signaling. Overall, these patterns suggest that this enhancer is responsible not for general ato regulation in SOPs, but specifically in situations where it depends on EGFR signaling. Moreover, the enhancer appears to mediate the EGFR signaling response in a variety of developmental situations. This element is referred as the ato recruitment enhancer (ato-RE). Significantly, ato-RE is not expressed in oenocytes, even though these are recruited by EGFR signaling from a primary chordotonal SOP (zur Lage, 2004).

How ato-RE-GFP expression responds to ectopic expression of pnt-P1 was analyzed using 109-68-Gal4, a Gal4 driver line that is expressed in many proneural clusters and SOPs in the imaginal discs. However, misexpression of UAS-pnt-P1 induced very little change in the ato-RE-GFP expression pattern in leg or wing discs. It was reasoned that another tissue-restricted factor is required for the response of ato-RE to Pnt. Ato protein itself may be such a factor, since it is already expressed at a low level in the leg PNC cells that are recruited. UAS-ato misexpression (109-68-Gal4 UAS-ato) results in a modest change in expression of ato-RE-GFP, even though such misexpression induces significant (nonrecruitment) chordotonal SOP commitment. In contrast, co-misexpression of both Pnt-P1 and Ato results in a significant increase in ectopic ato-RE-GFP expression in the leg and wing. This finding suggests two things: (1) Pnt/EGFR activation of ato-RE is contingent on the presence of Ato. This restricts its function to Ato-expressing PNC cells. (2) ato-RE is an autoregulatory enhancer, but unlike other proneural autoregulatory enhancers, autoregulation is contingent on the cell receiving an EGFR/Pnt signal (zur Lage, 2004).

These results suggest that the ato-RE is activated by the simultaneous presence of Ato and Pnt in recruited SOPs. As expected from previous evidence, Ato and Pnt-P1 are indeed both expressed in leg FCO SOPs. In the embryo, too, in double labeling experiments, cells that coexpress Ato and Pnt-P1 are clearly present at the time and location expected of recruited SOPs. Consistent with this, a GFP reporter transgene that responds directly to Ato regulation is expressed in both primary and recruited chordotonal SOPs and is coexpressed with Pnt-P1 (zur Lage, 2004).

ato-RE was tested for its ability to bind Pnt and Ato proteins in vitro (the latter as a heterodimer with Daughterless [Da] protein). In gel mobility shift assays using purified proteins and the entire ato-RE as a probe, Pnt and Ato/Da proteins both bind in a manner that is consistent with a single binding site each. In general, Ato/Da binds to the bHLH A-class E-box core consensus. On this criterion, there are two potential Ato/Da binding sites in ato-RE (E1 and E2). Ato/Da binding could be competed strongly by an E1-containing competitor oligonucleotide, but not strongly by an E2-containing competitor nor by a competitor with a mutated version of the E1 site [E1(M): CAGGTG→CCTAGG]. This suggests that Ato/Da binds to ato-RE largely via E1. Mobility shifts with site-specific oligonucleotides as probes supported this. To assess the in vivo function of these sites, site-directed mutagenesis was carried out on ato-RE and the effect on reporter gene expression was assessed in transgenic flies. Mutation of E2 had no discernable effect on ato-RE-GFP expression, but mutation of E1 completely abolished expression in the embryo, leg imaginal disc, and also the eye disc. Thus, E1 is likely to be a binding site through which Ato regulates its own expression in recruited chordotonal precursors (zur Lage, 2004).

Pnt binds to a consensus sequence around a GGAA core that has been characterized for vertebrate ETS-1, and a number of functional Pnt binding sites have been characterized that conform to this consensus. In ato-RE there are two potential Pnt binding sites (ETS-A and ETS-B). Purified Pnt-P1 protein binds to ato-RE, and this binding is competed efficiently with a competitor oligonucleotide containing the ETS-A site but not one containing the ETS-B site. This suggests that Pnt-P1 binds ato-RE via the ETS-A site. Mutating ETS-A abolished GFP reporter gene expression in the embryo, leg FCO region, and eye. A few GFP-expressing cells remaining in the leg may correspond to a tibial chordotonal organ. Mutation of ETS-B had no apparent effect. Thus, the ETS-A site is likely to be a binding site through which Pnt regulates ato expression in the recruited chordotonal precursors (zur Lage, 2004).

In summary, virtually the entire activity of the ato-RE requires the E1 and ETS-A sites, most likely by binding Ato/Da and Pnt, respectively (zur Lage, 2004).

A remarkable feature of the E1 and ETS-A sites is their proximity, their core sequences being separated by 4 bp. This proximity is maintained precisely in the sequence upstream of ato in the genomes of D. pseudoobscura and virilis, where the sites are within an identical stretch of 58 bp. The proximity and its conservation suggest that protein interactions between these transcription factors may contribute to the mechanism by which they specifically regulate ato. Molecular modeling suggests that the Pnt ETS domain and the E1 bHLH domains of Da/Ato heterodimer can bind simultaneously to this DNA sequence and may make direct contact with each other. Although no structures are known for any of these proteins, the conformations of the domains are likely to be highly similar to other proteins of similar sequence for which structures are available. The bHLH domains in the Ato:Da heterodimer were modeled using MyoD homodimer in complex with DNA, and Pnt was modeled using a structure of PU1's ETS domain in complex with DNA. The DNA molecules in each complex were superimposed such that the sites were the correct number of base pairs apart to resemble the E1-ETS-A sequence. The resultant model shows no serious steric clashes between Ato and Pnt domains and, indeed, the two proteins are close enough to form direct contacts (zur Lage, 2004).

This possibility was explored by investigating the binding of Ato/Da and Pnt in gel mobility shift assays. In the presence of all three proteins, a slower migrating protein-DNA complex was observed that represents all three proteins bound to the DNA. In a supershift assay, this complex is lost if antibodies to Ato or Pnt-P1 are included. Moreover, it appears that the triple binding is synergistic. In particular, although Pnt binds relatively poorly to ETS-A alone, the presence of all three proteins appears to drive strong binding of the ternary complex. Interestingly, the ternary complex also formed (albeit less efficiently) when the Pnt site was mutated such that it no longer bound Pnt when added alone. Thus, although Pnt requires the ETS-A site in vivo, in vitro Ato/Da can pull Pnt into the DNA-protein complex even when Pnt cannot interact as efficiently with the DNA itself. Consistent with this, Pnt can also interact with Ato in a GST pull-down assay in the absence of DNA. These data suggest that protein-protein interactions stabilize the DNA-protein complex and that cooperative binding may be important for this enhancer's function and specificity in vivo (zur Lage, 2004).

An interesting question is whether the synergistic interaction between Pnt and Ato/Da allows the E1/ETS-A sites to function in vivo outside the context of the ato-RE enhancer. A construct was made with GFP driven by two tandem repeats of a 35 bp fragment from the conserved ato-RE region, including the E1 and ETS-A sites [(E1+ETS-A)2-GFP]. In the embryo, expression of (E1+ETS-A)2-GFP was strikingly similar to ato-RE-GFP. It is strongly expressed in the precursors of vchAB, v'td2, and two sensilla of lch5. In the head there is particularly strong expression in cells giving rise to Bolwig's organ, as well as other ato-dependent locations. This construct, however, does not support any expression in the femoral precursors of the leg disc, suggesting that additional ato-RE sequences are required here for correct regulation (zur Lage, 2004).

To ascertain the contribution of the ETS-A site, a reporter transgene driven by six copies of the E1 site alone [(E1)6-GFP] was generated. Unlike the (E1+ETS-A)2-GFP construct, (E1)6-GFP is expressed in all ato-dependent SOPs in the embryo. Thus, the E1 site is capable of supporting Ato/Da-dependent regulation in all SOPs, but regulation is normally restricted to recruited SOPs by the need for Ato/Da to interact with Pnt. Presumably, this requirement is subverted when the E1 site is highly multimerized. A second possibility is that as well as binding Pnt, the ETS-A site can also bind a repressor. A likely candidate is the ETS repressor Yan. Yan acts in opposition to Pnt, and its repressor activity is relieved upon EGFR signaling by phosphorylation by ERK. Indeed, Yan protein is expressed during embryonic chordotonal recruitment, and recruitment is more extensive in yan mutant embryos. Consistent with this, Yan protein is able to bind the ETS-A site in vitro, and (E1+ETS-A)2-GFP is expressed in more cells in yan mutant embryos. This suggests that the ETS-A site is bound by Yan repressor. This repression of ato-RE is relieved by EGFR-dependent phosphorylation and by displacement by Pnt proteins. Interestingly, there is no evidence that Yan functions in leg disc SOP recruitment since its expression is undetectable during FCO development. However, FCO recruitment is susceptible to Yan function, since expression of a UAS-yanAct construct strongly inhibits chordotonal SOP recruitment (zur Lage, 2004).

A summary of the regulation of ato during recruitment is presented. Initially, Ato is expressed at a low level in PNC cells as a result of regulation by an enhancer(s) that is distinct from ato-RE. Such PNC enhancers are known for both the leg and the embryo. Coexpression of E(spl) in response to Notch signaling inhibits SOP commitment, but the low level of Ato provides the competence for these cells to respond to EGFR signaling from other SOPs. If an Ato-expressing cell receives this signal, the combined action of Pnt and Ato/Da acts via the ato-RE element to upregulate Ato expression, leading to SOP commitment. Although this model describes a mechanism for SOP recruitment, it is likely that the firstborn chordotonal SOPs (including the primary SOPs in the embryo) are selected from the PNC by mechanisms involving an interplay of ato regulation with Notch/E(spl) signaling, as described for ac/sc. This conventional SOP selection route would function via enhancers other than the ato-RE (zur Lage, 2004).

The combined response to Pnt and Ato/Da is mediated at the molecular level by cooperative binding of the transcription factors to adjacent sites that are evolutionarily conserved. The juxtaposition of sites allows high affinity of protein complex binding in vitro, and hence high specificity of enhancer activity in vivo even when the two sites form a synthetic enhancer in isolation from the rest of the enhancer. Binding by Pnt-P1 alone is rather poor but is much stronger in the presence of Ato/Da. Thus, cooperativity increases specificity. As in the case of Pnt, direct cooperative interaction with a selector gene product has been found to underlie the specificity of mammalian ETS-1 proteins, including interaction with Runx and Pax5. Significantly, cooperative binding has been characterized between ETS-1 and the bHLH protein, USF. Preliminary molecular modeling of bHLH and ETS domains on the ato-RE shows the feasibility of contact between the Ato HLH and Pnt ETS domains. Among proneural proteins, this interaction may be very specific for Ato: bHLH residues available for interaction with Pnt are uniquely conserved in Ato and its vertebrate homologs compared with Sc and its homologs. It is suggested that Sc is unable to make appropriate interactions with Pnt. Consistent with this, when the Ato/Da E1 site is altered to conform to the binding consensus for Sc/Da, there is a dramatic loss of ato-RE enhancer activity. In this light, Pnt can be thought of as a specificity cofactor that ensures that Ato is the only proneural protein that can regulate the ato-RE (zur Lage, 2004).

Positive autoregulation is a common transcriptional control mechanism. It is suggested that commonly, if not universally, positive autoregulation is contingent on other conditions being fulfilled in addition to the factor itself being present. In consequence, promotion or inhibition of autoregulation provides a sensitive nodal point of regulation that can be modulated by extrinsic factors. In the case of ato, autoregulation provides the switch through which EGFR signaling can drive the transition from SOP competence to commitment. In a related way, autoregulation plays an important part in conventional SOP determination by Ac and Sc proneural proteins. In current models, Notch inhibits SOP selection by antagonizing the activity of Ac and Sc autoregulatory enhancers. Genetically, chordotonal precursor recruitment is also inhibited by Notch signaling. This does not appear to be by direct DNA binding, however, since there are no good candidate consensus sites for Su(H) or E(spl) in ato-RE. One possibility is that E(spl) proteins interact directly with Ato/Da and that this inhibitory interaction is displaced by interaction with Pnt on the ato-RE. Other possibilities include direct interaction between Notch and EGFR pathways farther upstream than Pnt and Ato and the activation of yan expression by Notch signaling (zur Lage, 2004).

Autoregulation of ac and sc is relatively simple in the sense that a single autoregulatory enhancer functions in many or all of the locations in which these genes are expressed. In contrast, by requiring Pnt, ato-RE activity is limited to the subset of areas of ato expression in which recruitment signaling occurs. Such spatial restriction of autoregulatory enhancer activity appears to be an important part of ato regulation, since in addition to the ato-RE, the gene is proposed to have a number of distinct autoregulatory enhancers, with different ones required in different locations. Presumably, the autoregulatory action of each of these enhancers is contingent on spatially restricted factor(s) equivalent to Pnt. It will be important to find out what these factors are and whether they too interact directly with Ato/Da proteins at their respective binding sites (zur Lage, 2004).

Direct control of neurogenesis by selector factors in the fly eye: regulation of atonal by Ey and So

During eye development, the selector factors of the Eyeless/Pax6 or Retinal Determination (RD) network control specification of organ-type whereas the bHLH-type proneural factor Atonal drives neurogenesis. Although significant progress has been made in dissecting the acquisition of 'eye identity' at the transcriptional level, the molecular mechanisms underlying the progression from neuronal progenitor to differentiating neuron remain unclear. A recently proposed model for the integration of organ specification and neurogenesis hypothesizes that atonal expression in the eye is RD-network-independent and that Eyeless works in parallel or downstream of atonal to modify the neurogenetic program. This study shows that distinct cis-regulatory elements control atonal expression specifically in the eye and that the RD factors Eyeless and Sine oculis function as direct regulators. These transcription factors interact in vitro and indirect evidence is provided that this interaction may be required in vivo. The subordination of neurogenesis to the RD pathway in the eye provides a direct mechanism for the coordination of neurogenesis and tissue specification during sensory organ formation (Zhang, 2006).

This study found that regulatory elements controlling the early phase of ato expression in the eye lie within a 1.2 kb region located 3.1 kb downstream of the ato transcription unit. The early phase of ato transcription results from the integration of multiple regulatory inputs through separate cis-regulatory modules present within the 1.2 kb region (Zhang, 2006).

Cis-regulatory elements essential for gene activation map to the last 348 bp of the 1.2 kb region and include the So- and Ey-binding sites. Interestingly, the 348 bp region contains two relatively large (A1=99 bp and A2=140 bp) DNA sequences that are highly conserved from D. melanogaster to D. virilis. Based on this observation, constructs were generated containing only A1 or A2. However, neither region alone was sufficient to drive the stripe of reporter gene expression in the eye disc. Based on these results, it is conclude that the 348 bp region constitutes a 'core' or 'minimal' enhancer region for the transcriptional activation of ato in eye progenitor cells. Other factors undoubtedly bind to sequences within A1-A2 and regulate gene expression as neither A1 nor A2 alone are sufficient to drive expression in the eye disc. Genetic evidence suggests that signaling by the Bmp4-type factor Decapentaplegic (Dpp) also contributes to ato activation and two putative binding sites for Mad (a transcription factor shown to activate Dpp pathway targets) appear to be required for ato expression in all discs. However, a Mad consensus site present in the A2 box does not correspond to either of the two elements previously identified . Moreover, both the previously identified sites lie within the L fragment well upstream of the M'-M" interval containing the eye-disc enhancers. Future analyses of 3' enhancer-promoter interactions may resolve this issue (Zhang, 2006).

Separate cis-regulatory elements located within the conserved DNA regions IC1 and IC2 (IC1=88 bp and IC2=133 bp) control initial clusters formation. This feature of ato expression has been shown to require Notch (N) function. Sequence analysis of the IC1-IC2 region does identify a binding site for the effector of N signaling Suppressor of Hairless [Su(H)]. However, contradictory reports have been published on how Notch controls ato expression. Sun and colleagues found that transcription of ato is uniformly upregulated upon inactivation of Notch in Nts1 mutant discs. By contrast, early Ato protein expression is severely reduced in null Notch mutant clones . Since these experiments made use of different genetic reagents, it is difficult to interpret these results. Notch signaling may independently regulate ato expression at the mRNA and protein levels. Alternatively, the source of the discrepancy may lie in the use of different alleles, one hypomorphic (Nts1), and the other null (N54l9) (Zhang, 2006).

Lastly, activation of the 3'ato348gal reporter (core element) occurs prematurely as compared with endogenous ato. The 3'ato348gal mRNA is also found in cells lying just anterior to the proneural domain. Eye progenitors from this region are at a developmental stage referred to as pre-proneural and are characterized by the expression of the transcription factor Hairy (H) in addition to RD proteins. In the absence of Hairy and its partner Extra Macrochaetae, neurogenesis begins precociously within the eye disc. Thus, Hairy contributes to the downregulation of ato expression and prevents precocious neurogenesis. Activation of the reporters 3'ato348gal and 3'ato488gal (but not 3'ato1.2gal or 3'ato1.2-Δ298gal) in pre-proneural cells suggests that cis-elements mediating anterior repression lie within the 1.2 kb DNA fragment but outside the IC and A boxes. Although a search for canonical Hairy-binding sites does not identify potential regulatory elements, additional short stretches of evolutionarily conserved DNA are present and may contribute to this and/or other aspects of ato regulation (Zhang, 2006).

Over the last few years, Ey and So have been shown to play a crucial role in the deployment and maintenance of the RD network by directly regulating the transcription of several eye-specification genes [ey, so, eya, dachshund (dac) and optix]. However, little is known about downstream targets of the RD cascade. Although So also activates the post-MF expression of hedgehog and lozenge, this gene regulation is likely to reflect the late, differentiation-related functions of So. Thus, it is unclear how the RD factors induce eye formation and what aspects of the morphogenetic program they control directly (Zhang, 2006).

The results strongly suggest that the transcription factors Ey and So control activation of ato expression. This is the first example of a gene required during eye morphogenesis that is directly regulated by the RD network. The direct control of ato by Ey and So is a likely reason why ectopic eye induction by Eya+So or Dac depends on the activation of their upstream regulator ey. Other downstream targets may also be similarly controlled by multiple RD factors (Zhang, 2006).

The in vitro and in vivo evidence presented in this study also suggest that Ey and So may form a complex when bound to the adjacent cis-regulatory sites in the 3'ato core element. Together with the previously reported interactions of Eya-So and Eya-Dac, this finding raises the possibility that additional multimeric complexes involving several RD factors may also be involved in driving the transcriptional program for eye development. The observation that normal eye development is severely disrupted when one or another RD factor is over-expressed suggests that the RD proteins must be present at an appropriate level relative to one another. As all four proteins, Ey, Eya, So and Dac, have now been shown to interact in various combinations, the formation of such complexes and the recruitment of additional shared co-factors are likely to be sensitive to the relative concentration of RD factors present in eye progenitor cells (Zhang, 2006).

The model of gene regulation exemplified by the control of ato transcription provides a strong rationale for the feedback regulatory loops that link late and early RD gene expression. This regulation is likely to play a crucial role in ensuring the presence of appropriate levels of all four RD factors to optimize complex formation and co-regulation of downstream targets (Zhang, 2006).

Current models for the co-ordination of organ identity and neurogenesis in the eye place the Pax6 pathway either upstream of, or in parallel to, the control of neurogenesis. The findings presented in this paper favor the former model. Separate regions have been identified for the regulation of ato transcription in the eye versus other sensory organs (JO and CH). In addition, the presence of Ey- and So-binding sites that are required in vivo for reporter gene activation strongly suggests that endogenous ato expression is directly regulated by these factors. Thus, the RD network does not merely modify sensory organ development within the eye disc, but does, in fact, directly control it. In doing so, it also contributes to the co-ordination of selector and neurogenic inputs required to generate complex sensory structures such as the eye (Zhang, 2006).

Is this regulatory relationship between Ey-So and ato ancestrally derived? That is, was the direct link between ancestral Pax- and Ath-like genes already established in the protosensory organ that gave rise to today's ato-dependent sensory structures? The association of Pax-, Six- and Ath-type factors with sensory perception is not restricted to photic sensation but extends to mechanoreception in diverse organisms including mouse, jellyfish and mollusks. In the jellyfish P. carnea, which lacks eyes but responds to a variety of environmental stimuli including light, expression of a putatively ancestral-like PaxB gene, Six1/2, Six3/6 and atonal-like 1 is associated with neuronal precursors found in the medusa tentacles. Although the studies carried out in more basal metazoa consist mostly of analyses of gene expression and not function, this evidence does suggest that the association of Pax/Six/Ath-type factors and sensory organ development is ancient and may have been retained over more than 600 million years of evolutionary history (Zhang, 2006).

It is possible that the mechanisms of transcriptional regulation uncovered between Pax and Six genes and between Pax/Six and ato may have arisen early during evolution. Such regulatory interactions may have favored the continued association of Pax/Six/Ath as various modifications of their genetic cascades led to the development of more complex and diverse sensory organs. The investigation of ato/Ath gene regulation in other sensory organs and in basal metazoans is likely to clarify the evolutionary relationship among these pathways and the sensory modalities they control (Zhang, 2006).

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

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

In addition to RD factors, Dpp signaling is also known to be involved in eye development although little is known about its role in the activation of the ato 3′ enhancer. This study found that induction of the ato 3′ enhancer by ectopic expression of So and Eya under the 30A-GAL4 driver was limited mainly to specific regions near the A/P compartment boundary where endogenous Dpp is expressed. In addition, co-expression of Dpp with So and Eya led to expansion of ectopic ato 3′ reporter expression, indicating that Dpp signaling can synergize with So and Eya to activate the 2.8PB enhancer. As the 2.8PB enhancer does not contain Mad binding sites, it is unlikely that Dpp signaling regulates 2.8PB expression directly through binding of Mad protein to 2.8PB. It is hypothesized that some of the downstream targets of Dpp signaling may mediate the ability of Dpp signaling to synergize with So and Eya in the activation of the ato 3′ eye enhancer. Interestingly, Dac, a RD factor regulated by Dpp signaling, can also synergize with So and Eya in activating the ato 3′ eye enhancer, raising the possibility that induction of Dac contributes to the ability of dpp to synergize with so and eya in the activation of ato 3′ enhancer. The level of Dac in the posterior of the wing disc is significantly lower than that in the anterior in the absence of Dpp co-expression, while similar levels of Dac in the anterior and the posterior are observed when Dpp is co-expressed. Therefore the difference in the subset of cells induced to activate the ato 3′ enhancer by dpp + so + eya and by dac7c4 + so + eya expression could be in part due to differences in the level of Dac induced by Dpp expression and that reached with the 30A-GAL4 driver. Alternatively, it is possible that Dpp signaling has additional targets that contribute to its synergistic induction of the ato 3′ enhancer with So and Eya (Tanaka-Matakatsu, 2008).

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

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

Onset of atonal expression in Drosophila retinal progenitors involves redundant and synergistic contributions of Ey/Pax6 and So bindings sites within two distant enhancers

Proneural transcription factors drive the generation of specialized neurons during nervous system development, and their dynamic expression pattern is critical to their function. The activation of the proneural gene atonal (ato) in the Drosophila eye disc epithelium represents a critical step in the transition from retinal progenitor cell to developing photoreceptor neuron. This study shows that the onset of ato transcription depends on two distant enhancers that function differently in subsets of retinal progenitor cells. A detailed analysis of the crosstalk between these enhancers identifies a critical role for three binding sites for the Retinal Determination factors Eyeless (Ey) and Sine oculis (So). The study shows how these sites interact to induce ato expression in distinct regions of the eye field and confirms them to be occupied by endogenous Ey and So proteins in vivo. This study suggests that Ey and So operate differently through the same 3' cis-regulatory sites in distinct populations of retinal progenitors (Zhou, 2013).

Transcriptional Regulation

Atonal expression in the eye disc

atonal expression has been analyzed in eye discs which have mutant daughterless clones. In morphogenetic furrow spanning mutant da clones, no or very little ATO portein expression is observed in apical confocal sections. The apical position is normal for photoreceptor cell nuclei at the time of differentiation. Basal focal sections of these same clones reveal that ATO is still present but is is abnormal in two respects: either it is shifted posteriorly or expressed in a wider than normal stripe. R8 expression of ATO within mutant da clones is also abolished. These results demonstrate that loss of da function does not affect the activation of striped ATO expression at the anterior side of the furrow. However, da may only be required for the expression of ATO within R8 cells. Alternatively, da may function in another cellular process within the furrow upon which photoreceptor cell determination depends. Although mutant ato does not affect the expression of early striped DA in the furrow, DA is not found in late third instar and early pupal mutant atonal eye discs (Brown, 1996).

Fas II is required for the control of proneural gene expression. Clusters of cells in the eye-antennal imaginal disc express the achaete proneural gene and give rise to mechanosensory neurons; other clusters of cells express the atonal gene and give rise to ocellar photoreceptor neurons. In FasII loss-of-function mutants, the expression of both proneural genes is absent in certain locations, and as a result the corresponding sensory precursors fail to develop. In FasII gain-of-function conditions, extra sensory structures arise from this same region of the imaginal disc. Mutations in the Abelson tyrosine kinase gene show dominant interactions with FasII mutations, suggesting that Abl and Fas II function in a signaling pathway that controls proneural gene expression (Garcia-Alonso, 1995).

In the Drosophila eye, Notch antagonizes the basic helix-loop-helix (bHLH) protein Atonal, which is required for R8 photoreceptor determination.Analysis of the sensitivity of atonal expression to Notch signaling used a temperature-sensitive Notch allele to monitor either the expression of activated Notch or the ligand Serrate, as well as the expression of the atonal-dependant gene scabrous and the Notch-dependent Enhancer of split genes. The atonal expression pattern evolves from general prepattern expression, through transient intermediate groups to R8 precursor-specific expression. Successive phases of atonal expression differ in sensitivity to Notch. Prepattern expression of atonal is not inhibited. Inhibition begins at the intermediate group stage, corresponding to the period when atonal gene function is required for its own expression. At the transition to R8 cell-specific expression, Notch is activated in all intermediate group cells except the R8 cell precursor. R8 cells remain sensitive to inhibition in columns 0 and 1, but become less sensitive thereafter; non-R8 cells do not require Notch activity to keep atonal expression inactive. Thus, Notch signaling is coupled to atonal repression for only part of the atonal expression pattern. Accordingly, the Enhancer-of-split mdelta protein is expressed reciprocally to atonal at the intermediate group and early R8 stages, but is expressed in other patterns before and after. It is concluded that, in eye development, inhibition by Notch activity is restricted to specific phases of proneural gene expression, beginning when the prepattern decays and is replaced by autoregulation. It is suggested that Notch signaling inhibits atonal autoregulation, but not expression by other mechanisms, and that a transition from prepattern to autoregulation is necessary for patterning neural cell determination. Distinct neural tissues might differ in their proneural prepatterns, but use Notch in a similar mechanism (Baker, 1996).

A subset of Notch functions during Drosophila eye development require Su(H) and the E(spl) gene complex: Notch target atonal

The Notch signaling pathway is involved in many processes where cell fate is decided. Previous work has shown that Notch is required at successive steps during R8 specification in the Drosophila eye. Initially, Notch enhances atonal expression and promotes atonal function. After atonal autoregulation has been established, Notch signaling represses atonal expression during lateral specification. Once ato autoregulation is established, lateral specification starts to limit ato expression to R8 precursor cells (see Progression of the morphogenetic furrow across the eye disc). Thus Notch signaling is required at successive steps during R8 specification, initially to promote neural potential and later to suppress it through lateral specification. Consequently the phenotype of loss of Notch gene function varies with time. If Notch function is removed conditionally once ato expression has been enhanced, supernumerary R8 cells differentiate because lateral specification is affected. If N function is absent from the outset, such as in a clone of cells lacking N, little R8 specification can occur. For this reason clones of N null mutant cells in the eye disc almost completely lack neural differentiation, contrasting with the neurogenic phenotype of null mutant embryos. Using clonal analysis it is shown that Delta, a ligand of Notch, is required along with Notch for both proneural enhancement and lateral specification (Ligoxygakis, 1998).

E(spl) bHLH genes have been shown to be transcriptionally activated as a direct consequence of Notch signaling and, along with the corepressor protein Groucho, to mediate inhibition of proneural genes in the nucleus. In the eye, Notch-dependent expression of mdelta and mgamma accompanies repression of ato expression, suggesting that at least these two of the E(spl) bHLH genes contribute to R8 patterning during lateral specification. In addition, mdelta and perhaps mgamma are also transiently expressed prior to lateral specification, and the m7, m8 and mbeta genes are transcribed in distinct patterns that remain uncharacterized in detail for lack of specific antibodies. Thus, particular E(spl) bHLH proteins might mediate proneural Notch signaling as well as or instead of lateral specification. Clones of cells deleted for portions of the E(spl) complex were used to define its role more precisely. The E(spl)b32.2 deficiency deletes all seven bHLH genes and m4. Partial gro function was supplemented in the experiment by a linked gro + transgene. E(spl)b32.2 gro + homozygous cells display a cell autonomous neurogenic phenotype quite unlike that of N or Dl mutant clones. Antibodies against Boss or Elav proteins each label a much greater number of cells within the clone than in the surrounding wild-type tissue. Some clones were difficult to photograph because neurogenic regions often seem to fold in on themselves and crease the eye disc. Because neurogenesis can still occur, it appears that the proneural function of Notch can proceed without any E(spl)-C bHLH genes, whereas N function in lateral specification is severely impaired. Clones of cells homozygous for E(spl)BX22 are also neurogenic in phenotype. E(spl)BX22 affects gro and the bHLH genes m5, m7 and m8. It follows that gro is also dispensable for the proneural function of Notch, although it is probably required in lateral specification (Ligoxygakis, 1998).

Forced expression experiments were performed to further define the role of particular bHLH proteins. The hairy H10 enhancer trap was used to drive GAL4-dependent transgene expression anterior to and within the morphogenetic furrow. The double homozygote for both h H10 and UASmdelta was expressed during the requirement for ato. In the resulting phenotype, the eyes contain few facets and are greatly reduced in size. Eye imaginal discs contain few ommatidia. The defect is associated with reduction or absence of ato expression in the morphogenetic furrow. These findings indicate that mdelta protein is capable of repressing ato expression, as occurs during lateral specification. Not all E(spl)bHLH proteins repressed ato. Eye disc patterning occurred almost normally in h H10 /h H10;UASm5/UASm5 homozygotes and in h H10 /h H10; UASmbeta/UASmbeta homozygotes. Both of these homozygotes die as pupae without differentiating adult structures. It is concluded that the mdelta protein is qualitatively distinct from m5 and mbeta proteins in its ability to inhibit ato expression (Ligoxygakis, 1998).

Recent studies have identified Suppressor of Hairless as a common component in the Notch signal transduction pathway. Ligand binding (Delta or Serrate) to Notch activates Su(H), which can shuttle between the cytoplasm and the nucleus and act as a transcription factor. Activated Su(H) turns on a number of downstream target genes mediating Notch signaling in lateral specification or inductive processes. In order to investigate the role of Su(H), clones of cells homozygous for an apparent null allele of Su(H) were generated by FLP-mediated recombination. In the eye imaginal disc Su(H)- mutant cells are associated cell autonomously with neural hypertrophy. Many of the ectopic neural cells are R8 photoreceptors, based on expression of the R8- specific protein Boss. It appears that, like the E(spl)-C, Su(H) is required for lateral specification but not for R8 differentiation. To confirm this conclusion ato expression was examined. In wild type, initial broad expression of Ato protein is replaced by R8-specific expression that persists for 6-8 hours (3-4 columns of ommatidia) and then fades. Whereas ato expression begins normally in Su(H) mutant cells, ato expression is maintained in many more R8 cells than in wild type, indicating failure of lateral specification. Expression of ato then fades from Su(H) mutant R8 cells at the same time as from wild-type cells. Thus, like the E(spl)-C, Su(H) is required for lateral specification but not for the proneural function of Notch in the retina. Interestingly, although many extra R8 precursors form in Su(H) mutant clones, not all Su(H) mutant cells maintain ato expression or subsequently express the R8-specific Boss protein. Instead clusters of R8-like cells often seem interspersed with non-R8 neurons. ato expression in wild type first becomes patterned into regular 'intermediate groups' of about ten ato-expressing cells before resolving to individual R8 precursors. These results support the conclusions that initial spacing of intermediate groups is not part of the N-dependent lateral specification process, and so does not depend on E(spl) or Su(H) (Ligoxygakis, 1998).

Proneural enhancement by Notch overcomes Suppressor-of-Hairless repressor function in the developing Drosophila eye

The receptor protein Notch plays a conserved role in restricting neural-fate specification during lateral inhibition. Lateral inhibition requires the Notch intracellular domain to coactivate Su(H)-mediated transcription of the Enhancer-of-split Complex. During Drosophila eye development, Notch plays an additional role in promoting neural fate independent of Su(H) and E(spl)-C, and this finding suggests an alternative mechanism of Notch signal transduction. Genetic mosaics were used to analyze the proneural enhancement pathway. Proneural enhancement involves upregulation of proneural gene expression in single cells that will become neurons. In Drosophila eye development, Notch (N) is required for proneural enhancement in addition to lateral inhibition. The molecular mechanism of proneural enhancement has not been determined. As in lateral inhibition, the metalloprotease Kuzbanian, the EGF repeat 12 region of the Notch extracellular domain, Presenilin, and the Notch intracellular domain are required. By contrast, proneural enhancement becomes constitutive in the absence of Su(H), and this leads to premature differentiation and upregulation of the Atonal and Senseless proteins. Ectopic Notch signaling by Delta expression ahead of the morphogenetic furrow also causes premature differentiation. It is concluded that proneural enhancement and lateral inhibition use similar ligand binding and receptor processing but differ in the nuclear role of Su(H). Prior to Notch signaling, Su(H) represses neural development directly, not indirectly through E(spl)-C. During proneural enhancement, the Notch intracellular domain overcomes the repression of neural differentiation. Later, lateral inhibition restores the repression of neural development by a different mechanism, requiring E(spl)-C transcription. Thus, Notch restricts neurogenesis temporally to a narrow time interval between two modes of repression (Li, 2001).

In the developing eye, lateral inhibition restricts the proneural gene atonal (ato) to individual R8 photoreceptor cells, which found each ommatidium. Earlier, ato must first have reached levels of activity sufficient to sustain expression by autoregulation, in conjunction with its bHLH heterodimer partner encoded by daughterless (da) and with a zinc-finger protein encoded by senseless (sens). Such 'proneural enhancement' depends on N and Dl but not on Su(H) or E(spl)-C. Clones of cells mutant for the E(spl)-C or for Su(H) lead to neural hyperplasia because they lack lateral inhibition, but clones of cells mutant for N or Dl show reduced neural differentiation because they lack proneural enhancement. These divergent phenotypes show that proneural enhancement occurs by a mechanism distinct from that of lateral inhibition (Li, 2001).

Mosaic analysis with Notch pathway mutations have been used to elucidate the mechanism of proneural enhancement. Requirements similar to those of canonical N signaling for processed forms of Dl, Notch EGF repeats 10-12, and proteolytic processing of the N intracellular domain have been found. Proneural enhancement is independent of any Su(H)-mediated gene activation but is mimicked by the complete absence of Su(H) protein, and this indicates that proneural enhancement depends on the disruption of Su(H)-mediated gene repression (Li, 2001).

The phenotypes of other mutations can be compared to the E(spl) or N phenotypes. A neurogenic mutant phenotype indicates a role in lateral inhibition, not in proneural enhancement. A hyponeural phenotype indicates a requirement in proneural enhancement (Li, 2001).

The neurogenic phenotype of the metalloprotease kuz suggests that processed Dl might be important for lateral inhibition and that unprocessed, transmembrane Dl may not be sufficient. It is unknown what form of Dl is required for proneural signaling. Clones mutant for kuz show neural hyperplasia. The distribution of R8 cells labeled by Boss antibody is intermediate between the distributions of clones null for E(spl) and for N. This indicates either partial loss of lateral inhibition or a weak proneural phenotype that still permits some neurogenesis to occur. Ato expression was examined to distinguish these possibilities. In kuz clones, Ato protein appears at the same time as it does in neighboring wild-type regions, but it remains at a low level. Posterior to the furrow, small clusters of R8 cells express Ato at a higher level, but many fewer cells do so than in E(spl) clones. This shows that proneural enhancement is affected in kuz mutant clones, but to a lesser degree than in N null clones, so that more cells go on to take the R8 cell fate. An intermediate phenotype associated with small clusters of R8 cells results in combination with the kuz lateral-inhibition defect. This is consistent with a role for processed Dl in proneural enhancement as well as in lateral inhibition, although it is important to note that kuz might have roles besides Dl processing. Such roles might include other aspects of N function (Li, 2001).

EGF repeats 10-12 bind Dl and are important during lateral inhibition because a glutamic acid-to-valine substitution in EGF repeat 12 in the NM1 mutant is embryonic lethal and neurogenic. Clones of NM1 mutant cells in the eye affect proneural enhancement and lateral inhibition, as does kuz, and this finding indicates that Dl interacts with the EGF repeat 12 region of N for proneural enhancement as well as for lateral inhibition (Li, 2001).

Clones mutant for the psn mutation were examined to test whether the novel proneural pathway requires proteolytic processing of N. Clones of psn exhibit an intermediate phenotype. Small patches of R8 cells differentiate, as in NM1 or kuz clones but unlike in E(spl) clones. Ato expression initiates normally but never elevates to the same levels seen in the wild type. Lateral inhibition is deficient in psn clones as judged by the loss of E(spl) expression [E(spl) mDelta], so the intermediate psn phenotype indicates an effect on proneural enhancement in addition (Li, 2001).

In lateral inhibitory signaling, the processed intracellular domain enters the nucleus. Clones mutant for the NCO mutation were examined to test whether proneural enhancement is also mediated by the released intracellular domain or, alternatively, by other parts of the processed protein. In place of Gln-1865, NCO encodes a termination codon that truncates the N intracellular domain close to the transmembrane domain. Eye clones of NCO almost completely lack R8 cells or other neurons. Ato expression is greatly reduced, and only rare R8 cells form posterior to the furrow. Expression of the Senseless protein, a marker for Ato activity, is also greatly reduced, and this finding confirms the failure to establish high levels of Ato expression and function. These results show that the N intracellular domain is required for proneural enhancement. Similar results were obtained with N60g11, which truncates the intracellular domain carboxy-terminal to the ankyrin/CDC repeats (Li, 2001).

It is noteworthy that the NCO phenotype is 'stronger' than clones of the N protein null, in which occasional patches of neurogenesis are seen. If this is attributed to the dominant-negative effect of the protein encoded by NCO, then residual neurogenesis in N null clones must reflect residual N protein, perhaps persisting from before the mitotic recombination event (Li, 2001).

The N intracellular domain converts nuclear Su(H) protein from a transcriptional repressor into a transcriptional activator during lateral inhibition. What is its role in proneural enhancement? It has been concluded that proneural enhancement does not require Su(H) based on the neurogenic phenotype of Su(H) mutant clones. However, the original Su(H) mutants seem not to have eliminated the Su(H) repressor function. Recently, deletion alleles of the Su(H) gene have been recovered that eliminate all Su(H) function (Li, 2001).

Clones homozygous for the Su(H)Delta47 allele are neurogenic, as described previously for other alleles. In addition, however, Su(H)Delta47 mutant cells differentiate prematurely. Ato expression begin earlier in Su(H)Delta47 clones than in neighboring tissue, and it soon reaches high levels. The senseless gene is expressed in response to ato activity. Senseless is also expressed prematurely in Su(H)Delta47 clones. Daughterless protein is ubiquitous but upregulated in ato-expressing cells of the furrow. It was hard to see premature elevation of Daughterless in Su(H)Delta47 clones, and this must be subtle if it occurs (Li, 2001).

Premature differentiation in Su(H)Delta47 clones might be explained if Su(H) normally antagonizes proneural enhancement. Then, in the total absence of Su(H) protein, Ato would enhance prematurely and initiate eye differentiation. Accelerated differentiation would in turn accelerate the progress of the morphogenetic furrow, induce Atonal expression more anteriorly, and begin the cycle again. To investigate the effect of N signaling on this Su(H) function, Dl was misexpressed ahead of the morphogenetic furrow. A transposon insertion in the hairy gene provided GAL4 protein expression. Ato expression is expanded anteriorly throughout the domain of h expression in hGAL4; UAS-Dl eye discs. The sca gene, which is expressed in response to ato activity, is also expressed more anteriorly in response to ectopic Dl. Neural differentiation begins normally in the most posterior part of hGAL4;UAS-Dl eye discs but becomes progressively disorganized more anteriorly as differentiation accelerates (Li, 2001).

The similiarity between activating N signaling ahead of the morphogenetic furrow and deleting Su(H) indicates that N signaling overcomes repression mediated by Su(H). If Su(H) antagonizes proneural enhancement by activating gene transcription, activating N ahead of the furrow should have released the N intracellular domain, elevated gene transcription, and antagonized morphogenetic furrow progression and differentiation, opposite that of what was observed (Li, 2001).

Different forms or complexes of N intracellular domain might be required to antagonize Su(H)-mediated repression during proneural enhancement from those that coactivate Su(H)-mediated gene transcription. The possible role of bib, mam, and neur in proneural enhancement has not been assessed. The bib gene encodes a transmembrane protein required for lateral inhibition in embryonic neurogenesis. Ommatidia that are mutant for bib contain occasional extra photoreceptor cells, and some ommatidia have multiple R8 cells. Ato expression begins and progresses normally, but posterior to the morphogenetic furrow small clusters of two or three cells, instead of single cells as in the wild type, often retain Ato expression. Sections through the adult retinas often reveal ommatidia with extra photoreceptor cell rhabdomeres, both of the R8/R7 small rhabdomere class and of the larger R1-R6 outer photoreceptor class. Since bib affects lateral inhibition only slightly, it is possible that an equally subtle requirement for bib in proneural enhancement might be undetected in these experiments (Li, 2001).

These findings suggest a model for proneural enhancement. The release of N intracellular domain in response to Dl derepresses genes that are repressed by Su(H). The relevant targets do not require Su(H)-mediated transcriptional activation, so deletion of Su(H) mimics N signaling. The mechanism contrasts with lateral inhibition. N signaling provides N intracellular domain as a coactivator for Su(H), which is essential for the transcription of E(spl)-C. Lateral inhibition cannot proceed in the absence of Su(H) because blocking repression by Su(H) is not sufficient for E(spl)-C transcription (Li, 2001).

The ato gene could be a direct target of proneural enhancement. ato regulatory sequences have been examined for activity control regions, but possible repression sites have not been assessed. Another candidate is daughterless, which encodes a bHLH heterodimer partner of Ato that is required for Ato function in eye development. A third candidate is senseless, a zinc finger protein that enhances and maintains proneural gene expression. Expression of ato and sens is prematurely elevated in the absence of Su(H), which is consistent with regulation by Su(H)-R. However, each might depend on Su(H)-R only indirectly because elevated expression of ato or sens requires the function of all three genes (Li, 2001).

Why does proneural enhancement precede lateral inhibition if both depend on Su(H) and nuclear N intracellular domain? (1) Multiple lines of evidence indicate that proneural enhancement requires less N activation than does lateral inhibition. These include the greater sensitivity of lateral inhibition to the Nts mutation, nonautonomous rescue of proneural enhancement by Dl over distances for which lateral inhibition go unrescued, and neurogenesis in N mutant clones due to undetectably low levels of N protein (which is eliminated by dominant-negative protein from the NCO allele). Therefore proneural enhancement is expected to occur sooner in response to N signaling. (2) The evolving transcriptional regulation of ato changes sensitivity to lateral inhibition over time. Even recombinant N intracellular domain expression does not prevent initial ato expression ahead of the furrow, but ato is exquisitely sensitive later when its expression depends on autoregulation (Li, 2001).

The main result of this study is that neural development in the Drosophila eye depends on two functions of the N intracellular domain in response to ligand binding: (1) N relieves Su(H)-mediated repression to enhance ato expression and function and to permit neurogenesis (proneural enhancement); (2) later, another pathway requires N to coactivate Su(H)-dependent E(spl) transcription (lateral inhibition). No genes or regions of N have yet been found to be required to affect one function but not the other. By means of these two functions stimulated by the same ligand, N signaling coordinates the upregulation of ato in proneural cells and represses ato in cells not specified as neural precursor cells, and N restricts neural patterning to a narrow time interval between two distinct modes of repression (Li, 2001).

Regulation of EGF receptor signaling establishes pattern across the developing Drosophila retina

Regulation of Drosophila EGF receptor (Egfr) activity plays a central role in propagating the evenly spaced array of ommatidia across the developing Drosophila retina. Egfr activity is essential for establishing the first ommatidial cell fate, the R8 photoreceptor neuron. In turn, R8s appear to signal through Rhomboid and Vein to create a patterned array of 'proneural clusters' that contain high levels of phosphorylated ERKA and the bHLH protein Atonal. Secretion by the proneural clusters of Argos represses Egfr activity in less mature regions to create a new pattern of R8s. Propagation of this process anteriorly results in a retina with a precise array of maturing ommatidia (Spencer, 1998).

DERElp is a mutation that has been demonstrated to alter the spacing of ommatidia. It is a hypermorphic (gain-of-function) allele of Egfr. The eyes of DERElp /+ flies are rough and irregular: spacing between ommatidia is uneven and somewhat fewer ommatidia overall are present when compared to wild type. This loss of ommatidia appears to be due to repression of Atonal expression within the MF: the initial stripe of Atonal (Stage 0) is unaffected, but expression is lost in the region where proneural clusters normally form. Remarkably, Atonal expression reappears in a 1-3 cell group (Stages 2, 3), and the majority of R8s still form. In DERElp homozygotes, nearly all ommatidia fail to form, presumably due to a more extensive loss of Atonal. These results suggest that high levels of DER activity can repress Atonal expression and thereby alter the spacing of R8 photoreceptors and developing ommatidia. They also suggest that emergence of the R8 equivalence group may not depend on prior formation of a proneural cluster or Atonal expression within it. To further explore the role of Egfr signaling on R8 specification, an activated form of the downstream target Dras1 was employed. Flies containing Dras1Val12 fused to an inducible heat shock promoter were subjected to a 1 hour heat shock followed by a rest period at room temperature to assess the effects of transient, ubiquitous expression. Within 2 hours after the initiation of Dras1Val12 expression, Atonal expression was strongly upregulated throughout the MF, leaving a broad unpatterned band of Atonal in the region where it is normally partitioned into proneural clusters. This expansion in Atonal results in the production of ectopic R8s, as assessed by Boss expression 10 hours after heat-shock. Boss is an R8- specific protein that begins expression 6-8 hours after cells have left the MF; its ectopic expression 8-10 hours after heat-shock indicates the additional R8s are derived from cells within the MF at the time of heat-shock. Based on incorporation of the nucleotide analog BrdU, the presence of ectopic R8s is not due to cell proliferation within the MF, nor is any alteration in Atonal or Boss expression observed in wild-type flies receiving a similar heat shock regimen. Interestingly, not all cells prove sensitive to R8 induction, suggesting that not all cells within the MF are competent to respond to Ras pathway signaling in this manner. These results indicate that strong Dras1 signaling can upregulate Atonal expression; however, only cells within a restricted zone are competent to respond to increased Atonal and ras pathway signaling by differentiating as R8s. The upregulation of Atonal expression is followed by a 'rebound' downregulation. Loss of Atonal expression is observed 4-6 hours after transient expression of Dras1Val12, leaving only a few cells near the posterior edge of the MF that still retained Atonal. Loss of Atonal is accompanied by an expansion in the expression of two inhibitors of Atonal function, Rough and E(spl), and a stable loss of R8 cells as assessed by Boss expression (see above). The arrest in the addition of new R8s persists for approximately 20 hours before reinitiating. This diminished Atonal staining is similar to that seen in DERElp and argues that strong or chronic ras pathway signaling may induce factors that feed back to shut down endogenous DER/Dras1 activity (Spencer, 1998).

Notchspl is deficient for inductive processes in the eye, and E(spl)D Enhances split by interfering with proneural activity

Eye development in Drosophila involves the Notch signaling pathway at several consecutive steps. At first, Notch signaling is required for stable expression of the proneural gene atonal (ato), thereby maintaining the neural potential of the cells. Subsequently, in a process of lateral inhibition, Notch signaling is necessary to confine neural commitment to individual photoreceptor founder cells. Later on, the successive addition of cells to maturing ommatidia is under Notch control. In contrast to previous assumptions, the recessive Notch allele split (Nspl) specifically involves loss of the early proneural Notch activity in the eye, which is in agreement with bristle defects as well. As a result, fewer cells gain neural potential and fewer ommatidia are founded. Nspl alleles are characterized by a smaller number of ommatidia, which usually contain less than the normal set of photoreceptors. Enhancement of this phenotype by the dominant mutation Enhancer of split [E(spl)D] happens within the remaining proneural cells (in which Ato expression has been abolished). In line with genetic data, this process occurs primarily at the protein level due to altered protein-protein interactions between the aberrant E(spl)D and proneural proteins. Indeeed, in a yeast two-hybrid assay, the mutant M8*, representing the E(spl)D alteration, binds significantly more strongly to proneural proteins, especially Achaete and Atonal. The mutant M8* protein does not interfere with the establishment of high Ato levels within intermediate-group cells. In contrast, m8* transcripts accumulate to a much higher level due to increase of mRNA stability caused by the deletion. Heterodimerization of M8* with other E(spl) bHLH proteins is indistinguishable from that of the wild-type M8 protein. Therefore the Nspl mutation reduces the inductive, proneural activity of N, which is normally required to stabilize expression of the proneural gene atonal. As a consequence fewer intermediate groups arise: these groups serve as reservoirs for future R8 cells. E(spl)D potentiates the deficits of Nspl because in the compromized background the already lowered Ato levels in most presumptive R8 cells now drop below the threshold required to maintain neuronal fate. Nspl is the first Notch mutation known to specifically affect Notch inductive processes during eye development (Nagel, 1999).

Dual role for Hedgehog in the regulation of the proneural gene atonal during ommatidia development

The differentiation of cells in the Drosophila eye is precisely coordinated in time and space. Each ommatidium is founded by a photoreceptor (R8) cell. These R8 founder cells are added in consecutive rows. Within a row, the nascent R8 cells appear in precise locations that lie out of register with the R8 cells in the previous row. The bHLH protein Atonal determines the development of the R8 cells. The expression of atonal is induced shortly before the selection of a new row of R8 cells and is initially detected in a stripe. Subsequently, atonal expression resolves into regularly spaced clusters (proneural clusters) that prefigure the positions of the future R8 cells. The serial induction of atonal expression, and hence the increase in the number of rows of R8 cells, requires Hedgehog function. In addition to this role, Hedgehog signaling is also required to repress atonal expression between the nascent proneural clusters. This repression has not been previously described and appears to be critical for the positioning of Atonal proneural clusters and, therefore, the position of R8 cells. The two temporal responses to Hedgehog are due to direct stimulation of the responding cells by Hedgehog itself (Dominguez, 1999).

The initial expression of ato in the eye discs occurs in a strip of cells anterior to the morphogenetic furrow. The levels of Ato within this stripe vary, with enhanced Ato expression corresponding to the approximate position of proneural clusters. Behind the furrow, the only cells that express ato are the future R8 cells. In mature R8 cells, the expression of ato is repressed. When ato and hh expressions are compared, it appears that the refinement of ato expression occurs in cells close to the hh-expressing cells, whereas the continuous stripe of ato, which is believed to be induced by Hh, is 5-7 ommatidial rows in front of the first row of hh-expressing cells. This observation suggests that Hh acts at a distance to induce ato. Such a long-range action of Hh could either be direct or indirect (relay by a secondary signal) (Dominguez, 1999).

In the eye disc, the Ci protein is expressed dynamically, with the highest levels of Ci protein overlapping with Ato expression. Accordingly, misexpression of high levels of Ci in clones of cells showed that Ci is able to induce Ato. The Ci accumulation in cells ahead of the furrow depends on Hh, because cells lacking smo activity have low uniform levels of Ci. Loss of Hh reception in more posterior regions results in the failure to downregulate Ci levels and consequently mutant cells have inappropriately high Ci protein levels when compared to wild-type neighbors. This indicates that Hh stimulates (at long-range) and inhibits (at short-range) Ci accumulation (Dominguez, 1999).

The regulation of ato by the Hh-signaling pathway was studied further by generating clones of marked cells expressing a membrane-tethered Hh protein tagged with CD2 (Hh-CD2). ato expression in cells that have gained hh was examined. Misexpression of hh-CD2 can either activate (when clones are lying anteriorly) or repress (when they lie adjacent to the furrow) the expression of ato. Repression of ato is autonomous in the hh-CD2 cells, suggesting that Hh may repress ato directly. These observations suggest that Hh is secreted near the advancing furrow: close to the source ato expression is inhibited, further away it is induced. If hh-CD2 is misexpressed, naive cells begin to express ato prematurely and this ectopic ato initiates precocious ommatidial formation. However, slightly later (and within the region of influence of the endogenous hh), misexpression of hh-CD2 results in the premature repression of ato. Thus, cells experiencing the extra Hh exhibit no ato expression while the wild-type neighbors just begin to express ato. This model has been tested by manipulating the reception of the Hh signal using in vivo assays. Genetic evidence shows that Hh is required for both promoting and inhibiting ato expression (Dominguez, 1999).

In the proposed model, the induction of Hh has two effects in the responding cells: (1) as an ato inducing signal, through the activation (by upregulation) of the Zn-finger transcription factor Ci, and (2) as an inhibitory signal, through activation of Rough, to inhibit ato expression in the cells in and behind the furrow. The two responses occur in a cell sequentially, as monitored by ato and rough expression in the wild-type pattern and by analysis of their expression in marked clones. The expression domains of ato, Ci protein and rough and their relationship with Hh supports the model. Ci and rough are activated and expressed, respectively, by Hh in restricted spatial domains across the furrow and their expression either overlaps (in the case of Ci) or is complementary (in the case of rough) with ato, consistent with their respective roles in promoting or inhibiting ato expression (Dominguez, 1999).

ato expression is controlled by two enhancer elements located 5' or 3' to the coding sequences (Sun, 1998). A 3' enhancer directs initial expression in a stripe anterior to the furrow and a distinct 5' enhancer drives expression in the proneural clusters and R8 cells within and posterior to the furrow. The 5' enhancer, but not the 3' enhancer, depends on endogenous ato function. The identification of the factors that activate the 5' enhancer element will require refining the ato regulatory sequences followed by binding studies in vitro and in vivo. One of the factors binding to these ato promoters might be Ci. Preliminary results for the loss of ci in mitotic clones are consistent with Ci acting as a positive transcriptional regulator of ato (M. D. and E. Hafen, unpublished, cited in Dominguez, 1999). During furrow progression, Ci is upregulated in the cells anterior to the furrow and in groups of cells in the furrow that coincide with cells expressing ato. These high levels of Ci are then later downregulated to a low level behind the furrow. Ci is thought to act as a transcriptional factor activating or repressing target genes in a concentration-dependent manner. The transcriptional activator form of Ci is thought to correlate with high levels of full-length Ci protein induced by Hh. This upregulation of Ci proteins by Hh is a conserved feature of Hh signaling in all systems. Therefore it is surprising that in the eye Ci is not upregulated near to the Hh source but only in cells far away. The analysis of Ci distribution in smo3, hh AC and viable fused alleles (where the reception and transduction of the Hh signal is blocked or very reduced) suggests that high levels of Hh protein may inhibit Ci protein levels. Probably this regulation is required to restrict the domain of Ci activation and therefore, the cells that are competent to express ato. Thus, by combining a positive long-range inductive signal with short-range inhibition of Ci, Hh may act to pattern ato expression along the anteroposterior axis and refine the array of R8 cells (Dominguez, 1999 and references).

Progression of the morphogenetic furrow in the Drosophila eye: the roles of Hedgehog, Decapentaplegic and the Raf pathway

During Drosophila eye development, Hedgehog (Hh) protein secreted by maturing photoreceptors directs a wave of differentiation that sweeps anteriorly across the retinal primordium. The crest of this wave is marked by the morphogenetic furrow, a visible indentation that demarcates the boundary between developing photoreceptors located posteriorly and undifferentiated cells located anteriorly. Evidence is presented that Hh controls progression of the furrow by inducing the expression of two downstream signals. The first signal, Decapentaplegic (Dpp), acts at long range on undifferentiated cells anterior to the furrow, causing them to enter a 'pre-proneural' state marked by upregulated expression of the transcription factor Hairy. Acquisition of the pre-proneural state appears essential for all prospective retinal cells to enter the proneural pathway and differentiate as photoreceptors. The second signal, presently unknown, acts at short range and is transduced via activation of the Serine-Threonine kinase Raf. Activation of Raf is both necessary and sufficient to cause pre-proneural cells to become proneural, a transition marked by downregulation of Hairy and upregulation of the proneural activator, Atonal (Ato), which initiates differentiation of the R8 photoreceptor. The R8 photoreceptor then organizes the recruitment of the remaining photoreceptors (R1-R7) through additional rounds of Raf activation in neighboring pre-proneural cells. Dpp signaling is not essential for establishing either the pre-proneural or proneural states, or for progression of the furrow. Instead, Dpp signaling appears to increase the rate of furrow progression by accelerating the transition to the pre-proneural state. In the abnormal situation in which Dpp signaling is blocked, Hh signaling can induce undifferentiated cells to become pre-proneural but does so less efficiently than Dpp, resulting in a retarded rate of furrow progression and the formation of a rudimentary eye (Greenwood, 1999).

Hh, secreted by maturing photoreceptor cells, is normally responsible for inducing cells within and ahead of the morphogenetic furrow to initiate photoreceptor differentiation. Nevertheless, cells that lack Smoothened (Smo) function, and hence the ability to transduce Hh, can form normal ommatidia. These findings suggest that Hh can induce photoreceptor differentiation in Smo-deficient cells through the induction of other signaling molecules in neighboring wild-type tissue. As a first step toward identifying such secondary signals and analyzing their roles, the consequences of creating clones of cells homozygous for smo3, an amorphic mutation, have been examined on two early markers of retinal development, the expression of Ato and Hairy, which are expressed in adjacent dorso-ventral stripes within and anterior to the morphogenetic furrow. Ato expression has two prominent phases in the developing eye. In the first phase, Ato is expressed uniformly in a narrow dorso-ventral swath of cells that demarcates the anterior edge of the furrow. This uniform swath then breaks up into small clusters of Ato expressing cells and resolves into the second phase, a spaced pattern of single Ato expressing cells (the future R8 photoreceptor cells). The first phase of Ato expression is severely reduced or absent in clones of smo3 cells, similar to large clones that lack Hh. However, the second phase of expression still occurs, even though it is displaced posteriorly, indicating that it is delayed. This displacement is more severe in the middle of the clone than along the dorsal and ventral borders, producing a crescent shaped distortion of the line of spaced single cells that express Ato. It is concluded that cells within smo mutant clones can be induced to express Ato even though they cannot receive Hh, provided that they are located near to wild-type cells across the clone border. Equivalent effects have been observed for Hairy expression. Hairy is normally expressed at peak levels in a dorso-ventral stripe positioned immediately anterior to the Ato stripe, but is abruptly downregulated in more posteriorly situated cells. Clones of smo3 cells have only a modest effect on Hairy expression anterior to the furrow, causing a slight, but consistent, posterior displacement of the anterior edge of the stripe. However, they are associated with a pronounced failure to repress Hairy expression in some, but not all, posteriorly situated smo3 cells. As in the case of Ato expression, the exceptional mutant cells that retain the normal downregulation of Hairy are those positioned close to the lateral and posterior borders of the clones. Just within the lateral border, a line of cells is typically observed, one or two cell diameter lengths wide, where Hairy expression is repressed. Along the posterior border, the zone of mutant cells in which Hairy expression is repressed is usually wider (Greenwood, 1999).

These results are interpreted to indicate that (1) Hh normally induces cells to express a secondary signal (or signals) that can activate Ato expression and repress Hairy expression; (2) this signal acts non-autonomously, allowing it to move from wild-type cells where it is induced by Hh to nearby smo3 cells where it regulates Ato and Hairy expression; and (3) the range of this signal is short, restricting its action to only one or two cells across the lateral borders of smo3 mutant clones. A somewhat greater range of action is apparent along the posterior borders of such clones, perhaps because the adjacent wild-type cells were induced by Hh to send this signal at an earlier time than those along the lateral (more anterior) borders of the clone, allowing the signal more time to accumulate to higher levels and to move deeper into mutant tissue (Greenwood, 1999).

To examine how the posterior displacements in Ato and Hairy regulation in smo3 clones influence subsequent ommatidial development, the expression of the protein Elav, a marker of photoreceptor differentiation was examined. Clones of smo3 cells are capable of differentiating as photoreceptors, in agreement with previous findings. However, there is a significant delay. In wild-type tissue, Elav expression initiates immediately posterior to the morphogenetic furrow with the specification of the R8 cell and continues as other photoreceptors are recruited into the ommatidial cluster. In clones of smo3 cells, there is a clear posterior displacement in the onset of photoreceptor differentiation in mutant cells: photoreceptor differentiation is first seen at the posterior, and occasionally lateral, edges of the clone, correlating with the effects of neighboring wild-type tissue on Hairy and Ato expression and indicating a general delay in photoreceptor differentiation. However, as seen in more posteriorly situated clones, most or all of the smo3 tissue eventually differentiates as normally patterned ommatidia. Thus, Hh signal transduction is not autonomously required for presumptive eye cells to express Ato, downregulate Hairy, or differentiate as photoreceptors. This is in contrast to the general requirement for Hh signaling revealed by experiments in which Hh signaling is blocked throughout the entire disc using temperature-sensitive hh mutations. In the latter case, loss of Hh signaling causes a rapid and complete block in photoreceptor differentiation and furrow progression. Hh signaling appears to induce at least two secondary signals that are essential for the normal recruitment of undifferentiated cells to form the R8 photoreceptors. One of them appears to be the short-range signal that can induce Ato expression and repress Hairy in clones of smo minus cells. The second, Decapentaplegic (Dpp), appears to act at longer range to prime cells to receive this short range signal (Greenwood, 1999).

One candidate for a secondary signal, which acts downstream of Hh in the developing retina, is the TGF-beta homolog Dpp. Dpp is induced by Hh just anterior to the morphogenetic furrow. Moreover, experiments in other discs have established that Dpp can act at long range from its source to mediate the organizing activity of Hh on more anteriorly situated tissue. However, previous studies have shown that Dpp signaling is not essential for either photoreceptor differentiation or propagation of the furrow once photoreceptor differentiation initiates at the posterior edge of the eye primordium. These findings challenge the notion that Dpp mediates the organizing activity of Hh in front of the furrow. To test whether Dpp has such an organizing role, two kinds of experiments were performed. In the first, Dpp or activated Thickveins (Tkv), a type I TGFbeta receptor required for all known Dpp activities, was ectopically expressed anterior to the furrow. In the second, Dpp expression or Tkv activity was blocked. The results of these experiments indicate that Dpp signaling is both necessary and sufficient to upregulate Hairy expression anterior to the furrow and to maintain the normal rate of furrow progression, but that it is neither necessary nor sufficient to activate Ato expression and initiate photoreceptor differentiation in more posterior cells (Greenwood, 1999).

The dppblk mutation is associated with a deletion of cis-acting regulatory sequences that are essential for Hh-dependent transcription of dpp in the eye. As a result, Dpp signaling in the eye disc is abolished or severely reduced anterior to the furrow, and the resulting eye is greatly reduced in size in both the dorsal-ventral and antero-posterior axis. Hairy expression in wild-type and dppblk disks were compared, using the upregulation of Cubitus interruptis (Ci), a protein that is stabilized in response to Hh signaling, as a marker of the position at which the furrow should normally form. In wild-type eye discs, Ci accumulates to peak levels in a dorso-ventral stripe of cells just posterior to the stripe of peak Hairy expression, consistent with the finding that Hairy expression is repressed in response to Hh signaling within the furrow, but is activated by Dpp signaling anterior to the furrow. In contrast, the stripe of maximal Hairy expression is displaced posteriorly in dppblk discs relative to the stripe of maximal Ci expression. Moreover, the furrow appears to have moved only a small distance from the posterior edge of the presumptive eye primordium, even in eye discs from mature third instar larvae, consistent with the 'small eye' phenotype observed in the adult. These results indicate that Dpp signaling is normally required to activate high level Hairy expression in a stripe positioned just anterior to the furrow. They also indicate that Dpp signaling is necessary to sustain the normal rate of furrow progression. Finally, they suggest that Dpp signaling influences the response of cells to peak levels of Hh signal transduction: Hairy expression is downregulated in these cells in wild-type discs, but not in dppblk discs (Greenwood, 1999).

It is envisaged that pre-proneural cells are metastable, having a latent proneural capacity that is actively held in check by proneural repressors such as Hairy and Emc. How does activation of Raf precipitate the transition to the proneural state? Because the simultaneous loss of both Hairy and Emc activities causes an expansion of Ato expression similar to that resulting from the expression of activated Raf, it has been suggested that Raf activation may normally induce transition to the proneural state by blocking the expression or activity of these repressors. Consistent with this possibility, Hairy contains potential phosphorylation sites for MAPK, a kinase downstream of Raf in the signaling pathway. Daughterless expression is also upregulated in the furrow and is necessary to maintain Ato expression. Moreover, Daughterless, like Hairy, contains phosphorylation sites for MAPK, raising the possibility that Raf activity may directly potentiate proneural activators at the same time that it downregulates the activities of their repressors. Similar events may also occur in mammalian neural differentiation, as NGF-induced differentiation of the mammalian neuronal cell line PC12 is mediated by the phosphorylation of HES-1, a Hairy related protein (Greenwood, 1999).

A primary role for the epidermal growth factor receptor in ommatidial spacing in the Drosophila eye

The differentiation of regularly spaced structures within an epithelium is a common feature of developmental pattern formation. The regular spacing of ommatidia in the Drosophila eye imaginal disc provides a good model for this phenomenon. The correct spacing of ommatidia is a central event in establishing the precise hexagonal pattern of ommatidia in the Drosophila compound eye. The R8 photoreceptors are the founder cells of each of the ommatidia that comprise the adult eye and are specified by a bHLH transcription factor, Atonal. The epidermal growth factor receptor (Egfr) has a primary function in regulating R8 spacing. The receptor's activation within nascent ommatidia induces the expression of a secreted inhibitor that blocks atonal expression, and therefore ommatidial initiation, in nearby cells. The identity of the secreted inhibitor remains elusive but, contrary to previous suggestions, it has been shown that this inhibitor is not Argos. This Egfr-dependent inhibition acts in parallel to the inhibition of atonal by the secreted protein Scabrous. The activation of the Egfr pathway is dependent on Atonal function via the expression of Rhomboid-1. Therefore, it was concluded that Egfr's role in promoting cell survival is largely independent of its role in photoreceptor recruitment; even when cell death is blocked, most photoreceptors fail to form. Based on these data and those of others, a model is proposed for R8 spacing that comprises a self-organizing network of signaling molecules. This model describes how successive rows of ommatidia form out of phase with each other, leading to the hexagonal array of facets in the compound eye (Baonza, 2001a).

R8 cell spacing is disrupted in clones of cells mutant for Egfr. However, cell death is substantially elevated in these clones, and therefore it is not possible to tell whether the spacing defects are a direct consequence of Egfr loss or are secondary to cell death. To examine this, Egfr- clones were generated in a genetic background in which cell death was blocked in the eye by expressing the baculovirus p35 gene under the control of the eye-specific GMR enhancer. In these clones, R8 cells differentiate but their spacing is still disrupted. This result implies that the Egfr function in spacing is not secondary to cell death. The abnormal spacing is seen first as a failure of Atonal to become modulated into proneural clusters in the furrow; a broad band of fairly uniform Atonal is expressed until just posterior to the furrow. This eventually resolves into isolated Atonal-expressing cells that form a disorganized array. Since atonal expression does ultimately resolve to single cells despite the lack of proneural clusters, it is imagined that lateral inhibition mediated by Notch still occurs in the Egfr- clones (Baonza, 2001a).

The Drosophila Egfr signals principally through the Ras/MAPK signal transduction pathway. The observation that Egfr signaling has a direct role in spacing the proneural clusters in the eye imaginal disc is therefore consistent with the fact that MAPK activity within proneural clusters is necessary for the repression of atonal expression in cells between proneural clusters. The MAPK signal transduction pathway is activated by a wide range of receptor tyrosine kinases; therefore, tests were made to see whether Egfr is responsible for the observed MAPK activation in the furrow. In clones of cells carrying an Egfr null mutation the receptor is autonomously required for all MAPK activation. From this it is concluded that the Egfr is the only RTK that detectably activates MAPK in the morphogenetic furrow (Baonza, 2001a).

Interestingly, clones lacking the Egfr ligand, Spitz, have normal R8 cell spacing and MAPK activation. This finding suggests that another ligand for the receptor may be responsible for this function. A single novel Spitz-like ligand has recently been discovered in the completed Drosophila genome sequence (FlyBase ID FBgn0036744), and it is speculated that this could provide the missing function in R8 cell spacing. Testing this prediction awaits the identification of loss-of-function mutations in the spitz-2 gene (Baonza, 2001a).

The results described above imply that Egfr has a primary function in ommatidial spacing and suggest that it is the only RTK that activates MAPK in the proneural clusters. Since it has been shown that the transcription factor Atonal is also required for this MAPK activation, how Atonal and Egfr activation are related was investigated. One possibility is that Atonal directly activates the expression of rhomboid-1, a principal activator of Egfr signaling, as it does in the embryonic chordotonal organs. Therefore whether ectopic Atonal can activate rhomboid-1 expression was investigated. In wild-type cells, rhomboid-1-lacZ is expressed only in photoreceptors R8, R2 and R5. UAS-atonal was expressed under the control of sevenless-Gal4, which is expressed in all ommatidial cells except R8, R2 and R5. When atonal is thus misexpressed, it was found that rhomboid-1 expression (as detected by rhomboid-1-lacZ) is activated in ectopic photoreceptor cells. Thus, consistent with the model in which the activation of MAPK via Atonal depends on the activation of rhomboid-1 expression, atonal expression can induce rhomboid-1 expression, which in turn activates Egfr (Baonza, 2001a).

In some tissues, the expression of the Egfr activator, Rhomboid-1, is dependent on Egfr signaling itself. This dependency thereby constitutes a positive-feedback loop. If this were the case in the furrow, the Atonal-triggered expression of rhomboid-1 could not initiate Egfr signaling (as it would itself depend on prior signaling). Therefore, the expression of rhomboid-1-lacZ in Egfr- clones was examined. Since both the clone marker and the detector for rhomboid-1 expression are the lacZ gene, both are labeled in the same color. However, the ß-galactosidase that marks the clone is cytoplasmic, whereas the one that indicates rhomboid-1 expression is confined to the nucleus. In this way the rhomboid-1-expressing cells can be distinguished from the Egfr-positive cells; this is particularly obvious in transverse optical sections of the eye disc. Egfr- cells can initiate rhomboid-1 expression, and it is concluded that the initiation of rhomboid-1 expression in the furrow does not require Egfr activity (Baonza, 2001a).

The results described so far indicate that within proneural clusters, Atonal activates the expression of rhomboid-1, which in turn leads to the activation of the Egfr/Ras/MAPK pathway. This MAPK activity in proneural cells leads to a nonautonomous inhibition of atonal expression in the cells between proneural clusters. Scabrous is a secreted protein, expressed within clusters, that is also required for the inhibition of atonal expression between clusters. A possible prediction of the model is that scabrous expression in proneural cluster cells would be activated by Egfr/Ras/MAPK signaling -- in other words, that Scabrous is the inhibitory signal secreted by cluster cells in response to Egfr signaling. To test this, Scabrous expression was analyzed in Egfr- clones by using a monoclonal antibody against the Scabrous protein. Normal levels of Scabrous were observed in Egfr- clones, and this finding implies that Scabrous expression is not dependent on the Egfr pathway. The pattern of Scabrous expression was nevertheless altered, which reflects the abnormal spacing of cells in the furrow in Egfr- clones (Baonza, 2001a).

This result suggests that the inhibitory factor regulated by Egfr signaling works in parallel to Scabrous in repressing atonal between proneural clusters. A consequent prediction is that when both Scabrous and Egfr signaling are removed, the spacing defects in the furrow should be worse than those caused by either mutation alone. Conversely, if the Egfr-dependent inhibition is mediated by Scabrous, the double mutants should have the same phenotype as the single mutants. Complete loss of Scabrous alone causes a relatively mild defect in spacing. Clones doubly mutant for Egfr and scabrous were examined and they have reproducibly more severe spacing defects than do Egfr mutant clones alone. When the cells were stained with anti-Boss to label the R8 cells, this was particularly clear within the morphogenetic furrow; in Egfr- clones the spacing is irregular, but the overall number or R8s is not substantially increased over that of the wild type, while in Egfr-;sca- double-mutant clones, more R8s form in the furrow and typically produce a very closely spaced row of cells that is not seen in the single-mutant clones. This additive effect of removing Egfr and Scabrous supports the notion that they mediate two parallel pathways: each contributes to the inhibition of atonal expression between proneural clusters (Baonza, 2001a).

It has been proposed that the secreted Egfr antagonist, Argos, could be the inhibitor of R8 determination between preclusters. This suggestion has been directly tested in argos loss-of-function clones. The arrangement and spacing of the developing ommatidia are completely normal, even in very large clones induced in a minute background; some examples cover more than half of the eye disc. This result implies that Argos cannot be significantly involved in regulating ommatidial spacing. Consistent with this result, it has previously been shown that whole eyes mutant for eye-specific argos alleles do not have substantially disrupted precluster spacing (Baonza, 2001a).

A fairly simple model can be proposed for how R8 spacing is controlled; this model synthesizes the work of several groups. A key feature is that it is a self-organizing system; once atonal expression is initiated at the posterior of the disc, the pattern spreads across the whole retinal primordium without further input from signals other than those generated by the spacing mechanism itself. The first stage of ommatidial determination is the activation of a broad, uniform band of atonal expression anterior to the morphogenetic furrow. This is initiated at least in part by the secreted protein Hedgehog, which emanates from the more posterior, already differentiating, ommatidia. In the model, this band of Atonal expression becomes modulated by the combined action of two diffusible inhibitors, Scabrous and an unidentified inhibitory factor dependent on Egfr-induced signaling through the MAPK pathway, both of which are dependent on Atonal (Baonza, 2001 and references therein).

atonal expression is upregulated by an autoregulatory loop, just as the proneural clusters become apparent. It is at this point that it is proposed that Scabrous and the Egfr-dependent inhibitory factor act. They diffuse toward the anterior and inhibit atonal expression in those cells closest to the inhibitory source. By this mechanism, only the cells farthest from clusters in the previous row retain atonal expression. This produces the characteristic staggered arrangement of R8s in successive rows. Therefore, the central patterning event in establishing the overall arrangement of the ommatidia is the transformation of uniform Atonal expression into modulated expression, as controlled by a combination of Scabrous and the Egfr-dependent inhibitory signal (Baonza, 2001a).

Once Atonal expression is initially modulated by these inhibitory factors, well-defined proneural clusters are formed by a combination of the same inhibitory signals and the autoregulatory positive feedback loop that maintains and increases atonal expression within the clusters. It is suggested that this autoregulation makes the proneural cluster cells refractory to the inhibitory signals they themselves are producing (Baonza, 2001a).

There is no obvious candidate for either the Egfr-dependent inhibitory factor or for the signaling pathway it uses. It can be inferred, however, that it triggers the expression of the homeodomain protein Rough, since Rough expression is lost in Egfr- clones. Rough is a transcription factor that represses atonal expression and Rough is normally expressed in a complementary pattern to Atonal within the furrow. Rough expression is not affected by the loss of Scabrous, which is consistent with the idea that the Rough-mediated inhibition of atonal expression is instead controlled by the Egfr-dependent inhibitory factor. It was originally suspected that Scabrous, which regulates Notch signaling, could be the inhibitory factor. As described above, coupled with the phenotype of scabrous mutants alone, the results on Scabrous expression and the scabrous, Egfr double mutations imply that this is not the case. Nevertheless the possibility that Notch activity has a role in regulating precluster spacing has not been ruled out; it is clearly involved in the later process of lateral inhibition that inhibits atonal expression in all but one of the proneural clusters, so its function in nonautonomous atonal inhibition is well established. Despite this possibility, current evidence does not provide a convincing link between the Egfr-dependent inhibition and the Notch pathway (Baonza, 2001a).

An alternative Egfr-centered model of R8 spacing has been proposed. In this model, Argos would be the Egfr-dependent diffusible molecule that prevents atonal expression and R8 specification between the proneural clusters. However, R8 specification (as opposed to spacing) occurs normally in the absence of Egfr; an Egfr inhibitor such as Argos therefore would not be expected to prevent R8 determination. This is confirmed by results demonstrating that argos null mutant clones have normal ommatidial spacing, even when they are very large, as well as by data showing that eyes from viable, eye-specific argos mutants (i.e., in which the whole eye is mutant for argos) have reasonably normal ommatidial spacing. Another model attributes the crucial uniformity-breaking step to the secreted protein Hedgehog, which can inhibit atonal expression at high levels while activating it at lower levels. This view is conceptually distinct from that presented here, since the source of the diffusible inhibitor (Hedgehog) is not the proneural clusters but the much farther posterior differentiating ommatidia. According to this model, rough expression would be activated by inhibitory levels of Hedgehog. It is worth noting that this view of ommatidial spacing is not mutually inconsistent with the one presented here; several different pathways may contribute to the patterning of the ommatidial array (Baonza, 2001a).

Atonal influences the final differentiation of an R8 cell as well as its earlier selection. Reducing the amount of Atonal in already selected R8 cells leads to the reduced recruitment of subsequent ommatidial photoreceptors; conversely, overexpression of Atonal in R8 leads to excess recruitment. This result fits well with the proposed model and the observation that Atonal can activate the expression of Rhomboid-1 in R8 cells. During the recruitment phase of ommatidial development (which occurs posterior to the furrow after R8s are selected), Egfr signaling is required for triggering the determination of the non-R8 photoreceptors. This signaling is initiated by Rhomboid-1 and Rhomboid-3, which together allow the release of Spitz, the Egfr-activating ligand. A simple explanation is that the level of Atonal in R8 influences the level of Rhomboid-1 which, in turn, controls photoreceptor recruitment (Baonza, 2001a).

Rhomboid-1 is expressed not only in the R8 cell but also in the next two photoreceptors to be recruited, R2 and R5. The control of this expression can now be fully explained. The evidence in this paper suggests that in R8, rhomboid-1 is regulated by Atonal. In R2 and R5, but not in R8, rhomboid-1 expression has been shown to be under the control of Rough (which has a later role in photoreceptor specification as well as the function in spacing described here) (Baonza, 2001a).

These results emphasize the extraordinary diversity of functions that many proteins carry out in eye development. At least five distinct roles for Egfr have been discovered; Atonal is used first for specifying R8 cells and later for regulating their differentiation; Rough is an inhibitor of atonal in the furrow but in a later incarnation activates Rhomboid-1 expression in R2 and R5 and thereby triggers photoreceptor recruitment; Notch, too, has many functions, not all of which are fully understood. One implication of the repeated use of signaling molecules is that the signals themselves do not specify the outcome of signaling. Instead, the fate of a cell is largely determined by the 'state' of the cell that receives the signal, which can broadly be translated into the complement of transcription factors that a cell is expressing (Baonza, 2001a).

There are aspects of this concept, however, that remain unclear. For example, it has previously been shown that constitutively active Egfr is sufficient to trigger the differentiation of photoreceptors anterior to the morphogenetic furrow, even in the absence of Atonal. It is therefore not understood why Egfr signaling in the proneural clusters leads to the production of a diffusible inhibitor of atonal expression rather than photoreceptor determination. A possible explanation is that cells cannot become specified as photoreceptors by Egfr signaling while they are expressing Atonal. Alternative explanations include different effects caused by different Egfr ligands or by different levels of MAPK activation. More generally, a major goal will now be to understand how the successive Egfr signaling events in the eye fit together; for example, how are the transitions from furrow initiation to proneural cluster spacing to cell recruitment controlled? In the Drosophila oocyte, integrated regulation of multiple Egfr signaling events is a key aspect of developmental progression and coordinated patterning, and it is suspected that such linking of successive signaling events may be a general feature of complex developmental systems (Baonza, 2001a).

Notch signaling and the initiation of neural development in the Drosophila eye

Neural determination in the Drosophila eye occurs progressively. A diffusible signal, Dpp, causes undetermined cells first to adopt a 'pre-proneural' state in which they are primed to start differentiating. A second signal is required to trigger the activation of the transcription factor Atonal, which causes the cells to initiate overt photoreceptor neurone differentiation. Both Dpp and the second signal are dependent on Hedgehog (Hh) signaling. Previous work has shown that the Notch signaling pathway also has a proneural role in the eye (as well as a later, opposite function when it restricts the number of cells becoming photoreceptors -- a process of lateral inhibition). It is not clear how the early proneural role of Notch integrates with the other signaling pathways involved. Evidence suggests that Notch activation by its ligand Delta is the second Hh-dependent signal required for neural determination. Notch activity normally only triggers Atonal expression in cells that have adopted the pre-proneural state induced by Dpp. Notch drives the transition from pre-proneural to proneural by downregulating two repressors of Atonal: Hairy and Extramacrochaetae (Baonza, 2001b).

Loss of Notch signaling leads to a loss of neural differentiation. Cells within clones of a null allele of Notch fail to upregulate Atonal expression from its initial low, uniform level. This implies that Notch signaling is required for the initiation of neural development but not for the first low level expression of Atonal. To examine in detail the role of Notch signaling in promoting neural differentiation, clones of cells expressing the Notch ligand Delta were made and their ability to induce neural differentiation was examined. In the wing disc, similar ectopic expression of Delta in clones induces the activation of Notch signaling within the clone as well as non-autonomously in cells surrounding it (Baonza, 2001b).

Clones were generated using the Gal4/UAS system combined with the Flip-out technique and third instar larval eye discs were labelled with different markers to assess neural development. The phenotype of Delta-expressing clones depends on their position with respect to the morphogenetic furrow. Clones in the anterior part of the disc have no effect unless they are within 12-15 cell diameters of the furrow. Within this zone close to the furrow, Delta induces the ectopic expression of Atonal, both autonomously within the clone and non-autonomously, in cells surrounding the clone. In some of these clones there are also cells ectopically expressing the neural antigen Elav. This indicates that once Atonal expression is activated, the full neural program is initiated. Thus, the primary proneural function of Notch signaling is the activation of Atonal (Baonza, 2001b).

Consistent with the neural-promoting properties of Delta, clones that span the furrow from posterior to anterior cause the anterior displacement of Atonal and Elav expression. This displacement implies that the furrow accelerates as it moves through the clone. In the region of these clones that lies posterior to the furrow, the domain of Atonal expression is expanded and the Atonal-expressing cells are disorganized and more numerous. In this region repression of neural differentiation, visualized with the expression of Elav, is also observed. This later phenotype reflects the function of Notch signaling pathway in preventing neural differentiation posterior to the morphogenetic furrow (Baonza, 2001b).

Similar clones were also produced expressing the alternative Notch ligand, Serrate, and unlike Delta-expressing cells, these clones cause no neural induction ahead of the furrow. Conversely, when posterior to the furrow, Ser-expressing clones behave like those expressing Delta and prevent neural differentiation. This implies that anterior to the furrow, the two Notch ligands are not equivalent in their ability to activate the receptor. The reason for this has not been explored, but it is noted that the Notch glycosyltransferase Fringe, which makes Notch resistant to Serrate, is strongly expressed anterior to the furrow. The inability of Serrate to induce proneural Notch signaling is consistent with previous reports, which show that loss of Serrate caused no effects on eye development (Baonza, 2001b).

These results imply that there is a zone of about 12-15 cell diameters ahead of the morphogenetic furrow, where the activation of Notch signaling by Delta, but not by Serrate, is sufficient to trigger neural fate (Baonza, 2001b).

The progression of the morphogenetic furrow correlates with the modulated expression of the negative regulators of Atonal expression, Emc and Hairy. Hairy is expressed in a broad stripe anterior to the furrow and rapidly switched off in the furrow. Emc protein is present in all cells but the highest levels are present in a dorsoventral stripe of cells anterior to the domain of Hairy expression, whereas the lowest levels are observed in the furrow. Thus, the increase of Atonal expression in the proneural groups within the furrow is associated with the downregulation of both Emc and Hairy. Whether this downregulation of Emc and Hairy is mediated by Notch was tested by analyzing the expression of Emc and Hairy when Notch signaling is blocked and when it is ectopically activated (Baonza, 2001b).

In mitotic clones of the Notch null allele N54/9, the expression of Hairy is displaced posteriorly extending behind the morphogenetic furrow. The consequent ectopic expression of Hairy within the furrow is accompanied by a reduction in Atonal expression: Atonal levels remain at the low level normally observed anterior to the furrow. Similar results were obtained with Delta clones. Reciprocally, when Notch signaling is ectopically activated in clones of Delta-expressing cells, Hairy is downregulated, both within the clone and in the cells immediately surrounding it. In these clones Emc is also downregulated within the clone, although for reasons that are not understood, Emc levels are unusually high in the wild-type cells that border the clone. The downregulation of Emc and Hairy caused by the ectopic expression of Delta correlates with increased expression of Atonal ahead of the furrow. It is concluded from these results that Delta/Notch signaling promotes Atonal activation and neural differentiation by downregulating the repressors Hairy and Emc (Baonza, 2001b).

The most well characterized role of Notch signaling in R8 photoreceptor determination is mediating the process of lateral inhibition, which refines Atonal expression from a small group of cells to a single cell. However, an earlier and opposite role for Notch, this time promoting neural determination, has also been recognized, although how this 'proneural' function integrates with other pathways necessary for neural differentiation has been unclear. In this work, it has been shown that in normal eye development the proneural function of Notch signaling depends on prior Dpp signaling. Emc and Hairy, two negative regulators of Atonal expression, mediate the proneural function of Notch signaling in the eye. Thus, a model is proposed that links the upregulation of Atonal in the proneural groups with the downregulation of Hairy and Emc through the activation of Delta/Notch signaling (Baonza, 2001b).

Thus a model is proposed specifically to integrate proneural Notch signaling into the concept of a progression of cell states, from undetermined to pre-proneural to proneural. Hh in the cells posterior to the morphogenetic furrow activates the expression of Dpp in the furrow. The data support the idea that as Dpp acts at a longer range than Hh, this relays a signal to a zone extending about 15 cells anterior to the furrow, priming these cells for differentiation. This makes cells competent to receive a later signal that upregulates Atonal expression, thereby initiating overt neural differentiation. This second signal is also dependent on Hh, but operates only much closer to the furrow: the evidence implies that it consists of Delta activating Notch signaling. The initial 'pre-proneural' state is molecularly defined by the accumulation of the repressors of atonal transcription Hairy and Emc, as well as by the positive regulator of Atonal, the HLH transcription factor Daughterless. Therefore, although Atonal and Daughterless are both expressed in this pre-proneural zone, neural differentiation is not initiated, as Hairy and Emc ensure that Atonal activity remains below a threshold. The Hh-dependent activation of Delta/Notch signaling triggers the transition from this pre-proneural state to the proneural state by downregulating both Hairy and Emc. This negative regulation of the Atonal repressors is sufficient to allow the accumulation of active Atonal in the proneural groups to a level where R8 determination is initiated (Baonza, 2001b).

Notch can only trigger Atonal upregulation in a zone extending 12-15 cells anterior to the furrow, and this zone is defined as the cells that receive the diffusible factor Dpp, whose source is in the furrow. Dpp acts to define a ‘pre-proneural’ state that prepares cells for the imminent initiation of neural determination. This pre-proneural state is defined as the zone of cells that initiate Hairy and Atonal expression in response to Dpp signaling. A functional definition to this state can be added: all these cells are primed for neural differentiation because all can respond to Notch activation by upregulating Atonal levels (Baonza, 2001b).

Simultaneous loss of Hairy and Emc activity leads to the precocious differentiation of photoreceptors in a competent region ahead of the morphogenetic furrow, a phenotype that resembles that caused by ectopic expression of Delta. In addition, ectopic Notch signaling downregulates Hairy and Emc ahead of the morphogenetic furrow, causing the accumulation of Atonal at high levels; conversely, loss of function of Notch signaling increased the levels of Hairy. It is concluded that Delta/Notch signaling regulates the expression of these negative regulators in the eye. Consistent with this proposal, Emc is also regulated by Notch in the developing wing disc (Baonza, 2001b).

Although Notch signaling negatively regulates both Hairy and Emc, the ectopic expression of Delta does not affect both genes identically. Thus, whereas Hairy is removed both within the clone and in the neighboring cells, Emc is only downregulated autonomously within the clone. This distinction could be an artifact caused by the perdurance of ß-galactosidase. Alternatively, these differences may reflect a different requirement for Notch signaling in the regulation of both genes. Furthermore, the expression pattern of Hairy and Emc is different during the normal progression of the morphogenetic furrow. Hairy is precisely regulated, being expressed only in the cells anterior to the furrow, and is rapidly downregulated in the furrow. This precise regulation is crucial as shown by the ectopic expression of hairy. Emc has a much broader expression pattern in the eye disc, although it shows a similar upregulation followed by downregulation in the zone immediately anterior to the furrow (Baonza, 2001b).

It is also worth pointing out that not only does the expression pattern of Emc and Hairy differ, but their exact mechanism of repression is also distinct. Hairy regulates bHLH proteins by a mechanism of direct DNA binding and transcriptional repression. Emc, however, forms complexes with bHLH proteins, preventing their DNA binding. Thus, Emc can antagonize the proneural function of Atonal by two distinct mechanisms: (1) Emc presumably binds to Atonal, rendering it incapable of activating its targets; (2) Emc controls the levels of Atonal. By analogy to its regulation of two other bHLH transcriptional regulators, Achaete and Scute, it is expected that Emc interferes with the autoregulatory upregulation of atonal expression. This positive autoregulation is an essential component of its accumulation in cells within the morphogenetic furrow. In conclusion, the proneural action of Notch signaling increases Atonal activity by two mechanisms: atonal is transcriptionally upregulated, and at the same time a repressive co-factor is removed. These concerted actions lead to the accumulation of active Atonal and thereby the initiation of neural differentiation (Baonza, 2001b).

Novel function of the class I bHLH protein Daughterless in the negative regulation of proneural gene expression in the Drosophila eye

Two types of basic helix-loop-helix (bHLH) family transcription factor have functions in neurogenesis. Class II bHLH proteins are expressed in tissue-specific patterns, whereas class I proteins are broadly expressed as general cofactors for class II proteins. The Drosophila class I factor Daughterless (Da) is upregulated by Hedgehog (Hh) and Decapentaplegic (Dpp) signalling during retinal neurogenesis. The data suggest that Da is accumulated in the cells surrounding the neuronal precursor cells to repress the proneural gene atonal (ato), thereby generating a single R8 neuron from each proneural cluster. Upregulation of Da depends on Notch signalling, and, in turn, induces the expression of the Enhancer-of-split proteins for the repression of ato. It is proposed that the dual functions of Da--as a proneural and as an anti-proneural factor--are crucial for initial neural patterning in the eye (Lim, 2008).

Da is upregulated in the furrow region. Surprisingly, however, it was found that there are two distinct patterns of Da upregulation. The first pattern is a broad, low-level upregulation in the furrow (hereafter referred to as basal level). The second pattern is a stronger expression of Da (hereafter referred to as high level) selectively in the non-neural cells surrounding the Ato-positive R8 cells between proneural clusters. Tests were perfomed to see whether this previously unrecognized pattern of expression of Da is specific by examining eye discs containing da loss-of-function (LOF) clones. Both the basal and high-level expressions of Da in the furrow were lost in the LOF clones of da3, a null allele, showing the specificity of the pattern of Da expression (Lim, 2008).

The basal level of Da upregulation overlaps with the domain of Ato expression near the furrow, where they function together to regulate neurogenesis. As the furrow progression and expression of Ato are controlled by Hh and Dpp signalling, it was reasoned that regulation of Da expression in the furrow might be linked to these signalling pathways (Lim, 2008).

To test whether Hh signalling is required for the expression of Da, Da expression was examined in hh1 mutant eye discs in which the production of Hh ceases after the mid-third instar stage, resulting in reduced expression of Ato and arrest of furrow progression. The expression of Da was downregulated in hh1 mutant eye discs. LOF clones of smoothened (smo), a crucial component for Hh signal transduction, were generated. Da expression was significantly reduced in smo mutant clones spanning the furrow, suggesting that Hh signalling is required for the expression of Da. However, the expression of Da was not completely eliminated in hh1 mutant eye discs or in smo LOF clones. As Dpp signalling is partly required for the expression of Ato, whether Dpp signalling is also necessary for the expression of Da was tested by analysing LOF clones of mad (mothers against dpp), an essential factor for Dpp signalling transduction. Da expression showed little reduction in mad mutant clones, indicating that Dpp signalling by itself is not essential for Da expression. By contrast, the expression of Da was almost completely abolished in LOF clones of smo and mad double-mutant cells in the furrow region. Thus, the Hh and Dpp signalling pathways are crucial but partly redundant for the expression of Da. It was also found that loss of function of Ato reduced the level of Da expression in the furrow. Therefore, several factors, including Ato, coordinate the accumulation of Da in the furrow (Lim, 2008).

To test whether the upregulation of Da in the furrow has a function in neurogenesis, da3 LOF clones were generated and the effects of da mutation on the expression of Ato and neuronal differentiation were examined. Loss of da resulted in ectopic expansion of Ato expression in the mutant clone, suggesting that Da is crucial for repressing the expression of Ato (Lim, 2008).

Despite ectopic expression of Ato, most of the cells in da LOF mutant clones could not differentiate into photoreceptor cells, as indicated by the lack of neuronal markers such as Senseless (R8 marker) and Elav (pan-neural marker). Hence, the expression of ectopic Ato is insufficient to induce retinal differentiation in the absence of Da. However, local differentiation was occasionally detected near the posterior end of some clones. This might be due to the perdurance of Da in LOF clones, although other possibilities, such as partial non-autonomy or partial independence of photoreceptor differentiation from Da in the posterior region of the eye disc, cannot be excluded (Lim, 2008).

To support the idea that a high level of Da expression is required for the repression of Ato, a temperature-sensitive allele of da (dats) was examined that causes conditional partial loss of function of Da at the restrictive temperature. In dats mutant eye discs, Ato was expressed in several cells rather than a single R8 cell per proneural cluster. In addition, the effects of conditional expression of Da was tested by temperature shifts of heat-shock (hs)-da flies. Ato was repressed by the overexpression of Da after a longer heat shock but not after a shorter heat shock. These observations support the idea that enriched Da expression in the cells surrounding each R8 cell is required for generating a single R8 cell by the inhibition of Ato expression (Lim, 2008).

The expanded expression of Ato in da mutant clones might, in part, be due to the failure of da mutant cells to induce lateral inhibition of Ato expression. It is also possible that Da might be involved in the cell-autonomous repression of Ato expression. To test this possibility, Da was overexpressed in the dorsoventral margin of the eye disc using the optomotor blind (omb)-Gal4 driver. The overexpression of Da downregulated Ato expression in the expression domain of omb. Furthermore, the overexpression of Da in the antenna disc using the dpp-Gal4 driver resulted in Ato repression in the expression domain of dpp. Taken together, these data from LOF and overexpression analyses suggest that the high-level expression of Da is necessary and sufficient for the cell-autonomous repression of Ato during the selection of R8 (Lim, 2008).

Both Da and Notch (N) are essential for the selection of R8 by repressing Ato expression in non-R8 precursors within proneural clusters. Hence, Da might be involved in N-dependent lateral inhibition. Furthermore, the overexpression of ASC proneural factors, together with Da, can synergize with Suppressor of hairless and N to activate the expression of Enhancer-of-split (E(spl)) in cultured cells. Since E(spl) is expressed complementary to the expression of Ato in the same cells expressing a high level of Da, whether Da alone could regulate the expression of E(spl) was tested in vivo. The expression of E(spl) proteins was reduced in da3 mutant cells, showing that Da is required for the expression of E(spl) in vivo. Furthermore, the overexpression of Da with dpp-Gal4 could induce the expression of ectopic E(spl) in the dpp domain of the antenna disc. These results indicate that a high level of Da expression is necessary and sufficient for the activation of E(spl) expression (Lim, 2008).

Since E(spl) is the main mediator of N signalling, Ato repression by a high level of Da might be dependent on the expression of E(spl). To test this possibility, the MARCM method was used to generate E(spl) LOF clones in which the expression of Da is induced by tubulin (tub)-Gal4. Da overexpression in E(spl) LOF clones did not show a significant repression of Ato. Similarly, overexpression of E(spl)mδ in da LOF clones did not show noticeable repression of Ato. These data suggest that both Da and E(spl) are required for positive feedback regulation and for repression of Ato during lateral inhibition. However, it is also possible that other bHLH family genes of the E(spl) complex loci might be required, or that the overexpression of E(spl) or Da by tub-Gal4 in MARCM assays might not be strong enough to repress the expression of ato. By contrast, Da expression by dpp-Gal4 induces the expression of E(spl), even in the proximal sector of the antenna disc where Ato is not expressed. amos, the proneural gene for olfactory sensilla, is not expressed in the antenna disc at this time. Thus, a high level of Da can induce E(spl) in the absence of Ato, although Da might act with other class II proteins to promote the expression of E(spl) (Lim, 2008).

Since N signalling is activated in the same cells surrounding R8 founder neurons, whether Da expression is affected was examined by removing the function of N using a temperature-sensitive allele, Nts. The loss of function of N at the restrictive temperature resulted in several Ato-positive cells per proneural cluster. Furthermore, the transient loss of N activity abolished the high-level of Da expression between the proneural clusters but did not eliminate the basal level of Da expression in the same cells. This suggests that N signalling is essential for the high-level upregulation of Da expression. Since the expression of da is regulated by Hh and Dpp signalling, as well as Ato, it is possible that the regulation of Da by Hh and Dpp might be mediated by Ato-dependent N signalling in the non-R8 precursor cells (Lim, 2008).

To investigate further the role of N signalling in the expression of Da, whether E(spl) proteins mediate the function of N in inducing a high level of Da expression was examined. Loss of E(spl) caused ectopic expression of Ato in E(spl) mutant clones because of the lack of N-mediated lateral inhibition. Interestingly, the high level of Da expression was suppressed, but the basal level of Da expression was still detected in E(spl) mutant clones, as seen in Nts mutant eye discs. Thus, E(spl) is required for the high level but not for the basal level of Da expression. In contrast to da3 LOF mutant cells that fail to differentiate in spite of ectopic Ato expression, E(spl) LOF mutant cells not only expressed ectopic Ato but also differentiated into ectopic photoreceptors. Thus, the basal level of Da expression remaining in E(spl) LOF clones is sufficient for the formation of a functional complex with Ato to induce neural differentiation (Lim, 2008).

On the basis of the above observations, a model is proposed in which Da has dual functions as a proneural and as an anti-proneural factor depending on the expression level during early retinal neurogenesis . The anti-proneural function of Da proposed in this model provides an explanation for the abnormal upregulation of Ato in da mutant cells in the furrow, although the LOF experiments are also consistent with the pre-existing view that Da promotes the function of Ato. In Ato-positive neural precursors, low levels of Da expression are sufficient to form heterodimers with Ato to function as a proneural factor. In neighbouring cells, the N-E(spl) pathway further upregulates the expression of Da, which, in turn, induces more expression of E(spl). This putative feedback regulation might provide a mechanism for more effective lateral inhibition of Ato expression for the selection of R8. Interestingly, Da can form a homodimer and bind to DNA in vitro. Thus, in Ato-negative cells surrounding the R8 precursors, a high level of Da expression might enforce the formation of Da homodimers and/or heterodimers with other unknown bHLH proteins to repress the expression of ato. It would be interesting to see whether mammalian type I bHLH proteins such as E proteins might also be specifically regulated to have distinct developmental functions as seen in the case of Da (Lim, 2008).

Drosophila eyes absent is required for normal cone and pigment cell development

In Drosophila, development of the compound eye is orchestrated by a network of highly conserved transcriptional regulators known as the retinal determination (RD) network. The retinal determination gene eyes absent (eya) is expressed in most cells within the developing eye field, from undifferentiated retinal progenitors to photoreceptor cells whose differentiation begins at the morphogenetic furrow (MF). Loss of eya expression leads to an early block in retinal development, making it impossible to study the role of eya expression during later steps of retinal differentiation. Two new regulatory regions have been developed that control eya expression during retinal development. These two enhancers are necessary to maintain eya expression anterior to the MF (eya-IAM) and in photoreceptors (eya-PSE), respectively. Deleting these enhancers affects developmental events anterior to the MF as well as retinal differentiation posterior to the MF. In line with previous results, reducing eya expression anterior to the MF was found to affect several early steps during early retinal differentiation, including cell cycle arrest and expression of the proneural gene ato. Consistent with previous observations that suggest a role for eya in cell proliferation during early development, deletion of eya-IAM was found to lead to a marked reduction in the size of the adult retinal field. On the other hand, deletion of eya-PSE leads to defects in cone and pigment cell development. In addition it was found that eya expression is necessary to activate expression of the cone cell marker Cut and to regulate levels of the Hedgehog pathway effector Ci. In summary, this study uncovers novel aspects of eya-mediated regulation of eye development. The genetic tools generated in this study will allow for a detailed study of how the RD network regulates key steps in eye formation (Karandikar, 2014, PubMed ID: 25057928).

Scabrous complexes with Notch to mediate boundary formation

The mechanisms that establish and sharpen pattern across epithelia are poorly understood. In the developing nervous system, the first pattern elements appear as 'proneural clusters'. In the morphogenetic furrow of the immature Drosophila retina, proneural clusters emerge in a wave as a patterned array of 6 to 10 cell groups, which are recognizable by expression of Atonal, a basic helix-loop-helix transcription factor that is required to establish and pattern the first cell fate. The establishment and subsequent patterning of Atonal expression requires activity of the signaling transmembrane receptor Notch. In vivo and biochemical evidence is presented that the secreted protein Scabrous associates with Notch, and can stabilize Notch protein at the surface. The result is a regulation of Notch activity that sharpens proneural cluster boundaries and ensures establishment of single pioneer neurons (Powell, 2001).

In the morphogenetic furrow, Atonal's expression can be divided into four steps:(1) it is expressed as a broad, unpatterned stripe; (2) expression is then upregulated into evenly spaced proneural clusters;(3) in these proneural clusters, a 2 to 3 cell 'R8 equivalence group' emerges, and (4) expression narrows to identify a single cell in this group as the R8 photoreceptor neuron, the first cell type of the developing retina. In each step, as cells lose Atonal expression they concurrently gain expression of negative regulators, such as members of the E(spl) (Enhancer of Split) complex. Expression of E(spl) initially requires the presence of Atonal, and is subsequently amplified by the Notch signaling pathway to downregulate proneural bHLH expression and function. E(spl), therefore, represents one reporter of Notch activity (Powell, 2001).

Patterning of Atonal and E(spl) expression requires the normal activity of Scabrous, a secreted fibrinogen-related protein with a potential for association with components of the extracellular matrix. In the retina, Scabrous protein first appears in the proneural clusters, mirroring Atonal expression by narrowing to the R8 equivalence group, and eventually R8 alone. This expression is dependent on Atonal activity, which indicates that the scabrous locus may be a direct target of Atonal (Powell, 2001).

Genotypically null scaBP2 proneural clusters are poorly spaced with poorly defined borders. Broadened E(spl) expression throughout much of the proneural cluster region is one potential cause of this imprecision, suggesting that Notch activity is altered in sca BP2 mutants. These observations suggest that initial broad, low-level Atonal expression activates broad, low-level E(spl) expression, and that Scabrous is required to refine the complementary Atonal and E(spl) expression in the proneural cluster region -- events also associated with Notch activity (Powell, 2001).

A 1 hour pulse of ectopic Scabrous results in rapid loss of Atonal within 2 h; E(spl) shows low, diffuse expression that is lost within 4 h. This ectopic expression of Scabrous leads to aberrant patterning of R8s in a manner similar to that of the phenotypes observed in scabrous loss-of-function alleles. The loss of the initial broad stripe of Atonal, a Notch-dependent step, suggests that ectopic Scabrous can lead to a disruption of Notch function, a result consistent with overexpression studies in the Drosophila wing (Powell, 2001).

Nuclear translocation of activated MAP kinase is developmentally regulated in the developing Drosophila eye

In proneural groups of cells in the morphogenetic furrow of the developing Drosophila eye phosphorylated MAPK antigen is held in the cytoplasm for hours. A reagent has been developed to detect nuclear MAPK non-antigenically. Reported here is the use of this reagent, confirming that MAPK nuclear translocation is regulated by a second mechanism in addition to phosphorylation. This 'cytoplasmic hold' of activated MAPK has not been observed in cell culture systems. MAPK cytoplasmic hold has an essential function in vivo: if it is overcome, developmental patterning in the furrow is disrupted (Kumar, 2003).

Consistent with the suggestion that the MAPK pathway signal is blocked in intermediate proneural groups (phase 1), reporter gene expression is seen only later, in the future R8 cells in the last two columns of Atonal expression and later in cells as they are recruited into the assembling ommatidia (phase 2). This is consistent with normal Egfr pathway activity in the downregulation of Atonal at the end of phase 1 and then again at later stages (phase 2), when successive Ras pathway signals recruit each cell type that follows the founding R8 cell (which is specified by other means). Although no MAPK nuclear translocation was detected early in the furrow (in the intermediate groups) with either of the reagents (the transcription factor fusion or the epitope tag) the possibility that there is some lower level of nuclear MAPK at these stages that is below the limits of the two detection systems cannot be formally excluded. Similarly, the possibility that there are cytoplasmic functions for phosphorylated MAPK at these stages cannot be excluded. However, the results are consistent with two Egfr pathway functions in the developing R8 cells at this time (as Atonal expression ends): for the maintained expression of differentiation markers (Boss and Elav) and for later cell survival (Kumar, 2003).

Furthermore, through the addition of a constitutive NLS, MAPK is driven into the nuclei of cells in phase 1, thus overcoming MAPK cytoplasmic hold. This results in a rapid downregulation of Atonal and the precocious neural differentiation of the R8 photoreceptors. Taken together with the observation of the first nuclear translocation of MAPK at the time that Atonal is downregulated in normal development, it is suggested that the Egfr/Ras pathway may normally contribute to the end of phase 1 by ending Atonal expression (Kumar, 2003).

Others have suggested that Egfr pathway loss-of-function normally functions to downregulate Atonal expression at and after the intermediate group stage. This was not observed using the conditional mutation, Egfrtsla; however, since this experiment did not include a clone boundary a short delay (such as one column) could not have been detected. It may be that the pathway functions at this point through a much lower level signal (below the level of detection of the reagents) or it may be that it functions through cytoplasmic targets of phosphorylated MAPK (Kumar, 2003 and references therein).

What is the developmental purpose of this block of MAPK signaling in the furrow? Anterior to the furrow, MAPK cytoplasmic hold cannot function, or it would prevent the MAPK signaling required for the G1/S transition and thus halt cell proliferation. Perhaps this is one reason why all cells in phase 1 exit the cell cycle. However, new data suggest that the Egfr pathway does function in the furrow to maintain G1 arrest (visualized as increased cyclin B expression). This could be mediated through some low level of nuclear MAPK at this stage or possibly through cytoplasmic targets for MAPK signaling. However, although cyclin B expression is elevated posterior to the furrow in all cells other than R8 in Egfr pathway loss-of-function mutant clones, the leading edge of cyclin B expression does not advance (it is not expressed earlier). Thus, it may be that the role of Egfr pathway signals in maintaining G1 arrest is later than the end of Atonal expression (i.e., in phase 2, not in phase 1) (Kumar, 2003).

It is suggested that the founder cells have a special developmental function to fulfill in phase 1: they must act as organizing centers for lateral inhibition to produce the spaced pattern of R8 cells. If the founder cells did not inhibit their neighbors most or all cells in phase 1 might rapidly differentiate as photoreceptors, resulting in disorder. This type of disorder is observed when the Egfr/MAPK pathway is ectopically activated ahead of the furrow, when photoreceptor differentiation becomes independent of Atonal and R8 fate. The model may also explain the loss of ommatidia seen in EgfrElp gain-of-function mutants. Excess Ras/MAPK pathway signals may reduce Atonal expression and thus the number of R8 founder cells. The results led to a prediction that G1 cell-cycle arrest may be found in other cases in which a subset of progenitor cells is selected by lateral inhibition through active Notch pathway signaling and repression of Ras/MAPK signaling. In summary, the data are consistent with a model in which Egfr/MAPK signaling functions in ommatidial assembly but not directly in founder cell specification. It is proposed that MAPK cytoplasmic hold is restricted to the morphogenetic furrow, and does not happen anterior to the furrow (the proliferative phase) or posterior (during ommatidial assembly, or phase 2). It appears to be coincident with the regulated G1 arrest seen in the furrow (Kumar, 2003).

Atonal expression in Bolwig's organ

Bolwig's organ formation and atonal expression are controlled by the concerted function of hedgehog, eyes absent and sine oculis. Bolwig's organ primordium is first detected as a cluster of about 14 Atonal-positive cells at the posterior edge of the ocular segment in embryos and hence, atonal expression may define the region from which a few Atonal-positive founder cells (future primary photoreceptor cells) are generated by lateral specification. In Bolwig's organ development, neural differentiation precedes photoreceptor specification, since Elav, a neuron-specific antigen, whose expression is under the control of atonal, is expressed in virtually all early-Atonal-positive cells prior to the establishment of founder cells. Neither Atonal expression nor Bolwig's organ formation occurs in the absence of hedgehog, eyes absent or sine oculis activity. Genetic and histochemical analyses indicate that (1) the required Hedgehog signals derive from the ocular segment, (2) Eyes absent and Sine oculis act downstream of or in parallel with Hedgehog signaling and (3) the Hedgehog signaling pathway required for Bolwig's organ development is a new type and lacks Fused kinase and Cubitus interruptus as downstream components (Suzuki, 2000).

Prior to the establishment of Bolwig's organ founder cells, virtually all Bolwig's organ precursor (BOP) cells acquire neural fate. The earliest event of Bolwig's organ development may be ato expression at mid stage 10: this early ato expression defines the area of BOP. Early ato expression is regulated by the concerted action of Eya, So and Hh signals. During late stage 10 and early stage 11, Elav, a neuron-specific antigen, begins to be expressed in almost all BOP cells. This elav expression is likely to be regulated by Ato activity, since (1) BOP elav expression is reduced extensively in ato mutants and (2) the number of Elav-positive cells at stage 11 and Kr-positive Bolwig's organ neurons at stage 16 considerably increases upon ato misexpression. As with ato expression, eya, so and hh activity is essential for elav expression in BOP cells. In contrast to elav expression, ato expression is restricted to three founder cells at stage 12: this late ato expression disappears by the end of stage 12. Photoreceptor specification of putative founder cells may start during stage 11, since at late stage 11, 2-3 cells in a cluster start expressing Kr and/or Glass, which are specific markers for larval photoreceptors. Cells expressing Kr and/or Glass increase during stages 12-13 and all 12 photoreceptors express both Kr and Glass by stage 16. Similarly, a peripheral nervous system-specific signal recognized by mAb22C10 appears in a few BOP cells at stage 12 and becomes recognizable in all Bolwig's neurons by stage 16. Late ato expression may also be essential for normal photoreceptor formation. In ato mutants, neither Kr-positive nor mAb22C10-positive cells can be seen in stage-16 future larval eyes (Suzuki, 2000).

Hh signaling in Drosophila has been extensively analyzed in embryonic trunk segments and imaginal discs, and many common downstream components have been identified. In both systems, Ci activates target genes in response to hh signal. The pathway lying above Ci is thought to be bifurcated. Although the mechanism by which Smo passes signals to PKA or Fu remains unclear, PKA and Fu act under the direction of the putative Ptc/Smo receptor complex in parallel with each other. Ci is directly phosphorylated by PKA and cleaved to become a repressor, while Fu phosphorylates full-length Ci to make it a labile activator. Bolwig's organ development is regulated through the concerted action of Eya, So and Hh signaling. Although these three factors are essential for ato expression at stage 10, the earliest event in Bolwig's organ development so far identified, whether or not, they directly regulate other events of Bolwig's organ development remains to be clarified. Defects in stage-10 ato expression in BOP mutant for eya, so or hh are partially rescued by misexpression of the corresponding gene at late stage 9 and stage 10, suggesting that ato is a direct target of the putative Eya/So complex and an activator downstream of Hh signaling involved in Bolwig's organ development. Ci is expressed in BOP cells at stage 10. Fu is also ubiquitously expressed in the ectodermal head at stage 10. However, Fu and Ci are not involved in Hh signaling for Bolwig's organ development. Epistasis analysis has indicated that Eya and So act either downstream of or in parallel with Hh/Ptc signaling. Should the latter be the case, Hh signal must activate an unknown transcription activator (X) to positively regulate ato. This is the first demonstration of Hh signaling independent of both Fu and Ci. Hh signaling required for ocular-segment hh expression lacks Ci but not Fu, and this would imply the presence of another type of Hh signaling. The Hh signaling pathway required for ptc expression in cells posteroventral to Hh expression domains in the trunk has recently been shown to lack Fu but not Ci and consequently there must be considerable diversity in the downstream pathway of Hh signaling in Drosophila (Suzuki, 2000).

Atonal expression in the antennal disc

The bHLH transcription factor Atonal is sufficient for specification of one of the three subsets of olfactory sense organs on the Drosophila antenna. Misexpression of Atonal in all sensory precursors in the antennal disc results in their conversion to coeloconic sensilla. The mechanism by which specific sense organ fate is triggered remains unclear. The homeodomain transcription factor Cut, which acts in the choice of chordotonal-external sense organ does not play a role in olfactory sense organ development. The expression of atonal in specific domains of the antennal disc is regulated by an interplay of the patterning genes, Hedgehog and Wingless, and Drosophila epidermal growth factor receptor pathway (Jhaveri, 2000b).

neuralized-GalA101 was crossed to UAS-atonal in order to drive expression in all sensory precursors. Changes in the pattern of antennal sense organs are relatively easy to detect because of the rather invariant organization in the wild type. Trichoid sensilla and basiconic sensilla are located on the lateral and medial surfaces of the antennal surface respectively. The coeloconic sensilla occupy an intermediate region interspersed with the other two types of sense organs. Misexpression of ato using neu-GalA101 as a 'driver' results in a significant reduction of basiconica and trichoidea and a concomitant increase in coeloconic sensilla. It is believed that 'ectopic coeloconica' represent conversions of other sense organs because they are observed within the trichoid rich and basiconica rich domains. Some sensilla, which could not be definitively classified, have also been observed, although they possess some of the characteristics of the coeloconica. These results demonstrate that misexpression of ato in addition to the endogenous levels of proneural genes can subvert the fate of sense organs towards coeloconica. amos is the proneural gene for the trichoid and basiconic sensilla. Thus high levels of Ato can compete with normal amos function in sense organ type selection (Jhaveri, 2000b).

Pattern formation in the epidermis is regulated by a hierarchy of genes; the patterning genes -- engrailed, hh, dpp and wg -- specify co-ordinates of the disc and are expected to influence expression of prepatterning genes. Lz is a putative prepatterning gene in the antennal disc and has been shown to regulate expression of amos; genes regulating ato in the antenna are as yet unclear. The olfactory sense organs are located in a distinct pattern across the antenna, thus requiring co-ordinated control of the different proneural genes. Expression of a dominant negative Egfr results in a large number of ectopic atonal+ cells. Phosphorylated MAP kinase expression does not co-localize in ato+ cells and in the third instar antennal disc phosphorylated MAP kinase and Ato immunoreactivity occupy mutually exclusive domains. Hence the expression pattern of these two molecules is consistent with the model that signaling through the Ras/MAP kinase pathway acts to suppress ato expression. It is therefore proposed that signaling through Egfr and Ras/MAPK cascades plays a key role in linking positional information to the expression of proneural genes (Jhaveri, 2000b).

During Drosophila eye development, Hh and Dpp are required to initiate photoreceptors at the furrow while Wg inhibits differentiation at the lateral margins. Wg appears to act by antagonizing signaling through the Egfr pathway. In contrast, Hh may directly regulate ato expression, its diffusion ahead of the morphogenetic furrow turns on Ato, while higher levels behind the furrow lead to its downregulation. There is however evidence that Hh can also influence Egfr signaling since Ci has been shown to activate Mapk through the Egfr ligand Vein (Jhaveri, 2000b and reference therein).

During antennal development, suppression of Egfr activity by dominant negative strategies leads to ectopic ato expression. A possible candidate to link Egfr signaling and Ato is the homeodomain molecule Rough which plays such a role during photoreceptor development. Results from DN-Egfr misexpression, and the observation that Mapk levels are high in domains where Ato-expressing cells are absent leads the authors to suggest that signaling through Ras/MAPK determines the pattern of progenitor cells for coeloconic sensilla. This poses the question of how Egfr activation is regulated across the antennal disc (Jhaveri, 2000b).

Loss-of-function experiments have shown that Hh function is required for ato expression; misexpression analysis has demonstrated that low Hh levels turn on Ato, while higher levels suppress it. However the normal expression pattern of Hh in the antennal disc makes it unlikely that it could directly act to induce Ato in all domains. Ectopic expression of wg in the antennal disc has been shown to lead to induction of ato. Hh appears to act non-autonomously to induce Ato in neighboring cells; UAS-hh transgene produces the secreted form of Hh protein. The data suggests a dosage sensitivity in the regulation of ato by Hh. High levels of Hh produced within cells of the clone suppress ato expression, while low levels resulting from diffusion of protein outside the clone induce it. It is thus proposed that both Hh and Wg together pattern ato expression domains in the disc. The diffusible nature of Hh could allow its action at a long range to induce expression of ato as well as wg. Since Wg is also a secreted molecule, and can regulate ato through the Egfr cascade, it could serve to extend the range of Hh effect across the disc (Jhaveri, 2000b).

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

The bHLH transcription factor encoding gene, ato, is expressed in a ring in presumptive a2, but restricted to small spots in the dorsal leg disc. ato is required for the formation of most chordotonal organs in the fly. In the antenna, ato is required for formation of Johnston's organ (JO), a complex sense organ composed of a large number of chordotonal organs that is used for sensing acoustic vibrations transmitted from the arista through a3 (Dong, 2002).

The expression of ato is required for the formation of the JO. The JO is a structure unique to the antenna and is required to sense sound vibrations transmitted from the arista. ato function is generally associated with neuronal differentiation, so it is interesting that cuticular defects are associated with ato null antennae. It may be that formation of the JO is required for the normal morphology of the a2/a3 joint. The circular outline of the a2/a3 joint is lost in hth and Dll loss-of-function mutants, but is present in ss null mutants. Consistent with this, the antennal expression of ato is lost in hth null clones and in Dll hypomorphs, but persists in the ss null antenna discs. Thus although ss null mutants exhibit cuticular defects in a2 and a3, the a2/a3 joint to which the JO is attached is present. It is noted that the Dll hypomorphic combination used, DllGAL4/Dll3, does not lead to loss of a2. Thus the absence of ato expression in these antennae is not due to death of the cells that would normally express it (Dong, 2002).

Since ato is expressed within a subset of the sal/salr domain and is activated later than sal and salr in the antenna, tests were performed to see whether Dll and hth activate ato via sal/salr. No detectable reduction of ato expression is found in a2 either in Df(2L)32FP-5 clones or in salFCK–25/Df(2L)32FP-5 transheterozygous animals. This allelic combination lacks detectable sal and salr expression in the antenna, but retains sal and salr expression in the eye. The normal expression of ato in the antennae of these mutants suggests that the activation of ato expression by Dll and hth is independent of sal/salr. Antennal sal/salr expression is also unaffected in ato null imaginal discs. Therefore, sal/salr and ato are required in parallel for development of antennae that are functional in audition (Dong, 2002).

sal and salr, like ato, are required for normal auditory functions. Since both Dll and hth are required for the antennal expression of ato and sal, Dll and hth mutant antennae are also hearing defective. In contrast, ss null antennae exhibit normal expression of both ato and sal and normal morphology of the a2/a3 joint, leading to the idea that ss mutants are likely to be functional in audition (Dong, 2002).

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

In third instar imaginal discs, coexpression of Dll and Hth activates sal/salr and ato in a2 where they, in turn, are needed for JO development. The expression of ato is required for the formation of the JO and the a2/a3 joint to which it is attached. Although sal and salr are not required for the expression of ato, the a2/a3 joint is lost in the sal/salr null antenna. It is expected that this leads to improper formation of the JO, although it is also possible that defects in a2/a3 joint formation preclude JO differentiation. In addition, because sal is not lost in ato null antennae, it is concluded that sal/salr and ato are required in Drosophila parallel for proper formation of the JO. Furthermore, in the sal/salr null antenna, a3 cannot freely rotate within a2. This rotation is necessary for transmission of sound vibrations from the arista to the JO. Taken together, these findings implicate sal/salr in Drosophila audition. Interestingly, mutations associated with the human homolog of sal, SALL1 cause the human autosomal dominant developmental disorder, Townes-Brocks Syndrome (TBS). Auditory defects are also associated with the human genetic disorder, Split Hand/Split Foot Malformation (SHFM), and the SHFM1 locus is linked to the Dll homologs, DLX5 and DLX6. The sensorineural hearing defects associated with the Distal-less and spalt genes in both Drosophila and Homo sapiens, in conjunction with a recent finding that atonal functions in mouse as well as fly audition, leads to the proposal that insect and vertebrate hearing share a common evolutionary origin. Further developmental genetic dissection of the Drosophila auditory system should therefore provide additional insights into human ear development and suggest that Drosophila could provide a useful model system for studying both TBS and SHFM (Dong, 2002).

Atonal expression in bristles and sensilla of the wing

If Senseless is required for proneural expression, ectopic expression of Sens may induce proneural gene expression. Indeed, ectopic expression of Sens using the dpp-GAL4 driver causes robust expression of Sens in the expected wing stripe. This expression causes the formation of numerous bristles and sensilla campaniforma in the adult wing in proximity of the third wing vein where dpp is normally expressed. Similarly, ectopic expression of Sens in the leg disc causes many supernumerary bristles in the sternopleural area as well as in more distal portions of the leg. Ectopic bristles are observed with all UAS-sens reporters. Some UAS-sens transgenes driven by dpp-GAL4 cause very severe tufting in the adult notum, wings, and legs, and loss of tissues in other portions of imaginal discs, e.g., wing margins and distal leg structures. It is concluded that ectopic expression of Sens is sufficient to initiate ectopic external sensory organ development (Nolo, 2000).

To determine the molecular cascade underlying the formation of the extra external sensory organs, wing discs of UAS-sens; dpp-GAL4 larvae were stained with anti-Scute antibodies. Ectopic Sens expression causes ectopic activation of Scute and Asense. Hence, Sens is able to activate proneural gene expression. This provides a molecular basis for the generation of additional external sensory organs, since ectopic proneural gene expression has previously been shown to be sufficient to induce ectopic PNS organ formation (Nolo, 2000).

If Sens induces proneural gene expression and proneural genes are required for Sens production, a super-additive or synergistic interaction between sens and proneural genes may occur. Therefore, the weakest UAS-sens transgene (C1) was expressed in combination with an UAS-scute and an UAS-atonal transgene under the control of dpp-Gal4. Overexpression of Scute or Atonal alone causes a relatively mild phenotype with relatively few extra bristles. Scute expression induces Sens expression, but the expression levels of Sens are lower than those induced by dpp-Gal4; UAS-sens. Ectopic expression of Sens with the dpp-Gal4 driver causes a stronger phenotype when compared to ectopic expression of Scute or Atonal. However, simultaneous overexpression of Sens and Scute or Atonal causes severe tufting, including in many areas where Scute, Atonal, or Sens, when expressed individually, does not normally cause ectopic bristles. These areas do correspond to areas where the dpp-Gal4 driver has previously been shown to be expressed. It is therefore concluded that sens and the proneural genes can interact in a synergistic fashion (Nolo, 2000).

Drosophila lilliputian is required for proneural gene expression in retinal development

Proper neurogenesis in the developing Drosophila retina requires the regulated expression of the basic helix-loop-helix (bHLH) proneural transcription factors Atonal (Ato) and Daughterless (Da). Factors that control the timing and spatial expression of these bHLH proneural genes in the retina are required for the proper formation and function of the adult eye and nervous system. This study reports that lilliputian (lilli), the Drosophila homolog of the FMR2/AF4 family of proteins, regulates the transcription of ato and da in the developing fly retina. lilli controls ato expression at multiple enhancer elements. lilli was found to contributes to ato auto-regulation in the morphogenetic furrow by first regulating the expression of da prior to ato. FMR2 regulates the ato and da homologs MATH5 and TCF12 in human cells, suggesting a conservation of this regulation from flies to humans. It is concluded that lilli is part of the genetic program that regulates the expression of proneural genes in the developing retina (Distefano, 2012).

This study has shown that lilli, the Drosophila homolog of the FMR2/AF4 family of proteins, regulates the transcription of the bHLH proneural genes atonal and daughterless in the developing retina and antenna of flies. It was further shown that this transcriptional regulation is conserved from flies to human cells. The data suggest that lilli regulates ato differentially at the 5' and 3' enhancer elements. The 3' cis-regulatory element is a 1.2-kb region of DNA located approximately 3.1 kb downstream of the ato transcription unit, and controls the early phase of ato expression in the developing retina. At this element, lilli appears to regulate transcription of ato at multiple sites (Distefano, 2012).

While analysis of transcription driven by a 5.8-kb element described previously is significantly reduced to less than 25% of controls, transcription driven by a minimal 348-bp enhancer element found within the larger 5.8-kb 3' enhancer is only reduced to 55% of controls. An important question remains for lilli-mediated ato transcription: is lilli function required to directly induce ato expression, or rather to maintain expression once previously induced? Given its function as a transcriptional activator, lilli function may be directly required to turn on ato expression at the 348-bp enhancer element in the developing retina. However, lilli may also be required to maintain ato expression once activated by other factors (such as Sine oculis or Eyeless/ Pax6) at this enhancer element. Alternately, other cis-regulatory elements along the 5.8- kb enhancer region may require lilli function to modulate ato expression after activation. Further experimentation will be required to answer these questions (Distefano, 2012).

The data also show that lilli-mediated regulation of ato expression at the 5' enhancer region requires the expression of the Da protein. The ato protein auto-regulates its expression at the 50 enhancer region to sustain ato gene expression in the intermediate groups and single R8 photoreceptors. It is hypothesized that the lilli protein regulates ato expression indirectly, by affecting ato auto-regulation at the 5' enhancer element. This hypothesis on multiple observations. (1) ato protein expression and ato transcription from the 5' enhancer element is decreased, but not absent within lilli mutant clones, consistent with an indirect effect on ato transcription. (2) Da protein expression and da transcription is also decreased, but not absent within lilli mutant clones in the region corresponding to the 5' enhancer expression. (3) By replacing Da expression within lilli mutant clones, ato transcription directed by the 5' enhancer element can be fully rescued, but not ato transcription directed by the 3' enhancer element. This is also true for ato protein expression in these clones (Distefano, 2012).

Thus, the lilli protein must first induce da transcription in cells in the intermediate groups and R8s prior to ato expression within these cells. Then, Ato-Da heterodimers can form, and maintain the activation of ato expression within the intermediate groups and R8 cells. If lilli function is compromised, da expression decreases, as does ato's ability to auto-regulate (Distefano, 2012).

This study has shown that lilli is required for the proper expression of hairy, another bHLH factor that is expressed anterior to the furrow. Hairy is an inhibitory factor to furrow progression. Interestingly, ato and da expression does not expand anteriorly in lilli clones, suggesting that loss of hairy anterior to the furrow is not sufficient to remove all of hairy function in these clones. Still, it is interesting that this analysis has identified a third bHLH factor regulated by lilli, and may suggest that lilli protein functions broadly to regulate the expression of multiple bHLH factors in different tissues (Distefano, 2012).

lilli is homologous to the AF4/FMR2 family of nuclear proteins in humans. This family includes FMR2, LAF4, AF4, and AF5Q31. Members of this family are implicated in acute lymphoblastic leukemia and FRAXE nonsyndromic Fragile X mental retardation (Distefano, 2012).

This study has shown that FMR2 also regulates the expression of the proneural genes MATH5 and TCF12 in HEK293 cells, showing that the observations made in the fly retina are conserved in human cell. Patients with FRAXE exhibit various developmental and morphological problems, including mental retardation, delays in speech development, attention deficit disorder, hyperactivity, and impaired motor coordination. While the etiology of these symptoms remains unknown, defects in neurogenesis, and/or the regulation of critical transcription factors such as the human homologues of ato and Da may be related to these symptoms. Further, recent evidence has shown that ato also functions as a tumor suppressor gene. This may provide an additional link between the misregulation of bHLH protein in lilli mutants and the leukemia observed in AF4 mutants. Further research is required to determine the significance of this connection (Distefano, 2012).

Atonal Post-transcriptional Regulation

The 3' untranslated regions (3' UTRs) of Bearded, hairy, and many genes of the E(spl)-C contain a novel class of sequence motif, the GY box (GYB, GUCUUCC); extra macrochaetae contains the variant sequence GUUUUCC. The 3' UTRs of three proneural genes include a second type of sequence element, the proneural box (PB, AAUGGAAGACAAU). The full 13 nt PB is found once each in ac, l'sc, and ato, along with a second, variant version in both l'sc and ato. The presence of these motifs in such distantly related paralogs as hairy and certain bHLH genes of the E(spl)-C (for the GYB), and ato and two genes of the AS-C (for the PB), indicates that both classes of sequence element are subject to strong selection. Furthermore, both the PB and the GYB are conserved in the orthologs of ac and E(spl)m4 from the distantly related Drosophilids D. virilis and D. hydei, respectively, though these 3' UTRs are otherwise quite divergent from their D. melanogaster counterparts. These findings strongly suggest functional roles for both of these sequence elements (Lai, 1998).

Intriguingly, the central 7 nt of the PB and the GYB are exactly complementary, and are often located within extensive regions of RNA:RNA duplex predicted to form between PB- and GYB-containing 3' UTRs. Indeed, using in vitro assays, RNA duplex formation has been observed between the ato/Brd and ato/m4 3' UTR pairs that is PB- and GYB-dependent. It is noteworthy that the predicted duplex interactions involving the GYB of Brd are significantly stronger than those involving the GYBs of the other transcripts. For example, Brd and ato are perfectly complementary over 18 contiguous nucleotides. This difference in the degree of PB:GYB-associated complementarity is likely to have functional consequences (Lai, 1998).

In C. elegans, small antisense RNAs encoded by lin-4 mediate translational repression of lin-14 and lin-28 transcripts by binding to complementary sequences in their 3' UTRs. In Drosophila, PB- and GYB-bearing transcripts may likewise participate in a regulatory mechanism mediated by RNA:RNA duplexes, but with the feature that both partners are mRNAs that also direct the synthesis of functionally interacting proteins. The opportunity to form such duplexes clearly exists, since transcripts from proneural genes and their regulators very frequently accumulate in coincident or overlapping patterns. Moreover, while 7 nt is the minimum length of complementarity between any PB and any GYB, the longest possible uninterrupted duplex between a given GYB-bearing transcript and a given proneural partner is almost always considerably longer (8-12 nt). It is worth noting that in a lin-4/lin-14 duplex that has been shown to be sufficient for proper regulation in vivo, the longest region of uninterrupted complementarity is only 7 nt (Lai, 1998 and references therein).

The formation of the postulated RNA duplexes may serve to regulate proneural gene function, consistent with the known roles of hairy, emc, and the bHLH genes of the E(spl)-C. This might explain occasional C-to-U transitions in the GYB sequence (in emc and D. hydei m4); these variants retain complementarity with the PB due to G:U base-pairing. It is equally plausible that GYB-containing transcripts are regulated by duplex formation. A third very interesting possibility is that RNA:RNA duplexes formed between PB- and GYB-containing transcripts function to initiate a downstream regulatory activity affecting as-yet-unknown targets. Ample precedent exists establishing the trans-regulatory potency of double-stranded RNA. In any case, the apparent capacity of transcripts from the proneural genes and their regulators to form duplexes in their 3' UTRs suggests further complexity in the already complex regulatory interactions that control Drosophila neurogenesis (Lai, 1998).

roDom is a dominant allele of rough (ro) that results in reduced eye size due to premature arrest in morphogenetic furrow (MF) progression. The roDom stop-furrow phenotype is sensitive to the dosage of genes known to affect retinal differentiation, in particular members of the hedgehog (hh) signaling cascade. roDom interferes with Hh's ability to induce the retina-specific proneural gene atonal (ato) in the MF and normal eye size can be restored by providing excess Ato protein. roDom was used as a sensitive genetic background in which to identify mutations that affect hh signal transduction or regulation of ato expression. In addition to mutations in several unknown loci, multiple alleles of groucho (gro) and Hairless (H) were recovered. Analysis of their phenotypes in somatic clones suggests that both normally act to restrict neuronal cell fate in the retina, although they control different aspects of ato's complex expression pattern (Chanut, 2000).

Loss-of-function ro mutations cause eye roughness, due to mis-specification of photoreceptors R2 and R5, and the formation of ommatidia with more than one R8 photoreceptor. Repression of R8 cell fate has been attributed to inhibition of ato expression by the Ro homeodomain protein. In support of this proposal, Rough and Atonal proteins appear in complementary sets of cells behind the MF, and ato expression is expanded behind the MF in ro mutants. Generalized expression of ro under a heat-shock promoter (hs-ro) leads to loss of ato expression in the MF and eventually results in furrow arrest. Furrow arrest in roDom is also accompanied by loss of ato expression in the MF. By analogy to the hs-ro phenotype, it is proposed that roDom leads to excess Ro production, although that excess is not detectable by antibody staining (Chanut, 2000).

ro expression at the posterior edge of the MF is under the control of hh signaling. The position of ro-expressing cells, adjacent to hh-expressing cells, suggests that high levels of hh signaling are required for ro expression. By comparison, ato, also a target of hh signaling in the MF, is expressed further away from the hh source, suggesting a requirement for lower levels of hh signaling. It is proposed that the roDom rearrangement sensitizes the ro gene to hh signaling, either by removing a negative regulatory cis-element or by bringing in an additional hh-responsive enhancer element. The resulting anterior expansion of the ro expression domain would prevent ato expression and ultimately cause differentiation arrest (Chanut, 2000).

While this model cannot be proven at this point, it provides a simple explanation for the surprising genetic interactions between roDom and hh: if the rearranged ro gene is more sensitive to Hh, then increasing hh gene dosage will cause more Ro production and accelerate the differentiation arrest. However, reducing Hh signaling, by removing one copy of hh or by providing the inhibitor Ptc in excess, will diminish the amount of Ro protein made and restore Ato accumulation. Modifiers recovered in this screen should therefore include, among other things, components of hh signaling that affect ro or ato expression or partners of Ro in the inhibition of ato transcription (Chanut, 2000).

Expression of dpp is also sharply decreased in roDom. Like ato, dpp expression could be inhibited by ro directly. This may explain its sharp downregulation behind the MF in wild type at the location where ro begins to be expressed. Alternatively, its decrease in roDom could be a secondary consequence of decreased ato transcription. In support of the latter, dpp transcription is sharply reduced in the MF of ato1 homozygous larvae. Surprisingly, Hairy protein levels remain elevated ahead of the MF in roDom, although h has been shown to be a target of Dpp signaling. The same is true in ato1 mutants. This suggests that h is under the control of other, as yet unidentified, mechanisms that are not dramatically impaired by the roDom mutation and the accompanying loss of dpp transcription. In any case, effects of roDom on dpp and h expression are unlikely to explain the furrow arrest since the roDom phenotype is not detectably affected by changes of h and dpp gene dosage (Chanut, 2000).

In contrast, roDom is very sensitive to alterations of ato gene dosage, since it is enhanced by loss-of-function ato alleles and almost completely rescued when high levels of ato expression are restored ahead of the MF. The roDom phenotype therefore appears to result primarily from inhibition of ato expression due to excess Ro protein. On the basis of this understanding, the role of two of the strongest suppressors isolated in this screen, new alleles of gro and H, were analyzed on ato regulation and furrow progression (Chanut, 2000).

gro encodes a transcription inhibitor that combines with b-HLH genes of the E(spl) complex to inhibit expression of proneural genes such as achaete and scute. In gro mutant clones, expression of ato persists behind the MF longer than in wild-type tissue. This is consistent with a role for Gro in the N signaling events that lead to the refinement of ato expression behind the MF (Chanut, 2000).

However, Gro is also known to form inhibitory complexes with other transcription factors of the b-HLH class, such as Hairy, or of other classes, such as the c-Rel homolog Dorsal or the homeodomain, segment polarity regulator Engrailed. Association of Gro with Hairy deserves to be envisaged here, since Hairy has been implicated in inhibition of ato as well. It is found, however, that gro mutant clones expand ato expression posterior to the MF, whereas h inhibits ato expression anterior to the MF. Another intriguing possibility is that Gro associates with Ro to mediate inhibition of ato expression behind the MF. Although this hypothesis cannot be completely eliminated, it is unlikely, because complete loss-of-function phenotypes of ro and gro are different. While both of them lead to increased ato expression and imperfect R8 resolution, this effect is much more extensive and long lasting in gro mutant tissue than in ro mutant tissue. In addition, neuronal hyperplasia is not observed in ro mutant tissue, which suggests that at least this gro function must involve factors other than Ro. However, removal of E(spl) function results in neuronal hyperplasia and excess R8 development very similar to removal of gro function. Therefore, the hypothesis is favored that Gro restricts ato expression by combining with proteins of the E(spl) complex whose expression is induced by N signaling (Chanut, 2000).

Even in the complete absence of gro [or E(spl)] function, some refinement of ato expression still occurs, which indicates that factors independent of N and Gro play important roles in patterning Ato behind the MF. Candidates include Ro, the Egfr inhibitor Argos, and Hh. Moreover, outer photoreceptors differentiate in large excess between the R8 precursors and are the main cause of neuronal hyperplasia. Neuronal hyperplasia could occur as a direct consequence of the excess of R8 precursors in gro [and E(spl)] mutant tissue, which would, through the normal serial induction process, recruit an excess of neighboring cells into ommatidial clusters. However, differentiation of extra outer photoreceptors was observed with a hypomorphic gro allele in the absence of excess R8 differentiation. The excess of all photoreceptor types observed with a stronger gro allele may therefore reflect an involvement of gro in the restriction of cell fates at each step of ommatidial formation (Chanut, 2000).

gro mutant clones can also induce extensive overgrowth of head capsule and retinal tissues. In the wing, gro clones have been found to cause overgrowth via the induction of ectopic hh expression. Hh is also a powerful inducer of overgrowth in eye discs, when provided in excess or ectopically. However, overgrowth due to ectopic hh expression is accompanied by ectopic and premature photoreceptor differentiation, a phenotype not observed in overgrown gro mutant tissue. It is therefore unlikely that gro mutant clones cause ectopic hh expression in the eye. Besides, if gro mutations allowed increased Hh production, one would expect enhancement, rather than suppression, of the roDom phenotype. While scenarios cannot be eliminated for roDom in which a slight increase in cell proliferation allows the MF to progress further, it is more likely that gro mutations suppress roDom by allowing Ato protein to persist longer in the MF (Chanut, 2000).

Hairless inhibits N signaling by preventing Su(H), a transcription factor, from translocating to the nucleus and activating transcription of N targets such as the E(spl) complex genes. In the absence of H, Su(H) is free to enter the nucleus upon activation of N. Su(H) mutant clones lead to expanded Ato expression behind the MF, consistent with a role for Su(H) in the N-mediated lateral inhibition that leads to the refinement of ato expression. In H clones are found in which the refinement of ato expression to single cells appears accelerated. This is consistent with a role for H as an inhibitor of N and Su(H) in lateral inhibition. Surprisingly, however, individual clusters of Ato-expressing cells often persist in H mutant tissue behind the MF, instead of resolving to single R8 precursors; in adults as well, mutant ommatidia often contain more than one R8. This would suggest that at later stages H is required to resolve ato expression to single R8 precursors, a role that is not expected for an inhibitor of the N pathway. Anterior H mutant clones show precocious ato expression anterior to the MF. This might explain the patterning defects behind the MF, if precocious and excessive accumulation of Ato protein in the MF interferes with the proper execution of lateral inhibition via N or with downregulation by Ro. In this regard, it is noted that excess Ato protein, as provided under heat-shock control, is found to perturb the resolution of ato expression to single R8 precursors (Chanut, 2000).

It has been suggested that early ato expression, ahead of the MF, is in part the result of an as yet unsuspected 'proneural' effect of N signaling. The anterior expansion of ato expression in H mutant clones is consistent with this model, assuming that H would act as an inhibitor of N there as well. However, the proneural function of N must not be mediated by Su(H), since removal of Su(H) function does not abolish ato expression ahead of the MF. The results presented here may indicate that H antagonizes the proneural function of N via a mechanism that does not involve Su(H). Alternatively, the role of H on early ato expression may be independent of N signaling. Regardless of the exact mechanism, the enhanced expression of ato ahead of the MF in H mutants is likely to explain suppression of the roDom phenotype by counteracting the effect of ectopic Ro on ato expression in the MF (Chanut, 2000).

Finding that similar levels of suppression can be achieved by loss-of-function mutations in H and gro (which act in opposite directions in the N pathway) is not unique. A similar situation was encountered in another study where mutations in gro and H were both found to enhance the wing and bristle phenotypes associated with loss-of-function mutations in Egfr. The observation that mutations in both genes elevate ato expression in the vicinity of the MF, but at different stages of the differentiation process, helps resolve this paradox. The results also indicate that the exact timing (or location) of ato expression might not be crucial to MF progression, provided adequate levels are reached. This conclusion is supported by the finding that Ato supplied anterior to its normal expression domain, in the h expression domain, restores normal eye size in a roDom background. Whether proper R8 spacing and ommatidial patterning can be achieved under these conditions remains to be shown (Chanut, 2000).


atonal: Biological Overview | Evolutionary Homologs | Targets of Activity | Developmental Biology | Effects of Mutation | References

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