atonal
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).
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).
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).
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'ato348-ßgal reporter (core
element) occurs prematurely as compared with endogenous ato. The
3'ato348-ßgal 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'ato348-ßgal and
3'ato488-ßgal (but not
3'ato1.2-ßgal or
3'ato1.2-Δ298-ßgal) 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).
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).
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).
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 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).
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).
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).
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).
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).
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).