RESUMO
Cytoplasmic divisions are thought to rely on nuclear divisions and mitotic signals. We demonstrate in Drosophila embryos that cytoplasm can divide repeatedly without nuclei and mitotic CDK/cyclin complexes. Cdk1 normally slows an otherwise faster cytoplasmic division cycle, coupling it with nuclear divisions, and when uncoupled, cytoplasm starts dividing before mitosis. In developing embryos where CDK/cyclin activity can license mitotic microtubule (MT) organizers like the spindle, cytoplasmic divisions can occur without the centrosome, a principal organizer of interphase MTs. However, centrosomes become essential in the absence of CDK/cyclin activity, implying that the cytoplasm can employ either the centrosome-based interphase or CDK/cyclin-dependent mitotic MTs to facilitate its divisions. Finally, we present evidence that autonomous cytoplasmic divisions occur during unperturbed fly embryogenesis and that they may help extrude mitotically stalled nuclei during blastoderm formation. We postulate that cytoplasmic divisions occur in cycles governed by a yet-to-be-uncovered clock mechanism autonomous from CDK/cyclin complexes.
Assuntos
Citocinese , Embrião não Mamífero , Animais , Núcleo Celular , Centrossomo , Ciclinas/metabolismo , Drosophila , Mitose , Fuso Acromático/metabolismo , Embrião não Mamífero/citologia , Embrião não Mamífero/metabolismoRESUMO
The prevailing view of metazoan gene regulation is that transcription is facilitated through the formation of static activator complexes at distal regulatory regions. Here, we employed quantitative single-cell live-imaging and computational analysis to provide evidence that the dynamic assembly and disassembly process of transcription factor (TF) clusters at enhancers is a major source of transcriptional bursting in developing Drosophila embryos. We further show that the regulatory connectivity between TF clustering and burst induction is highly regulated through intrinsically disordered regions (IDRs). Addition of a poly-glutamine tract to the maternal morphogen Bicoid demonstrated that extended IDR length leads to ectopic TF clustering and burst induction from its endogenous target genes, resulting in defects in body segmentation during embryogenesis. Moreover, we successfully visualized the presence of "shared" TF clusters during the co-activation of two distant genes, which provides a concrete molecular explanation for the newly proposed "topological operon" hypothesis in metazoan gene regulation.
Assuntos
Proteínas de Drosophila , Fatores de Transcrição , Animais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Drosophila/genéticaRESUMO
Tissue morphogenesis is often controlled by actomyosin networks pulling on adherens junctions (AJs), but junctional myosin levels vary. At an extreme, the Drosophila embryo amnioserosa forms a horseshoe-shaped strip of aligned, spindle-shaped cells lacking junctional myosin. What are the bases of amnioserosal cell interactions and alignment? Compared with surrounding tissue, we find that amnioserosal AJ continuity has lesser dependence on α-catenin, the mediator of AJ-actomyosin association, and greater dependence on Bazooka/Par-3, a junction-associated scaffold protein. Microtubule bundles also run along amnioserosal AJs and support their long-range curvilinearity. Amnioserosal confinement is apparent from partial overlap of its spindle-shaped cells, its outward bulging from surrounding tissue and from compressive stress detected within the amnioserosa. Genetic manipulations that alter amnioserosal confinement by surrounding tissue also result in amnioserosal cells losing alignment and gaining topological defects characteristic of nematically ordered systems. With Bazooka depletion, confinement by surrounding tissue appears to be relatively normal and amnioserosal cells align despite their AJ fragmentation. Overall, the fully elongated amnioserosa appears to form through tissue-autonomous generation of spindle-shaped cells that nematically align in response to confinement by surrounding tissue.
Assuntos
Junções Aderentes , Proteínas de Drosophila , Desenvolvimento Embrionário , Animais , Proteínas de Drosophila/metabolismo , Proteínas de Drosophila/genética , Junções Aderentes/metabolismo , Microtúbulos/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/metabolismo , Embrião não Mamífero/citologia , alfa Catenina/metabolismo , Actomiosina/metabolismo , Morfogênese , Drosophila/embriologia , Forma Celular , Peptídeos e Proteínas de Sinalização IntracelularRESUMO
Morphogen gradients provide essential positional information to gene networks through their spatially heterogeneous distribution, yet how they form is still hotly contested, with multiple models proposed for different systems. Here, we focus on the transcription factor Bicoid (Bcd), a morphogen that forms an exponential gradient across the anterior-posterior (AP) axis of the early Drosophila embryo. Using fluorescence correlation spectroscopy we find there are spatial differences in Bcd diffusivity along the AP axis, with Bcd diffusing more rapidly in the posterior. We establish that such spatially varying differences in Bcd dynamics are sufficient to explain how Bcd can have a steep exponential gradient in the anterior half of the embryo and yet still have an observable fraction of Bcd near the posterior pole. In the nucleus, we demonstrate that Bcd dynamics are impacted by binding to DNA. Addition of the Bcd homeodomain to eGFP::NLS qualitatively replicates the Bcd concentration profile, suggesting this domain regulates Bcd dynamics. Our results reveal how a long-range gradient can form while retaining a steep profile through much of its range.
Assuntos
Proteínas de Drosophila , Proteínas de Homeodomínio , Animais , Padronização Corporal/genética , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Transativadores/genética , Transativadores/metabolismoRESUMO
Embryos repair wounds rapidly, with no inflammation or scarring. Embryonic wound healing is driven by the collective movement of the cells around the lesion. The cells adjacent to the wound polarize the cytoskeletal protein actin and the molecular motor non-muscle myosin II, which accumulate at the wound edge forming a supracellular cable around the wound. Adherens junction proteins, including E-cadherin, are internalized from the wound edge and localize to former tricellular junctions at the wound margin, in a process necessary for cytoskeletal polarity. We found that the cells adjacent to wounds in the Drosophila embryonic epidermis polarized Talin, a core component of cell-extracellular matrix (ECM) adhesions, which preferentially accumulated at the wound edge. Integrin knockdown and inhibition of integrin binding delayed wound closure and reduced actin polarization and dynamics around the wound. Additionally, disrupting integrins caused a defect in E-cadherin reinforcement at tricellular junctions along the wound edge, suggesting crosstalk between integrin-based and cadherin-based adhesions. Our results show that cell-ECM adhesion contributes to embryonic wound repair and reveal an interplay between cell-cell and cell-ECM adhesion in the collective cell movements that drive rapid wound healing.
Assuntos
Actinas , Integrinas , Animais , Actinas/metabolismo , Integrinas/metabolismo , Caderinas/metabolismo , Movimento Celular/fisiologia , Junções Intercelulares/metabolismo , Drosophila/metabolismo , Cicatrização/fisiologia , Adesão CelularRESUMO
Most piRNAs in the Drosophila female germline are transcribed from heterochromatic regions called dual-strand piRNA clusters. Histone 3 lysine 9 trimethylation (H3K9me3) is required for licensing piRNA production by these clusters. However, it is unclear when and how they acquire this permissive heterochromatic state. Here, we show that transient Piwi depletion in Drosophila embryos results in H3K9me3 decrease at piRNA clusters in ovaries. This is accompanied by impaired biogenesis of ovarian piRNAs, accumulation of transposable element transcripts, and female sterility. Conversely, Piwi depletion at later developmental stages does not disturb piRNA cluster licensing. These results indicate that the identity of piRNA clusters is epigenetically acquired in a Piwi-dependent manner during embryonic development, which is reminiscent of the widespread genome reprogramming occurring during early mammalian zygotic development.
Assuntos
Proteínas Argonautas/metabolismo , Metilação de DNA , Elementos de DNA Transponíveis , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Repressão Epigenética , Heterocromatina/metabolismo , Ovário/metabolismo , Interferência de RNA , RNA Interferente Pequeno/metabolismo , Fatores Etários , Animais , Proteínas Argonautas/genética , Proteínas Cromossômicas não Histona/genética , Proteínas Cromossômicas não Histona/metabolismo , Proteínas de Drosophila/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Feminino , Fertilidade , Regulação da Expressão Gênica no Desenvolvimento , Heterocromatina/genética , Histonas/metabolismo , Infertilidade Feminina/genética , Infertilidade Feminina/metabolismo , Infertilidade Feminina/fisiopatologia , Metilação , Morfogênese , Ovário/embriologia , Ligação Proteica , RNA Interferente Pequeno/genéticaRESUMO
Cell signaling plays an essential role in development, and knowledge of the identities of the cells sending the signal is critical. This can be a challenge, since signaling pathways and ligands are commonly used at multiple times and in multiple cell types during development. One solution to this problem is to create cell type-specific mutants using CRISPR/Cas9 to mutate enhancers that control different patterns of expression. In this issue of Genes & Development, Rogers and colleagues (pp. 634-638) provide the first use of this method in Drosophila to solve a long-standing issue in patterning of the embryonic central nervous system.
Assuntos
Proteínas de Drosophila/genética , Regulação da Expressão Gênica no Desenvolvimento , Animais , Padronização Corporal/genética , Sistema Nervoso Central , Drosophila/embriologia , MutagêneseRESUMO
The EGF signaling pathway specifies neuronal identities in the Drosophila embryo by regulating developmental patterning genes such as intermediate neuroblasts defective (ind). EGFR is activated in the ventral midline and neurogenic ectoderm by the Spitz ligand, which is processed by the Rhomboid protease. CRISPR/Cas9 was used to delete defined rhomboid enhancers mediating expression at each site of Spitz processing. Surprisingly, the neurogenic ectoderm, not the ventral midline, was found to be the dominant source of EGF patterning activity. We suggest that Drosophila is undergoing an evolutionary transition in central nervous system (CNS)-organizing activity from the ventral midline to the neurogenic ectoderm.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila/genética , Embrião não Mamífero/metabolismo , Fator de Crescimento Epidérmico/genética , Receptores ErbB/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Redes Reguladoras de Genes , Proteínas de Membrana/genética , Neurogênese/genética , Receptores de Peptídeos de Invertebrados/metabolismo , Animais , Sistemas CRISPR-Cas , Linhagem da Célula , Células Cultivadas , Sistema Nervoso Central , Drosophila/embriologia , Proteínas de Drosophila/antagonistas & inibidores , Embrião não Mamífero/citologia , Fator de Crescimento Epidérmico/antagonistas & inibidores , Fator de Crescimento Epidérmico/metabolismo , Receptores ErbB/genética , Feminino , Masculino , Proteínas de Membrana/antagonistas & inibidores , Proteínas de Membrana/metabolismo , Receptores de Peptídeos de Invertebrados/genética , Transdução de SinaisRESUMO
Hox genes encode transcription factors that play an important role in establishing the basic body plan of animals. In Drosophila, Antennapedia is one of the five genes that make up the Antennapedia complex (ANT-C). Antennapedia determines the identity of the second thoracic segment, known as the mesothorax. Misexpression of Antennapedia at different developmental stages changes the identity of the mesothorax, including the muscles, nervous system, and cuticle. In Drosophila, Antennapedia has two distinct promoters highly regulated throughout development by several transcription factors. Antennapedia proteins are found with other transcription factors in different ANTENNAPEDIA transcriptional complexes to regulate multiple subsets of target genes. In this review, we describe the different mechanisms that regulate the expression and function of Antennapedia and the role of this Hox gene in the development of Drosophila.
Assuntos
Proteínas de Drosophila , Fatores de Transcrição , Animais , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Proteínas de Homeodomínio/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Drosophila/genética , Drosophila/metabolismo , Drosophila melanogaster/genéticaRESUMO
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.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/genética , Biossíntese de Proteínas/genética , RNA Mensageiro Estocado/genética , Fatores de Transcrição/genética , Animais , Padronização Corporal/genética , Embrião não Mamífero/fisiologia , Regulação da Expressão Gênica no Desenvolvimento/genética , Morfogênese/genética , Zigoto/fisiologiaRESUMO
Half a century after Lewis Wolpert's seminal conceptual advance on how cellular fates distribute in space, we provide a brief historical perspective on how the concept of positional information emerged and influenced the field of developmental biology and beyond. We focus on a modern interpretation of this concept in terms of information theory, largely centered on its application to cell specification in the early Drosophila embryo. We argue that a true physical variable (position) is encoded in local concentrations of patterning molecules, that this mapping is stochastic, and that the processes by which positions and corresponding cell fates are determined based on these concentrations need to take such stochasticity into account. With this approach, we shift the focus from biological mechanisms, molecules, genes and pathways to quantitative systems-level questions: where does positional information reside, how it is transformed and accessed during development, and what fundamental limits it is subject to?
Assuntos
Padronização Corporal , Animais , Evolução Biológica , Padronização Corporal/genética , Humanos , Teoria da Informação , Modelos BiológicosRESUMO
Transcriptional repression is a pervasive feature of animal development. Here, we employ live-imaging methods to visualize the Snail repressor, which establishes the boundary between the presumptive mesoderm and neurogenic ectoderm of early Drosophila embryos. Snail target enhancers were attached to an MS2 reporter gene, permitting detection of nascent transcripts in living embryos. The transgenes exhibit initially broad patterns of transcription but are refined by repression in the mesoderm following mitosis. These observations reveal a correlation between mitotic silencing and Snail repression. We propose that mitosis and other inherent discontinuities in transcription boost the activities of sequence-specific repressors, such as Snail.
Assuntos
Drosophila melanogaster/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Mitose/fisiologia , Fatores de Transcrição da Família Snail/metabolismo , Animais , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Embrião não Mamífero , Elementos Facilitadores Genéticos/genética , Mitose/genética , Fatores de Transcrição da Família Snail/genética , Transgenes/genéticaRESUMO
Establishment of morphogen gradients in the early Drosophila embryo is challenged by a diffusible sextracellular milieu, and by rapid nuclear divisions that occur at the same time. To understand how a sharp gradient is formed within this dynamic environment, we followed the generation of graded nuclear Dorsal protein, the hallmark of pattern formation along the dorso-ventral axis, in live embryos. The dynamics indicate that a sharp extracellular gradient is formed through diffusion-based shuttling of the Spaetzle (Spz) morphogen that progresses through several nuclear divisions. Perturbed shuttling in wntD mutant embryos results in a flat activation peak and aberrant gastrulation. Re-entry of Dorsal into the nuclei at the final division cycle plays an instructive role, as the residence time of Dorsal in each nucleus is translated to the amount of zygotic transcript that will be produced, thereby guiding graded accumulation of specific zygotic transcripts that drive patterned gastrulation. We conclude that diffusion-based ligand shuttling, coupled with dynamic readout, establishes a refined pattern within the diffusible environment of early embryos.
Assuntos
Proteínas de Drosophila/fisiologia , Drosophila/embriologia , Gástrula/metabolismo , Gastrulação , Regulação da Expressão Gênica no Desenvolvimento , Morfogênese , Animais , Padronização Corporal , Núcleo Celular/fisiologia , Proteínas de Drosophila/genética , Embrião não Mamífero/fisiologia , Feminino , Peptídeos e Proteínas de Sinalização Intracelular/genética , Masculino , Mutação , Proteínas Nucleares/fisiologia , Fosfoproteínas/fisiologia , Transdução de Sinais , Fatores de Transcrição/fisiologiaRESUMO
Traditional studies of gene regulation in the Drosophila embryo centered primarily on the analysis of fixed tissues. These methods provided considerable insight into the spatial control of gene activity, such as the borders of eve stripe 2, but yielded only limited information about temporal dynamics. The advent of quantitative live-imaging and genome-editing methods permits the detailed examination of the temporal control of endogenous gene activity. Here, we present evidence that the pair-rule genes fushi tarazu (ftz) and even-skipped (eve) undergo dynamic shifts in gene expression. We observe sequential anterior shifting of the stripes along the anterior to posterior axis, with stripe 1 exhibiting movement before stripe 2 and the more posterior stripes. Conversely, posterior stripes shift over greater distances (two or three nuclei) than anterior stripes (one or two nuclei). Shifting of the ftz and eve stripes are slightly offset, with ftz moving faster than eve This observation is consistent with previous genetic studies, suggesting that eve is epistatic to ftz The precision of pair-rule temporal dynamics might depend on enhancer-enhancer interactions within the eve locus, since removal of the endogenous eve stripe 1 enhancer via CRISPR/Cas9 genome editing led to precocious and expanded expression of eve stripe 2. These observations raise the possibility of an added layer of complexity in the positional information encoded by the segmentation gene regulatory network.
Assuntos
Proteínas de Ligação a DNA/genética , Proteínas de Drosophila/genética , Drosophila/embriologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Fatores de Transcrição/genética , Animais , Drosophila/genética , Embrião não Mamífero/fisiologia , Elementos Facilitadores Genéticos/fisiologia , Edição de Genes , Redes Reguladoras de GenesRESUMO
Cascades of zygotic gene expression pattern the anterior-posterior (AP) and dorsal-ventral (DV) axes of the early Drosophila embryo. Here, we used the global run-on sequencing assay (GRO-seq) to map the genome-wide RNA polymerase distribution during early Drosophila embryogenesis, thus providing insights into how genes are regulated. We identify widespread promoter-proximal pausing yet show that the presence of paused polymerase does not necessarily equate to direct regulation through pause release to productive elongation. Our data reveal that a subset of early Zelda-activated genes is regulated at the level of polymerase recruitment, whereas other Zelda target and axis patterning genes are predominantly regulated through pause release. In contrast to other signaling pathways, we found that bone morphogenetic protein (BMP) target genes are collectively more highly paused than BMP pathway components and show that BMP target gene expression requires the pause-inducing negative elongation factor (NELF) complex. Our data also suggest that polymerase pausing allows plasticity in gene activation throughout embryogenesis, as transiently repressed and transcriptionally silenced genes maintain and lose promoter polymerases, respectively. Finally, we provide evidence that the major effect of pausing is on the levels, rather than timing, of transcription. These data are discussed in terms of the efficiency of transcriptional activation required across cell populations during developmental time constraints.
Assuntos
Padronização Corporal/genética , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , RNA Polimerase II/metabolismo , Transcrição Gênica , Animais , Proteínas de Drosophila/metabolismo , Feminino , Proteínas Nucleares , Fatores de Transcrição/metabolismo , Zigoto/metabolismoRESUMO
BACKGROUND: A feedforward loop (FFL) is commonly observed in several biological networks. The FFL network motif has been mostly studied with respect to variation of the input signal in time, with only a few studies of FFL activity in a spatially distributed system such as morphogen-mediated tissue patterning. However, most morphogen gradients also evolve in time. RESULTS: We studied the spatiotemporal behavior of a coherent FFL in two contexts: (a) a generic, oscillating morphogen gradient and (b) the dorsal-ventral patterning of the early Drosophila embryo by a gradient of the NF-κB homolog dorsal with its early target Twist. In both models, we found features in the dynamics of the intermediate node-phase difference and noise filtering-that were largely independent of the parameterization of the models, and thus were functions of the structure of the FFL itself. In the dorsal gradient model, we also found that proper target gene expression was not possible without including the effect of maternal pioneer factor Zelda. CONCLUSIONS: An FFL buffers fluctuation to changes in the morphogen signal ensuring stable gene expression boundaries.
Assuntos
Padronização Corporal/fisiologia , Proteínas de Drosophila/metabolismo , Embrião não Mamífero/metabolismo , Animais , Padronização Corporal/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Regulação da Expressão Gênica no Desenvolvimento/genética , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , NF-kappa B/metabolismo , Transdução de Sinais/genética , Transdução de Sinais/fisiologiaRESUMO
In many biological systems gene expression at mRNA and protein levels is not identical. Rigorous comparison of such differences on a spatio-temporal scale is still not feasible by high-throughput transcriptomic and proteomic analyses of early embryo development. Here, we characterize differences between mRNA and protein expression of Drosophila segmentation genes at the level of individual gene expression domains. We obtained quantitative imaging data on expression of gap genes gt and hb and pair-rule gene eve for Drosophila wild type embryos, Kr null mutants and Kr+/Kr- heterozygotes. To compare mRNA and protein expression we use several criteria including difference in amplitude and positions of expression domains, pattern shape and positional variability. For a number of gene expression domains we show examples where protein expression does not repeat mRNA expression even after a temporal delay. We calculated time delays between eve pattern formation at the level of mRNA and protein for wild type embryos, Kr mutants and Kr+/Kr- heterozygotes. We detect that in wild type embryos, the amplitudes of eve stripes 3 and 7 do not differ significantly at the level of mRNA, however, stripe 3 is higher than stripe 7 at the protein level. We further show that hb mRNA and protein expression in both anterior and posterior domains significantly differs at specific time points. The formation of hb PS4 stripe at the mRNA level proceeds five times faster than at the level of protein. With regard to spatial expression, we show that the offset between posterior gt mRNA and protein domains is much larger in Kr mutants than in wild type embryos and heterozygotes. Finally, we analyze differences in positional variability of eve stripe 7 expression in Kr mutants and Kr+/Kr- heterozygotes at the level of mRNA and protein. These results enable further perspectives to uncover mechanisms underlying discrepancies between mRNA and protein expression in early embryo.
Assuntos
Padronização Corporal/fisiologia , Embrião não Mamífero/embriologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Genótipo , RNA Mensageiro , Animais , Proteínas de Drosophila/biossíntese , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas de Homeodomínio/biossíntese , Proteínas de Homeodomínio/genética , Fatores de Transcrição Kruppel-Like/biossíntese , Fatores de Transcrição Kruppel-Like/genética , Microscopia Confocal , RNA Mensageiro/genética , RNA Mensageiro/metabolismo , Fatores de Transcrição/biossíntese , Fatores de Transcrição/genéticaRESUMO
It is long established that the graded distribution of Dorsal transcription factor influences spatial domains of gene expression along the dorsoventral (DV) axis of Drosophila melanogaster embryos. However, the more recent realization that Dorsal levels also change with time raises the question of whether these dynamics are instructive. An overview of DV axis patterning is provided, focusing on new insights identified through quantitative analysis of temporal changes in Dorsal target gene expression from one nuclear cycle to the next ('steps'). Possible roles for the stepwise progression of this patterning program are discussed including (i) tight temporal regulation of signaling pathway activation, (ii) control of gene expression cohorts, and (iii) ensuring the irreversibility of the patterning and cell fate specification process.
Assuntos
Padronização Corporal/genética , Drosophila melanogaster/genética , Desenvolvimento Embrionário/genética , Animais , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento/genética , Proteínas Nucleares/genética , Transdução de Sinais/genética , Fatores de Transcrição/genéticaRESUMO
Epigenetic patterns of histone modifications contribute to the maintenance of tissue-specific gene expression. Here, we show that such modifications also accompany the specification of cell identities by the NF-κB transcription factor Dorsal in the precellular Drosophila embryo. We provide evidence that the maternal pioneer factor, Zelda, is responsible for establishing poised RNA polymerase at Dorsal target genes before Dorsal-mediated zygotic activation. At the onset of cell specification, Dorsal recruits the CBP/p300 coactivator to the regulatory regions of defined target genes in the presumptive neuroectoderm, resulting in their histone acetylation and transcriptional activation. These genes are inactive in the mesoderm due to transcriptional quenching by the Snail repressor, which precludes recruitment of CBP and prevents histone acetylation. By contrast, inactivation of the same enhancers in the dorsal ectoderm is associated with Polycomb-repressed H3K27me3 chromatin. Thus, the Dorsal morphogen gradient produces three distinct histone signatures including two modes of transcriptional repression, active repression (hypoacetylation), and inactivity (H3K27me3). Whereas histone hypoacetylation is associated with a poised polymerase, H3K27me3 displaces polymerase from chromatin. Our results link different modes of RNA polymerase regulation to separate epigenetic patterns and demonstrate that developmental determinants orchestrate differential chromatin states, providing new insights into the link between epigenetics and developmental patterning.
Assuntos
Padronização Corporal/genética , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Epigênese Genética , Proteínas Nucleares/genética , Acetilação , Animais , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Histonas/metabolismo , Placa Neural/embriologia , Placa Neural/metabolismo , Proteínas Nucleares/metabolismo , Fosfoproteínas/genética , Fosfoproteínas/metabolismo , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Ativação Transcricional , Fatores de Transcrição de p300-CBP/genética , Fatores de Transcrição de p300-CBP/metabolismoRESUMO
Coordinated locomotion of an organism relies on the development of proper musculoskeletal connections. In Drosophila, the Slit-Robo signaling pathway guides muscles to tendons. Here, we show that the Slit receptor Roundabout 2 (Robo2) plays a non-cell-autonomous role in directing muscles to their corresponding tendons. Robo2 is expressed by tendons, and its non-signaling activity in these cells promotes Slit cleavage, producing a cleaved Slit N-terminal guidance signal that provides short-range signaling into muscles. Consistently, robo2 mutant embryos exhibited a muscle phenotype similar to that of slit, which could not be rescued by muscle-specific Robo2 expression but rather by ectodermally derived Robo2. Alternatively, this muscle phenotype could be induced by tendon-specific robo2 RNAi. We further show that membrane immobilization of Slit or its N-terminal cleaved form (Slit-N) on tendons bypasses the functional requirement for Robo2 in tendons, verifying that the major role of Robo2 is to promote the association of Slit with the tendon cell membrane. Slit-N tends to oligomerize whereas full-length uncleavable Slit does not. It is therefore proposed that Slit-N oligomers, produced at the tendon membrane by Robo2, signal to the approaching muscle by combined Robo1 and Robo3 activity. These findings establish a Robo2-mediated mechanism, independent of signaling, that is essential to limiting Slit distribution and which might be relevant to the regulation of Slit-mediated short-range signaling in additional systems.