RESUMO
In animals undergoing metamorphosis, the appearance of the nervous system is coincidently transformed by the morphogenesis of neurons. Such morphogenic alterations are exemplified in three types of intrinsic neurons in the Drosophila memory center. In contrast to the well-characterized remodeling of γ neurons, the morphogenesis of α/ß and α'/ß' neurons has not been adequately explored. Here, we show that mamo, a BTB-zinc finger transcription factor that acts as a terminal selector for α'/ß' neurons, controls the formation of the correct axonal pattern of α'/ß' neurons. Intriguingly, specific Mamo isoforms are preferentially expressed in α'/ß' neurons to regulate the expression of axon guidance molecule Semaphorin-1a. This action directs proper axon guidance in α'/ß' neurons, which is also crucial for wiring of α'/ß' neurons with downstream neurons. Taken together, our results provide molecular insights into how neurons establish correct axonal patterns in circuitry assembly during adult memory center construction.
Assuntos
Orientação de Axônios , Proteínas de Drosophila , Memória , Isoformas de Proteínas , Semaforinas , Animais , Axônios/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Memória/fisiologia , Metamorfose Biológica/fisiologia , Neurônios/metabolismo , Isoformas de Proteínas/metabolismo , Isoformas de Proteínas/genética , Semaforinas/metabolismo , Semaforinas/genética , Fatores de Transcrição/metabolismo , Fatores de Transcrição/genéticaRESUMO
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.
Assuntos
Ecdisona , Corpos Pedunculados , Animais , Axônios , Diferenciação Celular , Larva , Corpos Pedunculados/fisiologia , Neurônios/fisiologiaRESUMO
Neurogenesis in the Drosophila central brain progresses dynamically in order to generate appropriate numbers of neurons during different stages of development. Thus, a central challenge in neurobiology is to reveal the molecular and genetic mechanisms of neurogenesis timing. Here, we found that neurogenesis is significantly impaired when a novel mutation, Nuwa, is induced at early but not late larval stages. Intriguingly, when the Nuwa mutation is induced in neuroblasts of olfactory projection neurons (PNs) at the embryonic stage, embryonic-born PNs are generated, but larval-born PNs of the same origin fail to be produced. Through molecular characterization and transgenic rescue experiments, we determined that Nuwa is a loss-of-function mutation in Drosophila septin interacting protein 1 (sip1). Furthermore, we found that SIP1 expression is enriched in neuroblasts, and RNAi knockdown of sip1 using a neuroblast driver results in formation of small and aberrant brains. Finally, full-length SIP1 protein and truncated SIP1 proteins lacking either the N- or C-terminus display different subcellular localization patterns, and only full-length SIP1 can rescue the Nuwa-associated neurogenesis defect. Taken together, these results suggest that SIP1 acts as a crucial factor for specific neurogenesis programs in the early developing larval brain.
Assuntos
Encéfalo/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Neurogênese , Neurônios/metabolismo , Animais , Animais Geneticamente Modificados , Encéfalo/embriologia , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Larva/genética , Larva/metabolismo , Mutação com Perda de Função , Transdução de SinaisRESUMO
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.
Assuntos
Linhagem da Célula/genética , Animais , Repetições Palindrômicas Curtas Agrupadas e Regularmente Espaçadas , Reparo do DNA , Drosophila melanogaster , Técnicas de Introdução de Genes , Genes Reporter/genética , Proteínas de Choque Térmico/genética , Células-Tronco Pluripotentes Induzidas , Edição de RNA , Ativação Transcricional , Peixe-ZebraRESUMO
The transforming growth factor ß (TGF-ß) signaling pathway is evolutionarily conserved and widely used in the animal kingdom to regulate diverse developmental processes. Prior studies have shown that Baboon (Babo), a Drosophila type I TGF-ß receptor, plays essential roles in brain development and neural circuit formation. However, the expression pattern for Babo in the developing brain has not been previously reported. We generated a knock-in fly with a human influenza hemagglutinin (HA) tag at the C-terminus of Babo and assessed its localization. Babo::HA was primarily expressed in brain structures enriched with neurites, including the mushroom body lobe and neuropils of the optic lobe, where Babo has been shown to instruct neuronal morphogenesis. Since the babo 3' untranslated region contains a predicted microRNA-34 (miR-34) target sequence, we further tested whether Babo::HA expression was affected by modulating the level of miR-34. We found that Babo was upregulated by mir-34 deletion and downregulated by miR-34 overexpression, confirming that it is indeed a miR-34 target gene. Taken together, our results demonstrate that the baboHA fly permits accurate visualization of endogenous Babo expression during brain development and the construction of functional neural circuits.
Assuntos
Receptores de Ativinas/genética , Encéfalo/crescimento & desenvolvimento , Proteínas de Drosophila/genética , Drosophila/genética , Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento/genética , MicroRNAs/genética , Ativinas/metabolismo , Animais , Animais Geneticamente Modificados , Axônios/fisiologia , Proteínas de Transporte/metabolismo , Drosophila/crescimento & desenvolvimento , Proteínas de Drosophila/metabolismo , Técnicas de Introdução de Genes , Glicoproteínas de Hemaglutininação de Vírus da Influenza/genética , Corpos Pedunculados/crescimento & desenvolvimento , Neuritos/metabolismo , Transdução de Sinais/genética , Fator de Crescimento Transformador beta/metabolismoRESUMO
Mosaic analysis with a repressible cell marker (MARCM)-related technologies are positive genetic mosaic labeling systems that have been widely applied in studies of Drosophila brain development and neural circuit formation to identify diverse neuronal types, reconstruct neural lineages, and investigate the function of genes and molecules. Two types of MARCM-related technologies have been developed: single-colored and twin-colored. Single-colored MARCM technologies label one of two twin daughter cells in otherwise unmarked background tissues through site-specific recombination of homologous chromosomes during mitosis of progenitors. On the other hand, twin-colored genetic mosaic technologies label both twin daughter cells with two distinct colors, enabling the retrieval of useful information from both progenitor-derived cells and their subsequent clones. In this overview, we describe the principles and usage guidelines for MARCM-related technologies in order to help researchers employ these powerful genetic mosaic systems in their investigations of intricate neurobiological topics. © 2020 by John Wiley & Sons, Inc.
Assuntos
Drosophila melanogaster/genética , Neurônios/ultraestrutura , Animais , Divisão Celular , Linhagem da Célula , Células Clonais/ultraestrutura , Cor , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Expressão Gênica , Genes de Insetos , Genes Reporter , Genes Supressores , Discos Imaginais/ultraestrutura , Mosaicismo , Células-Tronco Neurais/citologia , Interferência de RNA , Recombinases , Recombinação GenéticaRESUMO
Stereotypic dendrite arborizations are key morphological features of neuronal identity, as the size, shape and location of dendritic trees determine the synaptic input fields and how information is integrated within developed neural circuits. In this review, we focus on the actions of extrinsic intercellular communication factors and their effects on intrinsic developmental processes that lead to dendrite patterning. Surrounding neurons or supporting cells express adhesion receptors and secreted proteins that respectively, act via direct contact or over short distances to shape, size, and localize dendrites during specific developmental stages. The different ligand-receptor interactions and downstream signaling events appear to direct dendrite morphogenesis by converging on two categorical mechanisms: local cytoskeletal and adhesion modulation and global transcriptional regulation of key dendritic growth components, such as lipid synthesis enzymes. Recent work has begun to uncover how the coordinated signaling of multiple extrinsic factors promotes complexity in dendritic trees and ensures robust dendritic patterning.
RESUMO
Diverse types of neurons must be specified in the developing brain to form the functional neural circuits that are necessary for the execution of daily tasks. Here, we describe the participation of Forkhead box class O (FOXO) in cell fate specification of a small subset of Drosophila ventral olfactory projection neurons (vPNs). Using the two-color labeling system, twin-spot MARCM, we determined the temporal birth order of each vPN type, and this characterization served as a foundation to investigate regulators of cell fate specification. Flies deficient for chinmo, a known temporal cell fate regulator, exhibited a partial loss of vPNs, suggesting that the gene plays a complex role in specifying vPN cell fate and is not the only regulator of this process. Interestingly, loss of foxo function resulted in the precocious appearance of late-born vPNs in place of early-born vPNs, whereas overexpression of constitutively active FOXO caused late-born vPNs to take on a morphology reminiscent of earlier born vPNs. Taken together, these data suggest that FOXO temporally regulates vPN cell fate specification. The comprehensive identification of molecules that regulate neuronal fate specification promises to provide a better understanding of the mechanisms governing the formation of functional brain tissue.
Assuntos
Diferenciação Celular/fisiologia , Proteínas de Drosophila/metabolismo , Fatores de Transcrição Forkhead/metabolismo , Neurônios/citologia , Condutos Olfatórios/citologia , Animais , Animais Geneticamente Modificados , Linhagem da Célula/fisiologia , Neurogênese/fisiologia , Neurônios/metabolismo , Condutos Olfatórios/metabolismoRESUMO
Elucidating how appropriate neurite patterns are generated in neurons of the olfactory system is crucial for comprehending the construction of the olfactory map. In the Drosophila olfactory system, projection neurons (PNs), primarily derived from four neural stem cells (called neuroblasts), populate their cell bodies surrounding to and distribute their dendrites in distinct but overlapping patterns within the primary olfactory center of the brain, the antennal lobe (AL). However, it remains unclear whether the same molecular mechanisms are employed to generate the appropriate dendritic patterns in discrete AL glomeruli among PNs produced from different neuroblasts. Here, by examining a previously explored transmembrane protein Semaphorin-1a (Sema-1a) which was proposed to globally control initial PN dendritic targeting along the dorsolateral-to-ventromedial axis of the AL, we discover a new role for Sema-1a in preventing dendrites of both uni-glomerular and poly-glomerular PNs from aberrant invasion into select AL regions and, intriguingly, this Sema-1a-deficient dendritic mis-targeting phenotype seems to associate with the origins of PNs from which they are derived. Further, ectopic expression of Sema-1a resulted in PN dendritic mis-projection from a select AL region into adjacent glomeruli, strengthening the idea that Sema-1a plays an essential role in preventing abnormal dendritic accumulation in select AL regions. Taken together, these results demonstrate that Sema-1a repulsion keeps dendrites of different types of PNs away from each other, enabling the same types of PN dendrites to be sorted into destined AL glomeruli and permitting for functional assembly of olfactory circuitry.
Assuntos
Antenas de Artrópodes/crescimento & desenvolvimento , Neurogênese/genética , Neurônios Receptores Olfatórios/metabolismo , Semaforinas/genética , Animais , Antenas de Artrópodes/metabolismo , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Dendritos/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Células-Tronco Neurais/metabolismo , Condutos Olfatórios/crescimento & desenvolvimento , Condutos Olfatórios/metabolismo , Semaforinas/metabolismoRESUMO
Mosaic analysis with a repressible cell marker (MARCM) is a positive mosaic labeling system that has been widely applied in Drosophila neurobiological studies to depict intricate morphologies and to manipulate the function of genes in subsets of neurons within otherwise unmarked and unperturbed organisms. Genetic mosaics generated in the MARCM system are mediated through site-specific recombination between homologous chromosomes within dividing precursor cells to produce both marked (MARCM clones) and unmarked daughter cells during mitosis. An extension of the MARCM method, called twin-spot MARCM (tsMARCM), labels both of the twin cells derived from a common progenitor with two distinct colors. This technique was developed to enable the retrieval of useful information from both hemi-lineages. By comprehensively analyzing different pairs of tsMARCM clones, the tsMARCM system permits high-resolution neural lineage mapping to reveal the exact birth-order of the labeled neurons produced from common progenitor cells. Furthermore, the tsMARCM system also extends gene function studies by permitting the phenotypic analysis of identical neurons of different animals. Here, we describe how to apply the tsMARCM system to facilitate studies of neural development in Drosophila.
Assuntos
Drosophila/genética , Mosaicismo , Neurogênese/genética , Células-Tronco/citologia , Animais , Linhagem da Célula , Drosophila/citologia , Mitose , Modelos Animais , Neurônios/fisiologiaRESUMO
MicroRNA-34 (miR-34) is crucial for preventing chronic large-scale neurite degeneration in the aged brain of Drosophila melanogaster. Here we investigated the role of miR-34 in two other types of large-scale axon degeneration in Drosophila: axotomy-induced axon degeneration in olfactory sensory neurons (OSNs) and developmentally related axon pruning in mushroom body (MB) neurons. Ectopically overexpressed miR-34 did not inhibit axon degeneration in OSNs following axotomy, whereas ectopically overexpressed miR-34 in differentiated MB neurons impaired γ axon pruning. Intriguingly, the miR-34-induced γ axon pruning defect resulted from downregulating the expression of ecdysone receptor B1 (EcR-B1) in differentiated MB γ neurons. Notably, the separate overexpression of EcR-B1 or a transforming growth factor- ß receptor Baboon, whose activation can upregulate the EcR-B1 expression, in MB neurons rescued the miR-34-induced γ axon pruning phenotype. Future investigations of miR-34 targets that regulate the expression of EcR-B1 in MB γ neurons are warranted to elucidate pathways that regulate axon pruning, and to provide insight into mechanisms that control large-scale axon degeneration in the nervous system.
Assuntos
Regulação para Baixo , Drosophila melanogaster/crescimento & desenvolvimento , MicroRNAs/genética , Corpos Pedunculados/citologia , Receptores de Esteroides/metabolismo , Animais , Axotomia , Diferenciação Celular , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Corpos Pedunculados/crescimento & desenvolvimento , Plasticidade Neuronal , Neurônios Receptores Olfatórios/citologiaRESUMO
In the Drosophila olfactory system, odorant information is sensed by olfactory sensory neurons and relayed from the primary olfactory center, the antennal lobe (AL), to higher olfactory centers via olfactory projection neurons (PNs). A major portion of the AL is constituted with dendrites of four groups of PNs, anterodorsal PNs (adPNs), lateral PNs (lPNs), lateroventral PNs (lvPNs) and ventral PNs (vPNs). Previous studies have been focused on the development and function of adPNs and lPNs, while the investigation on those of lvPNs and vPNs received less attention. Here, we study the molecular and cellular mechanisms underlying the morphogenesis of a putative male-pheromone responding vPN, the DA1 vPN. Using an intersection strategy to remove background neurons labeled within a DA1 vPN-containing GAL4 line, we depicted morphological changes of the DA1 vPN that occurs at the pupal stage. We then conducted a pilot screen using RNA interference knock-down approach to identify cell surface molecules, including Down syndrome cell adhesion molecule 1 and Semaphorin-1a, that might play essential roles for the DA1 vPN morphogenesis. Taken together, by revealing molecular and cellular basis of the DA1 vPN morphogenesis, we should provide insights into future comprehension of how vPNs are assembled into the olfactory neural circuitry.
Assuntos
Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Morfogênese/genética , Condutos Olfatórios/metabolismo , Neurônios Receptores Olfatórios/metabolismo , Atrativos Sexuais/metabolismo , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular , Dendritos/metabolismo , Dendritos/ultraestrutura , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/anatomia & histologia , Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Masculino , Moléculas de Adesão de Célula Nervosa/antagonistas & inibidores , Moléculas de Adesão de Célula Nervosa/genética , Moléculas de Adesão de Célula Nervosa/metabolismo , Condutos Olfatórios/ultraestrutura , Neurônios Receptores Olfatórios/ultraestrutura , Pupa/anatomia & histologia , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/metabolismo , RNA Interferente Pequeno/genética , RNA Interferente Pequeno/metabolismo , Semaforinas/antagonistas & inibidores , Semaforinas/genética , Semaforinas/metabolismo , Atrativos Sexuais/genética , Transdução de Sinais , Olfato/fisiologia , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The Drosophila central brain develops from a fixed number of neuroblasts. Each neuroblast makes a clone of neurons that exhibit common trajectories. Here we identified 15 distinct clones that carry larval-born neurons innervating the Drosophila central complex (CX), which consists of four midline structures including the protocerebral bridge (PB), fan-shaped body (FB), ellipsoid body (EB), and noduli (NO). Clonal analysis revealed that the small-field CX neurons, which establish intricate projections across different CX substructures, exist in four isomorphic groups that respectively derive from four complex posterior asense-negative lineages. In terms of the region-characteristic large-field CX neurons, we found that two lineages make PB neurons, 10 lineages produce FB neurons, three lineages generate EB neurons, and two lineages yield NO neurons. The diverse FB developmental origins reflect the discrete input pathways for different FB subcompartments. Clonal analysis enlightens both development and anatomy of the insect locomotor control center.
Assuntos
Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Locomoção/fisiologia , Rede Nervosa/crescimento & desenvolvimento , Células-Tronco Neurais/fisiologia , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados , Antígenos CD8/metabolismo , Linhagem da Célula/fisiologia , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Larva , Proteínas Luminescentes/genética , Proteínas Luminescentes/metabolismo , Rede Nervosa/metabolismo , Neurônios/citologiaRESUMO
BACKGROUND: The insect brain can be divided into neuropils that are formed by neurites of both local and remote origin. The complexity of the interconnections obscures how these neuropils are established and interconnected through development. The Drosophila central brain develops from a fixed number of neuroblasts (NBs) that deposit neurons in regional clusters. RESULTS: By determining individual NB clones and pursuing their projections into specific neuropils, we unravel the regional development of the brain neural network. Exhaustive clonal analysis revealed 95 stereotyped neuronal lineages with characteristic cell-body locations and neurite trajectories. Most clones show complex projection patterns, but despite the complexity, neighboring clones often coinnervate the same local neuropil or neuropils and further target a restricted set of distant neuropils. CONCLUSIONS: These observations argue for regional clonal development of both neuropils and neuropil connectivity throughout the Drosophila central brain.
Assuntos
Drosophila melanogaster/crescimento & desenvolvimento , Drosophila melanogaster/metabolismo , Animais , Encéfalo/citologia , Encéfalo/crescimento & desenvolvimento , Encéfalo/metabolismo , Linhagem da Célula , Células Clonais/citologia , Células Clonais/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Feminino , Larva/citologia , Larva/genética , Larva/crescimento & desenvolvimento , Larva/metabolismo , Masculino , Microscopia Confocal , Células-Tronco Neurais/citologia , Células-Tronco Neurais/metabolismo , Neurópilo/citologia , Neurópilo/metabolismoRESUMO
Binary cell fate decisions allow the production of distinct sister neurons from an intermediate precursor. Neurons are further diversified based on the birth order of intermediate precursors. Here we examined the interplay between binary cell fate and birth-order-dependent temporal fate in the Drosophila lateral antennal lobe (lAL) neuronal lineage. Single-cell mapping of the lAL lineage by twin-spot mosaic analysis with repressible cell markers (ts-MARCM) revealed that projection neurons (PNs) and local interneurons (LNs) are made in pairs through binary fate decisions. Forty-five types of PNs innervating distinct brain regions arise in a stereotyped sequence; however, the PNs with similar morphologies are not necessarily born in a contiguous window. The LNs are morphologically less diverse than the PNs, and the sequential morphogenetic changes in the two pairs occur independently. Sanpodo-dependent Notch activity promotes and patterns the LN fates. By contrast, Notch diversifies PN temporal fates in a Sanpodo-dispensable manner. These pleiotropic Notch actions underlie the differential temporal fate specification of twin neurons produced by common precursors within a lineage, possibly by modulating postmitotic neurons' responses to Notch-independent transcriptional cascades.
Assuntos
Linhagem da Célula , Proteínas de Drosophila/metabolismo , Drosophila/citologia , Regulação da Expressão Gênica no Desenvolvimento , Neurônios/citologia , Receptores Notch/metabolismo , Animais , Biomarcadores/metabolismo , Encéfalo/citologia , Encéfalo/embriologia , Encéfalo/metabolismo , Divisão Celular , Drosophila/embriologia , Drosophila/metabolismo , Proteínas de Drosophila/genética , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismo , Genes de Insetos , Imuno-Histoquímica , Larva/citologia , Larva/crescimento & desenvolvimento , Larva/metabolismo , Mecanotransdução Celular , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Neurogênese , Neurônios/metabolismo , Receptores Notch/genética , Análise de Célula Única/métodosRESUMO
The anterodorsal projection neuron lineage of Drosophila melanogaster produces 40 neuronal types in a stereotypic order. Here we take advantage of this complete lineage sequence to examine the role of known temporal fating factors, including Chinmo and the Hb/Kr/Pdm/Cas transcriptional cascade, within this diverse central brain lineage. Kr mutation affects the temporal fate of the neuroblast (NB) itself, causing a single fate to be skipped, whereas Chinmo null only elicits fate transformation of NB progeny without altering cell counts. Notably, Chinmo operates in two separate windows to prevent fate transformation (into the subsequent Chinmo-indenpendent fate) within each window. By contrast, Hb/Pdm/Cas play no detectable role, indicating that Kr either acts outside of the cascade identified in the ventral nerve cord or that redundancy exists at the level of fating factors. Therefore, hierarchical fating mechanisms operate within the lineage to generate neuronal diversity in an unprecedented fashion.
Assuntos
Encéfalo/citologia , Linhagem da Célula/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados , Padronização Corporal , Linhagem da Célula/genética , Proteínas de Ligação a DNA/genética , Proteínas de Ligação a DNA/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Fatores de Transcrição Kruppel-Like/genética , Fatores de Transcrição Kruppel-Like/metabolismo , Mutação/genética , Proteínas do Tecido Nervoso/genética , Fatores do Domínio POU/genética , Fatores do Domínio POU/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologia , Fatores de Transcrição/metabolismoRESUMO
Drosophila brains contain numerous neurons that form complex circuits. These neurons are derived in stereotyped patterns from a fixed number of progenitors, called neuroblasts, and identifying individual neurons made by a neuroblast facilitates the reconstruction of neural circuits. An improved MARCM (mosaic analysis with a repressible cell marker) technique, called twin-spot MARCM, allows one to label the sister clones derived from a common progenitor simultaneously in different colors. It enables identification of every single neuron in an extended neuronal lineage based on the order of neuron birth. Here we report the first example, to our knowledge, of complete lineage analysis among neurons derived from a common neuroblast that relay olfactory information from the antennal lobe (AL) to higher brain centers. By identifying the sequentially derived neurons, we found that the neuroblast serially makes 40 types of AL projection neurons (PNs). During embryogenesis, one PN with multi-glomerular innervation and 18 uniglomerular PNs targeting 17 glomeruli of the adult AL are born. Many more PNs of 22 additional types, including four types of polyglomerular PNs, derive after the neuroblast resumes dividing in early larvae. Although different offspring are generated in a rather arbitrary sequence, the birth order strictly dictates the fate of each post-mitotic neuron, including the fate of programmed cell death. Notably, the embryonic progenitor has an altered temporal identity following each self-renewing asymmetric cell division. After larval hatching, the same progenitor produces multiple neurons for each cell type, but the number of neurons for each type is tightly regulated. These observations substantiate the origin-dependent specification of neuron types. Sequencing neuronal lineages will not only unravel how a complex brain develops but also permit systematic identification of neuron types for detailed structure and function analysis of the brain.
Assuntos
Encéfalo/embriologia , Linhagem da Célula , Drosophila/citologia , Técnicas Genéticas , Mosaicismo , Neurogênese , Neurônios/citologia , Animais , Encéfalo/citologia , Encéfalo/metabolismo , Células Clonais , Drosophila/embriologia , Drosophila/metabolismo , Neurônios/metabolismo , Condutos OlfatóriosRESUMO
A comprehensive understanding of the brain requires the analysis of individual neurons. We used twin-spot mosaic analysis with repressible cell markers (twin-spot MARCM) to trace cell lineages at high resolution by independently labeling paired sister clones. We determined patterns of neurogenesis and the influences of lineage on neuron-type specification. Notably, neural progenitors were able to yield intermediate precursors that create one, two or more neurons. Furthermore, neurons acquired stereotyped projections according to their temporal position in various brain sublineages. Twin-spot MARCM also permitted birth dating of mutant clones, enabling us to detect a single temporal fate that required chinmo in a sublineage of six Drosophila central complex neurons. In sum, twin-spot MARCM can reveal the developmental origins of neurons and the mechanisms that underlie cell fate.
Assuntos
Linhagem da Célula , Neurogênese , Neurônios/fisiologia , Animais , Animais Geneticamente Modificados , Encéfalo/fisiologia , Sistema Nervoso Central/fisiologia , Drosophila , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Técnicas de Inativação de Genes , Técnicas Genéticas , Corpos Pedunculados/fisiologia , Proteínas do Tecido Nervoso/genética , Proteínas do Tecido Nervoso/metabolismo , Interferência de RNA , Fatores de TempoRESUMO
Drosophila Down syndrome cell adhesion molecule (Dscam) can be variably spliced to encode 152,064 distinct single-pass transmembrane proteins. In addition to 19,008 possible ectodomains and two alternative transmembrane segments, it may carry endodomains containing or lacking exons 19 and 23. Here, we determine the role of Dscam endodomain diversity in neural development. Dscam with full-length endodomain is largely restricted to embryogenesis. In contrast, most Dscams lack exons 19 and 23 at postembryonic stages. As implicated from the expression patterns, removal of Dscam exon 19-containing variants disrupts wiring of embryonic neurons while silencing of Dscam transcripts lacking exon 19 or exon 23 effectively blocks postembryonic neuronal morphogenesis. Furthermore, compared with exon 19-containing Dscam, transgenic Dscam without exon 19 is more efficiently targeted to neurites and more potently suppresses axon bifurcation in Dscam mutant neurons. In sum, Dscam with or without exon 19 in its endodomain is used to govern different stage-specific neuronal morphogenetic processes, possibly due to differences in protein targeting.
Assuntos
Proteínas de Drosophila/química , Proteínas de Drosophila/fisiologia , Morfogênese/fisiologia , Neurônios/fisiologia , Motivos de Aminoácidos , Sequência de Aminoácidos , Animais , Animais Geneticamente Modificados , Moléculas de Adesão Celular , Drosophila , Proteínas de Drosophila/genética , Marcação de Genes , Dados de Sequência Molecular , Morfogênese/genética , Neurogênese/genética , Neurogênese/fisiologia , Neurônios/química , Estrutura Terciária de Proteína/genética , Estrutura Terciária de Proteína/fisiologiaRESUMO
Understanding how a vast number of neuron types derive from a limited number of neural progenitors remains a major challenge in developmental neurobiology. In the post-embryonic Drosophila brain, specific neuron types derive from specific progenitors at specific times. This suggests involvement of time-dependent cell fate determinants acting as 'temporal codes' along with lineage cues to specify neuronal cell fates. Interestingly, such temporal codes might be provided not only by several regulators acting in sequence, but also by the differential protein levels of the BTB-zinc finger nuclear protein Chinmo. Identifying temporal codes and determining their origins should allow us to elucidate how neuronal diversification occurs through protracted neurogenesis.