InteractiveFly: GeneBrief

IGF-II mRNA-binding protein: Biological Overview | References

BIOLOGICAL OVERVIEW

The localization of specific mRNAs can establish local protein gradients that generate and control the development of cellular asymmetries. While all evidence underscores the importance of the cytoskeleton in the transport and localization of RNAs, there is only limited knowledge of how these events are regulated. Using a visual screen for motile proteins in a collection of GFP protein trap lines, the Drosophila IGF-II mRNA-binding protein (Imp), an ortholog of Xenopus Vg1 RNA binding protein and chicken zipcode-binding protein, was identified. In Drosophila, Imp is part of a large, RNase-sensitive complex that is enriched in two polarized cell types, the developing oocyte and the neuron. Using time-lapse confocal microscopy, it was establish that both dynein and kinesin contribute to the transport of GFP-Imp particles, and that regulation of transport in egg chambers appears to differ from that in neurons. In Drosophila, loss-of-function Imp mutations are zygotic lethal, and mutants die late as pharate adults. Imp has a function in Drosophila oogenesis that is not essential, as well as functions that are essential during embryogenesis and later development. Germline clones of Imp mutations do not block maternal mRNA localization or oocyte development, but overexpression of a specific Imp isoform disrupts dorsal/ventral polarity. Loss-of-function Imp mutations, as well as Imp overexpression, can alter synaptic terminal growth. These data show that Imp is transported to the neuromuscular junction, where it may modulate the translation of mRNA targets. In oocytes, where Imp function is not essential, a specific Imp domain in the establishment of dorsoventral polarity is implicated (Boylan, 2008).

The subcellular localization of mRNAs is a conserved means of localizing protein concentration gradients that underlie the establishment of cellular asymmetries and specialized cell functions. For example, the localization of β-actin mRNA to the leading edge of neurite growth cones and embryonic fibroblasts is important for cell motility and growth. Oligodendrocytes move the mRNA encoding myelin basic protein to distal processes, where it is required for myelination, and the accumulation of mRNAs in dendrites in response to synaptic activity has fostered the hypothesis that mRNA localization might mediate synaptic plasticity. In Drosophila and Xenopus oocytes, the localization of maternal mRNA is required to establish axial polarity of the embryo (Boylan, 2008).

The localization of mRNA requires cis-acting localization elements within the RNA, as well as associated trans-acting factors that bind the RNA and/or one another to form a ribonucleoprotein (RNP) complex. Localization elements, or 'zipcodes,' have been defined for a number of localized RNAs and generally reside in the UTR. The trans-acting proteins are required for multiple functions that include regulating mRNA translation and degradation, physically linking RNPs to the transport machinery, and tethering mRNAs to cortical anchors at specific locations. Not surprisingly, the assemblage of trans-acting factors that carry out these diverse functions is complex and dynamic (Boylan, 2008).

Drosophila oogenesis provides an excellent system in which to study the localization and transport of RNPs. Germline cysts are composed of 16 cells interconnected by cytoplasmic bridges; one cell in each cyst becomes the oocyte, while the others become nurse cells that support oocyte growth. RNAs transcribed in the nurse cells are assembled into RNPs and transported to the oocyte at early stages of development, and later are correctly positioned within the oocyte. Both transport to the oocyte and localization within the oocyte are microtubule-dependent processes, suggesting directed transport of RNP complexes by microtubule motors. In early egg chambers, a polarized array of microtubules extends through the cytoplasmic bridges that connect the nurse cells with the oocyte, while later in oogenesis, microtubule reorganization facilitates proper positioning of axial determinants within the oocyte. The microtubule motors, dynein and kinesin, as well as the dynactin regulatory complex, have been implicated in transport events during oogenesis, but the regulation of their interactions with specific RNP cargoes is only beginning to be addressed. Genetic screens in Drosophila have identified protein components of RNPs that are required for the proper localization of known mRNA axial determinants. The use of live imaging techniques has allowed the direct visualization of RNP transport during Drosophila oogenesis as well as apical localization of transcripts in the blastoderm embryo (Boylan, 2008).

While genetic and biochemical approaches have revealed the diversity and complexity of RNPs, understanding of their assembly, transport, localization and translational control is limited. To identify additional factors involved in these functions, a visual screen of GFP-tagged gene products that incorporate into motile particles in Drosophila egg chambers was undertaken. This study reports analysis of one line with a GFP insert in the Drosophila ortholog of mammalian IGF-II mRNA binding protein (Imp). Imp belongs to a conserved family of proteins that regulate mRNA localization, translation and stability (reviewed in Yisraeli, 2005. The chicken ZBP-1 and Xenopus Vg1-RBP were the founding members of this family, identified by their ability to bind to cis-acting localization elements in the 3' UTR of specific mRNAs. ZBP-1 targets the localization of β-actin mRNA to the leading edge of chicken fibroblasts and promotes cell migration (Farina, 2002). Recent work has identified ZBP1 as a potential suppressor of the invasive behavior of mammary carcinoma cells. In Xenopus, Vg1-RBP tethers Vg1 RNA at the vegetal pole to help establish embryonic polarity (Havin, 1998; Deshler, 1997; Ross, 1997), and also regulates the asymmetric translation of β-actin mRNA involved in axon guidance (Huttelmaier, 2005; Yao, 2006; Leung, 2006). In mice and humans, the related CRD-binding protein binds to and regulates the stability of mRNA, including c-Myc and CD44 (Leeds, 1997; Noubissi, 2006; Vikesaa, 2006). Elevated levels of Imp-related proteins in cancer cells, and their role in cell migration, have raised the level of interest in their function. Nonetheless, knowledge of the complement of mRNAs targeted by the Imp proteins and the cytoskeletal mechanisms involved in Imp transport and localization is uncertain (Boylan, 2008).

Two recent studies have provided initial characterizations of the RNA-binding functions of Imp in Drosophila oogenesis, with some conflicting results (Geng, 2006; Munro, 2006). Munro proposed that Imp associates with oskar mRNA through putative Imp-binding elements (IBE) that are required for proper localization of Imp, though not for the initial localization of oskar mRNA. Geng (2006) provided evidence that Imp binds to oskar mRNA with low affinity, and suggested instead that gurken mRNA is the major target to which Imp binds. They show by immunoprecipitation experiments that Imp associates with Squid and Hrp48, two RNP components with known roles in regulating gurken and oskar expression, respectively. This study work complements these analyses by examining Imp transport characteristics. A mutational analysis of Imp was conducted to investigate its functions in Drosophila, and in addition to its redundant function in oogenesis, have identified requirements for Imp in embryogenesis and in synaptic terminal growth (Boylan, 2008).

In a visual screen for gene products that are actively transported in oogenesis, Imp, the Drosophila ortholog of the chicken zipcode binding protein, ZBP-1, was uncovered. Recent reports have been confirmed that while Imp is essential, it is not required for oocyte development or the proper localization of maternal determinants. However, results of overexpression experiments are consistent with a redundant role for Imp in dorsoventral patterning, as previously reported (Geng, 2006). From this study of Imp function in neurons, Imp is shown to be required for the proper growth and/or maintenance of the synapse at the NMJ (Boylan, 2008).

Analysis of the GFP-Imp trap line extends previous studies to characterize transport of Imp in both ovaries and axons. Imp motility requires microtubule motor function. Similar to Exuperantia and Staufen RNPs (Misch, 2007), the number and velocity of GFP-Imp particles in nurse cells is significantly reduced in a hypomorphic dynein mutant background. Loss of dynein function lowers the velocities of all Imp particles, suggesting that dynein is the motor that actively transports Imp in the nurse cell compartment. Moreover, particle velocity is elevated in the kinesin null background, as was previously found for Stau-GFP and Exu-GFP RNP transport. This antagonistic interaction of dynein and kinesin suggests that both motors reside on the Imp RNP complex, but that in nurse cells only dynein actively promotes RNP translocation along microtubules (Misch, 2007). Consistent with this interpretation, saltatory, bidirectional movement of the Imp particles along microtubules in nurse cells. In contrast, Imp transport along microtubules within larval axons is saltatory, with frequent, short reversals in direction of transport. In axons, disruption of either dynein or kinesin reduces the velocity of dImp transport. It will be important to identify signaling pathways which regulate plus- and minus-end motor activities and consequently determine the directional bias of RNP transport in different tissues (Boylan, 2008).

The transport and localization of ZBP-1 particles at the leading edge of chicken fibroblasts is predominantly an actin-based process; localization of β-actin mRNA and ZBP-1 are disrupted by treatment with cytochalasin D, but not colchicine. Moreover, chemical inhibition of myosin ATPase activity blocks ZBP-1 accumulation at the leading edge of fibroblasts (Oleynikov, 2003). There is no evidence that actin mediates active transport of Drosophila Imp, but the retention of Imp at the posterior pole of the oocyte is cytochalasin-sensitive and may require actin. Although Drosophila Imp lacks the RNA-binding “RRM” domains present in vertebrate orthologs, it contains the four KH domains which are reportedly sufficient for ZBP-1 association with actin filaments, as well as for RNP granule formation (Farina, 2003). Nielsen (2002) showed the KH domains of human Imp1 were sufficient for its correct localization and association with microtubules. How the KH domain can mediate association with both actin and microtubules is not clear (Boylan, 2008).

The transport and localization of GFP-Imp during Drosophila oogenesis suggested a role for Imp in the localization of maternal RNA determinants that specify the embryonic axes. However, in germline clones of Imp loss-of-function mutations, there is no evidence of mispositioned mRNA determinants for either the anterior/posterior or dorsal/ventral axes (Geng, 2006; Munro, 2006). Expression of Oskar protein was also examined in Imp germline clones and no evidence was found that Imp is required for translational repression of oskar mRNA. This could reflect the existence of a functionally redundant factor that compensates for the loss of Imp during oogenesis (Boylan, 2008).

Geng (2006) report that reducing Imp function partially suppresses a gurken mRNA misexpression phenotype. In addition, Imp overexpression disrupts gurken mRNA localization and alters dorsoventral polarity, as reflected by defects in dorsal appendage formation. This study observed a similarly penetrant dorsalization of the eggshell upon overexpression of the Imp splice variant encoded by the transgene, UAS-SD. This isoform varies from the alternate Imp polypeptide only in a small N-terminal region preceding the four KH domains. According to a recent analysis, the polypeptide represented by UAS-SD is normally expressed within the germline. The isoform encoded by UAS-RE is apparently not abundantly expressed in the germline, and this study found that its ectopic expression there does not generate the dorsalized phenotype. It is proposed that a domain preceding the KH domains and unique to the isoform represented by UAS-SD mediates an interaction that is critical for the proper regulation of Imp function in the germline (Boylan, 2008).

The current observations also address the zygotic function of Imp and show that Imp is essential for embryonic development. In contrast to the lack of germline phenotypes in oogenesis, a significant proportion of embryos derived from homozygous Imp mutant eggs fail to hatch. This phenotype appears unrelated to the function of maternal determinants, and as suggested by Munro and colleagues (2006) may result from the misregulation of other IBE-containing transcripts in the absence of Imp. RNA targets are known to regulate cell migrations that contribute to tissue morphogenesis in other organisms. Similarly, the loss of Drosophila Imp function might disrupt cell migration in early embryonic development. Anterior defects in cuticle preparations were noted that could result from such defects. Imp is also required late in development; strong loss-of-function mutants die as pharate adults. The late lethal phase is consistent with neuronal function and the observed enrichment of Imp in the central nervous system (Nielsen, 1999). Strong mutations in Drosophila dFmr1 also die as pharate adults. dFMR1 is another KH-type RNA binding protein and overexpression or loss-of-function dFmr1 mutations are reported to generate neuronal and behavioral phenotypes. Consistent with the similar phenotype, dFMR1 associates with Imp in neuronal RNP granules (Barbee, 2006). This study reports that dFMR1 also associates with Imp in Drosophila ovaries, suggesting that the function of a dFMR1/Imp complex in RNA localization and translational control is not specific to neurons. The distribution of Imp and dFMR1 in neurons overlaps but is not identical, consistent with the interpretation that the two RNP components are not obligate partners. An earlier study has suggested that a fraction of the dFMR1 pool associates with PAR and lethal giant larvae proteins in a RNP complex. How broadly Imp distributes among different RNPs is not known (Boylan, 2008).

Previous studies have reported that the overexpression of Drosophila Imp in the larval nervous system generates defects in axonal pathfinding and neural development (Kraut, 2001; Norga, 2003). This study has extended the analysis of Imp function in neurons and provides evidence that Imp acts to modulate synaptic terminal growth. A decrease was observed in the size of the synaptic terminal at the NMJ in loss-of function mutant backgrounds, and an expansion of the terminal was detected when the UAS-RE transgene is overexpressed in neurons. The alterations in the size of synaptic termini correlate with aberrant neuromuscular activity. The defects in larval motility are mild, while eclosed adult flies exhibit severe neuromuscular defects, most notably very limited and uncoordinated sporadic leg movements. The defects observed are similar in both the loss-of-function Imp mutant background and in flies in which the RE isoform of Imp is specifically overexpressed in neurons. These results suggest that aberrant levels of Imp in neurons, either high or low, and the resultant increased or decreased size of synaptic termini can disrupt neuromuscular activity. The results suggest that Imp function in neurons is not restricted to the guidance of growth cones during embryogenesis, and it is speculated that Imp is also involved in the active delivery and translation control of transcripts at synaptic termini. The localization of Imp in synaptic boutons is not homogenously distributed, but appears to be enriched along the membrane. Thus, similar to the Drosophila oocyte, RNPs and the associated functional mRNA determinants become tethered to the cortical domains where local translation can quickly respond to changes in synaptic activities. Intriguingly, the delivery and local translation of mRNAs may regulate synaptic plasticity and homeostasis. However, the electrophysiological activity of synaptic terminals was not directly characterized, and it can only be speculated that the observed defects in neuromuscular activity result from the aberrant sizes and transmission of synapses in the Imp mutants. Moreover, it is worth noting that the electrophysiological activity and synaptic terminal size at the NMJ are not always tightly coupled. For example, highwire mutants have synaptic terminals that are double the size of controls, but the quantal content is reduced. Similarly, spinster mutants have a small or normal quantal content but a substantially increased synaptic terminal. The opposite situation is also possible; presynaptic rescue of gbb mutants results in normal quantal content with a small synaptic terminal size. All mutants in the BMP signaling pathway in Drosophila have a similar phenotype of decreased synaptic terminal size and quantal content. In wit mutants the synaptic terminal at the NMJ is 60% of normal size, while quantal content is about 20% of controls. Despite these significant defects at the morphological and electrophysiological level, wit larva do not show any obvious locomotion defects (Boylan, 2008).

A critical set of questions concerns how the transport and anchoring of Imp RNP complexes are influenced by signaling pathways. TGF-ß Bone Morphogenetic Protein signaling is required for synaptic development and plasticity in Drosophila. Mutations in either the ligand, glass bottom boat, or the corresponding type II receptor, Wit, impair synaptic growth and have a similar late lethal phase. The downstream consequence of Wit signaling is the nuclear accumulation of pMad. The current results establish that nuclear accumulation of pMad is not disrupted by the loss of Imp function and suggest that the mRNA targets of Imp do not function upstream of Wit. However, the data do not exclude the possibility that Imp acts downstream to communicate an anterograde cellular response to Wit signaling that modifies synaptic growth and plasticity. Synaptic development, homeostasis, and modulation of synaptic activities are important elements of functional neural circuits that depend on bi-directional signaling and communication between the synapse and cell body. While these observations underline the importance of Imp in synaptogenesis, the target transcripts regulated by Imp have not bee identified. The composition of neuronal Imp RNP particles is an important area to pursue in future experiments. Indeed, there are many cytoplasmic RNA structures present within neurons and our knowledge of their functions is rudimentary. Visual screens based on active cytoplasmic transport of RNP complexes should continue to provide an important tool for identifying additional gene products that regulate the transport and localization of mRNAs (Boylan, 2008).

Opposing intrinsic temporal gradients guide neural stem cell production of varied neuronal fates

Neural stem cells show age-dependent developmental potentials, as evidenced by their production of distinct neuron types at different developmental times. Drosophila neuroblasts produce long, stereotyped lineages of neurons.Factors that could regulate neural temporal fate were sought by RNA-sequencing of lineage-specific neuroblasts at various developmental times. Two RNA-binding proteins, IGF-II mRNA-binding protein (Imp) and Syncrip (Syp), were found to display opposing high-to-low and low-to-high temporal gradients with lineage-specific temporal dynamics. Imp and Syp promote early and late fates, respectively, in both a slowly progressing and a rapidly changing lineage. Imp and Syp control neuronal fates in the mushroom body lineages by regulating the temporal transcription factor Chinmo translation. Together, the opposing Imp/Syp gradients encode stem cell age, specifying multiple cell fates within a lineage (Liu, 2015).

Diverse neural stem cells produce distinct sets of specialized neurons. The suite of daughter neurons generated by common neural stem cells can further change as development progresses, which suggests that the neural stem cells themselves change over time. In Drosophila, the neuroblast, a type of neural stem cell, can bud off about 100 ganglion mother cells that each divide once to produce two, often different, daughter neurons. Mapping the serially derived neurons based on birth order/time has revealed that each individual neuroblast makes an invariant series of morphologically distinct neuronal types (Liu, 2015).

Drosophila central brain neuroblasts differ greatly in the number of neuronal types produced and the tempo at which changes occur. The four mushroom body neuroblasts divide continuously throughout larval and pupal development, but each produces only three classes of neurons. By contrast, the antennal lobe anterodorsal 1 (ALad1) neuroblast generates 22 neuronal types during larval development. Although sequential neuroblast expression of temporal transcription factors specifies neuronal cell fates in lineages of the Drosophila ventral nerve cord and optic lobes, this mechanism is not easily applied to central brain. This study analyzed fate determinants that direct neuronal diversification based on the age of the neuroblast (Liu, 2015).

Genes with age-dependent changes in expression levels throughout the life of a neuroblast could confer different temporal fates upon the neuronal daughter cells born at different times. Thus, this study aimed to find genes dynamically expressed in mushroom body and antennal lobe neuroblasts by sequencing transcriptomes over the course of larval and pupal neurogenesis. Mushroom body or antennal lobe neuroblasts were marked persistently and exclusively with green fluorescent protein (GFP) by genetic intersection and immortalization tactics. Approximately 100 GFP+ neuroblasts were isolated for each RNA/cDNA preparation. Quantitative polymerase chain reaction (qPCR) showed that known neuroblast genes, including deadpan (dpn) and asense, are enriched in mushroom body and antennal lobe neuroblasts compared with total larval brains. Samples passing this qPCR quality check were sequenced. The transcriptomes of mushroom body neuroblasts were obtained at 24, 50, and 84 hours after larval hatching (ALH), as well as 36 hours after puparium formation (APF), and the transcriptomes of antennal lobe neuroblasts at 24, 36, 50, and 84 hours ALH. The antennal lobe neuroblasts were not sequenced at 36 hours APF because they stop producing neurons around puparium formation. Also, mushroom body neuroblasts were not analyzed at 36 hours ALH because the mushroom body lineages do not undergo detectable fate or molecular changes between 24 and 50 hours ALH (Liu, 2015).

Strongly dynamic genes were defined as those with a greater than fivefold change in expression level across different time points and a maximum average abundance higher than 50 transcripts per million. Eighty-three strongly dynamic genes in the mushroom body and 63 in the antennal lobe. The two sets shared 16 genes in common. Among these 16 common genes, pncr002:3R, a putative noncoding RNA, ranks highest in absolute abundance, the importance of which remains unclear (Liu, 2015).

IGF-II mRNA-binding protein (Imp) and Syncrip (Syp), which code for two evolutionally conserved RNA-binding proteins, rank second and third in absolute abundance (McDermott, 2012; McDermott, 2014; Munro, 2006). Imp is expressed abundantly at 24 hours ALH and declines to a minimum at 84 hours ALH in antennal lobe and 36 hours APF in mushroom body neuroblasts, whereas Syp increases from minimal expression at 24 hours ALH to become one of the most abundant genes at late larval stages. Imp/Syp gradients with larger amplitudes and steeper slopes characterize antennal lobe neuroblasts, which yield more diverse neuron types at faster tempos than the mushroom body neuroblasts. Antibodies to Imp and Syp showed similar patterns in shifts of protein abundance in both mushroom body neuroblasts and neuronal daughter cells. The roles of Imp and Syp in neuronal temporal fate specification were investigated in both mushroom body and antennal lobe lineages (Liu, 2015).

The post-embryonic mushroom body neuroblasts sequentially produce γ, α'/β', and α/β neurons, which can be distinguished by a variety of markers. Fasciclin II (FasII) is expressed in the perimeter of the α/β lobes, is weakly expressed in the γ lobe, and is not expressed in the α'/β' lobes. Trio, a Dbl family protein, is expressed in the γ and α'/β' lobes. The γ neurons can also be identified in wandering larvae by expression of ecdysone receptor B1 isoform (EcR-B1). Moreover, one can predict the fate of newborn mushroom body neurons based on the protein levels of Chinmo, a known temporal transcription factor, as abundant Chinmo specifies the γ fate, weak Chinmo expression confers the α'/β' fate, and absence of Chinmo permits the α/β fates (Liu, 2015).

RNA interference (RNAi) aimed to reduce Imp expression resulted in up-regulated Syp, whereas knocking down Syp expression caused increased Imp expression. This reciprocal derepression was evident in protein and transcript content as well as phenotype. Silencing Imp triggered precocious production of α/β neurons throughout larval development; these neurons lacked EcR-B1 at the wandering larval stage and showed no Chinmo at 24 hours ALH. Imp-depleted neuroblasts ended neurogenesis prematurely: By 28 hours APF, no neuroblasts remained in the mushroom body. This resulted in small adult mushroom bodies with only α/β lobes. By contrast, silencing Syp extended the production of Chinmo-positive γ neurons through pupal development. The Syp-depleted adult mushroom bodies consisted of a single prominent γ lobe. The reciprocal temporal fate transformations were also seen in the mushroom body neuroblast clones homozygous for various Imp or Syp loss-of-function mutations. Thus, Imp specifies early γ neurons and Syp specifies late α/β neurons (Liu, 2015).

It was next asked whether prolonged coexpression of Imp and Syp can increase the number of α'/β' neurons, which are typically born at a time when Imp and Syp expression levels are similar. Ectopic induction of Imp or Syp transgenes enhanced Imp or Syp protein levels in mushroom body newborn neuronal daughter cells but not in the neuroblasts. In the case of Syp overexpression, the early larval neuroblasts still contained abundant Imp, as did their newborn neurons expressing ectopic Syp. Analogously, overexpressing Imp rendered the pupal-born neurons strongly positive for Syp as well as ectopic Imp. Therefore, after Imp or Syp overexpression, many more newborn neurons simultaneously expressed Imp and Syp, and the normally modest α'/β' neuronal lobes were enlarged in adult brains. Cell death was unlikely to distort the developmental outcomes, as rare sporadic cell death was only detected in mushroom body neurons expressing ectopic Syp at 50 hours ALH. Taken together, these data demonstrate that relative levels of Imp and Syp dictate mushroom body neuronal temporal fates (Liu, 2015).

The altered Chinmo protein levels upon Imp or Syp depletion prompted a look at whether Imp and Syp regulate chinmo expression. The abundance of chinmo transcripts normally decreases as neuroblasts age. This pattern was unperturbed in Imp or Syp-depleted neuroblasts. Together, these data suggest that Imp and Syp regulate chinmo expression at a posttranscriptional level (Liu, 2015).

Epistasis was used to explore whether Imp and Syp act to regulate mushroom body neuronal temporal fates through Chinmo. Overexpressing a chinmo transgene partially restored the production of γ neurons by the short-lived, Imp-depleted neuroblasts. Moreover, silencing chinmo transformed the supernumerary γ neurons made by the Syp-depleted neuroblasts into α/β neurons. Together, these observations place Chinmo downstream of Imp and/or Syp in the temporal fate specification of mushroom body (Liu, 2015).

To ascertain whether Imp/Syp gradients serve as a general temporal-fating mechanism, the roles of Imp and Syp were examined in the rapidly changing antennal lobe anterodorsal 1 (ALad1) lineage that yields ~60 larval-born neurons of 22 types. Although all 22 types express acj6-GAL4, only the first 12 types generated express GAL4-GH146. Imp depletion reduced the ALad1 daughter cell number (acj6+) but increased the ratio of late-type to early-type neurons. By contrast, Syp depletion increased total ALad1 daughter cells and increased the percentage of the early-type (GH146+) neurons. Despite precocious production of late-type neurons or prolonged generation of early-type neurons, 21 of the 22 neuron types were preserved. In summary, the opposing Imp/Syp gradients govern temporal fates in at least two different neuroblast lineages that produce mushroom body and antennal lobe neurons functioning in memory and olfaction, respectively (Liu, 2015).

The opposite temporal gradients of Imp and Syp in neuroblasts confer the serially derived daughter cells with graded levels of Imp/Syp. The acquisition of distinct daughter cell fates based on the Imp/Syp morphogens is reminiscent of early embryonic patterning by the opposite spatial gradients of maternally inherited Bicoid and Nanos. Different levels of Bicoid and Nanos are incorporated into cells along the anterior-posterior axis after cellularization of the blastoderm. Bicoid and Nanos function as RNA-binding proteins to initiate anterior-posterior spatial patterning via translational control of maternal transcripts that encode transcription factors. Analogously, temporal fate patterning of newborn neurons is orchestrated by post-transcriptional control of chinmo and potentially other genes by Imp and Syp. Because Imp and Syp may share common targets but show affinity for different RNA motifs, it is possible that Imp and Syp can both bind chinmo transcripts, but they may differentially target chinmo transcripts for translation versus sequestration. Descending Imp temporal gradients governs aging of Drosophila testis stem cell niche. Imp-1, the mammalian ortholog of Imp, is also needed to maintain mouse neural stem cells. It is proposed that graded Imp/Syp expression constitutes an evolutionally conserved mechanism for governing time-dependent stem cell fates, including temporal fate progression in neural stem cells and their derived neuronal lineages (Liu, 2015).

Mamo decodes hierarchical temporal gradients into terminal neuronal fate

Temporal patterning is a seminal method of expanding neuronal diversity. This study has unravel a mechanism decoding neural stem cell temporal gene expression and transforming it into discrete neuronal fates. This mechanism is characterized by hierarchical gene expression. First, Drosophila neuroblasts express opposing temporal gradients of RNA-binding proteins, Imp and Syp. These proteins promote or inhibit chinmo translation, yielding a descending neuronal gradient. Together, first and second-layer temporal factors define a temporal expression window of BTB-zinc finger nuclear protein, Mamo. The precise temporal induction of Mamo is achieved via both transcriptional and post-transcriptional regulation. Finally, Mamo is essential for the temporally defined, terminal identity of alpha'/beta' mushroom body neurons and identity maintenance. This study describes a straightforward paradigm of temporal fate specification where diverse neuronal fates are defined via integrating multiple layers of gene regulation. The neurodevelopmental roles of orthologous/related mammalian genes suggest a fundamental conservation of this mechanism in brain development (Liu, 2019).

The brain is a complicated organ which not only requires specific connections between neurons to form circuits, but also many neuronal types with variations in morphology, neurotransmitters and receptors. While mechanisms controlling neuronal diversity have not been globally examined, studying neural stem cells in the mouse and fruit fly have given insight into key aspects of neuronal specification. For example, in the mouse neocortex, radial glial progenitors (RGP) are multipotent-they produce a variety of neuron types organized sequentially into six layers, and then produce glia. In vivo lineage analysis demonstrated that after a stage of symmetric cell division, an individual neurogenic RGP produces an average of 8-9 progeny (range of 3-16) that can span all cortical layers. In Drosophila, clonal analysis has demonstrated a vast range of stem cell-specific lineage programs. On one extreme, lineage tracing of a single antennal lobe (AL) stem cell revealed a remarkable series of 40 morphologically distinct neuronal types generated sequentially. In light of these observations, a fundamental goal is to understand how distinct neuronal types correctly differentiate from a single progenitor. Despite a fundamental role for temporal patterning to create diverse neuronal lineages, understanding of neuronal temporal patterning is still limited. While scientists have discovered key temporal factors expressed in neural progenitors, much less is understood about how these signals are interpreted, that is what factors lie downstream of the specification signals to determine distinct neuronal temporal fates (Liu, 2019).

Despite its relatively small brain, Drosophila is leading the charge on studies of neuronal temporal fate specification. Many temporal transcription factors originally discovered in the fly have since been confirmed to have conserved roles in mouse retinal and cortical development. Moreover, temporal expression of an RNA binding protein, IGF-II mRNA-binding protein (Imp), that guides temporal patterning in the postembryonic fly brain is also implicated in mouse brain development. Drosophila brain development is an excellent model for studying neurogenesis; the neural stem cells, called neuroblasts (NB), are fixed in number, their modes of division are well characterized, and each NB produces a distinctive series of neurons which change fate based on birth order. Finally, the fruit fly is a genetically tractable system making it ideal for studying gene networks involved in cell fate decisions (Liu, 2019).

In Drosophila, cycling NBs express age-dependent genes that provide the serially derived newborn neurons with different temporal factors. In the embryonic ventral nerve cord and the optic lobe, the NBs express a rapidly changing series of four to six temporal transcription factors (tTF), some of which are directly inherited by the daughter neurons. Each tTF directly acts to specify a small number (two to four) of neuronal progeny. The neuronal progeny produced from one tTF window to the next can be quite different. The tTF series are intrinsically controlled, which ensures reliable production of all neuron types, but lacks the ability to adapt to complicated or changing conditions (Liu, 2019).

A separate mechanism is therefore required for adult brain development-both to produce very long series of related neuronal types and to coordinate with organism development. This can be accomplished utilizing protein gradients and hierarchical gene regulation, such as the mechanism used to pattern the fly's anterior/posterior (A/P) axis. In Drosophila A/P patterning, the embryo is progressively partitioned into smaller and smaller domains through layered gene regulation. This is initiated by asymmetric localization maternal mRNAs, bicoid (anterior) and nanos (posterior). The resulting opposing proteins gradients then act on maternal mRNA translation, and in the case of Bicoid, zygotic transcription. The embryo then progresses through expression of maternal morphogen gradients, then zygotic expression of gap genes to determine broad embryo regions, followed by progressive segmentation by the pair-rule and segment polarity genes, and finally specification by the homeotic selector genes (Liu, 2019).

Notably, in postembryonic brain development, two proteins in opposing temporal gradients expressed in NBs have been discovered. These proteins are Imp and Syncrip (Syp) RNA-binding proteins. Imp and Syp control neuronal temporal fate specification as well as the timing of NB termination (decommissioning). Imp promotes and Syp inhibits translation of the BTB-zinc finger nuclear protein, chinmo (chronologically inappropriate morphogenesis), so that protein levels in newborn neurons descend over time. The level of Chinmo correlates with the specification of multiple neuronal temporal fates. Discovering downstream layers in the Imp/Syp/Chinmo hierarchy is essential to fully comprehend the intricacies of temporal patterning in brain development (Liu, 2019).

Temporal regulation in the fly brain is easily studied in the relatively simple mushroom body (MB) neuronal lineages which are comprised of only three major cell types. These neuronal types are born in sequential order: beginning with γ neurons, followed by α'/β' neurons, and finally α/β neurons. Imp and Syp are expressed in relatively shallow, opposing temporal gradients in the MB NBs. Modulation of Imp or Syp expression results in shifts in the neuronal temporal fate. Imp and Syp post-transcriptionally control Chinmo so that it is expressed in a gradient in the first two temporal windows. γ neurons are produced in a high Chinmo window, α'/β' neurons are produced in a low Chinmo window, and α/β neurons are produced in a window absent of Chinmo expression. Moreover, altering Chinmo levels can shift the temporal fate of MB neurons accordingly, strongly implicating dose-dependent actions, similar to that of a morphogen. Despite its importance in temporal patterning, the mechanisms underlying the dosage-dependent effects of Chinmo on neuronal temporal identity is unknown (Liu, 2019).

This study describes Mamo (maternal gene required for meiosis), a BTB zinc finger transcription factor critical for temporal specification of α'/β' neurons. Mamo is expressed in a low Chinmo temporal window and Mamo expression can be inhibited both by high Chinmo levels and loss of chinmo. Additionally, Mamo is post-transcriptionally regulated by the Syp RNA binding protein. This layered regulation, which is utilized in both MB and AL lineages results in a discrete window of Mamo expression in young, post-mitotic neurons. In the MB lineages, this window corresponds to the middle window of neurogenesis and it was establish that Mamo codes for middle temporal fate(s); α'/β' neuronal characteristics are lost when Mamo levels are reduced and ectopic Mamo drives an increase in α'/β' neuron production. The temporal fate determination paradigm described in this study utilizes multiple levels of gene regulation. Temporal fate specification begins in the stem cell and proceeds in a hierarchical manner in successive stages where top and second-tier factors work together to specify neuronal temporal fate. These data suggest that Mamo deciphers the upstream temporal specification code and acts as a terminal selector to determine neuronal fate (Liu, 2019).

Chinmo levels in newborn neurons correlate with adult neuron identity. Based on smFISH, mamo transcription is initiated in newborn MB neurons around 72 hr ALH, which corresponds to weak Chinmo expression. Moreover, Mamo is only expressed when Chinmo levels are low, as Mamo is not expressed after either eliminating or overexpressing Chinmo. Together these data indicate that low Chinmo levels activate mamo transcription in young/maturing neurons (Liu, 2019).

Transcription initiation is not the only requirement for Mamo protein expression; Syp is also required as discussed below. This could explain why no Mamo expression turn on is seen in γ neurons, even as they age and Chinmo levels decrease, becoming quite low around wandering larval stage. γ neurons begin to express Mamo later, around pupation, despite lacking Syp. It has not yet been tested whether weak Chinmo levels are required for later Mamo expression in γ neurons. It is therefore possible that Mamo expression is controlled at this stage by an additional factor(s) (Liu, 2019).

ChIP-chip performed in embryos found five Chinmo binding sites within the mamo gene, consistent with direct activation of mamo transcription. However, the nature of Chinmo's concentration dependent actions is still unclear. Some morphogens such as Bicoid bind different targets at increasing concentrations based on the affinity of binding to different sites as well as the chromatin accessibility of the binding sites. This may also be the case with Chinmo, but would not easily explain why Mamo expression is inhibited at higher Chinmo concentrations. The gap gene Krüppel, on the other hand, has concentration dependent activities at the same binding site. Krüppel acts as an activator at lower concentrations and as a repressor at high concentrations. Krüppels C-terminus has the ability to activate genes and is also the location for dimerization. Upon dimerization, the C-terminus can no longer activate genes and Krüppel transforms from an activator to a repressor. The current data suggests that low concentrations of Chinmo activate mamo. However, in the testis, Chinmo is suspected to function as a transcriptional repressor. It is feasible that Chinmo, like Krüppel, could switch from an activator to a repressor. The protein concentration would affect whether Chinmo is a monomer (in the presence of other BTB proteins, a heterodimer) or a homodimer, and thus potentially which cofactors are recruited (Liu, 2019).

The ascending Syp RNA binding protein temporal gradient regulates Mamo expression both indirectly via its inhibition of Chinmo and also presumably directly, interacting with the mamo transcript and promoting its expression. The bi-modal, transcriptional (Chinmo) and post-transcriptional (Syp), regulation of the Mamo terminal selector is extremely advantageous. Given the finding that Mamo expression is positively autoregulated and that Mamo continues to be expressed into adult neurons, it is particularly important to control the timing of Mamo's onset. The additional layer of post-transcriptional regulation adds an extra safeguard, helping to guarantee that neuronal temporal patterning is a robust system. Indeed, as brain development needs to adapt to environmental conditions such as nutrient deprivation, it is crucial to ensure that there is no loss of neuronal diversity (Liu, 2019).

Syp is a homolog of mammalian SYNCRIP (synaptotagmin-binding cytoplasmic RNA-interacting protein) also known as hnRNP-Q. SYNCRIP is involved with multiple facets of mRNA regulation including mRNA splicing and maturation, mRNA localization and stabilization as well as inhibiting mRNA translation and miRNA-mediated repression via competition with Poly(A) binding proteins. The Drosophila ortholog seems to have corresponding functions. Drosophila Syp was isolated from the spliceosome B complex, indicating a conserved role in mRNA splicing. Syp has likewise been found to operate in mRNA localization and stabilization. Furthermore, it has clear roles in altering protein expression of its mRNA targets, both positively and negatively. The bidirectional influence on protein expression likely reflects different Syp modalities (Liu, 2019).

This study shows that Syp is required for Mamo protein expression in the MB and AL neuronal lineages. To determine the nature of this regulation, single molecule fluorescence in situ hybridization (smFISH) was performed. In the absence of Syp, mamo transcription was initiated prematurely in response to weak Chinmo levels, yet mature transcripts failed to accumulate. This leads to the belief that Syp directly binds mamo mRNA and aids in its splicing, maturation and/or stabilization. This is consistent with the finding that overexpressing a mamo cDNA (lacking 5' UTR, 3' UTR and introns) was able to promote cell fate changes despite repression of Syp (Liu, 2019).

Mamo is required to produce the α'/β' neurons in the middle temporal window of the MB lineages. Trio positive α'/β' neurons are clearly absent after RNAi depletion of Mamo during development. Cell production does not appear to be altered, as mamo-RNAi expressing MBs are a normal size. This begs the question of which, if any terminal fate the middle-born neurons adopt in the absence of Mamo. The limited markers for each MB cell type makes it difficult to determine whether the middle-born neurons undergo fate transformation or simply lack terminal fate. The presence of a Fas-II negative lobe hints that some middle-born neurons may not carry temporal fate information, but phenotypic analysis is complicated by defects in γ neuron maturation/remodeling. Removing the γ neurons with chinmo-RNAi eliminates this complication, but it is still unclear whether, without Mamo, the neurons are transformed to the α/β fate. The Fas-II positive, α/β lobe appears enlarged, but it is difficult to tell whether all axons are Fas-II positive or whether Fas-II negative axons are comingled with α/β axons. Without a cell type-specific, cell body marker for α/β neurons, it is ambiguous whether the middle-born cells are transformed to α/β or whether they simply lack α'/β' temporal fate. A transformation to α/β fate would suggest that either α/β is the default fate of MB neurons (requiring no additional terminal selector) or that Mamo expression inhibits α/β specific factors (Liu, 2019).

Mamo's role in promoting α'/β' fate is further supported by Mamo overexpression phenotypes. Overexpression of Mamo in the MB is able to transform α/β and γ neurons to α'/β' neurons. In an otherwise wildtype scenario, overexpression of mamo did not transform every cell to α'/β' fate. Instead the α'/β' lobe was expanded and the other lobe seemed to be an amalgam of α and γ like lobes. This could be due to incomplete penetrance/low expression levels of the mamo transgene or it is possible that the α/β and γ cells retain their own terminal selector driven, cell-type specific gene expression, thus complicating the fate of the differentiated neuron. Mamo overexpression does not alter the specification factors Imp, Syp or Chinmo and presumably there are terminal selector genes expressed downstream of high Chinmo and possibly in Chinmo-absent cells. This seems a likely possibility when overexpressing Mamo in γ neurons. With Syp-RNAi, NBs are 'forever young' and divide into adulthood, persistently producing 'early-born' γ neurons. Interestingly when combining Syp-RNAi with the Mamo transgene, the newborn cells begin to take on a γ-like fate (expressing Abrupt) before a majority transform into an α'/β'-specific, strong Trio expression pattern and adopt α'/β'-like axon morphology. This suggests that Mamo functions downstream of the temporal fate specification genes, but is capable of overriding downstream signals in α/β and γ neurons to promote α'/β' terminal fate (Liu, 2019).

What this study describes about the BTB-ZF transcription factor, Mamo's role in α'/β' cell fate easily fits into the definition of a terminal selector gene, coined by Oliver Hobert. Terminal selector genes are a category of 'master regulatory' transcription factors that control the specific terminal identity features of individual neuronal types. Key aspects of terminal selector genes are that they are expressed post-mitotically in neurons as they mature and they are continuously expressed (often via autoregulatory mechanisms) to maintain the terminal differentiated state of the neuron. Correspondingly, mamo transcription is initiated in newborn, post-mitotic neurons and Mamo protein expression is visible beginning in young/maturing neurons. After transcription initiation, Mamo positively regulates its own expression and continues to be expressed in α'/β' neurons into adulthood. The other quintessential feature of terminal selector genes is that they regulate a battery of terminal differentiation genes, so that removing a terminal selector gene results in a loss of the specific identity features of a neuron type and misexpression can drive those features in other neurons. Indeed, removing Mamo with RNAi results in the loss of α'/β' identity, both developmentally and into adulthood. Further, overexpressing Mamo in either α/β or γ MB neurons results in shift to α'/β' fate. Individual terminal selectors do not often function alone, but in combination with other terminal selectors. Therefore, there are likely terminal selectors downstream of the MB NB-specific genes that contribute to each of the MB neuron types. In this way, the lineage-specific and temporal patterning programs can combine to define individual neuron types. This feature enables the reutilization of terminal selector genes to create disparate neuron types when used in distinct combinations (Hobert, 2016). This further suggests that temporally expressed Mamo serves as a temporally defined terminal selector gene in other lineages, such as the AL lineages that are describe in this study (Liu, 2019).

Altering Chinmo levels via upstream RNA-binding proteins or miRNAs, or by reducing Chinmo with RNAi all result in shifts in the ratio of neurons with different neuronal temporal fates. This evidence suggests a mechanism where Chinmo acts in newborn neurons to promote temporal fate specification. A recent publication suggested that Chinmo affects temporal fate via a neuronal remodeling mechanism by controlling Ecdysone signaling . Marchetti demonstrates that Chinmo is required for EcR-B1 expression; however it remains unclear whether Chinmo directly affects EcR-B1 expression or if the Chinmo-dependent EcR-B1 expression is the sole mechanism for γ neuron temporal fate specification. Moreover, neuronal temporal fate is not accurately determined by neuronal morphology alone, particularly when ecdysone signaling has known effects on MB cell morphology and fate. Ecdysone receptor signaling is highly pleiotropic, including ligand-independent functions making dominant-negative and overexpression studies difficult to interpret. Therefore, further investigation is needed to clarify the roles of Ecdysone receptor signaling in MB neuronal temporal fate and remodeling. This will be addressed in a follow-up paper. This current manuscript strongly promotes the idea that Chinmo functions in newborn neurons to promote temporal fate as weak Chinmo expression directly precedes Mamo transcription and Mamo is essential for specification and maintenance of α'/β' fate (Liu, 2019).

This study describes a multilayered hierarchical system to define distinct neuronal temporal fate that culminates in the expression of a terminal selector gene. Analogous mechanisms likely underlie temporal patterning in mammalian brains. However, whether orthologous genes play equivalent roles in mammalian temporal patterning has not been fully investigated. The Imp and Syp RNA-binding proteins are evolutionarily conserved. Both homologs are highly expressed in the developing mouse brain and play vital roles in neural development and/or neuronal morphology. The opposing functions of Imp and Syp also appear to be conserved, as the murine orthologs IMP1 and SYNCRIP bind the identical RNA to either promote or repress axon growth, respectively. Moreover, IMP1 expression in fetal mouse neural stem cells plays important roles in stem cell maintenance and proper temporal progression of neurogenesis. It would likewise be very interesting to explore SYNCRIP in the context of temporal patterning (Liu, 2019).

While Chinmo and Mamo have no clear mammalian orthologs, they are both BTB-ZF (broad-complex, tram-track and bric-à-brac - zinc finger) transcription factors. The BTB domain is a protein interaction domain that can form homo or heterodimers and also binds transcriptional regulators such as repressors, activators and chromatin remodelers. The C2H2 (Krüppel-like) zinc fingers bind DNA-providing target specificity. BTB-ZF proteins have been found to be critical regulators of developmental processes, including neural development. Indeed, the BTB-zinc finger protein, Zbtb20, appears to be essential for early-to-late neuronal identity in the mouse cortex. Zbtb20 is temporally expressed in cortical progenitors and knockout results in cortical layering defects, as the inside-out layering of the cortex follows neuronal birth order. While mutations of other brain-expressed BTB-ZF proteins also show cortical layering phenotypes, potential roles in temporal patterning have not been explored (Liu, 2019).

This study illustrates a fate specification process in which a layered series of temporal protein gradients guide the expression of terminal selector genes. The first-tier temporal gradients are expressed in neural stem cells, followed by a restricted expression window in newborn neurons to finally induce a terminal selector gene in a subset of neurons as they mature. This time-based subdivision of neuronal fate can likely be further partitioned, finally resulting in sequentially born neurons with distinct cell fates. This study demonstrates that Mamo, a BTB-ZF transcription factor, delineates α'/β' neurons, the middle temporal window of the MB lineages. Corresponding data in the AL lineages suggest that Mamo may serve as a temporally defined, terminal selector gene in a variety of lineages in the Drosophila brain. Mamo expression is regulated transcriptionally by the descending Chinmo BTB-ZF transcription factor gradient and post-transcriptionally by the Syp RNA binding protein. This multi-tiered, bimodal regulation ensures that only the progeny in a precise temporal window (those with both weak Chinmo and significant Syp levels) can effectively activate the terminal selector gene, mamo. This discovery attests to the power of gradients in creating diverse cells from a single progenitor. Utilizing layers of temporal gradients to define discrete temporal windows mirrors how in early embryos the spatial gradients of RNA-binding proteins and transcription factors specify the fly's A/P axis. This paradigm provides considerable complexity of gene network regulation, leading to abundant neural cell diversity (Liu, 2019).

Seven-up-triggered temporal factor gradients diversify intermediate neural progenitors

Building a sizable, complex brain requires both cellular expansion and diversification. One mechanism to achieve these goals is production of multiple transiently amplifying intermediate neural progenitors (INPs) from a single neural stem cell. Like mammalian neural stem cells, Drosophila type II neuroblasts utilize INPs to produce neurons and glia. Within a given lineage, the consecutively born INPs produce morphologically distinct progeny, presumably due to differential inheritance of temporal factors. To uncover the underlying temporal fating mechanisms, type II neuroblasts' transcriptome was profiled across time. The results reveal opposing temporal gradients of Imp and Syp RNA-binding proteins (descending and ascending, respectively). Maintaining high Imp throughout serial INP production expands the number of neurons and glia with early temporal fate at the expense of cells with late fate. Conversely, precocious upregulation of Syp reduces the number of cells with early fate. Furthermore, this study reveals that the transcription factor Seven-up initiates progression of the Imp/Syp gradients. Interestingly, neuroblasts that maintain initial Imp/Syp levels can still yield progeny with a small range of early fates. It is therefore proposed that the Seven-up-initiated Imp/Syp gradients create coarse temporal windows within type II neuroblasts to pattern INPs, which subsequently undergo fine-tuned subtemporal patterning (Ren, 2017).

Temporal gradients of IGF-II mRNA-binding protein (Imp) and Syncrip (Syp) RNA-binding proteins have recently been described to promote early and late temporal fates, respectively, in mushroom body (MB) and antennal lobe (AL) lineages (Liu, 2015). Imp and Syp gradients oppose each other (Imp expression decreases and Syp increases over time), and they mutually inhibit each other's expression (Liu, 2015). It is unclear how the Imp-versus-Syp dominance is reversed and why the Imp-to-Syp switch occurs over different time courses in distinct NBs. Imp/Syp gradients control temporal fate in the MB by establishing a temporal gradient of Chinmo, a BTB-zinc finger nuclear transcription factor. In the AL lineages, while Imp/Syp gradients clearly promote early and late fates, it is not clear whether and how these RNA binding proteins specify all temporal fates in the rapidly changing AL lineages (Ren, 2017).

Canonical (type I) NBs produce post-mitotic neurons via budding off ganglion mother cells (GMCs), which each divides once into two neurons. By contrast, each of the 16 type II NBs (eight per brain hemisphere) first generates a series of intermediate neural progenitors (INPs). Each INP can, in turn, produce around five GMCs, thus giving rise to an INP sublineage that consists of a short sequence of neuronal and glial progeny. The complex type II pattern of neurogenesis mimics the production of neurons by mammalian neural stem cells through intermediate precursors (Ren, 2017).

Like type I NBs, type II NBs exhibit lineage identity and temporal fate. Labeling the progeny made by a type II NB (NB clone) reveals distinct lineage-characteristic morphology. Six type II NB lineages (DM1-DM6) originate from the dorsomedial posterior brain surface; the remaining two lineages (DL1 and DL2) arise from the dorsolateral posterior brain surface. An INP produces an invariant sequence of distinct sister neuron pairs, and successive INPs generate a similar, but not identical, neuronal series (Wang, 2014). These observations illustrate temporal fate diversification along both axes of NB and INP self-renewal. A cascade of three tTFs (Dichaete, Grainy head, Eyeless) governs temporal fates within the INP sublineages (Bayraktar, 2013). As to the extended axis of type II NB self-renewal, it is not clear whether a protracted tTF cascade exists or just gradients of proteins could guide the orderly derivation of variant INP sublineages (Ren, 2017).

In order to resolve the temporal fating mechanisms in type II NBs, this study profiled the transcriptome of type II NBs and uncovered 81 dynamic genes, including Imp and Syp. Imp and Syp were shown to promote early and late INP temporal fate, respectively. Two tTFs, Castor (Cas) and Seven-up (Svp), are critical for the initiation of Imp/Syp temporal gradients. Despite no progression of the Imp/Syp gradients, svp mutant clones carried INPs with multiple early fates. It is proposed that Cas/Svp-triggered Imp/Syp gradients confer coarse temporal fates to diversify INPs (Ren, 2017).

RNA-seq of type II NBs across larval development revealed 81 temporally dynamic genes, including Imp and Syp. Utilizing various neuronal classes characteristic of early or late INP sublineages, this study demonstrated that the opposing Imp/Syp gradients govern type II lineage temporal patterning. However, it is not clear whether absolute or relative levels of Imp and/or Syp confer specific temporal fates, or how a given Imp/Syp level is perceived by individual GMCs of the same INP origin. More sophisticated controls over Imp and Syp levels and finer temporal fate readouts are required to resolve these details. Nonetheless, svp mutant NBs, which maintain initial Imp/Syp levels, still undergo some limited temporal fate progression, as evidenced by the presence of various OL-elaboration neurons. It is therefore proposed that the rapidly progressing Imp/Syp gradients confer type II NBs with coarse temporal fates, which guide or permit subtemporal patterning among INPs born within a given Imp/Syp temporal window (Ren, 2017).

A brief series of Cas and Svp bursts initiates the prompt switch of Imp-versus-Syp dominance in type II NBs. Cas likely precedes Svp in its post-embryonic re-expression; however, the dynamic expression of Cas and Svp appear independently yet simultaneously controlled by an unknown temporal cue. Curiously, while Cas is dispensable, ectopic Cas delays the onset of Imp/Syp gradients. As the final NB tTF in the widely expressed embryonic cascade, Cas may be required for some embryonic INP fates that were not examined in this study. With regards to the role of Cas in Imp/Syp expression, it is hypothesized that the termination of Cas expression is important for proper Imp/Syp gradient progression. The Cas-GOF would thus expose the NBs to a prolonged Cas window. Precocious and continuous Svp expression did not accelerate or alter the Imp/Syp gradients, further implicating involvement of unknown temporal cues, not only in the regulation of dynamic Cas/Svp expressions, but also in confining the acute Svp action. It is speculated that the acute Svp signal can repress Imp as well as promote Syp and ultimately place Svp over Imp in their winner-take-all competition. Both the timing and intensity of the Cas/Svp bursts may vary among different NBs, which can potentially shape distinct Imp/Syp gradients in different neuronal lineages (Ren, 2017).

The Imp/Syp gradients in type II NBs are inherited by INPs born at distinct times and confer the INPs with their temporal identity. Each INP subsequently expresses a cascade of tTFs to assign its serially derived GMCs with distinct cell fates. Imp/Syp and their downstream effectors could interact with the INP tTFs to specify terminal temporal fates in post-mitotic cells. Multiple feedforward gene regulatory loops might also be involved to control the expression of terminal selector genes. Distinct Imp/Syp levels may specify different neuronal classes among the GMCs of the same birth order that inherit the same tTF from INPs. It is not possible to resolve such complex temporal fating mechanisms without further sophisticated single-cell lineage mapping tools (Ren, 2017).

Converging evidence indicates that conserved tTFs control neuronal diversity from Drosophila to mammalian species. Mammalian neural stem cells also show temporally patterned neurogenesis. The Ikaros family zinc finger 1, orthologs of Drosophila Hb, specify early temporal fate in both the retina and the cortex. A recent study showed that an ortholog of Drosophila castor, Casz1, promotes late neuronal fates in the mouse retina. The Chick Ovalbumin Upstream Promoter-Transcription Factors (COUP-TFI and II), mammalian orthologs of Drosophila svp, promote the temporal transition from neurogenesis to gliogenesis in neural stem cells. COUP-TFI also orchestrates the serial generation of distinct types of cortical interneurons. Loss of COUP-TFs resulted in overproduction of early-born neuronal fates, at the expense of late-born glial and interneuron fates. Thus, COUP-TFs seem to be functionally conserved to Drosophila svp (Ren, 2017).

Notably, a descending Imp gradient exists in mouse neural stem cells and governs temporal changes in stem cell properties. Given the use of INPs in both type II NB lineages and mammalian neurogenesis, it would be interesting to determine whether homologs of Imp/Syp/Chinmo play analogous roles in regulating the temporal fates of mammalian neural stem cells and whether these genes act with the conserved tTFs (Ren, 2017).

Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity

An important question in neuroscience is how stem cells generate neuronal diversity. During Drosophila embryonic development, neural stem cells (neuroblasts) sequentially express transcription factors that generate neuronal diversity; regulation of the embryonic temporal transcription factor cascade is lineage-intrinsic. In contrast, larval neuroblasts generate longer ~50 division lineages, and currently only one mid-larval molecular transition is known: Chinmo/Imp/Lin-28+ neuroblasts transition to Syncrip+ neuroblasts. This study shows that the hormone ecdysone is required to down-regulate Chinmo/Imp and activate Syncrip, plus two late neuroblast factors, Broad and E93. Seven-up triggers Chinmo/Imp to Syncrip/Broad/E93 transition by inducing expression of the Ecdysone receptor in mid-larval neuroblasts, rendering them competent to respond to the systemic hormone ecdysone. Importantly, late temporal gene expression is essential for proper neuronal and glial cell type specification. This is the first example of hormonal regulation of temporal factor expression in Drosophila embryonic or larval neural progenitors (Syed, 2017).

This study shows that the steroid hormone ecdysone is required to trigger a major gene expression transition at mid-larval stages: central brain neuroblasts transition from Chinmo/Imp to Broad/Syncrip/E93. Furthermore, it was shown that Svp activates expression of EcR-B1 in larval neuroblasts, which gives them competence to respond to ecdysone signaling, thereby triggering this gene expression transition. Although a global reduction of ecdysone levels is likely to have pleiotropic effects on larval development, multiple experiments were performed to show that the absence or delay in late temporal factor expression following reduced ecdysone signaling is not due to general developmental delay. First, the EcR gene itself is expressed at the normal time (~56 hr) in the whole organism ecdysoneless¹ mutant, arguing strongly against a general developmental delay. Second, a type II neuroblast seven-up mutant clone shows a complete failure to express EcR and other late factors, in the background of an entirely wild type larvae; this is perhaps the strongest evidence that the phenotypes that are described are not due to a general developmental delay. Third, lineage-specific expression of EcR dominant negative leads to loss of Syncrip and E93 expression without affecting Broad expression; the normal Broad expression argues against a general developmental delay. Fourth, live imaging was used to directly measure cell cycle times, and it was found that lack of ecdysone did not slow neuroblast cell cycle times. Taken together, these data support the conclusion that ecdysone signaling acts directly on larval neuroblasts to promote an early-to-late gene expression transition (Syed, 2017).

The role of ecdysone in regulating developmental transitions during larval stages has been well studied; it can induce activation or repression of suites of genes in a concentration dependent manner. Ecdysone induces these changes through a heteromeric complex of EcR and the retinoid X receptor homolog Ultraspiracle. Ecdysone is required for termination of neuroblast proliferation at the larval/pupal transition, and is known to play a significant role in remodeling of mushroom body neurons and at neuromuscular junctions. This study adds to this list another function: to trigger a major gene expression transition in mid-larval brain neuroblasts (Syed, 2017).

Does ecdysone signaling provide an extrinsic cue that synchronizes larval neuroblast gene expression? Good coordination of late gene expression is not seen, arguing against synchronization. For example, Syncrip can be detected in many neuroblasts by 60 hr, whereas Broad appears slightly later at ~72 hr, and E93 is only detected much later at ~96 hr, by which time Broad is low. This staggered expression of ecdysone target genes is reminiscent of early and late ecdysone-inducible genes in other tissues. In addition, for any particular temporal factor there are always some neuroblasts expressing it prior to others, but not in an obvious pattern. It seems the exact time of expression can vary between neuroblasts. Whether the pattern of response is due to different neuroblast identities, or a stochastic process, remains to be determined (Syed, 2017).

It has been shown preiously that the Hunchback-Krüppel-Pdm-Castor temporal gene transitions within embryonic neuroblasts are regulated by neuroblast-intrinsic mechanisms: they can occur normally in neuroblasts isolated in culture, and the last three factors are sequentially expressed in G2-arrested neuroblasts. Similarly, optic lobe neuroblasts are likely to undergo neuroblast-intrinsic temporal transcription factor transitions, based on the observation that these neuroblasts form over many hours of development and undergo their temporal transitions asynchronously. In contrast, this study shows that ecdysone signaling triggers a mid-larval transition in gene expression in all central brain neuroblasts (both type I and type II). Although ecdysone is present at all larval stages, it triggers central brain gene expression changes only following Svp-dependent expression of EcR-B1 in neuroblasts. Interestingly, precocious expression of EcR-B1 (worniu-gal4 UAS-EcR-B1) did not result in premature activation of the late factor Broad, despite the forced expression of high EcR-B1 levels in young neuroblasts. Perhaps there is another required factor that is also temporally expressed at 56 hr. It is also noted that reduced ecdysone signaling in ecd^ts mutants or following EcRDN expression does not permanently block the Chinmo/Imp to Broad/Syncrip/E93 transition; it occurs with variable expressivity at 120-160 hr animals (pupariation is significantly delayed in these ecd^ts mutants), either due to a failure to completely eliminate ecdysone signaling or the presence of an ecdysone-independent mechanism (Syed, 2017).

A small but reproducible difference was found in the effect of reducing ecdysone levels using the biosynthetic pathway mutant ecd^ts versus expressing a dominant negative EcR in type II neuroblasts. The former genotype shows a highly penetrant failure to activate Broad in old neuroblasts, whereas the latter genotype has normal expression of Broad (despite failure to down-regulate Chinmo/Imp or activate E93). This may be due to failure of the dominant negative protein to properly repress the Broad gene. Differences between EcRDN and other methods of reducing ecdysone signaling have been noted before (Syed, 2017).

Drosophila Svp is an orphan nuclear hormone receptor with an evolutionarily conserved role in promoting a switch between temporal identity factors. In Drosophila, Svp it is required to switch off hunchback expression in embryonic neuroblasts, and in mammals the related COUP-TF1/2 factors are required to terminate early-born cortical neuron production, as well as for the neurogenic to gliogenic switch. This study showed that Svp is required for activating expression of EcR, which drives the mid-larval switch in gene expression from Chinmo/Imp to Syncrip/Broad/E93 in central brain neuroblasts. The results are supported by independent findings that svp mutant clones lack expression of Syncrip and Broad in old type II neuroblasts (Tsumin Lee, personal communication to Chris Doe). Interestingly, Svp is required for neuroblast cell cycle exit at pupal stages, but how the early larval expression of Svp leads to pupal cell cycle exit was a mystery. The current results provide a satisfying link between these findings: Svp was shown to activate expression of EcR-B1, which is required for the expression of multiple late temporal factors in larval neuroblasts. Any one of these factors could terminate neuroblast proliferation at pupal stages, thereby explaining how an early larval factor (Svp) can induce cell cycle exit five days later in pupae. It is interesting that one orphan nuclear hormone receptor (Svp) activates expression of a second nuclear hormone receptor (EcR) in neuroblasts. This motif of nuclear hormone receptors regulating each other is widely used in Drosophila, C. elegans, and vertebrates (Syed, 2017).

The position of the Svp+ neuroblasts varied among the type II neuroblast population from brain-to-brain, suggesting that Svp may be expressed in all type II neuroblasts but in a transient, asynchronous manner. This conclusion is supported by two findings: the svp-lacZ transgene, which encodes a long-lived β-galactosidase protein, can be detected in nearly all type II neuroblasts; and the finding that Svp is required for EcR expression in all type II neuroblasts, consistent with transient Svp expression in all type II neuroblasts. It is unknown what activates Svp in type II neuroblasts; its asynchronous expression is more consistent with a neuroblast-intrinsic cue, perhaps linked to the time of quiescent neuroblast re-activation, than with a lineage-extrinsic cue. It would be interesting to test whether Svp expression in type II neuroblasts can occur normally in isolated neuroblasts cultured in vitro, similar to the embryonic temporal transcription factor cascade (Syed, 2017).

Castor and its vertebrate homolog Cas-Z1 specify temporal identity in Drosophila embryonic neuroblast lineages and vertebrate retinal progenitor lineages, respectively (Mattar, 2015). Although this study shows that Cas is not required for the Chinmo/Imp to Syncrip/Broad/E93 transition, it has other functions. Cas expression in larval neuroblasts is required to establish a temporal Hedgehog gradient that ultimately triggers neuroblast cell cycle exit at pupal stages (Syed, 2017).

Drosophila embryonic neuroblasts change gene expression rapidly, often producing just one progeny in each temporal transcription factor window. In contrast, larval neuroblasts divide ~50 times over their 120 hr lineage. Mushroom body neuroblasts make just four different neuronal classes over time, whereas the AD (ALad1) neuroblast makes ~40 distinct projection neuron subtypes. These neuroblasts probably represent the extremes (one low diversity, suitable for producing Kenyon cells; one high diversity, suitable for generating distinct olfactory projection neurons). This study found that larval type II neuroblasts undergo at least seven molecularly distinct temporal windows. If it is assumed that the graded expression of Imp (high early) and Syncrip (high late) can specify fates in a concentration-dependent manner, many more temporal windows could exist (Syed, 2017).

This study illuminates how the major mid-larval gene expression transition from Chinmo/Imp to Broad/Syncrip/E93 is regulated; yet many new questions have been generated. What activates Svp expression in early larval neuroblasts - intrinsic or extrinsic factors? How do type II neuroblast temporal factors act together with Dichaete, Grainy head, and Eyeless INP temporal factors to specify neuronal identity? Do neuroblast or INP temporal factors activate the expression of a tier of 'morphogenesis transcription factors' similar to leg motor neuron lineages? What are the targets of each temporal factor described here? What types of neurons (or glia) are made during each of the seven distinct temporal factor windows, and are these neurons specified by the factors present at their birth? The identification of new candidate temporal factors in central brain neuroblasts opens up the door for addressing these and other open questions (Syed, 2017).

Coopted temporal patterning governs cellular hierarchy, heterogeneity and metabolism in Drosophila neuroblast tumors

It is still unclear what drives progression of childhood tumors. During Drosophila larval development, asymmetrically-dividing neural stem cells, called neuroblasts, progress through an intrinsic temporal patterning program that ensures cessation of divisions before adulthood. Previous work has shown that temporal patterning also delineates an early developmental window during which neuroblasts are susceptible to tumor initiation. Using single-cell transcriptomics, clonal analysis and numerical modeling, this study now identifies a network of twenty larval temporal patterning genes that are redeployed within neuroblast tumors to trigger a robust hierarchical division scheme that perpetuates growth while inducing predictable cell heterogeneity. Along the hierarchy, temporal patterning genes define a differentiation trajectory that regulates glucose metabolism genes to determine the proliferative properties of tumor cells. Thus, partial redeployment of the temporal patterning program encoded in the cell of origin may govern the hierarchy, heterogeneity and growth properties of neural tumors with a developmental origin (Genovese, 2019).

Central nervous system (CNS) tumors are rare and constitute less than 2% of all cancers in adults. In contrast, they represent more than 25% of cancer cases in children (including medulloblastoma, retinoblastoma, rhabdoid tumors (AT/RT), gliomas etc), suggesting that the developing CNS is particularly sensitive to malignant transformation. Moreover, unlike most adult tumors, pediatric tumors are often genetically stable and their initiation and progression do not necessarily require the accumulation of mutations in multiple genes. For example, the biallelic inactivation of a single gene is sometimes sufficient to trigger malignant growth as illustrated by mutations in the RB1 and SMARCB1 genes in retinoblastoma and rhabdoid tumors respectively. Recent studies suggest that CNS pediatric tumors such as medulloblastomas recapitulate the fetal transcription program that was active in the cell of origin. However, it remains unclear how the invalidation of single genes during fetal stages can disrupt on-going developmental programs to trigger malignant growth, and whether these fetal/developmental programs influence the heterogeneity, composition, and proliferative properties of cells composing CNS tumors (Genovese, 2019).

Faced with the complexity of brain development and neural tumors in mammals, simple animal models can represent a powerful alternative to investigate basic and evolutionary conserved principles. The development of the CNS is undoubtedly best understood in Drosophila. The Drosophila CNS arises from a small pool of asymmetrically-dividing neural stem cells (NSCs), called neuroblasts (NBs). NBs possess a limited self-renewing potential. They divide all along development (embryonic and larval stages) to self-renew while generating daughter cells named Ganglion Mother Cells (GMCs). GMCs then usually divide once to produce two post-mitotic neurons or glia. NBs are the fastest cycling cells during development, able to divide every hour during larval stages when most of the neurons are produced. However, all NBs terminate during metamorphosis and are absent in adults. Two antagonistic RNA-binding proteins, IGF-II mRNA-binding protein (Imp) and Syncrip (Syp) are essential to first promote and then conclude this formidable period of activity. During early larval development (L1/L2), NBs express Imp that promotes NB self-renewal. Around late L2/early L3, NBs silence Imp to express Syp that remains expressed until NB decommissioning during metamorphosis. This Imp-to-Syp transition is essential to render NBs competent to respond to subsequent pupal pulses of the steroid hormone ecdysone and initiate a last differentiative division. Failure to trigger the transition results in NBs permanently dividing in adults. The Imp-to-Syp transition appears to be mainly regulated by a NB intrinsic timing mechanism driven by the sequential expression of transcription factors. This series of factors, also known as temporal transcription factors, has been first identified for its ability to specify different neuronal fates produced by NBs as they divide. In addition, temporal transcription factors also schedule the Imp-to-Syp transition to ensure that NBs will not continue cycling in adults. Recent transcriptomic analyses indicate that other genes are dynamically transcribed in NBs throughout larval stages, although their function and epistatic relationship with temporal transcription factors and the Imp/Syp module are unclear. All together, these studies highlight a complex, but still relatively unexplored, temporal patterning system in larval NBs (Genovese, 2019).

Perturbation of the asymmetric division process during early development can lead to NB exponential amplification. In such conditions, the NB-intrinsic temporal program limiting self-renewal appears to become inoperant, and uncontrolled NB amplification is observed. Serial transplantations of asymmetric division-defective NBs have revealed an ability to proliferate for months, if not years, demonstrating tumorigenic characteristics. Perturbation of asymmetric divisions can be induced by the inactivation of the transcription factor Prospero (Pros) in type I NB lineages (most lineages in the ventral nerve cord (VNC) and central brain (CB)). During development, Pros is strongly expressed in GMCs where it accumulates to induce cell cycle-exit and neuronal or glial differentiation. GMCs that lack pros fail to differentiate and revert to a NB-like state. This triggers rapid NB amplification at the expense of neuron production. Previous work has shown that inactivation of pros in NBs, and their subsequent GMCs, before mid-L3 (L3 being the last larval stage) leads to aggressive NB tumors that persist growing in adults. In contrast, inactivation of pros after mid-L3 leads to transient NB amplification and most supernumerary NB properly differentiate during metamorphosis, leading to an absence of growing tumors in adults. Interestingly, propagation of NB tumor growth beyond normal developmental stages is caused by the aberrant maintenance of Imp and the transcription factor Chinmo from early-born GMCs, the latter representing the cells of origin of such aggressive tumors (Narbonne-Reveau, 2016). Chinmo and Imp positively cross-regulate and inactivation of either in NB tumors stops tumorigenic growth. Because pros-/- NB tumors can only be induced during an early window of development, and are caused by the biallelic inactivation of a single gene, they represent an exciting and simple model to investigate the basic mechanisms driving the growth of tumors with an early developmental origin, such as in the case of pediatric CNS cancers (Genovese, 2019).

NB tumors can also be induced from type II NBs (a small subset of NBs in the central brain) or from neurons upon inactivation of the NHL-domain family protein Brat or Nerfin-1 respectively. In both cases, tumor growth appears to rely on the aberrant expression of the Chinmo/Imp module arguing for a general tumor-driving mechanism in the developing Drosophila CNS (Narbonne-Reveau, 2016). Interestingly, in the different types of NB tumors, Chinmo and Imp are only expressed in a subpopulation of cells, demonstrating heterogeneity in the population of tumor NBs (tNBs). However, the full repertoire of cells composing the tumor, the rules governing the cellular heterogeneity and the mechanisms determining the proliferative potential of each cell type remain to be investigated (Genovese, 2019).

This study used single-cell RNA-seq, clonal analysis and numerical modeling to investigate these questions. A subset of genes involved in the temporal patterning of larval NBs were identified that are redeployed in tumors to generate a differentiation trajectory responsible for creating tumor cell heterogeneity. This cellular heterogeneity results in NBs with different types of metabolism and different proliferative properties. This study also deciphered a robust hierarchical scheme that drives reproducible heterogeneity through the dysregulated but fine-tuned transition between the two RNA-binding proteins Imp and Syp. This work thus identifies a core larval NB temporal patterning program, the disruption of which not only causes unlimited growth but has an overarching role in governing the cellular hierarchy, heterogeneity and metabolism of NB tumors (Genovese, 2019).

This study demonstrates that temporal patterning, not only determines which cells are susceptible to cancer transformation during development (Narbonne-Reveau, 2016), but also has an overarching role in governing different aspects of CNS tumor organization such as hierarchy, heterogeneity and the proliferative properties of the different types of cells via the regulation of their metabolism (Genovese, 2019).

Given the recent discovery that temporal patterning is conserved in the developing mammalian brain (Telley, 2019), this study could shed light on an ancestral mechanism that governs the progression of CNS tumors with developmental origins (Genovese, 2019).

The rules governing the initiation and progression of CNS pediatric tumors that often exhibit stable genomes are still unclear. Previousl work has demonstrated that temporal patterning in Drosophila larval NBs delineates a window of time during which the Chinmo/Imp oncogenic module is expressed and makes early larval NBs prone to malignant transformation (Narbonne-Reveau, 2016). This study finds that after tumor initiation, temporal patterning is partly recapitulated in tNBs where it generates differentiation trajectories to constrain tumor composition and growth. This is illustrated by the presence of about 20 genes (Imp, chinmo, Lin-28, E23, Oatp74D, Gapdh2, Sip1/CG10939, plum/CG6490, SP1173, Chd64, CG10512, CG44325, CG5953, Syp, E93, lncRNA:noe, CG15646 and stg), previously identified to be temporally regulated in some larval NBs, that are differentially regulated along the pseudotime/differentiation trajectory reconstructed from single-cell RNA-seq analysis of tNBs, and/or differentially expressed in Imp+ vs Syp+ tNBs. Thus, this study identified what appears to be a subset of a core temporal patterning program encoded in central brain and ventral nerve cord NBs that becomes deregulated upon asymmetric-division defects during early development (Genovese, 2019).

Notably, the larval temporal transcription factor Cas and Svp, known to schedule the Imp-to-Syp transition during development are not enriched in Imp+ tNBs suggesting that they do not play a role in regulating the Imp-to-Syp transition along the trajectory in tumors. Interestingly, while Syp is transcriptionally regulated in larval NBs, it seems rather post-transcriptionally regulated in tNBs as Syp RNAs are present throughout all clusters. This suggests that different mechanisms may be operating in tumors than during development to regulate the Imp-to-Syp transition (Genovese, 2019).

This study observed that the proportions of Imp+ and Syp+ tNBs systematically reach an equilibrium over a few days with a 20/80 (+/-10) ratio in poxn > prosRNAi tumors. This suggests that the regulation of the Imp-to-Syp transition in tumors is not random and the predictability of the final proportions possibly implies robust underlying constraints. By investigating the population dynamics of Imp+ and Syp+ tNBs in prosRNAi tumors, this study has deciphered a finely tuned hierarchical division scheme that appears to constrain the growth and cellular heterogeneity of the tumor. Imp+ tNBs is shown in the tumorigenic context favor a symmetric self-renewing mode of divisions (in more than 60% of divisions) while unlikely to exit the cell-cycle. This allows the perpetuation of a small subset of Imp+ tNBs that are endowed with a seemingly unlimited self-renewing potential by the Imp/Chinmo module. Imp+ tNBs can also make symmetric and asymmetric divisions that generate Syp+ tNBs, leading to the production of a population of Syp+E93+ tNBs that accumulates through limited self-renewal, and have a high propensity for exiting the cell-cycle. Moreover, this study could shows that Syp+E93+ tNBs are unable to generate Imp+ tNBs, demonstrating a rigid cellular hierarchy reminiscent of development. In addition, in this context, Syp acts as a tumor suppressor by limiting tNB proliferation while Imp acts as an oncogene by promoting tNB proliferation and propagation of tumor growth. Together, these data argue for a scenario where cooption of the Imp-to-Syp transition is responsible for installing a hierarchical mode of tumor growth with Imp+ tNBs propagating unlimited growth in a CSC-like manner, while Syp+E93+ tNBs acts as transient amplifying progenitors with limited self-renewing abilities. Although the Imp/Syp RNA-binding proteins have an essential and antagonistic role in governing the proliferative properties of tumor cells, the function of the other redeployed temporal patterning genes is unknown (except for chinmo, downstream to Imp and Syp, that is essential for tumor growth). As many are linked with the Imp+ tNB state, it will be important in the future to decipher how they contribute to establish or maintain the CSC-like identity (Genovese, 2019).

The division parameters defined by clonal analysis and modeling approach could capture both the hierarchical aspect of tumor growth as well as the global population dynamics: from an initial homogenous pool of larval Imp+ tNBs to the stable heterogeneity observed during adulthood. It could also resolve the paradoxical observation that Chinmo+Imp+ tNBs end up in minority despite exhibiting a higher average mitotic rate. Although, like all models, it is not expected that this model would perfectly recapitulate all the parameters regulating tumor growth and heterogeneity (for example, this study has neglected apoptosis and neuronal differentiation that occur at low levels), it is thought that this model provides a reasonable and useful ground on which further studies can be performed for a more detailed understanding. On these lines, while the division pattern this study has described with a numerical model provides estimates of division probabilities in poxn > prosRNAi tumors, it says nothing as to how these probabilities are biologically set within the tumor. A possible scenario is that cell fate determination upon division relies on signals received by immediate neighboring tumor cells, resulting in effective probabilities at the scale of the whole tumor. Such a micro-environment dependent regulation of the Imp-to-Syp transition in tumors would strongly contrast with the cell-intrinsic regulation of the Imp-to-Syp transition that systematically occurs in NBs around early L3. Future studies will aim at deciphering the mechanisms that interfere with the developmental progression of the temporal patterning, upon asymmetric-division defects, to favor the self-renewing mode of divisions undergone by the Chinmo+Imp+ tNBs, allowing perpetuation of a population of CSC-like cells (Genovese, 2019).

Noteworthy, prosRNAi and snr1/dSmarcb1RNAi tumors exhibit different but reproducible ratios of Imp+ and Syp+ tNBs. This suggests the existence of tumor-specific mechanisms that fine-tune the Imp-to-Syp transition. Such mechanisms may be related to the tumor cell of origin, or to the genetic insult that initiated NB amplification. Further analysis will help identifying tumor-intrinsic signals regulating the balance between Chinmo+Imp+ tNBs and Syp+E93+ tNBs in various types of NB tumors (Genovese, 2019).

Until recently, the existence of temporal patterning in mammalian neural progenitors remained uncertain. Elegant single-cell transcriptomic studies of embryonic cortical and retinal progenitors in mice have now revealed that they transit through different transcriptional states that are transmitted to their progeny to generate neuronal diversity, similar to temporal patterning in Drosophila (Clark, 2019; Telley, 2019). However, it remains unknown whether temporal patterning determines the cell of origin and governs the growth of CNS tumors in children. Along these lines, the finding that the transcriptional programs operating in cerebellar progenitors during fetal development are recapitulated in medulloblastomas is promising (Vladoiu, 2019). By uncovering the overarching role of temporal patterning in governing tumor susceptibility during CNS development and in constraining tumor properties during cancer progression in Drosophila, this work thus possibly provides a new conceptual framework to better understand CNS tumors in children (Genovese, 2019).

Because of the difficulty to investigate metabolism at the single-cell level, it has been difficult to determine how heterogeneous is the metabolic activity of cells in tumors, and how it is controlled. Using a combination of single-cell and bulk RNA-seq approaches, this study has found that progression of temporal patterning provides a tumor-intrinsic mechanism that generates heterogeneity in the proliferative abilities of tumor cells through the progressive silencing of glucose and glutamine metabolism genes (Genovese, 2019).

Consequently, Chinmo+Imp+ tNBs, that lie at the top of the hierarchy, highly express glycolytic and respiratory/OXPHOS genes, as well as Gdh, that are down-regulated by the Imp-to-Syp transition. This default high expression of both glutamine and glucose metabolism genes in CSC-like Chinmo+Imp+ tNBs likely favors sustained self-renewal, but could also confer plasticity and a way to adapt cellular metabolism to different environmental conditions as frequently observed in CSCs (e.g., glutamine can compensate for glucose shortage) (Sancho, 2016) (Genovese, 2019).

This study showed that Syp+E93+ tNBs exhibit a reduced size, and that knock-down of glycolytic (Gapdh1 or Pglym78) or respiratory/OXPHOS genes (Cyt-c-p or Cyt-C1) prevented propagation of tumor growth in adults. Thus, reduction of biosynthesis and energy production through down-regulation of glucose and glutamine metabolism genes after the Imp-to-Syp transition could progressively exhaust Syp+E93+ tNB growth and self-renewing ability, ultimately leading to cell-cycle exit (Genovese, 2019).

With the demonstration that temporal patterning regulates glycolytic, TCA cycle and OXPHOS genes in NB tumors, this work provides a tumor-intrinsic mechanism that creates metabolic heterogeneity to control the proliferative potential of the various tumor cells. It was also observed that Syp+E93+ tNBs associated with lowest levels of metabolic and cell-cycle genes also upregulate genes of the E(spl) genes. Interestingly, expression of Hes genes (orthologs of Enhancer of split genes) in vertebrate neural stem cells is associated with the maintenance of a quiescent state in adults. Thus, E(spl) genes may promote the quiescent tNB state identified with the clonal and numerical analysis while preventing their differentiation in neurons (Genovese, 2019).

Down-regulation of the mRNA levels of metabolic genes after the Imp-to-Syp transition could be due to the silencing of a transcriptional activator or to an increased mRNA degradation. On one hand, Chinmo is a likely candidate for the first scenario, as its inactivation reduces growth in NBs (Narbonne-Reveau, 2016) and this study showed that it is a direct target of both Imp and Syp. On the other hand, the second scenario is consistent with Imp orthologs in human being able to promote OXPHOS and proliferation in glioma cells, through the post-transcriptional regulation of mitochondrial respiratory chain complex subunits (Genovese, 2019).

This study has also identified a small population of tNBs expressing various stress or growth arrest factors. One of these factors, Xrp1, is a transcriptional target of p53 in the response to irradiation. Xrp1 expression has also recently been linked to defects in translation rates, together with the expression of Irbp18 and GstE6. Thus, these factors may label a subset of tNBs undergoing DNA or translational stress. The reason and consequences of such cellular stresses in tumor progression need to be further investigated (Genovese, 2019).

Transcriptomic analyses have revealed strong similarities in the differentiation trajectories of tNBs in tumors and of NBs in larvae. Yet, it is surprising that the down-regulation of glutamine and glucose metabolism genes has not been detected in NBs during larval development, after the Imp-to-Syp transition (Ren, 2017). It is possible that the glial niche surrounding NBs, that is known to influence NB growth properties during larval stages, somehow sustains high levels of glucose metabolism genes in late Syp+E93+ NBs. Given that this glial niche is absent in tumors, Syp+E93+ tNBs may not be able to sustain the high expression of metabolic genes imposed by the Imp/Chinmo module, leading to progressive cell-cycle exit (Genovese, 2019).

Chinmo and Imp are reminiscent to oncofetal genes in mammals, in that their expression decrease rapidly as development progresses while they are mis-expressed in tumors. Along these lines, the three IMP orthologs in humans (also called IGF2BP1-3) are also known as oncofetal genes. They emerge as important regulators of cell proliferation and metabolism in many types of cancers including pediatric neural cancers. Along evolution, the ancestral Syncrip gene has been subjected to several rounds of duplication and has diverged into five paralogs in mammals, some of them emerging as tumor suppressors with an important role in tumor progression (Genovese, 2019).

Thus, the respective oncogenic and tumor suppressor roles of IMP and SYNCRIP gene families appear to have been conserved in humans and they may not be restricted to tumors of neural origin. This study therefore raises the exciting possibility that these two families of RNA-binding proteins form a master module at the top of the self-renewal/differentiation cascades, that regulates CSC populations and hierarchy in a spectrum of human cancers (Genovese, 2019).

Extrinsic activin signaling cooperates with an intrinsic temporal program to increase mushroom body neuronal diversity

Temporal patterning of neural progenitors leads to the sequential production of diverse neurons. To understand how extrinsic cues influence intrinsic temporal programs, Drosophila mushroom body progenitors (neuroblasts) were studied that sequentially produce only three neuronal types: γ, then α'β', followed by αβ. Opposing gradients of two RNA-binding proteins Imp and Syp comprise the intrinsic temporal program. Extrinsic activin signaling regulates the production of α'β' neurons but whether it affects the intrinsic temporal program was not known. This study shows that the activin ligand Myoglianin from glia regulates the temporal factor Imp in mushroom body neuroblasts. Neuroblasts missing the activin receptor Baboon have a delayed intrinsic program as Imp is higher than normal during the α'β' temporal window, causing the loss of α'β' neurons, a decrease in αβ neurons, and a likely increase in γ neurons, without affecting the overall number of neurons produced. These results illustrate that an extrinsic cue modifies an intrinsic temporal program to increase neuronal diversity (Rossi, 2020).

The building of intricate neural networks during development is controlled by highly coordinated patterning programs that regulate the generation of different neuronal types in the correct number, place and time. The sequential production of different neuronal types from individual progenitors, i.e. temporal patterning, is a conserved feature of neurogenesis. For instance, individual radial glia progenitors in the vertebrate cortex sequentially give rise to neurons that occupy the different cortical layers in an inside-out manner. In Drosophila, neural progenitors (called neuroblasts) also give rise to different neuronal types sequentially. For example, projection neurons in the antennal lobe are born in a stereotyped temporal order and innervate specific glomeruli. In both of these examples, individual progenitors age concomitantly with the developing animal (e.g., from embryonic stages 11-17 in mouse and from the first larval stage (L1) to the end of the final larva stage (L3) in Drosophila). Thus, these progenitors are exposed to changing environments that could alter their neuronal output. Indeed, classic heterochronic transplantation experiments demonstrated that young cortical progenitors placed in an old host environment alter their output to match the host environment and produce upper-layer neurons (Rossi, 2020).

The adult Drosophila central brain is built from ~100 neuroblasts that divide continuously from L1 to L3. Each asymmetric division regenerates the neuroblast and produces an intermediate progenitor called ganglion mother cell (GMC) that divides only once, typically producing two different cell types. Thus, during larval life central brain neuroblasts divide 50-60 times, sequentially producing many different neuronal types. All central brain neuroblasts progress through opposing temporal gradients of two RNA-binding proteins as they age: IGF-II mRNA binding protein (Imp) when they are young and Syncrip (Syp) when they are old. Loss of Imp or Syp in antennal lobe or Type II neuroblasts affects the ratio of young to old neuronal types. Imp and Syp also affect neuroblast lifespan. Thus, a single temporal program can affect both the diversity of neuronal types produced and their numbers (Rossi, 2020).

Since central brain neuroblasts produce different neuronal types through developmental time, roles for extrinsic cues have recently garnered attention. Ecdysone triggers all the major developmental transitions including progression into the different larval stages and entry in pupation. The majority of central brain neuroblasts are not responsive to ecdysone until mid-larval life when they begin to express the Ecdysone Receptor (EcR). Expressing a dominant-negative version of EcR (EcR-DN) in Type II neuroblasts delays the Imp to Syp transition that normally occurs ~60 hr after larval hatching (ALH). This leads to many more cells that express the early-born marker gene Repo and fewer cells that express the late-born marker gene Bsh (Rossi, 2020).

To further understand how extrinsic signals contribute to temporal patterning, Drosophila mushroom body neuroblasts were studied because of the deep understanding of their development. The mushroom body is comprised of ~2000 neurons (Kenyon cells) that belong to only three main neuronal types that have unique morphologies and play distinct roles in learning and memory. They receive input mainly from ~200 projection neurons that each relays odor information from olfactory receptor neurons. Each projection neuron connects to a random subset of Kenyon cells and each Kenyon cell receives input from ~7 different projection neurons. This connectivity pattern requires a large number of mushroom body neurons (~2,000) to represent complex odors. To produce this very large number of neurons, mushroom body development is unique in many respects. Mushroom body neurons are born from four identical neuroblasts that divide continuously (unlike any other neuroblast) from the late embryonic stages until the end of pupation (~9 days for ~250 divisions each). Furthermore, the two neurons born from each mushroom body GMC are identical. The neuronal simplicity of the adult mushroom body makes it ideal to study how extrinsic cues might affect diversity since the loss of any single neuronal type is obvious given that each is represented hundreds of times (Rossi, 2020).

The three main neuronal types that make up the adult mushroom body are produced sequentially during neurogenesis: first γ, followed by α'β', and then αβ neurons (see α'β' neurons are not generated from babo mutant neuroblasts), representing the simplest lineage in the central brain. The γ temporal window extends from L1 (the first larval stage) until mid-L3 (the final larval stage) when animals attain critical weight and are committed to metamorphosis; the α'β' window from mid-L3 to the beginning of pupation, and the αβ window from pupation until eclosion (the end of development). Like all other central brain neuroblasts Imp and Syp are expressed by mushroom body neuroblasts, but in much shallower gradients through time, which accounts for their extended lifespan. Imp and Syp are inherited by newborn neurons where they instruct temporal identity. Imp positively and Syp negatively regulate the translation of chronologically inappropriate morphogenesis (chinmo), a gene encoding a transcription factor that acts as a temporal morphogen in neurons. The first-born γ neurons are produced for the first ~85 cell divisions, when Imp levels in neuroblasts, and thus Chinmo in neurons, are high. α'β' neurons are produced for the next ~40 divisions, when Imp and Syp are at similar low levels that translate into lower Chinmo levels in neurons. Low Chinmo then regulates the expression in neurons of maternal gene required for meiosis (mamo), which encodes a transcription factor that specifies the α'β' fate and whose mRNA is stabilized by Syp. αβ neurons are generated for the final ~125 neuroblast divisions, when Syp levels are high, Imp is absent in neuroblasts, and thus Chinmo and Mamo are no longer expressed in neurons (Rossi, 2020).

Extrinsic cues are known to have important roles in regulating neuronal differentiation during mushroom body neurogenesis. The ecdysone peak that controls entry into pupation regulates γ neuron axonal remodeling. Ecdysone was also proposed to be required for the final differentiation of α'β' neurons. EcR expression in γ neurons is timed by activin signaling, a member of the TGFβ family, from local glia. Activin signaling from glia is also required for the α'β' fate (Marchetti, 2019): Knocking-down the activin pathway receptor Baboon (Babo) leads to the loss of α'β' neurons. It was proposed that activin signaling in mushroom body neuroblasts regulates the expression of EcR in prospective α'β' neurons and that when the activin pathway is inhibited, it leads to the transformation of α'β' neurons into later-born pioneer-αβ neurons (a subclass of the αβ class) (Marchetti and Tavosanis, 2019) (Rossi, 2020).

Although there is strong evidence that extrinsic cues have important functions in neuronal patterning in the Drosophila central brain, it remains unknown how extrinsic temporal cues interface with the Imp and Syp intrinsic temporal program to regulate neuronal specification. This question was addressed using the developing mushroom bodies. Activin signaling from glia was shown to be required for α'β' specification. However, this study also showed that activin signaling lowers the levels of the intrinsic factor Imp in mushroom body neuroblasts to define the mid-α'β' temporal identity window. Removing the activin receptor Babo in mutant clones leads to the loss of α'β' neurons, to fewer last-born αβ neurons, and to the likely generation of additional first-born γ neurons without affecting overall clone size. This appears to be caused by a delayed decrease in Imp levels, although the intrinsic temporal clock still progresses even in the absence of activin signaling. This study also demonstrated that ecdysone signaling is not necessary for the specification of α'β' neurons, although it might still be involved in later α'β' differentiation. These results provide a model for how intrinsic and extrinsic temporal programs operate within individual progenitors to regulate neuronal specification (Rossi, 2020).

Mushroom body neurogenesis is unique and programmed to generate many copies of a few neuronal types. During the early stages of mushroom body development, high Imp levels in mushroom body neuroblasts are inherited by newborn neurons and translated into high Chinmo levels to specify γ identity. As in other central brain neuroblasts, as development proceeds, inhibitory interactions between Imp and Syp help create a slow decrease of Imp and a corresponding increase of Syp. However, at the end of the γ temporal window (mid-L3), activin signaling from glia acts to rapidly reduce Imp levels in mushroom body neuroblasts without significantly affecting Syp, establishing a period of low Imp (and thus low Chinmo in neurons) and also low Syp. This is required for activating effector genes in prospective α'β' neurons, including Mamo, whose translation is promoted by Syp (Liu, 2019). The production of αβ identity begins when Imp is further decreased and Syp levels are high during pupation (see Model of how activin signaling defines the α'β' temporal identity window.). Low Chinmo in αβ neurons is also partly regulated by ecdysone signaling through the activation of Let-7-C, which targets chinmo for degradation. Based on this model, α'β' neurons could not be rescued by knocking-down Imp in babo clones, since low Imp is required for α'β' specification while the knockdown reduces its level below this requirement. It would be expected to rescue α'β' neurons if Imp levels were specifically reduced to the appropriate levels at L3. However, reducing Imp levels might not be the only function of activin signaling, which may explain why α'β' neurons are not simply made earlier (e.g., during L1-L2) when Imp is knocked-down (Rossi, 2020).

In babo mutant clones, it is speculated that additional γ neurons are produced at the expense of α'β' neurons since Imp levels in neuroblasts (as well as Chinmo in neurons) are higher for a longer time during development; There was also a significant decrease in the total number of αβ neurons in babo mutant clones that contrasts with a recent report by Marchetti (2019) that instead concluded that additional pioneer-αβ neurons are produced. It is believed that there is both an increase in the number of γ neurons and of the pioneer-αβ neuron subclass because pioneer-αβ neurons are the first of the αβ class to be specified (when Imp is still present at very low levels) during pupation. It is speculated that pioneer-αβ neurons are produced during the extended low Imp window that was detected during pupation in babo clones. However, this does not leave the time for the remaining population of αβ neurons to be formed, which explains why their number is reduced (Rossi, 2020).

This study has focused on the three main classes of mushroom body neurons although at least seven subtypes exist: 2 γ, 2 α'β' and 3 αβ. The subtypes are specified sequentially suggesting that each of the three broad mushroom body temporal windows can be subdivided further, either by fine-scale reading of the changing Imp and Syp gradients, by additional extrinsic cues, or perhaps by a tTF series as in other neuroblasts (Rossi, 2020).

Postembryonic central brain neuroblasts are long-lived and divide on average ~50 times. Unlike in other regions of the developing Drosophila brain, rapidly progressing series of tTFs have not yet been described in these neuroblasts. Instead, they express Imp and Syp in opposing temporal gradients. Conceptually, how Imp and Syp gradients translate into different neuronal identities through time has been compared to how morphogen gradients pattern tissues in space. During patterning of the anterior-posterior axis of the Drosophila embryo, the anterior gradient of the Bicoid morphogen and the posterior Nanos gradient are converted into discrete spatial domains that define cell fates. Since gradients contain unlimited information, differences in Imp and Syp levels through time could translate into different neuronal types. Another intriguing possibility is that tTF series could act downstream of Imp and Syp, similarly to how the gap genes in the Drosophila embryo act downstream of the anterior-posterior morphogens. This study has shown that another possibility is that temporal extrinsic cues can be incorporated by individual progenitors to increase neuronal diversity. In mushroom body neuroblasts activin signaling acts directly on the intrinsic program, effectively converting two broad temporal windows into three to help define an additional neuronal type. It is proposed that subdividing the broad Imp and Syp temporal windows by extrinsic cues may be a simple way to increase neuronal diversity in other central brain neuroblasts (Rossi, 2020).

This study has also shown that activin signaling times the Imp to Syp transition for mushroom body neuroblasts, similar to the function of ecdysone for other central brain neuroblasts. In both cases however, the switch still occurs, indicating that a separate independent clock continues to tick. This role for extrinsic cues during Drosophila neurogenesis is reminiscent of their roles on individual vertebrate progenitors. For example, hindbrain neural stem cells progressively produce motor neurons followed by serotonergic neurons before switching to producing glia. The motor neuron to serotonergic neuron switch is fine-tuned by TGFβ signaling. It would be interesting to determine if hindbrain neuronal subtypes are lost in TGFβ mutants, similar to how α'β' identity is lost in the mushroom bodies in babo mutants (Rossi, 2020).

The specification of α'β' neurons begins at mid-L3 with the onset of Mamo expression. In contrast, high levels of EcR are detected in mature mushroom body neurons starting at late L3. At this stage, both γ and α'β' neurons already exist and new α'β' neurons are still being generated. Thus, Mamo expression precedes EcR expression. These non-overlapping expression patterns suggest that ecdysone signaling does not regulate Mamo and therefore cannot control the specification of α'β' neurons. Furthermore, expression of UAS-EcR-RNAi or mutants for usp do not lead to the loss of α'β' neurons. It is noted that usp results contradict the loss of α'β' neuron reported by Marchetti (2017) in usp clones. However, α'β' neurons were seen in these clones based on the morphology of these neurons but the remodeling defect of γ neurons makes α'β' neurons difficult to identify. Nevertheless, ecdysone might still function later during α'β' differentiation, particularly during pupation when all mushroom body neurons express EcR (Rossi, 2020).

This study and that of Marchetti both show that expression of UAS-EcR-DN leads to the loss of α'β' neurons by acting in mushroom body neurons but not in neuroblasts. However, EcR must be first be expressed in the target cells of interest in order to make any conclusions about ecdysone function using UAS-EcR-DN. Since this study could not detect EcR protein in Mamo+ cells at L3, but expressing UAS-EcR-DN inhibits Mamo in those cells, it is concluded that EcR-DN artifactually represses Mamo and leads to the loss of α'β' neurons. This explains why expressing UAS-EcR-B1 does not rescue α'β' neurons in babo clones. However, Marchetti did rescue babo-RNAi by expressing EcR (Marchetti, 2019). This is likely because the current experiments were performed using babo MARCM clones in which the loss of α'β' neurons is much more severe than with babo-RNAi used in their experiments. Indeed, when attempts were made to eliminate α'β' neurons using a validated UAS-babo-RNAi construct, γ neurons did not remodel but there was only a minor (but significant) decrease in the number of α'β' neurons. This indicates that knocking-down babo with mb-Gal4 that is only weakly expressed in neuroblasts and newborn neurons is not strong enough to inhibit α'β' specification. Thus, it is speculated that the LexA line used by Marchetti (GMR26E01-LexA) may not be a reliable reporter for α'β' neurons upon babo knockdown, and that it might be ecdysone sensitive later in α'β' differentiation. Since EcR expression in all mushroom body neurons at L3 may be dependent on activin signaling directly in neurons, as it is in γ neurons for remodeling, expressing UAS-EcR-B1 together with UAS-babo-RNAi using OK107-Gal4 might both reduce the effectiveness of the RNAi while also allowing for the re-expression of GMR26E01-LexA (Rossi, 2020).

Glia are a source of the activin ligand myo, which is temporally expressed in brain glia starting at L3 to initiate the remodeling of mushroom body γ neurons (Awasaki, 2011) and α'β' specification (this study and Marchetti, 2019). However, knocking-down Myo from glia is not as severe as removing Babo from mushroom body neuroblasts. This might be due to incomplete knockdown of myo or to other sources of Myo, potentially from neurons. For example, in the vertebrate cortex, old neurons signal back to young neurons to control their numbers. It is also possible the Babo is activated by other activin ligands, including Activin and Dawdle. An intriguing hypothesis is that the temporal expression of myo in glia beginning at mid-L3 is induced by the attainment of critical weight and rising ecdysone levels. It would be interesting to determine whether blocking ecdysone signaling in glia leads to the loss of α'β' specification, similar to how blocking ecdysone reception in astrocytes prevents γ neuron remodeling (Rossi, 2020).

It is well established that extrinsic cues play important roles during vertebrate neurogenesis, either by regulating temporal competence of neural stem cells or by controlling the timing of temporal identity transitions. Competence changes mediated by extrinsic cues were demonstrated in classic heterochronic transplantation studies that showed that young donor progenitors produce old neuronal types when placed in older host brains. Recent studies show that the reverse is also true when old progenitors are placed in a young environment (Rossi, 2020).

Mechanisms of intrinsic temporal patterning are also conserved. For example, vertebrate retinal progenitor cells use an intrinsic tTF cascade to bias young, middle, and old retinal fates. Two of the factors (Ikaros and Casz1) used for intrinsic temporal patterning are orthologs to the Drosophila tTFs Hb and Cas. tTF series might also exist in cortical radial glia progenitors and even in the spinal cord. Recent results also show the importance of post-transcriptional regulation in defining either young or old cortical fates, which can be compared to the use of post-transcriptional regulators that are a hallmark of neuronal temporal patterning in Drosophila central brain neuroblasts. These studies highlight that the mechanisms driving the diversification of neuronal types are conserved (Rossi, 2020).

The let-7-Imp axis regulates aging of the Drosophila testis stem-cell niche

Adult stem cells support tissue homeostasis and repair throughout the life of an individual. During ageing, numerous intrinsic and extrinsic changes occur that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. In the Drosophila testis, ageing results in a marked decrease in the self-renewal factor Unpaired (Upd), leading to a concomitant loss of germline stem cells. This study demonstrates that IGF-II messenger RNA binding protein (Imp) counteracts endogenous small interfering RNAs to stabilize upd (also known as os) RNA. However, similar to upd, Imp expression decreases in the hub cells of older males, which is due to the targeting of Imp by the heterochronic microRNA let-7. In the absence of Imp, upd mRNA therefore becomes unprotected and susceptible to degradation. Understanding the mechanistic basis for ageing-related changes in stem-cell behaviour will lead to the development of strategies to treat age-onset diseases and facilitate stem-cell-based therapies in older individuals (Toledano, 2012).

Many stem cells lose the capacity for self-renewal when removed from their local microenvironment (or niche), indicating that the niche has a major role in controlling stem-cell fate. Changes to the local and systemic environments occur with age that result in altered stem-cell behaviour and reduced tissue maintenance and regeneration. The stem-cell niche in the Drosophila testis is located at the apical tip, where both germline stem cells (GSCs) and somatic cyst stem cells are in direct contact with hub cells. Hub cells express the self-renewal factor Upd, which activates the JAK-STAT signalling pathway to regulate the behaviour of adjacent stem cells. Ageing results in a progressive and significant decrease in the levels of upd in hub cells. However, constitutive expression of upd in hub cells was sufficient to block the age-related loss of GSCs, suggesting that mechanisms might be in place to regulate upd and maintain an active stem-cell niche (Toledano, 2012).

To identify potential regulators of upd, a collection of transgenic flies carrying green fluorescent protein (GFP)-tagged proteins was screened for expression in hub cells. The Drosophila homologue of Imp protein is expressed throughout the testis tip in young flies (Fabrizio, 2008); however, antibody staining revealed a decrease (~50%) in Imp expression in the hub cells of aged males. Imp is a member of a conserved family of RNA-binding proteins that regulate RNA stability, translation and localization (Yisraeli, 2005). Given the similarity in the ageing-related decline in Imp protein and upd mRNA in hub cells, it is proposed that Imp could be a new regulator of upd (Toledano, 2012).

To address whether Imp acts in hub cells to regulate upd, the bipartite GAL4-UAS system was used in combination with RNA-mediated interference (RNAi) to reduce Imp expression exclusively in hub cells. Fluorescence in situ hybridization (FISH) to detect upd mRNA was used in combination with immunofluorescence microscopy to determine whether the loss of Imp expression affects upd levels. The loss of Imp specifically in hub cells resulted in reduced expression of upd, as well as a significant reduction in GSCs and hub cell), when compared with controls. Consistent with a reduction in JAK-STAT signalling, decreased accumulation of STAT was observed when Imp levels were reduced by RNAi in hub cells (Toledano, 2012).

RNA-binding proteins characteristically target several RNAs; therefore, it was of interest to determine whether upd is a physiologically relevant target of Imp. Expression of upd together with an Imp RNAi construct was sufficient to completely rescue the defects caused by reduced Imp expression in hub cells, suggesting that Upd acts downstream of Imp to maintain GSCs and niche integrity. Importantly, the constitutive expression of upd alone in hub cells did not lead to an increase in GSCs in testes from 1-day-old males. These data suggest that Imp acts in hub cells to promote niche integrity and GSC maintenance, at least in part, by positively regulating upd (Toledano, 2012).

If Imp acts in hub cells in adult testes to regulate upd mRNA, it is speculated that the loss of Imp function during development might lead to a decrease in upd and a subsequent reduction in GSCs. Null mutations in Imp result in lethality at the pharate adult stage; therefore, testes from third instar larvae (L3) carrying Imp null alleles, Imp⁷ and Imp⁸, were examined. Deletion of the Imp locus was verified by PCR of genomic DNA. Combined immunofluorescence and FISH showed that although Fas3⁺ hub cells were easily detected, the expression of upd was significantly reduced: 24% of Imp⁷ mutants and 15% of Imp⁸ mutants had no detectable upd at this stage. In addition, the average number of GSCs and hub cells in testes from Imp mutants was significantly reduced when compared with control L3 testes. Notably, the re-expression of Imp in somatic niche cells was sufficient to rescue upd expression in Imp mutants to comparable levels to controls, and the reduction in the average number of GSCs and hub cells in Imp mutants was also reversed (Toledano, 2012).

Imp family members contain conserved KH domains that mediate direct binding to RNA targets. To determine whether Imp could associate directly with upd mRNA in vivo, testes were dissected from young flies expressing GFP-tagged Imp. Immunoprecipitation of Imp with anti-GFP antibodies, followed by quantitative reverse transcriptase PCR (qRT-PCR) analysis, showed a significant enrichment (~208-fold) of associated upd mRNA relative to control antibodies. Minimal enrichment for the ubiquitously expressed RNAs rp49 (also known as RpL32; ~4-fold) and GapDH (also known as Gapdh1; ~8 fold) or for the negative control med23 (~4-fold), was observed after Imp immunoprecipitation, indicating that the interaction between Imp and upd mRNA in hub cells is specific. Consistent with these observations, Imp protein and upd RNA co-localized in hub cells within perinuclear foci, probably ribonucleoprotein particles (Toledano, 2012).

An in vitro protein-RNA binding assay showed that Imp associates with the upd 3' untranslated region (UTR), specifically the first 250 base pairs (region 1), as no substantial binding to other portions of the upd 3'UTR was detected. Moreover, Imp did not bind the 5' untranslated or coding regions of upd or to the med23 3'UTR. Notably, a putative consensus binding sequence CAUH (in which H denotes A, U or C) for the mammalian IMP homologues (IGF2BP1- 3) occurs 22 times within the upd 3'UTR, including a cluster of four tandem repeats within the first 35 nucleotides of region 1. To test whether this motif mediates binding between Imp and upd, the first 33 nucleotides were removed to generate a sequence excluding the CAUH repeats, which resulted in a reduction in binding, compare domain 1 with domain 2. Point mutations in the third nucleotide of each motif (U = G) did not abolish the binding; however, point mutations in the consensus motif of MRPL9 RNA, a target of mammalian IGF2BPs, also did not abolish binding, suggesting that secondary structures probably mediate the association between IGFBP family members and their target RNAs. Altogether, the data identify the first 33 base pairs of the upd 3'UTR as a putative target sequence for Imp, and support observations that Imp associates specifically with upd in vivo (Toledano, 2012).

To gain further insight into the mechanism by which Imp regulates upd, a GFP reporter was constructed that contained the 3'UTR from either upd or med23. Transcript levels for gfp were fivefold higher in Drosophila Schneider (S2) cells that co-expressed Imp with the gfp-upd-3'UTR reporter than in cells that co-expressed Imp with the gfp-med23-3'UTR reporter. The significant increase in reporter mRNA levels indicates that it is likely that Imp regulates upd mRNA stability (Toledano, 2012).

RNA-binding proteins, including mammalian IGF2BP1, have been shown to counter microRNA (miRNA)-mediated targeting of mRNAs. However, no consensus miRNA seeds were located within the first 34 base pairs of domain 1 of the upd 3'UTR. It is speculated that if Imp binding blocks small RNA-mediated degradation of upd, polyadenylated, cleaved upd degradation intermediates would be detected in the testes of older males, when Imp expression in hub cells is reduced. Using a modified rapid amplification of complementary DNA ends (RACE) technique, a specific cleavage product was identifed starting at nucleotide 33 of the upd 3'UTR in the testes of 30-day-old flies, but not in RNA extracts from the testes of 1-day-old males. Importantly, the same degradation product of upd was also detected in the testes of young flies when Imp was specifically depleted from hub cells using RNAi-mediated knockdown. As a positive control, the esi-2-mediated cleavage product of mus308 was detected in testes from both 1- and 30-day-old flies (Toledano, 2012).

To test whether small RNAs might mediate upd cleavage, small RNA libraries generated from the testes of 1- and 30-day-old flies were cloned and deep-sequenced. Although no small RNAs with exact pairing to the upd degradation product were identified, two short interfering RNAs (siRNAs; termed siRNA1 and siRNA2) with high sequence complementarity to the predicted target site in the upd 3'UTR were present in the testis library generated from 30-day-old males. Using qRT- PCR for mature small RNAs, it was found that the siRNA2 levels in the testes, relative to the levels of the control small RNAs bantam and mir-184, were similar in young and old males (deep sequencing analysis demonstrated that expression of these two control miRNAs did not change with age). The source of siRNA2 is the gypsy5 transposon, which is inserted at several loci throughout the fly genome and is conserved in numerous Drosophila species (Toledano, 2012).

To gain further insight into the mechanism by which Imp and siRNA2 regulate upd, the levels of the upd GFP reporter (gfp-upd-3'UTR) in the presence or absence of Imp and siRNA2 was investigated in S2 cells. To generate a reporter that should not be susceptible to siRNA-mediated degradation, the cleavage site in the upd 3'UTR that was identified by RACE (³²AUU = CGG; gfp-upd-3'UTR^mut) was mutated. Cells were transfected with either of the GFP reporter constructs, with or without haemagglutinin-tagged Imp (Imp- HA), and subsequently transfected with siRNA2; gfp expression was quantified by qRT- PCR (Toledano, 2012).

The co-expression of siRNA2 and the gfp-upd-3'UTR reporter resulted in a significant decrease in gfp transcript levels. Conversely, the co-expression of Imp blocked siRNA2-mediated reduction of gfp mRNA such that gfp levels were higher than in control cells. Furthermore, mutation of the putative cleavage site rendered the upd 3'UTR resistant to siRNA2-mediated degradation. These data, in combination with the in vitro binding data, suggest that Imp binds to and protects the upd 3'UTR from endogenous and exogenous siRNA2 in S2 cells. Thus, endo-siRNA2 is a bona fide candidate that could direct upd degradation when Imp is absent or its levels are reduced, although targeting by other small RNAs cannot be excluded (Toledano, 2012).

In Drosophila, Argonaute-1 (AGO1) is the principle acceptor of miRNAs and primarily regulates targets in a cleavage-independent mode, whereas AGO2 is preferentially loaded with siRNAs and typically regulates targets by mRNA cleavage. AGO2 expression was detected throughout the tip of the testis, as verified by immunostaining of testes from transgenic flies expressing 3×Flag-HA-tagged AGO2. To test whether AGO2 binds to upd mRNA in vivo, thereby potentially regulating upd levels directly, testes were dissected from aged (30-day-old) 3×Flag- HA- AGO2 males. Immunoprecipitation of AGO2, followed by qRT- PCR, showed significant enrichment (~102-fold) of upd mRNA bound to AGO2. Negligible binding of a negative control, rp49, to AGO2 was detected, suggesting specific association of AGO2 with upd mRNA in vivo and supporting a previous findings that upd is probably targeted by the siRNA pathway (Toledano, 2012).

To test whether Imp can impede the binding of AGO2 to the upd 3'UTR, S2 cells stably expressing Flag-tagged AGO2 were transfected with the gfp-upd-3'UTR reporter. Consistent with our previous observations, transcript levels of gfp-upd-3'UTR increased ~18-fold when Imp was co-expressed. Despite increases in the overall levels of gfp mRNA, the presence of Imp markedly reduced the association of AGO2 with the upd 3'UTR, indicating that Imp antagonizes the ability of AGO2 to bind the upd 3'UTR (Toledano, 2012).

Similar to the AGO family, Drosophila encodes two Dicer proteins that seem to have distinct roles in small RNA biogenesis. Dicer-1 (Dcr-1) is essential for the generation of miRNAs, and Dcr-2 is required for siRNA production from exogenous and endogenous sources. If siRNAs were involved in upd degradation in older males, it would be predicted that the loss of Dcr-2 would suppress the ageing-related decline in upd and GSCs. Consistent with a role for Dcr-2 in the generation of siRNAs, siRNA2 levels were significantly reduced in Dcr-2 homozygous mutants relative to heterozygous controls. Testes from 30- and 45-day-old Dcr-2 mutant flies showed increased levels of upd by qRT- PCR when compared with controls. Whereas a ~90% reduction of upd is observed in the testes from aged Dcr-2 heterozygous controls, only a ~45% reduction in upd was observed in testes from age-matched, Dcr-2 homozygous mutants, indicating that upd levels are higher when Dcr-2 function is compromised. Furthermore, the testes from aged Dcr-2 mutants contained more GSCs, on average, when compared with controls. Conversely, the forced expression of Dcr-2 in hub cells resulted in a reduction in the average number of GSCs and led to a significant reduction in upd levels, as detected using qRT- PCR and combined immunofluorescence and FISH, which seemed to be specific, as no significant change in Imp transcript levels was observed. Expression of Imp in combination with Dcr-2 resulted in a significant increase in upd levels. These observations indicate that Imp can counter the decrease in upd levels resulting from forced Dcr-2 expression, providing further evidence that Imp protects upd from targeted degradation by the siRNA pathway (Toledano, 2012).

The data suggest that Imp has a role in stabilizing upd in hub cells; therefore, the ageing-related decline in Imp would be a major contributing factor to the decrease in upd mRNA in the hub cells of aged males. To investigate the mechanism that leads to the decline in Imp expression with age, the Imp 3'UTR was examined for potential instability elements. Within the first 160 base pairs there is a canonical seed sequence for the heterochronic miRNA let-7. Expression of a reporter gene under the control of the let-7 promoter showed that let-7 expression increases in hub cells of ageing male, which was confirmed by let-7 FISH of testes from aged males. In addition, mature let-7 miRNA was enriched twofold in the testes from 30-day-old flies, relative to 1-day-old males. Therefore, an age-related increase in let-7 is one mechanism by which Imp expression could be regulated in an ageing-dependent manner in testes from older males (Toledano, 2012).

Consistent with these observations, the forced expression of let-7 specifically in hub cells led to a decrease in Imp. In addition, let-7 expression in S2 cells reduced the levels of a heterologous gfp-Imp-3'UTR^WT reporter. S2 cells were transfected with a let-7 mimic or with negative control miRNA, and gfp expression was quantified by qRT- PCR. There was a 70% reduction in gfp-Imp-3'UTR^WT expression in the presence of let-7, relative to control miRNA. A gfp-Imp-3'UTR^mut reporter with mutations in the canonical seed for let-7 (at nucleotide 137) was unaffected by let-7 expression, indicating that mutation of the let-7 seed rendered the RNA resistant to degradation. These data confirm that let-7 can destabilize Imp through sequences in the 3'UTR. However, further increasing the levels of let-7 resulted in a decrease in gfp expression from the mutated 3'UTR, indicating that other, putative let-7 seeds in the Imp 3'UTR can be targeted by let-7 (Toledano, 2012)

If the age-related decrease in Imp contributes to a decline in upd and subsequent loss of GSCs, it is proposed that re-expression of Imp in hub cells would rescue the ageing-related decrease in upd. Therefore, flies in which Imp was constitutively expressed in hub cells were aged, and upd levels were quantified by qRT- PCR. The expression of an Imp construct containing a truncated 3'UTR (Imp-KH- HA) lacking let-7 target sequences specifically in hub cells was sufficient to suppress the ageing-related decline in upd, with concomitant maintenance of GSCs, similar to what was observed by re-expressing upd in the hub cells of aged males. Maintenance of Imp-KH- HA expression in aged males was verified by staining with an anti-HA antibody. Conversely, the expression of an Imp construct that is susceptible to degradation by let-7 (Imp^T21) did not lead to an accumulation of Imp in the testes of 30- and 50-day-old flies, as levels were similar to the levels of endogenous Imp at later time points. Consequently, the expression of this construct was not sufficient to block the ageing-related decline in GSCs. These data indicate that let-7-mediated regulation of Imp contributes to the decline in Imp protein in older flies, and supports a model in which an ageing-related decline in Imp, mediated by let-7, exposes upd to degradation by siRNAs. Thus, both the miRNA and siRNA pathways act upstream to regulate the ageing of the testis stem-cell niche by generating let-7 and siRNA2, which target Imp and upd, respectively (Toledano, 2012).

Drosophila has proven to be a valuable model system for investigating ageing-related changes in stem-cell behaviour. Cell autonomous and extrinsic changes contribute to altered stem-cell activity; thus, determining the mechanisms underlying the ageing-related decline of self-renewal factors, such as the cytokine-like factor Upd, may provide insight into strategies to maintain optimal niche function (Toledano, 2012).

The data indicate that Imp can regulate gene expression by promoting the stability of selected RNA targets by countering inhibitory small RNAs. Therefore, rescue of the aged niche by Imp expression may be a consequence of effects on Imp targets, in addition to upd, in somatic niche cells. Furthermore, as Imp is expressed in germ cells, it could also act in an autonomous manner to regulate the maintenance of GSCs. The canonical let-7 seed in the Imp 3'UTR is conserved in closely related species, and reports have predicted that the let-7 family of miRNAs target mammalian Imp homologues (IGF2BP1- 3). Given the broad role of the let-7 family in ageing, stem cells, cancer and metabolism, the regulation of Imp by let-7 may be an important, conserved mechanism in numerous physiological processes (Toledano, 2012).

Non-coding RNAs can ensure biological robustness and provide a buffer against relatively small fluctuations in a system. However, after a considerable change, a molecular switch is flipped, which allows a biological event to proceed unimpeded. In the current model, Imp preserves niche function in young flies until a time at which miRNAs and siRNAs act together to trigger an 'ageing' switch that leads to a definitive decline in upd and, ultimately, in stem-cell maintenance. Therefore, targeting signalling pathways at several levels using RNA-based mechanisms will probably prove to be a prevalent theme to ensure robustness in complex biological systems (Toledano, 2012).

Imp promotes axonal remodeling by regulating profilin mRNA during brain development

Neuronal remodeling is essential for the refinement of neuronal circuits in response to developmental cues. Although this process involves pruning or retraction of axonal projections followed by axonal regrowth and branching, how these steps are controlled is poorly understood. Drosophila mushroom body (MB) γ neurons provide a paradigm for the study of neuronal remodeling, as their larval axonal branches are pruned during metamorphosis and re-extend to form adult-specific branches. This study identified the RNA binding protein Imp as a key regulator of axonal remodeling. Imp is the sole fly member of a conserved family of proteins that bind target mRNAs to promote their subcellular targeting. Whereas Imp is dispensable for the initial growth of MB γ neuron axons, it is required for the regrowth and ramification of axonal branches that have undergone pruning. Furthermore, Imp is actively transported to axons undergoing developmental remodeling. Finally, it was demonstrated that profilin mRNA is a direct and functional target of Imp that localizes to axons and controls axonal regrowth. This study reveals that mRNA localization machineries are actively recruited to axons upon remodeling and suggests a role of mRNA transport in developmentally programmed rewiring of neuronal circuits during brain maturation (Medroni, 2014).

In cultured vertebrate neurons, ZBP1 mediates the transport of β-actin mRNA to axons, a process required for the chemiotropic response of growth cones to guidance cues. Whether these observations reflect a general requirement for ZBP1 and axonal mRNA transport during brain development has remained unclear. This study found that Imp, the Drosophila ZBP1 ortholog, accumulates in the cell bodies of a large number of neural cells in adult brain. Strikingly, Imp was additionally observed in the axonal compartment of a subpopulation of mushroom body (MB) neurons. MBs are composed of three main neuronal types (αβ, α'β', and γ) with specific axonal projection patterns and developmental programs. α'β' and αβ neurons are generated during late larval stage and early metamorphosis and are maintained until adulthood. γ neurons are born during late embryogenesis and early larval stages and undergo extensive remodeling during metamorphosis. Imp was found to be enriched in adult γ neuron axons where it colocalized with FasciclinII, but it was not detected in the axons of nonremodeling MB neurons (αβ and α'β' neurons). To test whether Imp is expressed in αβ and α'β' neurons, brains expressing GFP in γ and αβ-core neurons were labelled with antibodies against Imp and Trio, a protein specifically expressed in adult α'β' and γ neurons. Imp was not detected in αβ-core neurons but accumulated in the cell bodies of both α'β' and γ neurons. Thus, both the expression and subcellular distribution of Imp are tightly regulated in Drosophila MB neurons (Medroni, 2014).

To investigate whether Imp axonal translocation is developmentally regulated, the distribution of Imp within γ neurons was examined at different stages. In third-instar larvae, Imp accumulated exclusively in the cell bodies and was not observed in axons. During metamorphosis (pupariation), MB γ neurons first prune the distal part of their axons and then re-extend a medial branch to establish adult-specific projections. Six hours after puparium formation (APF), Imp was weakly detected in γ neuron axons. Such an axonal accumulation of Imp was visible at the time larval γ neurons have completed the pruning of their axonal processes (18 hr APF). During the subsequent intensive growth phase, Imp was enriched at the tip of axons, where it accumulated in particles. Thus, the translocation of Imp to axons is developmentally controlled, and correlates with axonal remodeling (Medroni, 2014).

To test whether Imp is required for γ axon developmental remodeling, the morphology was analyzed of adult homozygous mutant neurons generated using the MARCM (mosaic analysis with a repressible cell marker) system. Clones in which the entire progeny of a neuroblast was mutant exhibited a reduced number of cells and an altered morphology. Although wild-type adult γ axons typically span the entire medial lobe, a mixture of elongated and nonelongated axons was observed upon imp inactivation. To better visualize the morphology of mutant neurons, single labeled neurons were analyzed. Wild-type adult γ neurons extend one main axonal process that reaches the extremity of the MB medial lobe. Several secondary branches typically form along this main axonal process. In contrast, about 50% of imp γ axons failed to reach the end of the medial lobe. These defects did not result from axon retraction, as the proportion of defective axons did not increase with age. Interestingly, mutant axons of normal length but lost directionality were observed, suggesting that imp may be required for the response of γ axons to guidance cues during metamorphosis. imp mutant neurons also exhibited an overall decrease in the complexity of axonal arborization patterns characterized by a reduced number of terminal branches. Both phenotypes were significantly suppressed upon expression of a wild-type copy of Imp in γ neurons, revealing that imp acts cell autonomously to control axonal regrowth and branching (Medroni, 2014).

To determine whether Imp function in axonal growth correlates with its accumulation in axons, the requirement for Imp was investigated in two neuronal cell types where it is exclusively detected in cell bodies: larval γ neurons and α'β' neurons. Both single larval γ neurons and single adult α'β' neurons mutant for imp projected their axons normally. Furthermore, larval γ neuron neuroblast clones exhibited a normal morphology, confirming that imp is not necessary for initial axon growth. These results show that Imp is specifically required for the growth and branching of remodeling γ axons and suggest that its translocation to axons is critical for this function (Medroni, 2014).

To address whether Imp is transported actively to the axons of regrowing γ neurons, a live-imaging protocol was developed using cultured pupal brains expressing functional GFP-Imp fusions specifically in γ neurons). The culture conditions supported efficient axonal growth, as MB neurons from cultured brains grew similarly to their counterparts developing inside the pupa. Fast confocal imaging of axon bundles revealed that GFP-Imp fusions accumulated in particles undergoing bidirectional movement. In contrast, no particles could be detected upon expression of GFP alone. Motile GFP-Imp particles were distributed into three classes: particles with a strong net anterograde (56%) or retrograde (36%) movement and particles with little net bias (8%). Individually tracked particle trajectories were broken into segments to calculate velocities. Segmental velocities distributed over a wide range, with mean anterograde and retrograde segmental velocities of 0.98 ± 0.05 microm/s and 0.73 ± 0.03 microm/s, respectively. Furthermore, curves matching the graph of a quadratic function were obtained upon plotting of the mean square displacement (MSD) values over time, indicating that GFP-Imp particles undergo directed transport rather than diffusion. To assess the role of microtubules (MTs) in this process, brains were treated with colchicine. This treatment abolished MT dynamics, as revealed by the loss of EB1-GFP comets characteristic of growing MT plus ends. Strikingly, motile GFP-Imp particles were no longer observed under these conditions. These results demonstrate that Imp is a component of particles undergoing active MT-dependent transport during midpupariation, consistent with a role of Imp in the transport of selected mRNAs to regrowing γ axons (Medroni, 2014).

Previous in vitro studies have revealed that the axons of immature neurons are enriched in mRNAs encoding regulators of the actin cytoskeleton that play critical roles in axonal growth and guidance. To identify Imp mRNA targets, an immunoprecipitation RT-PCR-based screen was performed for mRNAs encoding actin regulators. Imp was found to selectively associate with chickadee (chic) mRNA, which encodes the G-actin binding protein Profilin. As revealed by affinity pull-down assays, endogenous Imp associated with the chic 3' untranslated region (UTR), but not with the chic coding sequence. To test whether Imp can interact with chic mRNA directly, the binding of recombinant MBP-Imp to the chic 3' UTR was analyzed in electrophoretic mobility shift assays. Retarded complexes formed in the presence of the chic 3' UTR, but not in the presence of a nonrelated RNA (y14). Furthermore, no significant interaction was observed when other MBP-tagged proteins were used. Notably, two discrete complexes were detected in the presence of low amounts of Imp, whereas higher-order complexes were formed with increasing amounts of Imp. Formation of these complexes was outcompeted by the addition of nonlabeled RNAs corresponding to the chic 3' UTR, but not to the chic coding sequence. Altogether, these results show that Imp associates with chic mRNA in vivo and that it can bind directly and specifically to the chic 3' UTR (Medroni, 2014).

To test whether chic mRNA localizes to the neurites of regrowing γ neurons, in situ hybridization was performed on pupal and adult brains. The poor signal-to-noise ratio obtained with this method at these stages, combined with the relatively low levels of axonally localized mRNAs, did not allow chic transcripts or reporters to be unambiguously detected in axons. Thus chic reporter constructs expressed under the control of the γ-specific 201Y-Gal4 driver was used and fluorescent in situ hybridizations was used on dissociated neurons extracted from 24 hr APF pupae and cultured for 3-4 days. chic reporter mRNAs could be observed in the neurites of γ neurons at a significantly higher frequency than control gfp mRNAs. Furthermore, chic mRNA and Imp colocalized in developing neurites, consistent with their association within mRNA transport complexes (Medroni, 2014).

To test whether the region of chic bound by Imp is required for chic mRNA localization to developing neurites, the distribution of reporters containing both the chic coding sequence and 3' UTR was compared with that of reporters lacking the chic 3' UTR. Transcripts with the chic 3' UTR localized more efficiently than transcripts lacking it, suggesting that Imp binding to the 3' UTR promotes chic axonal targeting. To exclude an effect of Imp on chic mRNA stability, the levels of chic transcripts were analyzed in cultured S2R+ cells. No significant differences in chic mRNA and Chic protein levels could be observed upon imp inactivation in these conditions (Medroni, 2014).

To functionally test the importance of chic regulation in vivo, the phenotypes associated with chic downregulation were examined. Consistent with described roles of Profilin in regulating F-actin polymerization and axonal pathfinding, it was observed that chic mutant γ neurons fail to properly extend their axons. More importantly, overexpression of chic significantly rescued the axonal growth defects observed in imp mutant neurons. Similar results were obtained with two independent UAS-chic transgenes, but not with overexpression of another regulator of F-actin polymerization (enabled). These results suggest that imp controls axonal remodeling by regulating chic expression in vivo and reveal that forced accumulation of Chic protein in axons can partially compensate for the loss of imp function (Medroni, 2014).

In conclusion, the finding that Drosophila Imp is required for γ axon regrowth but is dispensable for initial axonal growth suggests a novel and specific function of axonal mRNA targeting in developmental remodeling of the brain. Furthermore, these results highlight mechanistic similarities between developmental axonal regrowth and postinjury axonal regeneration, a process known to depend on axonal mRNA transport. Finally, this study uncovers that the translocation of Imp to γ axons is tightly linked to their developmental remodeling program. This reveals that mRNA transport machineries are subject to precise spatiotemporal regulation and may be specifically recruited in the context of developmental rewiring of the brain. It will now be interesting to identify the signals controlling the localization and the activity of mRNA transport machineries during this process (Medroni, 2014).

Imp (IGF-II mRNA-binding protein) is expressed during spermatogenesis in Drosophila melanogaster

Drosophila spermatogenesis results in the production of sixty‑four ~2-mm spermatozoa from an individual founder cell. Little is known, however, about the elongation of spermatids to such an extraordinary length. In a partial screen of a GFP-tagged protein trap collection, four insertions were uncovered that exhibit expression toward the tail ends of spermatid cysts and within the apical tip of the testis, suggesting that these protein traps may represent genes involved in spermatid elongation and pre-meiotic spermatogenesis, respectively. Inverse PCR followed by cycle sequencing and BLAST revealed that all four protein traps represent insertions within Imp (IGF-IImRNA binding protein), a known translational regulator. Testis enhancer trap analysis also reveals Imp expression in the cells of the apical tip, suggesting transcription of Imp prior to the primary spermatocyte stage. Taken together, these results suggest a role for Imp in the male germline during both spermatid elongation and premeiotic spermatogenesis (Fabrizio, 2008).

Imp associates with Squid and Hrp48 and contributes to localized expression of gurken in the oocyte

Localization and translational control of Drosophila gurken and oskar mRNAs rely on the hnRNP proteins Squid and Hrp48, which are complexed with one another in the ovary. Imp, the Drosophila homolog of proteins acting in localization of mRNAs in other species, is also associated with Squid and Hrp48. Notably, Imp is concentrated at sites of gurken and oskar mRNA localization in the oocyte, and alteration of gurken localization also alters Imp distribution. Imp binds gurken mRNA with high affinity in vitro; thus, the colocalization with gurken mRNA in vivo is likely to be the result of direct binding. Imp mutants support apparently normal regulation of gurken and oskar mRNAs. However, loss of Imp activity partially suppresses a gurken misexpression phenotype, indicating that Imp does act in control of gurken expression but has a largely redundant role that is only revealed when normal gurken expression is perturbed. Overexpression of Imp disrupts localization of gurken mRNA as well as localization and translational regulation of oskar mRNA. The opposing effects of reduced and elevated Imp activity on gurken mRNA expression indicate a role in gurken mRNA regulation (Geng, 2006).

Imp is the Drosophila homolog of a family of proteins that act in posttranscriptional regulation in a variety of animals. One of the founding members of the family, ZBP-1, binds to a localization element in the chicken beta-actin mRNA and appears to direct localization to the leading edge of embryonic fibroblasts. Another founding member, the Xenopus Vg1RBP/VERA protein, binds to signals directing localization of Vg1 and VegT mRNAs to the vegetal pole of the oocyte. Mammalian homologs, the Imp proteins, have been suggested to act in mRNA localization, mRNA stability, and translational regulation. A recent report (Munro, 2006) examined the RNA binding properties of Drosophila Imp protein, focusing specifically on the osk mRNA and its possible regulation by Imp. Although mutation of candidate Imp binding sites in the osk mRNA did block accumulation of Osk protein, loss of Imp activity did not cause a similar defect. This study shows that Imp interacts with Sqd and Hrp48, two proteins that regulate expression of osk and grk mRNAs. Mutation of the Imp gene does not substantially alter grk or osk expression. Nevertheless, the Imp mutant partially suppresses a grk misexpression phenotype, arguing that it does contribute to grk regulation but may act redundantly and does not have an essential role. Consistent with this interpretation, overexpression of Imp interferes with localization of grk mRNA (Geng, 2006).

Deployment of proteins that control patterning in the oocyte relies on coordinated programs of mRNA localization and translational control. Many RNA binding proteins contribute to these programs, and some interact with one another in regulatory RNPs. This study has shown that Imp is associated in an RNA-dependent manner with Sqd and Hrp48 and is thus part of a complex whose other members have clearly established roles in control of grk and osk expression. Imp does not have an essential role in regulation of either grk or osk mRNAs; both mRNAs are expressed with no obvious defects in Imp mutant ovaries. However, loss of Imp activity does partially suppress the grk misexpression defect in fs(1)K10 mutant oocytes, providing strong evidence that Imp contributes to regulation of grk. This view is reinforced by the colocalization of Imp with grk mRNA in vivo. Imp's role must be largely redundant, only becoming detectable when grk expression is perturbed. Overexpression of Imp has a much more dramatic effect, transiently blocking the dorsal localization of grk mRNA and disrupting localization and translational control of osk mRNA (Geng, 2006).

A repeated IMP-binding motif controls oskar mRNA translation and anchoring independently of Drosophila melanogaster IMP

Zip code-binding protein 1 (ZBP-1) and its Xenopus laevis homologue, Vg1 RNA and endoplasmic reticulum-associated protein (VERA)/Vg1 RNA-binding protein (RBP), bind repeated motifs in the 3' untranslated regions (UTRs) of localized mRNAs. Although these motifs are required for RNA localization, the necessity of ZBP-1/VERA remains unresolved. The role of ZBP-1/VERA was addressed through analysis of the Drosophila melanogaster homologue IGF-II mRNA-binding protein (IMP). Using systematic evolution of ligands by exponential enrichment, the IMP-binding element (IBE) UUUAY, was identified as a motif that occurs 13 times in the oskar 3'UTR. IMP colocalizes with oskar mRNA at the oocyte posterior, and this depends on the IBEs. Furthermore, mutation of all, or subsets of, the IBEs prevents oskar mRNA translation and anchoring at the posterior. However, oocytes lacking IMP localize and translate oskar mRNA normally, illustrating that one cannot necessarily infer the function of an RBP from mutations in its binding sites. Thus, the translational activation of oskar mRNA must depend on the binding of another factor to the IBEs, and IMP may serve a different purpose, such as masking IBEs in RNAs where they occur by chance. These findings establish a parallel requirement for IBEs in the regulation of localized maternal mRNAs in D. melanogaster and X. laevis (Monro, 2006).

IMP contains the four signature KH-type RNA-binding domains and the glutamine-rich COOH terminus that are present in the vertebrate orthologues. Affinity-purified antibodies against IMP reveal the protein in nurse cells and the oocyte early in oogenesis. However, the high concentration of IMP in the follicle cells blocks the penetration of the antibody into the oocyte after stage 4. Therefore, IMP localization was evaluated in a homozygous, viable, and fertile GFP-IMP protein trap line. GFP-IMP is enriched around the nurse cell nuclei and accumulates in the oocyte as soon as it is specified in the germarium, where it shows a uniform distribution until stage 7. IMP accumulates transiently at the anterior of the oocyte during stages 8-9 and then localizes in a crescent at the posterior pole at stage 9, where it remains for the duration of oogenesis. This pattern of localization is very similar to that observed for osk mRNA and Stau protein, which colocalize with IMP throughout oogenesis (Monro, 2006).

To ascertain whether IMP localization depends on osk, whether it is perturbed in various mutants that affect the posterior accumulation of osk mRNA and protein was examined. IMP does not localize to the posterior of the oocyte in staufen, barentsz, and hrp48 mutants, which block the transport of oskar mRNA to the posterior pole. Furthermore, IMP colocalizes with osk RNA to an ectopic dot in the center of the oocyte in a par-1 mutant that disrupts microtubule polarity. Together, these results demonstrate that the localization of IMP to the oocyte posterior pole requires the localization of osk mRNA (Monro, 2006).

IMP could localize to the posterior through a direct interaction with osk mRNA or protein or could be recruited to the posterior by a downstream component of the pole plasm. To distinguish between these possibilities, IMP localization was examined in a strong vasa hypomorph (vasa^PD/Df(2L) TW2), which prevents the posterior recruitment of Vasa by Osk and disrupts all subsequent steps in pole plasm assembly. IMP localizes normally to the posterior of these oocytes, suggesting that its posterior accumulation depends on osk directly. Finally, whether IMP localization depends on Osk protein rather than osk mRNA was addressed by examining a nonsense mutation (osk⁵⁴/Df) that disrupts the anchoring, but not the initial localization, of osk mRNA. IMP still localizes to the posterior of these oocytes at stage 9, but the posterior crescent is weaker than in wild type (WT) and disappears at stage 10. Thus, IMP behaves like osk mRNA in every mutant combination examined, suggesting that it localizes to the posterior in association with the mRNA (Monro, 2006).

The object of this study was to address whether D. melanogaster IMP is required for mRNA localization, because previous studies of its vertebrate homologues, ZBP-1 and VERA/Vg1RBP, had not resolved this question definitively. This study demonstrated that IMP binds directly to osk mRNA at well defined sites that are required for osk translation and anchoring. The best evidence that these sites are bona fide IBEs is that IMP is not recruited to the posterior by osk mRNA in which all 13 IBEs have been mutated with a single base change. Indeed, this is one of the only cases where it has been possible to demonstrate that an RBP interacts in vivo with well defined elements identified biochemically in vitro. In vitro, mutant RNA still competes for binding of IMP, albeit less effectively than the WT osk RNA, suggesting that the 3'UTR may contain other lower affinity sites. However, these sites are not involved in the recruitment of IMP to the posterior in vivo, nor are they sufficient for translational activation. Although the IBEs are thus bona fide in vivo IMP-binding sites, their role in osk RNA translation and anchoring is independent of IMP, which is not required for these activities (Monro, 2006).

Two outcomes of this investigation seem particularly surprising: (1) IBEs are required not for the initial localization of osk mRNA, but instead for its translational activation once it is localized and its subsequent anchoring at the posterior pole; (2) osk mRNA localization-dependent translation and anchoring require the IBEs in its 3'UTR, but not IMP itself (Monro, 2006).

Because Osk protein defines where the pole plasm forms, and hence where the pole cells and abdomen develop, it is essential that osk mRNA is only translated at the oocyte posterior. Indeed, translational control is arguably more important than localization in restricting Osk to the posterior, as normally only 18% of osk mRNA is actually localized, and osk mRNA localization mutants such as barentsz produce a normal abdomen. The translational repression of unlocalized osk mRNA occurs in different ways, depending on the stage of oogenesis. Mutants in RNA interference pathway components cause premature translation of osk mRNA during early oogenesis. Repression at later stages does not depend on these components, but instead requires the binding of Bruno and Hrp48 to three elements in the 3'UTR called Bruno response elements. This repression may occur at the level of translation initiation through the binding of Bruno to Cup protein and of Cup to the Cap-binding protein eIF4E, implying that the 5' and 3' ends of the mRNA are linked (Monro, 2006).

Much less is known about how osk mRNA translation is derepressed at the posterior, apart from the findings that a 297-nt element at the 5' end is required for the localization-dependent activation of a reporter RNA fused to the osk 3'UTR and that the osk 3'UTR, although sufficient to repress the translation of heterologous coding sequences, is insufficient to activate their translation at the posterior. The current data now provide direct evidence that the osk 3'UTR, through its IBEs, is required for translational derepression. Therefore, like activation, repression involves both the 5' and 3' ends. Moreover, three osk transgenes with only 3 out of 13 sites mutated at a single base prevent osk translational derepression. These are much more subtle mutations than the deletions that have previously been used to define osk derepression elements and these will be useful for identifying the corresponding derepressor proteins (Monro, 2006).

Although the CPEB homologue, Orb, and the RISC component, Aubergine, have been proposed to play a role in osk translational activation, mutants in these proteins also affect the initial localization of osk mRNA to the posterior, and this may account for the observed reduction in Osk protein levels. The only mutant combination that produces a similar phenotype to osk^13TTgAY, with only 3 out of 13 sites mutated at a single base, is stau-null mutants that have been rescued by a transgene-expressing Stau protein that lacks the fifth double-stranded RNA-binding domain. However, Stau is unlikely to be the putative factor that interacts with the IBEs in the osk 3'UTR to activate translation, both because Stau recognizes double-stranded RNA rather than short-sequence motifs and because the IBE mutations prevent osk mRNA translation without affecting Stau localization to the posterior pole at stage 9 (Monro, 2006).

This brings us to the most significant outcome of this investigation: osk RNA translational activation and anchoring is disrupted by mutants in the IBEs, but not by the loss of IMP itself. The possibility that the IBE mutations prevent osk mRNA derepression and IMP localization indirectly by altering the structure of the RNA seems extremely unlikely, since single-base substitutions within three nonoverlapping sets of three IBEs in widely separated regions of the >1-kb osk 3'UTR produce an identical and very specific defect in translation, without affecting any of the earlier functions of the 3'UTR, such as the maintenance of oocyte fate, the transport of the mRNA from the nurse cells into the oocyte, the translational repression of unlocalized mRNA, or its localization to the posterior pole. Thus, none of these mutations disrupt the binding of any of the factors that mediate these earlier steps, including Staufen, which is thought to recognize the secondary structure of the RNA through the interaction of its double-stranded RNA-binding domains with multiple stem loops. This strongly argues against the possibility that the single base changes to the IBEs inhibit osk RNA translation through a nonspecific effect on RNA folding. This leads to the conclusion that the IBEs play a direct role in the derepression of osk mRNA translation (Monro, 2006).

Because IMP itself is not necessary for derepression, this implies that the IBEs are also recognized by another factor, called factor X. IMP and factor X could function redundantly to derepress osk translation, i.e., the two proteins might share osk's IBEs and compensate for each other's loss. However, factor X cannot be a ZBP-1/VERA family member because, unlike mammals, no such relatives are evident in the D. melanogaster genome (Monro, 2006).

Alternatively, IMP and factor X might function independently, i.e., osk derepression might occur exclusively through factor X binding. Rather than implementing osk's translational derepression, IMP's actual function might be to compete with factor X for IBE binding. In support of this, it was found that overexpression of IMP reduces Osk protein levels at the posterior. Although the purpose of IMP competition is presently unclear, one possibility is that IMP serves to bind, and thereby mask, IBEs that occur by chance in RNAs, for which factor X binding would be unnecessary or even detrimental. According to this view, competition with IMP would restrict factor X binding to those mRNAs, such as osk, that contain many copies of IBEs clustered within a restricted region. In the absence of IMP, factor X could bind to mRNAs with fewer IBEs and inappropriately regulate their translation. This may explain why embryos from imp-null oocytes always die, but from defects that appear unrelated to Osk function (Monro, 2006).

This analysis of the interaction of IMP with osk mRNA closely parallels that of ZBP-1 and VERA/Vg1RBP with ß-actin and Vg1 mRNA, respectively. (1) In each case, the protein has been shown to colocalize with the localized mRNA and can be UV cross-linked to it in extracts; (2) the precise binding sites of each protein have been determined and reveal that each protein recognizes a repeated motif in the target mRNA; (3) the function of these sites has then been analyzed by introducing specific point mutations that abrogate the binding of the protein, and these have been found to have a dramatic effect on translation or localization. This study has gone one step further to compare the phenotype of the IBE mutants with that of mutations in IMP itself. The observation that the former gives a fully penetrant defect in osk mRNA translation, whereas the latter has no phenotype in the germline, conclusively demonstrates that IMP is not responsible for the function of the IBEs in the osk 3'UTR. This is important in light of the observation that many RBPs have been implicated in the posttranscriptional regulation of particular mRNAs by studying the effects of mutations in their binding sites. These results highlight the potential limitations of this approach by demonstrating that one cannot necessarily infer the function of a protein from the phenotype of mutations in the cis-acting sequences that it recognizes (Monro, 2006).

The clear similarities between the localizations and functions of Vg1 and VegT mRNAs in X. laevis oocytes, and of osk mRNA in D. melanogaster oocytes, suggest that binding motifs for ZBP-1 proteins have a fundamental role in embryogenesis. Vg1, VegT, and osk localize as mRNAs to one pole of the oocyte, which is the site where the germ or pole plasm forms, and all three proteins play key roles in the formation of the primary body axis. These findings extend this parallel by showing that the localized expression of all three proteins also depends on a repeated RNA motif, defined by its interaction with IMP or its homologues. Because the results rule out a function for IMP in the regulation of osk mRNA, this calls into question the role of VERA/Vg1RBP1 in the localization of Vg1 and Veg T mRNAs, and it may therefore be worth considering the possibility that there is also a factor X in X. laevis (Monro, 2006).

The biphasic expression of IMP/Vg1-RBP is conserved between vertebrates and Drosophila

The human IGF-II mRNA-binding proteins (IMPs) 1-3, and their Xenopus homologue Vg1 RNA-binding protein (Vg1-RBP) are RNA-binding proteins implicated in mRNA localization and translational control in vertebrate development. This study has sequenced the Drosophila homologue (dIMP) of these genes, and examined its expression pattern in Drosophila embryos by in situ hybridization. dIMP exhibits a biphasic expression pattern. In the early stages of development, a maternal pool of dIMP mRNA is evenly distributed in the embryo and degraded by the end of stage 4. Expression reappears in the developing central nervous system, where dIMP is expressed throughout neurogenesis. In addition, dIMP is present in the pole cells (Nielsen, 2000).

Search PubMed for articles about Drosophila Imp

Alsio, J. M., Tarchini, B., Cayouette, M. and Livesey, F. J. (2013). Ikaros promotes early-born neuronal fates in the cerebral cortex. Proc Natl Acad Sci U S A 110(8): E716-725. PubMed ID: 23382203

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Bayraktar, O. A. and Doe, C. Q. (2013). Combinatorial temporal patterning in progenitors expands neural diversity. Nature 498(7455): 449-455. PubMed ID: 23783519

Boylan, K. L., Mische, S., Li, M., Marques, G., Morin, X., Chia, W. and Hays, T. S. (2008). Motility screen identifies Drosophila IGF-II mRNA-binding protein--zipcode-binding protein acting in oogenesis and synaptogenesis. PLoS Genet 4: e36. Pubmed: 18282112

Clark, B. S., Stein-O'Brien, G. L., Shiau, F., Cannon, G. H., Davis-Marcisak, E., Sherman, T., Santiago, C. P., Hoang, T. V., Rajaii, F., James-Esposito, R. E., Gronostajski, R. M., Fertig, E. J., Goff, L. A. and Blackshaw, S. (2019). Single-cell RNA-Seq analysis of retinal development identifies NFI factors as regulating mitotic exit and late-born cell specification. Neuron 102(6): 1111-1126 e1115. PubMed ID: 31128945

Deshler, J. O., Highett, M. I. and Schnapp, B. J. (1997). Localization of Xenopus Vg1 mRNA by Vera protein and the endoplasmic reticulum. Science 276: 1128-1131. Pubmed: 9148809

Elliott, J., Jolicoeur, C., Ramamurthy, V. and Cayouette, M. (2008). Ikaros confers early temporal competence to mouse retinal progenitor cells. Neuron 60(1): 26-39. PubMed ID: 18940586

Fabrizio, J. J., Hickey, C. A., Stabrawa, C., Meytes, V., Hutter, J. A., Talbert, C. and Regis, N. (2008). Imp (IGF-II mRNA-binding protein) is expressed during spermatogenesis in Drosophila melanogaster. Fly (Austin) 2: 47-52. Pubmed: 18820448

Farina, K. L., Huttelmaier, S., Musunuru, K., Darnell, R. and Singer, R. H. (2003). Two ZBP1 KH domains facilitate beta-actin mRNA localization, granule formation, and cytoskeletal attachment. J Cell Biol 160: 77-87. Pubmed: 12507992

Geng, C. and MacDonald, P. M. (2006). Imp associates with Squid and Hrp48 and contributes to localized expression of gurken in the oocyte. Mol. Cell. Biol. 26: 9508-9516. PubMed ID: 17030623

Genovese, S., Clement, R., Gaultier, C., Besse, F., Narbonne-Reveau, K., Daian, F., Foppolo, S., Luis, N. M. and Maurange, C. (2019). Coopted temporal patterning governs cellular hierarchy, heterogeneity and metabolism in Drosophila neuroblast tumors. Elife 8. PubMed ID: 31566561

Havin, L., Git, A., Elisha, Z., Oberman, F., Yaniv, K., Schwartz, S. P., Standart, N. and Yisraeli, J. K. (1998). RNA-binding protein conserved in both microtubule- and microfilament-based RNA localization. Genes Dev 12: 1593-1598. Pubmed: 9620847

Huttelmaier, S., Zenklusen, D., Lederer, M., Dictenberg, J., Lorenz, M., Meng, X., Bassell, G. J., Condeelis, J. and Singer, R. H. (2005). Spatial regulation of beta-actin translation by Src-dependent phosphorylation of ZBP1. Nature 438: 512-515. Pubmed: 16306994

Kraut, R., Menon, K. and Zinn, K. (2001). A gain-of-function screen for genes controlling motor axon guidance and synaptogenesis in Drosophila. Curr Biol 11: 417-430. Pubmed: 11301252

Leeds, P., Kren, B. T., Boylan, J. M., Betz, N. A., Steer, C. J., Gruppuso, P. A. and Ross, J. (1997). Developmental regulation of CRD-BP, an RNA-binding protein that stabilizes c-myc mRNA in vitro. Oncogene 14: 1279-1286. Pubmed: 9178888

Leung, K. M., van Horck, F. P., Lin, A. C., Allison, R., Standart, N. and Holt, C. E. (2006). Asymmetrical beta-actin mRNA translation in growth cones mediates attractive turning to netrin-1. Nat Neurosci 9: 1247-1256. Pubmed: 16980963

Liu, Z., Yang, C. P., Sugino, K., Fu, C. C., Liu, L. Y., Yao, X., Lee, L. P. and Lee, T. (2015). Opposing intrinsic temporal gradients guide neural stem cell production of varied neuronal fates. Science 350: 317-320. PubMed ID: 26472907

Liu, L. Y., Long, X., Yang, C. P., Miyares, R. L., Sugino, K., Singer, R. H. and Lee, T. (2019). Mamo decodes hierarchical temporal gradients into terminal neuronal fate. Elife 8. PubMed ID: 31545163

Marchetti, G. and Tavosanis, G. (2017). Steroid hormone ecdysone signaling specifies mushroom body neuron sequential fate via Chinmo. Curr Biol 27(19): 3017-3024 e3014. PubMed ID: 28966087

McDermott, S. M., Meignin, C., Rappsilber, J. and Davis, I. (2012). Drosophila Syncrip binds the gurken mRNA localisation signal and regulates localised transcripts during axis specification. Biol Open 1: 488-497. PubMed ID: 23213441

McDermott, S. M., Yang, L., Halstead, J. M., Hamilton, R. S., Meignin, C. and Davis, I. (2014). Drosophila Syncrip modulates the expression of mRNAs encoding key synaptic proteins required for morphology at the neuromuscular junction. RNA 20: 1593-1606. PubMed ID: 25171822

Medioni, C., Ramialison, M., Ephrussi, A. and Besse, F. (2014). Imp promotes axonal remodeling by regulating profilin mRNA during brain development. Curr Biol 24(7): 793-800. PubMed ID: 24656828

Mische, S., Li, M., Serr, M. and Hays, T. S. (2007). Direct observation of regulated ribonucleoprotein transport across the nurse cell/oocyte boundary. Mol Biol Cell 18: 2254-2263. Pubmed: 17429069

Munro, T. P., Kwon, S., Schnapp, B. J. and St Johnston, D. (2006). A repeated IMP-binding motif controls oskar mRNA translation and anchoring independently of Drosophila melanogaster IMP. J Cell Biol 172: 577-588. Pubmed: 16476777

Narbonne-Reveau, K., Lanet, E., Dillard, C., Foppolo, S., Chen, C. H., Parrinello, H., Rialle, S., Sokol, N. S. and Maurange, C. (2016). Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila. Elife 5. PubMed ID: 27296804

Narbonne-Reveau, K., Lanet, E., Dillard, C., Foppolo, S., Chen, C. H., Parrinello, H., Rialle, S., Sokol, N. S. and Maurange, C. (2016). Neural stem cell-encoded temporal patterning delineates an early window of malignant susceptibility in Drosophila. Elife 5. PubMed ID: 27296804

Nielsen, F. C., Nielsen, J., Kristensen, M. A., Koch, G. and Christiansen, J. (2002). Cytoplasmic trafficking of IGF-II mRNA-binding protein by conserved KH domains. J Cell Sci 115: 2087-2097. Pubmed: 11973350

Nielsen, J., Christiansen, J., Lykke-Andersen, J., Johnsen, A. H., Wewer, U. M. and Nielsen, F. C. (1999). A family of insulin-like growth factor II mRNA-binding proteins represses translation in late development. Mol Cell Biol 19: 1262-1270. Pubmed: 9891060

Nielsen, J., Cilius Nielsen, F., Kragh Jakobsen, R. and Christiansen, J. (2000). The biphasic expression of IMP/Vg1-RBP is conserved between vertebrates and Drosophila. Mech Dev 96: 129-132. Pubmed: 10940634

Norga, K. K., Gurganus, M. C., Dilda, C. L., Yamamoto, A., Lyman, R. F., Patel, P. H., Rubin, G. M., Hoskins, R. A., Mackay, T. F. and Bellen, H. J. (2003). Quantitative analysis of bristle number in Drosophila mutants identifies genes involved in neural development. Curr Biol 13: 1388-1396. Pubmed: 12932322

Noubissi, F. K., Elcheva, I., Bhatia, N., Shakoori, A., Ougolkov, A., Liu, J., Minamoto, T., Ross, J., Fuchs, S. Y. and Spiegelman, V. S. (2006). CRD-BP mediates stabilization of betaTrCP1 and c-myc mRNA in response to beta-catenin signalling. Nature 441: 898-901. Pubmed: 16778892

Oleynikov, Y. and Singer, R. H. (2003). Real-time visualization of ZBP1 association with beta-actin mRNA during transcription and localization. Curr Biol 13: 199-207. Pubmed: 12573215

Ren, Q., Yang, C. P., Liu, Z., Sugino, K., Mok, K., He, Y., Ito, M., Nern, A., Otsuna, H. and Lee, T. (2017). Stem cell-intrinsic, Seven-up-triggered temporal factor gradients diversify intermediate neural progenitors. Curr Biol [Epub ahead of print]. PubMed ID: 28434858

Ross, A. F., Oleynikov, Y., Kislauskis, E. H., Taneja, K. L. and Singer, R. H. (1997). Characterization of a beta-actin mRNA zipcode-binding protein. Mol Cell Biol 17: 2158-2165. Pubmed: 9121465

Rossi, A. M. and Desplan, C. (2020). Extrinsic activin signaling cooperates with an intrinsic temporal program to increase mushroom body neuronal diversity. Elife 9. PubMed ID: 32628110

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Telley, L., Agirman, G., Prados, J., Amberg, N., Fievre, S., Oberst, P., Bartolini, G., Vitali, I., Cadilhac, C., Hippenmeyer, S., Nguyen, L., Dayer, A. and Jabaudon, D. (2019). Temporal patterning of apical progenitors and their daughter neurons in the developing neocortex. Science 364(6440). PubMed ID: 31073041

Toledano, H., D'Alterio, C., Czech, B., Levine, E. and Jones, D. L. (2012). The let-7-Imp axis regulates ageing of the Drosophila testis stem-cell niche. Nature 485: 605-610. Pubmed: 22660319

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date revised: 1 March 2024

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