BIOLOGICAL OVERVIEW

The achaete-scute complex of four genes is thought of as proneural, meaning that the genes initiate neural development. asense, the rogue member of the achaete-scute complex, has its own agenda, distinguishing it from the other three genes of the complex: achaete, scute and lethal of scute.

It has generally been thought that asense serve as a neural precursor gene, and not as a neurogenic gene, and acts downstream of achaete and scute only after the committment of cells to the neural fate (Brand, 1993). asense is known to be activated by Achaete and Scute in combination with Daughterless. asense transcription starts later in development and lasts longer in neuroblast progeny than that of the other proneural genes in the achaete-scute complex.

But the notion that asense acts only downstream of achaete and scute no longer appears valid (Dominguez, 1993). asense in fact functions as a proneural gene in bristle development and it regulates the expression of achaete and scute.

Developmental regulators and cell cycle regulators have to interface in order to ensure appropriate cell proliferation during organogenesis. An analysis of the roles of the pan-neural genes deadpan and asense defines critical roles for these genes in regulation of mitotic activities in the larval optic lobes. Loss of deadpan results in reduced cell proliferation, while ectopic deadpan expression causes over-proliferation. In contrast, loss of asense results in increased proliferation, while ectopic asense expression causes reduced proliferation. Consistent with these observations endogenous Deadpan is expressed in mitotic areas of the optic lobes, and endogenous Asense is expressed in cells that will become quiescent. Altered Deadpan or Asense expression results in altered expression of the cyclin dependent kinase inhibitor gene dacapo. Thus, regulation of mitotic activity during optic lobe development may, at least in part, involve deadpan and asense mediated regulation of the cyclin dependent kinase inhibitor gene dacapo (Wallace, 2000).

Hairy-like proteins can have opposite functions from proteins of the AS-C in neural development. ase, a member of the AS-C, has been reported to be expressed in the developing third instar optic lobes and loss of ase function results in disturbances of the adult optic lobe. It was asked whether the AS-C protein Ase can modulate mitotic activity. To this end, the effects of ectopic ase expression on mitotic activity in the developing larval optic lobe were investigated. As with Dpn, ectopic expression of Ase results in strong expression in most cells of the second and third larval optic lobes. This expression results in breaks in the normally continuous pattern of S phase positive cells in the outer proliferation center suggesting that increase and/or ectopic expression of the Ase protein decreases mitotic activity in the optic lobe (Wallace, 2000).

To analyze the possible involvement of ase gene function on mitotic activity in the larval optic lobes, the S phase activity in third instar larval brains was determined. In ase¹/sc^b57 mutants, there is an expansion of S phase activity to include the normally mitotic quiescent cells between the outer proliferation center and the lamina precursor cells (LPCs), as well as scattered S phase activity in the lamina. ase¹ is a deletion of the ase coding region and sc^B57 is a deletion of the entire AS-C as well as the proximal complementation group EC4 making the larva homozygous mutant for ase and heterozygous for the other members of the AS-C. In contrast, +/sc^B57 larval optic lobes show normal S phase activity. Thus, ase loss of function mutants show an increase in the S phase activity between the outer proliferation centers and the LPCs, and a random pattern of increased S phase activity in the lamina (Wallace, 2000).

If Ase is involved in the termination of mitotic activity in the larval optic lobes, then Ase expression would be expected in or near areas where the cell cycle is arresting. Ase protein expression was examined in the larval optic lobes; Ase was found to be present in a band at the posterior edge of the outer proliferation center that partially overlaps with Dpn expression. Ase is expressed just before the cells exit the outer proliferation center and cease S phase activity. These cells then arrest in G1 phase as they pass through the lamina furrow. Ase is also expressed in cells of the IPC and at a low level in the lamina furrow. The expression pattern of Ase, which comes to a maximum at the posterior edge of the outer proliferation center, is in agreement with a possible role for Ase in aiding cell cycle arrest as cells leave the outer proliferation center (Wallace, 2000).

In homozygous ase mutant third instar larvae, there is a strong reduction of dap RNA throughout the entire developing optic lobe while dap expression in the developing eye disk appears normal. The ase loss of function phenotype demonstrates that ase activity is necessary for the expression of dap throughout the developing optic lobe. When Ase is ectopically expressed in third instar optic lobes, ectopic activation of dap expression becomes evident. Therefore, ase activity has a positive regulatory effect on the dap RNA level (Wallace, 2000).

It was asked whether the phenotypical effects on cell proliferation produced by alterations of Dpn and Ase expression may be caused, at least in part, by changes in the levels of dap transcript. During embryogenesis, alteration in the levels of dap expression through either ectopic expression or by loss of function, result in dramatic changes in mitotic activity. Therefore, the mitotic activity in optic lobes of homozygous dap⁶ mutant third instar larvae were analyzed. While predominately recessive lethal, a few dap⁶ homozygous escapees can be viable to adulthood. Therefore, the larval optic lobes of homozygous dap⁶ mutant third instar larva can be analyzed. In such homozygous dap⁶ mutant larvae over-proliferation of the cells of the optic lobes is evident. There is a significant increase in the number of mitotically active cells and break down of mitotic domains, as compared to the wild type (Wallace, 2000).

A model is proposed for mitotic control in the developing third instar optic lobe in which cell proliferation is modulated by a positive regulator of mitotic activity such as Dpn and a negative regulator of mitotic activity such as Ase. In this model, one role of Dpn and Ase would be to interface with cell cycle regulation through the direct or indirect modulation of dap expression. Mitotic control during optic lobe development may involve the following events. Cells that give rise to the optic lobe delaminate from the neuroectoderm during embryogenesis and remain quiescent until first instar with the help of proteins such as Anachronism. The mitotic activity is then initiated through a process that requires the trol gene product and the developmental regulator and transcription factor Eve to begin the proliferation of neuroblasts to form the outer proliferation center. The mitotically active state of outer proliferation center cells would be maintained in part by Dpn. In the absence of Dpn, the cells in the outer proliferation center have a greater chance of exiting mitosis by allowing Dap to be expressed. As cells arrive at the edge of the outer proliferation center, Ase is expressed at high levels, allowing the neuroblasts to become quiescent only after they pass out of the region where Dpn is expressed. Suppression of dap by Dpn in the outer proliferation center would allow the neuroblasts to be mitotically active while the increased expression of Ase at the posterior edge of the outer proliferation center allows the neuroblasts to exit mitosis and begin differentiation. In addition, the resulting quiescent state needs to be maintained in the lamina; otherwise the cells may reenter mitosis (Wallace, 2000 and references therein).

cDNA clone length - 2.2kb

Bases in 5' UTR - 456

Exons - one

Bases in 3' UTR - 349

PROTEIN STRUCTURE

Amino Acids - 486

Structural Domains

In addition to the basic HLH motif, Asense contains a centrally located acidic region and a proline rich region near the N-terminal. Asense lacks the the N-terminal acidic domain found in the other three AS-C transcripts. The protein contains a PEST sequence involved in conferring a short half life, and an opa sequence (Gonzalez, 1989).

EVOLUTIONARY HOMOLOGS

The study of achaete-scute (ac/sc) genes is a paradigm to understand the evolution and development of the arthropod nervous system. The ac/sc genes have been identified in the coleopteran insect species Tribolium castaneum. Two Tribolium ac/sc genes have been identified -- 1) a proneural achaete-scute homolog (Tc-ASH) and 2) asense (Tc-ase), a neural precursor gene that reside in a gene complex. These genes reside 55 kb apart from each other and thus define the Tribolium ac/sc complex. Focusing on the embryonic central nervous system it is found that Tc ASH is expressed in all neural precursors and the proneural clusters from which they segregate. Through RNAi and misexpression studies it has been shown that Tc-ASH is necessary for neural precursor formation in Tribolium and sufficient for neural precursor formation in Drosophila. Comparison of the function of the Drosophila and Tribolium proneural ac/sc genes suggests that in the Drosophila lineage these genes have maintained their ancestral function in neural precursor formation and have acquired a new role in the fate specification of individual neural precursors. These studies, however, do not support a role for Tc-ASH in specifying the individual fate of neural precursors, suggesting that the ability of ac and sc to separately regulate this process may represent a recent evolutionary specialization within the Diptera. Furthermore, it is found that Tc-ase is expressed in all neural precursors, suggesting an important and conserved role for asense genes in insect nervous system development. This analysis of the Tribolium ac/sc genes indicates significant plasticity in gene number, expression and function, and implicates these modifications in the evolution of arthropod neural development (Wheeler, 2003).

Homologs of ac/sc genes have been described in a number of insect and non-insect species. These data support and augment the model in which the last common ancestor of arthropods contained a single prototypical ac/sc gene that carried out both proneural and asense functions. In support of this model, the sole Hydra ac/sc gene, CnASH, does not group with either the proneural or asense genes in phylogenetic analysis and contains motifs indicative of both the proneural and asense genes. In addition, phylogenetic analysis of the two ac/sc genes found in a spider, the chelicerate Cupiennius salei, indicates these genes are more closely related to each other than any other ac/sc genes. These data raise the possibility that a single ancestral ac/sc gene underwent independent duplication events in chelicerates and insects. Given this possibility, it is interesting that one of the Cupiennius ac/sc genes, Cs-ASH1, exhibits a proneural-like expression pattern and appears to carry out a proneural-like function and the other, Cs-ASH2, exhibits an asense-like expression pattern and appears to carry out an asense-like function. These data suggest that independent duplications of an ancestral ac/sc gene have independently given rise to proneural-like and asense-like functions in the chelicerate and insect groups. Alternatively, phylogenetic analysis may inappropriately partition chelicerate ac/sc genes from insect ac/sc genes because of evolutionary selection for species-specific amino acid changes in chelicerate as compared to insect proteins (Wheeler, 2003).

In contrast to the plasticity in proneural ac/sc genes within insects, asense genes appear to be well conserved. A single asense gene exists in Tribolium and Anopheles as well as in the derived dipteran species Ceratitis and Drosophila. In addition, Cupiennius contains a single non-orthologous ac/sc gene with asense-like properties (Cs-ASH2). Thus, the potential that the asense function evolved independently in insects and chelicerates suggests an important role for the asense function in arthropod neural development (Wheeler, 2003).

The existence of ac/sc genes in complexes in Drosophila, Anopheles and Tribolium suggests that this genomic arrangement has been conserved in most if not all holometabolous insects. Shared cis-regulatory regions probably explain why proneural ac/sc genes remain linked in insects and perhaps other species. However, this does not explain why asense is retained in the ac/sc complex as the regulation of asense expression is distinct from that of the proneural ac/sc genes. This phenomenon may be explained by the presence of proneural ac/sc gene cis-regulatory regions surrounding the asense gene. In this model, chromosomal rearrangements that separate asense from the ac/sc complex would probably disrupt proneural ac/sc gene expression and neural precursor formation, thus leading to decreased viability. Consistent with this idea, cis-regulatory regions that drive proneural ac/sc gene expression in the Drosophila PNS appear to flank the ase gene. Thus, the modular cis-regulatory regions that control proneural ac/sc gene expression may also be responsible for the evolutionary conservation of the ac/sc complex. Alternatively, other as yet unidentified genomic forces may preserve the linkage between asense and proneural ac/sc genes (Wheeler, 2003).

These findings raise a number of interesting points. (1) They highlight the potential for evolutionary plasticity of ac/sc genes. Significant changes in ac/sc gene number and expression have occurred over relatively short evolutionary distances and have been correlated with modifications to neural pattern and/or gene function. For example, alterations to ac/sc gene expression in Diptera appear to account for the different patterns of sensory organs found on dipteran species. In addition, data on the role of proneural genes in MP2 fate specification suggest that the increase in ac/sc gene number in Drosophila appears to have facilitated the evolution of new developmental roles for ac and sc in this lineage. (2) The possibility that independent duplication events in chelicerates and insects each gave rise to proneural-like and asense-like genes, indicates that dividing these genetic functions between two genes may be developmentally advantageous. (3) The hypothesis that the last common ancestor of all arthropods contained a single ancestral ac/sc gene suggests it may be possible to identify direct descendants of the prototypical ac/sc gene in extant basal members of each arthropod group. The recent emphasis on the development of genomic resources in non-model organisms should greatly aid progress along this line of inquiry. Thus, continued analysis of ac/sc gene expression, organization and function in arthropods should provide additional insight into the genetic basis of the development and evolution of nervous system pattern (Wheeler, 2003).

The work presented in this paper together with studies on ac/sc gene function in Drosophila provide strong evidence that serial duplications of proneural ac/sc genes in the dipteran lineage led to the diversification of proneural ac/sc gene function in Drosophila. In Drosophila, ac and sc carry out functions distinct from l'sc in specifying the individual fate of the MP2 precursor. Tc-ASH can function in Drosophila as a proneural gene but like Drosophila l'sc fails to specify efficiently the MP2 fate in the CNS. Together these results suggest the ability of ac and sc to specify MP2 fate in Drosophila arose after the divergence of Drosophila and Tribolium. These data provide an example whereby a subset of duplicated genes has evolved a new genetic function while the entire set of duplicate genes has retained the ancestral function (Wheeler, 2003).

In addition to functional changes, the generation of multiple proneural ac/sc genes in the insects was paralleled by modifications to the expression profiles of these genes. In Anopheles (a basal dipteran), and Tribolium a single proneural ac/sc gene is expressed in all CNS proneural clusters. In more derived Diptera the presence of multiple ac/sc genes allows for more complex proneural ac/sc gene expression patterns. For example, Ceratitis contains two proneural ac/sc genes, l'sc and sc; l'sc is expressed in all CNS proneural clusters while sc is expressed in a subset of these clusters. In Drosophila, ac and sc are expressed in the identical pattern of proneural clusters and their expression is largely complementary to that of l'sc. The sum of proneural ac/sc expression in each species then marks all CNS proneural clusters despite differences in the expression pattern of individual proneural ac/sc genes. Thus, in Drosophila, the complete expression pattern of proneural ac/sc genes is divided between the largely complementary expression profiles of ac and sc relative to l'sc. The division of labor between proneural ac/sc genes in Drosophila has resulted in mutually exclusive expression patterns for ac and sc relative to l'sc in proneural clusters like MP2. This spatial separation of proneural gene expression probably facilitated the potential for ac and sc to acquire developmental functions distinct from l'sc (Wheeler, 2003).

Together this work and that of others on arthropod ac/sc genes highlights the utility of studying ac/sc genes in elucidating the genetic basis of the development and evolution of arthropod nervous system pattern. These studies illustrate the dynamic nature of ac/sc gene number, expression and function over a relatively short evolutionary time. Based on this, future work on ac/sc genes in additional arthropod species should continue to provide insight into the molecular basis of the evolution of arthropod nervous system development (Wheeler, 2003).

The basic helix-loop-helix transcription factor NeuroD (Neurod1) has been implicated in neuronal fate determination, differentiation and survival. The expression of cnd-1, a C. elegans NeuroD homolog is first detected in neuroblasts of the AB lineage in 14 cell embryos and maintained in many neuronal descendants of the AB lineage during embryogenesis, diminishing in most terminally differentiated neurons prior to hatching. Specifically, cnd-1 reporter genes are expressed in the precursors of the embryonic ventral cord motor neurons and their progeny. A loss-of-function mutant, cnd-1(ju29), exhibits multiple defects in the ventral cord motor neurons. (1) The number of motor neurons is reduced, possibly caused by the premature withdrawal of the precursors from mitotic cycles. (2) The strict correlation between the fate of a motor neuron with respect to its lineage and position in the ventral cord is disrupted, as manifested by the variable expression pattern of motor neuron fate specific markers. (3) Motor neurons also exhibit defects in terminal differentiation characteristics including axonal morphology and synaptic connectivity. (4) The expression patterns of three neuronal type-specific transcription factors, unc-3, unc-4 and unc-30, are altered. These data suggest that cnd-1 may specify the identity of ventral cord motor neurons both by maintaining the mitotic competence of their precursors and by modulating the expression of neuronal type-specific determination factors. cnd-1 appears to have combined the functions of several vertebrate neurogenic bHLH proteins and may represent an ancestral form of this protein family (Hallam, 2000).

NeuroD has been proposed to function as a neural differentiation factor largely based on the following evidence. In Xenopus and mouse embryos NeuroD is primarily expressed in postmitotic neurons. Unlike early-acting bHLH neuronal determination genes, NeuroD is insensitive to lateral inhibition mediated by Notch and Delta. Moreover, in several CNS regions, including cortex, hippocampus, cerebellum and olfactory bulb, NeuroD expression persists into adulthood, suggesting unknown late-acting functions in terminally differentiated neurons. NeuroD loss-of-function studies in vertebrate retina, hippocampus and cerebellum have provided in vivo support for this function. In addition, retinal cells from NeuroD knockout mice exhibit a three- to four-fold increase in the number of Muller glia within the developing retina, suggesting that NeuroD plays a critical role in the neuron-glial fate determination step. Based on this observation the possibility has been raised that NeuroD may function in both fate determination and differentiation steps of postmitotic retinal neurons (Hallam, 2000).

There are similarities and differences between CND-1 and vertebrate NeuroD. By GFP reporter gene analysis, cnd-1 is expressed in a subset of postmitotic motor neurons where it appears to regulate axonal outgrowth and synaptic connectivity. However, cnd-1 appears to be expressed primarily in mitotically active neuronal precursors and may regulate both the mitotic competence of neuroblasts and neuronal sub-type selection such as neurotransmitter identity. In this regard, CND-1 shares some functional characteristics with vertebrate MASH1 and Drosophila Asense and Target of Pox-n (Tap). Tap is expressed exclusively in one of the neurons that innervate each larval chemosensory organ, possibly controlling the specific properties of that neuron (Gautier, 1997). MASH1 expression in both the CNS and PNS appears to play a dual role, coordinating generic programs of neuronal fate determination with sub-type-specific programs including neurotransmitter identity. Asense is detected in neural precursors and their progeny but is not expressed in proneural clusters. Tap, although more closely related to vertebrate Neurogenins, contains 33% sequence similarity in the extended homology region common to CND-1 and NeuroD. At present no Drosophila NeuroD homolog has been identified. CND-1 shares most sequence homology with vertebrate NeuroD proteins, however, the bHLH domain of CND-1 is also closely related to members of the vertebrate Neurogenin subfamily (Hallam, 2000).

The expression pattern of cnd-1 appears to resemble features of both neurogenins and NeuroD. A hybrid kind of homology is also evident in Y69A2AR, a C. elegans neurogenin-like gene that shows significant sequence similarity and conservation in the genomic structures to cnd-1. CND-1, Y69A2AR, Asense and Tap may therefore resemble ancestral types of bHLH proteins that later diverge into two or more subfamilies that function during sequential steps or in specific subprograms of neuronal fate determination and differentiation. Neurogenins function as neuronal determination factors in neural precursors and activate NeuroD expression in mouse and Xenopus. By analogy, cnd-1 may resemble an ancestral bHLH protein combining the functions of vertebrate Neurogenin and NeuroD (Hallam, 2000).

In mouse and Xenopus, Neurogenins have been shown to activate NeuroD; however, the downstream targets of NeuroD remain unknown. unc-30 (a homeodomain protein related to Drosophila Ptx1), unc-4 (see Drosophila Unc-4) and unc-3 (Drosophila homolog Knot/Collier) have been identified as potential targets of CND-1, because the expression of these three genes is reduced and spatially altered in cnd-1 mutants. Mammalian homologs of unc-30, unc-4 and unc-3 have been identified, and some aspects of their function appear to be conserved. The homeodomain of unc-30 is 83% identical to those of the vertebrate Pitx family, and Pitx-2 can transcriptionally activate GAD67. UNC-3 is a member of vertebrate O/E family, and has multiple roles in cholinergic motor neuron differentiation and sensory neuron function. O/E transcription factors have been implicated in the terminal differentiation of olfactory neurons. PHD1, a vertebrate paired homeodomain protein closely related to C. elegans unc-4, directly follows the expression of MASH1 and precedes the expression of terminal differentiation markers in the dorsal spinal cord. Several consensus E box sequences are present in the promoters of unc-3, unc-4 and unc-30. However, the fact that some cells still express these three genes in cnd-1(ju29) mutants and that ectopic cnd-1 expression fails to induce the expression of these three genes suggests that either CND-1 is an activator that is necessary but not sufficient for the activation of these neuronal-type-specific genes, or that cnd-1 may not act as a direct transcriptional activator or repressor. In the latter case, CND-1 may function as a transcriptional modulator to ensure the correct spatial and temporal expression of neuronal-sub-type-specific differentiation factors. Studies of MyoD and Myf5 bHLH proteins have shown that these bHLH proteins can influence chromatin structure at the target sequences to activate myogenic-specific genes. It will be of interest to see if neurogenic bHLH proteins employ similar functional strategies (Hallam, 2000).

REGULATION

Promoter Structure

The genes of the achaete-scute complex are regulated by intragenic enhancers, resembling locus control regions. These are promoter-like elements that function at a distance, and not as classical proximal promoter elements. The neural precursor regulatory enhancer element of asense has been identified. There are binding sites for heterodimers between Daughterless and Achaete/Scute. Deletion of these sites reduces but does not abolish asense expression (Jarman, 1993), indicating the existence of still more unidentified regulatory regions.

Prospero is a sequence-specific DNA-binding protein with novel sequence preferences that can act as a transcription factor. The consensus binding site for Pros protein is C A/C c/t N N C T/c. Pros binds to a 21-bp fragement of the asense promoter, which contains a CATTTCT sequence, resembling the consensus sequence. Pros binds to a synthetic oligomer containing multiple consensus sequences and activates transcription when this sequence is used as a promoter. The nervous system expression of even-skipped and fushi tarazu requires both these genes in addition to pros for normal function. Prospero can interact with homeodomain proteins to differentially modulate their DNA-binding properties (Hassan, 1997).

Transcriptional regulation

In addition to being a target of Achaete and Scute, asense is also regulated by hairy (Dominguez, 1993), a gene that regulates the pattern of sensilla (peripheral sense organs).

Neurons and glia are often derived from common multipotent stem cells. In Drosophila, neural identity appears to be the default fate of these precursors. Stem cells that generate either neurons or glia transiently express neural stem cell-specific markers. Further development as glia requires the activation of glial-specific regulators. However, this must be accompanied by simultaneous repression of the alternate neural fate. The Drosophila transcriptional repressor Tramtrack is a key repressor of neuronal fates. It is expressed at high levels in all mature glia of the embryonic central nervous system. Analysis of the temporal profile of Tramtrack expression in glia shows that it follows that of existing glial markers. When expressed ectopically before neural stem cell formation, Tramtrack represses the neural stem cell-specific genes asense and deadpan. Surprisingly, Tramtrack protein levels oscillate in a cell cycle-dependent manner in proliferating glia, with expression dropping before replication, but re-initiating after S phase. Overexpression of Tramtrack blocks glial development by inhibiting S-phase and repressing expression of the S-phase cyclin, cyclin E. Conversely, in tramtrack mutant embryos, glia are disrupted and undergo additional rounds of replication. It is proposed that Tramtrack ensures stable mature glial identity by both repressing neuroblast-specific genes and controlling glial cell proliferation (Badenhorst, 2001).

The timing of Ttk69 shows that it does not initiate glial determination. It has been proposed that Ttk69 is expressed in glia to repress neural identity genes. Lateral glioblasts transiently express neural stem cell markers during their development and can adopt the neuronal fate when the glial-determining pathway is not initiated in gcm mutants. Stable glial identity could require neuronal repression. To determine neuronal-specific genes repressed by Ttk69, an analysis was carried out of how ectopic expression of Ttk69 at various stages of nervous system development affects expression of the hierarchy of neuronal markers. This included the proneural genes of the achaete-scute complex, the pan-neural genes (for example asense) and the mature neuronal markers Elav and the antigen 22C10 (Badenhorst, 2001).

Ectopic expression of Ttk69 at any stage does not prevent neuroblast formation. Thus, expression of Ttk69 before neuroblast formation using Kr-Gal4 does not repress the proneural genes achaete or lethal of scute. Strikingly, however, it does inhibit the pan-neural genes asense, dpn and scratch. Consequently, further neuronal development is inhibited and expression of both mature neuronal markers Elav and 22C10 is ablated. Equivalent results were obtained by ectopically expressing Ttk69 in neuroblasts and their progeny using the sca-Gal4 driver. Such expression almost completely inhibits the normal expression of dpn in the embryonic CNS (Badenhorst, 2001).

If, however, Ttk69 is ectopically expressed after the normal neuroblast expression of asense and deadpan, neurons are not ablated. Thus, directed expression of Ttk69 using elav-Gal4 (which is expressed in all post-mitotic neurons after the phase of pan-neural gene expression does not repress the neural markers Elav, 22C210 or Fasciclin II. This indicates that the neural stem cell-specific genes asense and deadpan are the principal targets of Ttk69 repression in the hierarchy of neural determination. Moreover, neural identity, once conferred, cannot be reversed by Ttk69 overexpression, since Ttk69 expression cannot switch neurons to the alternative glial fate (Badenhorst, 2001).

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. ac^Hw1 and ac^Hw49c 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 ac^Hw49c 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 ac^Hw mutations. Adult emc/ttk transheterozygous flies develop ectopic bristles on the wing blade. Similarly, h^rM730, 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. ttk^1e11, 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).

Neural stem cell transcriptional networks highlight genes essential for nervous system development

Neural stem cells must strike a balance between self-renewal and multipotency, and differentiation. Identification of the transcriptional networks regulating stem cell division is an essential step in understanding how this balance is achieved. It has been shown that the homeodomain transcription factor Prospero acts to repress self-renewal and promote differentiation. Among its targets are three neural stem cell transcription factors, Asense, Deadpan and Snail, of which Asense and Deadpan are repressed by Prospero. This study identifies the targets of these three factors throughout the genome. A large overlap in their target genes was found, and indeed with the targets of Prospero, with 245 genomic loci bound by all factors. Many of the genes have been implicated in vertebrate stem cell self-renewal, suggesting that this core set of genes is crucial in the switch between self-renewal and differentiation. It was also found that multiply bound loci are enriched for genes previously linked to nervous system phenotypes, thereby providing a shortcut to identifying genes important for nervous system development (Southall, 2009).

Recent work on Drosophila neural stem cells (or neuroblasts) has provided important insights into stem cell biology and tumour formation. Neuroblasts divide in an asymmetric, self-renewing manner producing another neuroblast and a daughter cell that divides only once to give post-mitotic neurons or glial cells. During these asymmetric divisions the atypical homeodomain transcription factor, Prospero, is asymmetrically segregated to the smaller daughter cell, the ganglion mother cell (GMC), where it can enter the nucleus and regulate transcription. Neuroblasts lacking Prospero form tumours in both the embryonic nervous system and the larval brain. Using the chromatin profiling technique DamID, together with expression profiling, it has been showm that Prospero represses neuroblast genes and is required to activate neuronal differentiation genes. Therefore, Prospero acts as a binary switch to repress the genetic programs driving self-renewal (by directly repressing neuroblast transcription factors) and to promote differentiation. It was found that Prospero represses the neuroblast transcription factors, Asense, Deadpan and Snail, suggesting that these transcription factors may control genes involved in neural stem cell self-renewal and multipotency (Southall, 2009).

To identify the transcriptional networks promoting neural stem cell fate the binding sites of Asense, Deadpan and Snail were profiled, on a whole genome scale. These three proteins are members of a small group of transcription factors that are expressed in all embryonic neuroblast. The first, Asense, is a basic-helix-loop-helix protein, a member of the achaete-scute complex, and a homologue of the vertebrate neural stem cell factor, Ascl1 (Mash1). Unlike the other members of the achaete-scute complex, Asense is not expressed in proneural clusters in the embryo. Asense expression is initiated in the neuroblast and is maintained in at least a subset of GMC daughter cells. Asense is also expressed in most larval brain neuroblasts but is markedly absent from the DM/PAN neuroblast (Bello, 2008; Bowman, 2008). In these lineages, Asense expression is delayed and the daughter cells (secondary neuroblasts) of the Asense-negative DM/PAN neuroblasts undergo multiple cell divisions, expanding the stem cell pool before producing GMCs (Bello, 2008; Boone, 2008; Bowman, 2008). Ectopic expression of Asense limits the division potential of DM/PAN neuroblast progeny (Bowman, 2008). A study in the optic lobe showed that Asense expression coincides with the upregulation of dacapo and cell-cycle exit. Perhaps in combination, these results suggest that Asense may also have a pro-differentiation role (Southall, 2009).

The second transcription factor, Deadpan, is a basic-helix-loop-helix protein related to the vertebrate Hes family of transcription factors. Deadpan is expressed in all neuroblasts and has been shown to promote the proliferation of optic lobe neural stem cells. Unlike Asense, Deadpan is also expressed in the DM/PAN neuroblasts of the larval brain (Southall, 2009).

The third factor, Snail, is a zinc-finger transcription factor whose vertebrate homologues have roles in the epithelial to mesenchymal transition and in cancer metastasis. The Snail family members (Snail, Worniu and Escargot) are known to regulate neuroblast spindle orientation and cell-cycle progression (Southall, 2009).

To further understand the role of these pan neural stem cell transcription factors, their targets were mapped throughout the genome. This, combined with expression profiling, allows building of the gene regulatory networks governing neural stem cell self-renewal, and enhancement of knowledge of the function and mode of action of these transcription factors in neural stem cells (Southall, 2009).

To identify the genes regulated by Asense, Deadpan and Snail in the embryo, d their binding sites in vivo were mapped by DamID, as has been done previously for Prospero (Choksi, 2006). In brief, DamID involves tagging a DNA or chromatin-associated protein with a Escherichia coli DNA adenine methyltransferase (Dam). Wherever the fusion protein binds, surrounding DNA sequences are methylated. Methylated DNA fragments can then be isolated, labelled and hybridised on a microarray. This study expressed Dam fusion proteins in vivo, in transgenic Drosophila embryos. Methylated DNA fragments from transgenic embryos expressing Dam alone serve as a reference. Target sites identified by DamID have been shown to match targets identified by chromatin immunoprecipitation, by mapping to polytene chromosomes and by 3D microscopy data (Southall, 2009).

In comparing the results for Asense, Deadpan, Snail and Prospero, a high degree of overlap was seen between their targets. The average overlap for the four factors in pairwise comparisons is 40%, with the highest overlap between Deadpan and Snail (66%). The similarity in binding is illustrated by the binding of all four factors to the intronic regions of the cell-cycle regulation gene CycE. 245 genes are bound by all four proteins, including genes involved in neuroblast cell fate determination, cell-cycle control and differentiation. These loci are unlikely to represent regions of chromatin accessible to all transcription factors; only 17/245 (7%) were also bound by another neural transcription factor, Pdm1. The large overlap in the targets of Asense, Deadpan, Snail and Prospero implies that these may be a core set of genes involved in neuroblast self-renewal and differentiation (Southall, 2009).

Genome-wide analysis of Asense DamID peaks shows that Asense binding is associated with increased levels of DNA conservation (determined by the alignment of eight insect species. A representation of Asense binding around a generic gene shows an enrichment of ~2 kb upstream of the transcriptional start site, binding within intronic regions (32%) and also downstream of the gene (20%). This distribution is consistent with transcription factor-binding analysis and regulatory sequence studies in mice and humans (Southall, 2009).

The resolution of DamID is ~1 kb and there are currently no motif discovery tools available that can analyse the large amount of sequence data generated by full genome DamID. Therefore, a motif discovery tool, called MICRA (Motif Identification using Conservation and Relative Abundance) was developed to identify overrepresented motifs in low-resolution data. In brief, 1 kb of sequence from each binding site is extracted and filtered for conserved sequences. The relative frequency of each 6-10 mer is then calculated and compared with background frequency. Using MICRA the E-box, CAGCTG, was identified as the most overrepresented 6 mer in the regions of Asense binding (131% overrepresented using a conservation threshold of 0.6. In support of the in vivo binding data, in vitro studies had previously shown that Asense binds to CAGCTG, which is also the binding site of the vertebrate Asense homologue Ascl1 (Mash1) (Southall, 2009).

A GO annotation analysis of the genes bound by Asense shows a highly significant overrepresentation of genes involved in nervous system development and cell fate determination. Similar analyses were performed for Deadpan and Snail and for both transcription factors; DNA conservation was enriched surrounding their binding sites. Deadpan and Snail targets fall broadly into the same gene ontology classes as Asense and Prospero and the binding peaks show a similar distribution relative to gene structure as for Asense. Motif discovery using MICRA identifies sites consistent with previously published in vitro studies for Deadpan (CACGCG and CACGTG) and Snail (CAGGTA). These analyses provide unbiased support for the Deadpan and Snail DamID experiments (Southall, 2009).

When comparing the data sets for Asense, Deadpan, Snail and Prospero genomic loci in which multiple transcription factors bind were found. This phenomenon has been described previously in a Drosophila cell line and, more recently, in mouse embryonic stem (ES) cells in which these loci are termed 'multiple transcription factor-binding loci' (MTL). The ES cell MTLs are associated with ES-cell-specific gene expression and are thought to identify genes important for stem cell self-renewal. The data provide an independent and direct, in vivo demonstration of the phenomenon described in these two earlier studies. Analysis of neural MTLs (as determined by binding of Asense, Deadpan, Prospero and Snail within a 2 kb window) shows increased sequence constraint, correlating with the number of transcription factors bound. The increase in conservation is higher than expected solely based on the combined binding sites of the factors studied. This suggests that further factors may bind to these loci. The loci associated with MTLs are enriched for genes required for proper neural development and for viability (Southall, 2009).

To investigate further the relationship between the number of transcription factors bound at a locus and the importance of the associated target gene in neural development, a database (www.neuroBLAST.org) was assembled comprising DamID data, expression profiling of neural transcription factors, and data on Drosophila nervous system development collated from genetic screens, expression screens, gene homology and text mining screens. Using a random permutation algorithm and training sets of known nervous system development genes weighted scores were assiged to each screen. A total score is calculated for each gene, providing an indication of the gene's involvement in nervous system development. Multiple gene lists can be searched in the database, which is a useful method to pinpoint key genes in user generated gene lists (e.g. expression array results) (Southall, 2009).

Using the data collected for the database, a correlation was consistently found between gene sets bound by increasing numbers of transcription factors and genes in Drosophila genetic screens for defects in nervous system development, eye development and cell-cycle progression or in text mining screens (occurrence of the gene or its homologue with neural or stem cell terms; r=0.98) (Southall, 2009).

This study has shown that Asense, Deadpan, Prospero and Snail bind to genes essential for neural development. This finding enables highlighting of novel genes that may be involved in neural development. The neuroBLAST database ranks genes based on the number of transcription factors bound, together with their appearance in external screens. In this way it identifies known key players in neural development such as prospero, brain tumour, miranda, seven up and glial cells missing. The majority of these genes are identified by multiple binding information (DamID data), independent of external screens and weighted scores (Southall, 2009).

Interestingly, there are many high scoring genes that have not previously been characterised for a role in Drosophila neural development. These include CG32158, an adenylate cyclase known to be expressed in the CNS, two putative transcription factors (CG2052 and CG33291), an NADH dehydrogenase (CG2014) and an F-box protein (CG9772). There is also cenG1A, an ARF GTPase activator, is bound by all four transcription factors and is expressed in neuroblasts. CG9650 is bound by Prospero and Deadpan, and is a homologue of the BCL11b oncogene, which is essential for proper corticospinal neuron development in vertebrates. Another high scoring gene identified by this method is canoe (bound by all four transcription factors, neuroBLAST score of 33.7), which has recently been shown to regulate neuroblast asymmetric divisions (Southall, 2009).

Using the binding data for these four transcription factors as a foundation, attempts were made to construct the transcriptional networks governing neural stem cell self-renewal and differentiation. Although DamID reports protein-binding sites, it cannot show how individual target genes are regulated in response to binding. Expression profiling of neuroblasts and GMCs from wild type and mutant embryos can provide this information, and provide greater insight into the biological function of each of the transcriptional regulators (Southall, 2009).

Expression profiling of asense mutants was performed on 50-100 neuroblasts and GMCs microdissected from the ventral nerve cord of stage 11 wild type and mutant embryos. Genes that are bound by Asense exhibited a significant change in expression level in asense mutant neuroblasts and GMCs. In many cases, neuronal differentiation and Notch pathway genes (enhancer of split complex [E(spl)-C] and bearded complex) are upregulated in the mutant, suggesting that Asense normally represses them, whereas neuroblast genes are downregulated, suggesting they require Asense for expression. This contrasts with the data for Prospero, which represses neuroblast genes and is required for the activation of differentiation genes. Combined with the fact that Prospero represses expression of Asense, these data support an antagonistic relationship between Prospero and Asense. For example, the neuroblast genes miranda and grainy head are activated by Asense and repressed by Prospero, whereas transcription of the differentiation gene Fasciclin I is promoted by Prospero but inhibited by Asense. Interestingly, however, there are also examples of differentiation and cell-cycle exit genes activated by Asense, such as commissureless, hikaru genki and dacapo. Furthermore, when the full expression array data from prospero mutants and asense mutants are compared by cluster analysis two clusters were found in which genes are regulated antagonistically, but also two clusters in which genes are similarily regulated. These data suggest a dual role for Asense: activating the expression of neuroblast genes and repressing differentiation genes in the neuroblast, whereas promoting differentiation when present in the GMC (Southall, 2009).

This study has combined in vivo chromatin profiling and cell-specific expression profiling to identify the gene regulatory networks directing neural stem cell fate and promoting differentiation in the Drosophila embryo. Asense, Deadpan, Snail and Prospero were found to bind to many of the same target genes. The targets of Asense, Deadpan and Snail include neuroblast genes but also many differentiation genes. The binding of these neural stem cell factors to differentiation genes is not entirely unexpected. In vertebrates, stem cell transcription factors bind to and repress differentiation genes to maintain the stem cell state. Additionally, it is becoming apparent that transcription factors can have roles in both activation and repression, in Drosophila and in vertebrate stem cell transcriptional networks. The ability to either repress or activate is likely to be due to interaction with co-factors, and the ability to recruit chromatin remodelling complexes to specific loci (Southall, 2009).

It was shown previously that Prospero represses the expression of Asense and Deadpan in GMCs, supporting a model whereby a core set of genes involved in neuroblast self-renewal and multipotency is activated by the neuroblast transcription factors and repressed by Prospero. This study has shown that, in part, Asense acts oppositely to Prospero, promoting the expression of neuroblast genes and repressing certain differentiation genes. However, the data also indicate that Asense can promote the expression of some genes required for differentiation, including the cell-cycle inhibitor dacapo, which is a member of the p21/p27 family of cdk inhibitors. dacapo expression inititates in the GMC; a reduction was observed in levels of dacapo mRNA in the asense mutant neuroblasts and GMCs, similar to what has been reported in the developing optic lobe. asense mRNA is known to be expressed in at least a subset of GMCs, and Asense protein is present in larval GMCs. This suggests that Asense has a secondary role, to promote GMC cell-cycle exit and differentiation. Asense is absent in larval PAN neuroblasts whose progeny, unlike GMCs, divide in a stem cell-like manner. Ectopic expression of Asense prevents formation of these daughter cells, which can undergo extra divisions (Bowman, 2008), possibly by the upregulation of dacapo, and other differentiation genes (Southall, 2009).

The expression pattern, function and binding site specificity of Asense all correlate strongly with its vertebrate counterpart, Ascl1 (Mash1). Mash1 is expressed in neural precursors in vertebrates, is known to regulate genes involved in Notch signalling (Delta, Jag2, Lfng and Magi1), cell-cycle control (Cdc25b) and neuronal differentiation (Insm1)and recognises the E-box sequence, CAGCTG. Furthermore, Mash1 is consistently found to promote neuronal differentiation, consistent with a pro-differentiation role for Asense. Conversely, it was shown that Asense activates the expression of certain neuroblast genes, such as miranda, which is expressed in all neuroblasts and repressed by Prospero. Deadpan and Snail bind to many neuroblast genes. Given that the expression of deadpan and snail is restricted to pan-neural neuroblasts, it is likely that they can also activate the expression of neuroblast genes. However, confirmation of this awaits expression profiling of deadpan and snail mutant neuroblasts and GMCs (Southall, 2009).

Finally, this study has shown that multiple transcription factor binding is associated with genes that have critical functions in neural development. This relationship can be used to identify novel genes involved in neural development, including those with vertebrate counterparts. A similar gene network and data mining study, using two pair-rule genes in Drosophila, has recently been used to identify a new marker for kidney cancer. Therefore, large-scale analysis of gene regulatory networks, as used here, provides a powerful approach to identifying key genes involved in development and disease (Southall, 2009).

Targets of Activity

In bristle development, asense regulates the expression of achaete and scute (Dominguez, 1993).

DEVELOPMENTAL BIOLOGY

Embryonic

See the embryonic expression pattern of ase at the Berkeley Drosophila Genome Project Patterns of Gene Expression Site

asense expression is first seen in the neural primordium, presumably in neuroblasts. Later, Asense RNA is detected in most cells of the CNS primordium as well as in the labrum, optic lobe rudiment, procephalic neurogenic region and the posterior midgut rudiment. Expression lasts into germ band retraction [Images]. Expression in the brain occurs in isolated clusters of cells and single cells. Expression is thought to identify actively proliferating cells (Gonzalez, 1989).

The neuroblasts that give rise to the brain segregate from the procephalic neurectoderm and form three neuromeres: the protocerebrum (P), deuterocerebrum (D) and tritocerebrum (T). The first two neuromeres can each be further subdivided into three regions: the anterior, central and posterior protocerebral domains (Pa, Pc and Pp) and the anterior central and posterior deuterocerebral domains (Da, Dc and Dp). With respect to their position and the expression of the markers asense and seven-up, 23 small groups of neuroblasts consisting of from one to five neuroblasts per group have been identified. In Drosophila there are a total of 75-80 neuroblasts, 19 identified in Pa, 14 in Pc and 18 in Pp. There are 22 identified in the deuterocerebral domain and 6 in the tritocerebrum. The first seven groups of cells that segregate (Pc1 to 4, Dc1 to 3 and Dp1; collectively called SI/II) arise from the central domain of the protocerebral and deuterocerebral neurectoderm, respectively. Later groups form anterior and posterior to the earlier ones, leading to a centrifugal increase in the procephalic neuroblast population. SIII neuroblasts (Pa1 to 4, Pp1 and 2, and Dp2) arise during stage 10. SIV neuroblasts (Pa5 and 6, Pp3 and 4, Da1 and T1 and 2) arise during early stage 11, and SV neuroblasts (Pp5, Pdm) during stage 11 and early stage 12 (Younossi-Hartenstein, 1997).

Neuroblasts delaminate from the procephalic neurectoderm in a stereotyped spatiotemporal pattern that is tightly correlated with the expression of lethal of scute. The pattern of neuroblasts was reconstructed by using the marker asense; similar to its expression in the ventral neuroblasts, asense labels all brain neuroblasts. seven-up, expressed in specific subsets of neuroblasts making up approximately one-third of the total, is also used as a marker. For most, if not all, of these clusters the number of neuroblasts and the time of onset of svp expression are absolutely invariant. Neuroblast groups expressing svp are the following (number of cells in each group is given): Pa1 (1), Pa3 (2), Pa5 (2), Pc1 (1), Pc3 (4-6), Pp1 (1-2), Pp3 (3), Dc1 (1), Dc3 (5-6), Dp1 (2-3), and T1 (2) (Younossi-Hartenstein, 1996).

Larval

asense participates in the allocation of the sensory mother cells that give rise to the bristles of the anterior wing margin. asense is required for the development of hairy-induced extra sensilla. Ectopically expressed asense generates extra sense organs (Dominguez, 1993). asense expression arises in one large cluster in each leg disc. Expression is strong in the CNS, especially in a cap of cells over each optic lobe, destined to generate ganglion mother cells of the lamina and medulla. asense is expressed in the precursors of all adult sensory organs, the sensory mother cells, and their immediate progeny. Its deletion causes loss of some sensory organs and the abnormal differentiation of some of the remaining ones (Dominguez, 1993).

The selection of Drosophila sense organ precursors (SOPs) for sensory bristles is a progressive process: each neural equivalence group is transiently defined by the expression of proneural genes (proneural cluster), and neural fate is refined to single cells by Notch-Delta lateral inhibitory signalling between the cells. Unlike sensory bristles, SOPs of chordotonal (stretch receptor) sense organs are tightly clustered. It has been shown that for one large adult chordotonal SOP array (the adult femoral chordotonal sense organ), clustering results from the progressive accumulation of a large number of SOPs from a persistent proneural cluster. This is achieved by a novel interplay of inductive epidermal growth factor- receptor (EGFR) and competitive Notch signals. EGFR acts in opposition to Notch signaling in two ways: it promotes continuous SOP recruitment despite lateral inhibition, and it attenuates the effect of lateral inhibition on the proneural cluster equivalence group, thus maintaining the persistent proneural cluster. SOP recruitment is reiterative because the inductive signal comes from previously recruited SOPs (zur Lage, 1999).

The adult femoral chordotonal sense organ arises from a group of some 70-80 SOPs. A developmental analysis of Ato expression has revealed that these SOPs accumulate over an extended period of time in the dorsal region of each leg imaginal disc during the third larval instar and early pupa. The continued expression of Ato implies a sustained requirement for proneural function throughout the process of SOP accumulation. Unusually, Ato is persistently expressed in a group of ectodermal cells identified as the proneural cluster (PNC). From this PNC, cells are funnelled inward into a cavity formed by the folding of the disc. This invagination later becomes visible as a distinctive 2-cell wide intrusion, which is referred to as the 'stalk'. Cells at the deepest end of the stalk undergo shape changes to form an amorphous inner SOP mass. Invaginating cells are characterised by upregulation of Ato expression, a characteristic of SOP commitment. Surprisingly, SOP markers (Ase protein and the A101 enhancer trap line) are not expressed in all the stalk SOPs. Instead, these markers are only apparent in older cells, particularly at the time when they become part of the inner mass (which is therefore referred to as mature SOPs). Despite this, entry into the stalk seems to mark SOP commitment, since both the stalk and the mature SOPs are absent in discs from ato mutant larvae. This apparent intermediate stage may not have a counterpart in external sense organ precursor formation, although there is some evidence for multiple steps between the uncommitted cell and the SOP (the so-called pre-sensory mother cell state). Initially, Ato remains activated in all invaginated SOPs. This extended period of proneural gene expression is unusual since AS-C proneural expression is typically switched off in SOPs shortly after commitment. Later, at approximately 6 hours before puparium formation (BPF), Ato expression is switched off synchronously in the mature SOPs, although expression remains in the stalk SOPs and the PNC. At this point there is very little overlap between Ato and Ase or A101 (zur Lage, 1999).

The progressive accumulation of chordotonal SOPs suggests that a recruitment mechanism could explain the clustering of SOPs. The Drosophila Egfr signaling pathway is involved in a number of recruitment processes in development, and a role for Egfr signaling has been demonstrated in the induction of embryonic chordotonal precursors (zur Lage, 1997). Although there appear to be significant differences in the process of SOP formation in imaginal discs, as compared with the embryo, it was asked whether Egfr signaling is also involved in forming the femoral chordotonal cluster. To address this question, the pathway was conditionally disrupted by expressing a dominant negative form of Egfr protein. Expression of UAS-Egfr DN results in a dramatic loss of chordotonal SOPs in late third instar imaginal leg discs (as judged by Ase protein expression or the A101 enhancer trap line). This demonstrates that Egfr signaling is required for the process of femoral chordotonal SOP formation. In contrast, the appearance of bristle SOPs is unaffected, arguing against the possibility of a nonspecific effect on SOPs in general (zur Lage, 1999).

Effects of Mutation or Deletion

A deficiency of asense causes misrouted axons in the adult brain (Gonzalez, 1989).

klumpfuss shows genetic interactions with achaete, scute, lethal of scute and asense. l'sc is able to activate klu expression, but apparently only in the wing disc. There appears to be only a weak influence of the AS-C genes on klu expression, restricted to the wing area of the wing disc. However, the overall expression pattern of klu is largely independent of proneural genes. The assumption that SOPs enter apoptosis in klu mutants is supported by the observation of abundant cell death in other developing organs of klu mutants, like the legs. At certain bristle positions, such as that of the anterior sternopleura, klu is required during early bristle development immediately after proneural gene function, in order to allow a particular epidermal cell to develop as a SOP. It is suggested that klu is required only for initiation of bristle development, being downregulated once specification takes place (Klein, 1997).

In the expectation that more bHLH genes are required in neurogenesis in Drosophila, new bHLH genes have been sought in PCR experiments using degenerate primers based on homology to atonal. In addition to ato itself, the PCR product contained potential bHLH-encoding sequences from two new closely related genes, which have been termed amos and cousin of atonal (cato). cato is expressed widely in the developing PNS after neural precursor selection but before terminal differentiation. Consistent with this pattern, cato appears to be required for proper sensory neuron morphology (Golding, 2000).

There are widespread defects in neuronal morphology in cato-deficient embryos: the dendrites of chordotonal neurons are consistently malformed, appearing longer and often thicker than their wild-type equivalents. Although it is likely that cato mutant sensory neurons would be severely impaired functionally, the morphological defects observed are subtle. This might be the result of redundancy between cato and related gene functions. Of particular interest as a possible cato interactor is ase. because ase encodes a bHLH protein that is expressed in a postproneural pattern similar to that of cato. Although ase is widely expressed in the developing nervous system, most embryonic neurons appear normal in ase mutant embryos, and such individuals are indeed quite viable. Despite this, it was found that additional mutation of ase strongly enhances the neuronal differentiation defects of cato-deficient embryos. In particular, the dendritic defects described above are strongly exacerbated in these embryos. The scolopale is a glial structure that surrounds the PNS neuron. In the most striking cases it appears that the scolopale structure itself is inappropriately stained by neuronal antibodies, suggesting either that the scolopale cell was beginning to exhibit neuronal characteristics or that the dendrite extends around it rather than entering into it. Additionally, some neurons appear to have lost their particular neuronal characteristics and, although stained by 22C10, had either lost or not developed any distinctive neuronal morphology (Golding, 2000).

In addition to the worsening of neuronal morphology, these double-mutant embryos develop axon-related defects that are not observed in either cato or ase mutant embryos. In particular, many neurons of the dorsal and lateral sensory neuron groups have poorly formed or misrouted axons, and in extreme cases, axons are entirely missing. This suggests that cato and ase are required either for correct axon pathfinding to the CNS or for the general process of axon outgrowth itself. The axon outgrowth defects are similar to those observed in pros mutant embryos. Thus, as in pros mutants, dorsal sensory neurons are more strongly affected -- probably because they are more sensitive in their response to signals provided by the ventral CNS that aid their pathfinding (Golding, 2000).

Experimental misexpression of proneural genes in developing ectoderm results in ectopic sense organ formation owing to excessive SOP commitment. For instance, the scutellum (rear of thorax) normally bears four external sense organs (bristles) and no chordotonal organs. UAS-scute flies develop extra external sense organs when expression is driven by Gal4^109-68, a driver line specific for proneural cluster cells. Conversely, ato is functionally distinct in that its misexpression results in ectopic chordotonal organs within the scutellum, although numbers of external sense organs are also formed, which is postulated to be the result of dissociation of ato's SOP-determining and chordotonal identity-determining properties in this assay. Interestingly, when ase is misexpressed, phenotypes identical to those of sc are observed. Thus, although ase is not normally expressed during SOP commitment, it is capable of proneural function that is indistinguishable from ac and sc (Golding, 2000).

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date revised:  1 November 2010

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