RESUMEN
The brain consists of thousands of neuronal types that are generated by stem cells producing different neuronal types as they age. In Drosophila, this temporal patterning is driven by the successive expression of temporal transcription factors (tTFs)1-6. Here we used single-cell mRNA sequencing to identify the complete series of tTFs that specify most Drosophila optic lobe neurons. We verify that tTFs regulate the progression of the series by activating the next tTF(s) and repressing the previous one(s), and also identify more complex mechanisms of regulation. Moreover, we establish the temporal window of origin and birth order of each neuronal type in the medulla and provide evidence that these tTFs are sufficient to explain the generation of all of the neuronal diversity in this brain region. Finally, we describe the first steps of neuronal differentiation and show that these steps are conserved in humans. We find that terminal differentiation genes, such as neurotransmitter-related genes, are present as transcripts, but not as proteins, in immature larval neurons. This comprehensive analysis of a temporal series of tTFs in the optic lobe offers mechanistic insights into how tTF series are regulated, and how they can lead to the generation of a complete set of neurons.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Regulación del Desarrollo de la Expresión Génica , Lóbulo Óptico de Animales no Mamíferos , Factores de Transcripción , Visión Ocular , Percepción Visual , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Neuronas/citología , Neuronas/metabolismo , Lóbulo Óptico de Animales no Mamíferos/citología , RNA-Seq , Análisis de la Célula Individual , Factores de Transcripción/metabolismoRESUMEN
Deciphering how neuronal diversity is established and maintained requires a detailed knowledge of neuronal gene expression throughout development. In contrast to mammalian brains1,2, the large neuronal diversity of the Drosophila optic lobe3 and its connectome4-6 are almost completely characterized. However, a molecular characterization of this neuronal diversity, particularly during development, has been lacking. Here we present insights into brain development through a nearly complete description of the transcriptomic diversity of the optic lobes of Drosophila. We acquired the transcriptome of 275,000 single cells at adult and at five pupal stages, and built a machine-learning framework to assign them to almost 200 cell types at all time points during development. We discovered two large neuronal populations that wrap neuropils during development but die just before adulthood, as well as neuronal subtypes that partition dorsal and ventral visual circuits by differential Wnt signalling throughout development. Moreover, we show that the transcriptomes of neurons that are of the same type but are produced days apart become synchronized shortly after their production. During synaptogenesis we also resolved neuronal subtypes that, although differing greatly in morphology and connectivity, converge to indistinguishable transcriptomic profiles in adults. Our datasets almost completely account for the known neuronal diversity of the Drosophila optic lobes, and serve as a paradigm to understand brain development across species.
Asunto(s)
Drosophila melanogaster/citología , Drosophila melanogaster/crecimiento & desarrollo , Neuronas/clasificación , Neuronas/metabolismo , Lóbulo Óptico de Animales no Mamíferos/citología , Lóbulo Óptico de Animales no Mamíferos/crecimiento & desarrollo , Anatomía Artística , Animales , Apoptosis , Atlas como Asunto , Regulación del Desarrollo de la Expresión Génica , Masculino , Neuronas/citología , Pupa/citología , Pupa/crecimiento & desarrollo , Análisis de la Célula Individual , Sinapsis/metabolismo , Transcriptoma/genética , Vías Visuales , Vía de Señalización WntRESUMEN
Phage Ï29 DNA replication takes place by a protein-priming mechanism in which the viral DNA polymerase catalyses the covalent linkage of the initiating nucleotide to a specific serine residue of the terminal protein (TP). The N-terminal domain of the Ï29 TP has been shown to bind to the host DNA in a sequence-independent manner and this binding is essential for the TP nucleoid localisation and for an efficient viral DNA replication in vivo. In the present work we have studied the involvement of the TP N-terminal domain residues responsible for DNA binding in the different stages of viral DNA replication by assaying the in vitro activity of purified TP N-terminal mutant proteins. The results show that mutation of TP residues involved in DNA binding affects the catalytic activity of the DNA polymerase in initiation, as the Km for the initiating nucleotide is increased when these mutant proteins are used as primers. Importantly, this initiation defect was relieved by using the Ï29 double-stranded DNA binding protein p6 in the reaction, which decreased the Km of the DNA polymerase for dATP about 130-190 fold. Furthermore, the TP N-terminal domain was shown to be required both for a proper interaction with the DNA polymerase and for an efficient viral DNA amplification.
Asunto(s)
Fagos de Bacillus/metabolismo , Replicación del ADN , ADN Viral/metabolismo , Proteínas de Unión al ADN/metabolismo , Proteínas Virales/metabolismo , Fagos de Bacillus/genética , Sitios de Unión/genética , Biocatálisis , ADN Viral/genética , Proteínas de Unión al ADN/genética , ADN Polimerasa Dirigida por ADN/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Electroforesis en Gel de Poliacrilamida , Cinética , Modelos Genéticos , Mutación , Unión Proteica , Proteínas Virales/genética , Replicación ViralRESUMEN
During evolution, viruses have optimized the interaction with host factors to increase the efficiency of fundamental processes such as DNA replication. Bacteriophage 29 protein p1 is a membrane-associated protein that forms large protofilament sheets that resemble eukaryotic tubulin and bacterial filamenting temperature-sensitive mutant Z protein (FtsZ) polymers. In the absence of protein p1, phage 29 DNA replication is impaired. Here we show that a functional fusion of protein p1 to YFP localizes at the medial region of Bacillus subtilis cells independently of other phage-encoded proteins. We also show that 29 protein p1 colocalizes with the B. subtilis cell division protein FtsZ and provide evidence that FtsZ and protein p1 are associated. Importantly, the midcell localization of YFP-p1 was disrupted in a strain that does not express FtsZ, and the fluorescent signal was distributed all over the cell. Depletion of penicillin-binding protein 2B (PBP2B) in B. subtilis cells did not affect the subcellular localization of YFP-p1, indicating that its distribution does not depend on septal wall synthesis. Interestingly, when 29 protein p1 was expressed, B. subtilis cells were about 1.5-fold longer than control cells, and the accumulation of 29 DNA was higher in mutant B. subtilis cells with increased length. We discuss the biological role of p1 and FtsZ in the 29 growth cycle.
Asunto(s)
Fagos de Bacillus/fisiología , Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Proteínas del Citoesqueleto/metabolismo , Replicación del ADN/fisiología , Proteínas Virales/fisiología , Fagos de Bacillus/genética , ADN Viral/metabolismo , Microscopía FluorescenteRESUMEN
Protein-primed DNA replication constitutes a strategy to initiate viral DNA synthesis in a variety of prokaryotic and eukaryotic organisms. Although the main function of viral terminal proteins (TPs) is to provide a free hydroxyl group to start initiation of DNA replication, there are compelling evidences that TPs can also play other biological roles. In the case of Bacillus subtilis bacteriophage Ï29, the N-terminal domain of the TP organizes viral DNA replication at the bacterial nucleoid being essential for an efficient phage DNA replication, and it contains a nuclear localization signal (NLS) that is functional in eukaryotes. Here we provide information about the structural properties of the Ï29 TP N-terminal domain, which possesses sequence-independent DNA-binding capacity, and dissect the amino acid residues important for its biological function. By mutating all the basic residues of the TP N-terminal domain we identify the amino acids responsible for its interaction with the B. subtilis genome, establishing a correlation between the capacity of DNA-binding and nucleoid localization of the protein. Significantly, these residues are important to recruit the DNA polymerase at the bacterial nucleoid and, subsequently, for an efficient phage DNA replication.
Asunto(s)
Fagos de Bacillus/metabolismo , Bacillus subtilis/virología , Replicación del ADN , ADN Bacteriano/metabolismo , ADN Viral/metabolismo , Señales de Localización Nuclear/metabolismo , Proteínas Virales/metabolismo , Fagos de Bacillus/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Sitios de Unión , Núcleo Celular/metabolismo , Dicroismo Circular , ADN Viral/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Modelos Moleculares , Conformación Proteica , Estructura Secundaria de Proteína , Alineación de Secuencia , Proteínas Virales/química , Proteínas Virales/genéticaRESUMEN
Organization of replicating prokaryotic genomes requires architectural elements that, similarly to eukaryotic systems, induce topological changes such as DNA supercoiling. Bacteriophage 29 protein p6 has been described as a histone-like protein that compacts the viral genome by forming a nucleoprotein complex and plays a key role in the initiation of protein-primed DNA replication. In this work, we analyze the subcellular localization of protein p6 by immunofluorescence microscopy and show that, at early infection stages, it localizes in a peripheral helix-like configuration. Later, at middle infection stages, protein p6 is recruited to the bacterial nucleoid. This migrating process is shown to depend on the synthesis of components of the 29 DNA replication machinery (i.e., terminal protein and DNA polymerase) needed for the replication of viral DNA, which is required to recruit the bulk of protein p6. Importantly, the double-stranded DNA-binding capacity of protein p6 is essential for its relocalization at the nucleoid. Altogether, the results disclose the in vivo organization of a viral histone-like protein in bacteria.
Asunto(s)
Fagos de Bacillus/genética , Fagos de Bacillus/metabolismo , Bacillus subtilis/virología , Genoma Viral/genética , Histonas/metabolismo , Proteínas Virales/metabolismo , Bacillus subtilis/citología , ADN/metabolismo , Replicación del ADN/genética , Modelos Biológicos , Proteínas Mutantes/metabolismo , Unión Proteica , Transporte de ProteínasRESUMEN
A number of prokaryotic proteins have been shown to contain nuclear localization signals (NLSs), although its biological role remains sometimes unclear. Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. We predicted NLSs within the TPs of bacteriophages from diverse families and hosts and, indeed, the TPs of Φ29, Nf, PRD1, Bam35, and Cp-1, out of seven TPs tested, were found to localize to the nucleus when expressed in mammalian cells. Detailed analysis of Φ29 TP led us to identify a bona fide NLS within residues 1-37. Importantly, gene delivery into the eukaryotic nucleus is enhanced by the presence of Φ29 TP attached to the 5' DNA ends. These findings show a common feature of TPs from diverse bacteriophages targeting the eukaryotic nucleus and suggest a possible common function by facilitating the horizontal transfer of genes between prokaryotes and eukaryotes.
Asunto(s)
Bacteriófagos/metabolismo , Eucariontes/metabolismo , Señales de Localización Nuclear/metabolismo , Proteínas Virales/química , Secuencia de Aminoácidos , Animales , Fagos de Bacillus/metabolismo , Células COS , Núcleo Celular/metabolismo , Núcleo Celular/virología , Chlorocebus aethiops , ADN Viral/metabolismo , Transferencia de Gen Horizontal , Modelos Biológicos , Datos de Secuencia Molecular , Señales de Localización Nuclear/química , Células Procariotas/virología , Estructura Terciaria de Proteína , Proteínas Virales/metabolismoRESUMEN
Bacteriophage terminal proteins (TPs) prime DNA replication and become covalently linked to the DNA 5'-ends. In addition, they are DNA-binding proteins that direct early organization of phage DNA replication at the bacterial nucleoid and, unexpectedly, contain nuclear localization signals (NLSs), which localize them to the nucleus when expressed in mammalian cells. In spite of the lack of sequence homology among the phage TPs, these three properties share some common features, suggesting a possible evolutionary common origin of TPs. We show here that NLSs of three different phage TPs, Φ29, PRD1 and Cp-1, are mapped within the protein region required for nucleoid targeting in bacteria, in agreement with a previously proposed common origin of DNA-binding domains and NLSs. Furthermore, previously reported point mutants of Φ29 TP with no nuclear localization still can target the bacterial nucleoid, and Cp-1 TP contains two independent NLSs, only one of them required for nucleoid localization. Altogether, our results show that nucleoid and nucleus localization sequence requirements partially overlap, but they can be uncoupled, suggesting that conservation of both features could have a common origin but, at the same time, they have been independently conserved during evolution.
Asunto(s)
Bacteriófagos/metabolismo , Núcleo Celular/metabolismo , Proteínas de Unión al ADN/fisiología , Señales de Localización Nuclear , Proteínas Virales/química , Proteínas Virales/metabolismo , Secuencias de Aminoácidos , Animales , Fagos de Bacillus/metabolismo , Bacteriófago PRD1/genética , Bacteriófago PRD1/metabolismo , Bacteriófagos/genética , Células COS , Núcleo Celular/genética , Chlorocebus aethiops , Proteínas de Unión al ADN/genética , Escherichia coli/genética , Escherichia coli/metabolismo , Evolución Molecular , Mutación Puntual , Proteínas Virales/genéticaRESUMEN
The medulla is the largest neuropil of the Drosophila optic lobe. It contains about 100 neuronal types that have been comprehensively characterized morphologically and molecularly. These neuronal types are specified from a larval neuroepithelium called the Outer Proliferation Center (OPC) via the integration of temporal, spatial, and Notch-driven mechanisms. Although we recently characterized the temporal windows of origin of all medulla neurons, as well as their Notch status, their spatial origins remained unknown. Here, we isolated cells from different OPC spatial domains and performed single-cell mRNA-sequencing to identify the neuronal types produced in these domains. This allowed us to characterize in a high-throughput manner the spatial origins of all medulla neurons and to identify two new spatial subdivisions of the OPC. Moreover, our work shows that the most abundant neuronal types are produced from epithelial domains of different sizes despite being present in a similar number of copies. Combined with our previously published scRNA-seq developmental atlas of the optic lobe, our work opens the door for further studies on how specification factor expression in progenitors impacts gene expression in developing and adult neurons.
RESUMEN
The mechanism leading to protein-primed DNA replication has been studied extensively in vitro. However, little is known about the in vivo organization of the proteins involved in this fundamental process. Here we show that the terminal proteins (TPs) of phages Ï29 and PRD1, infecting the distantly related bacteria Bacillus subtilis and Escherichia coli, respectively, associate with the host bacterial nucleoid independently of other viral-encoded proteins. Analyses of phage Ï29 revealed that the TP N-terminal domain (residues 1-73) possesses sequence-independent DNA-binding capacity and is responsible for its nucleoid association. Importantly, we show that in the absence of the TP N-terminal domain the efficiency of Ï29 DNA replication is severely affected. Moreover, the TP recruits the phage DNA polymerase to the bacterial nucleoid, and both proteins later are redistributed to enlarged helix-like structures in an MreB cytoskeleton-dependent way. These data disclose a key function for the TP in vivo: organizing the early viral DNA replication machinery at the cell nucleoid.
Asunto(s)
Fagos de Bacillus/fisiología , Bacteriófago PRD1/fisiología , Replicación del ADN/fisiología , ADN Viral/biosíntesis , Proteínas Virales/metabolismo , Fagos de Bacillus/genética , Bacillus subtilis/genética , Bacillus subtilis/metabolismo , Bacillus subtilis/virología , Bacteriófago PRD1/genética , Replicación del ADN/genética , ADN Viral/genética , ADN Polimerasa Dirigida por ADN/metabolismo , Escherichia coli/genética , Escherichia coli/metabolismo , Escherichia coli/virología , Genes Bacterianos , Genes Virales , Modelos Biológicos , Modelos Moleculares , Mutación , Estructura Terciaria de Proteína , Proteínas Recombinantes de Fusión/genética , Proteínas Recombinantes de Fusión/metabolismo , Proteínas Virales/química , Proteínas Virales/genética , Replicación Viral/genética , Replicación Viral/fisiologíaRESUMEN
The large diversity of cell types in nervous systems presents a challenge in identifying the genetic mechanisms that encode it. Here, we report that nearly 200 distinct neurons in the Drosophila visual system can each be defined by unique combinations of on average 10 continuously expressed transcription factors. We show that targeted modifications of this terminal selector code induce predictable conversions of neuronal fates that appear morphologically and transcriptionally complete. Cis-regulatory analysis of open chromatin links one of these genes to an upstream patterning factor that specifies neuronal fates in stem cells. Experimentally validated network models describe the synergistic regulation of downstream effectors by terminal selectors and ecdysone signaling during brain wiring. Our results provide a generalizable framework of how specific fates are implemented in postmitotic neurons.
Asunto(s)
Proteínas de Drosophila , Drosophila melanogaster , Células-Madre Neurales , Neurogénesis , Neuronas , Lóbulo Óptico de Animales no Mamíferos , Factores de Transcripción , Animales , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulación del Desarrollo de la Expresión Génica , Neuronas/fisiología , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crecimiento & desarrollo , Células-Madre Neurales/citología , Células-Madre Neurales/metabolismo , Lóbulo Óptico de Animales no Mamíferos/citología , Lóbulo Óptico de Animales no Mamíferos/crecimiento & desarrollo , Lóbulo Óptico de Animales no Mamíferos/metabolismoRESUMEN
As neural circuits form, growing processes select the correct synaptic partners through interactions between cell surface proteins. The presence of such proteins on two neuronal processes may lead to either adhesion or repulsion; however, the consequences of mismatched expression have rarely been explored. Here, we show that the Drosophila CUB-LDL protein Lost and found (Loaf) is required in the UV-sensitive R7 photoreceptor for normal axon targeting only when Loaf is also present in its synaptic partners. Although targeting occurs normally in loaf mutant animals, removing loaf from photoreceptors or expressing it in their postsynaptic neurons Tm5a/b or Dm9 in a loaf mutant causes mistargeting of R7 axons. Loaf localizes primarily to intracellular vesicles including endosomes. We propose that Loaf regulates the trafficking or function of one or more cell surface proteins, and an excess of these proteins on the synaptic partners of R7 prevents the formation of stable connections.
New nerve cells in a developing organism face a difficult challenge: finding the right partners to connect with in order to form the complex neural networks characteristic of a fully formed brain. Each cell encounters many potential matches but it chooses to connect to only a few, partly based on the proteins that decorate the surface of both cells. Still, too many cell types exist for each to have its own unique protein label, suggesting that nerve cells may also use the amount of each protein to identify suitable partners. Douthit, Hairston et al. explored this possibility in developing fruit flies, focusing on how R7 photoreceptor cells present in the eye to detect UV light connect to nerve cells in a specific brain layer. It is easy to spot when the process goes awry, as the incorrect connections will be in a different layer. Experiments allowed Douthit, Hairston et al. to identify a protein baptized 'Lost and found' 'Loaf' for short which R7 photoreceptors use to find their partners. Removing Loaf from the photoreceptors prevented them from connecting with their normal partners. Surprisingly though, removing Loaf from both the eye and the brain solved this problem the cells, once again, formed the right connections. This suggests that R7 photoreceptors identify their partners by looking for cells that have less Loaf than they do: removing Loaf only from the photoreceptors disrupts this balance, leaving the cells unable to find their match. Another unexpected discovery was that Loaf is not present on the surface of cells, but instead occupies internal structures involved in protein transport. It may therefore work indirectly by controlling the movement of proteins to the cell surface. These findings provide a new way of thinking about how nerve cells connect. In the future, this may help to understand the origins of conditions in which the brain is wired differently, such as schizophrenia and autism.
Asunto(s)
Axones/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiología , Sinapsis/fisiología , Animales , Axones/clasificación , Proteínas de Drosophila/metabolismo , Expresión Génica , Células Fotorreceptoras de Invertebrados/metabolismoRESUMEN
To understand how neurons assemble to form functional circuits, it is necessary to obtain a detailed knowledge of their diversity and to define the developmental specification programs that give rise to this diversity. Invertebrates and vertebrates appear to share common developmental principles of neuronal specification in which cascades of transcription factors temporally pattern progenitors, while spatial cues modify the outcomes of this temporal patterning. Here, we highlight these conserved mechanisms and describe how they are used in distinct neural structures. We present the questions that remain for a better understanding of neuronal specification. Single-cell RNA profiling approaches will potentially shed light on these questions, allowing not only the characterization of neuronal diversity in adult brains, but also the investigation of the developmental trajectories leading to the generation and maintenance of this diversity.
Asunto(s)
Vías Nerviosas , Células-Madre Neurales/fisiología , Neuronas/fisiología , Corteza Somatosensorial/crecimiento & desarrollo , Animales , Recuento de Células , División Celular , Ratones , Especificidad de Órganos , Análisis de la Célula Individual , Corteza Somatosensorial/citología , Corteza Somatosensorial/fisiologíaRESUMEN
Bacillus subtilis phage Φ29 has a linear, double-stranded DNA 19 kb long with an inverted terminal repeat of 6 nucleotides and a protein covalently linked to the 5' ends of the DNA. This protein, called terminal protein (TP), is the primer for the initiation of replication, a reaction catalyzed by the viral DNA polymerase at the two DNA ends. The DNA polymerase further elongates the nascent DNA chain in a processive manner, coupling strand displacement with elongation. The viral protein p5 is a single-stranded DNA binding protein (SSB) that binds to the single strands generated by strand displacement during the elongation process. Viral protein p6 is a double-stranded DNA binding protein (DBP) that preferentially binds to the origins of replication at the Φ29 DNA ends and is required for the initiation of replication. Both SSB and DBP are essential for Φ29 DNA amplification. This review focuses on the role of these phage DNA-binding proteins in Φ29 DNA replication both in vitro and in vivo, as well as on the implication of several B. subtilis DNA-binding proteins in different processes of the viral cycle. We will revise the enzymatic activities of the Φ29 DNA polymerase: TP-deoxynucleotidylation, processive DNA polymerization coupled to strand displacement, 3'-5' exonucleolysis and pyrophosphorolysis. The resolution of the Φ29 DNA polymerase structure has shed light on the translocation mechanism and the determinants responsible for processivity and strand displacement. These two properties have made Φ29 DNA polymerase one of the main enzymes used in the current DNA amplification technologies. The determination of the structure of Φ29 TP revealed the existence of three domains: the priming domain, where the primer residue Ser232, as well as Phe230, involved in the determination of the initiating nucleotide, are located, the intermediate domain, involved in DNA polymerase binding, and the N-terminal domain, responsible for DNA binding and localization of the TP at the bacterial nucleoid, where viral DNA replication takes place. The biochemical properties of the Φ29 DBP and SSB and their function in the initiation and elongation of Φ29 DNA replication, respectively, will be described.
RESUMEN
Terminal proteins (TPs) of bacteriophages prime DNA replication and become covalently linked to the genome ends. Unexpectedly, we have found functional eukaryotic nuclear localization signals (NLSs) within the TP sequences of bacteriophages from diverse families and hosts. Given the role of bacteriophages as vehicles for horizontal gene transfer (HGT), we postulated that viral genomes that have covalently linked NLS-containing terminal proteins might behave as vectors for HGT between bacteria and the eukaryotic nucleus. To validate this hypothesis, we profited from the in vitro Φ29 amplification system that allows the amplification of heterologous DNAs producing linear molecules of DNA with TP covalently attached to both 5' ends. Interestingly, these in vitro-generated TP-DNA molecules showed enhanced gene delivery in mammalian cells, supporting a possible role in HGT by transferring genes between prokaryotes and eukaryotes. Moreover, these TP-DNA molecules are a useful tool to amplify and subsequently deliver genes efficiently into the eukaryotic nucleus. Here, we suggest various possible applications and further developments of the technique with biotechnological and therapeutic purposes.