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).

Functional genomics identifies neural stem cell sub-type expression profiles and genes regulating neuroblast homeostasis

The Drosophila larval central brain contains about 10,000 differentiated neurons and 200 scattered neural progenitors (neuroblasts), which can be further subdivided into ~95 type I neuroblasts and eight type II neuroblasts per brain lobe. Only type II neuroblasts generate self-renewing intermediate neural progenitors (INPs), and consequently each contributes more neurons to the brain, including much of the central complex. Six different mutant genotypes were characterized that lead to expansion of neuroblast numbers; some preferentially expand type II or type I neuroblasts. Transcriptional profiling of larval brains from these mutant genotypes versus wild-type allowed identification of small clusters of transcripts enriched in type II or type I neuroblast,s, and these clusters were validated by gene expression analysis. Unexpectedly, only a few genes were found to be differentially expressed between type I/II neuroblasts, suggesting that these genes play a large role in establishing the different cell types. A large group of genes predicted to be expressed in all neuroblasts but not in neurons were identified. A neuroblast-specific, RNAi-based functional screen was performed and 84 genes were identified that are required to maintain proper neuroblast numbers; all have conserved mammalian orthologs. These genes are excellent candidates for regulating neural progenitor self-renewal in Drosophila and mammals (Carney, 2012).

To identify genes expressed differentially between type I and type II neuroblasts, genes were sought clustered with pros and ase, the only two genes known to be differentially expressed in type II neuroblasts. It was found that pros and ase reside together in a small sub-cluster of only 11 genes within group B. This sub-cluster as a whole exhibits reduced expression in brat, lgl, and lgl lgd mutants and enrichment in aur, aPKC^CAAX, and lgl pins; remarkably, no other sub-cluster exhibits such a pattern. This suggests that the other nine genes in the cluster may also be specifically expressed in type I neuroblasts, like pros and ase, and that these are potentially the only genes that exhibit this unique pattern (Carney, 2012).

To test whether other genes in the small pros/ase cluster are also expressed in type I neuroblasts but not type II neuroblasts, an antibody was obtained to a candidate from this cluster, Retinal homeobox (Rx), a homeodomain-containing transcription factor. It was found that Rx is completely absent from type II neuroblasts, similar to Pros and Ase; Rx is detected in several type I neuroblasts as well as in a subset of differentiated type II progeny. Consistent with this expression pattern, it was found that brat mutants, which overproduce type II neuroblasts, show a loss of Rx staining. In contrast, lgl pins mutants, which have ectopic type I neuroblasts, show territories of strong Rx expression which is confined to Pros⁺ (likely type I-originating) cells. The fact that only a small patch of lgl pins mutant brain tissue is Rx⁺ is probably because Rx is normally expressed in a subset of type I neuroblasts. It is concluded that Rx, like Pros and Ase, is expressed in type I but not type II neuroblasts. Thus, most or all of the 11 genes in the pros/ase sub-cluster may be expressed in type I but not type II neuroblasts (Carney, 2012).

Genes expressed in type II neuroblasts but not type I neuroblasts were sought, as there are currently no known markers specifically expressed in type II neuroblasts. It was reasoned that transcripts expressed in type II neuroblasts should be enriched in genotypes that overproduce type II neuroblasts: brat, lgl and lgl lgd. One small cluster was found enriched in two of the three mutants (brat and lgl lgd). This cluster contains just 10 genes, seven encoding transcription factors. To verify the expression pattern of this gene cluster, the expression of one gene product, Optix, was examined. Optix is a conserved homeodomain-containing transcription factor required for eye development. Consistent with the microarray data, it was found that most of the Optix expression in the brain is indeed restricted to type II lineages; four of the six dorso-medial type II neuroblasts (DM1, 2, 3, and 6) express Optix, as do most of the INPs, GMCs, and neurons in these lineages. In addtion, recent work has shown that another gene in this cluster, pointedP1, is also preferentially expressed in type II neuroblasts (Sijun Zhu and Y.N. Jan, personal communication to Carney, 2012). The other two dorso-medial type II lineages (DM4 and 5) exhibit some expression of Optix in a subset of neuronal progeny, but it is absent from the neuroblasts and INPs in these lineages. In addition, a single dorsal type I neuroblast expresses Optix. Inspection of mutant brains further confirmed the type II-biased expression of Optix, in that brat mutant brains exhibit a marked increase in Optix⁺ neuroblasts, and in lgl pins, the increase in Optix is almost exclusively in a Pros⁻ (type II-originating) region of the brain. These results indicate that the clustering relationships can be used to predict type I/type II expression bias with good accuracy. It is concluded that Optix is primarily expressed in type II but not type I neuroblasts, and that Optix and the other five genes in this cluster are excellent candidates for regulators of type II neuroblast identity (Carney, 2012).

It has previously been shown that co-clustering of genes in expression profiling data is likely to reflect physical or genetic interactions and participation in the same pathway. The current results are consistent with these conclusions. For example, a small group of 11 genes was identified containing the only two genes known to be expressed in type I but not type II neuroblasts, and a third gene was shown to have a similar pattern of expression — thus all genes in this cluster are likely to be expressed in type I but not type II neuroblasts. Furthermore, the strong enrichment of GO terms in small sub-clusters within both group A and group C indicates that genes within these clusters are likely to share similar functions or processes (Carney, 2012).

At the outset of this study, a large group of genes was expected to be differentially expressed in type II versus type I neuroblasts, because these neuroblasts have such strikingly different cell lineages. However, only a few gene clusters were identified that were differentially regulated in such a type I/type II consistent manner — the 11 genes in the pros/ase cluster depleted in type II neuroblasts and the 10 genes enriched in type II neuroblasts. This suggests that the small number of genes identified may play a disproportionately large role in generating differences between type I and type II neuroblasts. Might pros and ase be the only genes regulating type I/type II differences? Both Ase and Pros can promote cell cycle exit, which may result in the Ase⁺ Pros⁺ type I progeny taking a GMC identity and undergoing just one terminal division and the Ase⁻ Pros⁻ type II progeny taking an INP identity and continuing to proliferate. Indeed, the misexpression of either Ase or low levels of Pros in type II neuroblasts is sufficient to cause the loss of INPs and/or their premature cell cycle exit, thereby decreasing lineage size toward the size of type I neuroblasts. However, it is unclear what is required to fully transform these cells into type I neuroblasts; addressing this question will require additional molecular markers and tracing the axon projections of the progeny of these 'transformed' neuroblasts (e.g., do they now fail to make intrinsic neurons of the adult central complex?). The fact that mutants in ase and pros do not transform type I neuroblasts into type II neuroblasts indicates that other genes, perhaps some in the pros/ase cluster described here, are also important for specification of type I neuroblast identity (Carney, 2012).

It was found that the neuroblasts in each mutant have remarkably similar expression profiles, as shown by the extensive list of similarly expressed genes in group A and by the list of genes with depleted expression in mutant brains, represented by group C. It is believed that these categories provide lists of genes that are representative of those expressed in neuroblasts and neurons, respectively, based on all known neuroblast-specific genes showing up in group A and all known neuron- or glial-specific genes being excluded from group A (Carney, 2012).

Group B genes apparently are not expressed in all neuroblasts like the group A genes, nor in all neurons or glia like group C genes. However, group B genes are more likely to be expressed in subsets of neurons, not neuroblasts, because group B genes as a whole have an over-representation of GO terms more similar to group C than to group A. Why then are group B genes excluded from group C, the neuron cluster? One possible explanation is that different neuroblast lineages are affected in each mutant, and thus different subsets of neurons are missing in each mutant. If different neuroblast lineages express different genes (which seems likely), then each mutant would be missing a unique subset of neural differentiation genes, leading to the cluster being excluded from group C. This model raises the intriguing possibility that group B sub-clusters may represent lineage-specific genes (Carney, 2012).

It is also possible that the mutant genotypes themselves may cause unique transcriptional differences, leading to a cluster of genes in group B. For example, several small sub-clusters in group B are expressed differently only in aPKC^CAAX brains. These transcriptional differences are not correlated with the number of type I or type II neuroblasts. Instead, these genes appear to be differentially expressed in response to elevated aPKC. Drosophila aPKC has been best studied as a component of the apical complex in mitotic neuroblasts, and its capacity for causing ectopic self-renewal has been shown to be reliant on both its catalytic activity and its membrane localizatio. However, aPKC has been ascribed a role in neuroblast proliferation as well as in polarit, and a vertebrate homolog, PKC-zeta, was shown to possess a nuclear role in both proliferation of neural progenitors and neuronal cell fate specification. These observations are consistent with a role of aPKC in causing transcriptional differences (Carney, 2012).

The findings of this study highlight the importance of expression profiling of multiple genotypes. This method gave a more reliable picture of the group A genes expressed in neuroblasts, because genes with lineage-specific or genetic background-specific changes in expression appeared to be focused into group B, where they do not interfere with the clustering of groups A and C. In addition, two small sub-clusters of genes were identified in group B that are excellent candidates for being preferentially expressed in type I or type II neuroblasts, for which there have been few examples to date. Finally, it is concluded that group A genes are likely to be expressed in neuroblasts, and functional studies have identified 84 genes that are conserved in mammals and required for regulating neuroblast numbers in Drosophila. Future phenotypic analysis in Drosophila will determine whether these genes regulate neuroblast survival, quiescence, asymmetric cell division, and/or self-renewal. Future studies on the expression and function of orthologous genes in mouse neural progenitors and human stem cells (IP or neural) will reveal whether they have conserved roles from flies to mammals (Carney, 2012).

A temporal transcriptional switch governs stem cell division, neuronal numbers, and maintenance of differentiation

The importance of producing the correct numbers of neurons during development is illustrated by both evolutionary enhancement of cognitive capacities in larger brains, and developmental disorders of brain size. In humans, increased neuronal numbers during development is speculated to partly derive from a unique subtype of neural stem cells (NSCs) that undergo a phase of expansion through symmetric self-amplifying divisions before generating neurons. Symmetric amplification also appears to underlie adult neural stem maintenance in the mouse. However, the mechanisms regulating this behavior are unclear. This study reports the discovery of self-amplifying NSCs in Drosophila and shows that they arise by a spatiotemporal conversion of classical self-renewing NSCs. This conversion is regulated by a temporal transition in the expression of proneural transcription factors prior to cell division. A causal link was found between stem cell self-amplification and increased neuronal numbers. It was further shown that the temporal transcriptional switch controls both stem cell division and subsequent neuronal differentiation (Mora, 2018).

The development of functional organs relies on the coordinated production of cells of different identities with temporal, spatial, and numerical precision. In the brain, where information processing depends on the output of interconnected neuronal circuits, not only the ratios of different neuronal subtypes, but also absolute numbers are important for optimal function. The number of neurons in the adult brain is a direct consequence of a spatiotemporally coordinated sequence of divisions of neural stem cells (NSCs) during development. However, it remains unclear how NSCs alter their division patterns over time and whether these alterations are causal to the generation of the correct number of neurons. Less clear still is whether and how the temporal transitions in NSC division influence the differentiation of their progeny (Mora, 2018).

In both mammals and insects NSCs regulate neurogenesis through a series of self-renewing divisions. NSC division patterns can be broadly classified in five categories. In three of these, NSCs divide asymmetrically renewing themselves and giving rise to daughters that differ in their proliferation potentials: daughters that do not divide, daughters that divide once, and daughters that divide multiple times. In the other two, NSCs divide symmetrically. One type of symmetric division common to vertebrates and invertebrates signals the end of stemness through the generation of two daughter cells committed to differentiation. A second, much rarer type, expands the progenitor pool through the generation of two cells, which retain the expression of NSC markers and the ability to generate neurons. In mouse, self-renewal by symmetric division has recently been reported to be predominant during adult neurogenesis, in contrast to what is observed in embryonic stages where most NSCs divide asymmetrically. In the primate brain, embryonic self-amplifying divisions have been detected in the NSCs known as outer radial glia (oRG). Multiple lines of evidence support the hypothesis that oRGs' high abundance and proliferative capacity are critical for the vast increase of brain size in primates. However, the direct evidence for the impact of symmetric amplification of NSCs on neuronal numbers, the mechanisms that mediate the switch from self-renewal to self-amplification and then to neurogenesis, and the impact of such a switch on terminal differentiation remain unexplored (Mora, 2018).

The fruit fly Drosophila melanogaster has long been a powerful model system for the discovery of the genetic, cellular, and molecular underpinnings of the behavior of NSCs, as well as the generation and differentiation of their neuronal progeny. Drosophila NSCs are called neuroblasts (Nbs), and two major modes of neurogenesis have been described. The type I Nbs self-renew while giving rise to committed daughters called ganglion mother cells (GMCs) that in turn divide terminally to produce two neurons or glia. The type II Nbs also self-renew but produce intermediate progenitors that in turn undergo a limited number of self-renewing divisions giving rise to GMCs, which give rise to neurons. Thus, to date, all Nbs in the fly brain are thought to produce neurons by asymmetric self-renewal and no symmetrically dividing, self-amplifying, NSCs have been found (Mora, 2018).

The majority of the fly brain is dedicated to visual processing. The higher-order visual centers called the optic lobes (OLs) receive the visual input from the retina and are arranged in four neuropils called lamina, medulla, lobula, and lobula plate (LP); all four organized in retinotopic maps. OL neurons derive from two major proliferation zones, called the outer proliferation center and the inner proliferation center (IPC), containing actively dividing Nbs. The organization of the OLs is constrained by the characteristics of the compound eye, which is composed of 1~750 repetitive units of 8 photoreceptors covering the visual field and projecting to the OL in a retinotopic order. This integration of the retinal map requires a tight control of the diversity and stoichiometry of the neuronal populations. While temporal and spatial cues required to generate different types of neurons have been identified, the control of the production of large numbers of neurons is much less understood. One striking example is the motion detection neurons of the LP called T4/T5. For each of the 750 retinal units, the LP contains 8 different T4/T5 direction sensitive neurons (T4a, b, c, d, and T5a, b, c, d, respectively). Thus, the direction-selective T4/T5 lineage generates approximately 12,000 neurons, representing more than 10% of all neurons in the fly brain. How such a massive proportion of neurons is generated is entirely unknown (Mora, 2018).

Another highly conserved feature of neurogenesis is that it is regulated by a small and highly conserved set of transcription factors known as the proneural proteins. First described in Drosophila, basic loop-helix proneural factors regulate neurogenesis in insects as well as in mammals. There are three families of proneural proteins named after their founding members; the Atonal (ATH), Achaete-scute (AS), and Neurogenin families. Proneural proteins most conserved function is to provide progenitors with the neuronal fate. In addition, they have been found to promote asymmetric division, exit from cell cycle and initiation of differentiation. Whether proneural proteins can promote symmetric proliferation or if they can combine their proliferation and differentiation functions in the same neuronal linage is still unclear (Mora, 2018).

This study has identified the first symmetrically self-amplifying NSCs in Drosophila giving rise to the population of T4/T5 neurons. These Nbs are generated by a temporal conversion of asymmetrically dividing Nbs, which is accompanied by a temporal transition in proneural protein expression from the AS protein Asense (Ase) to the ATH protein Atonal (Ato). Furthermore, it was discovered that the switch from Ase to Ato is necessary and sufficient for the switch in stem cell division pattern and the generation of the correct number of neurons. Lastly, it was demonstrated that Atonal creates a quantitative change in target gene expression that is propagated throughout the lineage to ensure the commitment of T4/T5 neurons to terminal differentiation (Mora, 2018).

This is the first example of transient amplification by symmetric division of NSCs in a non-mammalian animal, namely the Drosophila fruit fly. These cells are termed type III Nbs to distinguish them from previously described Nb types 0, I, and II. Embryonic NSC symmetric expansion is common in mammals, especially in gyrencephalic mammals, where oRG are highly abundant. This includes ferrets, non-human primates, and humans, but not rodents. oRG is thought to be in part responsible for the brain size expansion that is observed in these species. Having simpler models that recapitulate at least some aspects of oRG biology could be particularly relevant to the study of fundamental questions surrounding the control of brain size. Understanding the symmetry of self-renewal is also relevant for the study of adult neurogenesis where symmetric division have recently been shown to be predominant. In this context, limited rounds of symmetric self-renewal and consuming symmetric differentiation division can explain how neurogenesis is sustained for extended periods of time. This work finds that Drosophila symmetrically amplifying Nbs expand the progenitor pool while at the same time scheduling the future terminal differentiation of their progeny. This study describes genetic and regulatory control mechanisms of these features and the consequences of interfering with such mechanisms for brain development (Mora, 2018).

The type III Drosophila Nbs described in this study are located in the visual system anlagen in a region known as the IPC, where they generate two different neuronal populations: the C2, C3, T2, and T3 neurons (C/T neurons) and the T4/T5 neurons, in that specific temporal order. IPC Nbs transit through two distinct types of proliferation: an earlier phase of type I asymmetric divisions to generate C/T neurons and a later phase of symmetric transit amplification. These two phases coincide with a change in neuronal fate and number. While late born T4/T5 constitute one of the largest lineages in the Drosophila brain, the early born C/T neurons are two times less abundant. Although it is difficult to know the exact number of symmetric divisions each upper-Nb undergoes, it is interesting to note that one symmetric amplification before the terminal production of GMCs would account for the doubling of the number of upper-Nbs compared with lower-Nbs that were observed, resulting in exactly four T4/T5 neurons per upper-Nb. A concurrent study (Pinto-Teixeira, 2018) proposes that this particular stoichiometry may be accounted for through a single terminal Nb division. While the current observations do not contradict the stoichiometry, the suggestion that there is no amplifying step prior to the terminal division is difficult to reconcile with the multiple lines of evidence presented in this study. Together with a study by Apitz (2018), these authors further show that the layer specificity of T4/T5 neurons relies on Dpp and that the T4 versus T5 fate is Notch dependent. The Apitz study further shows how the Dpp signal is maintained from NE progenitors to neurons through a temporal relay mechanism. Together, these studies open the door for understanding precisely how this very large and complex lineage combines numerical expansion, cell fate, and layer-specific targeting over a series of successive temporal developmental transitions (Zhang, 2018).

This study focuses on the temporal transition of proliferation properties and shows that they are regulated by the serial expression of two proneural proteins, Ase and Ato. Interestingly Ase and Ato had not been involved in C/T versus T4/T5 fate decision, suggesting that lineage size can be controlled independently of cell fate. Previous studies in the IPC have shown that the switch in neuronal fate depends on another temporal series of two factors called Tailless and Dichaete. It would be interesting to investigate the crosstalk between these two temporal series as a model to further understand how neuronal numbers and neuronal fate are integrated during development. The current findings provide one of the first examples of Nbs changing their proliferation properties to achieve lineage size proportions, where NSC amplification is causally linked to an increase in the number of neurons generated (Mora, 2018).

Drosophila, Ato acts as a transcriptional activator regulating the commitment of different subsets of epithelial cells to the neuronal fate. However, in the IPC Nbs, Ato plays a dual role. On the one hand, it promotes the amplification of progenitors that express it, and on the other hand it ensures the terminal differentiation of their neuronal progeny. Curiously, Atoh1, the mammalian homolog of Ato, has been described both as a tumor suppressor in colorectal cancer and as an oncogene in medulloblastoma, the most common malignant brain tumor in children. It is suspected that this context-dependent function may be related to the dual role of Ato in amplification and differentiation characterized in this study (Mora, 2018).

It is important to note that, in the IPC, Ato can robustly impose symmetric division when ectopically expressed. However, only a fraction of Nbs divisions are affected in its absence. This demonstrates that Ato is sufficient, but not always necessary, for symmetric division, and suggests the existence of an overlapping and independent mechanism controlling the process. Ato in this context likely acts to ensure robust transitions first to symmetric amplification and later to differentiation. It is proposed that the strong reduction of T4/T5 neuron numbers in ato mutant brains is due to an incomplete transition from asymmetric to symmetric division. However, the effect of other functions of Ato yet to be characterized, for example in ensuring neuronal survival, cannot be excluded (Mora, 2018).

The fact that Ato expression in Nbs controls the differentiation of T4/T5 neurons is demonstrated by the ectopic expression of Nbs markers and the global downregulation of differentiation genes in neurons of ato mutant animals. This resembles the de-differentiation phenotype previously found in Drosophila mutants of longitudinal lacking (lola). However, unlike lola, Ato itself is never expressed in neurons, not even transiently. It is proposed that a stable cellular memory of differentiation is initiated transcriptionally in stem cells and inherited through successive cell divisions to ensure terminal differentiation of neuronal progeny. What the mechanisms of such a memory are, how they are activated in stem cells, and how they relate to stem cell division mode are exciting questions for future investigation (Mora, 2018).

A recurring observation throughout this analyses is that quantitative, rather than all or nothing, changes in gene expression downstream of Ato control the temporal progression of developmental events. For example, premature Ato expression causes a relatively modest reduction in Ase expression, and yet suffices to induce symmetric division prematurely. Similarly, quantitative regulation of Brat levels is required for a dose-dependent maintenance of terminal differentiation in postmitotic neurons. Brat is a member of a family of evolutionarily conserved tumor suppressor proteins that regulate differentiation and growth. In type I and II Nbs, Brat is asymmetrically inherited to promote differentiation. In IPC Nbs, Brat is symmetrically inherited during the transient amplification but it does not prevent Nb gene expression. It is therefore proposed that it is the progressive accumulation through temporal quantitative regulation, rather than its expression per se, that schedules the onset and maintenance of differentiation (Mora, 2018).

How cell division and differentiation are coordinated to determine organ size is a fundamentally important but poorly understood process. In Drosophila, the intrinsic activity life time of given proneural transcription factor is both a developmental and evolutionary strategy for the control of cell number in the peripheral nervous system. During the development of mammalian telencephalon, the expression of Ascl1, the mammalian homolog of Drosophila Achaete-scute proteins such as Ase, oscillates in NSCs. These oscillations promote proliferation, while sustained expression of Ascl1 promotes neuronal differentiation. Finally, there is evidence that spatiotemporal transitions in cross-regulatory transcription factors control root meristem growth in plants. This study shows that a similar logic regulates brain size. These observations suggest that the differential, temporally restricted and quantitative regulation of transcription factors and their target genes may serve a universal role as molecular clocks underlying the coordinated temporal order of developmental events (Mora, 2018).

Tailless/TLX reverts intermediate neural progenitors to stem cells driving tumourigenesis via repression of asense/ASCL1

Understanding the sequence of events leading to cancer relies in large part upon identifying the tumour cell of origin. Glioblastoma is the most malignant brain cancer but the early stages of disease progression remain elusive. Neural lineages have been implicated as cells of origin, as have glia. Interestingly, high levels of the neural stem cell regulator TLX correlate with poor patient prognosis. This study shows that high levels of the Drosophila TLX homologue, Tailless, initiate tumourigenesis by reverting intermediate neural progenitors to a stem cell state. Strikingly, tumour formation could be blocked completely by re-expressing Asense (homologue of human ASCL1), which is a direct target of Tailless. These results predict that expression of TLX and ASCL1 should be mutually exclusive in glioblastoma, which was verified in single-cell RNA-seq of human glioblastoma samples. Counteracting high TLX is a potential therapeutic strategy for suppressing tumours originating from intermediate progenitor cells (Hakes, 2020).

The results revealed the mechanism through which high levels of the orphan nuclear receptor Tll initiate tumours in the Drosophila CNS. Tll is expressed in Type II NSCs during larval development, where it is required for Type II NSC identity and subsequent lineage progression. In the absence of Tll, the proneural transcription factor Ase is derepressed in Type II NSCs. As a consequence, transit amplifying INPs are no longer generated and the resulting NSC lineages have a lower neurogenic potential (Hakes, 2020).

In mice, TLX is expressed in NSCs during embryonic development and in adulthood. Embryonic NSCs display defects in proliferation in the absence of TLX and the loss of TLX in adults results in the loss of transit-amplifying intermediates and reduction in neurogenesis. While these effects were previously attributed to changes in the NSC cell cycle, the current results suggest a cell fate change may occur due to the loss of TLX (Hakes, 2020).

High levels of TLX in human glioblastoma are correlated with tumour aggressiveness. High level expression of TLX results in glioblastoma-like lesions derived from SVZ NSC lineages in mouse models of glioblastoma indicating that TLX can also promote glioblastoma development. However, it was not known how high TLX leads to glioblastoma, nor had the cellular origin of TLX-induced tumours been identified. TLX and its Drosophila homologue, Tll, are highly conserved proteins and this study found that both genes are able to revert INPs to NSC fate as a first step in tumour initiation. Ectopic expression of Tll was also sufficient to induce the expansion of NSCs throughout the Drosophila CNS, demonstrating the widespread vulnerability of NSC and progenitor populations to ectopic Tll expression (Hakes, 2020).

This study found that the ectopic NSCs resulting from high Tll expression are negative for Ase. Tll binds to the ase locus, suggesting that Tll directly represses ase. The absence of Ase is a hallmark of Type II NSCs. Therefore, ectopic Tll promotes a cell fate change from INP/Type I NSC to Type II NSC and thereby initiates tumourigenesis (Hakes, 2020).

The capacity of Tll to induce NSC expansion had been reported previously as part of a study showing that Tll regulates the proliferation of larval mushroom body NSCs and GMCs (Kurusu, 2009). The authors showed that overexpressing Tll resulted in ectopic NSCs, but they did not identify the origin of these tumours and argued against a role for Tll in Type II NSC fate. Tll-induced tumourigenesis could be blocked by ectopic expression of Pros (Kurusu, 2009). However, ectopic Pros results in the loss of NSCs even in wild type brains. In contrast, Type I NSC lineages appear normal after Ase misexpression in wild type brains. Furthermore, it has been reported that high levels of the human homologue of Pros, PROX1, exacerbate glioblastoma, arguing against PROX1 expression as a therapeutic strategy (Hakes, 2020).

This study found that the tumourigenic capacity of Drosophila Tll and human TLX was highly conserved. Human TLX could also induce ectopic Type II NSCs from INPs through the repression of Ase. Analysis of scRNA seq from glioblastoma revealed that TLX and ASCL1 expression is mutually exclusive. It is notable that the origin of human glioblastoma has been mapped to the SVZ. While TLX positive NSCs have been identified in both the SVZ and dentate gyrus, high levels of TLX giving rise to glioblastoma has only been shown robustly in the SVZ. Furthermore, a recent study demonstrated that low expression levels of ASCL1 correlate with glioblastoma malignancy. Ectopic expression of ASCL1 in glioblastoma stem cells was sufficient to promote neuronal differentiation. Based on these results in Drosophila, it is predicted that introducing ASCL1 would override the repressive effect of TLX, induce neuronal differentiation and reduce tumour growth, thereby providing an effective treatment (Hakes, 2020).

The results indicate that INPs are the tumour initiating cells in Type II NSC lineages expressing high levels of the orphan nuclear receptor Tll and potentially implicate intermediate progenitors as one of the cells of origin in TLX+ glioblastomas, an aggressive and lethal brain tumour. This study found that Ase is a direct target of Tll and that Ase expression not only blocks Tll-induced tumourigenesis, but also reinstates a normal neural differentiation programme (Hakes, 2020).

Chen, R., Deng, X. and Zhu, S. (2022). The Ets protein Pointed P1 represses Asense expression in type II neuroblasts by activating Tailless. PLoS Genet 18(1): e1009928. PubMed ID: 35100262

The Ets protein Pointed P1 represses Asense expression in type II neuroblasts by activating Tailless

Intermediate neural progenitors (INPs) boost the number and diversity of neurons generated from neural stem cells (NSCs) by undergoing transient proliferation. In the developing Drosophila brains, INPs are generated from type II neuroblasts (NBs). In order to maintain type II NB identity and their capability to produce INPs, the proneural protein Asense (Ase) needs to be silenced by the Ets transcription factor pointed P1 (PntP1), a master regulator of type II NB development. However, the molecular mechanisms underlying the PntP1-mediated suppression of Ase is still unclear. This study utilized genetic and molecular approaches to determine the transcriptional property of PntP1 and identify the direct downstream effector of PntP1 and the cis-DNA elements that mediate the suppression of ase. The results demonstrate that PntP1 directly activates the expression of the transcriptional repressor, Tailless (Tll), by binding to seven Ets-binding sites, and Tll in turn suppresses the expression of Ase in type II NBs by binding to two hexameric core half-site motifs. This study further showa that Tll provides positive feedback to maintain the expression of PntP1 and the identity of type II NBs. Thus, thus this study identifies a novel direct target of PntP1 and reveals mechanistic details of the specification and maintenance of the type II NB identity by PntP1 (Chen, 2022).

This study has dissected the molecular mechanism of how PntP1 suppresses Ase expression to specify the type II NB identity. PntP1 was demonstrated to acts as a transcriptional activator to indirectly suppress Ase expression by activating Tll, whereas Tll provides positive feedback to maintain the expression of PntP1 and the type II NB identity. The cis-elements that mediate the suppression of Ase by Tll and the activation of Tll by PntP1 were mapped. Thus, this work reveals mechanistic details of PntP1-mediated suppression of Ase expression and specification of type II NBs and identifies a novel direct target of PntP1 in type II NBs (Chen, 2022).

This study has demonstrated that PntP1 functions as a transcriptional activator by showing that the artificial chimeric repressor protein EnR-Ets antagonizes the function of endogenous PntP1 proteins when expressed in type II NB lineages and that the artificial chimeric activator proteins VP16AD-pntP1^1/2C-Ets could functionally mimic endogenous PntP1 protein when expressed in type I NBs. These results are in line with a previous in vitro study showing that PntP1 substantially activates bacterial chloramphenicol acetyltransferase (CAT) reporter expression under the control of Ets binding sites. Interestingly, the results show that the chimeric protein VP16AD-Ets is not sufficient to functionally mimic endogenous PntP1 proteins even though the Ets domain can bind to PntP1 target DNAs as demonstrated by the antagonization of PntP1's function by the EnR-Ets protein. Only when the C-terminal sub-fragment (aa. 256-511) of PntP1 is included can the chimeric activator protein functionally mimic endogenous PntP1 proteins. The Ets family proteins usually recruit additional cofactors to activate/repress target gene expression. This C-terminal fragment likely contains protein-protein interaction domains that are essential to recruit its cofactors. Since neither the pntP1^1/2C-Ets truncated protein nor the chimeric protein VP16AD-pntP1^1/2N-Ets is able to mimic wild type PntP1 protein's function, it is unlikely that this C-terminal sub-fragment contains the activation domain of PntP1 and the other N-terminal sub-fragment [pntP1^1/2N] contains the protein-protein interaction domains instead. Otherwise, with the VP16AD functioning as an activation domain and the N-terminal sub-fragment recruiting cofactors, the chimeric VP16AD-pntP1^1/2N-Ets protein would be able to functionally mimic wild type PntP1 protein. Given that a large number of Ets family transcription factors share highly conserved Ets domains but have diverse functions and activities in distinct cell types, the C-terminal sub-fragment of PntP1 may recruit cell-type-specific cofactors to regulate the expression of specific target genes in type II NB lineages, such as Erm and Tll, as it has been proposed as a general strategy for Ets family proteins to regulate the expression of tissue-specific target genes. However, the regions of PntP1 subfragments that was included in chimeric repressor/activator constructs were arbitrarily defined. The results do not tell the precise boundaries of the activation domains or potential protein-protein interaction domains. To precisely map these functional domains will require more detailed and systemic analyses in the future (Chen, 2022).

PntP1 performs diverse functions in different cell types in type II NB lineages. For example, in type II NBs, PntP1 is required to suppress Ase expression. In newly generated imINPs, PntP1 prevents premature differentiation of INPs, whereas late during imINP development, it promotes INP cell fate commitment and prevents dedifferentiation of imINPs. Therefore, as a transcriptional activator, PntP1 must activate the expression of many different target genes in type II NB lineages. However, Erm is the only direct target that has been identified previously. This study identified Tll as another direct target of PntP1 that functions primarily in type II NBs to suppress Ase expression. PntP1 is both necessary and sufficient for Tll expression. Seven putative binding sites were identified and EMSAs and ChIP-qPCR assays verified that all these sites can bind to PntP1 both in vitro and in vivo. Although ChIP-qPCR was done using type II NB-enriched DNAs isolated from Brat knockdown larval brains, it is unlikely the results are artifacts. A previous study has demonstrated that type II NB-enriched chromatin isolated from brat mutant larval brains maintains similar transcriptional status for multiple genes examined, including pntP1, as in wild type type II NBs. Furthermore, Brat mainly functions as an RNA-binding protein in the imINPs to promote degradation of mRNAs of self-renewing genes such as dpn and klu. Therefore, loss of Brat unlikely affects the binding of PntP1 to its target DNAs in type II NBs, but this possibility was not fully verified particularly because the supernumerary type II NBs in Brat knockdown brains are derived from dedifferentiation of imINPs and it has never been extensively evaluated whether the dedifferentiated type II NBs have the exact same gene expression profiles as wild type type II NBs. It might be helpful to further verify the results by single-cell ChIP using isolated wild type type II NBs. However, since the bioinformatic prediction of PntP1 binding sites was limited to the enhancer region R31F04, it is not certain whether any additional PntP1 binding sites exist outside this enhancer region and contribute the activation of tll by PntP1. In any event, the seven binding sites identified within this enhancer region are sufficient for tll to be activated by PntP1 in type II NBs as demonstrated by the specific expression of R31F04-GAL4 in type II NBs (Chen, 2022).

This work further demonstrates that Tll is the direct target of PntP1 that mediates the suppression of Ase in type II NBs by showing that simultaneous knockdown of Tll essentially blocks the suppression of Ase by misexpressed PntP1 in type I NBs. By fine mapping the cis-repressive elements in ase regulatory regions, two Tll binding sites were idnetified located at -1,292bp ~ - 1,262bp upstream of the ase TSS, consistent with a recent study showing that Tll binds to a 5-kb region upstream of the ase TSS. Although the core hexameric sequences of these two binding sites are not exactly the same as the typical Tll binding hexamer 5'-AAGTCA, EMSA results demonstrate that Tll can indeed bind to these sites either as a monomer or a homodimer, the latter of which is common for orphan nuclear receptors (Chen, 2022).

In addition to PntP1 binding sites, other studies report that Suppressor of Hairless [Su(H)], a binding partner of the intracellular domain of Notch, and Zelda (Zld) also bind to the enhancer region of tll, implicating that Notch and Zld could be upstream activator of Tll. However, Su(H) and Zld are not just expressed in type II NBs but also in type I NBs. Thus, Su(H) and Zld are unlikely to be sufficient to activate Tll, but whether PntP1 acts together with Su(H) and Zld to activate Tll in type II NBs is worth further investigation (Chen, 2022).

A previous study shows that PntP1 is expressed not only in type II NBs but also strongly in imINPs. However, Tll is primarily expressed in type II NBs but only very weakly in imINPs, and ectopic expression of Tll in imINPs reverts imINPs to type II NBs. Therefore, there must be a mechanism to inhibit the activation of Tll by PntP1 in imINPs. A recent study proposed that Erm and Hamlet (Ham) function sequentially to suppresses Tll expression in imINPs based on 1) decreasing or increasing the copy number of the tll gene suppresses or enhances the supernumerary type II NB phenotype in ham erm double heterozygous mutants, respectively; and 2) overexpressing Erm or Ham in type II NBs inhibits the expression of Tll in type II NBs. However, these data could be also explained by changes in the expression of PntP1 as it is demonstrated in this study that there is a positive feedback loop between PntP1 and Tll. For example, the reduction in the Tll expression resulting from Erm overexpression in type II NBs could be due to inhibition of PntP1 by Erm in type II NB as was reported previously rather than direct inhibition of Tll by Erm. Furthermore, Erm and Ham are not expressed in the newly generated imINPs, in which Tll expression is already largely suppressed. Therefore, the suppression of Tll in the newly generated imINPs cannot be explained by the inhibition by Erm and Ham. Other mechanisms are likely involved in suppressing Tll in imINPs (Chen, 2022).

The current results not only demonstrate that PntP1 is a direct upstream activator of Tll but also show that Tll is required to maintain PntP1 expression in type II NBs. Therefore, there is a positive feedback loop between PntP1 and Tll that is essential for maintaining the type II NB identity. Considering that PntP1 misexpression induces Tll expression in all type I NBs and generation of mINPs in a subset of type I NB lineages, whereas Tll misexpression induces PntP1 expression only in a small subset of type I NB lineages and does not induce the generation of mINPs, it is thought that PntP1 functions as a master regulator of type II NB lineage development and acts upstream of Tll, which in turn suppresses Ase expression in type II NBs. Since Tll mainly functions as a transcriptional repressor as was also demonstrated in this study, it is unlikely that Tll directly activates PntP1 expression. A previous study suggests that there might be an unknown feedback signal from INPs that could be required for maintaining PntP1 expression. Thus, one potential explanation for the loss of PntP1 expression in Tll knockdown type II NBs could be the loss of INPs and their feedback signal resulting from the ectopic Ase expression in type II NBs. However, since Tll misexpression is able to induce PntP1 expression albeit only in a small subset of type I NBs, it is more likely that Tll suppresses the expression of another unknown transcriptional repressor that is normally suppressed by Tll in type II NBs. When Tll is knocked down, this unknown transcriptional repressor could be turned on in type II NBs to suppress PntP1 expression. Whereas in type I NBs, this unknown repressor may be normally expressed to suppress PntP1 expression and misexpression of Tll may relieve the suppression of PntP1 expression (Chen, 2022).

The GAL4 reporter assays also identified in the ase enhancer region a ~50-bp fragment that is sufficient for activating Ase expression in both type I and type II NBs. Earlier studies show that the Achaete-Scute (AS-C) complex proteins together with Daughterlesss (Da) activate Ase expression in NBs during the initial specification of NBs at embryonic stages by directly binding to four E-boxes in the 5'-UTR of ase. But how Ase is maintained in NBs once they are specified is not known. The current study identified a distinct enhancer region for activating/maintaining Ase expression in NBs, suggesting that factor(s) other than the AS-C proteins may be involved in activating/maintaining Ase expression in NBs after they are specified. Therefore, the lack of Ase expression in type II NBs is not because of the absence of an activation mechanism, but rather this activation mechanism is actively suppressed by Tll. Identifying the transcriptional activator(s) involved in activating/maintaining Ase expression after NBs are specified may shed a new light on the mechanisms regulating the development and maintenance of type I and type II NBs (Chen, 2022).

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).

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

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

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

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

Role of architecture in the function and specificity of two Notch-regulated transcriptional enhancer modules

In Drosophila melanogaster, cis-regulatory modules that are activated by the Notch cell-cell signaling pathway all contain two types of transcription factor binding sites: those for the pathway's transducing factor Suppressor of Hairless [Su(H)] and those for one or more tissue- or cell type-specific factors called 'local activators.' The use of different 'Su(H) plus local activator' motif combinations, or codes, is critical to ensure that only the correct subset of the broadly utilized Notch pathway's target genes are activated in each developmental context. However, much less is known about the role of enhancer "architecture"--the number, order, spacing, and orientation of its component transcription factor binding motifs--in determining the module's specificity. This study investigated the relationship between architecture and function for two Notch-regulated enhancers with spatially distinct activities, each of which includes five high-affinity Su(H) sites. The first, which is active specifically in the socket cells of external sensory organs, is largely resistant to perturbations of its architecture. By contrast, the second enhancer, active in the 'non-SOP' cells of the proneural clusters from which neural precursors arise, is sensitive to even simple rearrangements of its transcription factor binding sites, responding with both loss of normal specificity and striking ectopic activity. Thus, diverse cryptic specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. It is proposed that for certain types of enhancer, architecture plays an essential role in determining specificity, not only by permitting factor-factor synergies necessary to generate the desired activity, but also by preventing other activator synergies that would otherwise lead to unwanted specificities (Liu, 2012).

Detailed analysis of two different Notch-regulated transcriptional enhancer modules has revealed that they are very differently dependent on a particular architecture for their activity and specificity. The socket cell-specific ASE5 enhancer tolerates a variety of rearrangements of its required motifs without appreciable alteration of function in either nascent or mature sockets. Even when ASE5 is impaired quantitatively as a result of mutating all of its non-essential sequences, motif rearrangement generally has only modest effects on activity level, and never modifies the enhancer's specificity. In contrast, it was found that the mα enhancer is sensitive to simple exchanges in the positions of transcription factor binding motifs, responding with both loss of normal spatial specificity and ectopic activity (Liu, 2012).

Broadly speaking, then, one might say that ASE5 is more representative of a 'billboard' model of enhancer architecture (which posits that transcription factor binding motifs contribute to enhancer function largely independently of how they are organized), while the mα enhancer might be thought of as conforming more closely to an 'enhanceosome' model (which suggests that a module's function is crucially dependent on a particular configuration of transcription factor binding sites in order to create synergy between their inputs) (Liu, 2012).

It is useful to consider the characteristics that may determine whether a given module is more likely to lie at the 'billboard' or the 'enhanceosome' end of the spectrum. Though ASE5 and the mα enhancer are both Notch-activated, they function in different biological contexts, and it is suggested that this may be relevant to their respective architectural constraints. ASE5 acts in a single post-mitotic, differentiated cell type to establish and maintain autoregulation of Su(H) for several days. In this instance, due to the availability of cell type-specific 'local activators' such as Vvl, and the strong contribution that high Su(H) levels alone can make to the enhancer's activity, the need for a constrained architecture may be quite minimal. The mα enhancer, on the other hand, is faced with the challenging task of rapidly and transiently (over a period of hours) activating expression of the E(spl)mα gene in multiple non-SOP cells per PNC, while at the same time repressing its expression in each SOP. This might be expected to create a stringent requirement for constrained spacing between the lone proneural protein binding site and one or more Su(H) sites. At the same time, other aspects of the enhancer's normal specificity rely on inputs via POU-HD and/or homeodomain binding sites -- yet these must not be permitted to promote inappropriate activity in socket cells. Again, particular binding motif configurations may be called for as a preventative. The overall point is that two parameters -- an enhancer's specific biological task and context, and its particular combination of factor binding sites -- are likely to play a major role in determining the architectural constraints to which it may be subject (Liu, 2012).

The case of the mα enhancer serves to underscore the insufficiency, in many instances, of a transcription factor binding site 'code' in predicting the specificity of a cis-regulatory module. Despite the presence of five Su(H) sites and two motifs that can be bound by Vvl, the native mα enhancer shows no meaningful activity even in adult socket cells. Yet the mα-shuffle1 and mα-shuffle2 variants, in which the positions of the Vvl motifs are altered, do exhibit substantial adult socket cell activity. Thus, it is specifically the wild-type enhancer's architecture that normally prevents this from happening. A similar conclusion derives from examining the functionality of the proneural (E) plus Su(H) (S) 'code' embodied in the mα enhancer. When the lone E box site is in its native and evolutionarily conserved position 14 bp away from one of the Su(H) sites, it provides sufficient input to drive robust expression in all wing disc PNCs. But when it is moved instead to the location of one of the Vvl sites, the module's PNC activity is severely reduced. Again, the simple presence of Su(H) and proneural binding motifs in the mα enhancer does not suffice to predict its specificity; rather, the specific arrangement of these sites has a profound effect on its ability to generate the PNC specificity (Liu, 2012).

The critical role of binding site spacing and organization in generating the transcription factor synergies necessary for the normal activity of many enhancers is becoming increasingly clear. But the mutational analyses of both ASE5 and the mα enhancer demonstrate an equally important role for architecture in preventing inappropriate synergies and hence inappropriate specificities (Liu, 2012).

Two ASE5 variants are particularly informative in illuminating the importance of motif spacing in restraining enhancer activity. ASE5M2, in which only the five Su(H) sites are intact but spacing is preserved, is completely inactive in both pupal and adult socket cells. By contrast, the ABm version of ASE5-shrink, which likewise retains only the five Su(H) sites but now places them much closer together, is strongly active in adult sockets. Thus, ASE5's native architecture serves in part to prevent the Su(H) sites from responding on their own, and in this way maintains the enhancer's dependence on inputs from the box A and/or box B sequence elements, even in adult socket cells (Liu, 2012).

Next, the wholly ectopic responsiveness of mα-shrink in both pupal and adult socket cells demonstrates clearly that the potential for unrelated and unwanted specificities can be inherent in an enhancer's particular combination of transcription factor binding motifs. Even as it functions in an inappropriate cell type, mα-shrink follows a recognizable regulatory logic. Its activity in nascent socket cells is fully dependent, as expected from ASE5, on its POU-HD and/or homeodomain sites (and not on its 'E box' proneural protein binding site), while its robust adult socket activity -- as in the case of the ABm version of ASE5-shrink -- requires only the five Su(H) sites (Liu, 2012).

Finally, the far more modest alterations represented by the 'shuffle' versions of the mα enhancer explicitly demonstrate the critical role that motif placement and spacing may have in suppressing inappropriate specificities. Simply exchanging the position of one of the module's 'Vvl' sites with that of the E box proneural site creates novel activities in both the wing imaginal disc and the socket cell (Liu, 2012).

In a recent report, Swanson (2011) identified short-range transcriptional repression as the mechanism that prevents the cone cell-specific sparkling (spa) eye enhancer, which serves the Drosophila dPax2 gene, from being ectopically active in nearby photoreceptor cells. In this instance, moving the repression-mediating sequences out of their native context apparently eliminated their ability to exert a repressive effect, permitting the module to be active in an inappropriate cell type (Liu, 2012).

It is believed that these results with the mα enhancer are most simply consistent with a different mechanism for restraining unwanted enhancer specificities. In this model, the relative positions and spacings of transcription factor binding sites are organized so as to promote functional synergies between activators that generate the desired specificity, while at the same time preventing different activator synergies that would otherwise create undesirable specificities. Note that, while this mechanism places definite constraints on the allowable motif locations in the module, it does not require that the enhancer be transcriptionally repressed in the incorrect cell type(s) (Liu, 2012).

The possibility that, despite their simplicity, both of the 'site switches' embodied in the mα-shuffle1 and mα-shuffle2 constructs have disrupted the interaction of a short-range repressor with its target activator(s) cannot strictly rulef out. However, this is thought unlikely for a number of reasons. For example, such a repressor would have to be active in both a broad zone of wing disc tissue and in socket cells — two very different settings. It is suggested instead that the most parsimonious explanation for these findings is the synergy promotion/prevention model described above (Liu, 2012).

What might determine whether a given enhancer makes use of active repression to limit its specificity, or instead utilizes a simpler synergy prevention mechanism? One reasonable possibility is that repression is required, or more common, when the ectopic specificity that must be prevented consists of a cell or cells that are very closely related developmentally to those in which expression is wanted. Such inappropriate cells may be spatially very close to the correct cells, and/or may have a high degree of similarity in their developmental histories and gene expression profiles. In such cases, it may be difficult or impossible to evolve a motif architecture that simultaneously allows the proper activity and prevents the improper. On the other hand, when the ectopic specificity is a very different cell type or tissue, distant both temporally and spatially from the correct one, and sharing very little developmental history, perhaps motif arrangements that act to prevent inappropriate synergies are easier to evolve. Under this rubric, the use of repression by modules as different as the eve stripe 2 and spa enhancers is readily understood, just as the mα enhancer might instead be expected to inhibit socket cell activity by prevention of the necessary activator synergy. Indeed, the mα module appears to make use of both mechanisms: Activity of this enhancer in the SOP cell is antagonized by repression mediated by Su(H). As a member of the PNC, the SOP is of course surrounded by, and very closely related to, the non-SOPs (Liu, 2012).

Finally, it is interesting to consider what characteristics of an enhancer might put it particularly at risk for ectopic activity, which in turn would require the use of the preventive mechanisms that this study consider. Certainly utilizing transcription factors that are broadly expressed and active [such as Su(H)] would contribute to such a need, as would using inputs from factors that are members of paralogous families with very similar DNA-binding specificities (e.g., POU-HD proteins) (Liu, 2012).

The results described in this study, have important implications for understanding of enhancer evolution. It appears that, due to the specific combination of transcription factor binding motifs they employ, some (perhaps most) enhancers harbor the hidden potential to generate certain novel specificities that can be revealed through comparatively simple sequence changes. In a sense, such enhancers are 'poised' to express these silent specificities. Depending on how widespread this phenomenon is among enhancers in the whole genome, a tremendous potential may exist to explore a vast 'specificity space' through modest mutational events. Moreover, when applied to an individual enhancer, this perspective suggests that a particular novel specificity -- one that requires only relatively minor changes in motif placement to be expressed -- might be seen to evolve independently in more than one lineage (Liu, 2012).

These results also suggest that the minimum size of a given enhancer module may be subject to significant constraints, due to the need to prevent unwanted activator synergies through motif spacing. Thus, even if not all sequences in the enhancer mediate transcription factor inputs, some may be preserved evolutionarily in order to maintain distance between transcription factor binding sites (Liu, 2012).

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).

Ets transcription factor Pointed promotes the generation of intermediate neural progenitors in Drosophila larval brains

Intermediate neural progenitor (INP) cells are transient amplifying neurogenic precursor cells generated from neural stem cells. Amplification of INPs significantly increases the number of neurons and glia produced from neural stem cells. In Drosophila larval brains, INPs are produced from type II neuroblasts (NBs, Drosophila neural stem cells), which lack the proneural protein Asense (Ase) but not from Ase-expressing type I NBs. To date, little is known about how Ase is suppressed in type II NBs and how the generation of INPs is controlled. This study shows that one isoform of the Ets transcription factor Pointed (Pnt), PntP1, is specifically expressed in type II NBs, immature INPs, and newly mature INPs in type II NB lineages. Partial loss of PntP1 in genetic mosaic clones or ectopic expression of the Pnt antagonist Yan, an Ets family transcriptional repressor, results in a reduction or elimination of INPs and ectopic expression of Ase in type II NBs. Conversely, ectopic expression of PntP1 in type I NBs suppresses Ase expression the NB and induces ectopic INP-like cells in a process that depends on the activity of the tumor suppressor Brain tumor. These findings suggest that PntP1 is both necessary and sufficient for the suppression of Ase in type II NBs and the generation of INPs in Drosophila larval brains (Zhu, 2011).

This study has demonstrated that Drosophila PntP1 is a key molecule that suppresses Ase expression in type II NBs and promotes the generation of INPs. PntP1 is specifically expressed in type II NB lineages. Ectopic PntP1 expression suppresses Ase expression in type I NBs and induces ectopic INP-like cells. The generation of ectopic INP-like cells requires prior suppression of Ase in the NBs and Brat activity. Conversely, partial loss of PntP1 or inhibiting PntP1 activity results in the reduction or elimination of INPs and activation of Ase expression in type II NBs (Zhu, 2011).

How does PntP1 suppress Ase expression? Given that Pnt was known to function mainly as a transcriptional activator, it is likely PntP1 suppresses Ase expression indirectly by activating the expression of yet to be identified target genes. However, Ets family proteins with transcriptional activation activity, such as Pnt homolog Ets1, could also function as transcriptional repressors in a cell type- and promoter-dependent manner. It cannot be entirely ruled out that PntP1 acts as a transcriptional repressor to suppress Ase expression in type II NBs (Zhu, 2011).

Although results from this study as well as others suggest that suppressing Ase expression in NBs is necessary for the generation of INPs, loss of Ase expression alone in type I NB lineages does not lead to the generation of ectopic INPs. Therefore, in addition to suppressing Ase expression, PntP1 must activate other target genes to promote the generation of INPs. One of the major features that distinguish INPs from GMCs is that INPs undergo several rounds of self-renewing divisions, whereas GMCs divide terminally. Thus, among PntP1 target genes could be cell-cycle regulators that promote INPs to undergo self-renewing divisions. This finding is consistent with the well-established function of Ets transcription factors in cell-cycle control and tumorigenesis. Ets proteins stimulate cell proliferation by inducing the expression of cell-cycle regulators, such as Cyclin D1, Cdc2 kinase, and Myc. In the developing Drosophila eye disk, Pnt up-regulates the expression of String, the Drosophila homolog of Cdc25, to promote the G2-M transition. Therefore, PntP1 likely activates the expression of cell-cycle regulators to promote the self-renewing division of INPs. A partial loss of PntP1 in pntδ33 mutant clones or inhibition of PntP1 activity by Yan may lead to reduced expression of cell-cycle regulators and subsequent precocious termination of self-renewing divisions of INPs, resulting in a reduction or elimination of INPs in type II NB lineages (Zhu, 2011).

Like in type II NB lineages, the results show that the induction of INP-like cells by ectopic expression of PntP1 involves a Brat-mediated maturation process. Brat functions as a translational repressor, but exact targets of Brat in immature INPs remain unknown. The results show that when Brat is knocked down via RNAi, ectopic PntP1 expression leads to overproliferation of type II NB-like cells in type I NB lineages, similar to the phenotype observed in type II NB lineages when Brat is lost (Zhu, 2011).

However, neither ectopic PntP1 expression nor Brat knockdown alone causes similar overproliferation phenotypes in type I NB lineages. Therefore, it is possible that Brat promotes the maturation of INPs in part by translationally suppressing the expression of PntP1 target genes, particularly cell cycle-related genes, in immature INPs, thus preventing PntP1 from activating cell cycle progression in immature INPs before they fully mature (Zhu, 2011).

Although this study shows that PntP1 is able to induce the generation of ectopic INP-like cells, not every type I NB lineage ectopically expressing PntP1 produces INP-like cells. One explanation could be that additional factors may be required for the generation of INPs. These factors could be specifically expressed in type II NB lineages and function either independently, or together with PntP1 as cofactors, to promote the generation of INPs. In the absence of these factors, PntP1 promotes the generation of INPs at a much reduced efficiency. Ets transcription factors often regulate target-gene expression by recruiting other proteins. For example, Pnt interacts with Jun to induce R7 cell-fate specification in the Drosophila retina. It is possible that PntP1 needs cofactors to efficiently activate the expression of target genes that are involved in the generation of INPs. However, it is also possible that Ase and Pros in GMCs in type I NB lineages might counteract the role of PntP1 in cell-cycle progression by activating the expression of the cell-cycle inhibitor Dacapo, thus preventing the generation of self-renewing INP-like cells (Zhu, 2011).

In conclusion, this study demonstrates that PntP1 is responsible for the suppression of Ase in type II NBs and the generation of INPs. Suppression of Ase is likely a prerequisite for PntP1 to induce the generation of INPs. Furthermore, PntP1 possibly activates yet-to-be identified target genes, including cellcycle regulators, to induce the generation of INPs and to promote their self-renewing divisions. In addition, the data suggest that, at least in part, Brat promotes the maturation of INPs likely by suppressing the expression PntP1-regulated cell cycle-related genes in immature INPs, thus preventing immature INPs from entering self-renewing divisions before they fully mature (Zhu, 2011).

A regulatory transcriptional loop controls proliferation and differentiation in Drosophila neural stem cells

Neurogenesis is initiated by a set of basic Helix-Loop-Helix (bHLH) transcription factors that specify neural progenitors and allow them to generate neurons in multiple rounds of asymmetric cell division. The Drosophila Daughterless (Da) protein and its mammalian counterparts (E12/E47) act as heterodimerization factors for proneural genes and are therefore critically required for neurogenesis. This study demonstrates that Da can also be an inhibitor of the neural progenitor fate whose absence leads to stem cell overproliferation and tumor formation. This paradox can be explained by demonstrating that Da induces the differentiation factor Prospero (Pros) whose asymmetric segregation is essential for differentiation in one of the two daughter cells. Da co-operates with the bHLH transcription factor Asense, whereas the other proneural genes are dispensible. After mitosis, Pros terminates Asense expression in one of the two daughter cells. In da mutants, pros is not expressed, leading to the formation of lethal transplantable brain tumors. These results define a transcriptional feedback loop that regulates the balance between self-renewal and differentiation in Drosophila optic lobe neuroblasts. They indicate that initiation of a neural differentiation program in stem cells is essential to prevent tumorigenesis (Yasugi, 2014).

To further characterize the overproliferation caused by da RNAi, da RNAi was induced by insc-Gal4 in all larval NBs. The number of Deadpan (Dpn) expressing NBs increased at the expense of Embryonic lethal abnormal vision (Elav) expressing neurons. Although da was expressed in all NBs of the central brain and in some progenitor cells, no phenotype was found in these lineages when da³ amorphic mutant clones were induced using mosaic analysis with a repressible cell marker (MARCM) technique (Yasugi, 2014).

The visual processing centers of the fly brain consist of the so-called optic lobes. The medial surface of the optic lobes is surrounded by medulla NBs that differentiate from NE cells and generate medulla neurons on the inner side of the brain. In the optic lobe, da is expressed in NE cells and in medulla NBs. To induce daRNAiin the optic lobe, a dpn-Gal4 driver line was used that showed strong Gal4 expression in NE cells and medulla NBs and weak expression in medulla neurons (called dpnOL-Gal4). Expression of daRNAi using dpnOL-Gal4 caused a strong increase of Dpn positive NBs. The da RNAi phenotype was examined with the mitotic marker Phospho-Histone H3 (PH3), the NB marker Miranda (Mira) and the neuronal marker Elav. In the wild type, PH3 positive mitotic cells (NBs and GMCs) were restricted to the periphery of the optic lobe. In da RNAi samples, PH3 positive cells were mislocalized and ectopically found in the inner side of the brain. To confirm this phenotype, da³ mutant clones were induced in the optic lobe. In da³clones, Dpn positive NBs were found in the region that was normally occupied by medulla neurons. Thus, da is required for cell fate determination in medulla NBs (Yasugi, 2014).

To test whether the ectopic NBs in da RNAi brains have unlimited growth potential and can induce malignant tumors, optic lobes expressing GFP under the control of insc-Gal4, were dissected and implanted into the abdomen of wild type adult host flies. Transplanted cells from da RNAi brains proliferated and GFP positive cells were observed in the host flies, while no substantial growth was observed in control samples. PH3 positive mitotically active cells were observed in the tissue from transplanted daRNAi samples, and this tumor tissue consisted of both Dpn-expressing NB-like cells and Elav-expressing neuron-like cells. This suggests that the da tumor cells proliferate and some of the cells keep the stem cell state, but these cells also produce differentiating cells. This is consistent with the result from da³ clones, in which both ectopic NBs and differentiated neurons were observed. From these results, it is concluded that da acts as a tumor suppressor in optic lobe NB lineages (Yasugi, 2014).

Da is an E-box protein that heterodimerizes with other bHLH type transcription factors, such as the proneural proteins of the AS-C. The AS-C is composed of four transcription factors called Achaete (Ac), Scute (Sc), Lethal of Scute (L(1)sc), and Asense (Ase). While Ac is not expressed in the optic lobe, three of four AS-C proteins show specific expression. Sc is expressed in the NE cells and NBs, L(1)sc is transiently expressed in the transition zone between NE cells and NBs, and Ase is expressed in NBs and GMCs in the developing medulla. To test which of the AS-C genes might act with da during cell fate determination in medulla NBs, clones of several deletion lines were induced that uncover the AS-C region. Ectopic NBs were observed in clones of Df(1)260-1 uncovering all AS-C genes or in ase¹ that uncovers the ase coding region. On the other hand, no phenotype was observed in clones of Df(1)sc19, which deletes ac, sc, and l(1)sc. Since the phenotype of Df(1)260-1 or ase¹ clones was similar to the phenotype of da³ mutant clones and heterodimerization between Ase and da has been shown, it is concluded that da acts together with Ase to regulate cell fates in the optic lobe. It has been reported that da is required for the timely differentiation from NE cells to NBs and L(1)sc is involved in this transition during the optic lobe development. From the expression pattern of AS-C genes and results from the clonal analysis using deficiency lines, a dual function is proposed for Da: As a heterodimer with L(1)sc, da promotes the transition from NE cells to NBs. Later, da acts with Ase in NBs to promote differentiation and prevent tumor formation (Yasugi, 2014).

To identify the downstream targets of da and Ase, the expression was tested of candidate genes. The homeodomain transcription factor Pros acts as a cell fate determinant in embryonic and larval NBs and is regulated by da and Ase in embryos. In the larval optic lobe, Pros is localized to the basal cortex of dividing NBs and nuclear in GMCs and newly born medulla neurons. Whether Pros expression is dependent on da and/or Ase was tested. Pros expression decreased in da³ or ase¹ mutant clones suggesting that Pros acts downstream of da and Ase. To test whether pros is required for cell fate determination in the optic lobe, induced pros¹⁷ mutant clones were induced. In pros¹⁷ mutant clones, ectopic NBs were observed in the medulla neuron layer, which was similar to the phenotype of da³ or ase¹mutant clones. Overexpression of Pros, on the other hand, resulted in a decrease of medulla NBs. To test whether Pros acts downstream of Da, Pros was overexpressed in a da RNAi background. A reduced number of medulla NBs were observed in optic lobes overexpressing Pros in a da RNAi background, indicating that pros is epistatic to da. Thus, Pros is a key downstream target of da and Ase in optic lobe NBs (Yasugi, 2014).

Next, it was asked whether Pros expression is regulated by da in the central brain where da is not required for NB self-renewal. Nuclear Pros expression was found in differentiating daughter cells in the wild type. Pros expression remained in da³ mutant clones. Thus, unlike in the optic lobe, da is not essential for Pros expression in the central brain. This explains why the da phenotype is specific to the optic lobe NBs, while pros mutations cause overproliferation in all larval NBs. It is speculated that other factors may act redundantly to regulate Pros expression in the central brain (Yasugi, 2014).

If Pros is induced by da and Ase, then how are their functions turned off after asymmetric division? To test whether Pros can terminate the expression of ase, Ase expression was examined in pros¹⁷ clones. While Ase expression was restricted to the periphery of the optic lobe in wild type, Ase expression continued on the inner side of the optic lobe in pros¹⁷ clones. Thus, Pros turns off Ase expression and this transcriptional negative feedback loop regulates the proliferation and differentiation of NBs (Yasugi, 2014).

A prevailing view in stem cell biology is that a self-renewal program allows prolonged proliferation in stem cells and is turned off upon differentiation. The current data challenge this view and demonstrate that the ability to differentiate is pre-programmed in neural stem cells. This explains why transcription factors like da and Ase that are thought to be required for NB specification can be required for proper differentiation and act as tumor suppressors. It is proposed that a regulatory transcriptional loop assures cell fate determination and inhibits tumor formation. In a medulla NB, Da and Ase heterodimers induce Pros expression but Pros is excluded from the nucleus and therefore can not terminate Ase expression. After asymmetric cell division, however, Pros enters the nucleus of the GMC where it initiates differentiation and cell cycle exit. In the GMC, Pros terminates Ase expression and therefore triggers an irreversible decision towards differentiation. The data from embryonic NBs suggest that Pros can directly bind to the ase region and regulates its expression. In the absence of this regulation, GMCs maintain the stem cell fate and continue to grow into malignant tumors (Yasugi, 2014).

The role of Da, Ase, and Pros in neural stem cells could be conserved in mammals. Mammalian class I bHLH genes, namely E2A (encoding the E12 and E47 proteins), E2-2, and HEB are expressed in the developing brain. E2A, HEB, or E2A/HEB transheterozygous mutant mice show a brain size defect, suggesting that class I factors also regulate mouse brain development. Mash1 and Prox1, the vertebrate orthologs of Ase and Pros, are expressed in proliferating neural precursor cells of the developing forebrain and spinal cord. Like in Drosophila, Mash1 induces Prox1 and Mash1 promotes an early step of differentiation in neural stem cells. Like in vertebrates, NE cells in the Drosophila optic lobe first proliferate by symmetric cell division and then become asymmetrically dividing NBs. From these molecular and developmental similarities, it is speculated that the transcriptional regulatory mechanism this study identified might be well conserved in mammalian brains (Yasugi, 2014).

The data are of particular relevance in light of the recently postulated role of stem cells in the formation of malignant tumors. Failure to limit self-renewal capacity in stem cells or defects in progenitor cell differentiation can both lead to the formation of cells that continue to proliferate and ultimately form tumors. While genes acting in stem cells are thought to promote self-renewal, genes required in differentiating cells are thought to promote differentiation and limit proliferation and are therefore candidate tumor suppressors. The current data challenge this view and show that the path to differentiation is initiated in the stem cell and therefore even genes specific to stem cells can act as tumor suppressors. It will be interesting to determine whether a similar mechanism acts in mammalian neural stem cells as well. If it does, the expression pattern of a gene can no longer be used as a main criterion for whether it promotes or inhibits self-renewal in stem cell lineages (Yasugi, 2014).

Targets of Activity

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

DEVELOPMENTAL BIOLOGY

Embryonic

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).

A region-specific neurogenesis mode requires migratory progenitors in the Drosophila visual system

Brain areas each generate specific neuron subtypes during development. However, underlying regional variations in neurogenesis strategies and regulatory mechanisms remain poorly understood. In Drosophila, neurons in four optic lobe ganglia originate from two neuroepithelia, the outer (OPC) and inner (IPC) proliferation centers. Using genetic manipulations, this study found that one IPC neuroepithelial domain progressively transformed into migratory progenitors that matured into neural stem cells (neuroblasts) in a second domain. Progenitors emerged by an epithelial-mesenchymal transition-like mechanism that required the Snail-family member Escargot and, in subdomains, Decapentaplegic signaling. The proneural factors Lethal of scute and Asense differentially controlled progenitor supply and maturation into neuroblasts. These switched expression from Asense to a third proneural protein, Atonal. Dichaete and Tailless mediated this transition, which was essential for generating two neuron populations at defined positions. It is proposed that this neurogenesis mode is central for setting up a new proliferative zone to facilitate spatio-temporal matching of neurogenesis and connectivity across ganglia. (Apitz, 2014).

Recent studies have distinguished three neurogenesis modes in the Drosophila CNS. First, type I neuroblasts arise from neuroepithelia and generate GMCs, which produce neuronal and glial progeny. Second, Dpn+ type II neuroblasts in the dorsomedial central brain go through a transit-amplifying Dpn+, Ase+ population, called intermediate neural precursors, which generate GMCs and postmitotic offspring. Third, lateral OPC neuroepithelial cells bypass the neuroblast stage and generate lamina precursor cells (LPCs) that divide once to produce lamina neurons. The current results provide evidence for a fourth strategy: p-IPC neuroepithelial cells give rise to progenitors that migrate to a second neurogenic domain, where they mature into type I neuroblasts. These progenitors are distinct, as they originate from the neuroepithelium, do not express markers for neuroblasts, intermediate neural precursors, GMCs or postmitotic neurons, and acquire NSC properties after completing their migration (Apitz, 2014).

Migratory progenitors arise from the p-IPC by a mechanism that shares cellular and molecular characteristics with EMT. On the basis of data on gastrulation and neural crest formation, EMT is commonly associated with cells adopting a mesenchymal state, enabling them to leave their epithelial tissue and migrate through the extracellular matrix to new locations. A recent study also reported an EMT-like process in the mammalian neocortex, whereby newborn neurons and intermediate progenitors delaminate from the ventricular neuroepithelium and radially migrate to the pial surface. This study observed that neuroepithelial cells at the p-IPC margins and migratory progenitors upregulated the Snail homolog Esg, whereas E-cad levels were decreased. Moreover, esg knockdown caused the formation of ectopic E-cad-expressing clusters adjacent to the p-IPC. Although this is a previously uncharacterized role of Drosophila esg, these findings are consistent with the requirement of two Snail transcription factors, Scratch1 and 2, and downregulation of E-cad in cortical EMT migration (Apitz, 2014).

Although TGFβ signaling is well known to induce EMT, it was unclear whether it could have such a role in the brain. Two lines of evidence are consistent with a requirement of the Drosophila family member Dpp. First, it is expressed and downstream signaling is activated in dorsal and ventral p-IPC subdomains and emerging cell streams. Second, tkv mutant cells form small neuroepithelial clusters in p-IPC vicinity. Similar to the neural crest, where distinct molecular cascades control delamination in the head and trunk, region-specific regulators may also be required in p-IPC subdomains. Because neuroblasts derived from Dpp-dependent cell streams map to defined areas in the d-IPC, this pathway could potentially couple EMT and neuron subtype specification (Apitz, 2014).

Cell migration is an essential feature of vertebrate brain development. Commonly, postmitotic immature neurons migrate from their proliferation zones to distant regions, where they further differentiate and integrate into local circuits. Examples include the radial migration of projection neurons and tangential migration of interneurons in the embryonic cortex, as well as migration of interneuron precursors in the rostral migratory stream to the olfactory bulb in adults. In contrast, IPC progenitors develop into NSCs (neuroblasts) after they migrated. A recent study found that NSCs relocating from the embryonic ventral hippocampus to the dentate gyrus act as source for adult NSCs in the subgranular zone. In addition, cerebellar granule cell precursors migrate from the rhombic lip to the external granule layer, where they proliferate during early postnatal development. The migration of neural cell types that become proliferative in a new niche could therefore constitute a more general strategy. IPC progenitors form streams of elongated, closely associated cells. Despite their different developmental state, their organization is notably similar to the neuronal chain network in the lateral walls of the subventricular zone and the rostral migratory stream in mammals, or of migratory trunk neural crest cells in chick. Further studies will need to identify the determinants directing migratory progenitors into the d-IPC (Apitz, 2014).

Several constraints could shape a neurogenesis mode that requires migratory progenitors in the larval optic lobe. The OPC is located superficially and the IPC is positioned centrally. If medulla and lobula neurons arose by neuroepithelial duplications, these new populations would need to be integrated into an ancestral visual circuit consisting of lamina and lobula plate neurons. Cellular migration may therefore be a derived feature and serve as an essential spatial adjustment of the IPC to the newly added medulla. In principle, the migratory population could consist of immature neurons. However, migratory progenitors help to establish a new superficial proliferative niche, and to align OPC and d-IPC neuroblast positions. This in turn enables the OPC and IPC to use spatially matching birth order-driven neurogenesis patterns for establishing functionally coherent connections across ganglia (Apitz, 2014).

IPC progenitors were primed to mature into neuroblasts, but were prevented to do so in cell streams. Consistently, progenitors showed weak cytoplasmic Mira expression and prematurely differentiated into neuroblasts following loss of Pcl. Although Dichaete has been shown to repress ase to maintain embryonic neuroectodermal cells in an undifferentiated state, this study did not identify such a role in the IPC. Future studies are therefore required to distinguish whether this block in neuroblast maturation is released in the d-IPC by cell-intrinsic mechanisms or locally acting signals (Apitz, 2014).

The p-IPC and d-IPC consecutively expressed three proneural factors. esg-positive p-IPC neuroepithelial cells transiently expressed L'sc as they converted into progenitors. Following arrival in the d-IPC, progenitors matured into neuroblasts, which switched bHLH protein expression from Ase to Ato. This correlated with a change in cell division orientations from toward the lamina to the optic lobe surface and the generation of two lineages, distal cells and lobula plate neurons. The progression of neuroblasts through two stages is supported by the observations that progenitors solely entered the lower d-IPC, all neuroblasts were labeled with Ase in this area, and idpp reporter gene expression in a progenitor subset persisted in both lower and upper d-IPC neuroblasts and their progeny (Apitz, 2014).

Late l'sc knockdown reduced the number of d-IPC neuroblasts and both neuron classes, whereas p-IPC formation and EMT of progenitors appeared to be unaffected. This supports the idea that l'sc promotes neuroblast formation by controlling the rate of conversion and the progenitor supply. In contrast, ase loss severely decreased the amount of lower d-IPC neuroblasts and distal cells. This revealed a central role in the maturation of progenitors into neuroblasts, endowing them with the potential to proliferate and generate a specific lineage. Although these functions are the opposite of those observed in the OPC, they align with the role of a murine Ase homolog, Achaete-scute homolog 1 (Ascl1), in the embryonic telencephalon. Ase^- neuroblasts with type I proliferation patterns have not previously been described. Further underscoring the context-dependent activities of proneural bHLH factors, ato does not have the equivalent role of ase in conferring neurogenic properties to upper d-IPC neuroblasts, but acts upstream of differentiation programs controlling the projections of lobula plate neurons (Apitz, 2014).

Although Ase and Ato each regulated distinct aspects of d-IPC development, they were not required for either the transition or the extent of their expression domains. These functions were fulfilled by Dichaete and tll, whose cross-regulatory interactions were essential for the transition from Ase⁺ to Ato⁺, Dac⁺ expression. To link birth order and fate, temporal identity transcription factors are sequentially expressed by neuroblasts and inherited by GMCs and their progeny born during a given developmental window. Acting as the final two members of the OPC-specific series of temporal identity factors, Dichaete is required for Tll expression, whereas tll is sufficient, but not required, to inhibit Dichaete Although OPC and d-IPC neuroblasts shared the sequential expression of Dichaete and Tll, key differences include the fact that d-IPC progeny did not maintain Dichaete, that Tll was transiently expressed in newborn progeny of the upper d-IPC and was not maintained in older lineages, that Dichaete in the lower d-IPC was not required in its own expression domain for neurogenesis, and that Dichaete was required to activate tll, and tll to repress Dichaete and ase, as well as to independently upregulate Ato and Dac. Although the mechanisms that trigger the timing of the switch require further analysis, these observations support the notion that, in the d-IPC, Dichaete and tll do not function as temporal identity factors, but as switching factors between two sequential neuroblast stages. The vertebrate homologs of Dichaete and tll, Sox2 and Tlx, are essential for adult NSC maintenance and Sox2 positively regulates Tlx expression, suiggesting that core regulatory interactions between Dichaete and tll family members may be conserved (Apitz, 2014).

These studies uncovered molecular signatures for generating a migratory neural population by EMT and subsequent NSC development that are in part shared between the fly optic lobe and vertebrate cortical neurogenesis. The unexpected parallels suggest that ancestral gene regulatory cassettes imparting specific cellular properties may have been re-employed during vertebrate brain development. Analysis of p-IPC and d-IPC neurogenesis in the Drosophila optic lobe therefore opens new possibilities for systematically identifying genes regulating EMT, cell migration and sequential NSC specification (Apitz, 2014).

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:  25 April 2019

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