prospero
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 lobe. 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 M¸ller 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).
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).
Lee, C.-Y., et al. (2006). Brat is a Miranda cargo protein that promotes neuronal differentiation and inhibits neuroblast self-renewal. Dev. Cell 10: 441-449. 16549393
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).
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