Targets of Activity

Distinction between color photoreceptor cell fates is controlled by Prospero in Drosophila; Prospero regulates rhodopsin expression

The Drosophila compound eye consists of ~750 independently functioning ommatidia, each containing two photoreceptor subpopulations. The outer photoreceptors participate in motion detection, while the inner photoreceptors contribute to color vision. Although the inner photoreceptors, R7 and R8, terminally differentiate into functionally related cells, they differ in their molecular and morphological makeup. Several aspects of R7 versus R8 cell fate determination are regulated by the transcription factor Prospero (Pros). pros is specifically expressed in R7 cells, and R7 cells mutant for pros derepress R8 rhodopsins, lose R7 rhodopsins and acquire an R8-like morphology. This suggests that R7 inner photoreceptor cell fate is acquired from a default R8-like fate that is regulated, in part, via the direct transcriptional repression of R8 rhodopsins in R7 cells. Furthermore, this study provides transcriptional targets for pros that may lend insight into its role in regulating neuronal development in flies and vertebrates (Cook, 2003).

Photoreceptors absorb light with Rhodopsins (Rhs), the pigment proteins that are present in the stack of membranes that form the rhabdomeres. Several rhodopsin gene (rh) products exist with varying absorption spectra to ensure a broad range of light sensitivity. Outer PRs all express Rh1, while R7 cells express either Rh3 or Rh4 (UV-sensitive opsins), and R8 cells primarily express blue-sensitive Rh5 or green-sensitive Rh6. Inner PR rh expression is coordinately regulated within an ommatidium with Rh3-expressing R7 cells associated with underlying Rh5-expressing R8 cells (termed 'pale' ommatidia), and Rh4-expressing R7 cells associated with underlying Rh6-expressing R8 cells ('yellow' ommatidia). One exception to this is found in the two dorsal-most rows of ommatidia, the dorsal rim area (DRA), in which Rh3 is expressed in both R7 and R8 cells. The study of rh regulation has revealed that promoters of less than 300 bp can recapitulate endogenous rh expression in vivo, and are organized in a simple bipartite structure: a conserved proximal element (RCSI, rhodopsin conserved sequence I) common to all rh promoters, and upstream rh-specific sequences (RUS, rhodopsin upstream sequences) unique to each rh promoter. This organization predicts that common as well as unique factors function together to dictate rh gene expression, and consequently, to properly specify different ommatidial subtypes (Cook, 2003).

Advantage was taken of the subtype-specific expression of the inner PR rh genes as a tool for understanding later events in PR development. This study reports the identification of a conserved element that is specifically present in the R8 rh promoters, rh5 and rh6, which is responsible for repressing these genes in R7 cells. A yeast one-hybrid screen revealed that this element provides a binding site for Prospero (Pros), an atypical homeodomain transcription factor important for neuronal specification in the developing Drosophila embryo. Pros is specifically expressed in R7 photoreceptors in the adult. Furthermore, genetic studies demonstrate that prospero (pros) is both necessary and sufficient for the repression of R8 rh genes in R7 cells. pros mutant R7 cells not only gain expression of R8 rhodopsins, but also acquire R8-like morphological features and lose R7-specific markers such as Rh3 and Rh4. These findings suggest that once inner PRs are coordinately distinguished from outer PRs through Sal, Pros then helps to further distinguish R7 from R8, with R8 being the ground state of differentiation for inner PRs. Since the vertebrate homolog of prospero, prox1, is also expressed in subsets of retinal cell populations, it is proposed that a role for this transcription factor is evolutionarily conserved to provide cell type diversification in the eye (Cook, 2003).

During eye disc development, R7 cells are specified by three different signaling cascades: Sevenless (Sev), EGFR, and Notch. Previous studies have demonstrated that normal levels of pros expression in R7 cells require Ras pathway activation via EGFR and Sev signaling, as well as Notch activation. These data suggest that pros could be a critical target for inducing R7 differentiation. However, no changes in early R7-specific markers in pros mutant eye discs could be detected, and morphological characteristics such as correct projections to the medulla and rhabdomere positioning on top of the R8 rhabdomere within the ommatidial center are largely unaffected even in the adult retina. The findings reported in this study, however, indicate that while pros mutant R7 cells maintain some R7-specific gene products (e.g., Rh4) and lack R8-specific markers such as Sens, other aspects of R7/R8 differentiation involving R7 nuclear positioning and correct rh gene expression are dramatically affected. It was also found that pros expression was lost in sal mutants, while sal expression remained in pros mutants. These findings suggest that R7 cells acquire their functional identity through several distinct stages. Initially, all eight PRs are recruited at distinct times within the imaginal disc, allowing each to be influenced by a unique cell signaling environment. This recruitment/specification results in the proper establishment of projections to the optic lobes. At some point afterwards, a common pathway involving sal converges onto both the R7 and R8 cells that allows them to continue to develop as inner PRs, rather than adopting an outer PR state. These cells, based on pros loss-of-function experiments, are likely to adopt an R8-like morphology in the absence of additional signals, but must also remain distinct in their expression of cell-specific markers such as pros and sens. In R7 cells, pros helps to promote additional R7-specific characteristics, including the direct repression of R8 rhodopsins and nuclear positioning. Other factors must participate in events leading to their distal positioning in the retina as well as the subtype-specific expression of rh3 and rh4. Similarly, additional factors such as Sens are likely to contribute to equivalent aspects of R8 development. The findings reported in this study for pros, however, are particularly exciting as they provide a genetic inroad for blocking PR differentiation at an intermediate step. Future studies aimed at investigating these later events should be useful for understanding the pathways that transform eight unique cell types into the two functional visual systems in the adult (Cook, 2003).

prospero is critical for neuronal cell specification in the developing central and peripheral nervous system, and is transiently expressed in the nucleus of neuronal precursor cells. Despite extensive work aimed at understanding Pros function, little is known regarding its direct molecular targets. As exit from the cell cycle often precedes terminal differentiation, several studies have correlated the expression of cell-cycle regulators with Pros function. Indeed, the expression of genes such as decapo, string, cyclin E, E2F, and cyclin A is reduced during pros-mediated differentiation, and increased in the absence of pros. However, it has been difficult to assess which genes are directly controlled by Pros, and those whose expression changes as an indirect requirement to exit the cell cycle, as the Pros target sites within these genes are not known (Cook, 2003).

This study identified a functional Prospero target sequence, (T)AAGACG. The only other known Pros binding site, CA/TC/TNNCC/T was identified with a site selection assay (SELEX). There is no significant similarity between the minimal seq56B and the SELEX consensus, but a loose SELEX consensus was found within the full seq56 element CGGCTAAGAC. However, gel shifts with the GGCTAAG sequence showed no binding to Pros-S or Pros-L, consistent with the findings that the 3' end of seq56 is critical for Pros binding; additional gel shifts revealed that both Pros-S and Pros-L could bind weakly to individual SELEX sites, but only when these sites were multimerized was binding similar to that observed with seq56. Thus, it is likely that Pros exhibits some flexibility in its binding specificity, and as additional Pros target sites are identified, a clearer consensus will develop (Cook, 2003).

This observation that is supported by both Pros binding site studies is that Pros is likely to regulate transcription in combination with other factors. For instance, the findings that seq56 is a conserved imperfect palindrome, and that mutations that do not disrupt Pros binding still lead to R7 derepression (e.g., seq56A mutants), imply that the entire seq56 element is necessary to mediate repression. Indeed, although a perfect seq56B binding site was found within the rh4 promoter, no significant changes in Rh4 expression were observed in pros17, rh6[1] mutant eyes, nor in flies misexpressing pros-L in all photoreceptors; furthermore, mutation of this site within the rh4 minimal promoter did not abolish reporter expression in R7 cells. Thus, these data suggest an important role for the 5' end of the seq56 element in mediating repression (Cook, 2003).

Similar to the Drosophila visual system, fate mapping in vertebrates has revealed that all retinal cell types are derived from the same precursor cell population, and that differentiation of these cell types occurs in a stereotyped order. For instance, cone, horizontal, and amacrine cell genesis occurs prior to rod photoreceptor formation, followed by bipolar and Müller glia genesis. Recent findings have demonstrated that Prox1, the vertebrate counterpart of Pros, is embryonically expressed in dividing retinal progenitor cells, and postnatally expressed in differentiating horizontal cells, AII amacrine cells, and at lower levels in bipolar cells. Thus, Prox1 expression in horizontal/amacrine precursors may serve to drive these cells toward terminal differentiation and prevent them from dividing to become later cell types such as PRs. Indeed, in the prox1 knockout mouse, horizontal cells fail to form, while the rod PR number is increased 50%-70%. Therefore, it is likely that Prox1/Pros plays an evolutionarily conserved role in specifying unique neuronal cell types in the eye. While a more precise comparison between vertebrate and invertebrate PRs is difficult, opsin gene expression represents a late differentiation step in all PR development. Thus, it will be interesting to explore the possibility that Prox1/Pros regulate opsin expression throughout evolution. Indeed, preliminary analyses have revealed the presence of putative Pros binding sites within the minimal promoter of the Xenopus rhodopsin promoter. Furthermore, these sites are located precisely in a region demonstrated to function as a repressor element both in cultured cells and in vivo. Thus, it is believed that Pros/Prox1 is a key regulator of both early and late stages of PR development, not only in insects but also in vertebrates (Cook, 2003).

Protein Interactions

Miranda protein can interact with both Prospero and Numb. The regions of Miranda that interact with these two proteins are distinct from each other and are mapped to the central and C-terminal portions of the Miranda protein, respectively (Shen, 1997).

In embryos deficient for miranda, Prospero is not associated with the membrane, but stays in the cytoplasm in prophase. Prospero remains in the cytoplasm in metaphase and anaphase and then is segregated into both daughter cells. Shortly after cell division, Prospero is translocated into the nuclei of both daughter cells. The orientation of the mitotic spindle in neuroblasts and cells of the procephalic neurogenic region is normal in miranda deficient embryos. A transduced miranda can rescue the Prospero localization defects in neuroblasts of miranda indicating that miranda is required for the correct positioning of Prospero in neuroblasts during mitosis (Shen, 1997).

The Numb protein is asymmetrically localized to the basal cell membrane in neuroblasts and cells of the procephalic neurogenic region during mitosis, in contrast to the cytoplasmic distributions of Prospero. After cell division, Numb is segregated into only the basal daughter cell, whereas Prospero is translocated into the nuclei of both cells (Shen, 1997).

The asymmetric localization of Miranda in neuroblasts of prospero and numb mutants is indistinguishable from that of wild-type embryos. Therefore, the asymmetric localization of Miranda does not require prospero or numb. In embryos homozygous for a null allele of inscuteable, both Miranda and Prospero are unable to form crescents or they form crescents that are randomly localized along the cell membrane. Therefore Miranda crescent formation and localization requires inscuteable (Shen, 1997).

In the GMC, Prospero translocates to the nucleus, where it establishes differential gene expression between sibling cells. miranda, which encodes a new protein that co-localizes with Prospero in mitotic neuroblasts, tethers Prospero to the basal cortex of mitotic neuroblasts, directing Prospero into the GMC, and releases Prospero from the cell cortex within GMCs. miranda thus creates intrinsic differences between sibling cells by mediating the asymmetric segregation of a transcription factor into only one daughter cell during neural stem-cell division. The expression of even-skipped was followed in embryos mutant for six miranda alleles. A stereotyped pattern of GMCs and neurons express eve. The well characterized aCC/pCC, RP2, CQ, U, and EL neurons all express eve. In prospero mutant embryos, the aCC/pCC and RP2 neurons fail to express eve and most U and CQ neurons also fail to express eve. It was expected that all miranda alleles would show reduced Prospero activity in the GMC either because Prospero inappropriately segregates into both neuroblasts and GMCs, or because Prospero fails to translocate efficiently into the nuclei of GMCs. This predicts that the EVE CNS phenotype of miranda mutant embryos might resemble the Eve CNS phenotype of prospero mutant embryos, should miranda exert its effect through its ability to bind, segregate and release Prospero. This is the case for two catagories of miranda mutants. For the five alleles in which Prospero falls off the cortex (the inner surface of the cell membrane) of neuroblasts, there is an observed reduction of about one-half, in the number of RP2, aCC/pCC, U and CQ neurons expressing eve. Consistent with a decrease in the level of Prospero protein distributed into GMCs, this phenotype resembles a weak prospero phenotype. A one-half reduction in the number of eve-expressing EL neurons is observed; in prospero mutants all EL neurons form normally. This additional eve phenotype may result from the ectopic expression of Pros in neuroblasts or from defects in the partition of other factors dependent on Miranda function. This study raises some interesting questions. Miranda is itself asymmetrically localized: (1) what proteins tether it to the basal cortex of neuroblasts? (2) What proteins regulate miranda so that it releases Prospero in the GMC once cytokinesis is complete? (Ikeshima-Kataoka, 1997).

Inscutable and Prospero interact physically. The C-terminal 108 amino acids of Insc are sufficient to confer an interaction with Staufen, while other residues of Insc appear to inhibit the interaction mediated by the C-terminal 108 amino acids. The C-terminal region (residues 769-1026) of Stau confers this specific interaction. Both Staufen and Inscuteable proteins are cortically localized in the apex of neuroblasts; the apical localization of Staufen protein requires the presence of Inscuteable (Li, 1997).

Cellular diversity in the Drosophila central nervous system is generated through a series of asymmetric cell divisions in which one progenitor produces two daughter cells with distinct fates. Asymmetric basal cortical localization and segregation of the determinant Prospero during neuroblast cell divisions play a crucial role in effecting distinct cell fates for the progeny sibling neuroblast and ganglion mother cell. Similarly asymmetric localization and segregation of the determinant Numb during ganglion mother cell divisions ensures that the progeny sibling neurons attain distinct fates. The most upstream component identified so far which acts to organize both neuroblast and ganglion mother cell asymmetric divisions, is encoded by inscuteable. The Inscuteable protein is itself asymmetrically localized to the apical cell cortex and is required both for the basal localization of the cell fate determinants during mitosis and for the orientation of the mitotic spindle along the apical/basal axis. The functional domains of Inscuteable have been defined. Amino acids 252-578 appear sufficient to effect all aspects of its function, however, the precise requirements for its various functions differ. The region aa288-497 is necessary and sufficient for apical cortical localization and for mitotic spindle (re)orientation along the apical/basal axis. A larger region (aa288-540) is necessary and sufficient for asymmetric Numb localization and segregation; however, correct localization of Miranda and Prospero requires additional sequences from aa540-578. The requirement for the resolution of distinct sibling neuronal fates appears to coincide with the region necessary and sufficient for Numb localization (aa288-540). These data suggest that apical localization of the Inscuteable protein is a necessary prerequisite for all other aspects of its function. Although Inscuteable RNA is normally apically localized, RNA localization is not required for protein localization or any aspects of inscuteable function (Tio, 1999).

Paradoxically, Staufen is required for the basal localization of Prospero mRNA during mitosis. Prospero mRNA is localized to the apical cortex during interphase, however the change in PROS mRNA localization from the apical cortex at interpahse to the basal cortex at prophase fails to occur in animals that lack zygotic staufen. In staufen and inscuteable mutant neuroblasts, the PROS mRNA remains primarily on the apical cortex during mitosis, indicating that the apical cortical localization of PROS mRNA during interphase requires neither insc nor stau function. However, the basal cortical relocalization that takes place at prophase requires both insc and stau function. Since staufen mutation fails to affect either Inscuteable protein localization or mitotic spindle orientation in neuroblasts, it is concluded that stau acts downstream of inscuteable (Li, 1997).

Staufen binds to the 3' untranslated regions of Prospero mRNA, suggesting that Staufen's role in Prospero mRNA redistribution is mediated through this interaction. How then does Prospero mRNA get to the basal cortex during mitosis? It is assumed that the Inscuteable/Staufen-independent mechanism that operates to effect localization of PROS mRNA to the apical cortex during interphase is normally overridden by the Insc/Stau-mediated process during mitosis. There is strong evidence that suggests that during early development specific signals localized to the 3' UTR of Bicoid mRNA can recruit Staufen to form ribonucleoprotein particles that are subsequently transported in a process that requires intact microtubules (Ferrandon, 1994). It is therefore appealing to suggest that Stau might play a similar role in the neuroblast to transport PROS mRNA from the apical to basal cortex in the transition between interphase and mitosis. With respect to PROS mRNA localization, the role of Insc may be to facilitate Stau protein/PROS mRNA interaction with perhaps other components necessary for the transport of PROS mRNA (Li, 1997).

The Drosophila central nervous system develops from stem cell like precursors called neuroblasts, which divide unequally to bud off a series of smaller daughter cells, called ganglion mother cells. Neuroblasts show cell-cycle-specific asymmetric localization of both RNA and proteins: at late interphase, Prospero mRNA and Inscuteable, Prospero and Staufen proteins are all apically localized; at mitosis, Inscuteable protein remains apical, whereas Prospero mRNA, Prospero protein and Staufen protein form basal cortical crescents. In vitro culture of neuroblasts was used to investigate the role of intrinsic and extrinsic cues and the cytoskeleton in asymmetric localization of Inscuteable, Prospero and Staufen proteins. Neuroblast cytokinesis is normal in vitro, producing a larger neuroblast and a smaller ganglion mother cell. Apical localization of Inscuteable, Prospero and Staufen in interphase neuroblasts is reduced or eliminated in vitro, but all three proteins are localized normally during mitosis (apical Inscuteable, basal Prospero and Staufen). Microfilament inhibitors result in delocalization of all three proteins. Inscuteable becomes uniform at the cortex, whereas Prospero and Staufen become cytoplasmic; inhibitor washout leads to recovery of microfilaments and asymmetric localization of all three proteins. Microtubule disruption has no effect on protein localization, but disruption of both microtubules and microfilaments results in cytoplasmic localization of Inscuteable. It is concluded that both extrinsic and intrinsic cues regulate protein localization in neuroblasts. Microfilaments, but not microtubules, are essential for asymmetric protein anchoring (and possibly localization) in mitotic neuroblasts. These results highlight the similarity between Drosophila, Caenorhabditis elegans, vertebrates, plants and yeast: in all organisms, asymmetric protein or RNA localization and/or anchoring requires microfilaments (Broadus, 1997).

The generation of cellular diversity is essential in embryogenesis, especially in the central nervous system. During neurogenesis, cell interactions or asymmetric protein localization during mitosis can generate daughter cells with different fates. The asymmetric localization of a messenger RNA and an RNA-binding protein is described that creates molecular and developmental differences between Drosophila neural precursors (neuroblasts) and their daughter cells, ganglion mother cells (GMCs). The Prospero (Pros) mRNA and the RNA-binding protein Staufen (Stau) are asymmetrically localized in mitotic neuroblasts and are specifically partitioned into the GMC, as is Pros protein. Stau is required for localization of Pros RNA but not of Pros protein. Loss of localization of Stau or of Pros RNA alters GMC development, but only in embryos with reduced levels of Pros protein, suggesting that Pros mRNA and Pros protein act redundantly to specify GMC fate. GMCs do not transcribe the pros gene, showing that inheritance of Pros mRNA and/or Pros protein from the neuroblast is essential for GMC specification (Broadus, 1998).

An important question in cellular and developmental biology is how a cell divides to produce daughter cells with different fates. Drosophila neuroblasts are a model system for studying asymmetric cell division: at each division, neuroblasts retain stem cell-like features, whereas their sibling ganglion mother cell (GMC) has a more restricted fate. Establishing neuroblast/GMC differences involves the asymmetric localization of proteins (Inscuteable, Miranda, Prospero, and Staufen) and RNA (Prospero). All of these factors are apically localized during interphase, and all except Inscuteable move to the basal cortex at mitosis prior to being partitioned solely into the GMC. Miranda is colocalized with Staufen and Prospero in neuroblasts, and is required for the asymmetric cortical localization of both proteins. Analysis of miranda mutants reveals three functional domains within the Miranda protein: (1) an N-terminal domain (1-290 aa) sufficient for association of Miranda with the cell cortex and basal localization in mitotic neuroblasts; (2) a central domain (446-727 aa) necessary for apical localization in interphase neuroblasts as well as for 'cargo binding' of Prospero, Staufen, and Prospero mRNA, and (3) a C-terminal domain (727-830 aa) necessary for the timely degradation of Miranda and release of its cargo from the cortex of the newborn GMC. In addition, Miranda is asymmetrically localized in epithelial cells that lack Inscuteable and divide symmetrically; thus the mechanism regulating Miranda localization is common to epithelial cells and neuroblasts, and Inscuteable is not an obligate component. A C-terminal domain of Staufen is defined that is sufficient for Miranda-dependent cortical localization in neuroblasts (Fuerstenberg, 1998).

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

The relevance of functional interactions between Prospero and homeodomain proteins is supported by the observation that Prospero, together with the homeodomain protein Deformed, is required for proper regulation of a Deformed-dependent neural-specific transcriptional enhancer. Deformed and mouse Hoxa-5 binding to this neuronal enhancer is increased more than 10 fold by Pros. Pros reduces Eve's DNA binding to this enhancer, but does not modulate the binding of Engrailed. This interaction is unidirectional and specific, since neither Dfd, Eve nor En has an effect on Pros binding. The modulation by Pros does not require Pros binding to DNA. Pros protein modifies the trypsin sensitivity of Dfd protein, suggesting that Pros binds Dfd and is able to induce a conformation change in Dfd. Nevertheless, Pros is able to bind the Dfd neuronal autoregulatory enhancer and enhances Dfd binding to this DNA sequence. The DNA-binding and homeodomain protein-interacting activities of Prospero are localized to its highly conserved C-terminal region, and the two regulatory capacities are independent (Hassan, 1997).

Neuroblasts undergo asymmetric stem cell divisions to generate a series of ganglion mother cells (GMCs). During these divisions, the cell fate determinant Prospero is asymmetrically partitioned to the GMC by Miranda protein, which tethers it to the basal cortex of the dividing neuroblast. Interestingly, Prospero mRNA is similarly segregated by the dsRNA binding protein, Staufen. Staufen interacts in vivo with a segment of the Prospero 3' UTR. To assay RNA binding in vivo, the Prospero 3' UTR was injected into embryos expressing a green fluorescent protein (GFP)-Staufen protein fusion and the formation of Staufen ribonucleoprotein particles (RNP) was monitored. The full-length Prospero 3' UTR forms particles, as does the Bicoid 3' UTR, but not the coding region of the Prospero mRNA even though it is able to form an extended secondary structure. These RNPs are associated with the nuclei of the precellular embryo, and move with them to the cortex at stage 4. However, unlike the RNP particles formed between Staufen and the Bcd 3' UTR, the Staufen/Prospero 3' UTR particles do not associate with the astral microtubules. Similar results are observed when the Prospero 3' UTR is injected into embryos expressing wild-type Staufen (detected with anti-Staufen antibodies), rather than a GFP fusion. To further map the region of the Prospero 3' UTR with which Staufen interacts, either the 3' half of the UTR, or the 5' half were injected into embryos. Whereas the 3' segment recruits Staufen into RNPs within 5-10 min of injection, the 5' segment does so only slightly, if at all, after 20-30 min. Therefore, the region of the Prospero mRNA recognized by Staufen lies in the terminal 650 bases of the mRNA (Schuldt, 1998).

Staufen colocalizes with Prospero protein at all stages of the cell cycle. In embryos, Staufen is concentrated on the apical side of the neuroblast at interphase, then forms a crescent on the basal side of the cell in prophase, where it remains through mitosis before partitioning to the GMC at division. A similar subcellular distribution is seen in living embryos. This dynamic pattern of localization shows Staufen to be correctly placed to bind the Prospero mRNA throughout the cell cycle, and to mediate its segregation into the GMC (Schuldt, 1998)

Miranda colocalizes with Staufen protein and Prospero mRNA during neuroblast divisions, and neither Staufen nor Prospero RNA are localized in miranda mutants. Like Staufen, Miranda concentrates predominantly on the apical side of the cell at interphase. Interestingly, Miranda mRNA is also localized predominantly on the apical side of the neuroblast. Miranda protein then forms a crescent on the basal side of the neuroblast at prophase, where it remains until after cell division. Therefore, the subcellular distribution of Miranda suggests that it might interact with Staufen at all stages of the cell cycle (Schuldt, 1998).

It is concluded that Miranda binds to Prospero protein and to Staufen, which in turn binds Prospero mRNA, to form a complex on the apical side of the neuroblast. The complex may be anchored by Inscuteable at interphase, and then released as the cell cycle progresses. In mirandaRR127, Staufen accumulates on the apical side of the cell, suggesting that Miranda may regulate release from the apical cortex. Miranda, Prospero, Staufen, and Prospero mRNA then move as a group to the basal side of the cell during mitosis, a process that appears to require actin microfilaments. Staufen and Miranda also associate with the apical centrosome, although the significance of this interaction is unclear. Once at the basal cortex, the complex is anchored by factors that have not, as yet, been identified. However, as Miranda acts as the adapter between protein and RNA localization, these factors may be isolated in screens for other Miranda binding proteins. After cytokinesis, Miranda is rapidly degraded in the GMC, and Prospero is released and enters the nucleus. It may be important, therefore, to minimize translation of new Miranda protein in the GMC. Whereas Prospero mRNA is specifically segregated to the GMC, Miranda mRNA remains tightly anchored on the apical side of the neuroblast. By tethering Miranda mRNA in this way, Miranda protein, but not Miranda mRNA, is partitioned to the GMC at cell division (Schuldt, 1998).

Several interesting questions remain to be answered. What regulates the release of Miranda from the apical side of the cell? How are Miranda, Prospero, Staufen, and Prospero mRNA transported to the basal side of the neuroblast? Do they move as a complex, and how are they anchored at the basal cortex? Prospero and Staufen bind to the same region of Miranda, but it is not known whether they bind to the same molecule simultaneously. The answers to these questions may help to elucidate the mechanism of asymmetric protein and RNA localization not only in the nervous system, but also in other tissues, and in other organisms (Schuldt, 1998).

Neuroblasts in the developing Drosophila CNS asymmetrically localize the cell fate determinants Numb and Prospero as well as Prospero RNA to the basal cortex during mitosis. The localization of Miranda to the apical cortex, its interaction with Inscuteable in vitro and its role in localizing several downstream factors suggests that Miranda occupies a central link between Inscuteable at the apical cortex and the localization of Prospero, Staufen, and Prospero RNA to the basal cortex. How, early in mitosis, the apically localized Inscuteable dictates basal localization of intrinsic factors for asymmetric cell division may be elucidated by further studies on the genetic and cell biological mechanisms of the asymmetric localization of Miranda. The localization of Prospero requires the function of inscuteable and miranda, whereas Prospero RNA localization requires inscuteable and staufen function (Shen, 1998).

Miranda forms a crescent on the apical cortex of neuroblasts in late interphase. Later in mitosis, Miranda forms a crescent on the basal neuroblast cortex. Asymmetric localization of both Numb and Prospero has been shown to be dependent on the actin cytoskeleton. The actin dependence of Miranda localization was tested using the actin depolymerizing drug latrunculin A. After treatment of Drosophila embryos with 200 µM latrunculin A for 20 min, asymmetric localization of Miranda is completely disrupted, while membrane association is unperturbed. It is concluded that the asymmetric localization of Miranda during mitosis is an actin-dependent process. All Miranda fragments that contain the amino-terminal 298 amino acids exhibit the same asymmetric localization pattern as wild type Miranda. In contrast, a fragment containing amino acids 114-298 localizes to the cytoplasm and fails to segregate preferentially into the basal daughter cell, as does a fragment containing all residues carboxy-terminal to amino acid 300 (Shen, 1998).

The observation that Miranda protein fragments form an apical crescent that may coincide with the apical Inscuteable crescent led to a test of the possibility that Miranda interacts physically with Inscuteable. In an in vitro binding assay, Inscuteable coprecipitates with Miranda. An Inscuteable fragment from amino acids 252 to 615 also interacts with Miranda (Shen, 1998).

Miranda contains multiple functional domains: an amino-terminal asymmetric localization domain, which interacts with Inscuteable; a central Numb interaction domain, and a more carboxy-terminal Prospero interaction domain. Miranda and Staufen have similar subcellular localization patterns and interact in vitro. miranda function is required for the asymmetric localization of Staufen. Miranda localization is disrupted by the microfilament disrupting agent latrunculin A. These results suggest that Miranda directs the basal cortical localization of multiple molecules, including Staufen and Prospero mRNA, in mitotic neuroblasts in an actin-dependent manner (Shen, 1998).

When neuroblasts divide, Prospero protein and Pros mRNA segregate asymmetrically into the daughter neuroblast and sibling ganglion mother cell. Miranda is known to localize Prospero protein to the basal cell cortex of neuroblasts while the Staufen RNA-binding protein mediates Prospero mRNA localization. miranda is shown to be required for asymmetric Staufen localization in neuroblasts. Miranda thus acts to partition both Prospero protein and mRNA. Furthermore, Miranda localizes Prospero and Staufen to the basolateral cortex in dividing epithelial cells, which express the three proteins prior to neurogenesis. Analyses using miranda mutants reveal that Prospero and Staufen interact with Miranda under the same cell-cycle-dependent control. The wild-type Mira protein localizes predominantly to the cortex in interphase NBs, especially to the apical cortex along with Pros at late interphase. At the onset of prophase, the majority of the wild-type Mira becomes localized to the basal cortex as a crescent, while a fraction of the protein distributes to the apical region in a punctate manner. As the mitotic stage proceeds, an increasing proportion of Mira appears to be incorporated in the basal crescent. While some Mira protein is still observed apically during anaphase, most Mira protein segregates to the basally budding GMC. This pattern of subcellular localization is equally evident using polyclonal and monoclonal antibodies against a C-terminal polypeptide. mira mutations define three distinct functional regions along the mira sequence. The N-terminal 290 amino acids region acts in the basal localization of mira at mitosis in the NB and the epithelial cell. The region between amino acid 447 and 727 includes a domain necessary for the binding with Pros as well as the domain(s) required for the asymmetric localization of Stau in the NB. The C-terminal 103 amino acids region confers the cell cycle dependence on the interaction with Pros/Stau; the absence of this region results in the prolonged association with Pros/Stau during interphase without rapid proteolytic degradation in the GMC and NB. These observations suggest that the epithelial cell and neuroblast (both of epithelial origin) share the same molecular machinery for creating cellular asymmetry (Matsuzaki, 1998).

Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal: Regulation of Prospero basal location

An important question in stem cell biology is how a cell decides to self-renew or differentiate. Drosophila neuroblasts divide asymmetrically to self-renew and generate differentiating progeny called GMCs. The Brain tumor (Brat) translation repressor is partitioned into GMCs via direct interaction with the Miranda scaffolding protein. In brat mutants, another Miranda cargo protein (Prospero) is not partitioned into GMCs, GMCs fail to downregulate neuroblast gene expression, and there is a massive increase in neuroblast numbers. Single neuroblast clones lacking Prospero have a similar phenotype. It is concluded that Brat suppresses neuroblast stem cell self-renewal and promotes neuronal differentiation (Lee, 2006).

The translational repressor Brat directly interacts with the Miranda central domain and is a Miranda cargo protein specifically partitioned into the GMC daughter cell during neuroblast asymmetric cell division. Brat is the first Miranda cargo protein identified since the original finding that Prospero and Staufen were shown to be Miranda cargo proteins over 8 years ago. Prospero is a homeodomain transcriptional repressor, and Staufen is an RNA binding protein that interacts with prospero mRNA. It is unknown whether Miranda has other cargo proteins in addition to Brat, Prospero, and Staufen, and it is unclear whether all three known cargo proteins can associate with a single Miranda protein (Lee, 2006).

It is unknown how Brat promotes Prospero basal localization. A model is favored in which Brat protein stabilizes Prospero/Miranda interactions, so that Prospero protein is cytoplasmic in the absence of Brat. An obviously elevated level of cytoplasmic Prospero is not seen in brat mutant neuroblasts, but delocalization of Prospero protein from the basal crescent might not be visible over background. Alternatively, brat mutant neuroblasts may fail to transcribe or translate prospero in neuroblasts. This would most likely be an indirect effect, since Brat has been shown to only have translational repressor function. It has not been possible to detect prospero mRNA in wild-type larval neuroblasts, despite robust levels in GMCs, so this possibility has not been tested (Lee, 2006).

Some brat mutant neuroblasts show expanded aPKC cortical crescents, in some cases reaching the basal cortex. This phenotype appears specific for aPKC, because other apical cortical proteins (e.g., Baz, Pins) are unaffected. Brat might repress aPKC translation, leading to increased aPKC protein levels in brat mutants. Alternatively, the absence of Prospero or other basal cortical proteins may indirectly affect aPKC localization (Lee, 2006).

brat mutant brains show a dramatic increase in the number of large, proliferating Dpn+ neuroblasts between 48 and 96 hr ALH. Where do these hundreds of extra neuroblasts come from? They are unlikely to come from outside the brain, or from dedifferentiation of neurons or glia, although these models can't formally be ruled out. They are likely to derive from the pool of Dpn+ neuroblasts in the brain, because these are the primary pool of proliferating cells in the larval central brain, and thus the best candidates to generate the thousands of extra cells found in the hypertrophied brat mutant brains (Lee, 2006).

A model is proposed in which a subset of brat mutant “GMCs” enlarge into proliferative neuroblasts. This model is supported by several lines of evidence. (1) brat mutant GMCs maintain neuroblast-specific gene expression (Dpn, Miranda, Worniu); (2) brat mutants show an inverse relationship between increasing neuroblast number and decreasing neuronal number over time, consistent with GMCs forming neuroblasts instead of neurons; (3) brat mutant GMCs can be labeled by a BrdU pulse at their birth, yet most lose BrdU incorporation during the chase interval, showing that they either reenter the cell cycle or undergo cell death, and that cell death is not consistent with the brain overgrowth phenotype; (4) brat mutant telophase profiles show that all GMCs are born as small Miranda+ cells, ruling out physically or molecularly symmetric neuroblast divisions as a mechanism for increasing the neuroblast population; and (5) brat mutants show cell enlargement in other tissues, and a similar cell growth phenotype has been observed in mutants in the C. elegans brat ortholog (Lee, 2006).

What is the cellular origin of the brat mutant phenotype? brat mutant GMCs are compromised in three ways: they lack Brat translational repression activity, lack Prospero, and some may have ectopic aPKC. Loss of Brat translational repression activity could well play a role in the ectopic neuroblast self-renewal phenotype, because all brat mutants disrupting the NHL translational repression domain exhibit a brain tumor phenotype, and Brat has been previously shown to negatively regulate cell growth. Loss of Prospero also plays a role in the brat phenotype: prospero mutant GMCs have a failure to downregulate neuroblast gene expression and a failure in neuronal differentiation, similar to brat mutants. prospero null mutant embryos also show a slight delay in neuronal differentiation, although they appear to undergo normal neuroblast self-renewal. Finally, ectopic aPKC can also mimic aspects of the brat phenotype, including formation of supernumerary large Dpn+ neuroblasts. Interestingly, the mammalian paralogs of Drosophila aPKC (aPKCλ/ζ) are expressed in neural progenitors of the ventricular zone, and the mammalian Prospero ortholog Prox1 is expressed in differentiating neurons of the subventricular zone. Thus, identifying Prospero transcriptional targets and aPKC phosphorylation targets may provide further insight into the molecular mechanism of neural stem cell self-renewal in both Drosophila and mammals (Lee, 2006).

Protein phosphatase 4 mediates localization of the Miranda complex during Drosophila neuroblast asymmetric divisions

Asymmetric localization of cell fate determinants is a crucial step in neuroblast asymmetric divisions. Whereas several protein kinases have been shown to mediate this process, no protein phosphatase has so far been implicated. In a clonal screen of larval neuroblasts, the evolutionarily conserved Protein Phosphatase 4 (PP4) regulatory subunit PP4R3/Falafel (Flfl) was identified as a key mediator specific for the localization of Miranda (Mira) and associated cell fate determinants during both interphase and mitosis. Flfl is predominantly nuclear during interphase/prophase and cytoplasmic after nuclear envelope breakdown. Analyses of nuclear excluded as well as membrane targeted versions of the protein suggest that the asymmetric cortical localization of Mira and its associated proteins during mitosis depends on cytoplasmic/membrane-associated Flfl, whereas nuclear Flfl is required to exclude the cell fate determinant Prospero (Pros), and consequently Mira, from the nucleus during interphase/prophase. Attenuating the function of either the catalytic subunit of PP4 (PP4C; Pp4-19C in Drosophila) or of another regulatory subunit, PP4R2 (PPP4R2r in Drosophila), leads to similar defects in the localization of Mira and associated proteins. Flfl is capable of directly interacting with Mira, and genetic analyses indicate that flfl acts in parallel to or downstream from the tumor suppressor lethal (2) giant larvae (lgl). These findings suggest that Flfl may target PP4 to the MIra protein complex to facilitate dephosphorylation step(s) crucial for its cortical association/asymmetric localization (Sousa-Nunes, 2009).

Drosophila neuroblasts (NBs) are stem-cell-like neural progenitors, which undergo repeated asymmetric divisions to self-renew and generate neurons and/or glia. During each round of division the cell fate determinants Pros (a homeodomain-containing transcription regulator), Numb (a negative regulator of Notch signaling), as well as Brain Tumor (Brat, whose mechanism of action in cell fate specification is unclear) are asymmetrically localized as protein crescents on the NB cortex. In the embryo, the NB mitotic spindle is oriented along the apicobasal axis, the cell fate determinants and their adapter proteins localize to the NB basal cortex and segregate exclusively to the smaller basal daughter, called ganglion mother cell (GMC). The GMC divides terminally to produce two neurons or glial cells. The coordination between the basal localization of the cell fate determinants and the apicobasal orientation of the spindle during mitosis is mediated by several evolutionarily conserved proteins that localize to the apical NB cortex during the G2 stage of the cell cycle. These comprise [1] the Drosophila homologs of the Par3/Par6/aPKC protein cassette, [2] several proteins involved in heterotrimeric G protein signaling—Gαi/Partner of Inscuteable (Pins)/Locomotion defects (Loco), [3] as well as Inscuteable (Insc). In contrast to the embryo, NBs in the larval central brain divide without an apparent fixed orientation. Nevertheless the majority of central brain NBs appear to utilize the same molecular machinery as embryonic NBs, with the apical and basal molecules sharing similar hierarchical relationships and localizing to opposite sides of the NB cortex (Sousa-Nunes, 2009).

Asymmetric localization of Pros and Brat on the one hand and Numb on the other, is mediated through direct interactions with their respective adapters, the coil-coil proteins Miranda (Mira) and Partner of Numb (Pon). Although mutations affecting any of the apical proteins compromise asymmetric localization of basal proteins to varying extents, only in the case of aPKC has any mechanistic insight emerged. aPKC facilitates basal localization of cell fate determinants either through phosphorylation of the cytoskeletal protein Lgl and/or through direct phosphorylation of the determinant. Lgl is uniformly localized throughout the NB cortex, and is essential for cortical association and asymmetric localization of the cell fate determinants and their adapters. aPKC phosphorylates Lgl on three conserved serine residues and the triphosphorylated form appears to be inactive due to a conformational change. The proposed model is that unphosphorylated, active Lgl is restricted to the basal cortex because of apically localized aPKC. Consistent with this model, a nonphosphorylatable version of Lgl, Lgl3A, in which the three target serines have been mutated to alanines, appears to be constitutively active and its expression leads to uniform cortical localization of the normally basally restricted cell fate determinants. Numb is a second protein that can be phosphorylated by aPKC and phosphorylation of three N-terminal serines causes it to become cytoplasmic (Sousa-Nunes, 2009).

How Lgl acts to facilitate the localization of cell fate determinants is less clear. Lgl can bind nonmuscle Myosin II (Zipper) and genetic experiments suggest that Myosin II and Lgl have antagonistic activities. Hence, one possible scenario would be that Myosin II is active at the apical cortex due to the presence of phosphorylated Lgl, which is incapable of binding to Myosin II. Myosin II can then act to exclude basal proteins from the apical cortex. Alternatively, since yeast Lgl orthologs function in exocytosis, it has been suggested that Lgl might act by regulating this process. It is possible that Lgl positively promotes delivery and cortical association of the basal molecules, and that this is antagonized by Myosin II apically. In this scenario, Lgl is inhibited apically both by aPKC and Myosin II, and only basal Lgl is active and able to promote cortical association of the basal proteins (Sousa-Nunes, 2009).

The unconventional Myosin VI (Jaguar, Jar) and Myosin II bind in a mutually exclusive manner to the basal adapter protein Mira. However, in contrast to Myosin II, which acts antagonistically to Lgl, Jar acts in a synergistical manner with Lgl to effect Mira basal localization. In mitotic NBs devoid of Jar, Mira is mislocalized to the cytoplasm. Jar possibly mediates association of Mira with the basal actin cytoskeleton (Sousa-Nunes, 2009).

In addition to aPKC, a few other serine/threonine protein kinases have been shown to play a role in facilitating asymmetric protein localization in NBs. These include Cdk1, required for the asymmetric localization of both apical and basal components during mitosis, Aurora A (AurA), and Polo, both of which mediate Numb and Pon asymmetric localization. With the exception of Polo kinase, which phosphorylates a serine residue within the Pon asymmetric localization domain, substrates for the other kinases have not been identified. The involvement of protein kinases in NB asymmetric divisions implies the involvement of protein phosphatases; however, to date, none have been implicated in the process (Sousa-Nunes, 2009).

In a clonal genetic screen designed to identify genes that mediate NB asymmetric divisions, multiple loss-of-function alleles of flfl. Falafel (Flfl) were identified as a regulatory subunit of the evolutionarily conserved Protein Phosphatase 4 (PP4) Phosphatase complex. PP4 belongs to the best-studied family of cellular protein serine/threonine phosphatases, PP2A (the other major families being PP1, PP2B, and PP2C). Similarly to other PP2A-like phosphatases, PP4 functions as a heterotrimeric complex comprising of a catalytic subunit, PP4C, associated with two regulatory subunits, PP4R2 and PP4R3. PP4, or specifically PP4R3/Flfl, has been implicated in a variety of molecular and cellular processes including regulation of MEK/Erk, insulin receptor substrate 4, Hematopoietic progenitor kinase 1, and Histone deacetylase 3 activities, centrosome maturation, cell cycle progression, apoptosis, DNA repair, cell morphology, and lifespan control (Sousa-Nunes, 2009 and references therein).

This study shows that loss of flfl, as well as attenuation of PP4C/Pp4-19C or PPR2/PPp4R2r function by RNAi specifically results in delocalization of Mira and its associated proteins throughout the cytoplasm in metaphase/anaphase NBs; in addition, both Mira and Pros localize to the NB nucleus prior to nuclear envelope breakdown. Excessive nuclear Mira is dependent on the presence of Pros. These results suggest that whereas cytoplasmic or membrane-associated PP4 is required for asymmetric cortical localization of Mira (and its associated proteins) during metaphase and anaphase, nuclear PP4 is required to exclude Pros (and as a consequence, Mira) from the NB nucleus prior to nuclear envelope breakdown. Moreover, Flfl can complex with Mira in vivo and directly interact with Mira, suggesting that Flfl targets PP4 activity to the Mira complex to facilitate its correct localization (Sousa-Nunes, 2009).

In a clonal screen on third-instar larval (L3) brains, designed to identify novel genes on chromosome arm 3R required for NB asymmetric division, a novel allele of flfl, flfl795 was isolated. In metaphase and anaphase flfl795 clone NBs, Mira displays weak cortical crescents but also a pronounced mislocalization throughout the cytoplasm, whereas in surrounding heterozygous NBs Mira is localized to a robust crescent like in wild type with little cytoplasmic accumulation. As with many mutations that disrupt NB asymmetry during metaphase and anaphase, flfl795 NBs display telophase rescue: The majority of the cytoplasmic Mira relocalizes asymmetrically to the NB cortex at telophase, resulting in asymmetric segregation of Mira into the GMC. Using the flfl795 allele, two additional alleles [flfl795(2), flfl795(3)] were identified via complementation screening of an independent collection of ethylmethane sulfonate (EMS) mutant stocks. Sequencing of these three EMS-induced flfl alleles revealed single point mutations resulting in premature stop codons at positions 324 (flfl795) and 630 [flfl795(2)] of the longest isoform (980 amino acids) and a disruption to the splice acceptor site at the 3′ end of the fourth intron [flfl795(3)]. All three alleles display a mislocalization of Mira to the cytoplasm of metaphase NBs and form an allelic series in terms of phenotypic severity: flfl795 > flfl795(3) > flfl795(2) (Sousa-Nunes, 2009).

Homozygous flfl795 animals survive to pharate adults whereas hemizygous flfl795 animals [using Df(3R)Exel6170 to remove one copy of the flfl coding region] only survive until L3. Furthermore, although the cytoplasmic Mira phenotype of flfl795 homozygotes is highly penetrant, the majority of metaphase NBs still display weak Mira crescents, whereas the majority of metaphase NBs of flfl795 hemizygotes display no crescents. These results suggest that the strongest EMS allele (flfl795) is nevertheless a hypomorph. Therefore a flfl-null allele (flflN42) was generated by imprecise excision of the P-element P{EPgy2}flflEY03585, located ∼1 kb upstream of the flfl translational start site. This allele was confirmed to be a genetic null by the similar expressivities of NB phenotypes in flflN42 homozygotes and flflN42 hemizygotes, as well as in flfl795/flflN42 and flfl795/Df(3R)Exel6170. Consistently, flflN42 NBs are antigen-minus (see below) and molecular analysis indicates that it is a deletion extending into the coding region, deleting the first 1075 base pairs of the coding sequence. Subsequent analyses of the phenotype were carried out using the flflN42 allele, hereafter referred to simply as flfl (Sousa-Nunes, 2009).

In addition to the mislocalization of Mira, the Mira-associated proteins Pros, Brat and Staufen (Stau), are similarly mislocalized to the cytoplasm of metaphase/anaphase flfl NBs. Pros mislocalization occurs in Asense (Ase)-positive NB lineages which comprise the majority of lineages in the central brain; Ase-negative NBs are Pros-negative in flfl as well as in wild-type brains. In contrast, the localization of members of the other basal complex, Pon and Numb, and of apical complexes is unaffected. Hence, during NB division, flfl loss of function specifically affects the localization of the Mira complex (Sousa-Nunes, 2009).

Flfl homologs have been identified in several species, from yeast to humans. They all possess the same domain architecture: a Ran-binding domain (RanBD) at the N terminus, similar in three-dimensional structure to the Ena/VASP homology domain 1 (EVH1, which derives its name from the founding members Enabled and Vasodilator-stimulated phosphoprotein) and to the pleckstrin homology domain; followed by a conserved domain of unknown function (DUF625), a region containing armadillo/HEAT repeats, and a region of low complexity. Within the DUF625 domain, Flfl contains two putative NLSs (NLS1 and NLS2) as well as a nuclear export signal (NES); close to the C terminus Flfl contains a short conserved stretch of acidic and basic amino acid residues that has been shown to be required for nuclear localization of the Dictyostelium discoideum homolog, SMEK (NLS3). Flfl contains many putative target sites for O-linked N-acetylglucosamine (O-GlcNAc) glycosylation in its C-terminal 300 amino acids and numerous putative phosphorylation sites throughout, some of which are predicted to be PKC targets (Sousa-Nunes, 2009).

In conclusion, loss of function or RNAi knockdown of the regulatory subunits flfl/PP4R3 or PPP4R2r/PP4R2 as well as knockdown of the catalytic subunit Pp4C-19C/PP4C of PP4 causes mislocalization of Mira/Pros/Brat/Stau to the cytoplasm of metaphase and anaphase NBs (Sousa-Nunes, 2009).

Attenuation of PP4 function above also causes increased frequency of nuclear Mira/Pros prior to nuclear envelope breakdown. The observation that depletion of the catalytic subunit of PP4 results in identical phenotypes to the depletion of its regulatory subunits, suggests that phosphatase activity plays a role in the localization of Mira/Pros throughout the NB cell cycle (Sousa-Nunes, 2009).

Nuclear mislocalization of Mira seen in flfl, jar, or mira2L150 single-mutant NBs requires pros function. This suggests that, when transport of Mira toward or its tethering to the cortex is defective, Pros can take Mira into the nucleus. In this context, the normal relationship between Mira and Pros is reversed, with Pros instructing Mira localization rather than the converse. In the absence of pros, Mira is not localized to the nucleus, even when PP4 function is attenuated. Thus, the role of PP4 on these two temporally distinct localizations of Mira/Pros appears to involve distinct targets since one is a Mira-dependent localization and the other is Pros-dependent (Sousa-Nunes, 2009).

In contrast to serine-threonine kinases, substrate specificity for serine/threonine protein phosphatases is thought to be conferred not primarily by sequences adjacent to the target residues but rather by interaction between the substrate and regulatory subunits of the phosphatase complex. This is the case for the founding family member PP2A, whose variable subunit composition can also target the complex to distinct subcellular domains and is thought to be the case also for PP4. Flfl, a regulatory subunit of PP4, is able to bind Mira and Flfl and Mira are found in a complex in vivo. No binding was detected between Flfl and Pros but since Mira and Pros still colocalize when PP4 function is attenuated, these results also suggest that PP4 function is not required for the Mira-Pros interaction. Therefore, Pros could be recruited to PP4 by its association with Mira, which in turn binds Flfl (Sousa-Nunes, 2009).

Flfl is nuclear before and cytoplasmic after nuclear envelope breakdown. The results from nuclear excluded and membrane targeted versions of Flfl suggest that nuclear Flfl is required to exclude Mira/Pros from the nucleus when inefficiently bound to the cytoskeleton/cortex, whereas cytosolic or membrane-associated Flfl is required for the cortical association and asymmetric localization of Mira/Pros/Brat/Stau at metaphase and anaphase. The localization of Mira/Pros prior to and after nuclear envelope breakdown by PP4 may involve different phosphatase substrates. It is tempting to entertain the possibility that Mira dephosphorylation by PP4 in the cytoplasm is required for its asymmetric cortical localization during mitosis, and that Pros dephosphorylation by PP4 in the nucleus is required for its nuclear exclusion/progression through prometaphase. Indeed, a previous study has shown that cortical Pros is highly phosphorylated relative to nuclear Pros. To test this hypothesis, attempts were made to detect enrichment of a lower mobility band of Mira::3GFP in flfl larval extracts compared with wild type but this it could not be detected, working at the limits of detectability (Sousa-Nunes, 2009).

Asymmetric cortical localization of proteins during NB asymmetric division is dependent on an intact actin cytoskeleton. Although flfl is required for Mira cortical association, at no point in the NB cell cycle does Flfl exhibit cortical enrichment. However, modified versions of Flfl that are either uniformly cytoplasmic or cortically enriched can both drive asymmetric cortical localization of Mira and its associated proteins. Moreover, the Mira mislocalization phenotypes of flfl are strikingly similar to those of Myo VI/jar. Both mutants exhibit nuclear Mira/Pros prior to and cytoplasmic Mira and associated proteins following NB nuclear envelope breakdown; both Flfl and Jar are cytoplasmic at metaphase/anaphase; and genetically, both Jar and Flfl act parallel to or downstream from Lgl. Further propelled by the presence of a putative actin-binding domain in Flfl (the RanBD domain, which is an EVH1-like domain), it was asked whether Flfl too might facilitate association of Miranda with the actin cytoskeleton either separately from or in association with Jar. However, in vitro assays clearly showed that Flfl does not bind F-actin, although Mira alone does, with comparable strength to that of α-Actinin and Jar, used as controls. Furthermore, Jar could not be detected in Flfl containing protein immunoprecipitates. Therefore, it seems unlikely that Flfl acts either directly or in a complex with Jar to facilitate Mira transport along or tethering to the actin cytoskeleton. Still, Flfl could act indirectly; for example, by stabilization of the Mira-Jar association. It is speculated that Flfl may act by targeting PP4 to the Mira complex and that the consequent dephosphorylation of a component of this complex facilitates Jar-Mira association (Sousa-Nunes, 2009).

In Dictyostelium, mutants in the flfl homolog, smkA, exhibit phenotypes similar to strains defective in Myo II assembly, suggesting that smkA may regulate Myo II function. However, in flfl NBs the Mira mislocalization phenotype does not resemble that of Myo II loss of function, which has been described to lead to Mira mislocalization to the mitotic spindle in embryonic NBs (Sousa-Nunes, 2009).

The reduced proliferation seen in flfl NBs correlates with nuclear localization of Pros/Mira. Nuclear Pros negatively regulates transcription of cell cycle genes and positively regulates differentiation genes, and has been shown to limit NB proliferation. Therefore, ectopic nuclear Pros is likely to be at least one cause of the NB underproliferation observed in flfl brains. Still, it is possible that flfl has additional functions in promoting proliferation, independent of its role in excluding Pros/Mira from the NB nucleus. Indeed, an excessive proportion of phospho-histone H3-positive flfl NBs was detected relative to wild type. These NBs typically had a nucleus but the cell morphology was not spheroid, as would be expected in prophase cells. This suggests that flfl NBs either have a block or delay in prometaphase or that PP4 may be required for dephosphorylation of Histone H3; in either case, it seems to be required for dephosphorylation of other proteins involved in cell cycle progression. Nonetheless, pros,flfl double-mutant NB clones are indistinguishable from those of pros single mutants, both showing extensive overproliferation, suggesting that the loss of flfl is unable to override the overproliferation induced by loss of pros (Sousa-Nunes, 2009).

A transient expression of Prospero promotes cell cycle exit of Drosophila postembryonic neurons through the regulation of Dacapo

Cell proliferation, specification and terminal differentiation must be precisely coordinated during brain development to ensure the correct production of different neuronal populations. Most Drosophila neuroblasts (NBs) divide asymmetrically to generate a new NB and an intermediate progenitor called ganglion mother cell (GMC) which divides only once to generate two postmitotic cells called ganglion cells (GCs) that subsequently differentiate into neurons. During the asymmetric division of NBs, the homeodomain transcription factor Prospero is segregated into the GMC where it plays a key role as cell fate determinant. Previous work on embryonic neurogenesis has shown that Prospero is not expressed in postmitotic neuronal progeny. Thus, Prospero is thought to function in the GMC by repressing genes required for cell-cycle progression and activating genes involved in terminal differentiation. This study focused on postembryonic neurogenesis and shows that the expression of Prospero is transiently upregulated in the newly born neuronal progeny generated by most of the larval NBs of the OL and CB. Moreover, evidence is provided that this expression of Prospero in GCs inhibits their cell cycle progression by activating the expression of the cyclin-dependent kinase inhibitor (CKI) Dacapo. These findings imply that Prospero, in addition to its known role as cell fate determinant in GMCs, provides a transient signal to ensure a precise timing for cell cycle exit of prospective neurons, and hence may link the mechanisms that regulate neurogenesis and those that control cell cycle progression in postembryonic brain development (Colonques, 2011). During development, cell cycle progression must be coordinated with the regulation of cell specification and differentiation. The underlying mechanisms of coordination are likely to be particularly complex during neural development due to the enormous cell diversity in the brain. In Drosophila, these mechanisms have been well studied during embryonic CNS development. In embryonic neurogenesis, the homeodomain transcription factor Pros is expressed in the NB but it does not enter the nucleus due to its binding to the carrier protein Mira, which localizes to the cell cortex. This interaction facilitates the segregation of Pros from the parent NB to the GMC during asymmetric NB division. In the GMC, Pros is released from its carrier and translocates to the nucleus where it plays a binary role as a cell fate determinant, and as a promoter of terminal differentiation (Colonques, 2011).

It has been reported that Pros is similarly expressed and asymmetrically segregated during the proliferative activity of (type I) NBs in the larval CB although it does not seem to be expressed in CB dorso-medial lineages (type II) NBs. However, as this study shows, during postembryonic neurogenesis, in the majority of larval CB and OPC (outer proliferation center) neuronal lineages, pros expression is transiently upregulated in new born prospective neurons (GCs), in addition to its earlier expression and asymmetric segregation in some larval NBs. This is clearly different from the situation in embryonic lineages where pros is only transcribed in NBs, and Pros protein is downregulated in GCs after the division of their parent GMC (see Summary of cellular expression pattern of PROS in the larval CNS and lineage alterations in pros mutant clones) (Colonques, 2011).

This transient expression in most newborn postembryonic neurons shortly after the division of the GMC implies a novel role of Pros in postmitotic cells. It is postulated that this role is to inhibit cell cycle progression and promote cell cycle exit. The Pros GoF and LoF experiments support this notion. Pros GoF induces proliferation arrest and Pros LoF results in supernumerary cells with sustained expression of cell cycle markers, indicating an inability to withdraw from the cell cycle (Colonques, 2011).

In addition to the marked difference in Pros expression in postmitotic GCs during embryogenesis versus postembryonic neurogenesis (Pros is undetectable in embryonic GCs and high in postembryonic GCs), there are other functional differences in Pros action during embryonic versus postembryonic CNS development. For example, in pros mutant embryos, overproliferation is followed by abundant apoptotic cell death among the supernumerary cells. By contrast, no increas cell death was found in the larval OL of pros mutants. Moreover, while Pros and Dap seem to act in parallel to end the cell cycle in the embryonic CNS, Dap appears to act downstream of Pros in larval CNS neurons. These initial findings suggest that further differences between the functions of Pros during embryonic and postembryonic CNS neurogenesis may exist and should be considered (Colonques, 2011).

The fact that Pros protein is present in embryonic GMCs (intermediate progenitors) but not in embryonic GCs (prospective neurons), suggests that in the embryonic CNS, Pros initiates the end of mitotic activity in the GMC rather than in the GC. Accordingly, it has been proposed that the GMC is a transition state between the proliferating NB and the differentiating neuron that provides a window in which Pros represses stem cell-specific genes and activates differentiation genes. Nevertheless, it is not well understood how the GMC can go through its terminal cell cycle in spite of the repressive action of Pros on cell cycle regulators (Colonques, 2011).

The results strongly suggest that in postembryonic neurogenesis Pros acts not only in the GMC progenitor but also in the postmitotic GCs produced by the GMC. Thus, this analysis indicates that there are two main pros expression pattern subclasses among CB type I and OPC NB lineages. For the shake of simplicity they have been called them A and B. In type A, Pros is expressed in GCs after the division of GMCs while in type B, Pros is first expressed at low level in the NB and asymmetrically segregated to the GMC, and afterwards, upregulated in new born GCs. These two subsets of expression patterns correlate well with the two main phenotypes found in pros mutant clones. Thus, the LOF of pros in NBs with type A Pros expression appears to preclude cell cycle exit of GCs which, consequently, continue dividing and do not differentiate, yielding a type A clone composed of a single NB, a GMC and several small mitotic cells. By contrast, in lineages with type B Pros expression, the LOF of pros seems to cause primarily a change in the fate of the putative GMC that behaves like a NB maintaining the expression of asymmetric division genes (such as Mira) and overproliferating, to yield a type B clone composed of multiple large NB like cells (Colonques, 2011).

Hence, it is postulated that during postembryonic neurogenesis Pros functions in two sequential phases in type I NB lineages, first as cell fate determinant in some GMCs and later as cell cycle repressor in most GCs. Furthermore, the idea is favored that the different roles of Pros in postembryonic GMCs versus postembryonic GCs might be related to the higher level of expression observed in GCs compared to GMCs. Thus, high levels of Pros might be required to definitively withdraw the GCs from the cell cycle, while low levels might be sufficient to specify GMCs and modulate their cell cycle. The higher level of Dap protein in postembryonic GCs in relation to their parent GMCs and NBs is consistent with this hypothesis. The strong burst of Pros at the end of NB proliferation in ventral ganglia of early pupae is also in agreement with the idea that high levels of Pros are required to stop proliferation. Furthermore, it has been recently shown that the missexpression of Pros at high level suppresses proliferation in type II larval brain NBs lineages without apparent change in their identity (Colonques, 2011).

Taken together, all of these findings imply that different developmental strategies have been selected to couple cell fate decisions and cell cycle regulation during embryonic and postembryonic neurogenesis through the same effector, Pros. It is possible that this change in strategy is a consequence of the evolutionary adaptation to regulate the production of a large number of equivalent neurons in postembryonic lineages in contrast to embryonic neurogenesis where a much more limited set of specific neurons are generated in each lineage through GMC divisions (Colonques, 2011).

This study has shown that Pros is coexpressed with Dap in new born prospective neurons and, moreover, it was found that pros is sufficient and it is required for the expression of dap in these larval brain neuronal precursors. The dap gene encodes a member of the Cip/Kip family of CKIs with homology to mammalian p27kip1. This family of CKIs has been implicated in mediating cell cycle exit prior to terminal differentiation. They function by binding and inhibiting G1/S cyclin dependent kinase complexes. There is compelling data supporting a role of Dap in cell cycle exit during Drosophila embryogenesis. In Drosophila embryonic NB lineages, dap expression becomes apparent just before the terminal neurogenic division of the GMC. In contrast, this study has shown that dap is upregulated in new born postembryonic neurons. Consistent with a role in the termination of cell proliferation, dap expression in the larval OL has been tightly correlated with cells ending proliferation. Interestingly, Pros is required to terminate cell proliferation during embryonic neurogenesis and it has been shown to be involved in the regulation of dap expression in the embryonic nervous system. Thus, the results provide support to the idea that Pros promotes the cell cycle exit of post-embryonic GCs by upregulating the expression of dap. The data also suggest that this upregulation of dap is mediated by inhibiting the expression of Dpn. Dpn is an essential panneural bHLH transcription factor, which has been previously shown to be a suppressor of dap expression in the larval OL. Indeed, the dpn gene contains consensus Pros binding sites and Pros has been shown to be required to terminate the expression of dpn in the embryo (Colonques, 2011).

A new Prospero and microRNA-279 pathway restricts CO2 receptor neuron formation

CO2 sensation represents an interesting example of nervous system and behavioral evolutionary divergence. The underlying molecular mechanisms, however, are not understood. Loss of microRNA-279 in Drosophila leads to the formation of a CO2 sensory system partly similar to the one of mosquitoes. This study shows that a novel allele of the pleiotropic transcription factor Prospero resembles the miR-279 phenotype. A combination of genetics and in vitro and in vivo analysis was used to demonstrate that Pros participates in the regulation of miR-279 expression, and that reexpression of miR-279 rescues the pros CO2 neuron phenotype. Common target molecules of miR-279 and Pros were identified in bioinformatics analysis, and it was shown that overexpression of the transcription factors Nerfin-1 and Escargot (Esg) is sufficient to induce formation of CO2 neurons on maxillary palps. These results suggest that Prospero restricts CO2 neuron formation indirectly via miR-279 and directly by repressing the shared target molecules, Nerfin-1 and Esg, during olfactory system development. Given the important role of Pros in differentiation of the nervous system, it is anticipated that miR-mediated signal tuning represents a powerful method for olfactory sensory system diversification during evolution (Hartl, 2011).

The RanGEF Bj1 promotes Prospero nuclear export and neuroblast self-renewal

Drosophila larval neuroblasts are a model system for studying stem cell self-renewal and differentiation. This study report a novel role for the Drosophila gene Bj1 in promoting larval neuroblast self-renewal. Bj1 is the guanine-nucleotide exchange factor for Ran GTPase, which regulates nuclear import/export. Bj1 transcripts are highly enriched in larval brain neuroblasts (in both central brain and optic lobe), while Bj1 protein is detected in both neuroblasts and their neuronal progeny. Loss of Bj1 using both mutants or RNAi causes a progressive loss of larval neuroblasts, showing that Bj1 is required to maintain neuroblast numbers. Loss of Bj1 does not result in neuroblast apoptosis, but rather leads to abnormal nuclear accumulation of the differentiation factor Prospero, and premature neuroblast differentiation. It is concluded that the Bj1 RanGEF promotes Prospero nuclear export and neuroblast self-renewal (Joy, 2014).

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

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