RESUMEN
Mushroom bodies (MB) of adult Drosophila have a core of thousands of Kenyon neurons; axons of the early-born g class form a medial lobe and those from later-born α'ß' and αß classes form both medial and vertical lobes. The larva, however, hatches with only γ neurons and forms a vertical lobe 'facsimile' using larval-specific axon branches from its γ neurons. MB input (MBINs) and output (MBONs) neurons divide the Kenyon neuron lobes into discrete computational compartments. The larva has 10 such compartments while the adult has 16. We determined the fates of 28 of the 32 MBONs and MBINs that define the 10 larval compartments. Seven compartments are subsequently incorporated into the adult MB; four of their MBINs die, while 12 MBINs/MBONs remodel to function in adult compartments. The remaining three compartments are larval specific. At metamorphosis their MBIN/MBONs trans-differentiate, leaving the MB for other adult brain circuits. The adult vertical lobes are made de novo using MBONs/MBINs recruited from pools of adult-specific neurons. The combination of cell death, compartment shifting, trans-differentiation, and recruitment of new neurons result in no larval MBIN-MBON connections being maintained through metamorphosis. At this simple level, then, we find no anatomical substrate for a memory trace persisting from larva to adult. The adult phenotype of the trans-differentiating neurons represents their evolutionarily ancestral phenotype while their larval phenotype is a derived adaptation for the larval stage. These cells arise primarily within lineages that also produce permanent MBINs and MBONs, suggesting that larval specifying factors may allow information related to birth-order or sibling identity to be interpreted in a modified manner in the larva to allow these neurons to acquire larval phenotypic modifications. The loss of such factors at metamorphosis then allows these neurons to revert to their ancestral functions in the adult.
Asunto(s)
Drosophila , Neuronas , Animales , Drosophila/fisiología , Larva/fisiología , Neuronas/fisiología , Encéfalo , Axones , Cuerpos Pedunculados/fisiología , Drosophila melanogaster/fisiología , Metamorfosis BiológicaRESUMEN
During development, regulatory factors appear in a precise order to determine cell fates over time. Consequently, to investigate complex tissue development, it is necessary to visualize and manipulate cell lineages with temporal control. Current strategies for tracing vertebrate cell lineages lack genetic access to sequentially produced cells. Here, we present TEMPO (Temporal Encoding and Manipulation in a Predefined Order), an imaging-readable genetic tool allowing differential labeling and manipulation of consecutive cell generations in vertebrates. TEMPO is based on CRISPR and powered by a cascade of gRNAs that drive orderly activation and inactivation of reporters and/or effectors. Using TEMPO to visualize zebrafish and mouse neurogenesis, we recapitulated birth-order-dependent neuronal fates. Temporally manipulating cell-cycle regulators in mouse cortex progenitors altered the proportion and distribution of neurons and glia, revealing the effects of temporal gene perturbation on serial cell fates. Thus, TEMPO enables sequential manipulation of molecular factors, crucial to study cell-type specification.
Asunto(s)
Neuronas , Pez Cebra , Animales , Ratones , Linaje de la Célula/fisiología , Neuronas/fisiología , Neuroglía , Diferenciación Celular/genética , Neurogénesis/genética , Regulación del Desarrollo de la Expresión GénicaRESUMEN
Cell lineage defines the mitotic connection between cells that make up an organism. Mapping these connections in relation to cell identity offers an extraordinary insight into the mechanisms underlying normal and pathological development. The analysis of molecular determinants involved in the acquisition of cell identity requires gaining experimental access to precise parts of cell lineages. Recently, we have developed CaSSA and CLADES, a new technology based on CRISPR that allows targeting and labeling specific lineage branches. Here we discuss how to better exploit this technology for lineage studies in Drosophila, with an emphasis on neuronal specification.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Drosophila , Animales , Linaje de la Célula/genética , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Drosophila/genética , Neuronas/fisiologíaRESUMEN
While we think of neurons as having a fixed identity, many show spectacular plasticity.1-10 Metamorphosis drives massive changes in the fly brain;11,12 neurons that persist into adulthood often change in response to the steroid hormone ecdysone.13,14 Besides driving remodeling,11-14 ecdysone signaling can also alter the differentiation status of neurons.7,15 The three sequentially born subtypes of mushroom body (MB) Kenyon cells (γ, followed by α'/ß', and finally α/ß)16 serve as a model of temporal fating.17-21 γ neurons are also used as a model of remodeling during metamorphosis. As γ neurons are the only functional Kenyon cells in the larval brain, they serve the function of all three adult subtypes. Correspondingly, larval γ neurons have a similar morphology to α'/ß' and α/ß neurons-their axons project dorsally and medially. During metamorphosis, γ neurons remodel to form a single medial projection. Both temporal fate changes and defects in remodeling therefore alter γ-neuron morphology in similar ways. Mamo, a broad-complex, tramtrack, and bric-à-brac/poxvirus and zinc finger (BTB/POZ) transcription factor critical for temporal specification of α'/ß' neurons,18,19 was recently described as essential for γ remodeling.22 In a previous study, we noticed a change in the number of adult Kenyon cells expressing γ-specific markers when mamo was manipulated.18 These data implied a role for Mamo in γ-neuron fate specification, yet mamo is not expressed in γ neurons until pupariation,18,22 well past γ specification. This indicates that mamo has a later role in ensuring that γ neurons express the correct Kenyon cell subtype-specific genes in the adult brain.
Asunto(s)
Ecdisona , Cuerpos Pedunculados , Animales , Axones , Diferenciación Celular , Larva , Cuerpos Pedunculados/fisiología , Neuronas/fisiologíaRESUMEN
Cells in many tissues, such as bone, muscle, and placenta, fuse into syncytia to acquire new functions and transcriptional programs. While it is known that fused cells are specialized, it is unclear whether cell-fusion itself contributes to programmatic-changes that generate the new cellular state. Here, we address this by employing a fusogen-mediated, cell-fusion system to create syncytia from undifferentiated cells. RNA-Seq analysis reveals VSV-G-induced cell fusion precedes transcriptional changes. To gain mechanistic insights, we measure the plasma membrane surface area after cell-fusion and observe it diminishes through increases in endocytosis. Consequently, glucose transporters internalize, and cytoplasmic glucose and ATP transiently decrease. This reduced energetic state activates AMPK, which inhibits YAP1, causing transcriptional-reprogramming and cell-cycle arrest. Impairing either endocytosis or AMPK activity prevents YAP1 inhibition and cell-cycle arrest after fusion. Together, these data demonstrate plasma membrane diminishment upon cell-fusion causes transient nutrient stress that may promote transcriptional-reprogramming independent from extrinsic cues.
Asunto(s)
Proteínas Adaptadoras Transductoras de Señales/metabolismo , Membrana Celular/metabolismo , Núcleo Celular/metabolismo , Glicoproteínas de Membrana/metabolismo , Factores de Transcripción/metabolismo , Transcripción Genética/genética , Proteínas del Envoltorio Viral/metabolismo , Proteínas Quinasas Activadas por AMP/genética , Proteínas Quinasas Activadas por AMP/metabolismo , Proteínas Adaptadoras Transductoras de Señales/genética , Animales , Transporte Biológico , Fusión Celular , Línea Celular , Línea Celular Tumoral , Células Cultivadas , Células Gigantes/metabolismo , Células HEK293 , Humanos , Glicoproteínas de Membrana/genética , Ratones , RNA-Seq/métodos , Transducción de Señal/genética , Factores de Transcripción/genética , Proteínas del Envoltorio Viral/genética , Proteínas Señalizadoras YAPRESUMEN
Reconstructing the genealogy of every cell that makes up an organism remains a long-standing challenge in developmental biology. Besides its relevance for understanding the mechanisms underlying normal and pathological development, resolving the lineage origin of cell types will be crucial to create these types on-demand. Multiple strategies have been deployed towards the problem of lineage tracing, ranging from direct observation to sophisticated genetic approaches. Here we discuss the achievements and limitations of past and current technology. Finally, we speculate about the future of lineage tracing and how to reach the next milestones in the field.
Asunto(s)
Linaje de la Célula , HumanosRESUMEN
We present CLADES (cell lineage access driven by an edition sequence), a technology for cell lineage studies based on CRISPR-Cas9 techniques. CLADES relies on a system of genetic switches to activate and inactivate reporter genes in a predetermined order. Targeting CLADES to progenitor cells allows the progeny to inherit a sequential cascade of reporters, thereby coupling birth order to reporter expression. This system, which can also be temporally induced by heat shock, enables the temporal resolution of lineage development and can therefore be used to deconstruct an extended cell lineage by tracking the reporters expressed in the progeny. When targeted to the germ line, the same cascade progresses across animal generations, predominantly marking each generation with the corresponding combination of reporters. CLADES therefore offers an innovative strategy for making programmable cascades of genes that can be used for genetic manipulation or to record serial biological events.
Asunto(s)
Linaje de la Célula/genética , Animales , Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas , Reparación del ADN , Drosophila melanogaster , Técnicas de Sustitución del Gen , Genes Reporteros/genética , Proteínas de Choque Térmico/genética , Células Madre Pluripotentes Inducidas , Edición de ARN , Activación Transcripcional , Pez CebraRESUMEN
Gene targeting is an incredibly valuable technique. Sometimes, however, it can also be extremely challenging for various intrinsic reasons (e.g. low target accessibility or nature/extent of gene modification). To bypass these barriers, we designed a transgene-based system in Drosophila that increases the number of independent gene targeting events while at the same time enriching for correctly targeted progeny. Unfortunately, with particularly challenging gene targeting experiments, our original design yielded numerous false positives. Here, we deliver a much-improved technique, named Enhanced Golic+ (E-Golic+). E-Golic+ incorporates genetic modifications to tighten lethality-based selection while simultaneously boosting efficiency. With E-Golic+, we easily achieve previously unattainable gene targeting. Additionally, we built an E-Golic+-based, high-efficiency genetic pipeline for transgene swapping. We demonstrate its utility by transforming GAL4 enhancer-trap lines into tissue-specific Cas9-expressing lines. Given the superior efficiency, specificity and scalability, E-Golic+ promises to expedite development of additional sophisticated genetic/genomic tools in Drosophila.
Asunto(s)
Repeticiones Palindrómicas Cortas Agrupadas y Regularmente Espaciadas/genética , Drosophila/metabolismo , Marcación de Gen/métodos , Transgenes/genética , Animales , Animales Modificados Genéticamente/genética , Animales Modificados Genéticamente/metabolismo , Drosophila/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Femenino , Células Germinativas/citología , Células Germinativas/metabolismo , Masculino , Regiones Promotoras Genéticas , ARN Guía de Kinetoplastida/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismoRESUMEN
Wiring a complex brain requires many neurons with intricate cell specificity, generated by a limited number of neural stem cells. Drosophila central brain lineages are a predetermined series of neurons, born in a specific order. To understand how lineage identity translates to neuron morphology, we mapped 18 Drosophila central brain lineages. While we found large aggregate differences between lineages, we also discovered shared patterns of morphological diversification. Lineage identity plus Notch-mediated sister fate govern primary neuron trajectories, whereas temporal fate diversifies terminal elaborations. Further, morphological neuron types may arise repeatedly, interspersed with other types. Despite the complexity, related lineages produce similar neuron types in comparable temporal patterns. Different stem cells even yield two identical series of dopaminergic neuron types, but with unrelated sister neurons. Together, these phenomena suggest that straightforward rules drive incredible neuronal complexity, and that large changes in morphology can result from relatively simple fating mechanisms.
Asunto(s)
Encéfalo/fisiología , Linaje de la Célula , Drosophila melanogaster/citología , Células-Madre Neurales/citología , Neurogénesis , Animales , Encéfalo/citología , Drosophila melanogaster/genética , Larva , Neuronas/citologíaRESUMEN
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. Here, we use single molecule fluorescent in situ hybridisation to show that larval neurons selectively transcribe a long prospero mRNA isoform containing a 15â kb 3' untranslated region, which is bound in the brain by the conserved RNA-binding protein Syncrip/hnRNPQ. Syncrip binding increases the stability of the long prospero mRNA 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. Our findings highlight a regulatory strategy involving alternative polyadenylation followed by differential post-transcriptional regulation.This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Proteínas de Drosophila/genética , Drosophila/fisiología , Regulación del Desarrollo de la Expresión Génica , Proteínas del Tejido Nervioso/genética , Neuronas/metabolismo , Proteínas Nucleares/genética , Poliadenilación , ARN Mensajero/genética , Factores de Transcripción/genética , Regiones no Traducidas 3' , Animales , Proteínas de Drosophila/metabolismo , Inmunohistoquímica , Proteínas del Tejido Nervioso/metabolismo , Proteínas Nucleares/metabolismo , Especificidad de Órganos/genética , Estabilidad del ARN , Proteínas de Unión al ARN/genética , Proteínas de Unión al ARN/metabolismo , Factores de Transcripción/metabolismoRESUMEN
The genome is the blueprint for an organism. Interrogating the genome, especially locating critical cis-regulatory elements, requires deletion analysis. This is conventionally performed using synthetic constructs, making it cumbersome and non-physiological. Thus, we created Cas9-mediated Arrayed Mutagenesis of Individual Offspring (CAMIO) to achieve comprehensive analysis of a targeted region of native DNA. CAMIO utilizes CRISPR that is spatially restricted to generate independent deletions in the intact Drosophila genome. Controlled by recombination, a single guide RNA is stochastically chosen from a set targeting a specific DNA region. Combining two sets increases variability, leading to either indels at 1-2 target sites or inter-target deletions. Cas9 restriction to male germ cells elicits autonomous double-strand-break repair, consequently creating offspring with diverse mutations. Thus, from a single population cross, we can obtain a deletion matrix covering a large expanse of DNA at both coarse and fine resolution. We demonstrate the ease and power of CAMIO by mapping 5'UTR sequences crucial for chinmo's post-transcriptional regulation.
Asunto(s)
Sistemas CRISPR-Cas , Drosophila/genética , Edición Génica , Mutagénesis , Regiones no Traducidas 5' , Animales , Animales Modificados Genéticamente , Proteína 9 Asociada a CRISPR , Proteínas de Drosophila/genética , Genoma de los Insectos , Mutación INDEL , Masculino , Proteínas del Tejido Nervioso/genética , Espermatozoides/metabolismoRESUMEN
The first meeting exclusively dedicated to the 'High-throughput dense reconstruction of cell lineages' took place at Janelia Research Campus (Howard Hughes Medical Institute) from 14 to 18 April 2019. Organized by Tzumin Lee, Connie Cepko, Jorge Garcia-Marques and Isabel Espinosa-Medina, this meeting echoed the recent eruption of new tools that allow the reconstruction of lineages based on the phylogenetic analysis of DNA mutations induced during development. Combined with single-cell RNA sequencing, these tools promise to solve the lineage of complex model organisms at single-cell resolution. Here, we compile the conference consensus on the technological and computational challenges emerging from the use of the new strategies, as well as potential solutions.
Asunto(s)
Linaje de la Célula/genética , Rastreo Celular , Perfilación de la Expresión Génica , Secuenciación de Nucleótidos de Alto Rendimiento , Imagen Molecular , Animales , Sistemas CRISPR-Cas , Rastreo Celular/métodos , Biología Computacional , Código de Barras del ADN Taxonómico , Perfilación de la Expresión Génica/métodos , Secuenciación de Nucleótidos de Alto Rendimiento/métodos , Humanos , Imagen Molecular/métodos , Mutación , Filogenia , Análisis de la Célula Individual/métodosRESUMEN
Temporal patterning is a seminal method of expanding neuronal diversity. Here we 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 α'/ß' mushroom body neurons and identity maintenance. We describe 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.
Asunto(s)
Encéfalo/crecimiento & desarrollo , Diferenciación Celular , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Regulación de la Expresión Génica , Redes Reguladoras de Genes , Células-Madre Neurales/fisiología , Neuronas/fisiología , Factores de Transcripción/metabolismo , Animales , Drosophila , Perfilación de la Expresión GénicaRESUMEN
Gaining independent genetic access to discrete cell types is critical to interrogate their biological functions as well as to deliver precise gene therapy. Transcriptomics has allowed us to profile cell populations with extraordinary precision, revealing that cell types are typically defined by a unique combination of genetic markers. Given the lack of adequate tools to target cell types based on multiple markers, most cell types remain inaccessible to genetic manipulation. Here we present CaSSA, a platform to create unlimited genetic switches based on CRISPR/Cas9 (Ca) and the DNA repair mechanism known as single-strand annealing (SSA). CaSSA allows engineering of independent genetic switches, each responding to a specific gRNA. Expressing multiple gRNAs in specific patterns enables multiplex cell-type-specific manipulations and combinatorial genetic targeting. CaSSA is a new genetic tool that conceptually works as an unlimited number of recombinases and will facilitate genetic access to cell types in diverse organisms.
Asunto(s)
Sistemas CRISPR-Cas , Reparación del ADN , Marcación de Gen/métodos , Animales , Drosophila , Técnicas Genéticas , ARN Guía de Kinetoplastida , Recombinasas/genética , Pez CebraRESUMEN
The vast majority of the adult fly ventral nerve cord is composed of 34 hemilineages, which are clusters of lineally related neurons. Neurons in these hemilineages use one of the three fast-acting neurotransmitters (acetylcholine, GABA, or glutamate) for communication. We generated a comprehensive neurotransmitter usage map for the entire ventral nerve cord. We did not find any cases of neurons using more than one neurotransmitter, but found that the acetylcholine specific gene ChAT is transcribed in many glutamatergic and GABAergic neurons, but these transcripts typically do not leave the nucleus and are not translated. Importantly, our work uncovered a simple rule: All neurons within a hemilineage use the same neurotransmitter. Thus, neurotransmitter identity is acquired at the stem cell level. Our detailed transmitter- usage/lineage identity map will be a great resource for studying the developmental basis of behavior and deciphering how neuronal circuits function to regulate behavior.
Asunto(s)
Linaje de la Célula/fisiología , Sistema Nervioso Central/citología , Sistema Nervioso Central/crecimiento & desarrollo , Neuronas/metabolismo , Neurotransmisores/metabolismo , Animales , Diferenciación Celular , Drosophila , Células Madre/fisiologíaRESUMEN
A complex nervous system requires precise numbers of various neuronal types produced with exquisite spatiotemporal control. This striking diversity is generated by a limited number of neural stem cells (NSC), where spatial and temporal patterning intersect. Drosophila is a genetically tractable model system that has significant advantages for studying stem cell biology and neuronal fate specification. Here we review the latest findings in the rich literature of temporal patterning of neuronal identity in the Drosophila central nervous system. Rapidly changing consecutive transcription factors expressed in NSCs specify short series of neurons with considerable differences. More slowly progressing changes are orchestrated by NSC intrinsic temporal factor gradients which integrate extrinsic signals to coordinate nervous system and organismal development.
Asunto(s)
Células-Madre Neurales , Animales , Sistema Nervioso Central , Proteínas de Drosophila , Drosophila melanogaster , NeuronasRESUMEN
Macroglial cells in the central nervous system exhibit regional specialization and carry out region-specific functions. Diverse glial cells arise from specific progenitors in specific spatiotemporal patterns. This raises an interesting possibility that glial precursors with distinct developmental fates exist that govern region-specific gliogenesis. Here, we have mapped the glial progeny produced by the Drosophila type II neuroblasts, which, like vertebrate radial glia cells, yield both neurons and glia via intermediate neural progenitors (INPs). Distinct type II neuroblasts produce different characteristic sets of glia. A single INP can make both astrocyte-like and ensheathing glia, which co-occupy a relatively restrictive subdomain. Blocking apoptosis uncovers further lineage distinctions in the specification, proliferation and survival of glial precursors. Both the switch from neurogenesis to gliogenesis and the subsequent glial expansion depend on Notch signaling. Taken together, lineage origins preconfigure the development of individual glial precursors with involvement of serial Notch actions in promoting gliogenesis.
Asunto(s)
Encéfalo/embriología , Proteínas de Drosophila/metabolismo , Drosophila/embriología , Células-Madre Neurales/citología , Neurogénesis/fisiología , Receptores Notch/metabolismo , Animales , Apoptosis/fisiología , Astrocitos/citología , Encéfalo/citología , Linaje de la Célula/fisiología , Proliferación Celular/fisiología , Neuronas/citologíaRESUMEN
In their classic experiments, Olds and Milner showed that rats learn to lever press to receive an electric stimulus in specific brain regions. This led to the identification of mammalian reward centers. Our interest in defining the neuronal substrates of reward perception in the fruit fly Drosophila melanogaster prompted us to develop a simpler experimental approach wherein flies could implement behavior that induces self-stimulation of specific neurons in their brains. The high-throughput assay employs optogenetic activation of neurons when the fly occupies a specific area of a behavioral chamber, and the flies' preferential occupation of this area reflects their choosing to experience optogenetic stimulation. Flies in which neuropeptide F (NPF) neurons are activated display preference for the illuminated side of the chamber. We show that optogenetic activation of NPF neuron is rewarding in olfactory conditioning experiments and that the preference for NPF neuron activation is dependent on NPF signaling. Finally, we identify a small subset of NPF-expressing neurons located in the dorsomedial posterior brain that are sufficient to elicit preference in our assay. This assay provides the means for carrying out unbiased screens to map reward neurons in flies.
Asunto(s)
Proteínas de Drosophila/metabolismo , Neuronas/metabolismo , Neuropéptidos/metabolismo , Transducción de Señal/fisiología , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Neuropéptidos/genéticaRESUMEN
The termination of the proliferation of Drosophila neural stem cells, also known as neuroblasts (NBs), requires a 'decommissioning' phase that is controlled in a lineage-specific manner. Most NBs, with the exception of those of the mushroom body (MB), are decommissioned by the ecdysone receptor and mediator complex, causing them to shrink during metamorphosis, followed by nuclear accumulation of Prospero and cell cycle exit. Here, we demonstrate that the levels of Imp and Syp RNA-binding proteins regulate NB decommissioning. Descending Imp and ascending Syp expression have been shown to regulate neuronal temporal fate. We show that Imp levels decline slower in the MB than in other central brain NBs. MB NBs continue to express Imp into pupation, and the presence of Imp prevents decommissioning partly by inhibiting the mediator complex. Late-larval induction of transgenic Imp prevents many non-MB NBs from decommissioning in early pupae. Moreover, the presence of abundant Syp in aged NBs permits Prospero accumulation that, in turn, promotes cell cycle exit. Together, our results reveal that progeny temporal fate and progenitor decommissioning are co-regulated in protracted neuronal lineages.
Asunto(s)
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/metabolismo , Células-Madre Neurales/metabolismo , Proteínas de Unión al ARN/metabolismo , Animales , Animales Modificados Genéticamente , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica , Larva/metabolismo , Complejo Mediador/metabolismo , Modelos Biológicos , Cuerpos Pedunculados/citología , Cuerpos Pedunculados/metabolismo , Células-Madre Neurales/citología , Unión Proteica , Pupa/metabolismo , Proteínas de Unión al ARN/genéticaRESUMEN
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, 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 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, we reveal 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. We therefore 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.