RESUMEN
Transcription factors play an essential role in pattern formation during early embryo development, generating a strikingly fast and precise transcriptional response that results in sharp gene expression boundaries. To characterize the steps leading up to transcription, we performed a side-by-side comparison of the nuclear dynamics of two morphogens, a transcriptional activator, Bicoid (Bcd), and a transcriptional repressor, Capicua (Cic), both involved in body patterning along the anterior-posterior axis of the early Drosophila embryo. We used a combination of fluorescence recovery after photobleaching, fluorescence correlation spectroscopy, and single particle tracking to access a wide range of dynamical timescales. Despite their opposite effects on gene transcription, we find that Bcd and Cic have very similar nuclear dynamics, characterized by the co-existence of a freely diffusing monomer population with a number of oligomeric clusters, which range from low stoichiometry and high mobility clusters to larger, DNA-bound hubs. Our observations are consistent with the inclusion of both Bcd and Cic into transcriptional hubs or condensates, while putting constraints on the mechanism by which these form. These results fit in with the recent proposal that many transcription factors might share a common search strategy for target genes regulatory regions that makes use of their large unstructured regions, and may eventually help explain how the transcriptional response they elicit can be at the same time so fast and so precise.
RESUMEN
Mutations in signal transduction pathways lead to various diseases including cancers. MEK1 kinase, encoded by the human MAP2K1 gene, is one of the central components of the MAPK pathway and more than a hundred somatic mutations in the MAP2K1 gene were identified in various tumors. Germline mutations deregulating MEK1 also lead to congenital abnormalities, such as the cardiofaciocutaneous syndrome and arteriovenous malformation. Evaluating variants associated with a disease is a challenge, and computational genomic approaches aid in this process. Establishing evolutionary history of a gene improves computational prediction of disease-causing mutations; however, the evolutionary history of MEK1 is not well understood. Here, by revealing a precise evolutionary history of MEK1, we construct a well-defined dataset of MEK1 metazoan orthologs, which provides sufficient depth to distinguish between conserved and variable amino acid positions. We matched known and predicted disease-causing and benign mutations to evolutionary changes observed in corresponding amino acid positions and found that all known and many suspected disease-causing mutations are evolutionarily intolerable. We selected several variants that cannot be unambiguously assessed by automated prediction tools but that are confidently identified as "damaging" by our approach, for experimental validation in Drosophila. In all cases, evolutionary intolerant variants caused increased mortality and severe defects in fruit fly embryos confirming their damaging nature. We anticipate that our analysis will serve as a blueprint to help evaluate known and novel missense variants in MEK1 and that our approach will contribute to improving automated tools for disease-associated variant interpretation.
Asunto(s)
Displasia Ectodérmica , Cardiopatías Congénitas , Humanos , Animales , Mutación , Displasia Ectodérmica/genética , Mutación Missense , Cardiopatías Congénitas/genética , Aminoácidos/genética , MAP Quinasa Quinasa 1/genéticaRESUMEN
Germline mutations upregulating RAS signaling are associated with multiple developmental disorders. A hallmark of these conditions is that the same mutation may present vastly different phenotypes in different individuals, even in monozygotic twins. Here, we demonstrate how the origins of such largely unexplained phenotypic variations may be dissected using highly controlled studies in Drosophila that have been gene edited to carry activating variants of MEK, a core enzyme in the RAS pathway. This allowed us to measure the small but consistent increase in signaling output of such alleles in vivo. The fraction of mutation carriers reaching adulthood was strongly reduced, but most surviving animals had normal RAS-dependent structures. We rationalize these results using a stochastic signaling model and support it by quantifying cell fate specification errors in bilaterally symmetric larval trachea, a RAS-dependent structure that allows us to isolate the effects of mutations from potential contributions of genetic modifiers and environmental differences. We propose that the small increase in signaling output shifts the distribution of phenotypes into a regime, where stochastic variation causes defects in some individuals, but not in others. Our findings shed light on phenotypic heterogeneity of developmental disorders caused by deregulated RAS signaling and offer a framework for investigating causal effects of other pathogenic alleles and mild mutations in general.
Asunto(s)
Transducción de Señal , Proteínas ras , Animales , Proteínas ras/genética , Proteínas ras/metabolismo , Transducción de Señal/genética , Mutación , Drosophila/genética , Drosophila/metabolismo , FenotipoRESUMEN
Terminal regions of Drosophila embryos are patterned by signaling through ERK, which is genetically deregulated in multiple human diseases. Quantitative studies of terminal patterning have been recently used to investigate gain-of-function variants of human MEK1, encoding the MEK kinase that directly activates ERK by dual phosphorylation. Unexpectedly, several mutations reduced ERK activation by extracellular signals, possibly through a negative feedback triggered by signal-independent activity of the mutant variants. Here we present experimental evidence supporting this model. Using a MEK variant that combines a mutation within the negative regulatory region with alanine substitutions in the activation loop, we prove that pathogenic variants indeed acquire signal-independent kinase activity. We also demonstrate that signal-dependent activation of these variants is independent of kinase suppressor of Ras, a conserved adaptor that is indispensable for activation of normal MEK. Finally, we show that attenuation of ERK activation by extracellular signals stems from transcriptional induction of Mkp3, a dual specificity phosphatase that deactivates ERK by dephosphorylation. These findings in the Drosophila embryo highlight its power for investigating diverse effects of human disease mutations.
Asunto(s)
MAP Quinasa Quinasa 1/genética , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Fosfatasas de Especificidad Dual , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Humanos , MAP Quinasa Quinasa 1/metabolismo , Sistema de Señalización de MAP Quinasas/genética , Sistema de Señalización de MAP Quinasas/fisiología , Mutación , Fosforilación/efectos de los fármacos , Transducción de SeñalRESUMEN
Clonal dominance arises when the descendants (clones) of one or a few founder cells contribute disproportionally to the final structure during collective growth [1-8]. In contexts such as bacterial growth, tumorigenesis, and stem cell reprogramming [2-4], this phenomenon is often attributed to pre-existing propensities for dominance, while in stem cell homeostasis, neutral drift dynamics are invoked [5,6]. The mechanistic origin of clonal dominance during development, where it is increasingly documented [1,6-8], is less understood. Here, we investigate this phenomenon in the Drosophila melanogaster follicle epithelium, a system in which the joint growth dynamics of cell lineage trees can be reconstructed. We demonstrate that clonal dominance can emerge spontaneously, in the absence of pre-existing biases, as a collective property of evolving excitable networks through coupling of divisions among connected cells. Similar mechanisms have been identified in forest fires and evolving opinion networks [9-11]; we show that the spatial coupling of excitable units explains a critical feature of the development of the organism, with implications for tissue organization and dynamics [1,12,13].
RESUMEN
To bridge the gap between qualitative and quantitative analyses of the epidermal growth factor receptor (EGFR) in tissues, we generated an sfGFP-tagged EGF receptor (EGFR-sfGFP) in Drosophila The homozygous fly appears similar to wild type with EGFR expression and activation patterns that are consistent with previous reports in the ovary, early embryo, and imaginal discs. Using ELISA, we quantified an average of 1100, 6200 and 2500 receptors per follicle cell (FC) at stages 8/9, 10 and ≥11 of oogenesis, respectively. Interestingly, the spatial localization of the EGFR to the apical side of the FCs at early stages depended on the TGFα-like ligand Gurken. At later stages, EGFR localized to basolateral positions of the FCs. Finally, we followed the endosomal localization of EGFR in the FCs. The EGFR colocalized with the late endosome, but no significant colocalization of the receptor was found with the early endosome. The EGFR-sfGFP fly is an exciting new resource for studying cellular localization and regulation of EGFR in tissues.
Asunto(s)
Proteínas de Drosophila/metabolismo , Células Epiteliales/metabolismo , Receptores ErbB/metabolismo , Folículo Ovárico/metabolismo , Receptores de Péptidos de Invertebrados/metabolismo , Transducción de Señal , Animales , Proteínas de Drosophila/genética , Drosophila melanogaster , Endosomas/genética , Endosomas/metabolismo , Células Epiteliales/citología , Epitelio/metabolismo , Receptores ErbB/genética , Femenino , Folículo Ovárico/citología , Receptores de Péptidos de Invertebrados/genética , Factor de Crecimiento Transformador alfa/genética , Factor de Crecimiento Transformador alfa/metabolismoRESUMEN
Optogenetic perturbations, live imaging, and time-resolved ChIP-seq assays in Drosophila embryos were used to dissect the ERK-dependent control of the HMG-box repressor Capicua (Cic), which plays critical roles in development and is deregulated in human spinocerebellar ataxia and cancers. We established that Cic target genes are activated before significant downregulation of nuclear localization of Cic and demonstrated that their activation is preceded by fast dissociation of Cic from the regulatory DNA. We discovered that both Cic-DNA binding and repression are rapidly reinstated in the absence of ERK activation, revealing that inductive signaling must be sufficiently sustained to ensure robust transcriptional response. Our work provides a quantitative framework for the mechanistic analysis of dynamics and control of transcriptional repression in development.
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Proteínas de Drosophila/metabolismo , Proteínas HMGB/metabolismo , Sistema de Señalización de MAP Quinasas , Proteínas Represoras/metabolismo , Transporte Activo de Núcleo Celular , Animales , Núcleo Celular/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster , Quinasas MAP Reguladas por Señal Extracelular/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas HMGB/genética , Unión Proteica , Proteínas Represoras/genéticaRESUMEN
Proper tissue patterning is an essential step during organ formation. During this process, genes are expressed in distinct patterns, defining boundaries for future functional domains. The bone morphogenetic protein (BMP) signaling pathway sets the anterior domain during eggshell patterning. Previously, the Drosophila melanogaster homolog of BMPR2, Wishful thinking (WIT), was shown to be required for BMP signaling and patterning during eggshell formation. Expressed in a conserved anterior pattern, the width of wit patterning in the follicular epithelium is evolutionarily divergent between Drosophila species. We used genome editing to demonstrate how the gene pattern divergence is controlled in cis within the wit locus of D. virilis Furthermore, unlike direct targets of BMP signaling, we demonstrate how one transcription factor binding site shapes the pattern of WIT in D. melanogaster by negative regulation. However, changes in this site are not sufficient to explain the evolution of wit patterning, suggesting that a positive regulatory element that controls pattern divergence remains to be discovered.
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Tipificación del Cuerpo/fisiología , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/metabolismo , Proteínas Morfogenéticas Óseas/metabolismo , Proteínas de Drosophila/metabolismo , Evolución Molecular , Receptores de Superficie Celular/metabolismo , Animales , Receptores de Proteínas Morfogenéticas Óseas de Tipo II/genética , Proteínas Morfogenéticas Óseas/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Receptores de Superficie Celular/genética , Transducción de Señal/fisiología , Especificidad de la EspecieRESUMEN
Exponential increase of cell numbers in early embryos requires large amounts of DNA precursors (deoxyribonucleoside triphosphates (dNTPs)). Little is understood about how embryos satisfy this demand. We examined dNTP metabolism in the early Drosophila embryo, in which gastrulation is preceded by 13 sequential nuclear cleavages within only 2 hr of fertilization. Surprisingly, despite the breakneck speed at which Drosophila embryos synthesize DNA, maternally deposited dNTPs can generate less than half of the genomes needed to reach gastrulation. The rest of the dNTPs are synthesized "on the go." The rate-limiting enzyme of dNTP synthesis, ribonucleotide reductase, is inhibited by endogenous levels of deoxyATP (dATP) present at fertilization and is activated as dATP is depleted via DNA polymerization. This feedback inhibition renders the concentration of dNTPs at gastrulation robust, with respect to large variations in maternal supplies, and is essential for normal progression of embryogenesis.
Asunto(s)
Desoxirribonucleótidos/metabolismo , Drosophila/metabolismo , Retroalimentación Fisiológica , Animales , Desoxirribonucleótidos/biosíntesis , Drosophila/embriología , Drosophila/genética , Desarrollo EmbrionarioRESUMEN
Eggshell patterning has been extensively studied in Drosophila melanogaster However, the cis-regulatory modules (CRMs), which control spatiotemporal expression of these patterns, are vastly unexplored. The FlyLight collection contains >7000 intergenic and intronic DNA fragments that, if containing CRMs, can drive the transcription factor GAL4. We cross-listed the 84 genes known to be expressed during D. melanogaster oogenesis with the â¼1200 listed genes of the FlyLight collection, and found 22 common genes that are represented by 281 FlyLight fly lines. Of these lines, 54 show expression patterns during oogenesis when crossed to an UAS-GFP reporter. Of the 54 lines, 16 recapitulate the full or partial pattern of the associated gene pattern. Interestingly, while the average DNA fragment size is â¼3 kb in length, the vast majority of fragments show one type of spatiotemporal pattern in oogenesis. Mapping the distribution of all 54 lines, we found a significant enrichment of CRMs in the first intron of the associated genes' model. In addition, we demonstrate the use of different anteriorly active FlyLight lines as tools to disrupt eggshell patterning in a targeted manner. Our screen provides further evidence that complex gene patterns are assembled combinatorially by different CRMs controlling the expression of genes in simple domains.
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Drosophila melanogaster/genética , Regulación del Desarrollo de la Expresión Génica , Oogénesis/genética , Secuencias Reguladoras de Ácidos Nucleicos/genética , Animales , Animales Modificados Genéticamente , Mapeo Cromosómico , Genes de Insecto , Proteínas Fluorescentes Verdes/metabolismo , Modelos Genéticos , Especificidad de Órganos/genética , Factores de TiempoRESUMEN
Changes in gene regulation are associated with the evolution of morphologies. However, the specific sequence information controlling gene expression is largely unknown and discovery is time and labor consuming. We use the intricate patterning of follicle cells to probe species' relatedness in the absence of sequence information. We focus on one of the major families of genes that pattern the Drosophila eggshell, the Chorion protein (Cp). Systematically screening for the spatiotemporal patterning of all nine Cp genes in three species (Drosophila melanogaster, D. nebulosa, and D. willistoni), we found that most genes are expressed dynamically during mid and late stages of oogenesis. Applying an annotation code, we transformed the data into binary matrices that capture the complexity of gene expression. Gene patterning is sufficient to predict species' relatedness, consistent with their phylogeny. Surprisingly, we found that expression domains of most genes are different among species, suggesting that Cp regulation is rapidly evolving. In addition, we found a morphological novelty along the dorsalmost side of the eggshell, the dorsal ridge. Our matrix analysis placed the dorsal ridge domain in a cluster of epidermal growth factor receptor associated domains, which was validated through genetic and chemical perturbations. Expression domains are regulated cooperatively or independently by signaling pathways, supporting that complex patterns are combinatorially assembled from simple domains.
Asunto(s)
Drosophila melanogaster/genética , Drosophila/clasificación , Drosophila/genética , Proteínas del Huevo/genética , Regulación del Desarrollo de la Expresión Génica , Animales , Tipificación del Cuerpo/genética , Clonación Molecular , Drosophila melanogaster/clasificación , Proteínas del Huevo/metabolismo , Receptores ErbB/genética , Receptores ErbB/metabolismo , Evolución Molecular , Oogénesis , Filogenia , Análisis de Secuencia de ADN , Transducción de SeñalRESUMEN
The Drosophila eggshell is an elaborate structure that is derived from a monolayer of follicular epithelium surrounding the developing oocyte within the female ovary. The bone morphogenetic protein (BMP) signaling pathway is essential for controlling the patterning and morphogenesis of the eggshell. During oogenesis, the roles of patterning and morphogenesis by the BMP type I receptor thickveins (tkv) have been studied extensively. However, signaling through this pathway requires both type I and II receptors, and the latter has yet to be established in oogenesis. We focus on wishful thinking (wit), the Drosophila homolog to the mammalian BMP type II receptor, BMPRII. We found that wit is expressed dynamically in the FCs of D. melanogaster in an evolutionary conserved pattern. The expression patterns are highly correlated with the dynamics of the BMP signaling, which is consistent with our finding that wit is a target of BMP signaling. Furthermore, we established that WIT is necessary for BMP signaling, and loss of WIT is associated with cell autonomous loss of BMP responses. Of importance, we demonstrated that perturbations in WIT led to changes in eggshell morphologies in domains that are patterned by BMP signaling. Previous studies have shown a role for WIT in BMP signaling during neurogenesis; however, our results reveal a role for WIT in epithelial cells' development.