Promoter Structure

The exact positioning of neuroblasts in the neuroectodermal region that gives rise to the CNS is regulated by a combination of pair-rule genes. Proneural achaete-scute genes are controlled by combinations of axis-patterning genes through a common intergenic control region. Specifically, the odd-numbered segments in paired mutants lack achaete producing cells in the fourth row of neuroblasts. paired mutants also exhibit fusion of second and fourth rows in even-numbered segments. In fushi tarazu mutants, the fourth row of achaete clusters is removed in each segment. In every segment, the loss of odd-paired function removes achaete expression from the second row of clusters in each segment. In embryos mutant for sloppy-paired the second and fourth rows of achaete expression fuse in odd-numbered segments (Skeath, 1992b).

See Chris Doe's Hyper-Neuroblast map site for information on the expression of achaete in specific neuroblasts.

For more information on Drosophila neuroblast lineages, see Linking neuroblasts to their corresponding lineage, a site carried by Flybrain, an online atlas and database of the Drosophila nervous system.

The highly complex pattern of proneural clusters of imaginal discs is constructed piecemeal, by the action on ac and sc of site-specific, enhancer-like elements distributed along most of the AS-C (approximately 90 kb). Fragments of AS-C DNA containing these enhancers drive reporter lacZ genes in only one or a few proneural clusters. This expression is independent of the ac and sc endogenous genes, indicating that the enhancers respond to local combinations of factors (prepattern). The cross-activation between ac and sc, discovered by means of transgenes containing either ac or sc promoter fragments linked to lacZ, and thought to explain the almost identical patterns of ac and sc expression, does not occur detectably between the endogenous ac and sc genes in most proneural clusters. Coexpression is accomplished by activation of both ac and sc by the same set of position-specific enhancers (Gomez-Skarmeta, 1995).

The genes araucan and caupolican code for two divergent homeodomain proteins that regulate transcription from the position-specific enhancers of ac-sc. Expression in the wing imaginal disc starts during the second larval instar at the presumptive notum region and is increased in two large areas of the presumptive lateral heminotum. From the mid-third instar, expression occurs at the presumptive distal tegula, the dorsal radium, proximal vein L1, veins L3 and L5, the allula, and the pleura. This distribution suggests that ARA and CAUP in fact establish the prepattern for proneural clusters in the wing, and interact with the position-specific enhancers to regulate ac-sc in the very precise pattern displayed by these proneural clusters (Gomez-Skarmeta, 1996).

Hairy (h) acts as a negative regulator in both embryonic segmentation and adult peripheral nervous system (PNS) development (Ohsako, 1994). Achaete is a direct downstream target of H regulation in vivo. Mutation of a single, evolutionarily conserved, high-affinity Hairy binding site in the upstream region of ac results in the appearance of ectopic sensory organs in adult flies, forming a pattern that strongly resembles the phenotype of h mutants. This indicates that direct repression of ac by H plays an essential role in pattern formation in the PNS (van Doren, 1994). Thus h, like ara and caup, seems to be involved in setting the prepattern for transcription of ac-sc in proneural clusters.

Complex patterns of ac and sc expression are constructed by separable cis-controlling elements present within a large (ca. 90-kb) region. The yellow gene located 10 kb from ac has completely different expression patterns and is activated by different enhancers. Therefore, these genes may serve as a good model system for the analysis of proper enhancer-promoter recognition. Autologous recognition between genes and their respective promoters may depend on the existence of an interdomain boundary between AS-C and the yellow locus, or it may be determined by the specificity of the proteins assembled on a certain enhancer and promoter. An inversion is described that puts the yellow gene between the ac and sc genes and almost all of their cis-regulatory elements. This inversion shows only weak interference with the expression of the ac and sc genes. When the suppressor of Hairy wing-binding region of the su(Hw) insulator is deleted or inactivated by the su(Hw) mutation, the sc phenotype of the flies is practically indistinguishable from that of the wild type. The presence of the yellow gene between the AS-C enhancers and the promoters of the ac and sc genes does not interfere with ac and sc expression in most areas. This work shows, however, that it is not required that the su(Hw) insulator separate promoters from enhancers to allow inhibition of transcription by the su(Hw) protein. The presence of the su(Hw) insulator, located more than 20 kb away from the inversion, facilitates strong suppression of achaete and scute gene expression, although is does not separate the promoters from the AS-C enhancers (Golovnin, 1999).

The mechanism of direct interaction between AS-C enhancers and su(Hw) insulator is not yet clear. One possibility is that the pairing between the P elements located at the breakpoints of the inversion facilitates such interaction. However, deletions of the P elements on both sides of the inversion fail to influence the repression mediated by the su(Hw) insulator. Another possibility is that the inversion brings the su(Hw)-binding region into a close contact with the AS-C cis-regulatory elements due to changes in chromatin folding, which then leads to new long-range contacts between certain chromatin regions. As a result, the su(Hw)-mod(mdg4) complex formed on the su(Hw) insulator becomes capable of interacting directly with enhancer-bound transcription activators or with proteins responsible for enhancer-promoter interactions. The fact that the inactivation of AS-C control elements by the su(Hw)-binding region in the inversion is only partial may be explained by reversible interactions between the insulator and enhancers similar to the normal dynamic interactions observed between enhancers and promoters (Golovnin, 1999).

The Drosophila LIM-homeodomain protein Islet acts at the dorsocentral enhancer of ac/sc to antagonize proneural cell specification in the peripheral nervous system

The pattern of the external sensory organs (SO) in Drosophila depends on the activity of the basic helix-loop-helix (bHLH) transcriptional activators Achaete/Scute (Ac/Sc) that are expressed in clusters of cells (proneural clusters) and provide the cells with the potential to develop a neural fate. In the mesothorax, the GATA1 transcription factor Pannier (Pnr), together with its cofactor Chip, activates ac/sc genes directly through binding to the dorsocentral enhancer (DC) of ac/sc. The LIM-homeodomain (LIM-HD) transcription factor Islet (Isl) was identified by genetic screening and its role in the thoracic prepatterning was investigated. isl loss-of-function mutations result in expanded Ac expression in DC and scutellar (SC) proneural clusters and formation of ectopic sensory organs. Overexpression of Isl decreases proneural expression and suppresses bristle development. Moreover, Isl is coexpressed with Pnr in the posterior region of the mesothorax. In the DC proneural cluster, Isl antagonizes Pnr activity both by dimerization with the DNA-binding domain of Pnr and via competitive inhibition of the Chip-bHLH interaction. It is proposed that sensory organ prepatterning relies on the antagonistic activity of individual Chip-binding factors. The differential affinities of these binding-factors and their precise stoichiometry are crucial in specifying prepatterns within the different proneural clusters (Biryukova, 2005).

During Drosophila development, the expression of transcription factors divides the dorsal thorax into three domains -- one median and two lateral domains. The lateral domains are specified by the homeobox-containing proteins of the iroquois-complex (iro), whereas the GATA factor Pnr is required to establish the median domain. Within the mesothorax, Pnr together with U-shaped (Ush) and Chip plays a key role in dorsal closure. This report presents evidence that Isl is an essential regulator of the dorso-median patterning of the thorax. isl clones generated adjacent to the thoracic midline, induce a strong cleft, suggesting that Isl is required for proper dorsal closure during metamorphosis. Ectopic expression of Pnr leads to wing-to-thorax transformations, consistent with its role as medio-dorsal patterning factor. Ectopic Isl expression does not exhibit this phenotype, excluding the LIM-HD factor from a direct function as a prothoracic selector. Pnr is also known to activate wingless (wg) in dorsal thorax. isl loss-of-function has no significant effect on wg expression. However, overexpressed Isl strongly reduces the size of the wg thoracic stripe. This result is consistent with a repressive activity of Isl on Pnr (Biryukova, 2005).

Iro proteins and Pnr are direct activators of the proneural genes in their respective domains. Pnr binds directly to the DC enhancer of ac/sc, providing therefore region-specific control of the proneural prepatterning. Flies with reduced or lack of Pnr function fail to activate ac/sc and to develop DC and SC sensory organs. The proneural activity of Pnr is antagonized by Ush, the vertebrate homologue of the FOG (friend of GATA). Ush is expressed only in the dorsal-most cells of the medial region. As a consequence, the segregation of the sensory organ precursors occurs along two stripes at the border of the medial domain of Pnr expression, where Ush is absent or insufficient to repress Pnr (Biryukova, 2005).

Several lines of evidence indicate that Isl interferes with the proneural activity of Pnr as a repressor. (1) isl loss-of-function mutants show an opposite phenotype with regard to Pnr or Chip loss-of-function mutants: an excess of DC and SC sensory organs. (2) A genetic synergism exists between PnrD and isl alleles. This genetic interaction is less sensitive than that between PnrD and ush, implying an alternative route for Isl to modulate the Pnr proneural activity. (3) Isl is coexpressed with Pnr within the posterior mesothorax. (4) Isl modulates the activity of a DC:ac-lacZ reporter. Loss-of-function isl mutants expand the DC:ac-lacZ expression as in ush or PnrD constitutive mutants, whereas overexpressed Isl reduces the DC:ac-lacZ expression (Biryukova, 2005).

In the DC region, the regulation of Pnr concentration is critical for the proper position and shape of the DC proneural cluster. Isl expression overlaps with the dorsal-most domain of Pnr and DC proneural activity coincides with the posterior border of Isl expression. Therefore, it proposed that both Isl and Ush restrict Pnr activity in the mesothorax. Interestingly, the regulation of the concentration of the mammalian Pnr ortholog, GATA-1, is similarly critical for proper erythroid, megakaryocytic, eosinophilic and mast cell lineages (Biryukova, 2005).

Ush behaves as either an activator or a repressor of Pnr, depending on developmental context. No evidence was found for a direct Isl-Ush interaction by GST pull down assay: Ush, Pnr and Isl could be co-immunoprecipitated from transient transfected S2 cells. Both Ush and Isl may behave as positive cofactors of Pnr for nonneural activities, such as cardiac development, embryonic dorsal closure and metamorphosis. Several reports emphasize the role of the Pnr homolog, GATA-1 and Isl1 in human blood disorders. It seems likely that GATA:Islet interactions represent a conserved mechanism to specify different cell fates in humans and other organisms (Biryukova, 2005).

Isl proteins are known as positive regulators of transcription in vertebrates. In flies, Isl mediates repression of Pnr-driven proneural activity via binding to the DNA-binding domain of Pnr. Interestingly, these interactions are less specific than for the Pnr-Ush interaction, where the amino-terminal zinc finger of Pnr is specifically involved (Biryukova, 2005).

Genetic analyses of mutants reveal that the DC and the SC proneural clusters show differential sensitivities during neurogenesis. Ush mutants display ectopic DC bristles and a few additional SC bristles. This phenotype is similar to PnrD constitutive mutants, in which Pnr-Ush interactions are greatly reduced. In contrast, isl mutants show the opposite phenotype, with a large excess of SC bristles and a few additional DC bristles. The ChipE mutant exhibits antagonistic phenotypes: lack of DC bristles, reflecting Pnr loss-of-function and an excess of SC bristles, reflecting Isl loss-of-function. The differential topography of DC and SC enhancer binding sites presumably underlies differential transcription-complex binding affinities (Biryukova, 2005).

Chip is the ortholog of Ldb factors that are ubiquitous multiadaptor proteins in vertebrates. Each Ldb-dependent developmental event is specified by modification of the transcriptional complex and is dependent on the stoichiometry of the region-specific Ldb partners. During normal development of the thorax, different partners of Chip (i.e., Isl, Ap and Pnr) are expressed in the same region. The ChipE mutant is highly sensitive to the dosage of these factors. In ChipE flies, removing one copy of either Pnr or Isl causes pupal lethality associated with extreme morphogenetic phenotypes. Removing one copy of Ap, however, rescues the Pnr-dependent phenotypes of ChipE flies. Taken together, these results indicate selective competition between the different partners of Chip, suggesting that hierarchical protein interactions depending on differential affinities and the strict stoichiometry of Chip and its partners, are critical to establish proper transcriptional codes within different proneural fields (Biryukova, 2005).

isl mutants were isolated in genetic screens for dominant enhancers of the ChipE phenotype. This study demonstrates that the LIM-HD transcription factor Isl can bind to the LID of Chip. The binding of the LID domain of Chip with LIM domains has been conserved throughout evolution as has Chip binding with bHLHs proteins. LID contains two subdomains: a small N-terminal hydrophobic β patch (VMVV) followed by a large α helix. ChipE mutation has a single substitution that changes an Arg to Trp (R504W) in the middle of the α helix. This residue is highly conserved among species and mediates high-affinity contact with the LIM domains. Interestingly, the R504W substitution in Chip abolishes, or strongly reduces, both interactions with the bHLHs and also interactions with Isl. This result implies that bHLHs and Isl recognize the same site within the LID domain of Chip. The data argue that competition between bHLHs and Isl for the LID domain of Chip may be critical for modulating the activity of transcription complexes during development. In vertebrates, the NLI homolog of Chip mediates direct coupling of the proneural bHLH factors Ngn2, NeuroM and the LIM-HD transcription factors (Isl1 and Lhx3). This interaction leads to transcriptional synergism and the synchronization of motor neuron subtype specification with neurogenesis in the embryonic spinal cord of chicken. This work demonstrates that Isl is able to interfere with proneural activity of Chip-Pnr-bHLH transcription complex and therefore, Isl is thought to be able to antagonize proneural specification (Biryukova, 2005).

Interestingly, the ChipE mutation has little or no effect on interactions with other LIM-containing factors, such as Ap and dLMO (Beadex), suggesting that different factors have different affinities with the Chip LID domain. Therefore, the ChipE mutation changes the hierarchy of the affinities among the different partners of Chip in the mesothorax (Biryukova, 2005).

A transcription-complex 'cassette' model is proposed for the specification of region-specific patterns of specialized cell types. In this model, the presence of one of a number of alternative binding factors modifies the specificity of a core transcription complex. This model makes the prediction that, while the core components of the transcription complex will be strongly conserved in evolution, the specificity cassette components will vary significantly between species showing divergent morphogenetic patterns. Comparison of these variable components in related species should provide insights into the fundamental mechanisms of encoding the pattern of differentiated cell types within morphogenetic fields (Biryukova, 2005).

A major bristle QTL from a selected population of Drosophila uncovers the zinc-finger transcription factor Poils-au-dos, a repressor of achaete-scute

Traditional screens aiming at identifying genes regulating development have relied on mutagenesis. A new gene has been identified involved in bristle development, identified through the use of natural variation and selection. Drosophila melanogaster bears a pattern of 11 macrochaetes per heminotum. From a population initially sampled in Marrakech, a strain was selected for an increased number of thoracic macrochaetes. Using recombination and single nucleotide polymorphisms, the factor responsible was mapped to a single locus on the third chromosome, poils au dos (French for 'hairy back'), that encodes a zinc-finger-ZAD protein. The original, as well as new, presumed null alleles of poils au dos are associated with ectopic achaete-scute expression that results in the additional bristles. This suggests a possible role for Poils au dos as a repressor of achaete and scute. Ectopic expression appears to be independent of the activity of known cis-regulatory enhancer sequences at the achaete–scute complex that mediate activation at specific sites on the notum. The target sequences for Poils au dos activity were mapped to a 14 kb region around scute. In addition, pad has been shown to interact synergistically with the repressor hairy and with Dpp signaling in posterior and anterior regions of the notum, respectively (Gibert, 2005).

Expression of ac-sc in proneural clusters is regulated by independently-acting cis-regulatory enhancers. The enhancer responsible for activation of ac-sc in the cluster giving rise to the DC bristles has been characterized in detail. The activity of this enhancer in a reporter construct was examined using lacZ expression. The activity of this enhancer is modified in pad1. The domain of expression of lacZ appears wider. At the same time, the anterior limit of the cluster is retracted in a posterior direction. It is possible that this is in part due to the slight distortion of the overall shape of the notum seen in pad1 mutants. Interestingly, the ectopic bristles do not arise within the misshapen proneural cluster. They are therefore formed independently of the activity of the DC enhancer used for activation. In fact, the aDC, as well as the ectopic DC precursors, are both clearly situated outside the DC cluster. Another characterized enhancer of ac-sc, the L3-TSM enhancer involved in the formation of the sensilla on the anterior wing margin, anterior cross vein and third vein was examined and no significant modification was observed. These results suggest that poils au dos does not act through the cis-regulatory sequences controlling expression in the proneural clusters (Gibert, 2005).

To determine which regions of the AS-C are required for the formation of the ectopic bristles in pad, the pad1 mutant was placed in various ac-sc mutant backgrounds. These included several deletions generated by excision of the P-element in the line NP-6066. In(1)ac3, an inversion separating sequences located 1 kb upstream of ac, including the DC enhancer, was used, as well as Df(1)91B (which deletes 45 kb from a position 10.3 kb upstream of sc that includes ac and the DC enhancer); Df(1)115 (which deletes 7.8 kb between the positions 14.5 and 6.7 kb upstream of the scute ATG), and In(1)sc4 (an inversion with a breakpoint 7-8 kb downstream of sc). None of these rearrangements prevent formation of the ectopic bristles present in pad1. In(1)sc4 causes a loss of all scutellar bristles, because the relevant enhancer, located 40 kb downstream of sc, is translocated elsewhere and is thus not able to drive the expression of ac-sc in the scutellum. However, occasional scutellar bristles form in In(1)sc4; pad1 flies at the position normally occupied by the anterior scutellar bristle. In contrast to the rearrangements cited above, no, or very few, ectopic bristles are formed in scbald; pad1 flies. This hypomorphic sc allele carries the remains of a P element located 10 kb upstream of sc and displays a high frequency of missing SC, aDC and orbital bristles. Together, these results indicate that the target sequences are probably located in a fragment that extends 6.7 kb upstream and 7-8 kb downstream of sc (Gibert, 2005).

In order to visualize the precursors of the ectopic bristles in pad1, an antibody against Senseless, a marker of neural precursors, was used. A transgene was used driving the expression of LacZ under the control of the achaete/scute Sensory Organ Precursor enhancer (SOP-lacZ). The minimal SOP enhancer of 500 bp drives expression of lacZ exclusively in the bristle precursors and contains binding sites for Ac-Sc/Da (E boxes), as well as sites for the binding of repressors. It was observed that the precursors of ectopic bristles appear between 0 and 2 h after puparium formation. This is about the same time as the formation of the precursors for the anterior DC (aDC) bristles in wild-type flies. The posterior DC (pDC) precursors appear much earlier, around 24 to 12 h before puparium formation. In situ hybridization with a probe to sc, indicated that sc is expressed ectopically in third instar wing discs. Expression of ac was examined using an anti-Achaete antibody and is also significantly up-regulated in pad1. In both cases, the proneural clusters that give rise to the wild-type bristle precursors are clearly visible at wild-type locations, but they appear to be enlarged. In addition, many more cells express high levels of ac-sc outside the proneural clusters. These are mainly located in the future anterior and central regions of the notum, consistent with the fact that ectopic macrochaetes are found here. Weak sc expression can be detected in these areas in wild-type discs but does not give rise to sense organs. Ectopic expression in pad1 is particularly visible in the region of the presutural, DC and PSA bristles where many ectopic bristles form (Gibert, 2005).

To better visualize the regions of ectopic expression, the reporter construct EE4 containing an artificial SOP enhancer composed of four E-boxes and the binding sites for the Ac and Sc proteins was used. The EE4 construct lacks the sequences required for repression and so it is very sensitive to the levels of Ac-Sc and can be used to measure the increased amounts of Ac-Sc in the pad mutant. It was observed that expression driven by this enhancer in pad1 is significantly different from that seen in the wild type. In the wild type, it is expressed exclusively in the cells of the proneural clusters where it is present at high levels. In pad1, expression in the PSA region expands medially and expression in the DC region expands anteriorly. Some of the ectopic precursors appear within this expanded anterior region (Gibert, 2005).

Linking pattern formation to cell-type specification: Dichaete and Ind directly repress achaete gene expression in the Drosophila CNS

Mechanisms regulating CNS pattern formation and neural precursor formation are remarkably conserved between Drosophila and vertebrates. However, to date, few direct connections have been made between genes that pattern the early CNS and those that trigger neural precursor formation. Drosophila has been used to link directly the function of two evolutionarily conserved regulators of CNS pattern along the dorsoventral axis, the homeodomain protein Ind and the Sox-domain protein Dichaete, to the spatial regulation of the proneural gene achaete (ac) in the embryonic CNS. A minimal achaete regulatory region that has been identified that recapitulates half of the wild-type ac expression pattern in the CNS; multiple putative Dichaete-, Ind-, and Vnd-binding sites have been found within this region. Consensus Dichaete sites are often found adjacent to those for Vnd and Ind, suggesting that Dichaete associates with Ind or Vnd on target promoters. Consistent with this finding, Dichaete can physically interact with Ind and Vnd. Finally, the in vivo requirement of adjacent Dichaete and Ind sites in the repression of ac gene expression has been demonstrated in the CNS. These data identify a direct link between the molecules that pattern the CNS and those that specify distinct cell-types (Zhao, 2007).

Sox-domain proteins physically associate with other transcription factors to regulate gene transcription. Thus, the identification that Dichaete genetically interacts with Vnd and Ind suggested that Dichaete associates with Vnd and Ind to regulate gene expression in the CNS. To test this model, it was asked whether Dichaete can interact with Ind or Vnd in the yeast two-hybrid assay. Control experiments revealed that the full-length Dichaete protein as well as the region C-terminal to the high-mobility-group (HMG) DNA-binding domain (amino acids 221–384) activate transcription on their own when fused to the Gal4 DNA-binding domain, suggesting that the C-terminal region contains transcriptional activation activity. As a result, a number of distinct Dichaete fusion constructs were tested for self-activation of transcription and four were identified that were transcriptionally inert. One of these contained the HMG domain and the C-terminal region, indicating that the presence of the HMG domain may mask the transactivation properties of the C-terminal region. A prior study mapped a transactivation domain to the N-terminal region of Dichaete (Ma, 1998), yet no transactivation properties of this domain were identified in this study. Consistent with a transactivation domain residing in the C-terminal region of Dichaete, all other identified transactivation domains in Sox-family proteins map C-terminal to the HMG domain (Zhao, 2007).

By using the four Dichaete bait constructs, it was found that the N-terminal region of Dichaete (amino acids 1–141) specifically interacted with full-length Ind protein. In a reciprocal manner, the ability of the Dichaete N-terminal region to interact with two different regions of Ind was tested: the region N-terminal to the homeodomain (amino acids 1–302) and the region including the homeodomain and all residues C-terminal to it (296–391). Both regions of Ind interacted strongly with the Dichaete N-terminal region, suggesting that this region of Dichaete can interface with two distinct regions of Ind (Zhao, 2007).

In a similar manner, two distinct regions of Dichaete, the regions N-terminal (amino acids 1–141) and C-terminal (amino acids 221–384) to the HMG domain, interact with the full-length Vnd protein. Three different Vnd prey constructs were used to localize the regions of Vnd that interact with Dichaete. It was determined that the region of Vnd located between the TN domain (a domain common to Tinman/NK-2 proteins) and the homeodomain (amino acids 217–536) interacts with the Dichaete N-terminal domain. This result confirms and extends those of Yu (2005) who found that Vnd and Dichaete coprecipitate and that a Vnd deletion lacking the first 408 amino acids interacts with Dichaete. It was not possible to define the region of Vnd that interacts with the Dichaete C-terminal region, perhaps because the constructs interrupt the domain to which the C-terminal region of Dichaete binds or disrupt the general topology of this domain. Nonetheless, the yeast two-hybrid results indicate that Dichaete can interact with Ind and Vnd consistent with the model that Dichaete complexes with Ind and Vnd on target gene promoters to regulate transcription in the CNS (Zhao, 2007).

A molecular understanding of how Dichaete, Ind, and Vnd pattern the CNS requires the identification and characterization of the regulatory regions of candidate direct target genes. One such candidate is the ac gene. Prior studies on ac suggested that regulatory regions important for its spatial regulation exist both 5' and 3' to the ac gene. Thus, an 8.15-kb minigene was generated that contains the ac transcription unit as well as ~4.8 kb of DNA 5' to the transcription start and ~2.4 kb of DNA 3' to the polyadenylation site and its ability to drive ac expression in an In (1)y3PLsc8R mutant background was tested. This genetic background carries a deletion of ac and also deletes the regulatory regions necessary to drive sc expression in row 3. Thus, it allows visualization of ac expression as driven by the minigene in the absence of endogenous ac/sc gene expression in row 3. The ac minigene drives ac expression in half of its wild-type CNS pattern because ac is expressed normally in the medial and lateral clusters of row 3 but is not expressed in row 7. The dynamics of ac expression as driven by the minigene in row 3 mirror those of endogenous ac expression because ac expression in each cluster quickly becomes restricted to a single cell, the presumptive neuroblast, which then delaminates into the interior of the embryo and extinguishes ac gene expression before its first division. Thus, the DNA contained within the minigene is sufficient to activate ac in its wild-type expression pattern in row 3 and to mediate the Notch-dependent restriction of ac to the presumptive neuroblast (Zhao, 2007).

By creating a series of 5' and 3' deletions of the initial minigene, the regulatory regions sufficient to drive ac expression in row 3 was delimited to a 2.84-kb genomic fragment (pG7), which is referred to as the row 3 element. This element contains the ac transcription unit, 1.34 kb of DNA 5' to the start of transcription and 542 base pairs of DNA 3' to the end of the transcription unit. ac minigenes were characterized for their ability to respond to the functions of Dichaete, ind, and vnd and for the presence and in vivo relevance of putative binding sites for these factors (Zhao, 2007).

In support of Dichaete, Vnd, and Ind acting directly on the row 3 element to regulate ac expression, loss of Dichaete, vnd, or ind function affects ac expression as driven by ac-pG4 or ac-pG7 in the same way, and these defects are identical to those observed for endogenous ac expression in these mutant backgrounds. For example, loss of ind or Dichaete causes, respectively, strong or modest derepression of ac expression in the intermediate column, whereas loss of vnd results in the absence of ac expression in the medial column (Zhao, 2007).

To see whether Dichaete, Ind, or Vnd act directly on the row 3 element to control ac expression, this element was searched for perfect matches to the consensus Vnd [CAAGTG], Sox-domain [(A/T)(A/T)CAA(A/T)G and homeodomain (TAATGG) binding sites. The canonical Sox-domain and homeodomain binding site sequences were used because the consensus sites for Dichaete and Ind have not been determined. This search identified one match for Vnd (V) and three each for Dichaete (S1, S3, and S4) and Ind (H1, H3, and H4). Notably, predicted Dichaete/Sox-binding sites tend to reside close to predicted Vnd or Ind sites, consistent with Dichaete acting with Vnd and Ind to regulate ac expression. The sole exception is the Ind site (H1) located upstream of the transcriptional start site of ac. However, gel-shift assays identify a Dichaete-binding site 11 bp 5' of this Ind site (S2) (Zhao, 2007).

Because the precise binding specificity of Ind is unknown, whether Ind can bind the predicted sites was tested by using gel-shift assays. Focused was placed on the predicted Ind site located upstream of the transcription start site because it is the only location where Dichaete and Ind sites are found adjacent to each other. It was found that Ind specifically binds this site in vitro. During these experiments, a second Ind-binding site (TAAATG) 8 bp 3' to this site was found, that differs slightly from the consensus homeodomain site. Thus, Ind can bind to two sites located within 1 kb of the ac promoter, suggesting a possible molecular mechanism for Ind-dependent repression of ac (Zhao, 2007).

The initial search for Dichaete-binding sites required a perfect match to the consensus Sox-binding site. However, bona fide transcription factor-binding sites often differ from the experimentally defined consensus by a few base pairs, indicating that the search likely underpredicted possible Dichaete-binding sites. Because of this, gel-shift assays were used to search for Dichaete-binding sites throughout the entire row 3 element (pG7). Three sites were identified to which Dichaete bound specifically. Two of these correspond to sites identified in the consensus sequence search (sites S1 and S3); whereas the third resides 11 bp 5' of the first of the two Ind sites near the transcriptional start of ac (S2); this site (GACAATG) differs from the consensus by one base pair. No binding was detected of Dichaete to one predicted Sox site (S4). Because Dichaete and ind are known to repress ac expression, the three binding sites for Ind and Dichaete upstream of the ac promoter identify a likely site of action through which these factors repress ac (Zhao, 2007).

The clustering of binding sites for Dichaete, Vnd, and Ind, together with the ability of Dichaete to interact with Vnd and Ind, supports the idea that Dichaete acts with these factors to regulate ac expression in the CNS. To test this model directly, the in vivo relevance was assayed of the adjacent Vnd and Dichaete sites as well as the adjacent Dichaete and Ind sites on ac expression. ac expression was unaltered when the Vnd-binding site, the adjacent Dichaete site, or both sites were mutated. Thus, vnd either does not regulate ac expression directly or other Vnd binding sites in the row 3 element compensate for the loss of this site (Zhao, 2007).

The relevance of the three Dichaete- and Ind-binding sites located ~850 bp upstream of the start of ac transcription was assayed. Mutating any single site or any combination of two sites had no effect on ac expression. However, mutating all three sites derepressed ac expression in the intermediate column, a phenotype similar to that found in embryos mutant for ind or Dichaete. This result provides direct link between genes that pattern the CNS and those that specify distinct cell types. Because the derepression of ac is less severe than that observed in ind mutant embryos, Ind and Dichaete likely act through additional sites in this element to repress ac expression fully in the intermediate column (Zhao, 2007).

Unexpectedly, derepression of ac expression posterior to row 3 was observed upon mutation of the three sites. This posterior expansion of ac mimics the effect that removal of gooseberry function has on the expression of ac, suggesting that Gooseberry, another homeodomain protein, may bind the same sites as Ind and act with Dichaete to repress ac expression in its expression domain (Zhao, 2007).

Conserved properties of the Drosophila homeodomain protein, Ind: Regulation of achaete

Ind-Gsh-type homeodomain proteins are critical to patterning of intermediate domains in the developing CNS; yet, the molecular basis for the activities of these homeodomain proteins is not well understood. This study identifies domains within the Ind protein that are responsible for transcriptional repression, as well as those required for its interaction with the co-repressor, Groucho. To do this, a combination of chimeric transient transfection assays, co-immunoprecipitation and in vivo expression assays are utilized. IndÂ’s candidate Eh1 domain is shown to be essential to the embryonic repression activity of this protein, and that Groucho interacts with Ind via this domain. However, when activity is assayed in transient transfection assays using Ind-Gal4 DNA binding domain chimeras to determine domain activity, the repression activity of the Eh1 domain is minimal. This result is similar to previous results on the transcription factors, Vnd and Engrailed. Furthermore, the Eh1 domain is necessary, but not sufficient, for binding to Groucho; the C terminus of Ind, including the homeodomain also affects the interaction with this co-repressor in co-immunoprecipitations. Finally, this study shows that aspects of the cross-repressive activities of Ind/Gsh2-Ey/Pax6 are evolutionarily conserved. Taken together, these results point to conserved mechanisms used by Gsh/Ind-type homeodomain protein in regulating the expression of target genes (Van Ohlen, 2007).

The data presented in this study indicate that the capacity of ind to repress target gene expression is conferred not only by its ability to interact with Groucho through its Eh1 domain, but also by secondary domains, which include the C terminus of the protein, wherein resides the homeodomain. Indeed, deletion of Ind's C terminus affects the repressor activity of Ind in Gal4-Ind chimeric assays in tissue culture. Ind's physical interaction with Groucho suggests that this transcription factor uses redundant protein-protein interactions to exert maximal repressor activity (Van Ohlen, 2007).

Ind's candidate Eh1 domain is required for Ind-mediated repression in embryos and in vitro. Apart from the homeodomain, this is the only Ind region that is highly conserved between flies and vertebrates. Moreover, the co-immunoprecipitation data indicate that Ind's secondary structure is important for efficient Groucho binding. The fact that the full requirement for Ind's Eh1 domain is masked in the transient transfection assay can be explained by this observation, which coincidentally parallels previous findings for the Eh1 domain of Engrailed, Nkx6, and Vnd using chimeric transfection assays. Previous studies have shown that the sequestering of Groucho to its DNA-bound transcription factor target, Dorsal, requires secondary DNA binding proteins, including Dead Ringer and Cut. Potentially, the binding of Ind to DNA via the Gal4 DBD, rather than the homeodomain, results in an altered Ind conformation relative to when the native protein contacts its DNA target via its homeodomain. This could in turn result in less efficient Groucho binding to the chimeric Gal4-Ind proteins in the transfection assay. Indeed, the transcription factor, Pax 2, must be bound to its bone fide Pax 2 target for Groucho recruitment (Van Ohlen, 2007).

Dichaete and Sox neuro interact genetically with ind. An ac enhancer represses expression of that gene, when tested in a reporter assay in transgenic embryos. It contains 3 Ind binding sites adjacent to a single Dichaete binding site. When all four sites are mutated, the reporter is partially de-repressed relative to the wild-type reporter in transgenic embryos. In addition, Ind physically interacts with Dichaete in a yeast two-hybrid expression assay. These results, and the demonstration that Ind interacts with the co-repressor, Groucho, possibly explain the relatively weak Ind over-expression phenotype, despite strong expression of the transgene. Perhaps the limited (wild-type) availability of Groucho and Dichaete in cells that over-express Ind leads to the titration, and depletion, of these essential co-repressors, such that some ectopic Ind molecules cannot exert their regulatory effects maximally (Van Ohlen, 2007).

A major function of Ind/Gsh-type transcription factors is the restriction of the expression domains of proneural genes to distinct subsets of progenitors. The proneural gene, ac, is ectopically expressed in ind mutants, and this ectopic expression of ac expression leads to the loss of intermediate neuroblasts. This study shows that over-expression of ind causes down-regulation of ac in both ventral and lateral neuroblasts. Similarly, the proneural genes, neurogenenin 1 and 2, are ectopically expressed in gsh2 mutants. Moreover, just as Gsh2 represses Pax 6 in an adjacent domain, it was similarly found that Ind can repress eyeless, the Drosophila Pax 6 homologue. Ind and its vertebrate homologues differ however in their capacity to repress msh/msx genes. Whereas, the ability of ind to repress msh expression is critical to maintaining the tri-columnar organization of the neuroectoderm in Drosophila , Msx 1 expression is unaffected in gsh1; gsh2 double mutants, and the expression domains of these two proteins overlap. Thus, Ind shares many common properties with its vertebrate homologues, but also has repression targets that are not evolutionarily conserved. The non-conserved repression domains identified in Ind, additional to the Eh1 domain, may explain the divergence in the capacity of Ind/Gsh homeodomain proteins to repress Msx-msh gene expression. Further work is required to address whether the secondary repression domains in Ind are functionally significant in the embryo. In addition, whether primary protein structure alone accounts for some of the divergent activities of ind and gsh1 or gsh2 needs to be addressed, by determining whether ind's vertebrate homologues can functionally substitute for ind function in the Drosophila embryo (Van Ohlen, 2007).

An arthropod cis-regulatory element functioning in sensory organ precursor development dates back to the Cambrian

An increasing number of publications demonstrate conservation of function of cis-regulatory elements without sequence similarity. In invertebrates such functional conservation has only been shown for closely related species. This study demonstrates the existence of an ancient arthropod regulatory element that functions during the selection of neural precursors. The activity of genes of the achaete-scute (ac-sc) family endows cells with neural potential. An essential, conserved characteristic of proneural genes is their ability to restrict their own activity to single or a small number of progenitor cells from their initially broad domains of expression. This is achieved through a process called lateral inhibition. A regulatory element, the sensory organ precursor enhancer (SOPE), is required for this process. First identified in Drosophila, the SOPE contains discrete binding sites for four regulatory factors. The SOPE of the Drosophila asense gene is situated in the 5' UTR. Through a manual comparison of consensus binding site sequences, SOPE was identified in UTR sequences of asense-like genes in species belonging to all four arthropod groups (Crustacea, Myriapoda, Chelicerata and Insecta). The SOPEs of the spider Cupiennius salei and the insect Tribolium castaneum are shown to be functional in transgenic Drosophila. This would place the origin of this regulatory sequence as far back as the last common ancestor of the Arthropoda, that is, in the Cambrian, 550 million years ago. The SOPE is not detectable by inter-specific sequence comparison, raising the possibility that other ancient regulatory modules in invertebrates might have escaped detection (Ayyar, 2010).

Regulatory sequences involved in the restriction of proneural gene expression from proneural domains to selected neural precursors have mostly been studied in Drosophila, in particular with respect to the ac-sc genes and their role in the development of sensory bristles of the adult peripheral nervous system. The D. melanogaster ac-sc gene complex (AS-C) comprises four genes, three of which are required for bristle development. ac and sc are expressed in discrete proneural clusters through the activity of a number of independently acting cis-regulatory modules that are scattered throughout the approximately 150 kb of the AS-C and respond to positional cues. Subsequently, the expression of ac and sc refines to single sensory organ precursors (SOPs) where high levels of Ac/Sc activate the third gene, asense (ase), whose expression is limited to SOPs. Lateral inhibition and SOP expression is mediated by a specific cis-regulatory element, the SOP enhancer (SOPE). The SOPE contains binding sites for a number of transcription factors. Auto-regulation in the SOP relies on E boxes, binding sites for Ac, Sc and Ase, which activate their own transcription. The E boxes also mediate repression in cells not selected to be SOPs: products of the Enhancer of split (E(spl)) genes activated by Notch signaling associate with Ac-Sc, leading to transcriptional repression. Binding sites for NF-κB proteins, α boxes, are present and also mediate both activation and repression. It is likely that low levels of NF-κB and high levels of Ac-Sc activate, whereas high levels of NF-κB and low levels of Ac-Sc repress, the neural program. In addition, the SOPEs contain AT-rich sequences, β boxes, of unknown function and N boxes that, in the case of the ac-SOPE, have been shown to bind the transcriptional repressor Hairy. All three genes bear their own SOPE. That of ac is in the promoter close to the transcription start site and differs from the others in being devoid of α boxes. It drives expression of reporter genes first in proneural domains and then in SOPs. The SOPE of sc, positioned 3 kb upstream of the transcriptional start site, and that of ase, positioned in the 5' UTR, drive expression of reporter genes exclusively in the SOP. The SOPEs are strongly conserved in other Drosophilidae (Ayyar, 2010).

Proneural genes of both the ac-sc and ato classes have undergone independent duplication events in different taxa. The ato gene family is much expanded in vertebrates whereas duplication of ac-sc genes has taken place in different groups of arthropods. Previous data from available insect genomes have shown that while ac-sc genes have undergone a number of duplication events, all species analyzed bear a single ase gene. Conservation of both specific amino acid sequences and the SOPE in the 5' UTR suggest that the insect ase genes are derived from a common ancestor. This study shows that achaete-scute homologue (ASH) and ase-like genes are present in arthropods other than insects. Evidence is presented that gene duplications separating proneural from precursor-specific (ase-like) functions possibly occurred independently in different arthropod groups and that a SOPE in UTR sequences in ase-like genes of all groups has been inherited from an ancestral ASH/ase precursor gene in the last common ancestor of the Arthropoda (Ayyar, 2010).

Differential regulation of transcription through distinct Suppressor of Hairless DNA binding site architectures during Notch signaling in proneural clusters

In Drosophila, achaete (ac) and m8 are model basic helix-loop-helix activator (bHLH A) and repressor genes, respectively, that have the opposite cell expression pattern in proneural clusters during Notch signaling. Previous studies have shown that activation of m8 transcription in specific cells within proneural clusters by Notch signaling is programmed by a 'combinatorial' and 'architectural' DNA transcription code containing binding sites for the Su(H) and proneural bHLH A proteins. The study shows that the ac promoter contains a similar combinatorial code of Su(H) and bHLH A binding sites but contains a different Su(H) site architectural code that does not mediate activation during Notch signaling, thus programming a cell expression pattern opposite that of m8 in proneural clusters (Cave, 2011).

The results provide important new insights into the DNA transcription codes that program cell-specific gene expression in response to Notch signaling. The ac promoter contains an S-site architecture that mediates repression, not activation, during Notch signaling in proneural clusters. Given that there are unpaired S sites in the promoters of many other proneural and panneural genes, it is predicted that some, or potentially all, of these S sites could mediate repression in cells where Notch is activated. This differential activation versus repression of gene transcription programmed by distinct S-site architectures greatly expands the potential regulatory complexity of pathways mediated by Notch signaling. Previous studies suggested that specific S-site architectures (S-site 'subcodes') programmed specific interactions between Notch complexes on S sites and specific combinatorial coactivator proteins bound to nearby DNA sites. Together with these previous findings, the current study provides an important and novel understanding of the role that S-site architecture plays in mediating differential transcriptional responses to Notch signaling. Given that at least some aspects of the S-site architectural codes are functionally conserved in mammals, it will be interesting and important to test whether the same differential regulation mechanisms are conserved in mammals (Cave, 2011)

Transcriptional Regulation (part 1/3)

Achaete regulation in the neurectoderm

achaete and scute transcription become confined to the neurectoderm through the action of pair-rule, dorsal-ventral polarity and segment polarity genes.

Extramachrochaete negatively regulates achaete (van Doren, 1994).

Daughterless/Achaete-Scute complexes bind at three sites of achaete, thus reinforcing an autoregulatory loop (Ohsako, 1994).

Although ac and scute have similar patterns of expression, deletion of either gene removes specific subsets of sensory organs. This specificity was shown to reside in the peculiar regulation of ac and sc expression. These genes are first activated in complementary spatial domains in response to different cis-regulatory sequences. Each gene product then stimulates expression of the other gene, thus generating similar patterns of expression. Therefore, removal of one gene leads to the absence of both proneural gene products and sensory organs in the sites specified by its cis-regulatory sequences (Martinez, 1991).

ventral nervous system condensation defective (VND), also known as NK2, is absolutely required to activate ac, sc and l'sc gene expression in proneural clusters in specific domains along the medial column of the earliest arising neuroblasts. VND controls proneural gene expression at two distinct steps during neuroblast formation through separate regulatory regions. First, VND is required to activate proneural cluster formation within the medial column of every other neuroblast row, through regulatory elements located 3' to ac. Second, VND functions to increase or maintain proneural gene expression in the cell within the proneural cluster that normally becomes the neuroblast. It does this through a 5' regulatory region (Skeath, 1994).

Neurogenesis in Drosophila melanogaster starts by an ordered appearance of neuroblasts arranged in three columns (medial, intermediate and lateral) in each side (right and left) of the neuroectoderm. In the intermediate column, the receptor tyrosine kinase Egfr represses expression of proneural genes achaete and scute, and is required for the formation of neuroblasts. Most of the early function of Egfr is likely to be mediated by the Ras-MAP kinase signaling pathway, which is activated in the intermediate column, since a loss of a component of this pathway leads to a phenotype identical to that of Egfr mutants. MAP-kinase activation is also observed in the medial column where escargot (esg) and proneural gene expression are unaffected by Egfr. The homeobox gene ventral nerve system defective (vnd) is required for the expression of esg and scute in the medial column. vnd acts through the negative regulatory region of the esg enhancer that mediates the Egfr signal, suggesting vnd's role is to counteract Egfr-dependent repression. Thus, the nested expression of vnd and the Egfr activator Rhomboid is crucial to subdivide the neuroectoderm into the three dorsoventral domains (Yagi, 1998).

To investigate the involvement of Egfr in neurogenesis, mutant phenotypes of Efgr and its activator rho were examined at various stages of neurogenesis. The dorsoventral subdivision of the neuroectoderm in stage-6 embryos is detectable by expression of esg, which is expressed in the lateral and medial columns but not in the intermediate column. A loss-of-function, temperature-sensitive mutation of Egfr and a null mutation of rhomboid were used for analysis throughout this work. Egfr and rho mutations cause ectopic expression of esg in the intermediate column. Repression of esg in the intermediate column is likely to require a relatively high dose of Egfr signal. To examine the potential role of Egfr in neurogenesis, expression of the proneural genes ac and sc was carried out. These two proneural genes begin expression in the neuroectoderm of stage-7 embryos in a DV pattern of expression similar to that of esg in the previous stage. In Egfr and rho mutant embryos, ac and sc become ectopically expressed in the intermediate column. This phenotype is less penetrant and, occasionally, gaps of ac and sc expression are observed in the intermediate column. Since sc expression was similarly derepressed in Egfr mutant embryos, these phenotypes are likely to represent the near null phenotype of Egfr in the neuroectoderm. These data indicate that, in the intermediate column, the Egfr signal represses not only esg but also proneural genes, known to play key roles in neurogenesis. The effect of Egfr on neuroblast formation was monitored by the neuroblast marker Snail. Anti-Sna staining reveals three columns of SI neuroblasts in the control embryo: the intermediate column is distinguishable by the delayed onset of formation and number of Sna-positive cells. In Egfr and rho mutants, Sna-positive neuroblasts in the intermediate position are frequently missing, with a higher frequency of loss in Egfr embryos. In rho mutant embryos, the frequency of the loss of intermediate column neuroblasts is variable among embryos (Yagi, 1998).

The Sox-domain-containing gene Dichaete/fish-hook plays a crucial role in patterning the neuroectoderm along the DV axis. Dichaete is expressed in the medial and intermediate columns of the neuroectoderm, and mutant analysis indicates that Dichaete regulates cell fate and neuroblast formation in these domains. Molecular epistasis tests, double mutant analysis and dosage-sensitive interactions demonstrate that during these processes, Dichaete functions in parallel with ventral nerve cord defective and intermediate neuroblasts defective, and downstream of EGF receptor signaling to mediate its effect on development. These results identify Dichaete as an important regulator of dorsoventral pattern in the neuroectoderm, and indicate that Dichaete acts in concert with ventral nerve cord defective and intermediate neuroblasts defective to regulate pattern and cell fate in the neuroectoderm (Zhao, 2002).

Double labeling Dichaete mutant embryos for ac and ind, and double labeling ind mutant embryos for ac and Dichaete reveals an interdependent relationship between Dichaete and ind. In Dichaete mutant embryos, a significant number of row 3 and 7 intermediate column cells and NBs co-express ac and ind -- an occurrence never observed in wild-type embryos. Thus, the ability of ind to repress ac in the intermediate column requires Dichaete activity. Reciprocally, in ind mutant embryos, all row 3 and 7 intermediate column cells co-express ac and Dichaete. Thus, the ability of Dichaete to repress ac in the intermediate column requires ind activity (Zhao, 2002).

Genetic interactions between Dichaete and ind were tested. The partial derepression of ac expression and the incomplete loss of an Eve-positive RP2 neuron are the most sensitive assays for Dichaete function in the intermediate column. However, strong alleles of ind cause a complete derepression of ac expression, and a complete loss of RP2 neurons in this domain. Thus, an analysis of Dichaete ind double mutant embryos using these markers would be uninformative. To circumvent this problem, a test was performed to see whether ind dominantly enhances the Dichaete intermediate column ac and RP2 phenotypes. Embryos heterozygous for ind exhibit wild-type ac expression and RP2 formation. However, Dichaete ind/Dichaete + mutant embryos exhibit enhanced derepression of ac expression and an approximately threefold enhancement of the RP2 loss phenotype relative to Dichaete mutant embryos. The dominant enhancement of the Dichaete phenotype by ind reveals a genetic interaction between Dichaete and ind (Zhao, 2002).

Dichaete is expressed and regulates cell fate in the medial and intermediate neuroectodermal columns. However, Dichaete carries out distinct functions in each domain: Dichaete represses ac expression in the intermediate column but has no effect on ac expression in the medial column where Dichaete and ac are co-expressed (Zhao, 2002).

Sox proteins form a family of HMG-box transcription factors related to SRY, the mammalian testis determining factor. Sox-mediated modulation of gene expression plays an important role in various developmental contexts. Drosophila SoxNeuro, a putative ortholog of the vertebrate Sox1, Sox2 and Sox3 proteins, is one of the earliest transcription factors to be expressed pan-neuroectodermally. SoxNeuro is essential for the formation of the neural progenitor cells in the central nervous system. Loss of function mutations of SoxNeuro are associated with a spatially restricted hypoplasia: neuroblast formation is severely affected in the lateral and intermediate regions of the central nervous system, whereas ventral neuroblast formation is almost normal. Evidence is presented that a requirement for SoxNeuro in ventral neuroblast formation is masked by a functional redundancy with Dichaete, a second Sox protein whose expression partially overlaps that of SoxNeuro. SoxNeuro/Dichaete double mutant embryos show a severe neural hypoplasia throughout the central nervous system, as well as a dramatic loss of achaete expressing proneural clusters and medially derived neuroblasts. Genetic interactions of SoxNeuro and the dorsoventral patterning genes ventral nerve chord defective (vnd) and intermediate neuroblasts defective (ind) underlie ventral and intermediate neuroblast formation. Expression of the Achaete-Scute gene complex suggests that SoxNeuro acts upstream and in parallel with the proneural genes. The finding that Dichaete and SoxN exhibit opposite effects on achaete expression within the intermediate neuroectoderm demonstrates that each protein also has region-specific unique functions during early CNS development in the Drosophila embryo (Buescher, 2002 and Overton, 2002).

The differential loss of NBs and their progeny in the DV axis in SoxN mutants may result from the failure of neuroectodermal cells to be specified to a neural fate. Since SoxN is expressed throughout the neuroectoderm prior to neuroblast delamination and Dichaete is reported to have effects on proneural gene expression (Zhao, 2002), proneural gene expression was examined in SoxNU6–35 mutants. In SoxNU6–35 a loss of lateral column ac expression is seen as well as an overall reduction in ac levels. When the double mutants are examined for ac expression, a synergistic and an additive effect is seen. As with SoxN, the overall level of ac expression is lower compared with heterozygous siblings and there is a marked loss of lateral column ac expression. In addition, the double mutants display the Dichaete phenotype since ectopic intermediate column expression is seen in some rows (6%). However, in 21% of segments no ac expression is seen, suggesting that both Sox genes are principally able to positively regulate ac expression. Taken together, it is concluded that in the neuroectoderm, the elimination of group B Sox expression results in an early failure in neural specification and subsequent loss of neural progenitors (Overton, 2002).

ac is a marker for certain medial and lateral proneural clusters but is not normally expressed in the intermediate column. A striking reduction was observed in the SoxN mutant in the number of lateral column proneural clusters expressing ac. In 70% of these clusters, ac expression is no longer detected, compared with 12% of medial column proneural clusters. The loss of lateral column ac expression suggests that SoxN functions early in the neuroectoderm to specify proneural clusters correctly. In addition to this, there is an overall reduction in ac expression levels in the medial proneural clusters compared with the heterozygous sibling embryos stained in the same reaction. This implies that SoxN is required more generally in the neuroectoderm to establish the appropriate level of ac expression. The expression was examined of the related proneural gene l'sc in the neuroectoderm prior to neuroblast delamination and, in contrast to the results with ac, no appreciable effect was seen. Thus it appears that SoxN is selectively required in the neuroectoderm for the regulation of some proneural gene expression. The loss of ac expression in lateral proneural clusters partly explains why such a dramatic loss of lateral NBs is seen in SoxNU6–35 mutants. However, all lateral NBs are strongly affected in SoxNU6–35 embryos, including those that express l'sc and not ac. Hence, the normal expression of l'sc in SoxNU6–35 mutant embryos argues against a simple linear pathway in which SoxN acts only upstream of proneural genes, and suggests a mechanism in which SoxN functions both upstream and in parallel to the proneural genes to promote neuroblast formation. This parallel function of SoxN is additionally supported by the observation that the severe hypoplasia of SoxN mutant embryos resembles the phenotype in AS-C mutants, and is more severe than can be accounted for by the effect on ac expression, as loss of ac alone does not produce severe phenotypes (Overton, 2002).

A derepression of ac expression in the intermediate column has been reported in Dichaete mutants has been reported (Zhao, 2002) and those observations were confirmed. Therefore, whereas both SoxN and Dichaete mutants show loss of neuroblasts, the effect in the neuroectoderm differs: SoxN mutants display loss of ac expression but Dichaete mutants show some ac derepression (Overton, 2002).

Interestingly, the manner in which NBs are lost in SoxN and AS-C mutants appears mechanistically different. In AS-C mutants only a small proportion of NBs fails to be singled out and fails to delaminate from the NE (~25% of early NBs). The majority of NBs still segregate and later may be subject to cell death. By contrast, in SoxN mutant embryos, neuroectodermal cells fail to be singled out as NBs and delamination does not take place. Thus, it appears that loss of SoxN affects NBs formation at an earlier step than the loss of proneural genes. Proneural gene expression is regulated largely independently of SoxN, since loss of SoxN does not affect the neuroectodermal expression of L'sc and does not abolish that of Ac. It is suggested that SoxN acts upstream and in parallel to the proneural genes. Comparison of the NB phenotypes of AS-C mutant and SoxN mutant embryos has revealed that overlapping but not identical subsets of NBs were affected. This result suggests that SoxN function -- as it is understood it at this time -- does not explain why some NBs do not require the proneural genes of the AS-C. The binary decision of neuroectodermal cells to adopt the neural or the epidermal fate requires Notch signaling (Buescher, 2002).

Loss of SoxN results in a severe loss of NBs. Expression studies show that SoxN protein is present in the NE before and during the entire process of neurogenesis. Hence, the expression pattern provides no clue as to which step(s) depend on SoxN function. To approach this question, two key steps in neurogenesis were studied: (1) the establishment cell clusters with neural potential and (2) the 'singling out' of NBs (Buescher, 2002).

The proneural genes of the AS-C have been shown to be essential for the promotion of NB formation and deletion of the entire gene complex results in the loss of ~75% of all NBs. Many NBs that normally derive from clusters of neuroectodermal cells, which express either ac, sc, l'sc or a combination of these genes, fail to form in SoxN mutant embryos. This raises the question of whether proneural genes are still expressed in a SoxN mutant background in clusters of ectodermal cells, and, if so, do they still confer neural potential to these cells? In wild-type embryos, prior to NB segregation (stage 8), Ac protein is found in cell clusters in rows 3 and 7 in the ventral and lateral column of the NE, while L'sc is found in stripes of two to three cell widths that transverse the entire NE. Staining of stage 8 SoxN mutant embryos with anti-L'sc antibody revealed no appreciable difference from wild-type L'sc expression. Staining with anti-Ac antibody showed that Ac expression is initiated in both ventral and lateral clusters, but expression levels appear reduced and show significant variation in lateral cell clusters (Buescher, 2002).

In wild-type embryos, the process of lateral inhibition results in the singling out of one cell per proneural cluster that will enter the neural pathway. This process is accompanied by an upregulation of proneural gene expression, delamination of the NB from the neuroectodermal layer and the initiation of expression of a set of neuronal precursor genes. In stage 9 SoxN mutant embryos, a failure in the upregulation of Ac expression was frequently observed in lateral proneural clusters. In those instances in which Ac was still upregulated, expression was less robust than in wild type and varied significantly among different hemisegments. Variation of Ac expression levels was also apparent in ventrally delaminating cells. The failure to upregulate Ac expression was accompanied by a failure in cell delamination. Moreover, the expression of neuronal precursor genes was severely affected: in wild type, one of the earliest precursor genes to be expressed is asense (ase); ase is expressed in all delaminating NBs. In SoxN mutant embryos, ase expression was strongly reduced. These results suggest that in SoxN mutant embryos the establishment of proneural clusters is impaired but not abolished. The subsequent process of singling out NBs is severely defective (Buescher, 2002).

Prospero, targeting achaete, acts as a binary switch between self-renewal and differentiation in Drosophila neural stem cells

Stem cells have the remarkable ability to give rise to both self-renewing and differentiating daughter cells. Drosophila neural stem cells segregate cell-fate determinants from the self-renewing cell to the differentiating daughter at each division. This study shows that one such determinant, the homeodomain transcription factor Prospero, regulates the choice between stem cell self-renewal and differentiation. The in vivo targets of Prospero have been identified throughout the entire genome. Prospero represses genes required for self-renewal, such as stem cell fate genes and cell-cycle genes. Surprisingly, Prospero is also required to activate genes for terminal differentiation. In the absence of Prospero, differentiating daughters revert to a stem cell-like fate: they express markers of self-renewal, exhibit increased proliferation, and fail to differentiate. These results define a blueprint for the transition from stem cell self-renewal to terminal differentiation (Choksi, 2006).

To identify sites within the Drosophila genome to which Prospero binds, use was made of an in vivo binding-site profiling technique, DamID. DamID is an established method of determining the binding sites of DNA- or chromatin-associated proteins. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation (ChIP). DamID enables binding sites to be tagged in vivo and later identified on DNA microarrays. In brief, the DNA or chromatin-binding protein of interest is fused to an Escherichia coli adenine methyltransferase (Dam), and the fusion protein is expressed in vivo. The DNA-binding protein targets the fusion protein to its native binding sites, and the Dam methylates local adenine residues in the sequence GATC. The sequences near the protein-DNA interaction site are thereby marked with a unique methylation tag, over approximately 2-5 kilobase pairs (kb) from the binding site. The tagged sequences can be isolated after digestion with a methylation-sensitive restriction enzyme, such as DpnI (Choksi, 2006).

Dam was fused to the N terminus of Prospero, and transgenic flies were generated. The fusion protein is expressed from the uninduced minimal Hsp70 promoter of the UAS vector, pUAST, as high levels of expression of Dam can result in extensive nonspecific methylation and cell death. As a control for nonspecific Dam activity, animals expressing Dam alone were generated. To assess the sites to which Prospero binds during neurogenesis, genomic DNA was extracted from stage 10-11 embryos, approximately 4-7 hr after egg laying (AEL), expressing either the Dam-Prospero fusion protein or the Dam protein alone. The DNA was digested with DpnI and amplified by PCR. DNA from Dam-Prospero embryos was labeled with Cy3, and control DNA with Cy5. The samples were then cohybridized to genomic microarrays. Microarrays were designed that tile the entire euchromatic Drosophila genome. A 60 base oligonucleotide was printed for approximately every 300 bp of genomic DNA, resulting in roughly 375,000 probes on a single array (Choksi, 2006).

Log-transformed ratios from four biological replicates (two standard dye configurations plus two swapped dye configurations) were normalized and averaged. Regions of the genome with a greater than 1.4-fold log ratio (corresponding to approximately a 2.6-fold enrichment) of Dam-Prospero to the control over a minimum of four adjacent genomic probes were identified as in vivo Prospero binding sites. Using these parameters, a total of 1,602 in vivo Prospero binding sites were identified in the Drosophila genome. This work demonstrates that it is possible to map in vivo binding sites across the whole genome of a multicellular organism (Choksi, 2006).

Prospero is known to regulate the differentiation of photoreceptors in the adult eye, and recently sites have been characterized to which Prospero can bind upstream of two Rhodopsin genes, Rh5 and Rh6. A variant of the Prospero consensus sequence is found four times upstream of Rh5 and four times upstream of Rh6. Prospero was shown to bind this sequence in vitro, by band shift assay, and also by a 1-hybrid interaction assay in yeast. In addition, deletion analysis demonstrated that the consensus sequence is required for the Pros-DNA interaction both in vivo and in vitro. It was found that 67% of in vivo binding sites contain at least one Prospero binding motif. Combining in vivo binding-site data with searches for the Prospero consensus sequence reveals 1,066 distinct sites within the Drosophila genome to which Prospero binds during embryogenesis (Choksi, 2006).

A total of 730 genes have one or more of the 1,066 Prospero binding sites located within 1 kb of their transcription unit. Statistical analyses to determine GO annotation enrichment on the members of the gene list that had some associated annotation (519) was performed by using a web-based set of tools, GOToolbox. Using Biological Process (GO: 0008150) as the broadest classification, a list was generated of overrepresented classes of genes (Choksi, 2006).

The three most significant classes of genes enriched in the list of putative Prospero targets are Cell Fate Commitment, Nervous System Development, and Regulation of Transcription. Utilizing GO annotation, it was found that nearly 41% of all annotated neuroblast fate genes (11 of 27) are located near Prospero binding sites and that approximately 9% of known cell-cycle genes are near Prospero binding sites. These include the neuroblast genes achaete (ac), scute (sc), asense (ase), aPKC, and mira and the cell-cycle regulators stg and CycE. In addition, it was found that the Drosophila homolog of the mammalian B lymphoma Mo-MLV insertion region 1 (Bmi-1) gene, Posterior sex combs, is located near a Prospero binding site. Bmi-1 is a transcription factor that has been shown to regulate the self-renewal of vertebrate hematopoetic stem cells. It is concluded that Prospero is likely to regulate neuroblast identity and self-renewal genes as well as cell-cycle genes directly, repressing their expression in the GMC (Choksi, 2006).

Prospero enters the nucleus of GMCs, and its expression is maintained in glial cells but not in neurons . Therefore the list of targets was searched for genes annotated as glial development genes. Prospero binds near 45% of genes involved in gliogenesis. Among the glial genes, it was found that the master regulator of glial development, glial cells missing (gcm), and gilgamesh (gish), a gene involved in glial cell migration, are both near Prospero binding sites and are likely directly activated by Prospero in glia (Choksi, 2006).

In summary, Prospero binds near, and is likely to regulate directly, genes required for the self-renewing neural stem cell fate such as cell-cycle genes. It was also found that Prospero binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh) and to genes required for glial cell fate. The in vivo binding-site mapping experiments are supportive of a role for Prospero in regulating the fate of Drosophila neural precursors by directly controlling their mitotic potential and capacity to self-renew (Choksi, 2006).

The Drosophila ventral nerve cord develops in layers, in a manner analogous to the mammalian cortex. The deepest (most dorsal) layer of the VNC comprises the mature neurons, while the superficial layer (most ventral) is made up of the mitotically active, self-renewing neuroblasts. Neuroblast cell-fate genes and cell-cycle genes are normally expressed only in the most ventral cells, while Prospero is found in the nucleus of the more dorsally lying GMCs. If in GMCs, Prospero normally acts to repress neuroblast cell-fate genes and cell-cycle genes, then in a prospero mutant, expression of those genes should expand dorsally. Conversely, ectopically expressed Prospero should repress gene expression in the neuroblast layer.

The neuroblast genes mira, ase, and insc and the cell cycle genes CycE and stg show little or no expression in differentiated cells of wild-type stage 14 nerve cords. Expression of these neuroblast-specific genes was examined in the differentiated cells layer of prospero embryos and it was found that they are derepressed throughout the nerve cord of mutant embryos. mira, ase, insc, CycE, and stg are all ectopically expressed deep into the normally differentiated cell layer of the VNC. To check whether Prospero is sufficient to repress these genes, Prospero was expressed with the sca-GAL4 driver, forcing Prospero into the nucleus of neuroblasts. Prospero expression is sufficient to repress mira, ase, insc, CycE, and stg in the undifferentiated cell layer of the VNC. These data, combined with the Prospero binding-site data, demonstrate that Prospero is both necessary and sufficient to directly repress neuroblast genes and cell-cycle genes in differentiated cells. This direct repression of gene expression is one mechanism by which Prospero initiates the differentiation of neural stem cells (Choksi, 2006).

Having shown that Prospero directly represses genes required for neural stem cell fate, it was asked whether Prospero also directly activates GMC-specific genes. Alternatively, Prospero might regulate a second tier of transcription factors, which are themselves responsible for the GMC fate. Of the few previously characterized GMC genes, it was found that Prospero binds to eve and fushi-tarazu (ftz). In the list of targets several more GMC genes were expected to be found, but not genes involved in neuronal differentiation, since Prospero is not expressed in neurons. Surprisingly, however, it was foudn 18.8% of neuronal differentiation genes are located near Prospero binding sites (Choksi, 2006).

To determine Prospero's role in regulating these neuronal differentiation genes, in situ hybridization was carried out on prospero mutant embryos. Prospero was found to be necessary for the expression of a subset of differentiation genes, such as the adhesion molecules FasciclinI (FasI) and FasciclinII (FasII), which have roles in axon guidance and/or fasciculation. Netrin-B, a secreted protein that guides axon outgrowth, and Encore, a negative regulator of mitosis, also both require Prospero for proper expression. Therefore, in addition to directly repressing genes required for neural stem cell self-renewal, Prospero binds and activates genes that direct differentiation. These data suggest that Prospero is a binary switch between the neural stem cell fate and the terminally differentiated neuronal fate (Choksi, 2006).

To test to what extent Prospero regulates the genes to which it binds, genome-wide expression profiling was carried out on wild-type and prospero mutant embryos. While the DamID approach identifies Prospero targets in all tissues of the embryo, in this instance genes regulated by Prospero were assayed in the developing central nervous system. Small groups of neural stem cells and their progeny (on the order of 100 cells) were isolated from the ventral nerve cords of living late stage 12 embryos with a glass capillary. The cells were expelled into lysis buffer, and cDNA libraries generated by reverse transcription and PCR amplification. cDNA libraries prepared from neural cells from six wild-type and six prospero null mutant embryos were hybridized to full genome oligonucleotide microarrays, together with a common reference sample. Wild-type and prospero mutant cells were compared indirectly through the common reference (Choksi, 2006).

In the group of Prospero target genes that contain a Prospero consensus sequence within 1 kb of the transcription unit, 91 show reproducible differences in gene expression in prospero mutants. Seventy-nine percent of these genes (72) exhibit at least a 2-fold change in levels of expression. Many of the known genes involved in neuroblast fate determination and cell-cycle regulation (e.g., asense, deadpan, miranda, inscuteable, CyclinE, and string) show increased levels in a prospero mutant background, consistent with their being repressed by Prospero. Genes to which Prospero binds, but which do not contain an obvious consensus sequence, are also regulated by Prospero: CyclinA and Bazooka show elevated mRNA levels in the absence of Prospero, as does Staufen, which encodes a dsRNA binding protein that binds to both Miranda and to prospero mRNA (Choksi, 2006).

Expression of genes required for neuronal differentiation is decreased in the prospero mutant cells, consistent with Prospero being required for their transcription. These include zfh1 and Lim1, which specify neuronal subtypes, and FasI and FasII, which regulate axon fasciculation and path finding (Choksi, 2006).

The stem cell-like division of neuroblasts generates two daughters: a self-renewing neuroblast and a differentiating GMC. Prospero represses stem cell self-renewal genes and activates differentiation genes in the newly born GMC. In the absence of prospero, therefore, neuroblasts should give rise to two self-renewing neuroblast-like cells (Choksi, 2006).

The division pattern of individual neuroblasts was studied in prospero mutant embryos by labeling with the lipophilic dye, DiI. Individual cells were labeled at stage 6, and the embryos allowed to develop until stage 17. S1 or S2 neuroblasts were examined, as determined by their time of delamination. Wild-type neuroblasts generate between 2 and 32 cells, producing an average of 16.2 cells. Most of the clones exhibit extensive axonal outgrowth. In contrast, prospero mutant neuroblasts generate between 8 and 51 cells, producing an average of 31.8 cells. Moreover, prospero mutant neural clones exhibit few if any projections, and the cells are smaller in size. Thus, prospero mutant neuroblasts produce much larger clones of cells with no axonal projections, suggesting that neural cells in prospero mutants undergo extra divisions and fail to differentiate (Choksi, 2006).

Recently it was shown, in the larval brain, that clones of cells lacking Prospero or Brat undergo extensive cell division to generate undifferentiated tumors. Given that Prospero is nuclear in the GMC but not in neuroblasts, the expanded neuroblast clones in prospero mutant embryos might arise from the overproliferation of GMCs: the GMCs lacking Prospero may divide like neuroblasts in a self-renewing manner. It is also possible, however, that neuroblasts divide more frequently in prospero mutant embryos, giving rise to supernumerary GMCs that each divide only once. To distinguish between these two possibilities, the division pattern of individual GMCs was followed in prospero mutant embryos (Choksi, 2006).

S1 or S2 neuroblasts were labeled with DiI as before. After the first cell division of each neuroblast, the neuroblast was mechanically ablated, leaving its first-born GMC. All further labeled progeny derive, therefore, from the GMC. Embryos were allowed to develop until stage 17, at which time the number of cells generated by a single GMC was determined (Choksi, 2006).

To determine whether mutant GMCs are transformed to a stem cell-like state, stage 14 embryos were stained for the three neuroblast markers: Miranda (Mira), worniu (Wor), and Deadpan (Dpn). In wild-type embryos at stage 14, the most dorsal layer of cells in the VNC consists mostly of differentiated neurons. As a result, few or none of the cells in this layer express markers of self-renewal. Mira-, Wor-, and Dpn- expressing cells are found on the midline only or in lateral neuroblasts of the differentiated cell layer of wild-type nerve cords. In contrast, a majority of cells in the differentiated cell layer of stage 14 prospero mutant embryos express all three markers: Mira is found cortically localized in most cells of the dorsal layer of prospero nerve cords; Wor is nuclear in most cells of mutant VNCs; Dpn is ectopically expressed throughout the nerve cord of prospero mutants (Choksi, 2006).

Expression of neuroblast markers in the ventral-most layer of the nerve cord (the neuroblast layer), to exclude the possibility that a general disorganization of cells within the VNC contributes to the increased number of Mira-, Wor-, and Dpn-positive cells in the dorsal layer. The number of neuroblasts in a prospero mutant embryo is normal in stage 14 embryos, as assayed by Wor, Dpn, and Mira expression. Thus, the increased expression of neuroblast markers in prospero mutants is the result of an increase in the total number of cells expressing these markers in the differentiated cell layer. It is concluded that prospero mutant neuroblasts divide to give two stem cell-like daughters. GMCs, which would normally terminate cell division and differentiate, are transformed into self-renewing neural stem cells that generate undifferentiated clones or tumors (Choksi, 2006).

Therefore, Prospero directly represses the transcription of many neuroblast genes and binds near most of the temporal cascade genes: hb, Kruppel (Kr), nubbin (nub/pdm1), and grainyhead (grh), which regulate the timing of cell-fate specification in neuroblast progeny. Prospero maintains hb expression in the GMC, and it has been suggested that this is through regulation of another gene, seven-up (svp). Prospero not only regulates svp expression directly but also maintains hb expression directly. In addition, Prospero maintains Kr expression and is likely to act in a similar fashion on other genes of the temporal cascade. Intriguingly, Prospero regulates several of the genes that direct asymmetric neuroblast division (baz, mira, insc, aPKC). aPKC has recently been shown to be involved in maintaining the self-renewing state of neuroblasts (Choksi, 2006).

Prospero initiates the expression of genes necessary for differentiation. This is particularly surprising since prospero is transcribed only in neuroblasts, not in GMCs or neurons. Prospero mRNA and protein are then segregated to the GMC. Prospero binds near eve and ftz, which have been shown to be downstream of Prospero, as well as to genes required for terminal neuronal differentiation, including the neural-cell-adhesion molecules FasI and FasII. Prospero protein is present in GMCs and not neurons, suggesting that Prospero initiates activation of neuronal genes in the GMC. The GMC may be a transition state between the neural stem cell and the differentiated neuron, providing a window during which Prospero functions to repress stem cell-specific genes and activate genes required for differentiation. There may be few, or no, genes exclusively expressed in GMCs (Choksi, 2006).

Prospero acts in a context-dependent manner, functioning alternately to repress or activate transcription. This implies that there are cofactors and/or chromatin remodeling factors that modulate Prospero's activity. In support of this, although Prospero is necessary and sufficient to repress neuroblast genes, forcing Prospero into the nuclei of neuroblasts is not sufficient to activate all of the differentiation genes to which it binds (Choksi, 2006).

Neuroblasts decrease in size with each division throughout embryogenesis. By the end of embryogenesis, they are similar in size to neurons. A subset of these embryonic neuroblasts becomes quiescent and is reactivated during larval life: they enlarge and resume stem cell divisions to generate the adult nervous system. Neuroblasts in prospero mutant embryos divide to produce two self-renewing daughters but still divide asymmetrically with respect to size, producing a large apical neuroblast and a smaller basal neuroblast-like cell. The daughter may be too small to undergo more than three additional rounds of division during embryogenesis. prospero mutant cells eventually stop dividing, and a small number occasionally differentiate. This suggests that there is an inherent size limitation on cell division. The segregation of Brat, or an additional cell fate determinant, to the daughter cell may also limit the potential of the prospero mutant cells to keep dividing (Choksi, 2006).

The Prox family of atypical homeodomain transcription factors has been implicated in initiating the differentiation of progenitor cells in contexts as varied as the vertebrate retina, forebrain, and lymphatic system. Prospero/Prox generally regulates the transition from a multipotent, mitotically active precursor to a differentiated, postmitotic cell. In most contexts, Prox1 acts in a similar fashion to Drosophila Prospero: to stop division and initiate differentiation (Choksi, 2006).

It is proposed that Prospero/Prox is a master regulator of the differentiation of progenitor cells. Many of the vertebrate homologs of the Drosophila Prospero targets identified in this study may also be targets of Prox1 in other developmental contexts. Prospero directly regulates several genes required for cell-cycle progression, and it is possible that Prox1 will regulate a similar set of cell-cycle genes during, for example, vertebrate retinal development. In addition, numerous Prospero target genes have been identified whose orthologs may be involved in the Prox-dependent differentiation of retina, lens, and forebrain precursors (Choksi, 2006).

Achaete function in the formation of the stomatogastric nervous system

The gut-innervating stomatogastric nervous system of Drosophila, unlike the central and the peripheral nervous system, derives from a compact, single layered epithelial anlage. This anlage is initially defined during embryogenesis by the expression of proneural genes of the ac-sc complex in response to the maternal terminal pattern forming system. Within the stomatogastric nervous system anlage, the wingless-dependent intercellular communication system adjusts the cellular range of Notch-dependent lateral inhibition to single-out (specify) three achaete-expressing cells. Those cells define distinct invagination centers that orchestrate the behavior of neighboring cells to form epithelial infoldings, each headed by an achaete-expressing tip cell. The wingless pathway appears to act not as an instructive signal, but as a permissive factor which coordinates the spatial activity of morphoregulatory signals within the stomatogastric nervous system anlage (Gonzalez-Gaitan, 1995).

The segmented portion of the Drosophila embryonic central nervous system develops from a bilaterally symmetrical, segmentally reiterated array of 30 unique neural stem cells, called neuroblasts. The first 15 neuroblasts form about 30-60 minutes after gastrulation in two sequential waves of neuroblast segregation and are arranged in three dorsoventral columns and four anteroposterior rows per hemisegment. Each neuroblast acquires a unique identity, based on gene expression and the unique and nearly invariant cell lineage that this expression produces. Little is known as to the control of neuroblast identity along the DV axis. The Drosophila Egfr receptor (Egfr) has been shown to promote the formation, patterning and individual fate specification of early forming neuroblasts along the DV axis. Molecular markers identify particular neuroectodermal domains, composed of neuroblast clusters or single neuroblasts, and show that in Egfr mutant embryos (1) intermediate column neuroblasts do not form; (2) medial column neuroblasts often acquire identities inappropriate for their position, while (3) lateral neuroblasts develop normally. Active Egfr signaling occurs in the regions from which the medial and intermediate neuroblasts will later delaminate. The concomitant loss of rhomboid and vein yields CNS phenotypes indistinguishable from Egfr mutant embryos, even though loss of either gene alone yields minor CNS phenotypes. These results demonstrate that Egfr plays a critical role during neuroblast formation, patterning and specification along the DV axis within the developing Drosophila embryonic CNS (Skeath, 1998).

In a screen to identify mutations that disrupt embryonic CNS development, one P element mutation, l(2)03033, was identified that causes a loss of essentially all Eve-positive RP2/RP2 sib neurons. This P element maps to cytological position 57F1-2 in the right arm of the second chromosome and is known to be inserted within the Egfr locus. To verify that lesions in Egfr result in a nearly complete loss of RP2 motoneurons, three additional Egfr mutants were obtained, including the Egfr null allele, flb 1K35. Essentially all Eve-positive RP2 motoneurons are absent from embryos homozygous mutant for each Egfr allele (Skeath, 1998).

The first phase of CNS development, as gastrulation commences, involves the activation of the Ac-S proneural genes in a precise pattern of proneural clusters. To investigate whether Egfr regulates As-C expression in the neuroectoderm, the expression patterns of the achaete (ac) and lethal of scute (l'sc) genes were followed in Egfr mutant embryos. Loss of Egfr causes specific defects to the DV registration of ac and l'sc gene expression in the neuroectoderm; however, no defects to the AP registration for either ac or l'sc gene expression were found. In wild-type embryos during stages 8/9, ac is expressed in the medial and lateral, but not intermediate, clusters of rows 3 and 7; l'sc is expressed in the medial and lateral, but not intermediate, clusters of row 7 and in the medial, intermediate and lateral clusters of rows 1 and 5. A single neuroblast subsequently forms from each proneural cluster. In Egfr mutant embryos, ac gene expression expands into the intermediate column in rows 3 and 7 and l'sc expression expands into the intermediate column in row 7; l'sc is expressed normally in rows 1 and 5. The lateral limits of ac and l'sc gene expression in the neuroectoderm are unaltered in Egfr mutant embryos. The changes to the DV registration of ac and l'sc gene expression in Egfr mutant embryos suggest that neuroectodermal cells in the intermediate column change their fate. Both ac and l'sc are normally expressed in the medial and lateral columns in the affected rows, thus the phenotype is consistent with intermediate cells acquiring either a lateral or a medial fate. msh-1, which is expressed exclusively in the lateral column, expands into the intermediate column in Egfr mutant embryos. In this context, it appears that ac and l'sc expression expand from the lateral column into the intermediate column in the absence of Egfr (Skeath, 1998).

Achaete function in sensory organ formation

The receptor encoded by the Notch gene plays a central role in preventing cells from making decisions about their fates until appropriate signals are present. This function of Notch requires the product of the Suppressor of Hairless gene. Loss of either Notch or Suppressor of Hairless function results in cells making premature and incorrect cell fate decisions, while increases in Notch signaling prevent cells from making these decisions. The proneural clusters are not established correctly in certain Abruptex mutations of Notch and this failure to establish proneural clusters correctly is not due to increased Notch signaling during lateral inhibition. The overexpression of certain dominant negative Notch molecules can disrupt the initiation of proneural cluster development in a manner similar to the Abruptex mutants (Brennan, 1999).

Genetic analysis has revealed three regions of the extracellular domain which are functionally important. One is centered around EGF repeats 11 and 12 and is involved in the binding of Delta. A second one, revealed by the Abruptex mutations, is centered around EGF repeats 24 and 25. Mutations within this region have been proposed to either increase Delta signaling via Notch and consequenctly suppressing Achaete expression or, alternatively, to affect a distinct function of Notch that is involved in the initiation of Achaete expression rather than its suppression. The third region is highlighted by mutations in a cysteine-rich region known as the LNG repeats. Mutation or deletion of this region generates gain of function Notch molecules that appear to increase Delta signaling. While work in different systems has indicated a role for Notch in lateral inhibition, analysis of different Notch mutations has raised the possibility that there is a Notch signaling event different from the one that mediates lateral inhibition. This hypothesis stems from the identification, within the Abruptex(Ax) class of Notch alleles, of loss of function mutations, such as the Ax59d and AxM1 alleles. The Abruptex alleles are characterized, in addition to other phenotypes, by the loss of sense organs on the adult thorax. Since many of the Abruptex alleles behave phenotypically as gain of function mutations, the loss of sense organs is typically thought to be due to an increase in signaling during lateral inhibition. However, this hypothesis cannot explain the loss of bristles observed with the loss of function Abruptex alleles. Consequently it has been proposed that the Notch protein has an additional role in the formation of the sense organs and in particular a role in the establishment of the proneural clusters that is disrupted by alleles like Ax59d, which leads to the observed loss of sense organs. This analysis of Notch alleles that reduce bristle number has also highlighted a purely gain of function class of alleles, that includes the l(1)NB mutation. In these mutants the loss of bristles appears to be due to increased signaling during lateral inhibition (Brennan, 1999 and references therein).

It has been shown by (1) examination of the expression of the proneural gene, achaete, in the presence and absence of the Su(H) gene in the third larval instar wing disc of the Ax59d mutant and (2) examination of achaete expression in flies in which Notch function has been disrupted by expressing dominant negative forms of the Notch protein, that the initiation of the proneural clusters is disrupted in Ax59d mutants and flies in which dominant negative forms of Notch are over expressed. In addition, these experiments show that this failure to define proneural clusters correctly is not due to increased Notch signaling during lateral inhibition (Brennan, 1999).

One explanation for the suppression of Achaete expression caused by ECNdelta10-12 molecules, that is, expressed proteins in which the extracellular domain of Notch contain a deletion in EGF domains 10-12, might be that they interact with, and 'transactivate', the endogenous full-length Notch protein to generate a strong lateral inhibition signal. To test this possibility the ECNdelta10-12 protein was expressed in a genetic background where lateral inhibition does not occur, such as in larvae homozygous for null mutations of Su(H). In third instar discs that lack Su(H), each proneural cluster contains a large number of cells expressing high levels of Achaete. Expression of the ECNdelta10-12 molecule in Su(H) mutant discs, under the control of ptcGAL4, in many cases eliminates the expression of Achaete in the scutellar cluster. In those cases where Achaete expression is seen the cluster consists of two or three cells that express high levels of Achaete. However, in the same discs, Achaete expression is not affected in clusters not covered by the stripe of ECNdelta10-12 expression and therefore these clusters act as an internal control for the experiment. These results indicate that the suppression of Achaete is not dependent upon Su(H) and therefore it is unlikely that expression of the ECNdelta10-12 protein disrupts Achaete expression by generating a strong lateral inhibition signal. In support of this hypothesis, similar results have been obtained by expressing the ECNdelta10-12 protein in a kuzbanian mutant that also prevents lateral inhibition signaling (Brennan, 1999).

Previous experiments have shown that the loss of bristles characteristic of Abruptex mutations is suppressed if there is a simultaneous loss of shaggy function; this is likely to be due to the expression of Achaete caused by removing sgg/zw3 function. In light of the similarity observed in the phenotype caused by the Ax59d and AxM1 mutations and the effects of the ECNdelta10-12 molecule on the development of the PNS, the results with shaggy led the authors to test whether or not the phenotype generated by overexpressing the ECNdelta10-12 depends on sgg/zw3. Indeed, the phenotype generated by expressing the ECNdelta10-12 protein can be rescued by reinitiating the expression of Achaete by removing shaggy/zeste white 3 function. This suggests that the phenotype generated by expressing the ECNdelta10-12 is due to the loss of Achaete expression and not due to any secondary effect the ECNdelta10-12 protein may have on the development of the disc (Brennan, 1999).

The observed loss of Achaete expression in the Abruptex mutants and the overexpression of the ECNdelta10-12 protein could occur in a number of ways. For example, the Notch protein may transduce a signal into the cell that turns on the expression of Achaete in much the same way as it transduces a Delta signal that up-regulates Atonal expression during the development of the eye. In this scenario loss of Notch function should lead to the loss of Achaete expression. This fits well with the expected dominant negative behaviour of the ECN proteins and with the genetic analysis of the Abruptex mutations, which suggests that the Ax59d mutation is a loss of function mutation. However, it is unlikely that this hypothesis is the explanation for the current results, because clones of cells lacking Notch function have a neurogenic phenotype and express Achaete strongly. Alternatively the observed loss of Achaete expression could be due to the ECN molecules and the protein encoded by the Ax59d allele disrupting signaling through a distinct pathway that is required for the initiation of Achaete expression rather than altering signaling through the Notch pathway. In this situation, wild-type Notch function is only required during lateral inhibition and not in the establishment of the proneural clusters. Therefore, the removal of wild-type Notch function would be expected to lead to the failure of lateral inhibition signaling and a neurogenic phenotype, such as is observed in clones of lacking Notch function. One possible pathway with which the ECNdelta10-12 and Ax59d proteins may be interfering is the Wingless signaling pathway, since it is required for the initiation of Achaete expression in both the scutellar and the dorsocentral proneural clusters. The rescue of sensory bristle development by the removal of Shaggy function in Abruptex mutants and in flies in which the ECNdelta10-12 protein has been overexpressed fits with this hypothesis. The removal of Shaggy function from a cell constitutively activates Wingless signaling within that cell regardless of the presence or absence of Wingless. Therefore, the removal of Shaggy should reinitiate a Wingless signal attenuated by the ECNdelta10-12 and Ax59d proteins leading to sense organ development as observed (Brennan, 1999b). Finally, experiments where the ECN and ECNdelta10-12 proteins have been overexpressed throughout the developing wing suggest that these proteins can sequester the Wingless protein (Brennan, 1999).

The extracellular domain of Notch is essential for its interaction with extracellular molecules. Previous experiments have indicated that EGF-like repeats 11 and 12 are necessary for the interaction of Notch with its ligands Delta and Serrate. However, at least two aspects of the current results suggest that a region of the extracellular domain centered around EGF repeats 24-26 is required for Notch molecules to disrupt the establishment of the proneural clusters: (1) the Ax59d allele maps to this region within EGF-like repeat 25; (2) the dominant negative Notch molecules will only disrupt the initiation of Achaete expression as long as they contain this domain. It is likely that EGF-like repeats 17-19 and 24-26, present in the ECNdelta10-12 protein are necessary for the ECN molecules to disrupt proneural development. Interestingly, a comparison of EGF-like repeats indicates that the primary sequences of EGF-like repeats 24-26 and 17-19 are similar which suggests that either of these sequences may be sufficient for the ECN molecules to disrupt the establishment of the proneural clusters. Such a possibility can explain the observation that overexpression of ECN molecules deleted for either 17-19 or 24-26 produce antineurogenic phenotypes, similar to those of ECN and ECND10-12. EGF-like repeats 24-26 or repeats 17-19 which are still present in ECND17-19, and ECND24-26 proteins, respectively, would allow the proteins to antagonize the establishment of the proneural clusters (Brennan, 1999).

Sensory organ formation in Drosophila is activated by proneural genes that encode basic-helix-loop-helix (bHLH) transcription factors. These genes are antagonized by Hairy and other proline-bHLH proteins. Hairy may function to form inactive heterodimers with proneural activator proteins. Hairy does bind to DNA and has novel DNA-binding activity: Hairy prefers a noncanonical site, CACGCG, although it also binds to related sites. Mutation of a single CACGCG site in the achaete proneural gene blocks hairy-mediated repression of ac transcription in cultured Drosophila cells. Moreover, the same CACGCG mutation in an ac minigene transformed into Drosophila creates ectopic sensory hair organs like those seen in hairy mutants. Together these results indicate that Hairy represses sensory organ formation by directly repressing transcription of the ac proneural gene (Ohsako, 1994).

Each sensory organ of the Drosophila peripheral nervous system is derived from a single sensory organ precursor cell (SOP). These originate in territories defined by expression of the proneural genes of the Achaete-Scute complex (AS-C). Formation of ectopic sensilla outside these regions is prevented by transcriptional repression of proneural genes. The BTB/POZ-domain transcriptional repressor Tramtrack (Ttk) co-operates in this repression. Ttk is expressed ubiquitously, except in proneural clusters and SOPs. Ttk over-expression represses proneural genes and sensilla formation. Loss of Ttk enhances bristle-promoting mutants. Using neural repression as an assay, functional domains of Ttk have been dissected, confirming the importance of the Bric-a-brac-Tramtrack-Broad complex (BTB) motif. The Ttk BTB domain is a protein-protein interaction motif mediating tetramer formation (Badenhorst, 2002).

Since Ttk is excluded from proneural territories tests were perfomed to see if ectopic expression of Ttk could repress proneural genes and, hence, sensilla formation. Ttk isoforms were over-expressed prior to the formation of SOPs using the Gal4-UAS system under the control of MS1096-Gal4. Ectopic expression of Ttk69 removes the external structures (bristles and sockets) of all wing sensilla with the exception of the ventral mechanosensory bristles (these sensilla arise during pupal stages, at a time and in an area in which MS1096-Gal4 drives negligible expression). Antibody staining of pupal wings using mAb 22C10 shows that loss of external structures is accompanied by neuron ablation. Furthermore, in third instar larval wing discs, SOPs are ablated (revealed using A101). Over-expression of Ttk88 using MS1096-Gal4 has equivalent, albeit milder, effects on sensory organ formation. Ttk88 over-expression removes all dCh bristles but only reduces vCh and medial mechanosensory bristle numbers (Badenhorst, 2002).

The ablation of SOPs is caused by the repression of proneural genes. Ectopic expression of Ttk69 under the control of a heat-shock promoter inhibits achaete and scute transcription. Accumulation of Asense protein is also blocked. Over-expression of Ttk88 also perturbs achaete, scute and asense expression showing that both isoforms of Ttk can repress the AS-C. Significantly, though, the extent of repression is lower. This could reflect differences in protein stability of the two isoforms. Both are targeted for ubiquitin-dependent proteolysis. However, Ttk69, unlike Ttk88, is post-translationally modified by the small ubiquitin-like molecule dSmt3. This modification has been shown to protect IkappaBalpha from ubiquitin-dependent degradation (Badenhorst, 2002).

Both the expression profile of Ttk and its ability to repress proneural genes when expressed ectopically suggest that Ttk functions like the transcriptional repressors hairy and emc as a global regulator to limit AS-C expression to proneural clusters. To confirm this, tests were made to determined if loss, or reduction, of Ttk induces excess SOP production. Dominant interactions between ttk and known bristle-promoting mutants were sought. A series of ttk alleles were used: mutations that reduce both Ttk69 and Ttk88. Mutations that affect both isoforms of Ttk exacerbate ectopic bristle production seen in excess function achaete mutations. acHw1 and acHw49c induce ectopic Ac expression and cause the development of extra bristles, particularly along the wing veins L2, L3 and L5. Reduction of both Ttk69 and Ttk88 levels enhances these phenotypes. In contrast, slightly elevating Ttk69 levels through basal expression from a hs-ttk69 transgene decreases the strength of the acHw49c phenotype (Badenhorst, 2002).

ttk mutants that affect both isoforms of Ttk also interact dominantly with hairy and emc alleles to cause a phenotype that mimics the gain-of-function acHw mutations. Adult emc/ttk transheterozygous flies develop ectopic bristles on the wing blade. Similarly, hrM730, h/ttk transheterozygotes exhibit many ectopic bristles on the L2, L3 and L5 wing veins and a variable number of additional dorsocentral and scutellar bristles. However, reduction of both Ttk69 and Ttk88 is required for ectopic bristle production (Badenhorst, 2002).

Ttk blocks SOP recruitment by repressing transcription of the proneural genes. In the developing PNS, Ttk completely inhibits achaete and asense expression and blocks part of the scute expression profile. Surprisingly, in the embryonic central nervous system (CNS), Ttk over-expression only represses asense but has no effect on achaete. Inspection of the promoters of the proneural genes reveals that the immediate 5' promoter region of asense contains many clustered consensus Ttk69-binding sites, suggesting that Ttk inhibits asense by directly repressing the proximal promoter. In contrast, the upstream promoter region of achaete does not contain large numbers of consensus sites. A cluster of Ttk69-recogntion sites is found downstream of achaete. It is conceivable that specific repression of achaete in the PNS is achieved by blocking PNS-specific enhancers while not affecting regulatory elements required for expression in the CNS. The existence of separate enhancers directing expression of achaete and scute in the CNS and PNS has been inferred from deletions and inversions that affect subsets of the achaete expression profile (Badenhorst, 2002).

Alternatively, Ttk may repress achaete in cooperation with other factors that are only present in the wing imaginal disc and not expressed in the CNS. Ttk69 binds to the dMi-2 subunit of NURD - the nucleosome remodeling deacetylase . Recruitment of histone deacetylases to the Ttk69-binding sites downstream of achaete could establish an acetylation-free domain covering achaete if deacetylase activity spreads from the initial site of recruitment to modify flanking nucleosomes. Such deacetylation may be required to allow other factors to repress achaete (Badenhorst, 2002).

Over-expression of either isoform of Ttk can repress achaete and asense expressions. However, Ttk69 consistently has stronger effects. One explanation for this difference is that the isoforms may have different protein stabilities. Both isoforms are subject to ubiquitin-dependent proteolysis, but Ttk69 is also modified by the ubiquitin-like protein Smt3. Smt3-modification has been proposed to protect target proteins from ubiquitin-dependent proteolysis. Further evidence that Ttk69 and Ttk88 co-operate to repress proneural genes is provided by the genetic interactions between ttk and bristle-promoting mutants. Only ttk alleles that reduce expression of both Ttk69 and Ttk88 levels show a strong interaction. ttk1e11, which only affects Ttk69 but does not enhance phenotypes significantly. Flies mutant for ttk1, which reduces Ttk88 expression, are homozygous viable and do not show ectopic wing bristles. Although this mutation also has a slight Ttk69 gain-of-function phenotype caused inappropriate translation of Ttk69 in some microchaete daughter cells, this effect is largely confined to abdominal and thoracic microchaete and wing sensilla are unaffected (Badenhorst, 2002).

In the wing discs of Drosophila, the mechanosensory precursor cells are singled out from clusters of cells blocked at the G2 phase of the cell cycle. This mitotic quiescence and the selection of the precursors are under strict spatio-temporal control. G2 cells were forced to enter mitosis by overexpression of string, the Drosophila homolog of the cdc25 gene. Premature entrance in the cell cycle is associated with a loss of precursor cells. Precursors are lost consecutively to a transcriptional down-regulation of the determinant proneural achaete/scute genes. This down-regulation results from an over-activation of the Enhancer of Split genes, known as effectors of the Notch signalling pathway. It is concluded that exit from the cell cycle is required for proper neural cell fate determination (Nègre, 2003).

Thus, forcing G2 arrested cells into mitosis results in a loss of adult sense organs. The corresponding precursors are also lost. This result was obtained by using two distinct transgenic systems to control the timing and spatial location of stg-overexpression. In both cases, precursors are not selected because ac/sc proneural expression is repressed. This repression occurs at a transcriptional level. Noteworthy is the fact that bristles are lost using either the sca-Gal4 driver to overexpress stg, or the klu-Gal4 driver; this demonstrates that overexpression of stg not only prevents the early accumulation of Ac/Sc (klu-Gal4 driver), but can also downregulate Ac/Sc after the levels of these proteins have started to rise (sca-Gal4 driver). Thus, it is concluded that the arrest in G2 is necessary for proper determination of precursor cells. The complexity of the 5' regulatory sequences of stg indicates that this mitotic regulator might itself integrate information from patterning genes. For instance, the regulatory regions of the stg gene possess putative recognition sites for Achaete and Scute transcription factors. Here, it has been shown that stg can itself control the expression of developmental genes. The effect of stg on cell determination is unlikely to be direct, however, since the only known function of stg is to dephosphorylate the CDK1- cyclin B mitotic kinase. Future genetic approaches may reveal whether or not String has other biochemical targets (Nègre, 2003).

After stg overexpression using the klu-Gal4 driver, it was observed that E(Spl) expression is maintained in proneural regions in absence of Ac/Sc. It was also observed that stg can cause accumulation of the E(Spl) bHLH genes outside of proneural clusters, in a cell-autonomous mode. Maintenance of expression of E(Spl), a transcriptional repressor of the ac/sc expression, is relevant. It can functionally justify the loss of precursor cells. Nevertheless, it has been reported that E(Spl) transcription is dependent on the ac-sc genes in the proneural clusters. One explanation could be that deregulation of the cell cycle directly or indirectly increases transcription of the E(Spl) genes by modifying activity of upstream activators of the E(Spl) expression. Considering this hypothesis, E(Spl) should sometimes be expressed in incorrect positions compared to its wild-type expression. On the contrary, because E(Spl) genes are expressed at the exact positions for proneural clusters, it is suggested that forcing cell cycle more likely affects E(Spl) expression at a post-translational level rather than at a transcriptional level. In the mutants, initial transcription of E(Spl) genes would still have been dependent on Ac/Sc, which begin to accumulate in proneural domains. But, it is known that at least E(Spl) m5, m7 and m8 isoforms contain a PEST-rich motif that harbors an invariant Serine residue, which is phosphorylated by the casein kinase II. Casein kinase II is a ubiquitous serine/threonine kinase whose activity fluctuates with cell cycle progression. Phosphorylation usually regulates protein stability via activation of PEST motifs. Modification in the phosphorylation status of some E(Spl) proteins could exhibit a longer half-life in vivo, thus leading to their predominance over the proneural proteins, and therefore to an inhibition of neurogenesis. In other words, premature entry in the cell cycle would introduce an external bias in the highly dynamic process that opposes the antagonistic E(Spl) and Ac/Sc proteins and which normally occurs in cells of proneural clusters. It would confer an advantage to E(Spl) over proneural activity and would explain persistence of E(Spl) proteins after proneural products have disappeared (Nègre, 2003).

Altogether, these results suggest that proneural competence can only develop in mitotically arrested cells. The programmed incompatibility between cell cycling and proneural product accumulation may have several general, and not mutually exclusive, functional correlates. In proneural clusters, keeping cells together in a continuous group may be necessary. Indeed, cell interactions could be required to maintain Ac/Sc levels via indirect autoregulation through cell- cell signalling. Furthermore, a G2 arrest may be necessary to preserve a balance between the levels and/or activities of E(Spl) and Ac/Sc products that could directly or indirectly be dependent on post-transductional modifications. The relative strength of the signal impinging on a given cell determines whether products of the proneural genes or products of the E(Spl) become finally predominant. Changing the cell cycle phase could disrupt this equilibrium. Finally, divisions that underly normal cell proliferation and those involved in the fixed lineage of the precursor cell, make different demands on the cytoskeletal machinery. The asymmetric divisions of the precursor cell are strictly controlled in orientation and in time. These controls are presumably essential to realize a correct lineage. A period of mitotic quiescence may give the precursor cell the time and/or conditions required to reorganize its cytoskeleton in order to shift to an asymmetric mode of division. Although a quiescent period systematically precedes the emergence of neural precursors, re-entry into mitosis is independently controlled in the precursor and the surrounding epidermis. This suggests that quiescence is a necessary step preceding the lineage of the precursor. Moreover, the decision of the precursor to enter in its lineage is made independently of the mitotic state of its surrounding cells (Nègre, 2003).

In this study, causal relationship has been demonstrated to exist between cell cycle and neural determination in an endogenous system: the Drosophila wing imaginal discs, in which E(Spl) effectors of the Notch pathway behave as integrative sensors of the cell cycle status (Nègre, 2003).

Achaete function in sensory organ formation: Senseless acts on achaete as a binary switch during sensory organ precursor selection

During sensory organ precursor (SOP) specification, a single cell is selected from a proneural cluster of cells. Evidence is presented that Senseless (Sens), a zinc-finger transcription factor, plays an important role in this process. Sens is directly activated by proneural proteins in the presumptive SOPs and a few cells surrounding the SOP in most tissues. In the cells that express Sens low levels, Sens acts in a DNA-binding-dependent manner to repress transcription of proneural genes. In the presumptive SOPs that express Sens at high levels, Sens acts as a transcriptional activator and synergizes with proneural proteins. It is therefore proposed that Sens acts as a binary switch that is fundamental to SOP selection (Jafar-Nejad, 2003).

Proneural genes have been shown to be required for sens expression. To determine whether proneurals directly activate sens expression, the putative enhancers of sens were identified and were scanned for proneural protein-binding sites (E boxes). An 11-kb genomic fragment containing the sens locus is able to rescue the sens mutant phenotype. To identify the embryonic and imaginal disc enhancers, three genomic DNA fragments were used to create lacZ reporter transgenes. Both 5.9-kb and 3.4-kb fragments are sufficient to drive expression in the embryonic PNS in a pattern similar to endogenous sens. To refine sens enhancers, the 3.4-kb enhancer was divided into nine overlapping fragments. Fragments 8 and 9 induced lacZ expression in a pattern similar to the original 3.4-lacZ line, indicating that both contain regulatory elements sufficient for sens expression in the embryonic PNS. Fragments 8 and 9 were further divided into overlapping fragments. Only 9-1-lacZ expresses the reporter in a pattern similar to the 3.4-lacZ. Inspection of the 9-1 sequence showed that it contains a single E box. The recently sequenced genome of Drosophila pseudoobscura, a species 25-30 myr divergent from Drosophila melanogaster was used to align the genomic regions. The alignment showed that the E box, as well as several other elements in the 9-1 enhancer, is fully conserved. Upon mutation of this E box from CAGGTG to CCGGTG, most of the PNS cells failed to express lacZ, and staining in other cells was much weaker than for the wild-type transgene. These data indicate that proneural genes directly regulate the transcription of sens (Jafar-Nejad, 2003).

It is thought that the two core nucleotides of the E box as well as its flanking sequences are involved in the specificity of each E box for its cognate bHLH transcription factor. It was intriguing that expression of the lacZ marker is almost abolished in chordotonal organs that are dependent on atonal (ato) as well as in external organs and multiple dendritic organs that are dependent on ac, sc, and amos. Because the 9-1 fragment contains only a single E box, the data suggest that different proneural proteins can bind the same E box in vivo. Therefore EMSA was performed to determine whether a variety of Da-proneural heterodimers can shift a wild-type or an E box-mutated probe taken from the 9-1 sequence. Da homodimer, Ato/Da heterodimer, Ac/Da heterodimer, and Sc/Da heterodimer were all able to bind to this E box. Mutation from A to C in the second position of the E box abolished binding for all protein combinations tested, suggesting that these interactions are sequence specific. It is concluded that at least three proneural proteins (Ac, Sc, and Ato) directly regulate sens expression in the embryonic PNS, and that they may bind the same site in vivo (Jafar-Nejad, 2003).

To examine whether sens regulation in the precursors of the adult PNS is also under direct proneural regulation, the 9-1-lacZ and 9-1-mut-lacZ expression patterns were compared in the SOPs of the thoracic microchaetae. Similar to what was observed in embryos, a single-nucleotide change in the 9-1 E box abolishes most of the lacZ expression in pupae of the same age, again suggesting direct regulation of sens by proneurals (Jafar-Nejad, 2003).

The effects of loss- and gain-of-function of proneural genes on sens expression were assessed in the imaginal discs of third instar larvae. Because fragments 9 and 9-1 do not drive lacZ at this stage, enhancer 8 was used. The 8-lacZ transgene drives lacZ expression in several wing SOPs in late third instar larvae. To determine whether proneural genes are able to control 8-lacZ expression, Sc was overexpressed in the wing pouch using the C5-GAL4 driver. Many more cells express lacZ in the wing pouch than in wild type, indicating that the Sc protein is able to induce lacZ expression ectopically. However, removal of the activity of both ac and sc genes results in loss of lacZ expression in all of the ac/sc-dependent SOPs. The precursors of the ventral radius and the femoral chordotonal organs still express lacZ, since these cells are dependent on Ato expression. Moreover, upon Ato overexpression driven by dpp-GAL4, 8-lacZ is strongly induced at the A/P boundary. Together, these data indicate that proneural proteins regulate sens expression in the precursors of the adult PNS. Fragment 8 contains two E boxes, one of which is fully conserved between D. pseudoobscura and D. melanogaster. Band-shift experiments show that the Ac/Da heterodimer can bind to a radioactive probe that contains the conserved E box of fragment 8, further suggesting that proneurals directly regulate sens expression (Jafar-Nejad, 2003).

E(spl) proteins are known to prevent SOP formation through transcriptional repression of proneural gene expression. Whether they affect sens expression was examined. scabrous (sca)-GAL4 was used to express E(spl)m8 in the SOPs and a few cells around the SOPs in third instar imaginal discs. lacZ expression is abolished in most or all cells. Moreover, misexpression of an 'activator' version of E(spl)m7 (m7ACT), in which the Gro-binding motif is replaced with the VP16 transactivator domain, caused numerous extra lacZ-positive cells when driven in the wing pouch. These observations suggest that E(spl)m7 and E(spl)m8 proteins are also involved in the transcriptional regulation of sens and that proneural proteins and E(spl) proteins have an antagonistic relationship in transcriptional control of sens. E(spl) proteins are known to bind to proneural gene enhancers and m7ACT is able to activate ac and sc transcription. Therefore, it is formally possible that m7ACT is indirectly activating the sens enhancer through its up-regulation of proneural proteins. However, it has been shown that even in the absence of endogenous ac and sc, overexpression of m7ACT causes extra bristle formation, suggesting that the E(spl) proteins not only regulate proneural gene expression, but also regulate the expression of one or more of proneural target genes. Is m7ACT able to induce sens expression in the absence of ac and sc? To address this question, it was confirmed that overexpression of m7ACT can produce several extra bristles in a sc10-1 background. Staining of the imaginal wing discs of these flies shows that there are many Sens-positive cells in the anterior part of the presumptive notum, where the Eq-GAL4 driver used in this experiment is expressed. The data suggest that sens is one of the targets of the E(spl) proteins. Altogether, sens enhancers seem to be able to integrate the positive and negative inputs from proneural and E(spl) proteins, respectively (Jafar-Nejad, 2003).

To explore the mechanism by which Sens promotes SOP specification, how Sens regulates proneural gene expression was studied. In sens mutant clones, proneural proteins fail to accumulate in the SOPs. A strong synergism exists in the ability of Sens and Sc to promote extra bristle formation. Whether there is an in vivo synergy between Sens and Ac was examined. acHw-1 exhibits an occasional extra macrochaetae on the notum because of an increase in ac transcript level. Overexpression of Sens with sca-GAL4 also causes a number of extra micro- and macrochaetae on the notum, and overexpression of Sens in an acHw-1 background causes many more extra macrochaetae than the sum of the two genotypes alone. Therefore, there is a synergy between the bristle-promoting effects of the two proteins in vivo (Jafar-Nejad, 2003).

An assay to determine whether Sens can affect ac gene transcription was established in Drosophila S2 cells. The reporter construct used in this assay was a 470-nucleotide fragment of the ac gene containing the ac promoter region fused to the firefly luciferase. This fragment contains the Hairy-E(spl)-binding site and the three E boxes that are involved in ac regulation by proneural and E(spl) proteins. Moreover, during the course of microchaetae SOP specification, expression driven by the ac proximal enhancer/promoter refines from the proneural cluster to a single cell, supporting the notion that it can serve as an SOP-specific regulatory region. The constitutively active actin5 promoter was used to drive the expression of Da, Ac, Sens, or E(spl)m8. Sens alone does not activate the ac470-luc construct. Cotransfection of minimal amounts of actin5-da and -ac activates the luciferase expression about 10-fold. However, adding an additional 20 ng of the actin5-sens leads to a dramatic activation of the ac promoter (>500-fold). It is concluded that Sens can activate ac transcription through synergism with Ac/Da heterodimer in Drosophila S2 cells. These findings suggest that there is a parallel between the in vivo and transcriptional synergy observed between Sens and Ac (Jafar-Nejad, 2003).

Because E(spl)m8 strongly antagonizes SOP specification, it was postulated that E(spl)m8 may decrease the synergistic activation of ac by Sens. Cotransfection of 100 ng of actin5-E(spl)m8 with 1 ng actin5-da and -ac does not significantly repress the luciferase activity induced by these proneural proteins. However, cotransfection of actin5-E(spl)m8 together with sens, da, and ac constructs inhibits the synergy in a dose-dependent manner. In summary, Sens is able to strongly synergize with the proneural proteins in vivo and in vitro, and this synergism is antagonized by E(spl) proteins in a dose-dependent manner (Jafar-Nejad, 2003).

To determine whether the E(spl) antagonism of the Sens synergism operates in vivo, it was first documented that overexpression of Sens at high levels in the wing pouch produces a vast excess of bristles in the wing. In addition, extra vein tissue and thickening of the wing veins is observed. E(spl)m8 overexpression with the C5-GAL4 driver causes loss of wing vein tissue, as well as loss of some of the dorsal wing margin bristles. When Sens and E(spl) are coexpressed, the two proteins suppress each other's phenotypes; the number of extra bristles is decreased significantly, and many wing veins are restored. A higher magnification shows that there are still extra bristles as well as some aberrant vein tissue. Taken together, these data support the notion that Sens and E(spl)m8 have antagonistic effects at the level of proneural gene expression, in agreement with their in vivo effects on bristle formation. Finally, whether the physical interaction between Sens and E(spl) plays a role in their antagonism on ac enhancer was examined in S2 cells. The Sens-del, which lacks the 15-amino acid E(spl)-interacting motif, can synergize with Ac and Da similar to wild-type Sens. However, the ability of E(spl)-m8 to antagonize the synergy between Sens-del and proneural proteins is impaired when compared with its effect on the wild-type Sens/Ac/Da synergy, suggesting that the physical interaction between Sens and E(spl) plays a role in their antagonistic effect (Jafar-Nejad, 2003).

Sens can bind the consensus binding site of its vertebrate homolog Gfi-1. Examination of the ac proximal enhancer showed that only one putative Sens-binding site is present between two of the E boxes in this enhancer (S box). Band-shift assays show that Sens can bind to S box in a sequence-specific manner. Mutating the core sequence from AATC to GGTC abolished Sens binding in this assay. As a positive control, another oligo with 92% identity to the consensus (R21) was used. Significantly more R21 probe was shifted by Sens than the endogenous oligo. These data indicate that there is a binding site for Sens in the ac promoter (Jafar-Nejad, 2003).

To determine whether S box mutations affect the synergy between Sens and Da/Ac, the transfections were repeated with an S box-mutant version of the ac-luc reporter. Quite unexpectedly, the mutant reporter construct consistently showed a three- to fourfold increase in synergism when compared with wild-type reporter. This suggests that DNA-binding has a negative regulatory role in the transcriptional activation of ac by Sens in S2 cells. To test the in vivo relevance of this observation, whether the S box in the ac enhancer has a role in bristle formation in vivo was examined. It is well established that sc10-1 mutant flies are devoid of thoracic bristles. It has also been shown that two wild-type copies of a 2.2-kb ac minigene can restore some of the microchaetae on the notum of sc10-1 mutant flies. Therefore the S box core from AATC to GGTC was mutated in a 2.2-kb ac genomic fragment and transgenic animals were created. Six wild-type and nine mutant transgenic strains were obtained. For each strain, at least five flies containing the transgene in a sc10-1 background were scored. Comparison of the number of bristles restored by one copy of mutant versus wild-type transgene showed that, in agreement with the transcription assay results, the mutant ac minigene is more potent in promoting bristle formation than the wild-type transgene. Interestingly, one of the mutant transgenes rescued almost all microchaetae on the notum, along with the three ac-dependent macrochaetae on each side. It is worth mentioning that none of the six wild-type transgenic lines show macrochaetae rescue. These data suggest that DNA binding is a negative modulator of the synergy between Sens and Ac/Da heterodimer in vivo (Jafar-Nejad, 2003).

Because proneural gene expression precedes sens expression in most proneural clusters, one can postulate that at least in a transitional period, Sens levels will be lower than proneural protein levels. Because a 1 proneural:20 sens ratio was used in previous experiments, it was decided to reverse the ratio. Ac and Da can strongly induce luciferase gene expression. Since the amount of sens construct is increased, a gradual repression in luciferase activity was observed that reaches 50% of the Ac/Da activation. When the ratio is 1 proneural:20 sens, synergism is observed (~2000-fold activation of baseline). It is concluded that Sens can act both as a repressor and as an activator of ac transcription, depending on the ratio between Sens and Ac/Da (Jafar-Nejad, 2003).

Since previous transfection and in vivo assays suggested a negative role for Sens DNA-binding in ac transcription and bristle promotion, it was of interest to determine whether the repressive role of low-level Sens is mediated via DNA binding. A similar transfection assay was therefore performed using the AATC to GGTC mutated ac enhancer as the reporter. The results show that upon removal of the Sens-binding site, its ability to repress the luciferase level is lost. Moreover, the synergy between Sens and Ac/Da begins at a much lower sens:proneural ratio and reaches significantly higher levels. Therefore, the repressive effect of Sens seems to depend on its DNA binding (Jafar-Nejad, 2003).

The above findings prompted a reexamination of Sens expression and its colocalization with proneural proteins. Sens protein expression is not confined to the SOPs, in which it is abundantly expressed; it is also expressed at lower levels in cells surrounding the SOPs. This domain of expression is smaller than the proneural cluster and seems to be confined to the proneural field or even fewer cells. This occurs in the wing margin, the eye, and the microchaetae field of the pupal notum. In all of these extended proneural fields, low levels of Sens and proneural proteins are expressed in numerous cells that fail to become SOPs. However, in all of these tissues, cells that exhibit high levels of Sens also accumulate large amounts of proneural proteins. It is worth mentioning that it has not been possible to detect similar low-level Sens expression in the typical single-SOP fields of notum macrochaetae, which could either be a technical issue or suggest that in these proneural fields, Sens expression is confined to SOPs (Jafar-Nejad, 2003).

In summary, the data suggest that low levels of Sens are present in cells that surround the presumptive SOPs of the notum microchaetae, wing margin, embryonic PNS, as well as in cells that surround the presumptive R8 photoreceptors. Although all of the cells with low-level Sens expression also express low levels of proneural proteins, many of them will later lose proneural gene expression and adopt a non-neural fate. These observations are in agreement with the hypothesis that whereas high levels of Sens are required for proneural up-regulation in the SOP, low levels of Sens might repress proneural gene expression, and thus suppress neural potential (Jafar-Nejad, 2003).

Because Sens is expressed in the posterior wing margin, and because ac, sc, ato, and amos are not expressed in the posterior wing margin, it was of interest to determine whether expression of Sens in these cells is dependent on proneural gene expression by removing da. Large clones of da do not cause a loss of early Sens expression at the anterior or posterior wing margin, suggesting that early Sens expression in these cells is under the control of other signaling pathways. This early expression of Sens is not affected in a sc10-1 animal either. However, Sens expression is lost wherever the da clone encompasses an area other than the wing margin from which SOPs would normally arise. Finally, no Sens protein was detected in the developing notum of a 10-12-hour-old sc10-1 pupa, suggesting that in the pupal microchaetae field, proneural proteins are the primary transcriptional activators of sens. Together, these data suggest that whereas the initiation and up-regulation of Sens in the majority of presumptive SOPs are under direct transcriptional control of proneural proteins, other proteins seem to be involved in the initiation of Sens expression in the wing margin (Jafar-Nejad, 2003).

So far, evidence has been provided that low levels of Sens can act as a transcriptional repressor of ac in cell culture, and that in most proneural fields, low levels of Sens are present in the cells surrounding the presumptive SOP. To strengthen the hypothesis that Sens acts as a transcriptional repressor in the cells that express low levels of Sens, sens clones were generated in the wing imaginal discs of third instar larvae. If low levels of Sens repress proneural gene expression, and high levels promote SOP development, lack of Sens protein should lead to continued or slightly increased expression of proneural proteins in proneural fields, whereas causing a loss of up-regulation of proneural proteins in SOPs. Therefore broad low levels of proneural proteins are expected to be observed in sens mutant clones. Two different clones were selected, one parallel to the dorso-ventral midline and the other perpendicular to this midline. In both cases, the Sc expression fails to become restricted to single cells, as is observed in adjacent heterozygous or wild-type tissue. These observations provide further evidence that Sens is necessary to down-regulate proneural expression in the cells that will not adopt the SOP fate (Jafar-Nejad, 2003).

Finally, to provide additional evidence that Sens may act as a binary switch, varying levels of Sens were ectopically expressed to determine whether low levels of Sens expression prior to its normal onset of expression might cause bristle loss. Expression of Sens in the wing margin using the C96-GAL4 driver can result in wing-margin tissue loss, including bristles, similar to what is observed in Lyra mutants. C96-GAL4 was crossed to the weakest UAS-sens transgene, which is inserted on the X chromosome, and females and males were compared with one copy of the transgene in an otherwise identical genetic background and environment. Because males display dosage compensation, they should express more Sens protein than their sisters. Most male progeny have a few extra bristles along the margin when reared at 25°C. However, most female progeny display patches of wing margin bristle loss. These data suggest that lower amounts of exogenous Sens can preferentially lead to bristle loss (Jafar-Nejad, 2003).

A model for selection of an SOP from a proneural cluster is proposed in which an intricate set of feedback loops between various transcription factors determines, through the action of Sens and E(spl), the selection of the adult SOP. Most cells of a proneural cluster first express relatively low levels of proneural proteins. This leads to transcriptional activation of E(spl) genes in the cluster. E(spl) proteins, together with the corepressor Gro, then prevent the up-regulation of proneural gene expression in the cluster. It is thought that prepattern factors then lead to a higher level of proneural protein expression in a smaller group of cells of the proneural cluster, the proneural field. It is proposed that this higher level of proneural expression, probably together with the prepattern factors, induces low levels of Sens expression in the proneural field or an area that is even smaller. Consistent low levels of Sens staining are observed in groups of cells in the pupal microchaetae field, embryos, wing, and eye discs. These domains that are part of the proneural cluster colabel with proneural proteins, and a single or a few cells are typically selected from these domains to induce higher levels of Sens. It is proposed that Sens plays a critical role in the SOP through transcriptional synergy with proneural proteins. In addition, the data suggest that Sens plays a role in repressing proneural expression in non-SOP cells. Hence, it is proposed that Sens acts as a binary switch in the refinement of the proneural field that will lead to SOP selection (Jafar-Nejad, 2003).

The data also suggest that sens transcription is mediated directly through proneural binding to E boxes in the sens enhancers. In addition, sens enhancers integrate two opposing forces, the positive regulation by proneural and the negative regulation mediated by E(spl) proteins, similar to SOP-specific enhancers of the proneural genes (Jafar-Nejad, 2003).

Because E(spl) prevents the up-regulation of the proneural gene and sens expression, this repressive effect must be overcome if some cells of the proneural field are to be selected as SOPs. In fact, it has been shown that by repressing E(spl)m8 and other repressors of sens, Su(H) plays a positive role in the SOP fate promotion. It is also known that proneural proteins positively regulate E(spl) gene expression, which will prevent further up-regulation of proneural proteins. This negative feedback has prompted the idea that to accumulate large amounts of proneural proteins in the SOP, the equilibrium between the proneural and E(spl) proteins should be displaced in favor of proneurals. It is proposed that the synergy between Sens and Da/Ac on the ac regulatory region is a key mechanism for the up-regulation of ac transcription. In this model, Sens accelerates proneural gene expression and proneural protein accumulation, overruling the negative feedback conferred by E(spl). This hypothesis is supported by the observation that the synergy between Sens and proneurals is highly sensitive to the levels of E(spl) protein in the transcription assay, as well as in vivo. Ac up-regulation will lead to further Sens production and increased synergistic activation of ac transcription. In the absence of Sens, the presumptive SOPs fail to up-regulate proneural gene expression. Hence, Sens will render the presumptive SOP less sensitive to N signaling. This is also supported by the observation that coexpression of Sens and proneurals is able to produce closely spaced bristles, indicating highly inefficient N signaling. In summary, it is proposed that the balance between the levels of the Sens and E(spl) proteins determines the SOP selection (Jafar-Nejad, 2003).

The synergistic model of proneural gene activation predicts that low levels of Sens and proneural proteins may suffice to override the E(spl) inhibition. However, many cells that express sens and proneural genes fail to up-regulate proneural gene expression. At low levels, Sens acts as a repressor of ac transcription, suggesting that in addition to the relative levels of E(spl), the relative levels of proneural proteins and Sens also play a critical role in SOP selection. In those areas of the proneural field in which Sens and proneural protein levels are low, not only is the transcriptional synergy absent, but there is also a weak repression of proneural gene expression. This should lead to a rapid loss of Sens expression and a failure to adopt the SOP fate. Analysis of the Sc expression pattern in sens clones that include the wing margin confirms that in the absence of Sens function, the broad Sc expression in the wing margin persists, and at the same time, the presumptive SOPs fail to up-regulate Sc protein. This is further supported by the observation that overexpression of low levels of Sens causes bristle loss in the wing margin (Jafar-Nejad, 2003).

The mechanism by which Sens represses transcription of proneural genes is probably through DNA binding. When the S box is mutated, Sens is unable to repress ac transcription. This finding is corroborated with in vivo observations that the ac minigene with the mutated Sens-binding site is a more potent inducer of bristle formation than the wild-type minigene. It is therefore concluded that the transcriptional repression of the ac promoter by Sens is mediated through DNA binding (Jafar-Nejad, 2003).

Altogether, these data support a model in which Sens promotes the SOP fate in one cell by activating ac transcription, whereas it prevents SOP fate in the neighboring cells by repressing ac transcription. The relative levels of Sens, proneural, and E(spl) proteins seem to be the major determinants of these fate decisions. Therefore, it is proposed that Sens acts as a binary switch in SOP determination by affecting a series of interconnected positive and negative regulatory loops to refine the potential for a specific fate from a group of cells to a single cell, the SOP (Jafar-Nejad, 2003).

Achaete and Malpighian tubules

A unique cell, the tip mother cell, arises in the primordium of each Drosophila Malpighian tubule by lateral inhibition within a cluster of achaete-expressing cells. This cell maintains achaete expression and divides to produce daughters of equivalent potential, of which only one, the tip cell, adopts the primary fate and continues to express achaete, while in the other, the sibling cell, achaete expression is lost. In this paper the mechanisms are charted by which achaete expression is differentially maintained in the tip cell lineage to stabilize cell fate. Initially, wingless is required to maintain the expression of achaete in the tubule primordium so that wingless mutants lack tip cells. Conversely, increasing wingless expression results in the persistence of achaete expression in the cell cluster. Then, Notch signaling is restricted by the asymmetric segregation of Numb, as the tip mother cell divides, so that achaete expression is maintained only in the tip cell. In embryos mutant for Notch, tip cells segregate at the expense of sibling cells, whereas in numb neither daughter cell adopts the tip cell fate resulting in tubules with two sibling cells. Conversely, when numb is overexpressed two tip cells segregate and tubules have no sibling cells. Analysis of cell proliferation in the developing tubules of embryos lacking Wingless, after the critical period for tip cell allocation, reveals an additional requirement for wingless for the promotion of cell division. In contrast, alteration in the expression of numb has no effect on the final tubule cell number (Wan, 2000).

The tip cell progenitor is selected from a group of competent cells by lateral inhibition and is demarcated by the continued expression of ac. Further extrinsic and intrinsic cues (Wg signaling and the asymmetric distribution of Nb) operate to ensure the continued expression of ac and so confirm tip cell potential. The selection of cell fate from an equivalence group by lateral inhibition alone relies on chance fluctuations in the equilibrium of signaling between cells and therefore may not be completely reliable. The activity of other genes, by biasing lateral inhibition, serves to make the selection of cells to specific fates more robust. Such mechanisms have been shown to confirm cell fate in the PNS and of the anchor cell in the nematode gonad. The results presented here indicate that wg and nb are required for the specification of the tip cell and sibling cell fate in the Malpighian tubules. The activity of these two genes biases the outcome of intercellular signaling at separate stages in this process, resulting in the reliable allocation of tip and sibling cell fates, suggesting that this distinction is important to the development of the tubules. However, it is clear that continued cell division in the tubules relies only on the allocation of the tip cell progenitor and not on the differentiation of fate between the tip cell's daughter cells, in which nb plays an important role (Wan, 2000).

This result is surprising, since Nb is active where sister cells of specific lineages are allocated to separate cell fates, for example, in the PNS, in the CNS, and in myogenesis. Separation between sister cell fates involves the maintenance of gene expression in one sibling and its repression in the other, for example, of Kr, eve, and S59 in sibling muscle founder cells. This pattern is also seen in the tubules: ac, Kr, and Dl continue to be expressed in the tip cell but are repressed in the tip cell's sibling. In the neural and myogenic lineages the correct allocation of sibling cell fates underpins normal tissue differentiation. In the tubules, the separate roles of the tip cells and their siblings are not yet known; they both appear to be active in regulating cell proliferation but later only the tip cell expresses genes characteristic of neuronal cells. The later function of both cell types has yet to be elucidated. By manipulating nb, Malpighian tubules that lack sibling cells can be generated, but these have two tip cells or have two sibling cells but lack tip cells, thus providing an important tool for this analysis (Wan, 2000).

A screen for modifiers of notch signaling uncovers Amun, a protein with a critical role in sensory organ development functioning upstream of achaete

Notch signaling is an evolutionarily conserved pathway essential for many cell fate specification events during metazoan development. A large-scale transposon-based screen was conducted in the developing Drosophila eye to identify genes involved in Notch signaling. 10,447 transposon lines from the Exelixis collection were screened for modifiers of cell fate alterations caused by overexpression of the Notch ligand Delta, and 170 distinct modifier lines were identified that may affect up to 274 genes. These include genes known to function in Notch signaling, as well as a large group of characterized and uncharacterized genes that have not been implicated in Notch pathway function. A gene was further analyzed that has been named Amun, and it encodes a protein that localizes to the nucleus and contains a putative DNA glycosylase domain. Genetic and molecular analyses of Amun show that altered levels of Amun function interfere with cell fate specification during eye and sensory organ development. Overexpression of Amun decreases expression of the proneural transcription factor Achaete, and sensory organ loss caused by Amun overexpression can be rescued by coexpression of Achaete. Taken together, these data suggest that Amun acts as a transcriptional regulator that can affect cell fate specification by controlling Achaete levels (Shalaby, 2009).

Drosophila continues to play a leading role in the discovery of genes and mechanisms implicated in developmental processes mediated by, or associated with, the Notch signaling pathway. This study presents the results of a transposon screen for the effects of loss-of-function and gain-of-function mutations in a genetic background sensitized for Delta-mediated cell fate changes. In addition, Amun, a nuclear protein identified as a suppressor in the screen was characterized. Amun suppresses a dominant-negative effect of Delta overexpression on cone cell induction in the eye, suggesting that Amun can positively regulate Notch signaling in this context. Alternatively, Amun may function in a parallel or intersecting pathway to affect cone cell development. Evidence is provided that Amun can function early during the cellular patterning underlying mechanosensory bristle development by downregulating the expression of the proneural transcription factor Achaete. The identification and initial characterization of Amun function reflect the potential of the ensemble of 170 transposon insertions identified in the screen for discovery of additional factors that affect Notch signaling mediated development (Shalaby, 2009).

The Exelixis collection covers ~50% of Drosophila genes and contains many new alleles for genes that may prove to be involved in the Delta–Notch signaling pathway or other developmental pathways. The collection has also been screened in a search for modifiers of a Notch loss-of-function signaling phenotype in the wing margin using C96-driven MamDN. Among the 170 modifiers that were identified, 29 lines were also recovered by Kankel (2007) and 141 lines were recovered only in the current screen. Among the putative genes recovered in both screens are several known Notch pathway members and genes that have been previously recovered from Notch-based screens (e.g., numb, wingless, puckered, and Ras85D). In addition, several genes that had not been implicated previously in Notch signaling were identified in both screens, supporting roles for their encoded products during Notch-mediated development. These genes include peanut (a septin), Oatp30B (an ion channel), Indy (a transporter), and Hr38 (a hormone receptor). Of potentially equal interest are the 11 transposon lines that modified phenotypes in secondary tests in this work. Genes potentially disrupted by these transposons include karst (βHeavy-spectrin), bifocal (a cytoskeletal regulator), diaphanous (an actin-binding protein), and caudal (a transcriptional regulator). Further characterization of these genes, as well as other genes recovered in the screen, will help provide a deeper understanding of the mechanisms that govern the Notch signaling pathway (Shalaby, 2009).

A number of the results suggest that Amun is required for cell fate determination during Notch-mediated bristle organ development. Reduction of Amun function and Amun protein overexpression in the developing notum, using several Gal4 drivers including pnr, ptc, sca, and sr, generate defects during microchaeta and macrochaeta development. Substantial loss of microchaetae is observed in the nota of adults that express Amun under pnr-Gal4 or sr-Gal4 control during development. Immunohistochemical analysis of developing nota and the Achaete expression rescue experiments demonstrates that this loss of microchaetae is due to loss of the bHLH transcription factor Achaete. The expression patterns of the proneural proteins Achaete and Scute are best characterized for the dorsocentral macrochaetae, for which cis-regulatory elements control the expression of these genes in specific patterns to establish proneural clusters. These enhancer elements are thought to be activated directly by members of several signaling pathways, including Decapentaplegic and Wingless, as well as by other factors including Pannier (Pnr), Daughterless (Da), Chip, and members of the Iroquois complex (Araucan and Caupolican). The expression of achaete/scute is antagonized by several factors, including U-shaped and dCtBP, both of which bind Pnr to form a transcriptional corepressor complex; Extramacrochaetae (Emc), which forms a heterodimer with Da to inactivate it; and the E(spl)-C proteins, which are downstream targets of Notch signaling. In microchaeta proneural groups, Achaete is also known to be repressed by Hairy, as well as by Notch signaling. This study demonstrates that the effect of Amun overexpression on Achaete levels is cell autonomous, suggesting that the action of Amun on achaete expression could be direct. However, while it is tempting to speculate that Amun regulates Achaete levels by directly binding to cis-regulatory elements that affect achaete expression, it cannot be ruled out that Amun functions by repressing an activator of achaete (e.g., Da or Chip), by activating a repressor of achaete (e.g., Emc, Hairy, or the Notch pathway), or by destabilizing achaete mRNA or protein (Shalaby, 2009).

Reductions in Amun function by RNA interference result in small and disorganized microchaetae. In contrast to the Amun overexpression phenotype, the small microchaeta phenotype is not easily attributable to changes in Achaete expression, given that Achaete has no known roles in bristle development subsequent to SOP specification. It has been shown that bristle shaft size can be correlated with several processes. First, both the shaft and socket cells undergo endoreplication to form polyploid nuclei that are required to form the elongated shaft structure. The degree of endoreplication has been correlated with shaft size. Second, shaft length can be affected by mutations in genes that affect actin bundle formation necessary for proper elongation of the shaft. Third, there is a period of rapid protein synthesis during sensory bristle development that enables the shaft and socket cells to generate the high levels of protein required for the development of the socket and shaft structures. Genes necessary for this process include small bristles [which exports mRNA from the nucleus into the cytoplasm and the Minute loci (genes encoding ribosomal proteins), which can affect bristle shaft length. Preliminary data suggest that Amun is unlikely to affect endoreplication. Nuclei of microchaetae that develop in regions of the notum expressing sr-driven AmunRNAi were investigated and no consistent effects on nuclear size were found as compared to the nuclei of cells of microchaetae in regions devoid of AmunRNAi. Therefore the notion is favored that Amun may be required for transcriptional regulation of specific genes involved in growth and elongation of the shaft or for the elevated levels of mRNA and protein synthesis required for shaft development (Shalaby, 2009).

The finding that Amun can affect Achaete expression levels, together with the identification of Amun as a nuclear protein with a putative DNA glycosylase domain, are consistent with the hypothesis that Amun functions as a transcriptional regulator. While DNA glycosylases are best known for repair of damaged and mismatched bases, recent work indicates that they also play roles in transcriptional regulation. The mammalian DNA glycosylase thymine DNA glycosylase (TDG) acts as a transcriptional co-activator, when bound to CREB-binding protein (CBP) and p300 (Tini, 2002), to enhance CBP-activated transcription in cell culture (Cortazar, 2007). It also acts as a transcriptional corepressor when bound to thyroid transcription factor-1 (TTF1) to repress TTF1-activated transcription in cell culture (Cortazar, 2007; Kovtun, 2007). The Arabidopsis DNA glycosylase DEMETER is required to activate expression of the maternal MEDEA allele, an imprinted maternal gene essential for viability. In light of these studies, the nuclear localization of Amun is suggestive of a function for Amun as a transcriptional regulator (Shalaby, 2009).

In summary, this study demonstrated that Amun is a nuclear protein essential for organismal viability and proper cell fate specification during metamorphosis of Drosophila tissues, including the eye and mechanosensory organs. It is suggested that Amun affects at least two distinct processes during bristle organ development because of the distinct loss-of-function and gain-of-function bristle phenotypes associated with Amun. One pathway is critical for regulation of Achaete protein levels, and the other pathway affects sensory organ bristle shaft size. Because the sequence of Amun contains a putative DNA glycosylase domain, it was reasoned that Amun may act as a transcriptional regulator, as previously demonstrated for other DNA glycosylases. Further characterization of Amun is necessary to identify distinct transcriptional targets and pathways on which it may act and to decipher its potential function as a DNA glycosylase during Drosophila development (Shalaby, 2009).

Achaete regulation in imaginal discs

Continued: Achaete Transcriptional regulation part 2/3 | part 3/3

achaete: Biological Overview | Evolutionary Homologs | Targets of activity | Protein Interactions and Post-transcriptional Regulation | Developmental Biology | Effects of Mutation | References

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