Opposite Imp/Syp temporal gradients govern birth time-dependent neuronal fates
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ABSTRACT: Generating diverse neurons involves spatially distinct neural stem cells that show age dependent developmental fates. Drosophila neuroblasts produce long, diverse yet stereotyped series of distinct neurons, called lineages. We searched for novel temporal factors that could pattern such extended lineages by RNA-sequencing of specific neuroblasts at various developmental times. We found that two RNA-binding proteins, Imp and Syp, display opposing high-to-low and low-to-high temporal gradients with distinct dynamics in specific lineages. Manipulating Imp/Syp levels in mushroom body neuroblasts revealed opposing roles in the specification of early and late temporal fates, primarily via regulation of Chinmo translation. This study implicates the opposing Imp/Syp gradients as temporal morphogens that encode stem cell age and govern birth time-dependent offspring cell fate through post-transcriptional regulation of temporal dentity genes.
Project description:One neural stem cell can produce multiple transiently amplifying, intermediate neural progenitors (INP), which collectively yield diverse neuronal types. It is unclear if and how serially derived INPs contribute to neuron fate diversification. Drosophila type II neuroblasts, like mammalian neural stem cells, deposit neurons and also glia through INPs. The consecutively born INPs in a given lineage produce morphologically distinct progeny, presumably due to their inheritance of different temporal factors from the INP-producing progenitor. To uncover the underlying temporal fating mechanisms, we profiled type II neuroblasts' transcriptome across time. Our 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/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. Further, we reveal that the transcription factor, Seven-up initiates progression of the Imp/Syp gradients. Interestingly, neuroblasts apparently locked in their beginning Imp/Syp levels can still yield progeny with a small range of early fates. We propose 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.
Project description:The temporal patterning of neural stem cells is a powerful mechanism to generate neural diversity. In Drosophila, the progression of post-embryonic neurogenesis is driven by RNA-binding proteins Imp (IGF2BP) and Syp (SYNCRIP), however how they achieve their function is not well understood, since little is known about their RNA targets in the brain. Here, we present comprehensive RNA interactomes of Imp and Syp at different time points during larval brain development. Imp and Syp target highly overlapping sets of mRNAs whose encoded proteins impact fate specification, stem cell maintenance and tumourigenesis. Within these transcripts, Imp and Syp footprints exhibit limited overlap, instead we show their binding sites have co-evolved, suggesting a combinatorial or cooperative mode of regulation rather than competitive binding. Further, we identify RNAs dynamically interacting with Imp/Syp across development, consisting of existing and potentially new candidates of the neurogenesis program, and loss of Imp or Syp disrupts expression of key transcripts denotative of early or late-born neurons. Together, our findings highlight a post-transcriptional regulatory network that influences temporal order, and our study is a valuable resource for future studies finely-dissecting molecular mechanisms of neuronal fate specifications.
Project description:A brain consists of numerous distinct neurons arising from a limited number of progenitors, called neuroblasts in Drosophila. Each neuroblast makes a specific neuronal lineage. To unravel the transcriptional networks that underlie the development of distinct neuroblast lineages, we marked and isolated lineage-specific neuroblasts for RNA sequencing. We labeled particular neuroblasts throughout neurogenesis by activating a conditional neuroblast driver in specific lineages using various intersection strategies. The targeted neuroblasts were efficiently recovered using a custom-built device for robotic single cell picking. Transcriptome analysis on the mushroom body, antennal lobe, and type II neuroblasts besides non-selective neuroblasts, neurons, and glia revealed a rich repertoire of transcription factors expressed among neuroblasts in diverse patterns. In addition to those likely pan-neuroblast transcription factors, there exist many transcription factors selectively enriched or repressed in certain neuroblasts. The unique combinations of transcription factors present in different neuroblasts may govern the diverse lineage-specific neuron fates
Project description:As a newly-invented and highly-efficiency strobilurin fungicide, pyraoxystrobin (SYP-3343) has been recognized as a highly poisonous toxin for a variety of aquatic organisms. Nevertheless, the developmental toxicity and potential mechanism of SYP-3343 have not been well-documented. The results showed that SYP-3343 was relatively stable and maintained within the range of 20 % in 24 h, and the LC50 value to embryos at 72 hpf was 17.13 μg/L. The zebrafish embryotoxicity induced by 1, 2, 4, and 8 μg/L SYP-3343 is demonstrated by repressive embryo incubation, enhancive mortality rate, abnormal heart rate, malformed morphological characteristic, and impaired spontaneous coiling, indicating SYP-3343 mostly exerted its toxicity in a dose- and time-dependent manner. Besides SYP-3343 was critically involved in regulating cell cycle, mitochondrial membrane potential, and reactive oxygen species production as well as zebrafish primary cells apoptosis, which can be mitigated using antioxidant N-acetyl-L-cysteine. A significant change occurred in total protein content, the biochemical indices, and antioxidant capacities owing to SYP-3343 exposure. Additionally, SYP-3343 altered the mRNA levels of heart development-, mitochondrial function-, and apoptosis-related genes in zebrafish embryos. These results indicated that SYP-3343 induced apoptosis accompanying reactive oxygen species-initiated mitochondrial dysfunction in zebrafish embryos.
Project description:The numerous neurons and glia that form the brain originate from tightly controlled growth and division of neural stem cells, regulated systemically by known extrinsic signals. However, the intrinsic mechanisms that control the characteristic proliferation rates of individual neural stem cells are unknown. Here, we show that the size and division rates of Drosophila neural stem cells (neuroblasts) are controlled by the highly conserved RNA binding protein Imp (IGF2BP), via one of its top binding targets in the brain, myc mRNA. We show that Imp stabilises myc mRNA leading to increased Myc protein levels, larger neuroblasts, and faster division rates. Declining Imp levels throughout development limit myc mRNA stability to restrain neuroblast growth and division, while heterogeneous Imp expression correlates with myc mRNA stability between individual neuroblasts in the brain. We propose that Imp-dependent regulation of myc mRNA stability fine-tunes individual neural stem cell proliferation rates.
Project description:During development, neural stem cells are temporally patterned to sequentially generate a variety of neural types before exiting the cell cycle. Temporal patterning is well-studied in Drosophila, where neural stem cells called neuroblasts sequentially express cascades of Temporal Transcription Factors (TTFs) to control the birth-order dependent neural specification. However, currently known TTFs were mostly identified through candidate antibody screening and may not be complete. In addition, many fundamental questions remain concerning the TTF cascade initiation, progression, and termination. It is also not known why temporal progression only happens in neuroblasts but not in their differentiated progeny. In this work, we performed single-cell RNA sequencing of Drosophila medulla neuroblasts of all ages to study the temporal patterning process with single-cell resolution. Our scRNA-seq data revealed that sets of genes involved in different biological processes show high to low or low to high gradients as neuroblasts age. We also identified a list of novel TTFs, and experimentally characterized their roles in the temporal progression and neural fate specification. Our study revealed a comprehensive temporal gene network that patterns medulla neuroblasts from start to end. Furthermore, we found that the progression and termination of this temporal cascade also require transcription factors differentially expressed along the differentiation axis (neuroblasts -> -> neurons). Lola proteins function as a speed modulator of temporal progression in neuroblasts; while Nerfin-1, a factor required to suppress de-differentiation in post-mitotic neurons, acts at the final temporal stage together with the last TTF of the cascade, to promote the switch to gliogenesis and the cell cycle exit. Our comprehensive study of the medulla neuroblast temporal cascade illustrated mechanisms that might be conserved in the temporal patterning of neural stem cells.