Effects of Mutation or Deletion

The absence of fushi tarazu and even-skipped, due to the absence of prospero produces abnormal axon pathfinding by neurons (Doe, 1991 and Vaessin, 1991).

In the absence of the Drosophila Abl oncogene protein-tyrosine kinase (PTK), loss-of-function mutations in either Disabled or prospero have dominant phenotypic effects on embryonic development. Molecular and genetic characterizations indicate that the products of these genes interact with the Abl PTK by different mechanisms. Prospero encodes a cytoplasmic and nuclear protein required for correct axonal outgrowth. The product of disabled may be a substrate for the Abl PTK. The Disabled protein is colocalized with Abl in axons, its predicted amino acid sequence contains 10 motifs similar to the major autophosphorylation site of Abl, and the protein is recognized by antibodies to phosphotyrosine (Gertler, 1993).

Given the mild phenotypes of abl mutant animals, it is possible to design genetic screens to identify mutations in genes that enhance or suppress the abl mutant phenotypes. It has been hypothesized that in a genetic background sensitized by abl mutations, a 50% reduction in the level of a protein that is regulated by Abl might be sufficiently detrimental to shift the lethal phase from the pharate adult stage to an embryonic or early larval stage. This effect is called haploinsufficiency dependent on an abl mutant background (HDA). The genes identified are not haploinsufficient themselves but manifest their effects when the fly is also mutant for abl. disabled and prospero are two of the genes identified by this strategy. Although abl mutants exhibit no visible defects in the embryonic central nervous system (CNS), animals that are doubly mutant for abl die as embryos and fail to form proper axonal connections in the CNS. Heterozygous deletions of pros in the absence of abl cause embryonic and larval lethality. Examination of these embryos reveals, for the most part, normal axonal architecture in the CNS and peripheral nervous system, with variably penetrant subtle defects in the CNS, including fusion of the anterior and posterior commissural axon bundles. prosM4 homozygous mutant embryos display a segmentally repeated pattern of disrupted axon bundles in each neuromere. The longitudinal axons, which extend to the anterior and posterior between segments, are absent. The midline space between the two halves of the nervous system is wider than normal, with a loss of some midline cells of unknown identity. The anterior and posterior commissural axon bundles that cross the midline in each segment are replaced by a single axon bundle (Gertler, 1993).

The longitudinal glia (LG), progeny of a single glioblast, form a scaffold that presages the formation of longitudinal tracts in the ventral nerve cord (VNC) of the Drosophila embryo. The LG are used as a substrate during the extension of the first axons of the longitudinal tract. The differentiation of the LG has been examined in six mutations in which the longitudinal tracts are absent, displaced, or interrupted to determine whether the axon tract malformations may be attributable to disruptions in the LG scaffold. Embryos mutant for the gene prospero have no longitudinal tracts, and glial differentiation remains arrested at a preaxonogenic state. Two mutants of the Polycomb group also lacked longitudinal tracts; here the glia fail to form an oriented scaffold, but cytological differentiation of the LG is unperturbed. The longitudinal tracts in embryos mutant for slit fuse at the VNC midline and scaffold formation is normal, except that it is medially displaced. Longitudinal tracts have intersegmental interruptions in embryos mutant for hindsight and midline. In hindsight, there are intersegmental gaps in the glial scaffold. In midline, the glial scaffold retracts after initial extension. LG morphogenesis during axonogenesis is abnormal in midline. Commitment to glial identity and glial differentiation also occurs before scaffold formation. In all mutants examined, the early distribution of the glycoprotein Neuroglian is perturbed. This is indicative of early alterations in VNC pattern present before LG scaffold formation begins. Therefore, some changes in scaffold formation may reflect changes in the placement and differentiation of other cells of the VNC. In all mutants, alterations in scaffold formation precedes longitudinal axon tract formation (Jacobs, 1993).

Genes regulating dendritic outgrowth, branching, and routing in Drosophila

Signaling between neurons requires highly specialized subcellular structures, including dendrites and axons. Dendrites exhibit diverse morphologies yet little is known about the mechanisms controlling dendrite formation in vivo. Methods have been developed to visualize the stereotyped dendritic morphogenesis in living Drosophila embryos. Dendrite development is altered in prospero mutants and in transgenic embryos expressing a constitutively active form of the small GTPase cdc42. From a genetic screen, several genes have been identified that control different aspects of dendrite development including dendritic outgrowth, branching, and routing. These genes include kakapo, a large cytoskeletal protein related to plectin and dystrophin; flamingo, a seven-transmembrane protein containing cadherin-like repeats; enabled, a substrate of the tyrosine kinase Abl; and nine potentially novel loci. These findings begin to reveal the molecular mechanisms controlling dendritic morphogenesis (Gao, 1999).

The peripheral neurons in each hemisegment of the Drosophila embryo are grouped into dorsal, lateral, and ventral clusters. The neurons within each cluster can be further classified on the basis of their dendritic morphology; these categories are external sensory (es) neurons and chordotonal (ch) neurons, each containing a single dendrite; bipolar dendrite (bd) neurons, each with two simple unbranched dendritic projections; and multiple dendrite (md) neurons with extensive dendritic arborizations. The md neurons are thought to function as touch receptors or proprioceptors to sense body surface tension or deformation. The dendritic branching of md neurons does not begin until 16 hr after egg laying (AEL) and continues until and beyond hatching. Because impermeable cuticle already forms at 16 hr AEL, md neuron dendrites can not be visualized by standard antibody staining of whole mount embryos. It is possible to manually dissect individual embryos to allow antibody access; however, this technique is too laborious to be useful for a large-scale mutant screen. To circumvent these technical problems, an assay system was developed on the basis of expression of GFP in living embryos. First, a panel of Gal4 enhancer trap lines was screened to identify those that allow high levels of UAS-driven GFP expression in a subset of PNS neurons at the appropriate developmental stages. Of these, the Gal4 line 109(2)80 was chosen. Recombination was performed to create a second chromosome harboring both the Gal4 109(2) 80 transgene and a UAS-GFP transgene, but no background lethal mutations. A fly line homozygous for the Gal4 109(2) 80/GFP chromosome (denoted as Gal4 80/GFP) was then introduced. In the dorsal clusters of abdominal segments A1-A7, GFP expression labels both axons and dendrites of all six md neurons, one bd neuron, and one tracheal innervating neuron, but not the es neurons. In addition, high levels of GFP expression are detected in the lateral and ventral clusters, and in the antennomaxillary complex. Low levels of GFP fluorescence are also observed in a small subset of neurons in the central nervous system (CNS). The dendrites of dorsal cluster md neurons elaborate just underneath the epidermal layer. In larvae, these dendrites as revealed by Gal4 80/GFP are in tight association with a layer of epidermal cells labeled by Kruppel-Gal4/GFP. It is thus possible to visualize the md neuron dendrites in the dorsal cluster in living animals. A focus was placed on the development of these md neuron dendrites in wild-type as well as mutant embryos. To simplify this description, two types of easily detectable dendrites were defined: dorsal branches grow toward the dorsal midline and lateral branches grow along the approximate anterior-posterior axis toward segment boundaries (Gao, 1999).

The projection pattern of md neuron dendrites in a specific hemisegment is largely invariant from embryo to embryo, on the basis of observations on over thousands of embryos. The major characteristics of dendritic morphogenesis are summarized here. By 12-13 hr AEL, ch and es neurons have already sent out their initial dendritic projections. At this stage, bd neurons have also extended their dendrites. The primary dendrites of md neurons emerge at 13-14 hr AEL, 2 hr after the axons of PNS neurons have reached the CNS. The location of initial dendritic outgrowth and the orientation of this outgrowth are fairly invariant for md neurons. At 13 hr AEL, a dorsal dendrite first emerges from one md neuron in the anterior of the dorsal cluster; shortly after, a second dorsal dendrite emerges from a posterior md neuron of the same cluster. Both dorsal dendrites extend perpendicular to the anterior-posterior axis towards the dorsal midline. Each md neuron (in the dorsal cluster only) sends out one dorsally oriented primary dendrite; however, some md neurons have additional primary lateral dendrites. The dorsal extension essentially stops between 15 and 16 hr AEL, before the lateral branches start to develop. Between 15 and 17 hr AEL, numerous transient lateral branches extend and retract. These branches undergo constant remodeling. Only a subset is eventually stabilized between 18 and 20 hr AEL to become the final lateral branches. At this stage, dorsal and lateral branches are clearly distinguishable. The number of lateral branches in a particular segment is similar from embryo to embryo. In addition, the anterior and posterior dorsal branches within a hemisegment are clearly separated by an area devoid of dendrites. Before hatching (23-24 hr AEL), most lateral branches further elaborate tertiary branches before and after they reach the segment boundary, but only a small number of branches cross over into neighboring segments. At hatching, the dorsal branches have not yet reached the dorsal midline so there is a clear dendrite-free zone near the dorsal midline. After hatching, the dorsal branches resume elongation and reach the dorsal midline by the second instar stage. The length and the thickness of dendritic processes continue to increase with increasing larval body size (Gao, 1999).

Before hatching, the lateral branches are regularly spaced and project toward the segment boundaries. This pattern is relatively invariant from embryo to embryo for a specific hemisegment. To investigate how the dendritic patterning develops, dendrite formation was monitored in living embryos from 15 to 16 hr AEL and time-lapse analysis was carried out. Numerous lateral growth buds emerge anterior or posterior to the dorsal branches and then retract. Only a subset of these lateral branches elongates toward the segment boundaries and becomes stabilized. During this process, the length and orientation of dorsal branches remains largely unchanged. Numerous thin processes at the tips of the lateral branches undergo rapid extension and retraction. These thin processes are not labeled by a Tau-GFP fusion protein, indicating that they might not contain microtubules. This analysis reveals that dendritic development is a dynamic process (Gao, 1999).

Two approaches were used to identify genes involved in dendritic morphogenesis: (1) an investigation of the effects of previously isolated mutations, and (2) a systematic mutant screen. It was reasoned that dendrite development might share some common molecular mechanisms with axon and tracheal development, because all of these processes exhibit subcellular outgrowth and branching. Among the mutants that were examined, prospero mutants and embryos expressing a constitutively active form of Dcdc42 showed detectable dendrite phenotypes. prospero encodes a nuclear protein with multiple homopolymeric amino acid stretches and is expressed in neuronal precursor cells. It has been suggested that prospero controls the expression of neuronal precursor genes and is required for proper neuronal differentiation. Two different alleles of prospero were used in this study: pros17 and prosjo13. The prospero mutant embryos do not show any obvious cell fate change in the embryo PNS on the basis of available cell type-specific markers, instead they exhibit abnormal outgrowth and misrouting of axons from dorsal clusters of sensory neurons. In addition, abnormal dendritic patterning is observed in these mutant embryos. The anterior and posterior dorsal branches in wild-type embryos are roughly parallel to one another. However, in ~70% of pros17 embryos, these branches make dramatic turns and occasionally criss-cross each other. A similar phenotype is observed in ~10% of prosjo13 embryos. These studies indicate that proper development of both dendrites and axons requires the function of prospero (Gao, 1999).

Mechanism of glia-neuron cell-fate switch in the Drosophila thoracic neuroblast 6-4 lineage

During development of the Drosophila central nervous system, neuroblast 6-4 in the thoracic segment (NB6-4T) divides asymmetrically into a medially located glial precursor cell and a laterally located neuronal precursor cell. To understand the molecular basis for this glia-neuron cell-fate decision, the effects of some known mutations on the NB6-4T lineage were examined. prospero mutations lead to a loss of expression of Glial cells missing; this is essential to trigger glial differentiation in the NB6-4T lineage. In wild-type embryos, Pros protein is localized at the medial cell cortex of dividing NB6-4T and segregates to the nucleus of the glial precursor cell. miranda and inscuteable mutations alter the behavior of Pros, resulting in failure to correctly switch the glial and neuronal fates. These results suggest that NB6-4T uses the same molecular machinery in the asymmetric cell division as other neuroblasts in cell divisions producing ganglion mother cells. Furthermore, outside the NB6-4T lineage most glial cells appear independently of Pros (Akiyama-Oda, 2000).

In a null allele of pros no cells express Gcm or Repo in the NB6-4T lineage. In contrast, in a null allele of miranda all cells of the lineage express the glial proteins. The double mutant pros;mira produces no glial cells in the NB6-4T lineage, the same result obtained with the pros mutation. These results indicated that both pros and mira are involved in a pathway leading to the glia-neuron cell-fate switch, and that pros is epistatic to mira in this pathway. The effects of the insc mutation on the glia-neuron cell-fate switch in the NB6-4T lineage are slightly different from those of the pros or mira mutations. In insc mutants, both glial and non-glial cells are generated from NB6-4T in many of the hemisegments examined, but glial fate arises randomly from either of the daughter cells. These involvements of pros, mira and insc suggest an analogy between the first cell division of NB6-4T and NB cell divisions that produce GMCs and no glia. Pros, Mira and Insc proteins behave similarly during the first division of NB6-4T to the usual NB divisions producing GMCs. In the analyses of wild-type and mutant embryos, the high levels of expression of the earliest glial protein Gcm, and of the later glial protein Repo, are correlated with the nuclear localization of Pros in NB6-4T daughter cells. Consistent with this, in a pros mutant in which the mutant Pros protein does not enter the nucleus even after cell division, no glial cells are observed in the NB6-4T lineage. These observations suggest an important role for Pros in the onset of glial differentiation in the NB6-4T lineage (Akiyama-Oda, 2000).

A novel mode of asymmetric division identifies the fly neuroglioblast 6-4T

Asymmetric cell divisions and segregation of fate determinants are crucial events in the generation of cell diversity. Fly neuroblasts, the precursors that self-reproduce and generate neurons, represent a clear example of asymmetrically dividing cells. Less is known about how neurons and glial cells are generated by multipotent precursors. Flies provide the ideal model system to study this process. Indeed, neuroglioblasts (NGBs) can be specifically identified and have been shown to require the gcm fate determinant to produce glial cells, which otherwise would become neurons. The division of a specific NGB (NGB6-4T), which produces a neuroblast (NB) and a glioblast (GB), has been followed. To generate the glioblast, gcm RNA becomes progressively unequally distributed during NGB division and preferentially segregates. Subsequently, a GB-specific factor is required to maintain gcm expression. Both processes are necessary for gliogenesis, showing that the glial vs. neuronal fate choice is a two-step process. This feature, together with gcm subcellular RNA distribution and the behavior of the NGB mitotic apparatus identify a novel type of division generating cell diversity (Ragone, 2001).

The present study shows that Pros transcription factor is necessary to maintain gcm expression and thereby activate the glial program in the glioblast. Indeed, in the absence of Pros, gcm RNA progressively disappears from the GB. The gain-of-function phenotype also demonstrates that Pros is not sufficient to initiate gcm expression nor to induce the glial fate on its own. Pros protein and RNA most likely form a complex with Staufen and Miranda. In the absence of Miranda, which is necessary to localize them, both daughter cells inherit the RNA and the protein. In addition, they both inherit gcm RNA. As a consequence, the two daughter cells adopt the GB fate. It is speculated that Pros is required for both gcm-independent and gcm-dependent maintenance, since in its absence the levels of gcm expression are even lower than in the absence of autoregulation. The gcm promoter contains, indeed, several binding sites for the Pros protein. Finally, Pros does not affect all glial cells, therefore it is likely that specific factors will be required in other lineages (Ragone, 2001).

gcm displays several differences with respect to pros with regard to RNA localization. (1) asymmetric distribution is not evident before metaphase; (2) asymmetry occurs progressively during cell division rather than being sharply apical at interphase and basal at metaphase; (3) gcm transcripts are present at the cortex and in the cytoplasm. These differences suggest the existence of different RNA localization pathways in asymmetrically dividing cells. That Stau and Mira may participate to the process is suggested by the mislocalization of gcm RNA in stau and mira mutants. In addition, the gcm 3'UTR displays a stem-loop secondary structure, a conformation that is necessary for the interaction of Staufen with Bicoid 3'UTR. However, this mechanism is not sufficient to ensure a correct fate choice. Moreover, mira and stau are not fully penetrant with respect to gcm RNA distribution. Finally, and more importantly, the cytoplasmic localization of some gcm transcripts as well as the kinetics of asymmetry calls for a cortical microfilament independent mechanism. Thus, the same RNA may be the target of two localization pathways: this complements the observation that the same RNA binding protein may localize transcripts using pathways with different cytoskeletal requirements (Ragone, 2001).

Drosophila p27Dacapo expression during embryogenesis is controlled by a complex regulatory region independent of cell cycle progression

In the embryonic epidermis, dacapo expression starts during G2 of the final division cycle and is required for the arrest of cell cycle progression in G1 after the final mitosis. The onset of dacapo transcription is the earliest event known to be required for the epidermal cell proliferation arrest. To advance an understanding of the regulatory mechanisms that terminate cell proliferation at the appropriate stage, the control of dacapo transcription has been analyzed. dacapo transcription is not coupled to cell cycle progression. It is not affected in mutants where proliferation is arrested either too early or too late. Moreover, upregulation of dacapo expression is not an obligatory event of the cell cycle exit process. During early development of the central nervous system, Dacapo cannot be detected during the final division cycle of ganglion mother cells, while it is expressed at later stages. The control of dacapo expression therefore varies in different stages and tissues. The dacapo regulatory region includes many independent cis-regulatory elements. The elements that control epidermal expression integrate developmental cues that time the arrest of cell proliferation (Meyer, 2002).

p27DAP expression is detected in the Prospero-positive MP2 neuroblast. MP2 is an exceptional neuroblast that accumulates Prospero in the nucleus. Moreover, MP2 divides just once to produce two postmitotic neurons. The final division of this unusual neuroblast therefore is preceded by dap expression, as in the epidermis. However, anti-p27DAP labeling of prospero mutant embryos, indicates that dap expression in MP2 is not the result of Prospero translocation into the nucleus. At later stages, the pattern of anti-p27DAP labeling observed in the CNS is complex and highly dynamic. Anti-p27DAP signals of non-uniform intensity were detected in both Prospero-positive as well as Prospero-negative cells (Meyer, 2002).

Limited analyses of dap expression during CNS development in wild-type embryos demonstrates that cell cycle exit is not always preceded by dap expression. When the first GMCs are generated during embryonic CNS development and progress through their terminal division cycle, p27DAP cannot be detected in these cells, while expression in the unusual MP2 neuroblast is readily observed. This dap expression in the MP2 neuroblast occurs also in prospero mutant embryos. All the findings therefore argue against the suggestion favored by previous studies, which argues that the timely arrest of cell proliferation in GMC progeny might depend on the induction of dap expression by the transcription factor Prospero. The idea that nuclear Prospero might trigger dap expression in GMCs to bring about the G1 arrest observed in the two MP2 neurons generated by GMC division appeared very attractive. Moreover, in principle this hypothesis is also suggested by the correlation that MP2, an exceptional neuroblast that behaves like a GMC in that it divides just once to produce two postmitotic neurons, accumulates Prospero in the nucleus and expresses dap (Meyer, 2002).

The findings argue against a mechanism that operates generally in all GMCs to prevent further cell cycle progression after the terminal division by Prospero-mediated induction of dap expression; more complex mechanisms might have to be considered that might even vary in different neuroblast lineages. The regulation of dap expression in the nervous system certainly entails such complexity. This work does not exclude a dap-independent, general cell cycle arrest mechanism that operates in all GMCs and is perhaps even triggered by nuclear Prospero (Meyer, 2002).

Taste, movement, and death: varying effects of new prospero mutants during Drosophila development

The PGal4 transposon inserted upstream of the pan-neural gene prospero causes several neural and behavioral defects in the Voila1 strain. The precise excision of the transposon simultaneously rescued all these defects whereas its unprecise excision created new prosV alleles, including the null allele prosV17. The relationship between the genetic structure of pros locus, larval locomotion, and larval gustatory response were studied. These two behaviors showed varying degrees of variation depending upon the pros allele. A good relation was found between behavioral alteration, the level of Pros protein in the embryo, and the degree of disorganization in the larval neuromuscular junction. These data suggest that the complete development of the nervous system requires a full complement of Pros, and that a gradual decrease in the levels of this protein can proportionally alter the development and the function of the nervous system (Grosjean, 2003).

Prospero distinguishes sibling cell fate without asymmetric localization in the Drosophila adult external sense organ lineage

The adult external sense organ precursor (SOP) lineage is a model system for studying asymmetric cell division. Adult SOPs divide asymmetrically to produce IIa and IIb daughter cells; IIa generates the external socket (tormogen) and hair (trichogen) cells, while IIb generates the internal neuron and sheath (thecogen) cells. The expression and function of prospero has been examined in the adult SOP lineage. Although Prospero is asymmetrically localized in embryonic SOP lineage, this is not observed in the adult SOP lineage: Prospero is first detected in the IIb nucleus; during IIb division, it is cytoplasmic and inherited by both neuron and sheath cells. Subsequently, Prospero is downregulated in the neuron but maintained in the sheath cell. Loss of prospero function leads to double bristle sense organs (reflecting a IIb-to-IIa transformation) or single bristle sense organs with abnormal neuronal differentiation (reflecting defective IIb development). Conversely, ectopic prospero expression results in duplicate neurons and sheath cells and a complete absence of hair/socket cells (reflecting a IIa-to-IIb transformation). It is concluded that (1) despite the absence of asymmetric protein localization, prospero expression is restricted to the IIb cell but not its IIa sibling; (2) prospero promotes IIb cell fate and inhibits IIa cell fate, and (3) prospero is required for proper axon and dendrite morphology of the neuron derived from the IIb cell. Thus, prospero plays a fundamental role in establishing binary IIa/IIb sibling cell fates without being asymmetrically localized during SOP division. Finally, in contrast to previous studies, the IIb cell is found to divide prior to the IIa cell in the SOP lineage (Manning, 1999).

What mechanisms lead to prospero expression in the IIb cell but not in the IIa cell? Specification of IIa/IIb cell fates is determined by the relative activity of Notch signaling. Productive Notch signaling results in IIa cell fate; asymmetric localization of Numb protein into the IIb cell blocks Notch signaling and results in the IIb cell fate. It is proposed that productive Notch signaling prevents prospero expression in the IIa cell, whereas lack of Notch signaling allows prospero expression in the IIb cell. Consistent with this model, SOP lineages with unregulated Notch signaling produce a pair of IIa cells that both fail to express prospero, while SOP lineages lacking Notch function produce two IIb cells that both express prospero (Reddy, 1999a). One effector of Notch signaling in the IIa cell is the zinc-finger transcriptional repressor Tramtrack, which may directly or indirectly repress prospero expression. Interestingly, prospero is expressed in the R7 neuron during eye development and tramtrack mutants have supernumerary R7 neurons, while tramtrack misexpression reduces R7 differentiation. Thus, a similar Notch-, tramtrack-dependent pathway may repress prospero expression in both the R7 photoreceptor neuron and the IIa cell. It should be noted that a somewhat different mechanism must be involved in repressing prospero in the neuron but not the sheath cell; in this case, Notch signaling is required for sheath cell fate, the cell that maintains prospero expression. The lack of Notch-mediated repression of prospero expression in the sheath cell may reflect the fact that Notch signaling is SuH-dependent in the IIa cell, but SuH-independent in the sheath cell. prospero is essential for distinguishing IIa and IIb cell fates (Manning, 1999 and references).

A role for prospero in establishing different IIa/IIb cell fates has been demonstrated based on both loss-of-function and misexpression experiments. A significant fraction of the SOP lineages lacking prospero function show a duplication of the external bristle (a progeny of the IIa cell) and a loss of the neuron (a progeny of the IIb cell) (Reddy, 1999a). Socket cell fate could not be adequately scored, because multiple socket cells can generate a single, fused socket structure. The simplest interpretation of the double bristle prospero minus sense organs is that the IIb cell has become partially or fully transformed into a IIa cell, resulting in duplicate hair/sockets and loss of neuron/sheath cell. It is unlikely, but it cannot be rule out, that the neuron is transformed into a duplicate hair cell and the sheath cell is unaffected. In both notum and eye, however, there are still many single bristle sense organs that have an associated neuron and, in these sense organs, the IIb cell must have been specified relatively normally. Thus prospero is not strictly necessary for IIb cell specification, but its function is important for the high-fidelity specification of IIb cell fate. While the presence of prospero in the IIb cell is important for reliable IIb cell specification, the absence of prospero from the IIa cell is absolutely essential for IIa cell specification. Misexpression of prospero in the IIa cell and its progeny results in a fully penetrant loss of a socket cell marker (SuH) as well as the morphological external socket and hair structures; there is a corresponding increase in the internal Elav+ neurons and BarH1+ sheath cells. The misexpression experiments show that absence of Prospero in the IIa cell is required for normal IIa development, and that the presence of Prospero in the IIa transforms it partially or fully to the IIb cell fate. Thus, differential expression of prospero between IIa and IIb siblings is essential for normal SOP development. Similar results were observed using transient heat-shock-induced misexpression of prospero, although in these experiments a very low frequency of double and triple bristle sense organs at the borders of the bald areas was observer. The cell lineage of these rare sense organs is unknown (Manning, 1999).

It is interesting to consider the different mechanisms by which prospero acts to distinguish sibling cell fate. During embryonic neuroblast cell division, localization of Prospero into the daughter GMC is necessary for GMC development, but exclusion of Prospero from the neuroblast is relatively unimportant for neuroblast development (this is because neuroblast development is fairly normal in miranda mutants where Prospero remains in the neuroblast; Chris Doe, unpublished results cited in Manning, 1999). In contrast, during the adult SOP lineage, it appears equally important to remove Prospero from the IIa cell as well as provide it to the IIb cell. Another key difference between the adult SOP lineage and the embryonic SOP and neuroblast lineages is the timing of cell divisions. There are several hours between each cell division in the adult SOP lineage, considerably longer than the 40-60 minutes cell cycle of embryonic neuroblasts and SOPs. The shorter cell cycles of the embryonic lineages may require asymmetric localization of Prospero for efficient specification of sibling cell fate, whereas the longer adult SOP cell cycles may provide time for the action of other regulatory mechanisms (e.g. Notch-mediated repression of prospero expression) (Manning, 1999).

In single bristle prospero minus sense organs, a single neuron was observed with profound defects in neurite outgrowth. The defects in axon and dendrite outgrowth and connectivity could be due to lack of prospero function in the IIb cell, a non-autonomous effect due to lack of prospero function in the sheath cell, or the absence of prospero function in the neuron itself. The first possibility is unlikely because axon outgrowth defects can be observed in R7 neurons, which do not arise from a Prospero+ precursor cell. The second possibility is unlikely because lack of sheath cells (in glial cells missing embryos) does not generate similar axon outgrowth defects. A third model is favored, in which prospero has a direct function in the neuron, because many neurons with different origins (CNS, PNS, eye) transiently express prospero and all show a similar prospero mutant phenotype: stunted and misrouted axons (Manning, 1999).

Effects of Mutation: Regulated nuclear export of Prospero

Subcellular distribution of the Prospero protein is dynamically regulated during Drosophila embryonic nervous system development. Prospero is first detected in neuroblasts where it becomes cortically localized and tethered by the adapter protein, Miranda. After division, Prospero enters the nucleus of daughter ganglion mother cells where it functions as a transcription factor. A mutation has been isolated that removes the C-terminal 30 amino acids from the highly conserved 100 amino acid Prospero domain. Molecular dissection of the homeo- and Prospero domains, and expression of chimeric Prospero proteins in mammalian and insect cultured cells indicates that Prospero contains a nuclear export signal that is masked by the Prospero domain. Nuclear export of Prospero, which is sensitive to the drug leptomycin B (LMB), is mediated by Exportin. Mutation of the nuclear export signal-mask in Drosophila embryos prevents Prospero nuclear localization in ganglion mother cells. It is proposed that a combination of cortical tethering and regulated nuclear export controls Prospero subcellular distribution and function in all higher eukaryotes (Demidenko, 2001).

The l(3R)S8 locus was originally recovered during a selection for suppressors of a conditional lethal mutation in the largest subunit of RNA polymerase II, RpII215K1. A single nucleotide transformation was found in the prospero locus in l(3R)S8 mutants. The transformation is located near the 3' end of the pros gene, converting the tryptophan codon at amino acid 1378 into a stop codon (TGG->TGA). The resulting mutant Prospero protein lacks its C-terminal 30 amino acids, which reside in a 100 amino acid motif, known as the Prospero domain. This domain is highly conserved among Prospero proteins from Drosophila to mammals. This is the first reported mutation in the Prospero domain (PD) and has allowed its function to be determined (Demidenko, 2001).

The PD alone does not activate transcription. However, although the PD does not appear to contain transcription-stimulating activity, it does play a crucial role in the functioning of Prospero. Deletion of the C-terminal 30 amino acids always abrogates activation by Prospero when both the HD and the remainder of the PD are present in the protein. It is postulated that the PD might regulate either the subcellular localization or stability of the Prospero protein. To examine this, constructs were transfected into a mammalian cell and assayed for the subcellular localization and abundance of the fusion proteins. All of the constructs that contain an intact C terminus are expressed highly and are nuclear localized. In contrast, after deletion of the C-terminal 30 amino acids, the fusion proteins become restricted primarily to the cytoplasm (Demidenko, 2001).

The changes in subcellular distribution of Prospero caused by progressive C-terminal deletions were informative. They indicated that two previously undescribed domains, which regulate Prospero subcellular distribution, are present in the C terminus. One corresponds to a NES and/or cytoplasmic tether, and the second to a region that inhibits or masks this export signal/tether. The initial deletion derivatives allow the export signal/tether to be mapped roughly to the Prospero HD and the N-terminal 70 amino acids of the PD. This signal can overcome a functional NLS and cause Prospero derivatives to be restricted to the cytoplasm as long as the C-terminal 30 amino acids of the PD are removed. The C-terminal 30 amino acids include the second domain: the regulatory or masking region that blocks the nuclear export and/or tethering signal (Demidenko, 2001).

Additional deletion constructs were tested to delimit more precisely the NES and its mask. Internal deletions of the PD show that the entire domain is required for masking. Constructs lacking amino acids 1378/1407, 1340/78 or 1308/39 are all localized to the cytoplasm. To define the NES more precisely a further series of terminal deletions of Prospero are constructed. Each of these deletion constructs is exported from the nucleus. However, both the short and long isoforms of the terminal deletion IIIC are nuclear, demonstrating that the NES requires at least some of the amino acids between 1248 and 1281. An internal deletion removing helix two of the homeodomain, from amino acids 1266/81, has no effect on export. However, removal of amino acids 1252/65 abrogates nuclear export. This deletion removes helix one of the homeodomain. It is concluded that the region from 1252 to 1265 is necessary for functional nuclear export (Demidenko, 2001).

To demonstrate that this region is sufficient to act as an NES, a short peptide covering this region was fused to GFP. Alone, GFP expression is ubiquitous in CV1 cells. Addition of 28 amino acids from Prospero to GFP causes the fusion protein to be excluded from the nucleus. Note that the peptide used is derived from the short isoform of Prospero. By convention, amino acids of the short isoform are numbered relative to the long form; therefore, amino acids 1215/1271 lack 29 amino acids from the N terminus of the homeodomain and the NES is delimited to just 28 amino acids. It is concluded that amino acids 1215/1271 are sufficient to function as a nuclear export signal. Furthermore, nuclear export is blocked by LMB, demonstrating that this NES acts through the Exportin pathway. The PD was able to mask nuclear export of the GFP fusion protein. Addition of the region 1215/1407 to GFP restores the ubiquitous distribution observed using GFP alone. Thus the PD is sufficient to act as an NES mask in isolation from other regions of Prospero (Demidenko, 2001).

At least two mechanisms could account for the masking of the Prospero NES by the PD -- the masking region may interact directly with the NES to block association with the nuclear export receptor; alternatively, the PD may serve to recruit a co-factor analogous to Hth. As the motif is able to block nuclear export in both mammalian and Drosophila cells, it must be highly conserved and is likely to represent a regulatory mechanism common to all higher eukaryotes. The ability of the PD to inhibit the Prospero NES does not of itself indicate how differential nuclear export of Prospero is achieved. It does, however, indicate that alterations in export occur by regulating activity of the PD. The PD masking region contains numerous potential phosphorylation sites. Biochemical analysis has revealed that Prospero is more highly phosphorylated when cytoplasmic than nuclear. Phosphorylation status has been implicated in the function of the NESs found in other proteins such as Cyclin D1, Hdac5, Net, ternary complex factor and the yeast protein Far1p. The transition in Prospero subcellular distribution may be driven by similar kinase cascades acting to modify the masking domain. By regulating the activity of the NES at different stages of neuronal development it is possible to switch Prospero distribution from cytoplasmic to nuclear and back. In neuroblasts, an active NES will cause the rapid cycling of Prospero protein, which escapes interaction with Miranda and enters the nucleus, back into the cytoplasm. When the neuroblast buds off a GMC, concomitant inactivation of the Prospero NES and degradation of Miranda in the daughter GMC, allows Prospero to localize to the nucleus. After division of the GMC to produce two daughter neurons, reactivation of the NES could allow Prospero to be exported. Thus, regulated nuclear entry of the Prospero transcription factor could control the transcription programs characteristic of each cell type (Demidenko, 2001).

Identification of genes involved in Drosophila melanogaster geotaxis, a complex behavioral trait

Identifying the genes involved in polygenic traits has been difficult. In the 1950s and 1960s, laboratory selection experiments for extreme geotaxic behavior in fruit flies established for the first time that a complex behavioral trait has a genetic basis. But the specific genes responsible for the behavior have never been identified using this classical model. To identify the individual genes involved in geotaxic response, cDNA microarrays were used to identify candidate genes and fly lines mutant in these genes were assessed for behavioral confirmation. The identities of several genes that contribute to the complex, polygenic behavior of geotaxis have thus been determined (Toma, 2002).

Pioneering experiments on Drosophila melanogaster and Drosophila pseudoobscura investigated the nature of the genetic basis for extreme, selected geotaxic behavior. These experiments constituted the first attempt at the genetic analysis of a behavior. Selection and chromosomal substitution experiments successfully showed that there is a genetic basis for extreme geotaxic response in flies and, by implication, for behavior in general. These experiments also added to understanding of the role of variation in phenotypic evolution and selection. Despite their seminal contributions in behavioral genetics, population genetics and the study of selection, by their nature these experiments could not identify specific genes (Toma, 2002 and references therein).

These results highlight both the success and the limitation of behavioral selection experiments. Although selection results tend to be representative of the natural interactions of genes that produce behavior and can demonstrate that a trait has a genetic basis, they do not pinpoint specific genes that influence the trait. This is partly due to the involvement of many genes and the relatively minor role of each in complex polygenic phenotypes -- a problem that is especially acute for the intrinsically more variable phenotypes that are associated with behavior. The advent of cDNA microarray technology offers an easily generalized strategy for detecting gene expression differences and can complement other means of identifying the genes that underlie complex traits. An expression difference may occur in a gene that is not itself polymorphic, but that gene may contribute to the realization of the phenotypic difference (Toma, 2002).

As a starting point for identifying genes that affect a complex trait, the selected, established Hi5 and Lo extreme geotaxic lines were examined for changes in gene expression between strains of Drosophila melanogaster subjected to long-term selection and isolation. A two-step approach was used: (1) the differential expression levels of mRNAs isolated from the heads of Hi5 and Lo flies was determined using cDNA microarrays and real-time quantitative PCR (qPCR); (2) a subset of the differentially expressed genes was independently tested for their influence on geotaxis behavior by running mutants for these genes through a geotaxis maze. It was reasoned that some of the differences in gene expression between strains might be related to phenotypic differences and that it should therefore be possible, at least in part, to reconstruct the phenotype with independently derived mutations in some of the differentially expressed genes (Toma, 2002).

The findings indicate that differences in gene expression can be used to identify phenotypically relevant genes, even when no large, single-gene effects are detectable by classical, quantitative genetic analysis. Three of the four genes implicated by microarray and qPCR measurements caused differences in geotaxis, whereas none of the six control genes had an effect. Only those genes that had larger differences in expression according to the microarrays, or that were significantly different according to qPCR results (cry, Pdf and Pen), significantly changed geotaxis scores. The converse was not true, because altered geotaxis behavior did not always accompany larger differences in mRNA levels, as shown by pros, although this might reflect the sensitivity of pros to aspects of the genetic context. All of the genes tested for which there was little or no difference in mRNA levels between the selected Hi5 and Lo lines also showed no influence on geotaxis behavior (Toma, 2002).

The directionality of behavioral and mRNA differences proved to be consistent with predictions that were based on expression levels. Homozygous null mutants of Pdf and cry showed a significant increase in geotaxis score, which is consistent with a lower level of expression of these genes in Hi5 relative to Lo. Similarly, the heterozygous Pen mutant showed a significant downward shift in geotaxis score, which is consistent with a lower level of Pen expression in Lo relative to Hi5. Thus, the change in behavior of the tested mutants corresponds to the direction predicted by differences in transcript level in the selected Hi5 and Lo lines (Toma, 2002).

Whereas the cry, Pen and female Pdf mutants produced the anticipated effect on behavior, the magnitude of behavioral effect was smaller than in the original selected lines. This probably reflects the difference between the aggregate effect of an ensemble of genes in the selected lines as opposed to the individual effect of a single mutant gene in a neutral background. In addition, their relatively small effects are exactly the results that one would predict in a polygenic system such as geotaxis behavior, in which many genes have small contributions to the overall phenotype. The three genes identified in this study would not have been predicted on the basis of their previously defined functions (Toma, 2002).

These results show that the two separate approaches to behavioral genetics -- the classical Hirschian quantitative analysis of genetic architecture and the modern Benzerian approach of single-gene mutant analysis -- are complementary and can be unified. This study used the results of a Hirschian approach of laboratory selection for natural variants to identify single gene differences, such as one would find in a Benzerian approach. The results are consistent with the suggestion that naturally occurring variants in behavior correspond to mild lesions in pleiotropic genes (Toma, 2002).

Finally, the results show that differences in gene expression identified by cDNA microarray analysis can be used as a starting point for narrowing down the numbers of candidate genes involved in complex genetic processes. Such an approach is analogous, as well as complementary, to the current method of mapping quantitative trait loci to large chromosomal intervals and then making educated guesses about which genes within those intervals may be involved in the trait (Toma, 2002).

The combination of selection, with its ability to exaggerate natural phenotypic variation, and global analysis of differences in gene expression by cDNA microarray analysis offers a promising approach to previously intractable molecular analyses of behavior. The geotaxis genes that were identified might have been the direct targets of selection, or they might be downstream of the direct targets. Additional studies using the Hi5 and Lo selected lines will be required to distinguish between these possibilities and to determine the causal role that these genes have in the context of the selected lines (Toma, 2002).

This study has gone from the selection of a 'laboratory-evolved' behavioral phenotype, to screening for mRNA differences, to partially reconstituting the phenotype using mutants. This shows the feasibility of combining genomic and classical genetic approaches for the breakdown and partial reassembly of an artificially selected behavioral trait (Toma, 2002).

Combinatorial expression of Prospero, Seven-up, and Elav identifies progenitor cell types during sense-organ differentiation in the pupa

The Drosophila antenna has a diversity of chemosensory organs within a single epidermal field. Three broad categories of sense-organs are known to be specified at the level of progenitor choice. However, little is known about how cell fates within single sense-organs are specified. Selection of individual primary olfactory progenitors is followed by organization of groups of secondary progenitors, which divide in a specific order to form a differentiated sensillum. The combinatorial expression of Prospero, Elav, and Seven-up shows that there are three secondary progenitor fates. The lineages of these cells have been established by clonal analysis and marker distribution following mitosis. High Notch signaling and the exclusion of these markers identifies PIIa; this cell gives rise to the shaft and socket. The sheath/neuron lineage progenitor PIIb, expresses all three markers; upon division, Prospero asymmetrically segregates to the sheath cell. In the coeloconica, PIIb undergoes an additional division to produce glia. PIIc is present in multiinnervated sense-organs and divides to form neurons. An understanding of the lineage and development of olfactory sense-organs provides a handle for the analysis of how olfactory neurons acquire distinct terminal fates (Sen, 2003).

Development of single sensory unit s have been traced by using enhancer-trap insertions into the neurilized genes (neuA101 and neu-Gal4). Olfactory progenitor cells delaminate from the epithelium as single isolated cells with apically located nuclei and are arranged in distinct domains in the early antennal disc. By 8 h APF, these progenitors begin association with one to three additional cells forming well-defined clusters. These cell clusters do not arise by division of the olfactory progenitor since the first evidence of cell division as seen by phosophohistone-3 (PH3) immunoreactivity is after 12 h APF. The cells within the cluster are referred to as secondary progenitors, since their division gives rise to all the cells of an individual sense-organ. Most of the clusters divide between 16 and 22 h APF (Sen, 2003).

This analysis was restricted to clusters of secondary progenitors composed of three cells, although two and four cell clusters can also be identified by expression of GFP driven by neu-Gal4 (henceforth referred to as Neu-GFP). At 14 h APF, clusters are oriented in a single plane and have not yet begun cell division. Expression of Pros and Elav was examined by using specific antibodies, while Svp was monitored by following ß-galactosidase activity in the enhancer-trap line svpP1725. None of these markers express in primary olfactory progenitor cells but appear shortly after formation of groups of secondary progenitors. Double-labeling of 14-h APF discs with anti-Pros and anti-Elav reveals that two of the three cells within a cluster express both of these markers. Pros expression appears prior to that of Elav within the same cell. One of these cells also expresses the Svp reporter (henceforth called Svp-lacZ). The combinatorial expression of genes allowed identification of three progenitor types. PIIa does not express any of the markers and is recognized only by expression of Neu-GFP; PIIb expresses Neu-GFP, Pros, Elav, and SvplacZ, while PIIc expresses Neu-GFP, Pros, and Elav. Clusters composed of only two cells lack the PIIc progenitor and those with four cells contain two PIIc progenitors. Hence, differential expression of genes could provide cells within a single cluster the potential to exhibit independent fates (Sen, 2003).

The distribution of Pros, Elav, and SvplacZ was examined during division of the secondary progenitors. Staining with phenylene-diamine allowed identification of interphase nuclei, while entry and exit from mitosis was monitored by changes in Neu-GFP distribution. During mitosis, only one cell per cluster exhibits asymmetric cortical Pros crescents. The neighboring cell shows either compact or a uniform cytosolic localization. The failure to observe two cortical Pros crescents per cluster even in colcemid-arrested discs suggested either that PIIb and PIIc divide at different times or that Pros is asymmetrically segregated in only one of these cells (Sen, 2003).

By 36 h APF, postmitotic cells of the sensory units occupy positions comparable to those in the adult; the shaft and socket cells are identifiable by their external cuticular projections. Pros is present in sheath and socket cells, while Elav is exclusively neuronal. Clonal experiments have shown that the sheath cell arises from PIIb lineage possibly inheriting Pros asymmetrically from the progenitor. The socket, however, is derived from PIIa, which does not express Pros, indicating de novo synthesis (Sen, 2003).

The majority of peripheral antennal glia arise during development of the coeloconic sensilla. PIIb has been identified as the glial progenitor in a number of gliogenic sense-organs. In the olfactory sense-organs, PIIb can be unequivocally recognized by expression of Pros, Elav, and Svp-lacZ (Sen, 2003).

Clusters in the region of the antenna populated by coleoconic sense-organs were selected for detailed analysis. PIIb divides to produce a large cell that remains within the epithelial layer and a smaller basal cell. The basal cell transiently expresses Pros and low levels of the Svp reporter and also stains with antibodies against the glial cell marker Reverse Polarity. The nascent glial cell loses Pros and Svp expression and rapidly migrates away to become associated with the fasiculating sensory neurons (Sen, 2003).

The gliogenic lineage described above occurs only within the coeloconica sensilla (i.e., ~70 out of 450 sensilla). PIIIb, like PIIb in all other clusters, expresses Pros, Svp, and Elav. Mitosis of all secondary progenitors is completed by 22 h APF, and marker distribution in progeny was examined at 25 h APF. At this time, sensory cells orient along the apicobasal axis resembling positions in the mature sensillum (Sen, 2003).

Pros expression is detected in two subepidermally located accessory cells identified as sheath and socket. Upon division of PIIb, Svp-lacZ is distributed equally to both progeny. One of these is the sheath, which also expresses Pros, while the other stains with the neuron-specific antibody mAb22C10. The expression of ß-galactosidase fades from the sheath cell and is not apparent by 36 h APF. The perdurance of the reporter thus allows for the identification of sheath and neuron as siblings derived from PIIb (Sen, 2003).

In the pros-Gal4;UAS-GFP strain, GFP expression was observed in PIIb and PIIc but not PIIa. After division, all neurons were labeled, despite the fact that they did not express Pros protein, suggesting that all neurons within a sensory unit are derived from progenitors that express Pros (Sen, 2003).

Results from lineage experiments demonstrate that fates of cells within a sense organ are determined by the secondary progenitor from which they arise. What are the mechanisms that regulate secondary progenitor identity? Previous analysis in the mechanosensory lineage has shown that binary choice between PIIa and PIIb is mediated by N signaling. One of the effects of N activity is the downregulation of pros in PIIa. The possible role of N in determination of secondary progenitors of the antennal sense organs was tested using a temperature sensitive loss-of-function allele. Nts-1 animals pulsed at 32°C from 10 to 16 h APF show a significant reduction in the number of external sensory structures on the antennal surface. The presence of glial cells and neurons was visualized by staining 36-h APF antennae with anti-Repo and mAb22C10, respectively. The number of glial cells was increased over that in control animals (Sen, 2003).

Sensory neurons leave the antenna in three well-defined fascicles which are visualized by staining with mAb22C10. The diameter of the fascicles is somewhat increased in Nts-1 animals, suggesting an increase in neurons. An increase in internal cells (glia and neurons) concomitant with a reduction in external cells (shafts and sockets) can be explained by a switch of PIIa to PIIb/PIIc lineages (Sen, 2003).

In sibling cells, a bias in N signaling occurs because of an asymmetric distribution of the membrane-associated protein Numb (Nb), which binds the intracellular region of N antagonizing its function. N signaling can also be compromised by ectopic expression of a dominant negative (DN-N) construct of the N receptor which interferes with ligand-dependent signaling. The sca-Gal4P309 insertion strain was used to drive UAS-mediated Nb or DN-N activity in secondary progenitors. Expression of sca-Gal4P309-driven GFP is visualized in the proneural domains, the primary and secondary progenitors, but is not detected in the majority of sensory clusters after division (Sen, 2003).

Animals of sca-Gal4P309/UAS-Nb show a strong reduction in external structures on the adult antenna concomitant with an increase in glial cell numbers. The defect was also observed although significantly weaker in sca-Gal4P309/UAS-DN-N. In both genotypes, there appears to be an increase in neuronal number since the fascicles appeared thicker than in the controls. It is proposed that these phenotypes are caused by a transformation of PIIa to PIIb; in the coeloconic lineages, this would result in an increase of glial cells (Sen, 2003).

To test this hypothesis, pros-Gal4 was used to drive expression of DN-N in PIIb/PIIIb and PIIc but not PIIa. The numbers of external cells (sensillar shafts) from all three sense-organ types was counted and was found to be comparable to normal controls. This is consistent with the findings that tormogen and trichogen cells arise from the PIIa, which is not being manipulated in this genotype. There was also no change in glial cell number, even though N activity is being lowered in the PIIb progenitors. While it is possible that ectopically expressed DN-N is not sufficient to compromise N signaling, the favored explanation is that N is not required in PIIb/PIIIb. This would imply a PIIa-to-PIIb switch, which in the coeloconic lineages, results in excess of glial cells (in addition to neurons) (Sen, 2003).

If indeed N signaling plays a role in the binary choice between secondary progenitor types, then gain of N activity would be expected to increase the external cells (socket and shaft) that arise from the PIIa lineage. The truncated cytoplasmic domain of the N receptor (N-intra) is constitutively active, and its misexpression creates a dominant gain-of-function condition. Expression of Nintra early during sense organ development interfers with olfactory progenitor choice and subsequent recruitment of secondary progenitors. These effects of N could be avoided by exploiting the thermosensitive nature of the Gal4/UAS system (Sen, 2003).

sca-Gal4P309; UAS-Nintra animals were reared at 16°C until 10 h APF and then shifted to 28°C to activate N specifically in secondary progenitors. The adult antennae show a variety of defects affecting the external structures of the sensory units. There were cases of multiple sockets and sensilla with two shafts arising from a socket. While, in principle, such phenotypes could be explained by a role of N in binary choice between shaft and socket cells, the appearance of sensilla with four sockets or two shafts with a single socket can only be explained by invoking conversion of PIIb/PIIc to PIIa (Sen, 2003).

The PIIa lineage differs from PIIb by the absence of both Pros and Svp. Misexpression of Pros or Svp using sca-Gal4P309 results in a striking reduction in external cuticular structures on the antennal surface. This is consistent with a lack of PIIa identity. In order to test whether ectopic expression of Pros or Svp could convert PIIa to PIIb/c, 36-h APF antennae were stained with anti-Repo and mAb22C10. The numbers of glia or neurons were not altered. Coexpression of both Pros and Svp was achieved by heat-pulsing P(hs-Pros)/P(hs-Svp) pupae at 32°C for 6 h starting at 10 h APF. This did not result in an alteration in numbers of glia or neurons, although the antennae show a reduction in the numbers of external cuticular structures (Sen, 2003).

These data suggest that, while ectopic expression of either Pros or Svp interfere with PIIa fate, this is insufficient to convert cells to a PIIb identity. This means that N determines secondary progenitors through mechanisms other than and/or in addition to regulating the expression of Pros and Svp (Sen, 2003).

Pros is expressed in PIIb and PIIc and subsequently in the sheath and socket cells. The role of pros in these cells was tested by generating clones of the null allele pros17 using the FLP/FRT system. It was ascertained, in control clonal experiments, that the pwn mutation, which was used to mark pros minus clones, did not interfere with sensillar development. Several different morphological phenotypes were observed within the clones. The most abundant were sensillae with duplicated shafts or sockets. There were a few examples of three shafts arising from a single socket. The low clonal frequency coupled with variable phenotypes in clones made it difficult for examination of the fate of the progenitors themselves (Sen, 2003).

One possible explanation for these phenotypes is a partial conversion of PIIb/PIIc to a PIIa lineage, resulting in an increase of external cells at the expense of internal cells. A complete switch of PIIb and PIIc to PIIa would result in sensillae with three shafts and three sockets; this was not seen, suggesting that fate conversion is partial. These results suggest that Pros expression is necessary to bias cells toward a PIIb/PIIc fate; in pros minus secondary progenitors, the fate shifts toward PIIa (Sen, 2003).

Prospero maintains the mitotic potential of glial precursors enabling them to respond to neurons

During central nervous system development, glial cells need to be in the correct number and location, at the correct time, to enable axon guidance and neuropile formation. Repair of the injured or diseased central nervous system will require the manipulation of glial precursors, so that the number of glial cells is adjusted to that of neurons, enabling axonal tracts to be rebuilt, remyelinated and functional. To a large extent, the molecular mechanisms controlling glial precursor proliferative potential are unknown. This study shows that glial proliferation is regulated by interactions with axons and that the Drosophila gene prospero is required to maintain the mitotic potential of glia. During growth cone guidance, Prospero positively regulates cycE promoting cell proliferation. Neuronal Vein activates the MAPKinase signalling pathway in the glia with highest Prospero levels, coupling axon extension with glial proliferation. Later on, Prospero maintains glial precursors in an undifferentiated state by activating Notch and antagonising the p27/p21 homologue Dacapo. This enables prospero-expressing cells alone to divide further upon elimination of neurons and to adjust glial number to axons during development (Griffiths, 2004).

The longitudinal glia (LG) of the Drosophila CNS share some features with vertebrate oligodendrocyte precursors. Like oligodendrocytes, LG are also produced in excess and the excess cells are eliminated through apoptosis. The survival of both oligodendrocytes and at least some of the LG depends on contact with axons and on Neuregulin/Vein. There is also suggestive evidence that LG proliferation may be under non-autonomous control. The LG originate from the segmentally repeated longitudinal glioblasts (LGBs). DiI labelling of the LGB produces a clone of variable number of progeny cells, resulting in between 7 and 10 progeny cells. There is apoptosis in up to three cells in this lineage in normal embryos, meaning that the resulting progeny of the LG lineage if they were all to survive may be around 12 cells. This suggests that the mitotic profile of the LGB lineage is not simply symmetrical and/or perhaps LG precursor divisions are under non-autonomous control (Griffiths, 2004).

This study analyzed the mechanisms that regulate proliferation of the LG as they interact with pioneer axons. Proliferation of the LG is shown to be regulated by neurons and prospero (pros) is shown to play a key role in linking glial proliferation and axon guidance. Early on, Pros enables glial proliferation in response to pioneer neurons. Once the axonal bundles are formed, Pros maintains glial precursors in an arrested, immature state, enabling pros-expressing cells alone to divide further upon elimination of neurons (Griffiths, 2004).

This study has found novel roles of Pros in promoting cell proliferation and preventing cell cycle exit. The glia reach the extending growth cones in clusters of four cells, when cell division halts for some time. Normally, two of these LG then divide, resulting in a total of six, which then divide again, but since some LG die the real final number ranges between 8 and 11 cells. In pros mutants, the glia contact the pioneer neurons in clusters of eight cells rather than the normal four, suggesting that LG divided faster than normally in the presence of maternal CycE, skipping a G1 phase. The division of four LG into six is also missed, thus changing the mitotic pattern from its normal 4-6-12 to 4-8 (Griffiths, 2004).

Loss of pros function causes a reduction in LG proliferation, which is manifested in three ways. (1) In pros mutants, the first division of the two anterior LG with highest Pros levels is missed, because there is no dpERK. (2) LG do not divide at the normal times during axon guidance and fasiculation is not produced in pros mutants, because of the absence of CycE. Thus, although LG divided earlier in pros mutants, these divisions are uncoupled from axon guidance. Thus, Pros changes the mitotic profile in the LG from a simple symmetric pattern to a pattern in which the LG respond to incoming axons. (3) In the absence of Pros, LG do not have the potential to overproliferate when neurons are ablated (Griffiths, 2004).

Pros protein is present in all dividing LG and in LG that retain mitotic potential. During growth cone guidance and axonal fasciculation, Pros promotes LG proliferation of the two LG that are able to respond to Vein and activate the MAP kinase pathway. Vein induces LG cell division as well as cell survival of the two EGFR-positive LG. Knock-down of Vein function with targeted RNAi exclusively directed to the the MP2 neurons is sufficient to cause LG apoptosis. Loss of Vein function in genetic null embryos reduces mitosis, also when apoptosis is blocked. Thus, the EGFR/MAPKinase signalling pathway controls both cell survival and cell proliferation in these two LG. The EGFR also controls both cell survival and cell proliferation in the retina, in response to the ligand Spitz. Later on, when the axonal fascicles are formed, Pros maintains the mitotic potential in the LG by preventing them from exiting the cell cycle. In fact, only Pros-positive LG can enter S phase upon ectopic expression of cycE. In this way, at the end of embryogenesis, the LG are divided into Pros-positive G1-arrested LG and Pros-negative LG, which have exited the cell cycle and are in G0. Pros maintains the LG in the G1-arrested undifferentiated, immature precursor state by positively regulating Notch and by antagonising Dacapo (Griffiths, 2004).

These findings on the roles of Pros in the LG during axon guidance differ from Pros' neuroblast functions. In neuroblast lineages, Pros protein is located in a crescent and it is distributed asymmetrically to the daughter cell upon the division of the neuroblast. In the ganglion mother cell, Pros is internalised into the nucleus, where it determines cell fate and it restricts cell division. However, the progeny of the LGlioblast [from the time in which they contact the pioneer axons (four-cell stage)] divide apparently symmetrically, although asynchronously. During these divisions, Pros is present in the nuclei of all dividing LG, and not in crescents. Upon cell division, Pros is segregated symmetrically to the two daughter cells and it is downregulated after cell division, at the time that the posterior LG migrate with the axons. Finally, during axon guidance, pros mutations cause a reduction in LG proliferation rather than an excess, meaning that pros is necessary for cell division to proceed (Griffiths, 2004).

Pros and its vertebrate homologue Prox1 can inhibit cell proliferation and promote cell cycle exit. In fact, both in pros and Prox1 mutants, cell proliferation and the expression of cyclin increase, and both Prox1 and Pros can promote p27/dap expression. In the LG, Pros promotes cell proliferation and it prevents cell cycle exit by antagonising Dap. Therefore, Pros controls cell cycle genes in different ways in different cellular contexts. Moreover, temporal regulation is crucial and Pros can both promote and antagonise dap expression at different time points. Upon ectopic pros expression the LG divide less and do not express cycE. However, in the LG this may not be due to the promotion of cell cycle exit but to the earlier halt of precursors in cell cycle arrest (Griffiths, 2004).

These findings also contrast with the roles of Pros in mixed neuro-glioblast lineages, where Pros is segregated asymmetrically to the daughter cell that will become a glial cell. The LG is a glial-only lineage. In the LG, Pros may control the fate of the two LG with higher Pros levels, which signal through MAPKinase/dpERK. The results show that during axon guidance Pros plays a primary role in the maintenance of the proliferative and undetermined state (Griffiths, 2004).

The current findings on the non-autonomous regulation of glial proliferation contrast with previous work that envisioned a cell-autonomous proliferation profile determined by lineage identity. Accordingly, the LGB would divide in a straightforward symmetrical fashion, into 2-4-8 cells. This conclusion was based on the finding that BrdU is incorporated in four Repo-positive cells. The data show that the incorporation of BrdU into four LG represents a narrow time window in the LG lineage, and not the final division. In fact, mitosis is detected in up to five LG at the same stage (Griffiths, 2004).

The finding of a different LG profile has important implications. It means that the final number of LG is not fixed at eight cells, but variable between 8 and 11, depending on how many LG die. A final fixed number of eight LG could be achieved faster through simple symmetrical divisions without considerable influence on final glial cell mass. In fact, in Pros mutants a final number of eight cells is achieved at an earlier time point, and these eight cells stretch out to occupy the whole length of the segmental neuropile. However, the sequential increase and adjustment in LG number deploys a restricted number of LG at sequential steps in axonal patterning. This enables glia to be in the correct number at discrete time points to enable axon guidance and fasciculation (Griffiths, 2004).

The first event in growth cone guidance occurs at the four-cell stage, when LG stop dividing for some time and wait for the pioneer growth cones to extend. At this time, the LG are in the first G1 phase in the lineage. The G1 phase is a characteristic time in which cells respond to growth factors to signal through ERK, and in the retina axons approach selectively precursors that are in G1. As the growth cones approach, the two anterior LG (with higher Pros levels) of the four-cell clusters divide in response to Vein. Vein is produced by the MP2 pioneer neurons, which require LG for axon guidance. By regulating both cell survival and cell proliferation, Vein ensures that LG are present in the correct number to enable growth cone guidance. Pros regulates the zygotic expression of CycE in LG, thus introducing the first G1-S transition, and the fate of the EGFR signalling cells. In this way, Pros modulates the timing of the response of glia to a neuronal signal to divide. Subsequently, the LG continue to divide at times in which axons undergo fasciculation and defasciculation. In this way, LG are deployed in restricted numbers to enable sorting out of axons through time (Griffiths, 2004).

Later on, neurons prevent further glial proliferation. Thus, glial number is achieved by the dual response of glia to neuronal signals: earlier on neurons promote glial proliferation and later on they prevent it. At the later stages, Pros confers developmental plasticity by maintaining a subset of the LG in an undifferentiated state, since G1-arrested LG enable cell number adjustment. This confers robustness to the establishment of the axonal bundles through development (Griffiths, 2004).

Pros promotes G1 arrest; it prevents both cell cycle exit and terminal differentiation of LG precursors, and it maintains them in an immature state, with mitotic potential. Pros-positive LG might be the only ones to divide further during metamorphosis, in the restructuring of the neuropile to form the adult CNS. Or perhaps, Pros-positive LG constitute a population of glial precursors with the capacity to divide further if required, for instance upon variations occurring during development, in response to different environmental conditions or to limited damage (Griffiths, 2004).

The maintenance of a subset of the LG in an immature state by Pros allows them to divide further when neurons are ablated. The increase in LG proliferation upon neuronal ablation resembles an in vivo repair-like response in glia. In vertebrates, neuronal injury causes limited glial overproliferation and spontaneous remyelination. The therapeutic implementation of CNS repair will require the manipulation of oligodendrocyte precursors, and the controlled adjustment of their number relative to the regenerating axons. This requires knowledge of what controls oligodendrocyte precursor differentiation and proliferation relative to neuronal contact. The current results have demonstrated that Pros plays this role in Drosophila, and it invites research into the role of Prox1 in oligodendrocyte precursor proliferation and differentiation (Griffiths, 2004).

The adult mammalian brain has oligodendrocyte precursors, and just like dap induces cell cycle exit and terminal differentiation in the LG, p27 and p21 induce cell cycle exit and terminal differentiation of oligodendrocyte precursors. In fact, p27 is part of the cell cycle timer that restricts the number of times that oligodendrocyte precursors can divide. Interestingly, Notch maintains the stem cell state in vertebrates, and in Drosophila, Notch is restricted to the immature LG precursors. Remarkably, activation of Notch in oligodendrocyte precursors by Jagged1 from optic nerve axons prevents oligodendrocyte differentiation. Perhaps Prox1 maintains oligodendrocyte precursors in an immature state by regulating Notch and by antagonising p21 and p27. Upon injury or disease in the CNS, Prox1 could be a key molecule coordinating glial cell number and the re-establishment of axonal trajectories to enable repair (Griffiths, 2004).

This study has shown that Pros plays a fundamental role in adjusting glial cell number to the extending axons during guidance and fasciculation, and in maintaining glial precursors in an undifferentiated state that enables them to respond to neurons. This interactive mechanism provides robustness to the formation of the axonal trajectories, essential for the structural stability of the CNS during development. The finding that Pros enables the glia to respond to neuronal ablation by overproliferating provokes further research into the potential use of the Drosophila CNS for the study of repair (Griffiths, 2004).

Induction of tumor growth by altered stem-cell asymmetric division in Drosophila melanogaster

Loss of cell polarity and cancer are tightly correlated, but proof for a causative relationship has remained elusive. In stem cells, loss of polarity and impairment of asymmetric cell division could alter cell fates and thereby render daughter cells unable to respond to the mechanisms that control proliferation. To test this hypothesis, Drosophila larval neuroblasts were generated containing mutations in various genes that control asymmetric cell division and then their proliferative potential was assayed after transplantation into adult hosts. It was found that larval brain tissue carrying neuroblasts with mutations in raps (also called pins), mira, numb or pros grew to more than 100 times their initial size, invading other tissues and killing the hosts in 2 weeks. These tumors became immortal and can be retransplanted into new hosts for years. Six weeks after the first implantation, genome instability and centrosome alterations, two traits of malignant carcinomas, appeared in these tumors. Increasing evidence suggests that some tumors may be of stem cell origin. These results show that loss of function of any of several genes that control the fate of a stem cell's daughters may result in hyperproliferation, triggering a chain of events that subverts cell homeostasis in a general sense and leads to cancer (Caussinus, 2005).

Malignant transformation and loss of cell polarity are tightly correlated in human carcinomas. Likewise, Drosophila larval tissues with mutations in dlg1, l(2)gl or scrib have impaired apicobasal polarity and neoplastic growth in the imaginal epithelia and nervous system. There are several hypotheses to explain how loss of polarity contributes to neoplastic transformation. Most of them involve models in which changes in cellular architecture impinge directly on the cell cycle either by inhibiting signals that restrain cell proliferation or by enhancing mitogenic pathways. An alternative hypothesis is that loss of polarity in stem cells that divide asymmetrically impairs the mechanisms that specify the fate of the resulting daughter cells. If these daughter cells are unable to follow their normal developmental program, they may not respond to the mechanisms that control proliferation in the wild-type lineage (Caussinus, 2005).

Drosophila neuroblasts are stem cells whose asymmetric cell-division machinery is fairly well characterized and thus provide a good model to test this hypothesis. In the embryo, Insc integrates into the apical cortex of two neuroblast protein complexes, Baz-DmPar6-aPKC and Gialpha-Raps, by associating with Baz and Raps. These two complexes mediate the basal localization of Mira and Pon and their interacting proteins, Pros and Numb, whose segregation into the ganglion mother cell (GMC) is required for the unequal fate of the two neuroblast daughter cells. The basal localization of Mira and Pros, as well as the spindle orientation and asymmetry of daughter-cell sizes, require the functions provided by dlg1, l(2)gl and scrib. Larval neuroblasts originate from quiescent embryonic neuroblasts, and their asymmetric division seems to be controlled by the same molecular complexes, although minor differences have been reported (Caussinus, 2005).

To assess the effect of disrupted stem-cell asymmetric division on cell proliferation, larval neuroblasts were generated with mutations in aPKC, raps, mira, pros or numb and their proliferation potential was assayed after transplantation into adult hosts. No substantial growth of 101 pieces of wild-type larval brains were observed 2 weeks after transplantation. Similar results were observed for 109 implants that carried homozygous aPKCk06403 clones, none of which grew to any noticeable extent. In contrast, pieces of brains from rapsP89/raps P62 larvae or from larvae carrying homozygous numb03235, miraZZ176 or pros 17 clones grew to more than 100 times their original size, severely damaging and displacing the host's organs in the abdomen. Of the 103 flies studied in detail, 92% had one or more small tumor colonies derived from the implanted tissue but located at a long distance from the point of injection. The efficiency of tumor development ranged from 8% for numb03235 clones to 20% for rapsP89/rapsP62 tissue (Caussinus, 2005).

To assess further the growth potential of these tumors, they were cut into pieces and reimplanted into new hosts. More than 90% of these flies developed a tumor, even when they were implanted with numb 03235 tissue that had initially developed tumors in only 8% of implanted adults. This result suggests that the growing tumor mass adapts itself very rapidly to its new environment. Pieces of brain lobes from 9- to 12-d-old homozygous brat k06028 and l(3)mbt ts1 larvae, in which overgrowth was already apparent, developed tumors in 91% and 58%, respectively, of the implanted hosts (Caussinus, 2005).

All the tumors described here have been maintained in the laboratory, some for more than 2 years. This shows that the transformed cells became immortal and can proliferate without end, in contrast to cells of wild-type imaginal discs implanted into adult hosts, which remain alive after years of culture but very rarely proliferate. Among the established cell lines, substantial differences were observed in speed of growth, host lifespan or frequency or average number of additional tumor colonies, that could be attributed to the mutant background from which the tumors originated. Using the same criteria, these tumors were indistinguishable from dlg1, l(2)gl and scrib neuroblastomas (Caussinus, 2005).

Attempts were made to determine the kinds of cells that could be found in these tumors. Using green fluorescent protein as a clonal marker, it was observed that in tumors derived from tissue carrying numb 03235, miraZZ176 or pros17 clones induced by mitotic recombination, neither the wild-type twin nor the heterozygous background cells were able to proliferate upon implantation and were lost within 2 weeks. These cells accounted for most of the implanted mass, and so their inability to hyperproliferate provided a valuable internal control to substantiate the conclusion that tumor growth in this assay required the loss of the genes under study and was not just the result of dissection and transplantation into adult hosts. It also showed that the tumor growth induced by the loss of function of these genes was cell-autonomous (Caussinus, 2005).

Immunofluorescence staining for cell-specific markers identified the neuroblasts as relatively large cells, 8-12 microm in diameter, that expressed Mira. In miraZZ176 tumors, neuroblasts were identified by the expression of Wor. Ganglion cells were identified as small cells, 4-6 microm in diameter, that did not express Mira but did express Pros or, in pros 17-derived tumors, Numb. The intermediately sized cells that did not express Pros, some of which showed weak Mira staining, might be GMCs. Neuroblasts accounted for most of the mitotic activity observed in these tumors (86%). Daughter-cell size and Mira segregation during mitosis were symmetric in neuroblasts derived from rapsP89/rapsP62 tumors but asymmetric in those derived from numb03235 and pros 17 tumors. Daughter-cell size was also asymmetric in neuroblasts from miraZZ176 tumors (Caussinus, 2005).

Neither neuroblasts nor ganglion cells were markedly diluted or over-represented as the tumors aged from host to host. Therefore, like l(2)gl and dlg1 tumors, the tumors derived from numb03235, miraZZ176, pros17 and raps P89/rapsP62 were neuroblastomas that resulted from the uncontrolled division of neuroblast stem cells and were largely composed of the undifferentiated cell types that belong to this lineage. The mechanism by which these tumors grew is not understood, but it must account for the observed continuous expansion of both the neuroblast and the ganglion cell populations. One plausible mechanism could be a low frequency of neuroblast divisions that generate two neuroblast daughters. Real-time analysis of cell proliferation in these tumors may provide an answer to this issue (Caussinus, 2005).

In most solid human tumors, malignancy is very often correlated with genome instability, which is thought to contribute to multistage carcinogenesis. As in most animal cells, the frequency of natural cases of genome instability in wild-type Drosophila neuroblasts and GMCs is low (less than 10-3). This is also the case in numb03235, miraZZ176, pros 17 and rapsP89/raps P62 tumors shortly after transplantation. In 40-d-old tumors, however, 10%-15% of the cells presented different kinds of karyotype defects. Of the 340 karyotypes obtained from numb, mira, pros and raps tumors, 62% included segmental aneuploid; 9% were monosomic, trisomic or tetrasomic with respect to one or more chromosomes; 6% were triploid or tetraploid; and the remaining 23% included cells that could not be karyotyped owing to very high levels of ploidy, chromosome fragmentation or chromosome condensation (Caussinus, 2005).

The karyotypes obtained from cells in a single tumor were as different from one another as they were from the karyotypes of cells from other tumors, and none of the tumor lines that were established presented a distinct set of chromosome aberrations. Therefore, no substantial differences were observed attributable to the mutant condition that originated the tumor. In most tumor lines, the frequency of cells that contained abnormal karyotypes did not change noticeably over time, with one exception: 3 months after the first implantation, genome instability affected more than 95% of the cells in mirTF, a tumor line derived from miraZZ176. The absence or very low incidence of genome instability during the first round of implantation suggests that genome instability did not cause tumor formation in these tumor lines. But the onset of genome instability correlates well with a marked increase in the frequency of hosts that developed a tumor in later transplantations. Therefore, the possible contribution of genome instability to the evolution of these tumors remains to be assessed. Genome instability has also been reported in l(2)gl neuroblastomas (Caussinus, 2005).

In mammalian carcinomas, genome instability is tightly correlated with severe alterations of the centrosome cycle that affect the number of centrosomes per cell as well as centrosome size and shape. Supernumerary centrosomes can result in multipolar spindles and contribute to the generation of aneuploidy. Like the DNA cycle, the centrosome cycle is tightly controlled in wild-type neuroblasts, so that cells that have an abnormal number of centrosomes are exceptionally rare in wild-type tissue. This was not the case in numb03235, mira ZZ176, pros17 or raps P89/rapsP62 tumors: forty days after the first implantation, 15%-20% of those cells had more than two centrosomes. Some of these centrosomes were irregularly shaped, and their size range was much wider than that of control cells. A fraction of these could be centriole-less aggregates of pericentriolar material. The cells that had supernumerary centrosomes seemed to be hyperploid (Caussinus, 2005).

None of the mutant conditions from which these tumors originated has been reported to affect chromosome segregation or the centrosome cycle, which were both unaffected in early tumors. In addition, the cells of wild-type imaginal discs that have been kept for years in adult hosts maintain a stable genome and can differentiate into adult structures. Therefore, genome instability and impaired centrosome cycles in numb 03235, miraZZ176, pros17 and rapsP89/rapsP62 tumors cannot be considered a consequence of the mutant background or long-term exposure to the adult abdomen environment. Rather, the onset of genome instability and centrosome alterations suggests that once the mechanisms that control cell proliferation have been over-ridden, hyperproliferation triggers a chain of events that subverts cell homeostasis in a very general sense, including the DNA and centrosome cycles (Caussinus, 2005).

In summary, neoplastic transformation of Drosophila larval neuroblasts can be triggered by perturbing several of the functions that mediate asymmetric stem-cell division. In terms of growth rate, cell types, metastatic activity and extent of genome and centrosome instability, the resulting tumors are essentially indistinguishable from one another, regardless of the mutant from which they derive. The main conclusion that can be drawn from these data is that these tumors might have a common etiology: perturbation of neuroblast polarity and the resulting impairment of cell-fate determination. This argument is strengthened by the case of the homeobox-containing transcription factor Pros, which lies downstream of the other genes required for neuroblast asymmetric division (Caussinus, 2005).

The tumors in this study are practically indistinguishable from the neuroblastomas that arise in adults implanted with pieces of dlg1, l(2)gl or scrib mutant larval brains. Because these three neoplastic tumor suppressors are required for multiple aspects of neuroblast asymmetric cell division, including the basal localization of Mira, Numb and Pros, mislocalization of these proteins might explain, at least partially, the uncontrolled cell proliferation produced by loss of dlg1, l(2)gl or scrib function in larval neuroblasts (Caussinus, 2005).

The unequal segregation of cell-fate determinants resulting from asymmetric cell division is a fundamental mechanism for generating cellular diversity during development, organ homeostasis and repair. If impaired segregation of cell-fate determinants can cause the hyperproliferation of larval neuroblasts of Drosophila, it may similarly affect tissue stem cells in other species. At the moment, however, any parallel to stem-cell models of human cancer remains purely speculative. Consistent with this hypothesis, the inactivation of both Numb and Numb-like in the mouse dorsal forebrain leads to impaired neuronal differentiation, hyperproliferation of neural progenitors and delayed cell-cycle exit. In addition, loss of Lgl1 (also called Mlgl or Hugl), one of the two L(2)gl homologs in the mouse, results in a failure to asymmetrically localize Numb and leads to severe brain dysplasia (Caussinus, 2005).

In most human tumors, the identity of the first carcinogenic cell remains elusive. Indirect but growing evidence suggests that in some cases, the founders may be stem cells. Stem cells are self-renewing, have limitless replicative potential and produce differentiating cells, three features found in many cancers. Carcinomas occur in tissues that are maintained by a continuous supply of differentiating daughter cells originating from stem-cell division. Moreover, some of the signaling pathways that control stem-cell self-renewal, like the Notch, Wnt-ß-catenin and Hedgehog pathways, are known to have a role in carcinogenesis in these tissues. The results show that inactivation of any of several molecular mechanisms that control the asymmetry of the segregation of cell-fate determinants during stem-cell division may result in hyperproliferation of the stem-cell compartment and could contribute to cancer (Caussinus, 2005).

Partnership of Brat and Prospero in regulating self-renewal in Drosophila neural stem cells

How stem cells generate both differentiating and self-renewing daughter cells is unclear. This study shows that Drosophila larval neuroblasts - stem cell-like precursors of the adult brain - regulate proliferation by segregating the growth inhibitor Brat and the transcription factor Prospero into only one daughter cell. Like Prospero, Brat binds and cosegregates with the adaptor protein Miranda. In larval neuroblasts, both Brat and Prospero are required to inhibit self-renewal in one of the two daughter cells. While Prospero regulates cell-cycle gene transcription, Brat acts as a posttranscriptional inhibitor of dMyc. In brat or prospero mutants, both daughter cells grow and behave like neuroblasts leading to the formation of larval brain tumors. Similar defects are seen in lethal giant larvae (lgl) mutants where Brat and Prospero are not asymmetric. This study has identified a molecular mechanism that may control self-renewal and prevent tumor formation in other stem cells as well (Betschinger, 2006).

These data reveal a molecular mechanism that controls self-renewal in Drosophila larval neuroblasts. The growth regulator Brat segregates asymmetrically during neuroblast division and inhibits self-renewal in one of the two daughter cells. Together with the asymmetrically segregating transcription factor Prospero, Brat ensures that this daughter cell will stop growing, exit the cell cycle, and differentiate into neurons. In brat or prospero mutants, or in lgl mutants, where Brat and Prospero are not asymmetrically segregated, both daughter cells proliferate leading to the formation of a brain tumor and death of the animal. These tumors are neoplastic and can be transplanted into the abdomen of wild-type flies where they overgrow, invade other tissues, and eventually kill the host (Betschinger, 2006).

Asymmetric cell division has been studied in the Drosophila central and peripheral nervous systems. In the peripheral nervous system, the determinants Numb and Neuralized segregate into one of the two daughter cells, and in their absence, this cell is transformed into its sister cell. In the embryonic central nervous system, Prospero acts as a segregating determinant, but in prospero mutants, many GMCs are still correctly specified. The data suggest that this is because Prospero acts partially redundant with Brat. In embryos double mutant for prospero and brat, most GMCs expressing the marker Eve are missing and neuronal differentiation in the embryonic CNS is greatly impaired. These observations suggest that Brat and Prospero act together to specify GMC fate in Drosophila embryos (Betschinger, 2006).

Although cell-cycle markers are expressed longer and stronger in prospero and brat, prospero mutant embryos, uncontrolled overproliferation has not been described in Drosophila embryos so far. In larvae, however, both brat and prospero mutant neuroblasts can initiate tumor formation. It is proposed that this difference is due to distinct mechanisms of cell growth during the two stages. During embryogenesis, cell number increases dramatically but the total volume of the embryo remains constant. Embryonic neuroblasts therefore shrink with each division and they might exit the cell cycle simply because they become too small. Support for this model comes from mutations affecting cell size asymmetry during neuroblast divisions, like Gβ13F (Fuse, 2003) or Ric-8 (Hampoelz, 2005): in these mutants, GMCs are larger, neuroblasts shrink faster and, as a consequence, divide less often. In larval neuroblasts, the situation is quite different. Several results indicate that larval neuroblasts grow significantly while cell growth is inhibited in GMCs. First, the total volume of clones generated from individual neuroblasts is several times more than the initial volume of the neuroblast. Second, the size of 'old' and 'young' (earlier and more recently generated) GMCs is approximately the same, indicating that GMCs do not grow significantly during clone formation. Third, larval neuroblasts do not become progressively smaller during development indicating that the loss of cytoplasm from each division must be compensated for by growth. Taken together, these results suggest that larval neuroblasts might be more appropriate as a model for the control of self-renewal in stem cells (Betschinger, 2006).

These experiments show that the restriction of cell growth in the GMC requires the genes lgl, brat, and prospero. While lgl seems to be required indirectly due to its role in asymmetric protein segregation, Prospero and Brat act in the GMC to regulate several important events: They repress neuroblast fate, inhibit cell-cycle progression, and prevent cell growth. Prospero is a homeodomain transcription factor, and the cell-cycle genes Cyclin A, Cyclin E, and Dacapo (the fly homolog of the CDK inhibitor p21) were shown to be among its transcriptional targets. Similar to Drosophila Prospero, its vertebrate homolog Prox-1 has been shown to regulate cell-cycle genes, and loss of prox-1 leads to increased proliferation of retinal progenitor cells (Betschinger, 2006).

For Brat, two different functions have been described: First, it acts as a translational regulator of the gap-gene hunchback. Hunchback is expressed in the embryonic nervous system but is not present in wild-type or brat mutant larval neuroblasts and is unlikely to be relevant for the growth regulatory activity of Brat. More likely, Brat acts through its well-described inhibitory activity on ribosomal RNA synthesis. Cells mutant for brat or its C. elegans homolog ncl-1 have larger nucleoli, more ribosomal RNA, and higher rates of protein synthesis, and these activities have been made responsible for the cell size increase that is observed in C. elegans and Drosophila brat mutant cells. These data suggest that this second function of Brat is also linked to posttranscriptional gene regulation. It is proposed that Brat downregulates dMyc in one of the two daughter cells and thereby inhibits protein synthesis and cell growth. Whether Brat controls dMyc translation, protein stability, or RNA stability is unclear. Interestingly, the C. elegans Brat homolog ncl-1 has been identified as one of the genes required for RNAi (Kim, 2005). Since the microRNA pathway was shown to be involved in regulation of Drosophila stem cell proliferation (Hatfield, 2005), differential regulation of this pathway in neuroblasts and GMCs by Brat could provide another explanation for its mutant phenotype (Betschinger, 2006).

Brat is part of a protein family that is characterized by a C-terminal NHL domain, several zinc-finger like B boxes, and a coiled-coil region. While the vertebrate members of this family (TRIM-2, TRIM-3, and TRIM-32) are not well characterized, the mutant phenotype of the two other Drosophila members (Dappled and Mei-P26) suggests a common function as tumor suppressors. Mutations in dappled cause melanomic tumors of the fat body, and mei-P26 mutations lead to ovarian tumors. While dappled tumors have not been well characterized, the mei-P26 phenotype has been attributed to overproliferation of undifferentiated germ cells. It is similar to-and genetically interacts with-bag of marbles, a well-characterized repressor of proliferation in the daughter cells of germline stem cells. Thus, it is conceivable that proliferation control in stem cells is a common activity of NHL domain proteins (Betschinger, 2006).

Recent evidence suggests that some human brain tumors contain stem cell-like neural progenitors that are responsible for tumor formation. Together with the identification of stem cell-like subpopulations in leukaemia, multiple myeloma, and breast cancer, this has led to the so-called cancer stem cell hypothesis which proposes that only a small population of cells in a tumor have the ability to proliferate and self-renew. This discovery suggests mechanisms for tumorigenesis other than the simple loss of proliferation control, in particular dedifferentiation of cells into additional stem cells and symmetric division of stem cells. Animal models for tumor stem cells are essential for developing new therapeutic approaches that target these mechanisms. Although Drosophila can only mimic some aspects of tumorigenesis, it might contribute to the identification of the molecular pathways operating in tumor stem cells. Human Lgl has already been implicated in tumor progression, and the characterization of Brat homologs will verify the relevance of Drosophila as a tumor stem cell model (Betschinger, 2006).

Control of neuronal cell fate and number by integration of distinct daughter cell proliferation modes with temporal progression

During neural lineage progression, differences in daughter cell proliferation can generate different lineage topologies. This is apparent in the Drosophila neuroblast 5-6 lineage (NB5-6T), which undergoes a daughter cell proliferation switch from generating daughter cells that divide once to generating neurons directly. Simultaneously, neural lineages, e.g. NB5-6T, undergo temporal changes in competence, as evidenced by the generation of different neural subtypes at distinct time points. When daughter proliferation is altered against a backdrop of temporal competence changes, it may create an integrative mechanism for simultaneously controlling cell fate and number. This study identified two independent pathways, Prospero and Notch, which act in concert to control the different daughter cell proliferation modes in NB5-6T. Altering daughter cell proliferation and temporal progression, individually and simultaneously, results in predictable changes in cell fate and number. This demonstrates that different daughter cell proliferation modes can be integrated with temporal competence changes, and suggests a novel mechanism for coordinately controlling neuronal subtype numbers (Ulvklo, 2012).

The NB5-6T lineage utilizes two distinct mechanisms to control daughter cell proliferation. In the early part of the lineage, pros limits daughter cell (GMC) proliferation, whereas in the late part canonical Notch signaling in the neuroblast further restricts daughter cell proliferation, resulting in a switch to the generation of neurons directly. The switch in daughter cell proliferation is integrated with temporal lineage progression and enables the specification of different Ap neuron subtypes and the control of their numbers (Ulvklo, 2012).

The data on Notch activation in the NB5-6T lineage, using both antibodies and reporters, indicate progressive activation in the neuroblast: weak at St10-11 and more robust from St12 onward. Thus, Notch activity coincides with the proliferation mode switch. How is this gradual activation of Notch in the neuroblast controlled? NB5-6T undergoes the typical progression of the temporal gene cascade, with Cas expression preceding strong Notch activation. Thus, one possible scenario is that the late temporal gene cas activates the Notch pathway. However, analysis of the E(spl)m8- EGFP reporter shows that this Notch target is still activated at the proper stage in cas mutants. Although this does not rule out the possibility that other, unknown, temporal factors might regulate Notch signaling, it rules out one obvious player, cas. Alternatively, as Notch signaling is off when neuroblasts are formed (a prerequisite for neuroblast selection), Notch activation in the neuroblast at later stages might simply reflect a gradual reactivation of the pathway. Although such a reactivation might at a first glance appear too imprecise, it is possible that the specificity of this particular Notch output -- proliferation control -- might be combinatorially achieved by the intersection of Notch signaling with other, more tightly controlled, temporal changes (Ulvklo, 2012).

Pros and Notch control daughter proliferation in different parts of NB5-6T, and no evidence of cross-regulation between these pathways was found. The limited overproliferation of the lineage when each pathway is separately mutated results not from redundant functions, but rather stems from the biphasic nature of this lineage. Specifically, in pros mutants, Notch signaling is likely to be on in all 'A' type sibling daughter cells, as Numb continues to be asymmetrically distributed between daughter cells. Thus, Notch signaling in 'A' cells may preclude each 'A' cell from dividing even once. This notion is in line with recent studies showing that postmitotic Notch activated cells ('A' cells) within the Drosophila bristle lineages are particularly resilient to overexpression of cell cycle genes. Similarly, in Notch pathway mutants, as Ap cells now divide (in essence becoming GMC-type cells), Pros will still play its normal role in these 'GMCs' and limit their proliferation to a single extra cell division. However, in kuz;pros double mutants, Ap cells are relieved of both types of daughter cell proliferation control and can thus divide for many additional rounds. This notion also applies to early parts of the NB5-6T lineage and probably to the majority of other VNC lineages, as indicated by the extensive overproliferation of the entire NB5-6T lineage, and to the general overproliferation of the VNC. However, based on the findings that neither the Notch pathway nor pros controls neuroblast identity or its progression, it is postulated that these large clones contain a single, normally behaving NB5-6T neuroblast. In fact, the neuroblast is likely to exit the cell cycle and undergo apoptosis on schedule, as neither of these decisions depends upon pros or the Notch pathway. Of interest with respect to cancer biology is that the findings point to a novel mechanism whereby mutation in two tumor suppressors (e.g., Pros and Notch) cooperate to generate extensive overproliferation: not by acting in the same progenitor cell at the same time, but by playing complementary roles controlling daughter cell proliferation (Ulvklo, 2012).

As an effect of alternate daughter cell proliferation patterns, both vertebrates and invertebrates display variability in neural lineage topology. Similarly, progenitors in these systems undergo temporal changes in competence, as evident by changes in the types of neurons and glia generated at different time points. Hence, the temporal-topology interplay described in this study is likely to be extensively used and to be conserved in mammals. As a proof of principle of this novel developmental intersection, single and double mutants were examined for kuz and nab, thereby independently versus combinatorially affecting temporal progression and daughter cell proliferation. Strikingly, these mutants show the predicted combined effect, with the appearance of additional Ap1/Nplp1 neurons beyond those found in each individual mutant (Ulvklo, 2012).

If programmed proliferation switches are conserved, how might such a topology-temporal interplay become utilized in mammals? There are several examples in which different clusters/pools/nuclei of neurons of distinct cell fate are generated from the same progenitor domain in the developing mammalian nervous system. Such pools often contain different numbers of cells, but the underlying mechanisms controlling the precise numbers of each subtype are poorly understood. Based on previous studies in a number of models, at least three different mechanisms can be envisioned. Based on the current study, a novel fourth mechanism is proposed, whereby alteration of daughter cell proliferation is integrated with temporal progression to control subtype cell numbers. These four mechanisms are not mutually exclusive, and given the complexity of the mammalian nervous system it is tempting to speculate that all four mechanisms are utilized during development (Ulvklo, 2012).

Homeobox gene distal-less is required for neuronal differentiation and neurite outgrowth in the Drosophila olfactory system

Vertebrate Dlx genes have been implicated in the differentiation of multiple neuronal subtypes, including cortical GABAergic interneurons, and mutations in Dlx genes have been linked to clinical conditions such as epilepsy and autism. This study showed that the single Drosophila Dlx homolog, distal-less, is required both to specify chemosensory neurons and to regulate the morphologies of their axons and dendrites. distal-less was shown to be necessary for development of the mushroom body, a brain region that processes olfactory information. These are important examples of distal-less function in an invertebrate nervous system and demonstrate that the Drosophila larval olfactory system is a powerful model in which to understand distal-less functions during neurogenesis (Plavicki, 2012).

The phenotype exhibited by dll-null embryos is more severe than that of sc, amos, or ato single mutants or amos;ato double mutants, most closely resembling that of the sc;amos;ato triple mutants, indicating that dll might lie upstream of the proneural genes and regulate their expression in precursors of the Dorsal organ (DO), the larval olfactory organ (see Schematic of the larval chemosensory system). However, ato is expressed in the antennal segments of dll-null embryos, and dll is expressed in ato1 and amos1 mutants. Altogether, these data indicate that dll is likely to act in parallel with the proneural genes during DO development (Plavicki, 2012).

prospero (pros) is also required for DO development. pros encodes a homeodomain transcription factor that is asymmetrically distributed during SOP division. In other contexts, pros represses neuronal stem-cell proliferation while promoting neuronal differentiation. Mutations in pros result in gustatory behavioral deficits and disrupt axon and dendrite outgrowth from both DO and terminal organ (TO) neurons. The axon pathfinding defects exhibited by pros mutants resemble those seen in dll-null embryos, although it is unclear whether the same subsets of neurons are affected (Plavicki, 2012).

The axon scaffolding associated with the MBs in the embryonic brain is disrupted in dll-null embryos. Specifically, projections from the MB Kenyon cells across the supraesophageal commissure appear to be missing in late-stage embryos. MB defects also were observed in the brains of larvae in which postmitotic drivers such as elav-GAL4 and OR83b-GAL4 were used in conjunction with dll-RNAi to knock down activity. This finding indicates that dll also may be necessary for the later specification and/or differentiation of larval-born Kenyon cells. Because elav-GAL4 is not active until neurons are specified, the disruptions in the larval MBs observed in elav-GAL4;UAS-dll-RNAi animals are consistent with a role for dll in either axon guidance or viability of the postmitotic neurons (Plavicki, 2012).

The brain phenotypes detected in dll mutants resemble those of cephalic gap gene mutants. Specifically, loss of otd, ems, or btd results in embryonic brain segmentation defects and disrupts the formation of brain commissures and axon tracts. In otd mutants, protocerebral neuroblasts, including the MB precursors, are missing. In ems and/or btd mutants, subsets of neuroblasts are lacking in the deutocerebral neuromere, which harbors the larval antennal lobe (LAL), and the tritocerebral neuromere, which receives gustatory inputs. It therefore is possible that dll is a key effector of cephalic gap gene function during brain development (Plavicki, 2012).

pros mutants exhibit DO axon defects similar to those of dll. It therefore is possible that Dll and Pros collaborate to regulate other genes needed for axon pathfinding by DOG neurons. In the brain, but not in the DO Ganglion (DOG) or TO ganglion (TOG), mislocalization of Futsch protein was observed in dll mutants. Futsch is a microtubule-associated protein with functions in both axonogenesis and dendritogenesis. It therefore is possible that some of the defects observed in dll mutant MB lobes (which consist of axon tracts) and calyces (which contain Kenyon cell dendrites) are caused by misregulation of futsch. Other likely effectors of dll function during axon pathfinding in both DO and MB are Pak3 and Down syndrome cell adhesion molecule (Dscam). Both play important roles in axon guidance, including the targeting of adult Drosophila ORNs. Both also have been identified as putative downstream targets of vertebrate Dlx1/2. The loss-of-function Drosophila dll phenotypes in both DOG and MB are reminiscent of DSCAM phenotypes and consistent with dll regulation of DSCAM in multiple neuronal subtypes (Plavicki, 2012).

Given the dramatic reduction in Kenyon cell number in the dll hypomorphs, it might be expected that the peduncles and lobes would be even thinner than observed. However, a similar reduction in Kenyon cell number without concomitant thinning of the lobes has been observed in eyR mutants. In this case, ablation studies were used to demonstrate that, at late third instar, recently born Kenyon cells have not yet contributed to MB lobes. It is therefore anticipated that MB defects may be more pronounced in dll mutant adults (Plavicki, 2012).

prospero: Biological Overview | Evolutionary Homologs | Regulation | Protein Interactions | Developmental Biology | References

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