InteractiveFly: GeneBrief

Syncrip: Biological Overview | References

BIOLOGICAL OVERVIEW

Localized mRNA translation is thought to play a key role in synaptic plasticity, but the identity of the transcripts and the molecular mechanism underlying their function are still poorly understood. This study shows that Syncrip, a regulator of localized translation in the Drosophila oocyte and a component of mammalian neuronal mRNA granules, is also expressed in the Drosophila larval neuromuscular junction, where it regulates synaptic growth. RNA-immunoprecipitation followed by high-throughput sequencing and qRT-PCR were used to show that Syncrip associates with a number of mRNAs encoding proteins with key synaptic functions, including msp-300, syd-1, neurexin-1, futsch, highwire, discs large, and alpha-spectrin. The protein levels of MSP-300, Discs large, and a number of others are significantly affected in syncrip null mutants. Furthermore, syncrip mutants show a reduction in MSP-300 protein levels and defects in muscle nuclear distribution characteristic of msp-300 mutants. These results highlight a number of potential new players in localized translation during synaptic plasticity in the neuromuscular junction. It is proposed that Syncrip acts as a modulator of synaptic plasticity by regulating the translation of these key mRNAs encoding synaptic scaffolding proteins and other important components involved in synaptic growth and function (McDermott, 2014).

Localized translation is a widespread and evolutionarily ancient strategy used to temporally and spatially restrict specific proteins to their site of function and has been extensively studied during early development and in polarized cells in a variety of model systems. It is thought to be of particular importance in the regulation of neuronal development and in the plastic changes at neuronal synapses that underlie memory and learning, allowing rapid local changes in gene expression to occur independently of new transcriptional programs. The Drosophila neuromuscular junction (NMJ) is an excellent model system for studying the general molecular principles of the regulation of synaptic development and plasticity. Genetic or activity-based manipulations of synaptic translation at the NMJ has previously been shown to affect the morphological and electrophysiological plasticity of NMJ synapses. However, neither the mRNA targets nor the molecular mechanism by which such translational regulation occurs are fully understood (McDermott, 2014).

Previously work identified CG17838, the fly homolog of the mammalian RNA binding protein SYNCRIP/hnRNPQ, which was named Syncrip (Syp). Mammalian SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules that contain CamKIIα, Arc, and IP3R1 mRNAs (Bannai, 2004; Kanai, 2004; Elvira, 2006) and is thought to regulate translation via an interaction with the noncoding RNA BC200/BC1, itself a translational repressor (Duning, 2008). Moreover, SYNCRIP/hnRNPQ competes with poly(A) binding proteins to inhibit translation in vitro (Svitkin, 2013) and regulates dendritic morphology (Chen, 2012) via association with, and localization of, mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex. Drosophila Syp has a domain structure similar to its mammalian homolog, containing RRM RNA binding domains and nuclear localization signal(s), as well as encoding a number of protein isoforms. It was previously shown that Syp binds specifically to the gurken (grk) mRNA localization signal together with a number of factors previously shown to be required for grk mRNA localization and translational regulation (McDermott, 2012). Furthermore, syp loss-of-function alleles lead to patterning defects indicating that syp is required for grk and oskar (osk) mRNA localization and translational regulation in the Drosophila oocyte (McDermott, 2014).

This study shows that Syp is detected in the Drosophila third instar larval muscle nuclei and also postsynaptically at the NMJ. Syp is required for proper synaptic morphology at the NMJ, as syp loss-of-function mutants show a synaptic overgrowth phenotype, while overexpression of Syp in the muscle can suppress NMJ growth. Syp protein associates with a number of mRNAs encoding proteins with key roles in synaptic growth and function including, msp-300, syd-1, neurexin-1 (nrx-1), futsch, highwire (hiw), discs large 1 (dlg1), and α-spectrin (α-spec). The protein levels of a number of these mRNA targets, including msp-300 and dlg1, are significantly affected in syp null mutants. Furthermore, in addition to regulating MSP-300 protein levels, Syp is required for correct MSP-300 protein localization, and syp null mutants have defects in myonuclear distribution and morphology that resemble those observed in msp-300 mutants. It is proposed that Syp coordinates the protein levels from a number of transcripts with key roles in synaptic growth and is a mediator of synaptic morphology and growth at the Drosophila NMJ (McDermott, 2014).

The results demonstrate that Syp is required for the appropriate branching of the motoneurons and the number of synapses they make at the muscle. These observations are potentially explained by the finding that Syp is also required for the correct level of expression of msp-300, dlg1 and other mRNA targets. Given that it was previously shown that Syp regulates mRNA localization and localized translation in the Drosophila oocyte (McDermott, 2012), and studies by others have shown that mammalian SYNCRIP/hnRNPQ inhibits translation initiation by competitively binding poly(A) sequences (Svitkin, 2013), these functions of Syp as occurring at the level of translational regulation of the mRNAs to which Syp binds. The data are also consistent with other work in mammals showing that SYNCRIP/hnRNPQ is a component of neuronal RNA transport granules (Bannai, 2004; Kanai, 2004; Elvira, 2006) that can regulate dendritic morphology via the localized expression of mRNAs encoding components of the Cdc-42/N-WASP/Arp2/3 actin nucleation-promoting complex (Chen, 2012; McDermott, 2014 and references therein).

Translation at the Drosophila NMJ is thought to provide a mechanism for the rapid assembly of synaptic components and synaptic growth during larval development, in response to rapid increases in the surface area of body wall muscles or in response to changes in larval locomotion. The phenotypes observed in this study resemble, and are comparable to, those seen when subsynaptic translation is altered genetically or by increased locomotor activity. In syp null mutants, NMJ synaptic terminals are overgrown, containing more branches and synaptic boutons. Similarly, bouton numbers are increased by knocking down Syp in the muscle using RNAi. In contrast, overexpression of Syp in the muscle has the opposite phenotype, resulting in an inhibition of synaptic growth and branching. Furthermore, expressing RNAi against syp in motoneurons alone does not result in a change in NMJ morphology, indicating that Syp acts postsynaptically in muscle, but not presynaptically at the NMJ to regulate morphology. Interestingly, pan-neuronal syp knockdown or overexpression using Elav-GAL4 also results in NMJ growth defects, revealing that some of the defects observed in the syp null mutant may be attributed to Syp function in neuronal cell types other than the motoneurons, such as glial cells, which are known to influence NMJ morphology. Finally, while Syp is not required in the motoneuron to regulate synapse growth and is not detected in the motoneuron, the possibility cannot be excluded that Syp is present at low levels in the presynapse and regulates processes independent of synapse morphology. A further detailed characterization of the cell types and developmental stages in which Syp is expressed and functions is required to better understand the complex phenotypes that were observe (McDermott, 2014).

RNA binding proteins have emerged as critical regulators of both neuronal morphology and synaptic transmision, suggesting that protein production modulates synapse efficacy. Consistent with this, it has been shown in a parallel study that Syp is required for proper synaptic transmission and vesicle release and regulates the presynapse through expression of retrograde Bone Morphogenesis Protein (BMP) signals in the postsynapse (Halstead, 2014). In this role, Syp may coordinate postsynaptic translation with presynaptic neurotransmitter release. These observations provide a good explanation for how Syp influences the presynapse despite being only detectable in the postsynapse. This study has shown that Syp associates with a large number of mRNAs within third instar larvae, many of which encode proteins with key roles in synaptic growth and function. Syp mRNA targets include msp-300, syd-1, nrx-1, futsch, hiw, dlg1, and α-spec. Syp negatively regulates Syd-1, Hiw, and DLG protein levels in the larval body wall but positively regulates MSP-300 and Nrx-1 protein levels. Dysregulation of these multiple mRNA targets likely accounts for the phenotypes that were observed. Postsynaptically expressed targets with key synaptic roles that could explain the synaptic phenotypes that were observed in syp alleles include MSP-300, α-Spec, and DLG. For example, mutants in dlg1 and mutants where postsynaptic DLG is destabilized or delocalized have NMJ morphology phenotypes similar to those observed upon overexpression of Syp in the muscle. Presynaptically expressed targets include syd-1, nrx-1, and hiw. However, this study has shown that syp knockdown in presynaptic motoneurons does not result in any defects in NMJ morphology. The RIP-Seq experiments were carried out using whole larvae and will, therefore, identify Syp targets in a range of different tissues and cells, the regulation of which may or may not contribute to the phenotype that were observed in syp mutants. It is, therefore, possible that Syp associates with these presynaptic targets in other neuronal cell types such as the DA neurons of the larval peripheral nervous system. It is also possible that Nrx-1 or Hiw are expressed and required postsynaptically in the muscle, but this has not been definitively determined. syp alleles may provide useful tools to examine where key synaptic genes are expressed and how they are regulated (McDermott, 2014).

The identity of localized mRNAs and the mechanism of localized translation at the NMJ are major outstanding questions in the field. To date, studies have shown that GluRIIA mRNA aggregates are distributed throughout the muscle. The Syp targets identified in this study, such as msp-300, hiw, nrx-1, α-spec, and dlg1, are now excellent candidates for localized expression at the NMJ. Ultimately, conclusive demonstration of localized translation will involve the visualization of new protein synthesis of targets during activity-dependent synaptic plasticity. Biochemical experiments will also be required to establish the precise mode of binding of Syp to its downstream mRNA targets, the basis for interaction specificity, and the molecular mechanism by which Syp differentially regulates the protein levels of its mRNA targets at the Drosophila NMJ. Despite the fact that mammalian SYNCRIP is known to associate with poly(A) tails, this study and other published work have revealed that Syp can associate with specific transcripts (Chen, 2012; McDermott, 2012). How Syp associates with specific mRNAs is unknown, and future studies are needed to uncover whether the interaction of Syp with specific transcripts is dictated by direct binding of the three Syp RRM RNA binding domains or by binding to other specific mRNA binding proteins. It is also possible that specific mRNA stem–loops, similar to the gurken localization signal, are required for Syp to bind to its mRNA targets (McDermott, 2014).

This study shows that msp-300 is the most significant mRNA target of Syp. MSP-300 is the Drosophila ortholog of human Nesprin proteins. These proteins have been genetically implicated in various human myopathies. For example, Nesprin/Syne-1 or Nesprin/Syne-2 is associated with Emery-Dreifuss muscular dystrophy (EDMD) (Puckelwartz, 2009) as well as severe cardiomyopathies (Puckelwartz, 2010). Moreover, Syp itself is increasingly linked with factors and targets that can cause human neurodegenerative disorders. Recent work has revealed that SYNCRIP/hnRNPQ and Fragile X mental retardation protein (FMRP) are present in the same mRNP granule (Chen, 2012), and loss of expression of FMRP or the ability of FMRP to interact with mRNA and polysomes can cause cases of Fragile X syndrome. Separate studies have also shown that SYNCRIP interacts with wild-type survival of motor neuron (SMN) protein but not the truncated or mutant forms found to cause spinal muscular atrophy (Rossoll, 2002), and Syp genetically interacts with Smn mutations in vivo (Sen, 2013). Understanding Syp function in the regulation of such diverse and complex targets may, therefore, provide new avenues for understanding the molecular basis of complex disease phenotypes and potentially lead to future therapeutic approaches (McDermott, 2014).

Syncrip/hnRNP Q influences synaptic transmission and regulates BMP signaling at the Drosophila neuromuscular synapse

Synaptic plasticity involves the modulation of synaptic connections in response to neuronal activity via multiple pathways. One mechanism modulates synaptic transmission by retrograde signals from the post-synapse that influence the probability of vesicle release in the pre-synapse. Despite its importance, very few factors required for the expression of retrograde signals, and proper synaptic transmission, have been identified. This study identified the conserved RNA binding protein Syncrip as a new factor that modulates the efficiency of vesicle release from the motoneuron and is required for correct synapse structure. syncrip is required genetically and its protein product is detected only in the muscle and not in the motoneuron itself. This unexpected non-autonomy is at least partly explained by the fact that Syncrip modulates retrograde BMP signals from the muscle back to the motoneuron. Syncrip influences the levels of the bone morphogenic protein ligand Glass bottom boat from the post-synapse and regulates the pre-synapse. These results highlight the RNA-binding protein Syncrip as a novel regulator of synaptic output. Given its known role in regulating translation, it is proposed that Syncrip is important for maintaining a balance between the strength of presynaptic vesicle release and postsynaptic translation (Halstead, 2014).

Neuronal communication depends on the release of neurotransmitters from the pre-synaptic terminal that causes rapid depolarization in the postsynaptic cell. In tandem, retrograde signals emanating from the post-synapse signal back to the pre-synapse. These signals modulate synaptic output through changes in the structure and function of the pre-synaptic terminals. The retrograde signals in flies are crucial for regulating local synaptic strength during development). In mammalian central neurons, the release of retrograde signals from the dendrites has been implicated in long term potentiation and synapse growth. However, despite their importance, relatively few factors are known to regulate the expression of retrograde signals, and little is known about how retrograde signaling is coordinated with other synaptic processes, such as local translation (Halstead, 2014).

The Drosophila third instar neuromuscular junction (NMJ) is an excellent model to study synaptic function and significant progress has been made in discovering some of the players involved in retrograde signaling. The rapid growth of the muscle during the larval stage requires a concomitant expansion of the neuromuscular synapse to maintain contraction efficacy. The ability to correlate changes on either side of the synapse with neuronal activity has revealed that retrograde signaling regulates plastic growth and homeostasis at the larval NMJ. While the details of the retrograde signaling pathway has remained elusive, genetic and pharmacological studies have implicated calcium signaling, nuclear import pathways, presynaptic exosome secretion, postsynaptic vesicle trafficking via Synaptotagmin family members, and Bone Morphogenic Protein growth factor secretion (Halstead, 2014 and references therein).

The Bone Morphogenic Protein (BMP) pathway contains members of the conserved transforming growth factor β (TGF-β) family and is one of the best characterized retrograde signaling pathways. The retrograde BMP ligand, Glass bottom boat (Gbb) is secreted from the muscle and received by presynaptic receptors, which in turn phosphorylate the transcription factor Mothers against decapentaplegic (Mad) and interact with LIM kinase 1, which act in tandem to contribute to presynaptic stability and growth. P-Mad is then trafficked to the neuron nucleus where it programs transcription. BMP retrograde signaling serves to stabilise synapse structure and neurotransmission. This conserved signaling pathway is central to the development and function of synapses, and misregulation is associated with a number of diseases. Despite this, little is known about the factors that serve to regulate the BMP signaling pathway and coordinate it with other processes in the synapse (Halstead, 2014).

Genetic studies suggest that retrograde signaling proceeds through a complex pathway involving multiple processes. However, one class of gene that has not been well examined in retrograde signaling are the RNA-binding proteins (RBPs). This is surprising, as RBPs are highly expressed in neuronal tissues and are known to be central to the long-term changes that facilitate synapse plasticity through localised translation . Moreover, recent genome-wide analysis has revealed that RBPs can participate in multiple cell processes auxiliary to mRNA metabolism (Halstead, 2014).

This study has identified a new factor in retrograde signaling, Syncrip (Syp), which is required in the muscle to influence vesicle release and membrane integrity pre-synaptically. Syp is the fly homolog of hnRNP Q/SYNaptotagmin-binding Cytoplasmic RNA-Interacting Protein (SYNCRIP) and is a highly conserved heterogenous nuclear ribonucleoprotein (hnRNP). This study identified Syp in a biochemical screen for proteins that associate with the gurken localization signal (Van De Bor, 2005), an RNA signal that is necessary and sufficient for the localization of gurken mRNA to determine axial polarity in the oocyte and future embryo. Syp was found to be required for mRNA localization and local translation of gurken mRNA in oogenesis. Syp has also been detected in mammalian dendrites, where it is found in trafficked RNP granules containing mRNAs encoding synaptic receptors and non-coding regulatory RNAs (Duning, 2008; Bannai, 2004). Moreover, in vitro, mammalian SYNCRIP competes with Poly(A) binding proteins to inhibit translation (Svitkin, 2013) and is required to regulate dendritic morphology (Chen, 2012; Halstead, 2014 and references therein).

This study shows that Drosophila Syp is present in the muscle and required for correct vesicle biogenesis, docking, and neurotransmitter release from the pre-synapse. This unexpected non-autonomous requirement for an RNA binding protein is explained by the fact that Syp is necessary for the correct levels of the canonical BMP signaling pathway in both muscle and neuron. Upregulation of BMP signaling in syp mutants was shown to correlate with aberrant synapse structure and function. This results suggest that the conserved RNA binding protein Syp regulates synaptic output via retrograde signaling. Given that Syp is thought to have a known role in regulating translation, it is proposed that Syp serves to coordinate synaptic efficacy through retrograde signaling with postsynaptic localised translation (Halstead, 2014).

The results highlight the conserved mRNA-binding protein Syp as a novel factor required in the post-synapse for the modulation of synaptic output in the pre-synapse. During synaptic plasticity, neurotransmitter released from the pre-synapse is thought to signal changes in protein production at the post-synapse, which modulates the structure and efficacy of the post-synapse. In the reverse direction, retrograde signals from the postsynapse are thought to signal back to regulate the structure and the rate of secretion from the pre-synapse. Despite their importance, little is known about how these two processes are balanced and coordinated across the synapse during development and synaptic plasticity, which is crucial for memory and learning. This study has identified the RNA binding protein Syp as a new factor that influences both these processes. Given that the mammalian homologues of Drosophila syp (SYNCRIP/hnRNP Q1 and 2) restricts translation in both neuronal and non-neuronal cells (Chen, 2012; Svitkin, 2013), a model was proposed in which Syp co-ordinates translation in the post-synapse with retrograde signaling to the pre-synapse, thus fine-tuning both sides of the synapse. In support of this model, loss of Syp was found to lead to an upregulation of retrograde signaling factors. syp mutants also show enlarged post synaptic densities and a dramatic decrease in the rate of presynaptic vesicle release. In addition, in a parallel study it was revealed that Syp is genetically required only in the muscle to regulate NMJ morphology (McDermott, 2014), since loss of Syp leads to synapse overgrowth and over expression of Syp leads to synapse undergrowth. While the possibility that Syp also has a role in the motoneuron cannot be completely excluded, this seems unlikely. First, Syp protein cannot be detected in the motoneuron and second, RNAi depletion experiments show that Syp is only required postsynaptically and not presynaptically for correct NMJ morphology (Halstead, 2014).

Previous genetic studies have revealed a well characterised pathway for Gbb retrograde signaling. Loss of Glass bottom boat leads to NMJ undergrowth, defects in the integrity of the synaptic membrane, vesicle size, and a decrease in synaptic output, and many of these phenotypes are rescued by expression of Gbb expression in muscle. Accordingly, loss of the presynaptic Gbb receptors, Wishful thinking and Thick veins, and the downstream R-smads, also lead to similar phenotypes. Together these studies show that a decreased in Gbb signalling leads to synapse undergrowth and decrease in synaptic output. The results support a role for Syp in lowering Gbb retrograde signalling, since in syp mutants Gbb protein is elevated and the NMJ synapse is overgrown. However, one aspect of the syp phenotype that does not fit this model is a reduction in the efficiency of vesicle release, which is the opposite of that expected from an increase in Gbb signaling. There are several possible explanations to this discrepancy. While the phenotype of Gbb loss of function has been characterised in detail, overexpression of Gbb has only been used to rescue mutant phenotypes. It is therefore possible that overexpression of Gbb and P-MAD leads to similar effects on vesicle release as the loss of Gbb function. Consistent with this possibility, disruption of the ubiquitin ligase Highwire leads to upregulation of BMP signalling, and to a decrease in synaptic transmission and NMJ overgrowth similar to syncrip mutants. Another possibility is that syp could also be influencing the pre-synapse through roles in other cell types. Supporting this idea is the fact that Syp is present and required in many tissues, has many downstream targets (McDermott, 2014) and its mechanism of action is likely to be different in different individual targets and tissues. Syp could therefore influence vesicle release efficiency in the motorneurons through a role in glia or interneurons. Finally, in a parallel study, immunoprecipitation of Syp was performed followed by high-throughput sequencing to assess the RNA binding partners of Syp (McDermott, 2014). Syp associates with multiple transcripts encoding key synaptic regulators, as well as many mRNAs encoding proteins of unknown function. While Syp does not associate with glass bottom boat mRNA, it is quite possible that it regulates the BMP pathway by binding an mRNA encoding one of the pathway's many other components, a novel BMP regulator, or indeed a previously undiscovered parallel retrograde signalling pathway (Halstead, 2014).

Adding to the complexity of Syp's function, both mammalian SYNCRIP and Drosophila Syp contain three canonical RNA binding domains, RNA recognition motifs (RRMs), which are known to sometimes bind proteins as well as RNA and mammalian SYNCRIP contains an additional RGG/RG rich C-terminal domain that is likely to interact with proteins. This domain is thought to promote interaction between mammalian SYNCRIP and multiple Synaptotagmins in vitro through their common C₂B domain (Mizutani, 2000). While there are 15 known vertebrate Synaptotagmins, only two (Syt 4 and 7) are expressed in the Drosophila third instar larval muscle. Intriguingly, vesicle trafficking by Synaptotagmin 4 at the postsynapse has been implicated in retrograde signaling in both flies and mammals, while the role of Syt 7 is less clear. As a result, Synaptotagmin 4 may seem an attractive candidate to interact with Drosophila Syp. Inspection of the syp gene however shows that Drosophila Syp lacks the 161 amino acid C-terminal domain found in mammalian SYNCRIP that is both necessary and sufficient for association with the Synaptotagmin C₂B domain. The Synaptotagmin-interacting domain in mammalian SYNCRIP is rich in RGG/RG motifs, which can facilitate protein–protein interaction. However, while Syp's RRM domains are highly conserved between mammals and flies, Drosophila Syp contains no canonical RGG/RG motifs. Moreover, genome-wide searches for canonical RGG/RG motifs in flies and mammals detect mammalian SYNCRIP/hnRNP Q and R, but not Drosophila Syp. It is therefore unlikely that Syp mediates retrograde signaling through interaction with Synaptotagmins, though further analysis is required to rule this out conclusively (Halstead, 2014).

Interestingly, mammalian SYNCRIP has been shown to bind polyA sequences and interact with polyA binding protein (PABP) (Svitkin, 2013). PABP interacts with a wide variety of different complexes, including the microRNA-induced silencing complex (miRISC), to serve as a key regulator of global translation. Moreover, PABP has been shown to accumulate at the NMJ post-synapse to promote experience-dependent local translation that alters the efficacy of the synapse. Owing to the enrichment of Syp at the NMJ post-synapse, it is possible that Syp regulates the local expression of key members of the BMP pathway through interaction with the poly(A) tail. However, other studies have revealed Syp discriminates between different mRNAs and associates with specific transcripts (McDermott, 2012; McDermott, 2014; Chen, 2012). As loss of Syp leads to enlargement of the post synaptic densities, Syp may act to restrict translation of specific mRNAs encoding key synaptic proteins. The precise mechanism through which Syp binds specific mRNAs is unknown, but a greater understanding of the in vivo mRNA targets of Syp is likely to reveal common sequences or RNA structures, as well as identifying potential key regulatory targets for localised translation (Halstead, 2014).

In linking retrograde signaling to translation in the postsynaptic compartment, Syp fits with a growing body of evidence showing a role for RNA-binding proteins in integrating RNA metabolism with other key cellular processes. Through interaction with multiple mRNAs and possibly protein targets, Syp could integrate a number of different synaptic processes. Indeed, it is proposed that there are clear advantages for integrating trans-synaptic signaling with postsynaptic translation to balance output on both sides of the synapse. It is interesting to consider that other RBPs central to neurobiology may also be 'moonlighting' in seemingly unrelated processes. This diverse repertoire of RBP functions may explain why mutations in RBPs often lead to neurodegenerative diseases with complex phenotypes. One such highly complex disease phenotype is caused by Fragile X mental retardation protein (FMRP), which interacts with many mRNAs directly. Interestingly, recent work has also revealed that Syp and FMRP are present in the same mRNP granule (Chen, 2012), although they do not interact directly. Furthermore, separate studies have revealed that SYNCRIP binds with wildtype Survival of Motor Neuron (SMN) protein, but not the truncated or mutants forms found in Spinal Muscular Atrophy, and Syp genetically interacts with Smn mutations in vivo (Sen, 2013). While the functional significance of Syp's diverse interactions is not yet fully clear, such data highlight that Syp, like many other RBPs, has an elaborate set of molecular interactions that lead to a complex phenotype when the gene is mutated (Halstead, 2014).

While multiple studies have detected SYNCRIP in the dendrites of mammalian neurons, and experiments in cell culture have revealed the protein as a regulator of translation, the exact in vivo role of SYNCRIP remains untested in mammals. This work highlights a new role for Syp in regulating synapse function and BMP signaling in Drosophila. Given the high degree of similarity between Drosophila and mammalian Syp, the importance of BMP signaling in multiple human diseases and the well conserved features of the Drosophila NMJ as an in vivo model to investigate BMP pathways, it seems likely that Mammalian SYNCRIP will also be shown to have important function in BMP signaling and synapse biology (Halstead, 2014).

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).

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).

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).

The asymmetrically segregating lncRNA cherub is required for transforming stem cells into malignant cells

Tumor cells display features that are not found in healthy cells. How they become immortal and how their specific features can be exploited to combat tumorigenesis are key questions in tumor biology.This study describes the long non-coding RNA cherub (long non-coding RNA:CR43283) that is critically required for the development of brain tumors in Drosophila but is dispensable for normal development. In mitotic Drosophila neural stem cells, cherub localizes to the cell periphery and segregates into the differentiating daughter cell. During tumorigenesis, de-differentiation of cherub-high cells leads to the formation of tumorigenic stem cells that accumulate abnormally high cherub levels. cherub establishes a molecular link between the RNA-binding proteins Staufen and Syncrip. As Syncrip is part of the molecular machinery specifying temporal identity in neural stem cells, it is proposed that tumor cells proliferate indefinitely, because cherub accumulation no longer allows them to complete their temporal neurogenesis program (Landskron, 2018).

Throughout the animal kingdom, stem cells supply tissues with specialized cells. They can do this because they have the unique ability to both replicate themselves (an ability termed self-renewal) and to simultaneously generate other daughter cells with a more restricted developmental potential. Besides their role in tissue homeostasis, stem cells have also been linked to tumor formation. They can turn into so-called tumor stem cells that sustain tumor growth indefinitely. The mechanisms that endow tumor stem cells with indefinite proliferation potential are not fully understood (Landskron, 2018).

Most Drosophila brain tumors originate from the so-called type II neuroblasts (NBIIs). NBIIs divide asymmetrically into a larger cell that retains NB characteristics and a smaller intermediate neural progenitor (INP). Newly formed immature INPs (iINPs) go through a defined set of maturation steps to become transit-amplifying mature INPs (mINPs). After this, a mINP undergoes 3-6 divisions generating one mINP and one ganglion mother cell (GMC) that in turn divides into two terminally differentiating neurons or glial cells (Landskron, 2018).

During each NBII division, a set of cell fate determinants is segregated into the INP. Among those are the Notch inhibitor Numb and the TRIM-NHL protein Brain tumor (Brat). Loss of these cell fate determinants leads to the generation of ectopic NB-like cells at the expense of differentiated brain cells. Formation of malignant brain tumors has also been observed upon the depletion of downstream factors that normally maintain the INP fate (Landskron, 2018).

These features make Drosophila a model for the stepwise acquisition of tumor stem cell properties. When numb or brat are inactivated, the smaller NBII progeny fails to establish an INP fate and initially enters a long transient cell cycle arrest. Only after this lag period, the smaller cell regrows to a NB-sized cell that has acquired tumor stem cell properties and that it is therefore refered to as tumor neuroblast (tNB). NBIIs and ectopic tNBs are indistinguishable in terms of markers. Both cell populations are characterized by the expression of self-renewal genes and lack differentiation markers, but nevertheless behave differently. Shortly after entering pupal stages, NBs decrease their cell volumes successively with each NB division before they exit the cell cycle and differentiate. However, tNBs do not shrink during metamorphosis and continue to proliferate even in the adult fly brain. Moreover, in contrast to wild-type brains, the resulting tumor brains can be serially transplanted into host flies for years, indicating the immortality of these tumors (Landskron, 2018).

Similarly, mammalian homologues of numb and brat have been shown to inhibit tumor growth. Furthermore, the human brat homologue TRIM3 is depleted in 24% of gliomas and NUMB protein levels are markedly reduced in 55% of breast tumor cases. Therefore, results obtained in these Drosophila tumor models are highly relevant (Landskron, 2018).

This study used the Drosophila brat tumor model to investigate how tNBs differ from their physiological counterparts, the NBIIs. The results indicate that progression towards a malignant state is an intrinsic process in brat tNBs that does not correlate with stepwise acquisition of DNA alterations. Transcriptome profiling of larval NBs identified the previously uncharacterized long non-coding (lnc) RNA cherub as crucial for tumorigenesis, but largely dispensable for NB development. The data show that cherub is the first identified lncRNA to be asymmetrically segregated during mitosis into INPs, where the initial high cherub levels decrease with time. Upon the loss of brat, the smaller cherub-high cell reverts into an ectopic tNBs resulting in tumors with high cortical cherub. Molecularly, cherub facilitates the binding between the RNA-binding protein Staufen and the late temporal identity factor Syp and consequently tethers Syp to the plasma membrane. Depleting cherub in brat tNBs leads to the release of Syp from the cortex into the cytoplasm and represses tumor growth. These data provide insight into how defects in asymmetric cell division can contribute to the acquisition of tumorigenic traits without the need of DNA alterations (Landskron, 2018).

It is commonly assumed that cancer cells become malignant and gain replicative immortality by acquiring genetic lesions. Surprisingly, however, the current data indicate that brat tumors do not require additional genetic lesions for the transition to an immortal state. This is not a general feature of Drosophila tumors as genomic instability alone can induce tumors in Drosophila epithelial cells and intestinal stem cells. However, the current results are supported by previous experiments demonstrating that defects in genome integrity do not contribute to primary tumor formation in NBs. Similarly, tumors induced by loss of epigenetic regulators in Drosophila wing discs do not display genome instability. In addition, the short time it takes from the inactivation of brat to the formation of a fully penetrant tumor phenotype would most likely be insufficient for the acquisition of tumor-promoting DNA alterations. More likely, the enormous self-renewal capacity and fast cell cycle of Drosophila NBs requires only minor alterations for the adoption of malignant growth. Interestingly, epigenetic tumorigenesis has been described before in humans, where childhood brain tumors only harbor an extremely low mutation rate and very few recurrent DNA alterations. Comparable observations have been made for leukemia. The current results might help to understand mechanisms of epigenetic tumor formation, which are currently unclear in humans (Landskron, 2018).

cherub is the first lncRNA described to segregate asymmetrically during mitosis. Once cherub is allocated through binding to the RNA-binding protein Staufen into the cytoplasm of INPs, its levels decrease over time. The results show that the inability to segregate cherub into differentiating cells leads to its accumulation in tNBs. The increasing amount of tumor transcriptome data indicates that a vast number of lncRNAs show increased expression levels in various tumor types. Intriguingly, the mammalian homologue of cherub's binding partner Staufen has been also described to asymmetrically localize RNA in dividing neural stem cells. Hence, besides transcriptional upregulation, asymmetric distribution of lncRNAs between sibling cells might play a role in the accumulation of such RNAs in mammalian tumors (Landskron, 2018).

The data suggest a functional connection between cherub and proteins involved in temporal neural stem cell patterning. This study that tNBs retain the early temporal identity factor Imp even during late larval stages. However, IGF-II mRNA-binding protein (Imp) expression in brat mutants is heterogeneous and only a subpopulation of tNBs maintains young identity (Landskron, 2018).

Tumor heterogeneity has also been described for pros tumors, where only a subset of tNBs maintains expression of the early temporal factors Imp and Chinmo. Interestingly, it is this subpopulation that drives tumor growth in prospero tumors (Landskron, 2018).

Consistent with this, genetic experiments show that 'rejuvenating' tNBs enhances tumor growth and consequently increases the survival of tumor bearing flies, whereas 'aging' tNBs identity has the opposite effect. Although mammalian counterparts of Imp have not yet being shown to act as temporal identity genes, their upregulated expression has been implied in various cancer types. Therefore, temporal patterning of NBs has an essential role in brain tumor propagation in Drosophila (Landskron, 2018).

The subset of tNBs that retain early identity in tumors is lost in a cherub mutant background. This suggests that cherub itself might regulate temporal identity. In NBs and tNBs cherub regulates Syp localization by facilitating the binding of Syp to Staufen and thus recruiting it to the cell cortex. In tumors depleted of cherub, Syp localizes mainly to the cytoplasm and is no longer observed at the cortex. As the removal of Syp in tNBs leads to enhanced tumor growth and early lethality, those data suggest that cherub could control temporal NB identity by regulating the subcellular localization of Syp(Landskron, 2018).

How could cherub regulate the function of Syp? The RNA-binding protein Syp is a translational regulator and has been suggested to control mRNA stability. As mammalian SYNCRIP/hnRNP Q interacts with a lncRNA that suppresses translation, cherub might regulate Syp to inhibit or promote the translation of a subset of target mRNAs. In particular, in NBs Syp acts at two stages in NBs during development: Firstly, approximately 60 hr after larval hatching it represses early temporal NB factors, like Imp. Secondly, at the end of the NB lifespan Syp promotes levels of the differentiation factor prospero to facilitate the NB's final cell cycle exit. As cherub depletion in brat tumors leads to decreased tumor growth, it is possible that cherub inhibits the Syp-dependent repression of the early factor Imp, which this study shows to be required for optimal tumor growth. However, cherub mutant NBIIs do not show altered timing or expression of Imp during development. In accordance, brat tumors show high cortical cherub levels, but only a subset of NBs expresses Imp. Rather than rendering Syp completely inactive, it is suggested that cherub decreases the ability of Syp to promote factors important to restrict NB proliferation. As prospero is not expressed in NBIIs, it remains to be investigated which Syp targets are affected by cherub (Landskron, 2018).

Remarkably, cherub mutants are viable, fertile and do not affect NBII lineages. Neurons generated by NBIIs predominantly integrate into the adult brain structure termed central complex, which is important for locomotor activity. As cherub mutants show normal geotaxis, function of the lncRNA seems dispensable for NBIIs to generate their neural descendants (Landskron, 2018).

Nevertheless, the conserved secondary RNA structures of cherub and its conserved expression pattern in other Drosophila species suggest that it has a functional role. There are several possibilities why no phenotype is observed upon the loss of cherub. In wild-type flies cherub might confer robustness. A similar scenario was observed in embryonic NBs, in which Staufen segregates prospero mRNA into GMCs. The failure to segregate prospero mRNA does not result in a phenotype, but it enhances the hypomorphic prospero GMC phenotype. Thus segregation of prospero mRNA serves as support for Prospero protein to induce a GMC fate. Similarly, cherub could act as a backup to reliably establish correct Syp levels in NBIIs and in INPs. Alternatively, cherub might fine-tune the temporal patterning by regulating the cytoplasmic pool of Syp in the NBs. Increasing Syp levels have been suggested to determine distinct temporal windows, in which different INPs and ultimately neurons with various morphologies are sequentially born. Therefore, it cannot be excluded that changes in Syp levels lead to subtle alterations in the number of certain neuron classes produced by NBIIs that only reveal themselves in pathological conditions like tumorigenesis (Landskron, 2018).

This study illustrates how a lncRNA can control the subcellular localization of temporal factors. In addition to temporal NB identity, Syp regulates synaptic transmission and maternal RNA localization. While cherub is not expressed in ovaries or adult heads, Staufen has been implicated in these processes, suggesting that other RNAs might act similarly to cherub. Interestingly, the mammalian Syp homolog hnRNP Q binds the noncoding RNA BC200, whose upregulation is used as a biomarker in ovarian, esophageal, breast and brain cancer. In the future, it will be interesting to investigate whether the mechanism identified in Drosophila is involved in mammalian tumorigenesis as well (Landskron, 2018).

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).

Neuronal upregulation of Prospero protein is driven by alternative mRNA polyadenylation and Syncrip-mediated mRNA stabilisation

During Drosophila and vertebrate brain development, the conserved transcription factor Prospero/Prox1 is an important regulator of the transition between proliferation and differentiation. Prospero level is low in neural stem cells and their immediate progeny, but is upregulated in larval neurons and it is unknown how this process is controlled. This study used single molecule fluorescent in situ hybridisation to show that larval neurons selectively transcribe a long prospero mRNA isoform containing a 15 kb 3' untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the mRNA stability of the long prospero isoform, which allows an upregulation of Prospero protein production. Adult flies selectively lacking the long prospero isoform show abnormal behaviour that could result from impaired locomotor or neurological activity. These findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation (Samuels, 2020).

Many key regulators of NB proliferation and differentiation have been identified and characterised. Recently, an increasing number of RBPs, the key regulators of post-transcriptional processes, have been implicated in neurodevelopment, suggesting that the importance of post-transcriptional regulation in the brain has so far been underestimated. This study has applied smFISH to examine the regulation of the transcription factor (TF) Pros, a master switch promoting neuronal differentiation. Pros expression is regulated by the differential stability of its mRNA isoforms depending on their 3' UTR lengths. Unstable short mRNA isoforms produce sufficient Pros protein to prevent GMCs and their neuronal progeny from reverting back to NB identity, but a switch to the more stable proslong isoforms is required to upregulate Pros protein in larval neurons. These findings highlight the capacity of cell type-specific alternative 3' UTRs to mediate different modes of post-transcriptional regulation of mRNA isoforms (Samuels, 2020).

Surprisingly, it was found that neurons do not de-differentiate into NBs in syp mutants, despite the loss of neuronal Pros upregulation in the absence of proslong. Previous work has shown that Pros elimination in young or middle-aged larval neurons causes de-differentiation of neurons and their reversion to NBs. This work suggests that the low levels of Pros remaining in the syp mutant (provided by prosshort) are sufficient for neurons to maintain their identity. proslong is not required to drive differentiation of GMCs to neurons or to maintain neuronal identity. Although this study has not uncovered the function of proslong in larval/pupal brain development, the impaired locomotive activity of the proslong-REDr adult flies indicates a role of proslong in the adult brain, either because of an earlier neuronal specification event or due to a function of Pros in the adult brain (Samuels, 2020).

Exclusion of the 3' UTR extension from pros transcripts in the proslong-REDr brains reduces the number of pros transcripts and Pros protein in the neurons. However, the pros transcript levels are still much higher in proslong-REDr than in syp mutant brains. This residual pros signal suggests that Syp can stabilise pros mRNA through binding to additional regions of the transcript, perhaps the 5' UTR sequence that is also unique to the proslong transcripts, or some shared sequence included in all transcripts (Samuels, 2020).

The 3' UTR extension of proslong may mediate a second regulatory step, at the level of translation. The upregulation of Pros protein in the neurons is lost in the proslong-REDr brains, despite the relatively high levels of remaining pros mRNA transcripts. While the pros exon smFISH signal is much higher in the proslong-REDr brains, compared to the syp mutant, the Pros protein levels are similar between the two genotypes. This result suggests that the proslong 3' UTR extension includes additional regulatory sequences that promote increased pros translation, either via Syp or an unidentified second RBP. This hypothesis would explain why a moderate decrease in pros transcript levels in proslong-REDr brains leads to a loss of neural Pros protein upregulation (Samuels, 2020).

These experiments show that Pros expression is controlled at two levels: alternative polyadenylation and then differential mRNA stability, regulated through Syp binding. The molecular mechanism underlying the cell type-specific choice of pros isoform has not yet been identified. In Drosophila embryos, Elav is recruited at the promoter of extended genes and is required to extend the 3' UTR of brat. Future experiments will determine whether pros differential polyadenylation is regulated at the promotor region by a similar Elav-dependent mechanism (Samuels, 2020).

Many key regulators in the brain also have complex gene structures such as multiple isoforms and long 3' UTRs, hallmarks of post-transcriptional mechanisms. Such genes include the temporal regulator neuronal fate, chinmo, the driver of cell growth and division, myc and the mRNA-binding proteins, Brat and Imp. Quantitative smFISH approaches combined with genetics and biochemistry will allow the detailed disentanglement of the transcriptional and post-transcriptional mechanisms regulating these genes (Samuels, 2020).

Mammalian SYNCRIP/hnRNPQ is an important regulator of neural development and has a number of post-transcriptional roles including regulating mRNA stability through binding at the 3' end of transcripts. Prox1, the mammalian orthologue of Pros, is a tumour suppressor that regulates stem cell differentiation in the brain as well as many other organ systems. Like Drosophila pros, prox1 has several isoforms including alternative 3' UTRs, and a burst of Prox1 expression is required to drive the differentiation of immature granular neurons in the adult hippocampus. It is plausible that post-transcriptional regulation by RBPs helps determine the expression and translation of Prox1. Application of quantitative smFISH to mammalian systems will uncover whether Prox1 expression levels, like pros, are regulated through differential stabilisation of different 3' UTR isoforms (Samuels, 2020).

Drosophila Syncrip binds the gurken mRNA localisation signal and regulates localised transcripts during axis specification

In the Drosophila oocyte, mRNA transport and localised translation play a fundamental role in axis determination and germline formation of the future embryo. gurken mRNA encodes a secreted TGF-alpha signal that specifies dorsal structures, and is localised to the dorso-anterior corner of the oocyte via a cis-acting 64 nucleotide gurken localisation signal. Using GRNA chromatography, this study characterised the biochemical composition of the ribonucleoprotein complexes that form around the gurken mRNA localisation signal in the oocyte. A number of the factors already known to be involved in gurken localisation and translational regulation, such as Squid and Imp, were identified, in addition to a number of factors with known links to mRNA localisation, such as Me31B and Exu. Previously uncharacterised Drosophila proteins were identified, including the fly homologue of mammalian SYNCRIP/hnRNPQ, a component of RNA transport granules in the dendrites of mammalian hippocampal neurons. It was shown that Drosophila Syncrip binds specifically to gurken and oskar, but not bicoid transcripts. The loss-of-function and overexpression phenotypes of syncrip in Drosophila egg chambers show that the protein is required for correct grk and osk mRNA localisation and translational regulation. It is concluded that Drosophila Syncrip is a new factor required for localisation and translational regulation of oskar and gurken mRNA in the oocyte. It is proposed that Syncrip/SYNCRIP is part of a conserved complex associated with localised transcripts and required for their correct translational regulation in flies and mammals (McDermott, 2012).

This study has identified Drosophila Syp as a novel conserved component of localised RNP granules. Syp associates specifically with grk and osk and is required for their localisation and translational regulation in the Drosophila germline. Although SYNCRIP has been studied in mammalian cells using biochemical approaches, this study is the first to address the function of Syp in vivo, and particularly in generating cellular asymmetry in the germline. Despite a number of genetic screens that have been carried out to identify the genes required for axis specification in flies, Syp was not previously identified as being required for axis determination. This work together with a number of other biochemical based studies illustrates that there are many other essential factors that are still to be identified as having a role in axis specification (McDermott, 2012).

Evidence is presented in this study that Syp is required for axis specification and germline formation by affecting the localisation and translation of grk and osk mRNAs. The phenotypes of loss of function mutations in the gene, and overexpression of the protein support an interesting role for Syp in regulating grk and osk mRNAs. It is noted that the loss of function phenotypes are of low penetrance and are such that further studies are required to uncover the precise mechanism of Syp function. However, Syp is not the only component of grk and osk mRNPs that has a partially penetrant loss of function phenotype. Indeed, others such as IGF-II mRNA-binding protein (Imp) give a stronger phenotype only when in combination with other mutations. Unexpectedly, overexpression of Syp gives the same phenotype as its loss of function, but at a higher penetrance. These results are potentially interesting, but difficult to interpret with certainty. The interpretation is favoured that a certain stoichiometry is necessary within the RNP complexes in which Syp is found. Overexpression of Syp may cause the displacement of certain translational repressors, allowing grk mRNA to be prematurely translated in the nurse cells. In contrast, with loss of function of Syp, grk mRNA localisation and translational regulation may be disrupted in different ways because loss of Syp could lead to decreased stability of the RNP complex. This could in turn lead to the loss of certain components necessary for localization and translational regulation. The eggshell phenotypes observed in the syp germline clones also include some defects that are not typical of a disruption of grk mRNA localisation and translation. These results are interpreted as indicating that Syp has additional target mRNAs in the germline whose localisation and/or translation are affected in the syp mutant. On the basis of the morphological defects observed in a number of eggs these targets may include mRNAs involved in follicle cell migration or actin organisation during oogenesis, such as bullwinkle (bwk) or chickadee (chic). Mislocalisation and/or altered translation of these mRNAs could result in the shorter eggs and the bwk-like dorsal appendage defects that are observed (McDermott, 2012).

The physical association of Syp with these mRNAs further supports a function for Syp in the regulation of grk and osk mRNAs, although it is unclear whether this is through a direct or indirect interaction. Syp does not appear to colocalise with localised grk or osk in the oocyte, and so Syp may function before the mRNAs reach their final destination in the oocyte in order to influence localisation and translation. Syp was also identified biochemically with a number of the factors already known to be required for grk and osk mRNA localisation and translational regulation. These include Sqd, Imp, PTB, PABP, Me31B and BicC. The homologues of these factors were also identified in biochemical studies of SYNCRIP interactors in mammalian cells. Therefore, it is proposed that the current results have uncovered a conserved module of RNA binding proteins that are required for both mRNA transport and translational regulation. The Syp associated complex that this study has uncovered binds to grk via a relatively small stem loop sequence, the GLS. While Syp also associates with osk mRNA, the minimal region necessary and sufficient for osk mRNA localisation has not yet been defined. Therefore, it is unknown whether a small region is required in this case, and if so whether it is in any way similar to the GLS, structurally or in primary sequence (McDermott, 2012).

Taking the results of this study in the context of other data and the previous publications on SYNCRIP and its associated proteins in mammalian hippocampal neurons, it is proposed that Syp may be present at neuronal synapses in a complex with at least some of the same proteins that are required for grk mRNA localisation. This idea is supported by the fact that at least some of these factors, namely Sqd, Imp, PTB, PABP and Me31B are also present in the Drosophila nervous system. The expression studies show that Syp is absent from embryos but is highly expressed in the larval nervous system. Given the role of Syp in mRNA localization and translational regulation in the oocyte, and its presence in larval brains, it is proposed that Syp could have a similar function in the nervous system. In comparison with the oocyte, much less is known about localised transcripts and their translational regulation in the nervous system, and it remains to be determined to what extent this proposal is valid and in which neuronal tissues Syp is required in larvae. Nevertheless, this work is the first demonstration that Syp functions in mRNA localisation and translational control in the oocyte and coupled with the work on mammalian SYNCRIP showing association with RNP granules in the dendrites of hippocampal neurons it is attractive to propose that Syp protein also has a conserved function in regulation of neuronal mRNAs (McDermott, 2012).

Search PubMed for articles about Drosophila Syncrip

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

Bannai, H., Fukatsu, K., Mizutani, A., Natsume, T., Iemura, S., Ikegami, T., Inoue, T. and Mikoshiba, K. (2004). An RNA-interacting protein, SYNCRIP (heterogeneous nuclear ribonuclear protein Q1/NSAP1) is a component of mRNA granule transported with inositol 1,4,5-trisphosphate receptor type 1 mRNA in neuronal dendrites. J Biol Chem 279: 53427-53434. PubMed ID: 15475564

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

Chen, H. H., Chang, J. G., Lu, R. M., Peng, T. Y. and Tarn, W. Y. (2008). The RNA binding protein hnRNP Q modulates the utilization of exon 7 in the survival motor neuron 2 (SMN2) gene. Mol Cell Biol 28: 6929-6938. PubMed ID: 18794368

Chen, H. H., Yu, H. I., Chiang, W. C., Lin, Y. D., Shia, B. C. and Tarn, W. Y. (2012). hnRNP Q regulates Cdc42-mediated neuronal morphogenesis. Mol Cell Biol 32: 2224-2238. PubMed ID: 22493061

Duning, K., Buck, F., Barnekow, A. and Kremerskothen, J. (2008). SYNCRIP, a component of dendritically localized mRNPs, binds to the translation regulator BC200 RNA. J Neurochem 105: 351-359. PubMed ID: 18045242

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

Elvira, G., Wasiak, S., Blandford, V., Tong, X. K., Serrano, A., Fan, X., del Rayo Sanchez-Carbente, M., Servant, F., Bell, A. W., Boismenu, D., Lacaille, J. C., McPherson, P. S., DesGroseillers, L. and Sossin, W. S. (2006). Characterization of an RNA granule from developing brain. Mol Cell Proteomics 5: 635-651. PubMed ID: 16352523

Halstead, J. M., Lin, Y. Q., Durraine, L., Hamilton, R. S., Ball, G., Neely, G. G., Bellen, H. J. and Davis, I. (2014). Syncrip/hnRNP Q influences synaptic transmission and regulates BMP signaling at the Drosophila neuromuscular synapse. Biol Open. PubMed ID: 25171887

Kanai, Y., Dohmae, N. and Hirokawa, N. (2004). Kinesin transports RNA: isolation and characterization of an RNA-transporting granule. Neuron 43: 513-525. PubMed ID: 15312650

Landskron, L., Steinmann, V., Bonnay, F., Burkard, T. R., Steinmann, J., Reichardt, I., Harzer, H., Laurenson, A. S., Reichert, H. and Knoblich, J. A. (2018). The asymmetrically segregating lncRNA cherub is required for transforming stem cells into malignant cells. Elife 7. PubMed ID: 29580384



Lee, K. H., Woo, K. C., Kim, D. Y., Kim, T. D., Shin, J., Park, S. M., Jang, S. K. and Kim, K. T. (2012). Rhythmic interaction between Period1 mRNA and hnRNP Q leads to circadian time-dependent translation. Mol Cell Biol 32: 717-728. PubMed ID: 22124155

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

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(10): 1593-606. PubMed ID: 25171822

Mizutani, A., Fukuda, M., Ibata, K., Shiraishi, Y. and Mikoshiba, K. (2000). SYNCRIP, a cytoplasmic counterpart of heterogeneous nuclear ribonucleoprotein R, interacts with ubiquitous synaptotagmin isoforms. J Biol Chem 275: 9823-9831. PubMed ID: 10734137

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 ID: 16476777

Puckelwartz, M. J., Kessler, E., Zhang, Y., Hodzic, D., Randles, K. N., Morris, G., Earley, J. U., Hadhazy, M., Holaska, J. M., Mewborn, S. K., Pytel, P. and McNally, E. M. (2009). Disruption of nesprin-1 produces an Emery Dreifuss muscular dystrophy-like phenotype in mice. Hum Mol Genet 18: 607-620. PubMed ID: 19008300

Puckelwartz, M. J., Kessler, E. J., Kim, G., Dewitt, M. M., Zhang, Y., Earley, J. U., Depreux, F. F., Holaska, J., Mewborn, S. K., Pytel, P. and McNally, E. M. (2010). Nesprin-1 mutations in human and murine cardiomyopathy. J Mol Cell Cardiol 48: 600-608. PubMed ID: 19944109

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 27(9):1303-1313. PubMed ID: 28434858

Rossoll, W., Kroning, A. K., Ohndorf, U. M., Steegborn, C., Jablonka, S. and Sendtner, M. (2002). Specific interaction of Smn, the spinal muscular atrophy determining gene product, with hnRNP-R and gry-rbp/hnRNP-Q: a role for Smn in RNA processing in motor axons? Hum Mol Genet 11: 93-105. PubMed ID: 11773003

Sen, A., Dimlich, D. N., Guruharsha, K. G., Kankel, M. W., Hori, K., Yokokura, T., Brachat, S., Richardson, D., Loureiro, J., Sivasankaran, R., Curtis, D., Davidow, L. S., Rubin, L. L., Hart, A. C., Van Vactor, D. and Artavanis-Tsakonas, S. (2013). Genetic circuitry of Survival motor neuron, the gene underlying spinal muscular atrophy. Proc Natl Acad Sci U S A 110: E2371-2380. PubMed ID: 23757500

Syed, M. H., Mark, B. and Doe, C. Q. (2017). Steroid hormone induction of temporal gene expression in Drosophila brain neuroblasts generates neuronal and glial diversity. Elife 6 [Epub ahead of print]. PubMed ID: 28394252

Samuels, T. J., Arava, Y., Jarvelin, A. I., Robertson, F., Lee, J. Y., Yang, L., Yang, C. P., Lee, T., Ish-Horowicz, D. and Davis, I. (2020). Neuronal upregulation of Prospero protein is driven by alternative mRNA polyadenylation and Syncrip-mediated mRNA stabilisation. Biol Open. PubMed ID: 32205310

Van De Bor, V., Hartswood, E., Jones, C., Finnegan, D. and Davis, I. (2005). gurken and the I factor retrotransposon RNAs share common localization signals and machinery. Dev Cell 9: 51-62. PubMed ID: 15992540

Wang, Y. C., Yang, J. S., Johnston, R., Ren, Q., Lee, Y. J., Luan, H., Brody, T., Odenwald, W. F. and Lee, T. (2014). Drosophila intermediate neural progenitors produce lineage-dependent related series of diverse neurons. Development 141(2): 253-258. PubMed ID: 24306106

Xing, L., Yao, X., Williams, K. R. and Bassell, G. J. (2012). Negative regulation of RhoA translation and signaling by hnRNP-Q1 affects cellular morphogenesis. Mol Biol Cell 23: 1500-1509. PubMed ID: 22357624

date revised: 21 June 2024

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