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).

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).

REFERENCES

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date revised:  15 June 2008

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