RESUMEN

Cytoplasmic flows are widely emerging as key functional players in development. In early Drosophila embryos, flows drive the spreading of nuclei across the embryo. Here, we combine hydrodynamic modeling with quantitative imaging to develop a two-fluid model that features an active actomyosin gel and a passive viscous cytosol. Gel contractility is controlled by the cell cycle oscillator, the two fluids being coupled by friction. In addition to recapitulating experimental flow patterns, our model explains observations that remained elusive and makes a series of predictions. First, the model captures the vorticity of cytosolic flows, which highlights deviations from Stokes' flow that were observed experimentally but remained unexplained. Second, the model reveals strong differences in the gel and cytosol motion. In particular, a micron-sized boundary layer is predicted close to the cortex, where the gel slides tangentially while the cytosolic flow cannot slip. Third, the model unveils a mechanism that stabilizes the spreading of nuclei with respect to perturbations of their initial positions. This self-correcting mechanism is argued to be functionally important for proper nuclear spreading. Fourth, we use our model to analyze the effects of flows on the transport of the morphogen Bicoid and the establishment of its gradients. Finally, the model predicts that the flow strength should be reduced if the shape of the domain is more round, which is experimentally confirmed in Drosophila mutants. Thus, our two-fluid model explains flows and nuclear positioning in early Drosophila, while making predictions that suggest novel future experiments.

Asunto(s)

Proteínas de Drosophila , Drosophila , Animales , Drosophila/metabolismo , Citosol/metabolismo , Hidrodinámica , Citoplasma/metabolismo , Proteínas de Drosophila/metabolismo

RESUMEN

Nascent myotubes undergo a dramatic morphological transformation during myogenesis, in which the myotubes elongate over several cell diameters and are directed to the correct muscle attachment sites. Although this process of myotube guidance is essential to pattern the musculoskeletal system, the mechanisms that control myotube guidance remain poorly understood. Using transcriptomics, we found that components of the Fibroblast Growth Factor (FGF) signaling pathway were enriched in nascent myotubes in Drosophila embryos. Null mutations in the FGF receptor heartless (htl), or its ligands, caused significant myotube guidance defects. The FGF ligand Pyramus is expressed broadly in the ectoderm, and ectopic Pyramus expression disrupted muscle patterning. Mechanistically, Htl regulates the activity of Rho/Rac GTPases in nascent myotubes and effects changes in the actin cytoskeleton. FGF signals are thus essential regulators of myotube guidance that act through cytoskeletal regulatory proteins to pattern the musculoskeletal system.

Asunto(s)

Tipificación del Cuerpo/genética , Drosophila/embriología , Factores de Crecimiento de Fibroblastos/metabolismo , Fibras Musculares Esqueléticas/metabolismo , Proteínas de Unión al GTP rac/metabolismo , Citoesqueleto de Actina/metabolismo , Animales , Animales Modificados Genéticamente , Drosophila/genética , Drosophila/metabolismo , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Ectodermo/metabolismo , Femenino , Factores de Crecimiento de Fibroblastos/genética , Ligandos , Masculino , Desarrollo Musculoesquelético/genética , Proteínas Tirosina Quinasas/genética , Proteínas Tirosina Quinasas/metabolismo , Receptores de Factores de Crecimiento de Fibroblastos/genética , Receptores de Factores de Crecimiento de Fibroblastos/metabolismo , Transducción de Señal/genética , Proteínas de Unión al GTP rac/genética , Proteínas de Unión al GTP rho/metabolismo

RESUMEN

Interphase chromatin is organized into topologically associating domains (TADs). Within TADs, chromatin looping interactions are formed between DNA regulatory elements, but their functional importance for the establishment of the 3D genome organization and gene regulation during development is unclear. Using high-resolution Hi-C experiments, we analyze higher order 3D chromatin organization during Drosophila embryogenesis and identify active and repressive chromatin loops that are established with different kinetics and depend on distinct factors: Zelda-dependent active loops are formed before the midblastula transition between transcribed genes over long distances. Repressive loops within polycomb domains are formed after the midblastula transition between polycomb response elements by the action of GAGA factor and polycomb proteins. Perturbation of PRE function by CRISPR/Cas9 genome engineering affects polycomb domain formation and destabilizes polycomb-mediated silencing. Preventing loop formation without removal of polycomb components also decreases silencing efficiency, suggesting that chromatin architecture can play instructive roles in gene regulation during development. VIDEO ABSTRACT.

Asunto(s)

Cromatina/metabolismo , Proteínas de Drosophila/metabolismo , Silenciador del Gen , Proteínas del Grupo Polycomb/metabolismo , Animales , Sistemas CRISPR-Cas , Cromatina/genética , Proteínas de Drosophila/genética , Drosophila melanogaster , Proteínas del Grupo Polycomb/genética

RESUMEN

Cis-regulatory modules (CRMs) are defined by unique combinations of transcription factor-binding sites. Emerging evidence suggests that the number, affinity, and organization of sites play important roles in regulating enhancer output and, ultimately, gene expression. Here, we investigate how the cis-regulatory logic of a tissue-specific CRM responsible for even-skipped (eve) induction during cardiogenesis organizes the competing inputs of two E-twenty-six (ETS) members: the activator Pointed (Pnt) and the repressor Yan. Using a combination of reporter gene assays and CRISPR-Cas9 gene editing, we suggest that Yan and Pnt have distinct syntax preferences. Not only does Yan prefer high-affinity sites, but an overlapping pair of such sites is necessary and sufficient for Yan to tune Eve expression levels in newly specified cardioblasts and block ectopic Eve induction and cell fate specification in surrounding progenitors. Mechanistically, the efficient Yan recruitment promoted by this high-affinity ETS supersite not only biases Yan-Pnt competition at the specific CRM but also organizes Yan-repressive complexes in three dimensions across the eve locus. Taken together, our results uncover a novel mechanism by which differential interpretation of CRM syntax by a competing repressor-activator pair can confer both specificity and robustness to developmental transitions.

Asunto(s)

Diferenciación Celular/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citología , Drosophila melanogaster/embriología , Proteínas del Ojo/metabolismo , Regulación del Desarrollo de la Expresión Génica/genética , Miocardio/citología , Proteínas Represoras/metabolismo , Animales , Proteínas de Unión al ADN/química , Proteínas de Unión al ADN/genética , Proteínas de Unión al ADN/metabolismo , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Embrión no Mamífero , Elementos de Facilitación Genéticos/genética , Proteínas del Ojo/química , Proteínas del Ojo/genética , Proteínas de Homeodominio/química , Proteínas de Homeodominio/genética , Proteínas de Homeodominio/metabolismo , Modelos Moleculares , Proteínas del Tejido Nervioso/química , Proteínas del Tejido Nervioso/genética , Proteínas del Tejido Nervioso/metabolismo , Organogénesis/genética , Unión Proteica , Estructura Cuaternaria de Proteína , Transporte de Proteínas , Proteínas Proto-Oncogénicas/química , Proteínas Proto-Oncogénicas/genética , Proteínas Proto-Oncogénicas/metabolismo , Proteínas Represoras/química , Proteínas Represoras/genética , Factores de Transcripción/química , Factores de Transcripción/genética , Factores de Transcripción/metabolismo

RESUMEN

BACKGROUND: The transcriptional corepressor Groucho (Gro) is required for the function of many developmentally regulated DNA binding repressors, thus helping to define the gene expression profile of each cell during development. The ability of Gro to repress transcription at a distance together with its ability to oligomerize and bind to histones has led to the suggestion that Gro may spread along chromatin. However, much is unknown about the mechanism of Gro-mediated repression and about the dynamics of Gro targeting. RESULTS: Our chromatin immunoprecipitation sequencing analysis of temporally staged Drosophila embryos shows that Gro binds in a highly dynamic manner primarily to clusters of discrete (<1 kb) segments. Consistent with the idea that Gro may facilitate communication between silencers and promoters, Gro binding is enriched at both cis-regulatory modules, as well as within the promotors of potential target genes. While this Gro-recruitment is required for repression, our data show that it is not sufficient for repression. Integration of Gro binding data with transcriptomic analysis suggests that, contrary to what has been observed for another Gro family member, Drosophila Gro is probably a dedicated repressor. This analysis also allows us to define a set of high confidence Gro repression targets. Using publically available data regarding the physical and genetic interactions between these targets, we are able to place them in the regulatory network controlling development. Through analysis of chromatin associated pre-mRNA levels at these targets, we find that genes regulated by Gro in the embryo are enriched for characteristics of promoter proximal paused RNA polymerase II. CONCLUSIONS: Our findings are inconsistent with a one-dimensional spreading model for long-range repression and suggest that Gro-mediated repression must be regulated at a post-recruitment step. They also show that Gro is likely a dedicated repressor that sits at a prominent highly interconnected regulatory hub in the developmental network. Furthermore, our findings suggest a role for RNA polymerase II pausing in Gro-mediated repression.

Asunto(s)

Factores de Transcripción con Motivo Hélice-Asa-Hélice Básico/metabolismo , Regulación del Desarrollo de la Expresión Génica , Genómica , Proteínas Represoras/metabolismo , Animales , Cromatina/metabolismo , Drosophila melanogaster/embriología , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Embrión no Mamífero/metabolismo , Unión Proteica

RESUMEN

Gastrulation of the Drosophila embryo is one of the most intensively studied morphogenetic processes in animal development [1-4]. Particular efforts have focused on the formation of the ventral furrow, whereby ∼1,000 presumptive mesoderm cells exhibit coordinated apical constrictions that mediate invagination [5, 6]. Apical constriction depends on a Rho GTPase signaling pathway (T48/Fog) that is deployed by the developmental regulatory genes twist and snail [7-10]. It is thought that coordinate mesoderm constriction depends on high levels of myosin along the ventral midline, although the basis for this localization is uncertain. Here, we employ newly developed quantitative imaging methods to visualize the transcriptional dynamics of two key components of the Rho signaling pathway in living embryos, T48 and Fog. Both genes display dorsoventral (DV) gradients of expression due to differential timing of transcription activation. Transcription begins as a narrow stripe of two or three cells along the ventral midline, followed by progressive expansions into more lateral regions. Quantitative image analyses suggest that these temporal gradients produce differential spatial accumulations of t48 and fog mRNAs along the DV axis, similar to the distribution of myosin activity. We therefore propose that the transcriptional dynamics of t48 and fog expression foreshadow the coordinated invagination of the mesoderm at the onset of gastrulation.

Asunto(s)

Tipificación del Cuerpo , Drosophila melanogaster/crecimiento & desarrollo , Drosophila melanogaster/genética , Elementos de Facilitación Genéticos , Gastrulación , Animales , Proteínas de Drosophila/genética , Embrión no Mamífero/citología , Embrión no Mamífero/metabolismo , Regulación del Desarrollo de la Expresión Génica , Proteínas de la Membrana/genética , Mesodermo , Morfogénesis , Mutación , Transducción de Señal

RESUMEN

How the shape and size of tissues and organs is regulated during development is a major question in developmental biology. Such regulation relies upon both intrinsic cues (such as signaling networks) and extrinsic inputs (such as from neighboring tissues). Here, we focus on pattern formation and organ development during Drosophila embryogenesis. In particular, we outline the importance of both biochemical and mechanical tissue-tissue interactions in size regulation. We describe how the Drosophila embryo can potentially provide novel insights into how shape and size are regulated during development. We focus on gene expression boundary scaling in the early embryo and how size is regulated in three organs (hindgut, trachea, and ventral nerve cord) later in development, with particular focus on the role of tissue-tissue interactions. Overall, we demonstrate that Drosophila embryogenesis provides a suitable model system for studying spatial and temporal scaling and size control in vivo.

Asunto(s)

Embrión no Mamífero/embriología , Desarrollo Embrionario/fisiología , Regulación del Desarrollo de la Expresión Génica/fisiología , Animales , Drosophila , Embrión no Mamífero/citología , Tamaño de los Órganos/fisiología

RESUMEN

Because maturing oocytes and early embryos lack appreciable transcription, posttranscriptional regulatory processes control their development. To better understand this control, we profiled translational efficiencies and poly(A)-tail lengths throughout Drosophila oocyte maturation and early embryonic development. The correspondence between translational-efficiency changes and tail-length changes indicated that tail-length changes broadly regulate translation until gastrulation, when this coupling disappears. During egg activation, relative changes in poly(A)-tail length, and thus translational efficiency, were largely retained in the absence of cytoplasmic polyadenylation, which indicated that selective poly(A)-tail shortening primarily specifies these changes. Many translational changes depended on PAN GU and Smaug, and these changes were largely attributable to tail-length changes. Our results also revealed the presence of tail-length-independent mechanisms that maintained translation despite tail-length shortening during oocyte maturation, and prevented essentially all translation of bicoid and several other mRNAs before egg activation. In addition to these fundamental insights, our results provide valuable resources for future studies.

Asunto(s)

Drosophila/embriología , Oocitos/metabolismo , Biosíntesis de Proteínas , ARN Mensajero/metabolismo , Animales , Regulación de la Expresión Génica

RESUMEN

The class I phosphoinositide 3-kinase (PI3K) can be activated by a large variety of extracellular stimuli and is responsible for generating phosphatidylinositol-3,4,5-trisphosphate (PI(3,4,5)P(3)) from phosphatidylinositol-4,5-bisphosphate at the plasma membrane. The expression pattern of the class I PI3K and distribution of PI(3,4,5)P(3), visualized by its specific binding protein, GRP1-PH, were examined during Drosophila embryogenesis. We found that the RNA of Pi3K21B, encoding the Drosophila p60 regulatory subunit of the class I PI3Ks, was expressed maternally and expressed primarily in pole cells after cellularization until completion of germ band elongation. The RNA of Pi3K92E, encoding the Drosophila p110 catalytic subunit of the class I PI3Ks, was also expressed maternally. During gastrulation, its transcript level became lower and was slightly enriched in invaginating cells. Both Pi3K21B and Pi3K92E were expressed ubiquitously after germ band elongation and persisted during germ band shortening. PI(3,4,5)P(3) was distributed at the apical region of the invaginating cells during gastrulation. These findings suggest a possible involvement of class I PI3K and PI(3,4,5)P(3) in the regulation of invagination during gastrulation.

Asunto(s)

Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriología , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Fosfatos de Inositol/química , Fosfatidilinositol 3-Quinasas/metabolismo , Actinas/metabolismo , Animales , Animales Modificados Genéticamente , Dominio Catalítico , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Desarrollo Embrionario , Gástrula/metabolismo , Proteínas Fluorescentes Verdes/metabolismo , Hibridación in Situ , Lípidos/química , Microscopía Confocal , Fosfatidilinositol 3-Quinasas/genética , Transducción de Señal

RESUMEN

The transcription factors of the Snail family are key regulators of epithelial-mesenchymal transitions, cell morphogenesis, and tumor metastasis. Since its discovery in Drosophila ∼25 years ago, Snail has been extensively studied for its role as a transcriptional repressor. Here we demonstrate that Drosophila Snail can positively modulate transcriptional activation. By combining information on in vivo occupancy with expression profiling of hand-selected, staged snail mutant embryos, we identified 106 genes that are potentially directly regulated by Snail during mesoderm development. In addition to the expected Snail-repressed genes, almost 50% of Snail targets showed an unanticipated activation. The majority of "Snail-activated" genes have enhancer elements cobound by Twist and are expressed in the mesoderm at the stages of Snail occupancy. Snail can potentiate Twist-mediated enhancer activation in vitro and is essential for enhancer activity in vivo. Using a machine learning approach, we show that differentially enriched motifs are sufficient to predict Snail's regulatory response. In silico mutagenesis revealed a likely causative motif, which we demonstrate is essential for enhancer activation. Taken together, these data indicate that Snail can potentiate enhancer activation by collaborating with different activators, providing a new mechanism by which Snail regulates development.

Asunto(s)

Drosophila/genética , Drosophila/metabolismo , Factores de Transcripción/metabolismo , Secuencias de Aminoácidos , Animales , Drosophila/embriología , Proteínas de Drosophila/metabolismo , Embrión no Mamífero , Elementos de Facilitación Genéticos/genética , Regulación del Desarrollo de la Expresión Génica , Mesodermo/metabolismo , Unión Proteica , Factores de Transcripción de la Familia Snail , Factores de Transcripción/genética , Proteína 1 Relacionada con Twist/metabolismo

RESUMEN

The spectrum of lectin binding sites as it emerges during embryonic development of Drosophila was analysed by means of fluorescein-labelled lectins. As development and morphogenesis proceed, the reaction pattern becomes more and more complex. Mannose/glucose-, mannose-, N-acetylglucosamine- and poly-N-ace-tylglucosamine-specific lectins bind ubiquitously. Nuclear envelopes only have binding sites for wheat germ agglutinin. N-acetylgalactosamine-binding lectins are specific for ectodermal derivatives. Gaß-3-N-acetylgalac-tosamine-binding lectins are highly selective markers for neural structures, haemocytes and Garland cells. It is also shown that Drosophila laminin is differentially glycosylated. The possible implications of differential and germ layer-specific glycosylation are discussed.

RESUMEN

Our present detailed understanding of the genetic mechanisms controlling segmentation has been made possible, in large part, by comprehensive screens of cuticular morphology that identified genes involved in epidermal patterning. To systematically identify genes involved in internal morphogenesis, specifically development of the gut, we have screened mutant embryos produced by a collection of 53 embryonic lethal mutations affecting embryonic pattern formation or differentiation, and a collection of 161 deficiencies covering, in aggregate, approximately 70% of the genome. Staining with the anti-crumbs antibody was used to characterize the Malpighian tubules and hindgut, as well as other internal organs. The geneshuckebein, tailless andwingless, and two previously undescribed loci at 24C/D and 68D/E, are required to establish the primordia for the posterior midgut and hindgut/Malpighian tubules. A locus in region 30A/C is required for extension of the midgut epithelium to surround the yolk, and region 36E/37F is required for outbudding of the Malpighian tubule primordia. Several deficiencies were identified that uncover loci with specific effects on the morphogenesis (elongation, lumen formation) of the hindgut and Malpighian tubules and on the formation of constrictions in the midgut.

RESUMEN

Genetic controls regulating the establishment of the pharyngeal primordia in the anterior region of the Drosophila embryo were investigated through the analysis of the expression of thecnc gene, which is continuously expressed specificially in three pharyngeal segments. The spatial regulation ofcnc gene transcription was analyzed by in situ hybridization of CNC transcript-specific probes to embryos mutant for other cephalic patterning genes. The anterior domain of CNC expression (corresponding to the labral segment primordium) was found to be activated bybicoid andtorso maternal pathways, independently of known zygotic gap genes, and sequentially constricted to its final size by repression from neighboring region-specific genes. Control of the posterior domain (corresponding to the intercalary and mandibular segment primordia) involved combinatorial regulation by zygotic gap genes: activation by thebtd gap gene and repression from theotd gap gene anteriorly and thesna gene ventrally. Surprisingly, the posterior domain was shifted relative to the segmentation plan in mutants of theems gap gene. These regulatory controls establishing the limits of CNC expression in the pharyngeal primordia suggest that one mechanism for patterning within the anterior terminal region may involve direct activation of region-specific gene(s) by maternal factors over a relatively broad domain followed by constriction of that domain by repression from adjacently activated zygotic genes.

RESUMEN

The developmental patterns of embryos produced by female germ line cells homozygous for null-enzyme mutations of dunce and for dunce in combination with each of three different rutabaga mutations are compared with the normal pattern. At least three discrete developmental defects at progressive stages following fertilization can be identified and correlated with the loss of adenylate cyclase activity caused by rutabaga mutations, suggesting that the defects are caused by elevated cyclic AMP levels in female germ line cells. The earliest defect occurs soon after fertilization and affects DNA replication and mitosis, prevents nuclear migration, and leads to large polyploid nuclei. A later defect prevents cleavage nuclei from migrating into, or dividing in, the posterior region of the egg. The last affects the developmental behavior or fate of blastoderm cells. Some of these defects mimic those produced by previously described maternal-effect mutations.

RESUMEN

In order to investigate the localized requirements for the activity of genes that are required in a Drosophila embryo for segmentation, we have analyzed the patterns in genetic mosaics. In this paper, we describe our results with four different X-chromosome linked segmentation loci: armadillo, fused, giant and unpaired. For each locus, we first describe in detail the cuticle phenotype of mutant embryos. We then describe the segmentation patterns in embryos mosaic for these mutations, in each case utilizing the shavenbaby (svb) larval cuticle marker mutation to identify the regions of pattern made by genetically mutant cells. For all four loci, we can identify embryos containing large regions of both mutant and wildtype pattern. In these mosaics the regions of mutant pattern are marked with svb and the genetically wildtype (svb +) cells make wildtype pattern. The interpretations of the patterns in embryos mosaic for fused and unpaired are complicated by the variability of the phenotypes. However, after taking these complications into account, our principal conclusion is that the requirement for embryonic gene activity seems to be primarily cell autonomous. Based on the descriptions of the mutant phenotypes of these four loci and the analysis of the mosaics, we speculate on the possible roles these genes play in the process of segmentation.