RESUMEN
Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.
Asunto(s)
Proteínas Morfogenéticas Óseas , Transducción de Señal , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas Morfogenéticas Óseas/genética , Ratones , Humanos , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/química , Glicoproteínas/metabolismo , Glicoproteínas/genética , Péptidos y Proteínas de Señalización Intercelular/metabolismo , Péptidos y Proteínas de Señalización Intercelular/genética , Sitios de Unión , Dominios Proteicos , Unión Proteica , Organoides/metabolismo , Organoides/embriología , Células HEK293 , Gastrulación/genética , Mutación , Cristalografía por Rayos X , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Drosophila melanogaster/genética , ProteínasRESUMEN
Dosage compensation, the balancing of X-linked gene expression between sexes and to the autosomes, is critical to an organism's fitness and survival. In Drosophila, dosage compensation involves hypertranscription of the male X chromosome. Here, we use quantitative live imaging and modeling at single-cell resolution to study X chromosome dosage compensation in Drosophila. We show that the four X chromosome genes studied undergo transcriptional bursting in male and female embryos. Mechanistically, our data reveal that transcriptional upregulation of male X chromosome genes is primarily mediated by a higher RNA polymerase II initiation rate and burst amplitude across the expression domain. In contrast, burst frequency is spatially modulated in nuclei within the expression domain in response to different transcription factor concentrations to tune the transcriptional response. Together, these data show how the local and global regulation of distinct burst parameters can establish the complex transcriptional outputs underpinning developmental patterning.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Masculino , Femenino , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Factores de Transcripción/genética , Factores de Transcripción/metabolismo , Cromosoma X/metabolismo , Compensación de Dosificación (Genética)RESUMEN
Regulation of mRNA degradation is critical for a diverse array of cellular processes and developmental cell fate decisions. Many methods for determining mRNA half-lives rely on transcriptional inhibition or metabolic labelling. Here, we use a non-invasive method for estimating half-lives for hundreds of mRNAs in the early Drosophila embryo. This approach uses the intronic and exonic reads from a total RNA-seq time series and Gaussian process regression to model the dynamics of premature and mature mRNAs. We show how regulation of mRNA stability is used to establish a range of mature mRNA dynamics during embryogenesis, despite shared transcription profiles. Using single-molecule imaging, we provide evidence that, for the mRNAs tested, there is a correlation between short half-life and mRNA association with P-bodies. Moreover, we detect an enrichment of mRNA 3' ends in P-bodies in the early embryo, consistent with 5' to 3' degradation occurring in P-bodies for at least a subset of mRNAs. We discuss our findings in relation to recently published data suggesting that the primary function of P-bodies in other biological contexts is mRNA storage.
Asunto(s)
Drosophila , Cuerpos de Procesamiento , Animales , Drosophila/genética , Drosophila/metabolismo , Imagen Individual de Molécula , ARN Mensajero/genética , ARN Mensajero/metabolismo , Estabilidad del ARN/genéticaRESUMEN
A BMP gradient is essential for patterning the dorsal-ventral axis of invertebrate and vertebrate embryos. The extracellular BMP binding protein Short Gastrulation (Sog) in Drosophila plays a key role in BMP gradient formation. In this study, we combine genome editing, structural and developmental approaches to study Sog function in Drosophila. We generate a sog knockout fly stock, which allows simple reintegration of altered versions of the sog coding sequence. As proof-of-principle, we test the requirement for two cysteine residues that were previously identified as targets for palmitoylation, which has been proposed to enhance Sog secretion. However, we show that the sogC27,28S mutant is viable with only very mild phenotypes, indicating that these residues and their potential modification are not critical for Sog secretion in vivo. Additionally, we use experimental negative stain EM imaging and hydrodynamic data to validate the AlphaFold structure prediction for Sog. The model suggests a more compact shape than the vertebrate ortholog Chordin and conformational flexibility between the C-terminal von Willebrand C domains. We discuss how this altered compactness may contribute to mechanistic differences in Sog and Chordin function during BMP gradient formation. This article has an associated First Person interview with the first author of the paper.
Asunto(s)
Proteínas de Drosophila , Drosophila , Animales , Proteínas Morfogenéticas Óseas/metabolismo , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/metabolismo , Gastrulación , Humanos , Transducción de SeñalRESUMEN
MOTIVATION: The MS2-MCP (MS2 coat protein) live imaging system allows for visualization of transcription dynamics through the introduction of hairpin stem-loop sequences into a gene. A fluorescent signal at the site of nascent transcription in the nucleus quantifies mRNA production. Computational modelling can be used to infer the promoter states along with the kinetic parameters governing transcription, such as promoter switching frequency and polymerase loading rate. However, modelling of the fluorescent trace presents a challenge due its persistence; the observed fluorescence at a given time point depends on both current and previous promoter states. A compound state Hidden Markov Model (cpHMM) was recently introduced to allow inference of promoter activity from MS2-MCP data. However, the computational time for inference scales exponentially with gene length and the cpHMM is therefore not currently practical for application to many eukaryotic genes. RESULTS: We present a scalable implementation of the cpHMM for fast inference of promoter activity and transcriptional kinetic parameters. This new method can model genes of arbitrary length through the use of a time-adaptive truncated compound state space. The truncated state space provides a good approximation to the full state space by retaining the most likely set of states at each time during the forward pass of the algorithm. Testing on MS2-MCP fluorescent data collected from early Drosophila melanogaster embryos indicates that the method provides accurate inference of kinetic parameters within a computationally feasible timeframe. The inferred promoter traces generated by the model can also be used to infer single-cell transcriptional parameters. AVAILABILITY AND IMPLEMENTATION: Python implementation is available at https://github.com/ManchesterBioinference/burstInfer, along with code to reproduce the examples presented here. SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.
Asunto(s)
Algoritmos , Drosophila melanogaster , Animales , Drosophila melanogaster/genética , Factores de Tiempo , Simulación por ComputadorRESUMEN
Significant regulation of gene expression is mediated at the translation level. Here, we describe protocols for imaging and analysis of translation at single mRNA resolution in both fixed and living Drosophila embryos. These protocols use the SunTag system, in which the protein of interest is visualized by inserting a peptide array that is recognized by a single chain antibody. Simultaneous detection of individual mRNAs using the MS2/MCP system or by smFISH allows translation sites to be identified and quantified. For complete information on the generation and use of this protocol, please refer to Vinter et al. (2021).
Asunto(s)
Embrión no Mamífero/metabolismo , Biosíntesis de Proteínas/genética , ARN Mensajero/análisis , Imagen Individual de Molécula/métodos , Animales , Drosophila/genética , Drosophila/metabolismo , Embrión no Mamífero/química , Femenino , Hibridación Fluorescente in Situ , Masculino , ARN Mensajero/química , ARN Mensajero/genética , ARN Mensajero/metabolismo , Anticuerpos de Cadena ÚnicaRESUMEN
Visualizing transcription live in Drosophila is providing important new insights into the spatiotemporal regulation of transcription. Here, we describe a protocol to visualize and quantitate transcription from gene loci that are tagged with MS2 stem-loop sequences in the Drosophila embryo. MS2 stem-loop sequences are recognized by a coat protein fused to a fluorescent protein and visualized with microscopy. We also describe an analysis pipeline to extract and subsequently quantify transcription dynamics. For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020).
Asunto(s)
Drosophila/embriología , Perfilación de la Expresión Génica/métodos , Microscopía Fluorescente/métodos , Animales , Proteínas de la Cápside/genética , Secuencias Invertidas Repetidas/genética , ARN Mensajero/genética , Tomografía Computarizada por Rayos X/métodosRESUMEN
The maintenance of stem cell populations and the differentiation of their progeny is coordinated by specific communication with associated niche cells. Here, we describe a protocol for short-term live imaging of the Drosophila ovarian germline stem cell niche ex vivo. By immobilizing the ovarian tissue in a fibrinogen-thrombin clot, we are able to maintain the tissue for short-term high-temporal live imaging. This enables the visualization of dynamic cellular processes, such as the cytoskeletal dynamics that control stem cell niche communication. For complete details on the use and execution of this protocol, please refer to Wilcockson and Ashe (2019).
Asunto(s)
Células Madre Germinales Adultas/citología , Imagen Molecular/métodos , Animales , Diferenciación Celular , Drosophila/citología , Femenino , Células Germinativas/citología , Células Madre Oogoniales/citología , Ovario/citología , Nicho de Células Madre/fisiología , Células Madre/citologíaRESUMEN
The Hunchback (Hb) transcription factor is crucial for anterior-posterior patterning of the Drosophila embryo. The maternal hb mRNA acts as a paradigm for translational regulation due to its repression in the posterior of the embryo. However, little is known about the translatability of zygotically transcribed hb mRNAs. Here, we adapt the SunTag system, developed for imaging translation at single-mRNA resolution in tissue culture cells, to the Drosophila embryo to study the translation dynamics of zygotic hb mRNAs. Using single-molecule imaging in fixed and live embryos, we provide evidence for translational repression of zygotic SunTag-hb mRNAs. Whereas the proportion of SunTag-hb mRNAs translated is initially uniform, translation declines from the anterior over time until it becomes restricted to a posterior band in the expression domain. We discuss how regulated hb mRNA translation may help establish the sharp Hb expression boundary, which is a model for precision and noise during developmental patterning. Overall, our data show how use of the SunTag method on fixed and live embryos is a powerful combination for elucidating spatiotemporal regulation of mRNA translation in Drosophila.
Asunto(s)
Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Drosophila/genética , Biosíntesis de Proteínas/genética , ARN Mensajero Almacenado/genética , Factores de Transcripción/genética , Animales , Tipificación del Cuerpo/genética , Embrión no Mamífero/fisiología , Regulación del Desarrollo de la Expresión Génica/genética , Morfogénesis/genética , Cigoto/fisiologíaRESUMEN
CRISPR-Cas9 genome editing has transformed biology by enabling site-specific genome modifications to be simply engineered. Here, we describe two CRISPR-Cas9 approaches to introduce MS2 stem-loop sequences into endogenous gene loci in Drosophila. This can facilitate live imaging of nascent transcription in Drosophila. For complete details on the use and execution of this protocol, please refer to Hoppe et al. (2020).
Asunto(s)
Drosophila/embriología , Edición Génica/métodos , Perfilación de la Expresión Génica/métodos , Animales , Sistemas CRISPR-Cas , Genoma , Secuencias Invertidas Repetidas/genética , ARN Mensajero/genéticaRESUMEN
Morphogen gradients specify cell fates during development, with a classic example being the bone morphogenetic protein (BMP) gradient's conserved role in embryonic dorsal-ventral axis patterning. Here, we elucidate how the BMP gradient is interpreted in the Drosophila embryo by combining live imaging with computational modeling to infer transcriptional burst parameters at single-cell resolution. By comparing burst kinetics in cells receiving different levels of BMP signaling, we show that BMP signaling controls burst frequency by regulating the promoter activation rate. We provide evidence that the promoter activation rate is influenced by both enhancer and promoter sequences, whereas Pol II loading rate is primarily modulated by the enhancer. Consistent with BMP-dependent regulation of burst frequency, the numbers of BMP target gene transcripts per cell are graded across their expression domains. We suggest that graded mRNA output is a general feature of morphogen gradient interpretation and discuss how this can impact on cell-fate decisions.
Asunto(s)
Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/metabolismo , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Animales , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/metabolismo , Transducción de Señal/genética , Factores de Transcripción/metabolismoRESUMEN
In the Drosophila ovarian germline, Bone Morphogenetic Protein (BMP) signals released by niche cells promote germline stem cell (GSC) maintenance. Although BMP signaling is known to repress expression of a key differentiation factor, it remains unclear whether BMP-responsive transcription also contributes positively to GSC identity. Here, we identify the GSC transcriptome using RNA sequencing (RNA-seq), including the BMP-induced transcriptional network. Based on these data, we provide evidence that GSCs form two types of cellular projections. Genetic manipulation and live ex vivo imaging reveal that both classes of projection allow GSCs to access a reservoir of Dpp held away from the GSC-niche interface. Moreover, microtubule-rich projections, termed "cytocensors", form downstream of BMP and have additional functionality, which is to attenuate BMP signaling. In this way, cytocensors allow dynamic modulation of signal transduction to facilitate differentiation following GSC division. This ability of cytocensors to attenuate the signaling response expands the repertoire of functions associated with signaling projections.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Ovario/citología , Transducción de Señal , Nicho de Células Madre , Células Madre/metabolismo , Animales , Proteínas Morfogenéticas Óseas/genética , Diferenciación Celular , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster , Femenino , Regulación del Desarrollo de la Expresión Génica , Células Madre/citologíaRESUMEN
Tissue patterning, through the concerted activity of a small number of signaling pathways, is critical to embryonic development. While patterning can involve signaling between neighbouring cells, in other contexts signals act over greater distances by traversing complex cellular landscapes to instruct the fate of distant cells. In this review, we explore different strategies adopted by cells to modulate signaling molecule range to allow correct patterning. We describe mechanisms for restricting signaling range and highlight how such short-range signaling can be exploited to not only control the fate of adjacent cells, but also to generate graded signaling within a field of cells. Other strategies include modulation of signaling molecule action by tissue architectural properties and the use of cellular membranous structures, such as signaling filopodia and exosomes, to actively deliver signaling ligands to target cells. Signaling filopodia can also be deployed to reach out and collect particular signals, thereby precisely controlling their site of action.
Asunto(s)
Tipificación del Cuerpo , Desarrollo Embrionario , Transducción de Señal , Animales , Extensiones de la Superficie Celular/metabolismo , Vesículas Extracelulares/metabolismo , Humanos , LigandosRESUMEN
The bone morphogenetic protein (BMP) pathway is a major conserved signalling pathway with diverse roles in development and homeostasis. Given that cells exist in three-dimensional environments, one important area is to understand how the BMP pathway operates within such complex cellular environments. The extracellular matrix contains information regarding tissue architecture and its mechanical properties that is transmitted to the cell via integrin receptors. In this review, I describe various examples of modulation of the BMP pathway by integrins. In the case of the Drosophila embryo and some cell line-based studies, integrins have been found to enhance BMP responses through different mechanisms, such as enhancement of BMP ligand-receptor binding and effects on Smad phosphorylation or stability. In these contexts, BMP-dependent activation of integrins is a common theme. However, I also discuss examples where integrins inhibit the BMP pathway, highlighting the context-dependent nature of integrin-BMP cross-talk.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Matriz Extracelular/metabolismo , Integrinas/metabolismo , Transducción de Señal , Animales , Proteínas de la Matriz Extracelular/metabolismo , Humanos , Modelos Biológicos , Unión Proteica , Proteínas Smad/metabolismoRESUMEN
The BMP signaling pathway has a conserved role in dorsal-ventral axis patterning during embryonic development. In Drosophila, graded BMP signaling is transduced by the Mad transcription factor and opposed by the Brinker repressor. In this study, using the Drosophila embryo as a model, we combine RNA-seq with Mad and Brinker ChIP-seq to decipher the BMP-responsive transcriptional network underpinning differentiation of the dorsal ectoderm during dorsal-ventral axis patterning. We identify multiple new BMP target genes, including positive and negative regulators of EGF signaling. Manipulation of EGF signaling levels by loss- and gain-of-function studies reveals that EGF signaling negatively regulates embryonic BMP-responsive transcription. Therefore, the BMP gene network has a self-regulating property in that it establishes a balance between its activity and that of the antagonistic EGF signaling pathway to facilitate correct patterning. In terms of BMP-dependent transcription, we identify key roles for the Zelda and Zerknüllt transcription factors in establishing the resulting expression domain, and find widespread binding of insulator proteins to the Mad and Brinker-bound genomic regions. Analysis of embryos lacking the BEAF-32 insulator protein shows reduced transcription of a peak BMP target gene and a reduction in the number of amnioserosa cells, the fate specified by peak BMP signaling. We incorporate our findings into a model for Mad-dependent activation, and discuss its relevance to BMP signal interpretation in vertebrates.
Asunto(s)
Proteínas Morfogenéticas Óseas/genética , Proteínas de Unión al ADN/genética , Proteínas de Drosophila/genética , Proteínas del Ojo/genética , Proteínas de Homeodominio/genética , Proteínas Represoras/genética , Factores de Transcripción/genética , Animales , Tipificación del Cuerpo/genética , Proteínas Morfogenéticas Óseas/biosíntesis , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Embrión no Mamífero , Desarrollo Embrionario/genética , Factor de Crecimiento Epidérmico/genética , Regulación del Desarrollo de la Expresión Génica , Redes Reguladoras de Genes , Proteínas Nucleares , Transducción de Señal/genéticaRESUMEN
Within a 3D tissue, cells need to integrate signals from growth factors, such as BMPs, and the extracellular matrix (ECM) to coordinate growth and differentiation. Here, we use the Drosophila embryo as a model to investigate how BMP responses are influenced by a cell's local ECM environment. We show that integrins, which are ECM receptors, are absolutely required for peak BMP signaling. This stimulatory effect of integrins requires their intracellular signaling function, which is activated by the ECM protein collagen IV. Mechanistically, integrins interact with the BMP receptor and stimulate phosphorylation of the downstream Mad transcription factor. The BMP-pathway-enhancing function of integrins is independent of focal adhesion kinase, but it requires conserved NPXY motifs in the ß-integrin cytoplasmic tail. Furthermore, we show that an α-integrin subunit is a BMP target gene, identifying positive feedback between integrin signaling and BMP pathway activity that may contribute to robust cell fate decisions.
Asunto(s)
Proteínas Morfogenéticas Óseas/fisiología , Proteínas de Drosophila/fisiología , Drosophila melanogaster/metabolismo , Integrinas/fisiología , Animales , Línea Celular , Colágeno Tipo IV/genética , Colágeno Tipo IV/metabolismo , Drosophila melanogaster/embriología , Embrión no Mamífero/metabolismo , Desarrollo Embrionario , Retroalimentación Fisiológica , Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Transducción de SeñalRESUMEN
Precise control of the range of signalling molecule action is crucial for correct cell fate patterning during development. For example, Drosophila ovarian germline stem cells (GSCs) are maintained by exquisitely short-range BMP signalling from the niche. In the absence of BMP signalling, one GSC daughter differentiates into a cystoblast (CB) and this fate is stabilised by Brain tumour (Brat) and Pumilio (Pum)-mediated post-transcriptional repression of mRNAs, including that encoding the Dpp transducer, Mad. However, the identity of other repressed mRNAs and the mechanism of post-transcriptional repression are currently unknown. Here, we identify the Medea and schnurri mRNAs, which encode transcriptional regulators required for activation and/or repression of Dpp target genes, as additional Pum-Brat targets, suggesting that tripartite repression of the transducers is deployed to desensitise the CB to Dpp. In addition, we show that repression by Pum-Brat requires recruitment of the CCR4 and Pop2 deadenylases, with knockdown of deadenylases in vivo giving rise to ectopic GSCs. Consistent with this, Pum-Brat repression leads to poly(A) tail shortening and mRNA degradation in tissue culture cells, and we detect a reduced number of Mad and shn transcripts in the CB relative to the GSC based on single molecule mRNA quantitation. Finally, we show generality of the mechanism by demonstrating that Brat also attenuates pMad and Dpp signalling range in the early embryo. Together our data serve as a platform for understanding how post-transcriptional repression restricts interpretation of BMPs and other cell signals in order to allow robust cell fate patterning during development.
Asunto(s)
Células Madre Adultas/fisiología , Diferenciación Celular/fisiología , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila/crecimiento & desarrollo , Regulación del Desarrollo de la Expresión Génica/fisiología , Ovario/citología , Proteína Smad4/metabolismo , Factores de Transcripción/metabolismo , Animales , Western Blotting , Diferenciación Celular/genética , Femenino , Técnica del Anticuerpo Fluorescente , Regulación del Desarrollo de la Expresión Génica/genética , Hibridación in SituRESUMEN
Members of the Tolloid family of metalloproteinases liberate BMPs from inhibitory complexes to regulate BMP gradient formation during embryonic dorsal-ventral axis patterning. Here, we determine mechanistically how Tolloid activity is regulated by its non-catalytic CUB domains in the Drosophila embryo. We show that Tolloid, via its N-terminal CUB domains, interacts with Collagen IV, which enhances Tolloid activity towards its substrate Sog, and facilitates Tsg-dependent stimulation of cleavage. In contrast, the two most C-terminal Tld CUB domains mediate Sog interaction to facilitate its processing as, based on our structural data, Tolloid curvature positions bound Sog in proximity to the protease domain. Having ascribed functions to the Tolloid non-catalytic domains, we recapitulate embryonic BMP gradient formation in their absence, by artificially tethering the Tld protease domain to Sog. Our studies highlight how the bipartite function of Tolloid CUB domains, in substrate and ECM interactions, fine-tune protease activity to a particular developmental context.
Asunto(s)
Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Matriz Extracelular/metabolismo , Metaloproteinasas Similares a Tolloid/metabolismo , Animales , Dominio Catalítico , Colágeno Tipo IV/metabolismo , Proteínas de Drosophila/química , Modelos Moleculares , Proteínas Mutantes/metabolismo , Mutación Puntual , Unión Proteica , Ingeniería de Proteínas , Especificidad por Sustrato , Metaloproteinasas Similares a Tolloid/químicaRESUMEN
cis-regulatory modules (CRMs) act sequentially to regulate temporal expression of genes, but how the switch from one to the next is accomplished is not well understood. To provide insight, here we investigate the cis-regulatory system controlling brinker (brk) expression in the Drosophila embryo. Two distally located CRMs support expression at different times, while a promoter-proximal element (PPE) is required to support their action. In the absence of Brk protein itself or upon mutagenesis of Brk binding sites within the PPE, the late-acting CRM, specifically, is delayed. This block to late-acting CRM function appears to be removed when the early-acting CRM is also deleted. These results demonstrate that autoregulatory feedback is necessary for the early-acting CRM to disengage from the promoter so that the late-acting CRM may act. Autoregulation may be a commonly used mechanism to control sequential CRM action necessary for dynamic gene expression throughout the course of development.