klumpfuss


REGULATION

The development of the posterior spiracles of Drosophila may serve as a model to link patterning genes and morphogenesis. A genetic cascade of transcription factors downstream of the Hox gene Abdominal-B subdivides the primordia of the posterior spiracles into two cell populations that develop using two different morphogenetic mechanisms. The inner cells that give rise to the spiracular chamber invaginate by elongating into 'bottle-shaped' cells. The surrounding cells give rise to a protruding stigmatophore by changing their relative positions in a process similar to convergent extension. In the larvae the spiracular chamber forms a very refractile filter, the filzkorper. The opening of the spiracular chamber, the stigma, is surrounded by four sensory organs; the spiracular hairs. Clones labeling the spiracular hairs show that each one is formed by four cells related by lineage, two neural and two support cells, the typical structure of a type I external sensory organ. When the larva is buried in the semi-liquid medium on which it feeds, the stigmatophore periscopes out of the medium allowing the larva to continue breathing. The genetic cascades regulating spiracular chamber, stigmatophore, and trachea morphogenesis are different but coordinated to form a functional tracheal system. In the posterior spiracle, this coordination involves the control of the initiation of cell invagination that starts in the cells closer to the trachea primordium and spreads posteriorly. As a result, the opening of the tracheal system shifts back from the spiracular branch of the trachea into the posterior spiracle cells (Hu, 1999).

Downstream of Abd-B the cascade can be subdivided into various levels. The activation of six genes -- cut, empty spiracles (ems), nubbin (nub), klumpfuss (klu), and spalt (sal) -- does not require expression any of the other genes studied, suggesting that these six genes are at the top of the cascade under Abd-B regulation. The cut, ems, nub, and klu genes are expressed in the spiracular chamber in overlapping patterns. The sal gene is not expressed in the spiracular chamber but in the cells that surround it and will form the stigmatophore. The exclusion of sal from the spiracular chamber is partly due to repression by cut, because in cut mutants sal is expressed at low levels in the internal part of the spiracle. Downstream of these putative Abd-B targets other genes are activated. These include the transcription factors grainyhead (grh), trachealess (trh) and engrailed (en) (Hu, 1999).

The spiracle phenotypes in mutants for the early Abd-B downstream genes have been analyzed. In sal mutants the stigmatophore does not form, resulting in embryos with a normal spiraclular chamber that does not protrude. Conversely, mutations in ems and cut affect the spiracular chamber but not the stigmatophore. Mutations for ems result in a spiracular chamber that lacks a filzkorper and is not connected to the trachea. In cut mutants the filzkorper is almost completely missing, but the trachea is still connected to the spiracular chamber and the spiracular hairs are also missing. In trh mutants, where the tracheal pits do not form and there is no tracheal network, the spiracular chamber cells still invaginate, forming a filzkorper. However, this filzkorper is shorter than that of the wild type probably due to a secondary requirement of trh, which is also expressed in the spiracular chamber cells. These results show that the spiracular chamber, the stigmatophore, and the trachea develop independently of one another. No phenotypes for either klu or nub could be detected, indicting that although these genes are expressed in the spiracle, they are either redundant or their function is not required for spiracle morphogenesis (Hu, 1999).

Lozenge directly activates argos and klumpfuss to regulate programmed cell death

Reducing the activity of the Drosophila Runx protein Lozenge (Lz) during pupal development causes a decrease in cell death in the eye. Lz-binding sites were identified in introns of argos (aos) and klumpfuss (klu); these genes were shown to be directly activated targets of Lz. Loss of either aos or klu reduces cell death, suggesting that Lz promotes apoptosis at least in part by regulating aos and klu. These results provide novel insights into the control of programmed cell death (PCD) by Lz during Drosophila eye development (Wildonger, 2005).

These findings, together with what is known about aos and klu, support the following model: Lz induces aos expression in cone cells, wherefrom Aos diffuses to antagonize EGFR activity in the surrounding 2° and 3° cells. The expression pattern of aos923-lacZ indicates that Lz also regulates aos expression in 2° and 3° cells, suggesting that these cells may also send antisurvival signals. The data further suggest that within the 2° and 3° cells, Lz activates klu, which antagonizes EGFR signaling downstream of the receptor. Lz also activates klu expression in cone and 1° cells, but it is unclear what function klu has in these cells. Although two phases of PCD during retinal development have been proposed, these experiments support a role for Lz in promoting only the EGFR-dependent phase. An alternative possibility is that the decrease in cell death in lz mutant retinas is due to an increase in 2° and 3° cell differentiation stimulated by an increase in EGFR signaling. However, given the large body of evidence demonstrating that lz normally functions to promote differentiation, a model in which lz acts to suppress differentiation is not favored (Wildonger, 2005).

The mammalian homolog of Lz, Runx1 (also known as AML1), is also a transcriptional regulator. In humans, translocations that affect Runx1 are associated with acute myelogenous leukemia (AML), which is characterized by the proliferation of undifferentiated hematopoietic cells. Effects on cell cycle regulators have been implicated in contributing to this overproliferation, but it is likely that PCD also plays a role . Changes in the amount of the apoptotic regulator Wilms Tumor 1 (WT1) are often found in AML patients. lz promotes cell death in the Drosophila eye in part by activating the expression of klu, the Drosophila homolog of WT1. It is suggested that these findings may be relevant to how Runx1 chimeras lead to the development of AML in humans. Furthermore, they suggest that WT1 may be a direct target of Runx1 (Wildonger, 2005).

Hairless induces cell death by downregulation of EGFR signalling activity

Overexpression of the Notch antagonist Hairless (H) during imaginal development in Drosophila is correlated with tissue loss and cell death. Together with the co-repressors Groucho (Gro) and C-terminal binding protein (CtBP), H assembles a repression complex on Notch target genes, thereby downregulating Notch signalling activity. This study investigated the mechanisms underlying H-mediated cell death in S2 cell culture and in vivo during imaginal development in Drosophila. First, the domains within the H protein that are required for apoptosis induction in cell culture were mapped. These include the binding sites for the co-repressors, both of which are essential for H-mediated cell death during fly development. Hence, the underlying cause of H-mediated apoptosis seems to be a transcriptional downregulation of Notch target genes involved in cell survival. In a search for potential targets, transcriptional downregulation of rho-lacZ and EGFR signalling output were noted. Moreover, the EGFR antagonists lozenge, klumpfuss and argos were all activated upon H overexpression. This result conforms to the proapoptotic activity of H, as these factors are known to be involved in apoptosis induction. Together, the results indicate that H induces apoptosis by downregulation of EGFR signalling activity. This highlights the importance of a coordinated interplay of Notch and EGFR signalling pathways for cell survival during Drosophila development (Protzer, 2008).

This work allows two important conclusions: that overexpression of H induces cell-autonomous apoptosis, and that H requires the co-repressors Gro and CtBP for its proapoptotic activity. It is known that H assembles a repression complex together with the two co-repressors, resulting in transcriptional downregulation of Notch target genes. Hence, the ability of H to induce cell death is most likely a consequence of the repression of Notch target genes that are involved in cell survival. It is noted, however, that not every cell that receives an overdose of H dies. One simple explanation for this observation is that the only cells that die are those in which the relevant Notch target genes are normally active, as these cells require a Notch signal for survival. As H results in a repression of Notch activity, these cells would be driven into cell death, whereas those cells that do not depend on higher Notch levels for survival would be resistant to an H overdose. How is this effect of H realised at the molecular level? So far, it has not been possible to narrow down the analyses towards one target gene, the repression of which by the H repressor complex induces apoptosis. The most straightforward idea, repression of the anti-apoptotic protein Diap1, is not supported by the data. Instead, it was found that EGFR signalling activity is downregulated as a consequence of the upregulation of several negative regulators of EGFR (Protzer, 2008).

The existence of a densely woven network of genetic interactions between the EGFR and Notch signalling pathways is well established. This intensive cross-talk harmonises many developmental processes, such as proliferation, differentiation, cell fate specification, morphogenesis and programmed cell death. Still, the molecular basis of this genetic interplay remains largely obscure. So far, few molecular intersections between the Notch and EGFR pathways have been revealed. For example, EGFR signalling causes phosphorylation of the co-repressor Gro, thereby negatively modulating the transcriptional outputs of Notch signalling via the Enhancer of split [E(spl)] genes. Conversely, a myc-Gro complex was shown to inhibit EGFR signalling during neural development in the Drosophila embryo. Although mutual antagonism is probably the most prominent relationship in EGFR-Notch interactions, in some developmental situations both pathways cooperate to potentiate each other's signalling activities. One such example with regard to cell survival has been described in the retina of rugose mutant flies, where cell type-specific cell death could be reversed by an increase in Notch or EGFR signalling activity, indicating that both pathways adopt an anti-apoptotic function in this developmental context. Also, R7 photoreceptor cell specification requires the combined input of both Notch and EGFR signals. Moreover, Notch defines the scope of rho expression in the Drosophila embryo, thereby activating the EGFR pathway required for early ectodermal patterning. Also, during the development of mouse embryonic fibroblast, the Notch receptor-processing γ-secretase presenilin acts as a positive regulator of ERK basal level activity (Protzer, 2008).

A significant decrease was observed in the levels of activated MAPK (diP-ERK), which provides a good assessment of EGFR pathway activation, upon induction of H. Activated MAPK directly phosphorylates two transcription factors, Aop (Yan) and Pointed (Pntp2). Phosphorylation inactivates Aop, which in the unmodified state, represses EGFR targets. At the same time, phosphorylation activates Pointed, which then causes EGFR target gene transcription. As H is a well-defined transcriptional repressor of Notch target genes, it is most unlikely that it impedes EGFR activity at the level of phosphorylation. Moreover, it is not thought that H acts at the level of transcriptional regulation of EGFR target genes, even though combinatorial and antagonistic activities of the nuclear effectors of the EGFR and Notch signalling pathways have been described during eye development. Instead, the hypothesis is favored that H represses the transcription of EGFR activators, or might indirectly provoke the activation of EGFR repressors that affect, for example, the production of EGFR ligands or signal transduction (Protzer, 2008).

Rho activity is required for a timely and spatially regulated release of EGFR ligands. Accordingly, the expression of rho is highly dynamic during Drosophila development, and precedes the appearance of EGFR-induced activated MAPK. Hence, downregulation of rho by H would eventually result in lower levels of activated MAPK (diP-Erk). In contrast to other components of the EGFR signalling pathway, ectopic expression of rho results in EGFR activation in a wide range of tissues, indicating that Rho is an essential and limiting factor. So far, transcriptional control is the only known means of rho regulation. The complex array of enhancers regulating rho expression reflects the dynamic pattern of EGFR activation throughout Drosophila development (Protzer, 2008).

Interestingly, a transcriptional repression of rho-lacZ was observed in H gain-of-function clones that was dependent on the co-repressors Gro and CtBP. This effect might very well be direct, because it was shown previously that rho transcription is regulated by Su(H) in the neuroectoderm as well as in the gut of the Drosophila embryo. As mentioned above, Notch signalling has also been shown to regulate rho expression in the embryonic ectoderm. Moreover, during egg development, a band of Notch activity establishes the boundary between the two dorsal appendage tube cell types, whereby Notch levels are high in rho-expressing cells. In accordance with this, potential Su(H)-binding sites are present in the regulatory regions of rho1 and rho3, making a direct regulation of rho during eye development via the Notch-Su(H)-H complex very likely. It is noted, however, that the downregulation of rho-lacZ and of activated MAPK were focussed at the morphogenetic furrow, where primary photoreceptor cells are specified and ommatidia are founded. Regulation of rho by H would then be expected to interfere with photoreceptor formation rather than with cell survival, which is in agreement with the disturbed cellular architecture of H gain-of-function flies (Protzer, 2008).

Most interestingly, upon H overexpression, ectopic induction of lz, klu and aos was observed. All three genes are known to be involved in cell death induction during pupal eye development. There it was shown that the Runx protein Lz binds to the regulatory regions of klu and aos, resulting in the direct transcriptional activation of these target genes. Therefore, one might speculate that H executes its effect on klu and aos activity via the activation of lz. Moreover, as klu and aos are well-known inhibitors of EGFR signalling activity, this in itself suggests that H impedes EGFR signalling activity via these factors. This interpretation helps to explain why aos expression is induced in H gain-of-function clones, although it is well known that aos is triggered by EGFR signalling, thereby forming an inhibitory loop that acts on EGFR activity. The high levels of Lz still activate aos in H gain-of-function clones, keeping activity of the EGFR pathway low. Alternatively, aos and klu levels might be increased as a consequence of the downregulation, by H, of an as yet unknown repressor. Since H behaves as a kind of 'multi-adaptor protein', which not only recruits the transcriptional silencers Gro and CtBP to Notch targets but also binds other proteins such as Pros26.4, it is also possible that H interacts with positive regulators of lz, klu and aos (Protzer, 2008).

However, a model is favored whereby H influences EGFR signalling activity on two levels. On the one hand, through transcriptional repression of rho, H causes a loss of EGFR signalling output that interferes with cell specification. On the other hand, by interfering with their repressor(s), H relieves the restriction on lz, klu and aos expression, causing their accumulation. In consequence, the survival/death balance is tipped towards apoptosis in those cells that are susceptible to the effects of a lowered EGFR signal. Those cells that do not depend on high Notch and EGFR activity levels for survival would be resistant to an H overdose (Protzer, 2008).

Finally, one can envisage that a downregulation of Notch and EGFR signalling activities, resulting from the overexpression of H, might leave a cell in a state of 'uncertainty' that does not allow any further differentiation towards a certain cell type, but leaves the cell vulnerable to the apoptotic programme (Protzer, 2008).

Brain tumor specifies intermediate progenitor cell identity by attenuating beta-catenin/Armadillo activity

During asymmetric stem cell division, both the daughter stem cell and the presumptive intermediate progenitor cell inherit cytoplasm from their parental stem cell. Thus, proper specification of intermediate progenitor cell identity requires an efficient mechanism to rapidly extinguish the activity of self-renewal factors, but the mechanisms remain unknown in most stem cell lineages. During asymmetric division of a type II neural stem cell (neuroblast) in the Drosophila larval brain, the Brain tumor (Brat) protein segregates unequally into the immature intermediate neural progenitor (INP), where it specifies INP identity by attenuating the function of the self-renewal factor Klumpfuss (Klu), but the mechanisms are not understood. This study reports that Brat specifies INP identity through its N-terminal B-boxes via a novel mechanism that is independent of asymmetric protein segregation. Brat-mediated specification of INP identity is critically dependent on the function of the Wnt destruction complex, which attenuates the activity of β-catenin/Armadillo (Arm) in immature INPs. Aberrantly increasing Arm activity in immature INPs further exacerbates the defects in the specification of INP identity and enhances the supernumerary neuroblast mutant phenotype in brat mutant brains. By contrast, reducing Arm activity in immature INPs suppresses supernumerary neuroblast formation in brat mutant brains. Finally, reducing Arm activity also strongly suppresses supernumerary neuroblasts induced by overexpression of klu. Thus, the Brat-dependent mechanism extinguishes the function of the self-renewal factor Klu in the presumptive intermediate progenitor cell by attenuating Arm activity, balancing stem cell maintenance and progenitor cell specification (Komori, 2013).

Asymmetric stem cell division provides an efficient mechanism to simultaneously self-renew a stem cell and to generate a progenitor cell that produces differentiated progeny. Because self-renewal proteins segregate into both daughter progeny of the dividing parental stem cell through the inheritance of its cytoplasmic content, rapidly downregulating the activity of these proteins is essential for the specification of progenitor cell identity. Brat plays a central role in specifying INP identity in the Ase- immature INP by antagonizing the function of the self-renewal transcription factor Klu (Xiao, 2012). These previous findings have been extended to show that Brat specifies INP identity in the Ase- immature INP through two separable, but convergent, mechanisms. A novel Brat-dependent mode of Wnt pathway regulation was identified that prevents Ase- immature INPs from reverting into supernumerary neuroblasts. Brat specifies INP identity by attenuating the transcriptional activity of Arm through its N-terminal B-boxes. This negative regulation of Arm is achieved through the activity of Apc2 and the destruction complex. Because increased arm function alone is insufficient to induce supernumerary neuroblasts, the ability of Wnt signaling to promote neuroblast identity is dependent on other signaling mechanisms that act downstream of Brat. Indeed, Arm function is essential for Klu to induce supernumerary neuroblasts. These two Brat-regulated mechanisms function to safeguard against the accidental reversion of an uncommitted progenitor cell into a supernumerary stem cell and to ensure that an uncommitted progenitor cell can only adopt progenitor cell identity (Komori, 2013).

Physical interaction with the cargo-binding domain of Mira is essential for the unequal segregation of Brat into the immature INP following the asymmetric division of neuroblasts. Previous studies concluded that the NHL domain of Brat directly interacts with the cargo-binding domain of Mira, but the roles of the B-boxes and the coiled-coil domain in the asymmetric segregation of Brat were unknown due to a lack of specific mutant alleles. By combining a yeast two-hybrid interaction assay and in vivo functional validation, it is concluded that both the coiled-coil domain and the NHL domain are indeed required for the asymmetric segregation of Brat into the Ase- immature INP following the asymmetric division of neuroblasts. It is speculated that the coiled-coil domain and the NHL domain of Brat function cooperatively to provide a more stable binding platform for Mira to ensure efficient protein segregation (Komori, 2013).

The severity of the supernumerary neuroblast phenotype in various brat mutant allelic combinations correlates with the level of endogenous brat inherited by the Ase- immature INP. The brat DG19310 mutation carries a transposable P-element inserted in the 5′ regulatory region of the brat gene. The brat11 mutation, however, results in a premature stop codon at amino acid 779, leading to a truncated form of the protein that lacks most of the NHL domain and is predicted to be unable to interact with Mira. The brat DG19310 or brat DG19310/11allelic combination most likely reduces Brat expression without affecting its binding to Mira. Thus, the minimal threshold of Brat necessary for the proper specification of INP identity in Ase- immature INPs is met most of the time, leading to a mild supernumerary neuroblast phenotype in brat DG19310 or brat DG19310/11 brains. By contrast, the brat11 homozygous or brat11/Df mutant allelic combination impairs the binding of Brat to Mira, rendering the Mira-based asymmetric protein-sorting mechanism unable to segregate Brat into the Ase- immature INP. As such, the threshold of Brat necessary for proper specification of INP identity in Ase- immature INPs is rarely met, leading to a severe supernumerary neuroblast phenotype in brat11 or brat11/Df brains. Overexpression of the bratΔC-coil or bratΔNHL transgene using the UAS/Gal4 system almost certainly results in an abnormally high level of the transgenic protein in the cytoplasm of neuroblasts. Thus, inheriting a portion of the neuroblast cytoplasm containing an overwhelming abundance of the mutant transgenic protein is likely to be sufficient to reach the threshold of Brat necessary for proper specification of INP identity in Ase- immature INPs. It is concluded that the mechanism that causes Brat to asymmetrically segregate into the Ase- immature INP is functionally separable from the mechanism that specifies INP identity (Komori, 2013).

Could the asymmetric protein segregation mechanism promote the specification of INP identity by depleting Brat from the neuroblast? Type II neuroblasts overexpressing brat, bratΔC-coil or brat<ΔNHL maintained their identity and generated similar numbers of progeny as wild-type control neuroblast. Thus, it is unlikely that Brat-dependent specification of INP identity occurs through asymmetric depletion of Brat from the neuroblast. Whether Brat acts redundantly with other asymmetrically segregating determinants to specify INP identity in Ase- immature INPs was also tested. Numb also exclusively segregates into the immature INP during asymmetric divisions of type II neuroblasts. However, asymmetric segregation of Numb is not dependent on Brat, and Numb-dependent specification of INP identity also occurs independently of Brat. Thus, it is unlikely that Brat acts redundantly with other asymmetric segregating determinants to specify INP identity in Ase- immature INPs (Komori, 2013).

A surprising finding revealed by the current study is that the B-boxes are uniquely required for the specification of INP identity. This raises a series of interesting questions. What are the roles of B-boxes in the function of Brat in embryonic neuroblasts? Embryos lacking both maternal and zygotic function of brat often lack RP2 neurons but never possess supernumerary neuroblasts. Since brat mutant alleles that specifically affect the function of B-boxes are unavailable, the roles of B-boxes in the function of Brat during the asymmetric division of embryonic neuroblasts remain unknown. Brat regulates embryonic pattern formation by repressing mRNA translation through the ternary complex that also contains Nanos and Pumilio. However, it is unlikely that Brat specifies INP identity through the Nanos-Pumilio-Brat translational repression complex for the following reasons. First, the NHL domain of Brat is required for binding to Pumilio and Nanos and for the assembly of the translational repressor complex. However, the NHL domain is dispensable for Brat-dependent specification of INP identity. Second, Nanos expression is undetectable in larval brains, and pumilio mutant larval brains do not possess supernumerary type II neuroblasts. Together, these results are consistent with the conclusion that Brat specifies INP identity via a novel Arm-mediated mechanism (Komori, 2013).

The amino acid sequence of the B-boxes is highly conserved among all TRIM family proteins, including Brat, and is predicted to adopt a 'RING-like' fold tertiary structure. The RING-like fold might facilitate protein-protein interactions. This is a particularly intriguing hypothesis in light of the fact that Apc2 and Brat both localize to the basal cortex in type II neuroblasts, and overexpression of brat, but not bratδB-boxes, can restore Apc2 protein localization in neuroblasts. However, epitope-tagged Brat and endogenous could not be coprecipitated Apc2 from the brain lysate extracted from brat null mutant larvae overexpressing a Myc-tagged Brat transgenic protein. Thus, Brat might maintain Apc2 protein localization indirectly through other factors. Future biochemical analyses of the Brat protein and identification of the proteins that directly interact with the B-boxes will provide insight into how Brat controls Apc2 localization (Komori, 2013).

The destruction complex targets β-catenin/Arm for degradation during canonical Wnt signaling, so reduced function of the destruction complex will lead to an increase in β-catenin/Arm, which forms a complex with Tcf/LEF family transcription factors to activate Wnt target gene expression. This study has concluded that the Brat-Apc2 mechanism specifies INP identity by preventing aberrant activation of Wnt target gene expression in Ase- immature INPs. The role of the Wnt ligand was tested in the Brat-dependent specification of INP identity by removing the function of the Wnt ligand using a temperature-sensitive mutant allele or by overexpressing a dominant-negative form of Frizzled (FzDN or GPIdFz2) in brat DG19310/11 mutant brains. Interestingly, neither of these manipulations modified the supernumerary neuroblast phenotype in the sensitized brat genetic background (data not shown). These results suggest that the Wnt ligand and its receptor Fz are irrelevant in the Brat-dependent specification of INP identity and that the Brat- Apc2 mechanism prevents Wnt target gene expression in Ase- immature INPs by negatively regulating the activity of Arm. However, these data do not exclude the possibility that a novel activating mechanism of Wnt signaling might be present in type II neuroblasts in Drosophila larval brains (Komori, 2013).

Attempts were made to directly demonstrate that loss of brat function indeed leads to derepression of Wnt target gene expression in supernumerary neuroblasts.The expression was examined of two distinct Wnt reporter transgenes, WRE-lacZ and Notum-lacZ in brat mutant brains. However, it was not possible to detect the expression of these transgenes in supernumerary neuroblasts in brat null mutant brains. Because genetic manipulations altering the activity of Arm efficiently modify the supernumerary neuroblast phenotype in brat mutant brains, these two transgenes are unlikely to have the necessary regulatory elements to reflect Wnt target gene activity in this tissue. Thus, it is proposed that the Brat-Apc2 mechanism specifies INP identity by antagonizing the transcriptional activity of Arm in Ase- immature INPs via a receptor-independent mechanism (Komori, 2013).

Wnt signaling regulation plays key roles in both stem cell renewal and the differentiation of progenitor cell types (Merrill, 2012; Habib et al., 2013). In the mammalian intestinal epithelium, for example, loss of Apc and activation of Wnt signaling results in the maintenance of stem cell properties in the progenitor cells, a failure to differentiate, and the production of intestinal polyps that progress to malignant tumors. In the intestine, the inappropriate activation of Wnt signaling is sufficient to elicit stem cell properties. In the progenitor cells of larval type II neuroblasts, the activation of Wnt signaling alone, through either the expression of stabilized Arm or the loss of Apc2, does not drive stem cell renewal in otherwise wild-type immature INPs. In this system, Brat is the key regulator attenuating self-renewal through two independent, but convergent, mechanisms in its regulation of both Klu and Wnt signaling. Although Arm activity is required for Klu-dependent self-renewal in immature INPs, its inability to promote self-renewal alone suggests that Wnt signaling is likely to be playing a permissive role rather than an instructive role in eliciting the neuroblast identity. It is proposed that Brat downregulates the function of Klu through both Arm-dependent and -independent mechanisms. Previous studies have demonstrated that TRIM32 and TRIM3, vertebrate orthologs of Brat, are essential regulators of neural stem cells during brain development and brain tumor formation (Boulay, 2009; Schwamborn, 2009). It will be interesting to test whether TRIM32 and TRIM3 regulate neural stem cells via a β-catenin-dependent mechanism (Komori, 2013).


DEVELOPMENTAL BIOLOGY

Embryonic

The earliest Klu expression as detected with anti-Klu-antibody is nuclear and occurs before the syncytial blastoderm stage. The staining disappears after cellularization. At stage 10, Klu staining reappears in a subset of neuroblasts (NBs) and in the procephalic region. The staining becomes more prominent in the NBs and brain at stage 11; some epithelial cells start to express Klu and are stained as patches on the lateral sides of the embryos. The staining in the ventral cord and brain continues through stage 12. At stage 16, the staining seen in the ventral cord and the brain remains but is weaker; the epithelial cells are stained as three- to four-cell-wide stripes and the posterior spiracles are also Klu+. The earliest NB staining is seen at stage 10, when six NBs are initially stained by the antibody; they are row 5 NBs (NB5-2, NB5-3, and NB5-6); row 7 NBs (NB7-1 and NB7-4) and NB3-5. At stage 11, with the exception of the five NBs (NB2-4, NB3-3, NB4-4, NB5-4 and NB6-4), all NBs show nuclear staining. At late stage 11, only two NBs (NB2-3 and NB6-4) do not express Klu. Klu is also expressed in some GMCs (the progeny of neuroblasts), including GMC4-2b (Yang, 1997).

Klumpfuss acts to differentiate between the identities of the first two secondary precursor cells produced from one NB lineage. Klu is expressed in the NB4-2 lineage only after two rounds of NB cell division, in the second born GMC (GMC4-2b) (Yang, 1997).

The Drosophila brain develops from the procephalic neurogenic region of the ectoderm. About 100 neural precursor cells (neuroblasts) delaminate from this region on either side in a reproducible spatiotemporal pattern. Neuroblast maps have been prepared from different stages of the early embryo (stages 9, 10 and 11, when the entire population of neuroblasts has formed), in which about 40 molecular markers representing the expression patterns of 34 different genes are linked to individual neuroblasts. In particular, a detailed description is presented of the spatiotemporal patterns of expression in the procephalic neuroectoderm and in the neuroblast layer of the gap genes empty spiracles, hunchback, huckebein, sloppy paired 1 and tailless; the homeotic gene labial; the early eye genes dachshund, eyeless and twin of eyeless; and several other marker genes (including castor, pdm1, fasciclin 2, klumpfuss, ladybird, runt and unplugged). Based on the combination of genes expressed, each brain neuroblast acquires a unique identity, and it is possible to follow the fate of individual neuroblasts through early neurogenesis. Furthermore, despite the highly derived patterns of expression in the procephalic segments, the co-expression of specific molecular markers discloses the existence of serially homologous neuroblasts in neuromeres of the ventral nerve cord and the brain. Taking into consideration that all brain neuroblasts are now assigned to particular neuromeres and individually identified by their unique gene expression, and that the genes found to be expressed are likely candidates for controlling the development of the respective neuroblasts, these data provide a basic framework for studying the mechanisms leading to pattern and cell diversity in the Drosophila brain, and for addressing those mechanisms that make the brain different from the truncal CNS (Urbach, 2003).

In the trunk, the zinc-finger transcription factor Klumpfuss (Klu) is expressed from stage 10 onwards in an increasing number of NBs, and at stage 11, almost all NBs (except NB2-3 and NB6-4) show nuclear Klu staining. The expression of Klu in the procephalon was analyzed using an antibody against Klu and the P-lacZ enhancer trap strain klu P212 which basically shows an identical expression pattern. Klu is not expressed in the neuroectoderm. Similar to the situation in the trunk CNS, Klu protein is first found at a detectable level at stage 9, in a subset of (about 17) brain NBs and at late stage 11 in almost all brain NBs. For most NBs, there is a significant delay between birth and onset of klu expression. Klu also appears to be expressed in ganglion mother cells, as was shown for the trunk (Urbach, 2003).

Larval

klumpfuss is expressed in each of the imaginal discs. Expression starts in the wing disc within the prospective wing area early in the third larval instar. Shortly thereafter, expression becomes restricted to the prospective margin and the hinge of the wing: at about the same time, transcripts appear in the anlagen of notum and scutellum. klu is expressed in most proneural clusters at, or shortly after, the onset of achaete expression; klu expression precedes the appearance of sensory organ precursors. While cells of the proneural clusters express klu, sensory organ precursors (SOPs) themselves do not. Since klu expression is rather uniform before the appearance of SOPs, the gene must be switched off in cells that initiate neural development. Expression in the leg discs starts early during the third larval instar. At this time the expression domain occupies a wedge-like sector encompassing roughly one third of the circumference of the leg disc. Rings of expressing cells successively become visible distal to a knob-like central structure during the third larval stage. The rings correspond to the anlagen of the leg segments; the order of their appearence reflects the developmental pattern of the leg disc. Expression in the antennal and dorsal prothoracic discs also occurs in concentric domains. In the eye disc, expression starts behind the morphogenetic furrow and extends through the whole anlage. klu is further expressed in the regions that form the head capsule. Expression in the larval brain is restricted to the neuroblasts and the proliferation zone of the optic lobes (Klein, 1997).

A genetic cascade involving klumpfuss, nab and castor specifies the abdominal leucokinergic neurons in the Drosophila CNS

Identification of the genetic mechanisms underlying the specification of large numbers of different neuronal cell fates from limited numbers of progenitor cells is at the forefront of developmental neurobiology. In Drosophila, the identities of the different neuronal progenitor cells, the neuroblasts, are specified by a combination of spatial cues. These cues are integrated with temporal competence transitions within each neuroblast to give rise to a specific repertoire of cell types within each lineage. However, the nature of this integration is poorly understood. To begin addressing this issue, this study analyzed the specification of a small set of peptidergic cells: the abdominal leucokinergic neurons. The progenitors of these neurons were identified, along with the temporal window in which they are specified, and the influence of the Notch signaling pathway on their specification. The products of the genes klumpfuss, nab and castor were shown to play important roles in their specification via a genetic cascade (Benito-Sipos, 2010).

Recent findings on NB5-6 demonstrate that Cas and Grh act as crucial temporal genes to specify several cell fates at the end of this lineage. The current findings with NB5-5 reveal similar roles for Cas and Grh, and indicate that the ABLKs are specified in a Cas/Grh temporal window. It was observed that cas mutants generate no ABLKs, that cas misexpression leads to clusters of two to four ABLKs per hemisegment, and that Cas is expressed in all the ABLKs. Thus, these data confirm that cas plays a role as a temporal identity gene, which remains compatible with its proposed role as a switching temporal factor (Benito-Sipos, 2010).

The proposed role of grh as a temporal identity gene remains open to question. It has also been reported that it is required to regulate mitotic activity and apoptosis of post-embryonic NBs. However, recent evidence has emerged indicating that Grh also temporally regulates FMRFamide neuropeptide cell fate and can act in a combinatorial manner with dimm and apterous to trigger ectopic FMRFamide expression. Similarly, it was found that Grh is required for correct specification of the ABLKs. Together, these results suggest that Grh also plays an instructive role in ABLK specification. Thus, many neuropeptidergic neurons are generated late in several lineages, and depend upon the late temporal genes cas and grh for their specification (Benito-Sipos, 2010).

NB5-5 does not express the temporal genes hb and Kr, and genetic analysis confirms that these two genes are not required for specification of ABLK fate. It was observed that NB5-5 initially expresses Pdm at the time of delamination in late stage 11. Pdm is downregulated at early stage 12, when Cas is activated, and there is a brief period in which both proteins can be detected. The lack of molecular markers does not permit determination of whether the Pdm/Cas coexpression stage generates a GMC (Benito-Sipos, 2010).

It is of interest to note that the phenotype observed misexpressing cas with NB-specific drivers was very mildcompared with that obtained using a pan-neuronal driver. NB5-5 expresses cas soon after delamination and generates six to nine neurons. This suggests that NB5-5 probably has a broad Cas temporal window. Thus, the phenotype obtained upon misexpressing cas with elav-Gal4 indicates that Cas might have a later requirement in postmitotic cells that generates subtemporal windows. Consistent with this interpretation, cas misexpression rescues the grh phenotype of loss of ABLKs, which also suggests that Grh, in addition to being required as a temporal factor, would be indirectly required to activate cas expression in postmitotic cells (Benito-Sipos, 2010).

The Notch pathway is involved in many cell fate decisions in neural development. This study has shown that the ABLK and its sibling are equivalent cells committed to die, and that activation of the Notch pathway in the ABLK prevents its death. A similar situation has been described for specification of the anterior and posterior Corner Cells (CaCC/pCC) neurons in the grasshopper NB1-1 lineage, in which the siblings start as equivalent cells and interaction between them leads to different fates. By contrast, activation of Notch in the NB7-3 lineage drives PCD). Here, activation of the Notch pathway, or misexpression of p35 in the sibling cell, is sufficient to generate two ABLK neurons. A systematic analysis of the lineage of apoptotic cells in embryos in which apoptosis is prevented has shown that the lineage of abdominal NB5-5 contains twice the normal number of cells, but that they have wild-type-like axonal projections. It is concluded that in this lineage, Notch does not play an instructive role in specifying ABLK neuronal fate, but influences a fate decision by regulating the competence to respond to a program of cell death (Benito-Sipos, 2010).

A set of mutants were identified that produce an altered number of ABLKs. In most cases the effect is very mild. Among the mutants with the strongest phenotypes were jumu, nab and klu. The jumu phenotype was expected because it has been shown that Jumu is required in the NB4-2 lineage for normal segregation of Numb in the asymmetric cell divisions. Consistent with this interpretation, the fact that the phenotype of jumu in the NB5-5 lineage is similar to that seen in spdo explains its phenotype and indicates that in jumu embryos Notch is off in both siblings (Benito-Sipos, 2010).

nab and klu embryos display a strong reduction in the number of ABLKs, suggesting that both genes have direct roles in ABLK specification. Interestingly, Cas activates the expression of both genes via repression of Pdm. The lack of availability of markers for identifying ABLKs in earlier stages did not permit establishing whether Nab and Klu are required in the NB or in postmitotic cells (Benito-Sipos, 2010).

Misexpression of cas in nab embryos showed ectopic ABLKs, suggesting that Cas acts either parallel to, or downstream of, Nab. The lack of molecular markers specific to the NB5-5 lineage does not allow determination of whether all ectopic ABLKs are generated by the NB5-5 or by other lineages. Nevertheless, several results suggest that, most probably, all of them are produced by NB5-5. First, it has been observed that neurons that belong to one lineage form a coherent cluster. Second, all of them express gsb, which labels rows five and six NB, and do not express lbe, an NB5-6-specific marker. Third, ABLKs are the unique cells expressing Lk in the ventral ganglion. However, downregulation of cas was not observed in nab mutants, and the same has been reported in the better characterized lineages of NB3-3 and NB5-6; together, these results indicate that the molecular relationship between Cas and Nab requires a more complex interpretation than a linear genetic cascade (Benito-Sipos, 2010).

klu encodes a zinc-finger protein but does not appear to interact directly with Nab, and no evidence was found that nab and klu regulate each other. Surprisingly, nab misexpression rescues the phenotype of a lack of ABLKs observed in klu. By contrast, it was found that cas misexpression produces more ABLKs in grh, nab or klu than in wild-type background As proposed above, these results suggest that Cas plays a role in postmitotic cells that is crucial for ABLK specification (Benito-Sipos, 2010).

The sqz phenotype is epistatic over the nab phenotype. Thus, although nab embryos have no ABLKs, sqz and nab sqz show a normal pattern of ABLKs. It has been shown by pull-down assay that Nab physically interacts with Sqz, and in vertebrates the Nab homologs act as transcriptional co-factors. Since both genes, sqz and nab, are expressed in the ABLKs, it is proposed that the function of Sqz in NB5-5 lineage is to repress the ABLK fate. In normal development, as both genes are expressed in the ABLKs, Nab binds to Sqz and blocks its repressor activity; in nab embryos Sqz represses the ABLK fate, but in nab sqz the pattern is wild-type because there is no repression by Sqz. This intimate interplay between Sqz and Nab is also found in the NB 5-6 linage, in which sqz is first required to activate cell fate determinants, and then acts with nab to suppress the same determinants (Benito-Sipos, 2010).

The findings reported in this study extend understanding of the mechanisms of ABLK specification. However, more precise analysis of the genes and the mechanisms involved in specification of the different cell fates in the NB5-5 lineage will require additional molecular markers. This would permit identification of the different neurons generated from this NB and the genes required to specify their various fates (Benito-Sipos, 2010).

A temporal mechanism that produces neuronal diversity in the Drosophila visual center

The brain consists of various types of neurons that are generated from neural stem cells; however, the mechanisms underlying neuronal diversity remain uncertain. A recent study demonstrated that the medulla, the largest component of the Drosophila optic lobe, is a suitable model system for brain development because it shares structural features with the mammalian brain and consists of a moderate number and various types of neurons. The concentric zones in the medulla primordium that are characterized by the expression of four transcription factors, including Homothorax (Hth), Brain-specific homeobox (Bsh), Runt (Run) and Drifter (Drf/Vvl), correspond to types of medulla neurons. This study examined the mechanisms that temporally determine the neuronal types in the medulla primordium. For this purpose, transcription factors were sought that are transiently expressed in a subset of medulla neuroblasts (NBs, neuronal stem cell-like neural precursor cells) and identified five candidates [Hth, Klumpfuss (Klu), Eyeless (Ey), Sloppy paired (Slp) and Dichaete (D)]. The results of genetic experiments at least explain the temporal transition of the transcription factor expression in NBs in the order of Ey, Slp and D. The results also suggest that expression of Hth, Klu and Ey in NBs trigger the production of Hth/Bsh-, Run- and Drf-positive neurons, respectively. These results suggest that medulla neuron types are specified in a birth order-dependent manner by the action of temporal transcription factors that are sequential ly expressed in NBs (Suzuki, 2013).

In the embryonic central nervous system, the heterochronic transcription factors suchas Hb, Kr, Pdm, Cas and Grh are expressed in NBs to regulate the temporal specification of neuronal identity. They regulate each other to achieve sequential changes in their expression in NBs without cell-extrinsic factors. However, expression of the embryonic heterochronic genes was not detected in the medulla NBs.Instead this study found that Hth, Klu, Ey, Slp and D are transiently and sequentially expressed in medulla NBs. The expression of Hth and Klu was observed in lateral NBs, while that of Ey/Slp and D was observed in intermediate and medial NBs, respectively. These observations suggest that the expression of heterochronic transcription factors changes sequentially as each NB ages, as observed in the development of the embryonic central nervous system (Suzuki, 2013).

This study demonstrates that at least three of the temporal factors Ey, Slp and D regulate each other to form a genetic cascade that ensures the transition from Ey expression to D expression in the medulla NBs. Ey expression in NBs activates Slp, while Slp inactivates Ey expression. Similarly, Slp expression in NBs activates D expression, while D inactivates Slp expression. In fact, the expression of Slp is not strong in newer NBs in which Ey is strongly expressed, but is up regulated in older NBs in which Ey is weakly expressed in the wildtype medulla. A similar relationship is found between Slp and D, supporting the idea that Ey, Slp and D regulate each other's expression to control the transition from Ey-expression to D-expression. In the embryonic central nervous system, similar interaction is mainly observed between adjacent genes of the cascade hb-Kr-pdm-cas-grh, and this concept may also be applied to the medulla primordium. The expression pattern and function of Ey, Slp and D suggest that they are adjacent to each other in the cascade of transcription factor expression in medulla NBs (Suzuki, 2013).

However, no such relationship was found between Hth, Klu and the other temporal factors.The sequential expression of Hth and Klu could be regulated by an unidentified mechanism that is totally different from the genetic cascade that controls the transition through Ey-Slp-D. Or, there might be unidentified temporal factors that are expressed in lateral NBs which act upstream of Hth and Klu to regulate their expression. It is necessary to identify additional transcription factors that are transiently expressed in medulla NBs (Suzuki, 2013).

The expression of concentric transcription factors in the medulla neurons correlates with the temporal sequence of neuron production from the medulla NBs (Hasegawa, 2011). In the larval medulla primordium, the neurons are located in the order of Hth/Bsh-, Run- and Drf-positive cells from inside to outside, and these domains are adjacent to each other (Hasegawa, 2011). Given that NBs generate neurons toward the center of the developing medulla, Hth/Bsh-positive neurons are produced at first, and then Run-positive and Drf-positive neurons. Thus Hth/Bsh, Run and Drf were used as markers to examine roles of Hth, Klu, Ey, Slp and D expressed in NBs in specifying types of medulla neurons. The continuous expression of Hth and Ey from NBs to neurons and the results of clonal analyses that visualize the progeny of NBs expressing each one of the temporal transcription factors suggest that the temporal windows of NBs expressing Hth, Klu and Ey approximately correspond to the production of Hth/Bsh-, Run- and Drf- positive neurons, respectively. Indeed, the results of the genetic study suggest that Hth and Ey are necessary and sufficient to induce the production of Hth/Bsh- and Drf-positive neurons,respectively (Hasegawa, 2011, 2013). Ectopic Klu expression at least induces the produc tion of Run-positive neurons (Suzuki, 2013).

Slp and D expression in NBs may correspond to the temporal windows that produce medulla neurons in the outer domains of the concentric zones, which are most likely produced after the production of Drf-positive neurons. The results at least suggest that Slp is necessary and sufficient and D is sufficient to repress the production of Drf-positive neurons. Identification of additional markers that are expressed in the outer concentric zones compared to the Drf-positive domain would be needed to elucidate the roles of Slp and D in specification of medulla neuron types (Suzuki, 2013).

D mutant clones did not produce any significant phenotype except for derepression of Slp expression in NBs. Drf expression in neurons was not affected either. Since D is a Sox family transcription factor, SoxN, another Sox family transcription factor, is a potential candidate molecule that acts together with D in the medulla NBs. However, its expression was found in neuroepithelia cells and lateral NBs that overlap with Hth-positive cells but not with D-positive cells. All the potential heterochronic transcription factors examined in this study are expressed in three to five cell rows of NBs. Nevertheless, one NB has been observed to produce one Bsh- positive and one Run-positive neuron (Hasegawa, 2011). Therefore, the expression pattern of the heterochronic transcription factors is not sufficient to explain the stable production of one Bsh-positive and one Run-positive neuron from a single NB.The combinatorial action of multiple temporal factors expressed in NBs may play important roles in the specification of Bsh- and Run- positive neurons (Suzuki, 2013).

Another possible mechanism that guarantees the production of a limited number of the same neuronal type from multiple rows of NBs expressing a temporal transcription factor could be a mutual repression between concentric transcription factors expressed in medulla neurons. For example, Hth/Bsh, Run and Drf may repress each other to restrict the number of neurons that express either of these transcription factors. However, expression of Run and Drf was not essentially affected in hth mutant clones and in clones expressing Hth (Hasegawa, 2011). Similarly, expression of Hth and Drf was not essentially affected in clones expressing run RNAi under the control of AyGal4, in which Run expression is eliminated. Hth and Run expression was not affected in drf mutant clones (Hasegawa, 2011). These results suggest that Hth/Bsh, Run and Drf do not essentially regulate each other during the formation of concentric zones in the medulla (Suzuki, 2013).

During embryonic development, the heterochronic genes that are expressed in NBs (hb-Kr-pdm-cas-grh) are maintained and act in GMCs to specify neuronal type. Similarly, Hth and Ey are continuously expressed from NBs to neurons, suggesting that their expression may also be inherited through GMCs (Hasegawa, 2011). However, this type of regulatory mechanism may be somewhat modified in the case of Klu, Slp and D (Suzuki, 2013).

Klu is expressed in NBs and GMCs, but not in neurons. Slp and D are predominantly detected in NBs and neurons visualized by Dpn and Elav, respectively. Occasionally, however, expression of D was found in putative GMCs, which are situated between NBs and neurons. Additionally, both D-positive and D-negative cells were found among Miranda-positive GMCs. Slp expression was not found in Miranda-positive GMCs. Finally, D is expressed in medulla neurons forming a concentric zone in addition to its expression in medial NBs. However, D expression was abolished in slp mutant NBs but remained in the mutant neurons, suggesting that D expression in medulla neurons is not inherited from the NBs. These results suggest that Slp and D expression are not maintained from NBs to neurons and that not all the temporal transcription factors expressed in NBs are inherited through GMCs. However, it is possible to speculate that Klu, Slp and D regulate expression of unidentified transcription factors in NBs that are inherited from NBs to neurons through GMCs (Suzuki, 2013).

Effects of mutation or deletion

klumpfuss mutants show loss of bristles at some positions and fusion of tarsal segments. The strongest alleles are semilethal when homozygous, some animals developing to adulthood but dying shortly after hatching. Among the mutant larvae defects are detected in the mouth-hooks, where some teeth are missing and in the larval brain, the morphology of which is obviously abnormal. A number of macrochaetae are missing in head and thorax, particularly from the anterior margin of the wing, the wing veins, antenna and legs of homozygotes. Non-innervated bristles at the margin of the alula are also affected. The results suggest that in some positions klu is require in order for epidermal cells to develop as sensory organ precursors and, in other positions, for proper differentiation of the progeny cells (Klein, 1997).

The distal regions of the leg segments are preferentially affected. For all three leg pairs in homozygotes for hypomorphic alleles, tarsal segments 3-5 are fused together, as well as the fusion of trochanter and femur. For more severe alleles, the defects are stronger. It is thought that klu is not involved in the proximal-distal pattern formation of the leg disc; rather it is required for the differention of the distal tarsal region (Klein, 1997).

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

Klu is expressed in the NB4-2 lineage only after two rounds of NB cell division, that is, in the second born GMC (GMC4-2b). In loss-of-function mutant embryos, the first born GMC (GMC4-2a) as well as its progeny neurons are duplicated; this duplication of the GMC4-2a sublineage arises because GMC4-2b adopts the identity of GMC4-2a and divides to produce the GMC4-2a progeny. Moreover, when Klu is ectopically expressed in GMC4-2a, it fails to acquire its normal identity and fails to produce correctly specified progeny. klu therefore acts to specify the identity of GMC4-2b and to make it distinct from GMC4-2a. These findings further suggest that the determination of GMC cell fate occurs in two steps; the initial GMC identity is the consequence of inheritance from the maternal NB, however, the subsequent stabilization of this identity requires functions like klu in the GMC (Yang, 1997).

The neuropeptide gene hugin is altered in klu mutants and hug itself is regulated by food signals

Feeding is a fundamental activity of all animals that can be regulated by internal energy status or external sensory signals. A zinc finger transcription factor, klumpfuss, is required for food intake in Drosophila larvae. Microarray analysis indicates that expression of the neuropeptide gene hugin (hug) in the brain is altered in klu mutants and that hug itself is regulated by food signals. Neuroanatomical analysis demonstrates that hug-expressing neurons project axons to the pharyngeal muscles, to the central neuroendocrine organ, and to the higher brain centers, whereas hug dendrites are innervated by external gustatory receptor-expressing neurons, as well as by internal pharyngeal chemosensory organs. The use of tetanus toxin to block synaptic transmission of hug neurons results in alteration of food intake initiation, which is dependent on previous nutrient condition. These results provide evidence that hug neurons function within a neural circuit that modulates taste-mediated feeding behavior (Melcher, 2005).

In a screen for Drosophila mutant larvae defective in feeding, the P-element line P(9036) was identified. These animals fail to pump food from the pharynx into the esophagus; this is not due to a morphological block in the esophagus. The failure to feed is also not due to a general illness of the animal or global locomotory defects, because they can move around with the same vigor as wild-type or heterozygote siblings. P(9036) larvae also display wandering-like behavior, in which they move away from the food. During this wandering-like phase, P(9036) larvae move about with food lodged in their pharynx, further supporting the view that the feeding defect is not due to a general body movement defect. Wandering behavior is observed in wild-type larvae when they stop feeding and move away from food shortly before pupariation. These feeding behavior defects have also been observed for pumpless (ppl) mutants. ppl encodes an amino acid catabolizing enzyme that is expressed exclusively in the fat body, an organ analogous to the vertebrate liver. Thus, P(9036) and ppl mutants, as immature first instar larvae, display feeding behaviors characteristic of sated, full-grown, third instar larvae. The gene corresponding to P(9036) was characterized and found to be klu, a zinc finger protein-encoding gene that is expressed specifically in the developing nervous system. P(9036) fails to complement the lethality of all klu alleles tested, and trans-heterozygotes also show the characteristic feeding defect (Melcher, 2005).

To study the central control process that could underlie the feeding defect of klu mutants, microarray analysis of klu mutants was performed with a focus on neuropeptide genes. It was reasoned that their expression patterns in the brain would be specific enough for analysis at single-cell resolution. Furthermore, neuropeptides have been shown to influence food intake in different organisms, including mammals. RNA from klu mutant larvae and wild-type larvae were isolated and hybridized to three Affymetrix chips each and compared. In situ hybridizations were performed on wild-type larval brains with the six highest upregulated genes. Efforts were focused on hug because it had the most specific expression pattern in the larval brain. While all others showed staining in different parts of the brain or in the ventral nerve cord (VNC), hug showed staining in only a cluster of about 20 cells in the subesophageal ganglion (SOG) of the larval brain, with no staining anywhere else. hug expression in embryos is also highly restricted in the brain. hug encodes a prepropeptide capable of generating at least two neuropeptides, Drm-PK2 and hug-γ. The former encodes a myostimulatory peptide while the latter shows homology to ecdysis-triggering hormone-1, ETH-1. Both can activate a G-protein-coupled receptor belonging to the vertebrate neuromedin U group. A hug homolog is also found in Anopheles gambiae (Melcher, 2005).

To confirm the microarray data, semi-quantitative in situ hybridization was performed in wild-type and klu mutant larval brains. hug is upregulated in klu mutants. Whether hug expression is also regulated in ppl larvae, which display a similar feeding defect as klu, was examined. There is also an upregulation of hug in ppl mutants. Whether hug expression is regulated by different nutrient signals was examined. Wild-type larvae were placed in starvation and sugar-rich conditions (that is, both being amino acid-deficient diets) and hug expression was monitored. hug was downregulated in both conditions, indicating a response to nutrient signals distinct from simple lack of energy. Since hug is upregulated in klu and in ppl mutants, both of which do not feed and wander about, a higher hug level correlates with decrease of food intake and food-seeking behavior. Under starvation and sugar conditions, a lower hug level correlates with increased food-seeking behavior, since larvae become hyperactive and disperse when food is removed (Melcher, 2005).

Klumpfuss is involved in the determination of sensory organ precursors in Drosophila

The neural precursor cells [sensory organ precursor cell (SOP)] of the external sense organs of Drosophila arise from proneural clusters, which are defined through the expression pattern of proneural genes such as the genes of the achaete-scute complex (AS-C). The activities of these genes enable each cell within a cluster to become the SOP. A selection process mediated by the Notch signalling pathway and Extramacrochaetae selects a defined number of cells within the proneural cluster to realise the SOP fate, while it redirects the rest to the epidermoblast fate. This study reports a new function required for SOP determination mediated by the zinc finger transcription factor Klumpfuss (Klu). Klu participates in a novel mechanism that appears to regulate the expression as well as the activity of the proneural proteins. This analysis indicates that Klu is a repressor of transcription, which acts via a double-negative loop to promote SOP formation: it suppresses the expression of an unidentified antagonist of proneural activity. A detailed structure function analysis is reported that identifies functionally important domains within Klu (Kaspar, 2008).

The determination of the SOP of the mechano-sensory bristles of Drosophila is a well-known model system for studying neural development. Yet, despite of the extensive knowledge about the process, it is still not completely clear how the SOP is determined within a proneural cluster. This is substantiated by recent data that indicate that several mechanisms that are required are still unknown: for example, an unidentified NFkappaB-like-factor appears to be required for the determination of the SOP in addition to the previously known components. Furthermore, the zinc finger protein Charlatan (Chn) is involved in a novel layer of regulation that enhances the general expression of ac and sc during the development of macrochaetae (Kaspar, 2008).

This study shows that Klu is a novel factor required for SOP formation. Over-expression of Klu results in formation of supernumerary bristles, whereas loss of its function leads to loss of bristles due to the lack of determination of the corresponding SOP. Although this study has focussed on the development of the large macrochaetae of the notum, which are experimentally accessible, Klu is also thought to be required for the development of other types of bristles, since it has been previously shown that bristles are missing in several regions of the body of klu mutant flies. Furthermore previous analysis showed that bristles and their corresponding SOPs are absent in the proximal wing. Thus, it is likely that klu has a similar role in the development of bristles at many regions of the fly body (Kaspar, 2008).

Expression of DN-Klu results in stronger bristles loss than loss of its function. The bristle loss can be suppressed by co-expression of wild-type Klu indicating that the effect of DN-Klu is specific. One explanation for the stronger phenotypes of DN-Klu is that Klu might interact with other proteins in a transcription complex and expression of DN-Klu, which is probably unable to bind to DNA, titrates out a binding partner from this complex. In agreement with this notion is the observation that gro, which encodes a co-repressor, genetically interacts with klu. Whether Klu might titrate out Gro by is currently being tested by determining whether these proteins interact physically (Kaspar, 2008).

The ability of the Klu to induce SOP development is largely dependent on the proneural genes, such as Ac and Sc. This analysis suggests that the Klu appears to enhance the activity as well as expression of these proneural bHLH factors. Since Klu acts as a repressor of transcription, it must promote SOP development indirectly through the repression of an antagonist (or several antagonists), which suppresses expression as well as the activity of the proneural genes. The analysis allows some conclusions to be drawn about the mode of action of the antagonist to regulate the activity: EE4-lacZ consists of a basal promoter and four tandem repeats of the E boxes—the DNA target sequences for proneural factors of the bHLH family. The fact that expression of Klu promotes and expression of dominant negative Klu (HA-DNklu) suppresses expression of EE4-lacZ indicates that the antagonist can regulate the expression of EE4-lacZ. Assuming a direct regulation, it could be achieved either by binding of the antagonist to the E-boxes of EE4-lacZ or by direct interaction with the proneural proteins. Interestingly, both modes of actions have been recently demonstrated for the proteins of the E(spl)-C, which act as transcriptional repressors as well as regulators of the activity of the proneural proteins (Kaspar, 2008).

There are two obvious known candidates for the antagonist: the Notch signalling pathway, which activates the genes of the E(spl)-C and Emc. HA-DNklu was found to prevent SOP development in Psn mutant wing imaginal discs, where the expression of the genes of the E(spl)-C is absent. Furthermore, over-expression of Klu does not affect the expression of E(spl)m8. These results do not suggest that the proteins of the E(spl)-C are the antagonists. Likewise, the expression pattern of emc was unchanged upon Klu over-expression. No genetic interactions was detected between klu and emc during bristle development. These results and the fact that Emc is probably involved only in macrochaete development make it an unlikely candidate. A third candidate is hairy (h), another known antagonist of the activity of proneural genes. It is possible that Klu suppresses the activity of h in order to promote bristle development. This possibility is thought unlikely for the following reasons: 1. Loss of h function only affects the development of the microchaete, but not macrochaete, while Klu affects the development of both types. 2. The expression of h during the third larval instar stage in the notum is largely restricted to a stripe-like domain that is only partially overlapping with and much smaller than that of klu in third instar wing imaginal discs. Klu promotes SOP development at many locations outside the h domain. 3. No change was observed in expression of Hairy upon over-expression of Klu in third larval instar discs in anti-Hairy antibody staining. 4. Hairy suppresses SOP development by suppressing the expression of ac. However, the data indicate that Klu can enhance the activity of several proneural factors and, as discussed above, evidence suggests that the antagonist acts on the post-translational level. Considering these facts, it is unlikely that Klu suppresses the expression of h to promote SOP development. Thus, the antagonist remains to be identified and it is possible a so far unknown factor (Kaspar, 2008).

The fact that the Klu acts as a repressor of transcription that promotes SOP development through the repression of an unidentified antagonist of proneural activity, has some interesting implications for the state of the imaginal disc cells: The finding that expression of Enklu (dominant negative Klu) can force probably all wing imaginal disc cells to choose the SOP fate, suggests that the antagonist regulated by Klu must be present and active in all disc cells to prevent the neural fate. Thus, the results suggest that the default state of wing imaginal disc cells might be 'proneural' and must be actively hindered from realising this fate (Kaspar, 2008).

An interesting question is how over-expression of Klu can induce residual SOP formation in the absence of Ac/Sc or Da activity. In the case of ac/sc mutants (sc10.1 mutants), a weak proneural activity is obvious since they have a few bristles and weak expression of sca can be observed in their imaginal discs. This weak activity can be generated by various means: It has been recently shown that Da can induce SOP development in the absence of the proneural proteins of the AS-C or Sens if over-expressed. Furthermore, it has been shown that Da can form homodimers in vitro, which can bind to DNA target sequences, albeit with a weaker affinity than Da/Sc or Da/Ac heterodimers. This suggests that Da, which is ubiquitously expressed, can provide a weak proneural activity, even if it is not able to heterodimerise with proneural bHLH factors, probably by forming homodimers. This weak activity is not sufficient to induce SOP formation if Da is expressed at endogenous levels in ac/sc double mutants. Alternatively Da can dimerise with a non-bHLH factor to provide weak proneural activity in ac/sc double mutants. In this respect it is important that recent work has demonstrated that Da can dimerise also with the zinc finger protein Sens, a non-bHLH protein, to provide the proneural activity required for bristles development in the wing. This analysis shows that over-expression of Klu can enhance proneural activity. Thus, it is believed that over-expression of Klu enhances the weak proneural activity either provided by Da homo- or unknown heterodimers sufficiently to allow the formation of a few additional bristles in ac/sc double mutants (Kaspar, 2008).

Following a similar line of arguments, enhancement of a weak proneural activity provided by Ac or Sc homo-or heterodimers through Klu over-expression could explain the SOP formation observed in da cells. Indeed a residual proneural activity in da mutant embryos has been reported. Since only Hnt, which is also expressed in other cell types such as tracheal cells, was used as SOP marker in these experiments it is also possible that the observed in Hnt-positive cells in da clones have switched their fate. However, such a transformation was never observed in any of the other experiments where Klu was over-expressed: Hnt-positive cells were always also positive for other SOP markers tested (Kaspar, 2008).

A related question is why Klu can induce bristle development at ectopic sites such as the mesopleura, head and scutellum upon over-expression. Previous studies of the expression domains of the proneural genes ac and sc in the wing imaginal discs revealed that they are also expressed in regions where no SOP arises, sometimes at high levels. This is at least in part due to the high expression of Emc in these regions. It is believed that over-expression of Klu enhances the weak activity of the proneural proteins in these regions to a level sufficient to initiate bristle development. Indeed, bristles were found developing at the similar ectopic position in strong emc mutants as upon klu over-expression. Furthermore, over-expression of Klu in proneural areas with scaGal4 (a target of proneural factors) results in development of bristles at many ectopic sites of the head and thorax (e. g. the head) (Kaspar, 2008).

Furthermore, this study found that sca-lacZ, is weakly expressed in the notum in areas between the proneural clusters, suggesting that weak proneural activity is also present outside the proneural clusters. Indeed, the SOP of the pSA macrochaete is arising from such an area where no cluster is observed. In summary, these observations indicate that proneural activity exists in many regions of the fly body where bristles are normally absent and it is believed that over-expression of Klu enhances this weak activity sufficiently to allow formation of bristles there. Because of the existing positive feedback loop between proneural activity and expression of proneural genes, this enhancement leads to the observed weak ectopic expression of ac and sc, which further supports formation of ectopic bristles (Kaspar, 2008).

FACS purification and transcriptome analysis of drosophila neural stem cells reveals a role for Klumpfuss in self-renewal

Drosophila neuroblasts (NBs) have emerged as a model for stem cell biology that is ideal for genetic analysis but is limited by the lack of cell-type-specific gene expression data. This study describes a method for isolating large numbers of pure NBs and differentiating neurons that retain both cell-cycle and lineage characteristics. Transcriptional profiles were determined by mRNA sequencing and identify predicted NB-specific transcription factors were identified that can be arranged in a network containing hubs for Notch signaling, growth control, and chromatin regulation. Overexpression and RNA interference for these factors identify Klumpfuss as a regulator of self-renewal. Loss of Klumpfuss function causes premature differentiation, and overexpression results in the formation of transplantable brain tumors. The data represent a valuable resource for investigating Drosophila developmental neurobiology, and the described method can be applied to other invertebrate stem cell lineages as well (Berger, 2012).

Unlike their differentiating sibling cells, NBs regrow to their original size after each division and maintain their identity over many cell divisions. Therefore, NBs must express a regulatory transcriptional network that is highly robust over time but can be rapidly and irreversibly modified by Numb, Pros, and Brat. Previous loss-of-function experiments revealed a surprising level of redundancy among the known TFs acting in NBs (San-Juán, 2011; Zacharioudaki, 2012). Therefore, ther transcriptome data was used to identify a complete set of predicted TFs that are highly expressed in NBs and strongly downregulated during differentiation. In total, 28 TFs matched the criteria. Assuming that functionally related TFs are more likely to be coexpressed, stage- and tissue-specific microarray data was used to infer putative regulatory interactions (Berger, 2012).

The resulting hypothetical network contains six hubs that are connected to more than five genes in the network. The first hub is HLHmγ, a direct nuclear target of the Notch signaling pathway. HLHmγ connects to Dpn, and both were shown to act redundantly in controlling NB self-renewal (Zacharioudaki, 2012). It also connects to Wor and Klu, which have been linked to Notch signaling by both coexpression and functional studies. Finally, it connects to Grh, which can regulate proliferation in NBs. Surprisingly, Grh is actually a negative regulator of HLHmγ, but it has been proposed that such paradoxical elements are frequent components of transcriptional circuits and can maintain homeostatic concentrations or contribute to robust regulation (Berger, 2012).

HLHmγ and Wor are connected to two other hubs that consist of genes involved in ribosome biogenesis and growth control. Mod is the Drosophila homolog of Nucleolin, the major nucleolar protein of growing eukaryotic cells that is thought to play a role in rRNA transcription and ribosome assembly. CG10565 is the fly ortholog of MPP11, a chaperone of the DNA-J family that is involved in ribosome assembly and has been implicated in the regulation of cell growth. In NBs, CG10565 connects to Bigmax and TFAM, two factors that may be involved in growth control because they are direct downstream targets of the major growth regulators Myc and Max. The fourth interesting hub is Ssrp, a chromatin regulator that has been identified based on its RNAi overproliferation phenotype. Surprisingly, Ssrp RNAi causes gain rather than loss of NBs, and may therefore maintain a chromatin state that allows differentiation. Finally, Ken and Coop have been implicated in Jak/STAT and wingless signaling, respectively. Both of these pathways are linked to Notch signaling, although no such functional link has been described in NBs. Thus, this hypothetical transcriptional network provides potential explanations for several aspects of NB biology. For example, it could explain the direct effect of Notch signaling on cell growth that has been described in Drosophila NBs (Berger, 2012).

Previous studies of Drosophila NBs have provided important insights into the mechanism of asymmetric cell division. Despite this progress, however, the transcriptional circuits that control self-renewal and act downstream of segregating determinants have remained elusive. Functional redundancy has already been demonstrated for some TFs acting in NBs and may have hindered the genetic identification of others. These transcriptional data lay the foundation for a more targeted search for functional interconnections between redundantly acting factors. The hypothetical transcriptional network this study has identified provides testable hypotheses for how Notch signaling might connect to the machinery for growth regulation. It suggests that Notch target genes control other TFs that regulate ribosome biogenesis and assembly in a surprisingly direct manner. With the use of this established method to purify larval NBs, and the development of new techniques for chromatin immunoprecipitation even from limited numbers of cells, it will become technically feasible to test the proposed network. The ease with which individual TFs can be removed by RNAi may ultimately result in an understanding of the architecture of this network in unprecedented detail (Berger, 2012).

klumpfuss distinguishes stem cells from progenitor cells during asymmetric neuroblast division

Asymmetric stem cell division balances maintenance of the stem cell pool and generation of diverse cell types by simultaneously allowing one daughter progeny to maintain a stem cell fate and its sibling to acquire a progenitor cell identity. A progenitor cell possesses restricted developmental potential, and defects in the regulation of progenitor cell potential can directly impinge on the maintenance of homeostasis and contribute to tumor initiation. Despite their importance, the molecular mechanisms underlying the precise regulation of restricted developmental potential in progenitor cells remain largely unknown. This study used the type II neural stem cell (neuroblast) lineage in Drosophila larval brain as a genetic model system to investigate how an intermediate neural progenitor (INP) cell acquires restricted developmental potential. The transcription factor Klumpfuss (Klu) was identified as distinguishing a type II neuroblast from an INP in larval brains. klu functions to maintain the identity of type II neuroblasts, and klu mutant larval brains show progressive loss of type II neuroblasts due to premature differentiation. Consistently, Klu protein is detected in type II neuroblasts but is undetectable in immature INPs. Misexpression of klu triggers immature INPs to revert to type II neuroblasts. In larval brains lacking brain tumor function or exhibiting constitutively activated Notch signaling, removal of klu function prevents the reversion of immature INPs. These results led to a proposal that multiple mechanisms converge to exert precise control of klu and distinguish a progenitor cell from its sibling stem cell during asymmetric neuroblast division (Xiao, 2012).

Asymmetric stem cell division provides an efficient mechanism to preserve a steady stem cell pool while generating differentiated progeny within the tissue where the stem cells reside. Precise spatial control of the stem cell determinants inherited by both sibling cells in every asymmetric cell division ensures that a daughter cell maintains the stem cell characteristics while the sibling progeny acquires the progenitor cell identity. In mitotic type II neuroblasts, the basal proteins Brat and Numb segregate into immature INPs and are required for the formation of INPs. This study significantly extends the findings from previous studies and showed that Brat and Numb function in immature INPs to prevent them from acquiring a neuroblast fate while promoting the INP identity. Identification and characterization of the klu gene led to a proposal that Brat and Numb converge to exert precise control of Klu to distinguish an immature INP from its sibling type II neuroblast. Numb also prevents a GMC from reverting to a type I neuroblast by inhibiting Notch signaling in the type I neuroblast lineage. Interestingly, although overexpression of klu was insufficient to induce supernumerary type I neuroblasts, increased function of klu can drastically enhance the reversion of GMCs to type I neuroblasts in the presence of activated Notch signaling. Thus, it is proposed that aberrant activation of Notch signaling induces reversion of GMCs by activating multiple downstream genes including klu. Together, these data lead to the conclusion that precise regulation of klu by multiple signaling mechanisms distinguishes a progenitor cell from its sibling stem cell during asymmetric stem cell division (Xiao, 2012).

The essential role of Brat and Numb in regulating the formation of INPs is well established, but lack of insight into maturation has hindered investigation into the mechanisms by which these two proteins distinguish an immature INP from its sibling type II neuroblast. A previous study defined immature INPs by the following criteria: (1) being immediately adjacent to the parental type II neuroblast, (2) lacking Dpn expression and (3) displaying a very low level of CycE expression. Based on these criteria, analyses of the spatial expression pattern of various cell fate markers in the type II neuroblast lineage clones in wild-type brains revealed that onset of Ase expression correlates with an intermediate stage of maturation. In 16-hour clones, one type II neuroblast (Dpn+ Ase- CycE+), two to three Ase- immature INPs (Dpn- Ase- CycE-), two to three Ase+ immature INPs (Dpn- Ase+ CycE-) and INPs (Dpn+ Ase+ CycE+) were reproducibly observed. Furthermore, it was shown that Ase- immature INPs maintain expression of the type II neuroblast-specific marker PntP1, whereas Ase+ immature INPs showed virtually undetectable PntP1 expression. Thus, onset of Ase expression should serve as a useful marker for an intermediate stage during maturation (Xiao, 2012).

The data lead to a proposal that Brat distinguishes an immature INP from its sibling type II neuroblast by indirectly antagonizing the function of Klu based on the following evidence. First, Klu was undetectable in Ase immature INPs in the brat single-mutant or brat and numb double-mutant type II neuroblast clones. Thus, a Brat-independent mechanism must exist to downregulate Klu in immature INPs. Second, overexpression of a truncated Brat transgenic protein lacking the NHL domain, which is required for repression of mRNA translation, completely suppresses the formation of supernumerary neuroblasts. Thus, it is unlikely that downregulation of Klu in immature INPs occurs via a Brat-dependent translational repression of klu mRNA. It is proposed that Brat might suppress the expression of a co-factor necessary for the function of Klu, just as WT1 requires co-factors in order to regulate the expression of its target genes in vertebrates (Roberts, 2005). Further investigation will be necessary to discern how Brat establishes restricted developmental potential in immature INPs by antagonizing the function of Klu (Xiao, 2012).

WT1 requires its zinc-finger motifs to regulate transcription of its target genes and can function as an activator or a repressor of transcription in a context-dependent manner (Roberts, 2005). A previous study showed that overexpression of Klu can partially suppress the expression of a lacZ reporter transgene containing the cis-regulatory elements from the even-skipped gene, a putative direct target of Klu, in the fly embryonic central nervous system. Since Klu and WT1 display extensive homology in zinc-fingers 2-4, Klu is likely to recognize a similar DNA binding sequence as WT1. The even-skipped cis-regulatory element contains three putative WT1 binding sites, but nucleotide substitutions in these sites that were predicted to abolish Klu binding failed to render the lacZ reporter transgene unresponsive to overexpression of klu. These data led to a speculation that Klu might recognize a distinct consensus DNA binding sequence to WT1. To test this hypothesis, two UAS-WT1 transgenes were generated that encode the two most prevalent isoforms of the WT1 protein, WT1 −KTS and WT1 +KTS. Interestingly, neither WT1 transgene, when overexpressed by wor-GAL4, triggered the formation of supernumerary type II neuroblasts in larval brain. This is consistent with Klu recognizing a distinct consensus DNA binding sequence to WT1. However, it cannot be ruled out that the inability of the WT1 transgenic protein to induce supernumerary type II neuroblasts is simply due to the absence of necessary co-factors in the fly, as repression of target gene transcription by WT1 requires additional co-factors in vertebrates. More studies will be necessary to elucidate the molecular function of Klu in promoting type II neuroblast identity (Xiao, 2012).

Restricted developmental potential functionally defines progenitor cells and allows them to generate differentiated progeny through limited rounds of cell division without impinging on the homeostatic state of the stem cell pool. Despite their importance, the molecular mechanisms by which progenitor cells acquire restricted developmental potential remain experimentally inaccessible in most stem cell lineages. However, studies from various groups have paved the way for using fly larval brain neuroblast lineages as an in vivo model system for investigating how progenitor cells acquire restricted developmental potential (Xiao, 2012).

This study describes the expression pattern of additional molecular markers that allow unambiguous identification of two distinct populations of immature INPs. Furthermore, experimental evidence is provided strongly suggesting that these two groups of immature INPs possess distinct functional properties. More specifically, Ase- immature INPs readily revert to type II neuroblasts in response to misexpression of Klu, whereas Ase+ immature INPs appear much less responsive to Klu. These data lead to a proposal that the genome in immature INPs becomes reprogrammed during maturation such that these cells become progressively less responsive to neuroblast fate determinants such as Klu. As a consequence, an INP becomes completely unresponsive to Klu following maturation. Further experiments will be required to validate this model in the future (Xiao, 2012).


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klumpfuss: Biological Overview | Evolutionary Homologs | Regulation | Developmental Biology | Effects of Mutation

date revised: 10 August 2014  

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