RESUMO
Chromatin loops between gene pairs have been observed in diverse contexts in both flies and vertebrates. Combining high-resolution Capture-C, DNA fluorescence in situ hybridization, and genetic perturbations, we dissect the functional role of three loops between genes with related function during Drosophila embryogenesis. By mutating the loop anchor (but not the gene) or the gene (but not loop anchor), we disentangle loop formation and gene expression and show that the 3D proximity of paralogous gene loci supports their co-regulation. Breaking the loop leads to either an attenuation or enhancement of expression and perturbs their relative levels of expression and cross-regulation. Although many loops appear constitutive across embryogenesis, their function can change in different developmental contexts. Taken together, our results indicate that chromatin gene-gene loops act as architectural scaffolds that can be used in different ways in different contexts to fine-tune the coordinated expression of genes with related functions and sustain their cross-regulation.
Assuntos
Cromatina , Cromossomos , Animais , Hibridização in Situ Fluorescente , Cromatina/genética , Drosophila/genéticaRESUMO
Enhancers control gene expression and have crucial roles in development and homeostasis1-3. However, the targeted de novo design of enhancers with tissue-specific activities has remained challenging. Here we combine deep learning and transfer learning to design tissue-specific enhancers for five tissues in the Drosophila melanogaster embryo: the central nervous system, epidermis, gut, muscle and brain. We first train convolutional neural networks using genome-wide single-cell assay for transposase-accessible chromatin with sequencing (ATAC-seq) datasets and then fine-tune the convolutional neural networks with smaller-scale data from in vivo enhancer activity assays, yielding models with 13% to 76% positive predictive value according to cross-validation. We designed and experimentally assessed 40 synthetic enhancers (8 per tissue) in vivo, of which 31 (78%) were active and 27 (68%) functioned in the target tissue (100% for central nervous system and muscle). The strategy of combining genome-wide and small-scale functional datasets by transfer learning is generally applicable and should enable the design of tissue-, cell type- and cell state-specific enhancers in any system.
Assuntos
Aprendizado Profundo , Drosophila melanogaster , Embrião não Mamífero , Elementos Facilitadores Genéticos , Redes Neurais de Computação , Especificidade de Órgãos , Animais , Cromatina/genética , Cromatina/metabolismo , Conjuntos de Dados como Assunto , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Embrião não Mamífero/embriologia , Embrião não Mamífero/metabolismo , Elementos Facilitadores Genéticos/genética , Especificidade de Órgãos/genética , Reprodutibilidade dos Testes , Análise de Célula Única , Transposases/metabolismo , Biologia Sintética/métodosRESUMO
The T-box transcription factor Tbx1 and the LIM-homeodomain transcription factor Islet1 are key components in regulatory circuits that generate myogenic and cardiogenic lineage diversity in chordates. We show here that Org-1 and Tup, the Drosophila orthologs of Tbx1 and Islet1, are co-expressed and required for formation of the heart-associated alary muscles (AMs) in the abdomen. The same holds true for lineage-related muscles in the thorax that have not been described previously, which we name thoracic alary-related muscles (TARMs). Lineage analyses identified the progenitor cell for each AM and TARM. Three-dimensional high-resolution analyses indicate that AMs and TARMs connect the exoskeleton to the aorta/heart and to different regions of the midgut, respectively, and surround-specific tracheal branches, pointing to an architectural role in the internal anatomy of the larva. Org-1 controls tup expression in the AM/TARM lineage by direct binding to two regulatory sites within an AM/TARM-specific cis-regulatory module, tupAME. The contributions of Org-1 and Tup to the specification of Drosophila AMs and TARMs provide new insights into the transcriptional control of Drosophila larval muscle diversification and highlight new parallels with gene regulatory networks involved in the specification of cardiopharyngeal mesodermal derivatives in chordates.
Assuntos
Músculos Abdominais/citologia , Linhagem da Célula/fisiologia , Proteínas de Drosophila/metabolismo , Drosophila/anatomia & histologia , Regulação da Expressão Gênica no Desenvolvimento/fisiologia , Modelos Anatômicos , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismo , Músculos Abdominais/fisiologia , Animais , Animais Geneticamente Modificados , Imunoprecipitação da Cromatina , Drosophila/genética , Drosophila/fisiologia , Imuno-Histoquímica , Larva/anatomia & histologia , Larva/fisiologia , Imagem com Lapso de Tempo , Vísceras/anatomia & histologiaRESUMO
Members of the T-Box gene family of transcription factors are important players in regulatory circuits that generate myogenic and cardiogenic lineage diversities in vertebrates. We show that during somatic myogenesis in Drosophila, the single ortholog of vertebrate Tbx1, optomotor-blind-related-gene-1 (org-1), is expressed in a small subset of muscle progenitors, founder cells and adult muscle precursors, where it overlaps with the products of the muscle identity genes ladybird (lb) and slouch (slou). In addition, org-1 is expressed in the lineage of the heart-associated alary muscles. org-1 null mutant embryos lack Lb and Slou expression within the muscle lineages that normally co-express org-1. As a consequence, the respective muscle fibers and adult muscle precursors are either severely malformed or missing, as are the alary muscles. To address the mechanisms that mediate these regulatory interactions between Org-1, Lb and Slou, we characterized distinct enhancers associated with somatic muscle expression of lb and slou. We demonstrate that these lineage- and stage-specific cis-regulatory modules (CRMs) bind Org-1 in vivo, respond to org-1 genetically and require T-box domain binding sites for their activation. In summary, we propose that org-1 is a common and direct upstream regulator of slou and lb in the developmental pathway of these two neighboring muscle lineages. Cross-repression between slou and lb and combinatorial activation of lineage-specific targets by Org-1-Slou and Org-1-Lb, respectively, then leads to the distinction between the two lineages. These findings provide new insights into the regulatory circuits that control the proper pattering of the larval somatic musculature in Drosophila.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Regulação da Expressão Gênica no Desenvolvimento , Desenvolvimento Muscular/fisiologia , Proteínas com Domínio T/metabolismo , Animais , Padronização Corporal/fisiologia , Linhagem da Célula , Proteínas de Drosophila/genética , Drosophila melanogaster/anatomia & histologia , Proteínas de Homeodomínio/genética , Proteínas de Homeodomínio/metabolismo , Músculos/citologia , Músculos/embriologia , Músculos/fisiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/metabolismo , Células-Tronco/citologia , Células-Tronco/fisiologia , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismoRESUMO
The T-Box family of transcription factors plays fundamental roles in the generation of appropriate spatial and temporal gene expression profiles during cellular differentiation and organogenesis in animals. In this study we report that the Drosophila Tbx1 orthologue optomotor-blind-related-gene-1 (org-1) exerts a pivotal function in the diversification of circular visceral muscle founder cell identities in Drosophila. In embryos mutant for org-1, the specification of the midgut musculature per se is not affected, but the differentiating midgut fails to form the anterior and central midgut constrictions and lacks the gastric caeca. We demonstrate that this phenotype results from the nearly complete loss of the founder cell specific expression domains of several genes known to regulate midgut morphogenesis, including odd-paired (opa), teashirt (tsh), Ultrabithorax (Ubx), decapentaplegic (dpp) and wingless (wg). To address the mechanisms that mediate the regulatory inputs from org-1 towards Ubx, dpp, and wg in these founder cells we genetically dissected known visceral mesoderm specific cis-regulatory-modules (CRMs) of these genes. The analyses revealed that the activities of the dpp and wg CRMs depend on org-1, the CRMs are bound by Org-1 in vivo and their T-Box binding sites are essential for their activation in the visceral muscle founder cells. We conclude that Org-1 acts within a well-defined signaling and transcriptional network of the trunk visceral mesoderm as a crucial founder cell-specific competence factor, in concert with the general visceral mesodermal factor Biniou. As such, it directly regulates several key genes involved in the establishment of morphogenetic centers along the anteroposterior axis of the visceral mesoderm, which subsequently organize the formation of midgut constrictions and gastric caeca and thereby determine the morphology of the midgut.
Assuntos
Proteínas de Drosophila/genética , Proteínas de Drosophila/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Proteínas com Domínio T/genética , Proteínas com Domínio T/fisiologia , Animais , Padronização Corporal , Diferenciação Celular , Movimento Celular , Drosophila melanogaster , Elementos Facilitadores Genéticos , Genes Reporter , Imuno-Histoquímica/métodos , Mesoderma/metabolismo , Microscopia de Fluorescência/métodos , Modelos Genéticos , Morfogênese , Transdução de Sinais , Transcrição GênicaRESUMO
To regulate expression, enhancers must come in proximity to their target gene. However, the relationship between the timing of enhancer-promoter (E-P) proximity and activity remains unclear, with examples of uncoupled, anticorrelated and correlated interactions. To assess this, we selected 600 characterized enhancers or promoters with tissue-specific activity in Drosophila embryos and performed Capture-C in FACS-purified myogenic or neurogenic cells during specification and tissue differentiation. This enabled direct comparison between E-P proximity and activity transitioning from OFF-to-ON and ON-to-OFF states across developmental conditions. This showed remarkably similar E-P topologies between specified muscle and neuronal cells, which are uncoupled from activity. During tissue differentiation, many new distal interactions emerge where changes in E-P proximity reflect changes in activity. The mode of E-P regulation therefore appears to change as embryogenesis proceeds, from largely permissive topologies during cell-fate specification to more instructive regulation during terminal tissue differentiation, when E-P proximity is coupled to activation.
Assuntos
Elementos Facilitadores Genéticos , Regulação da Expressão Gênica no Desenvolvimento , Animais , Elementos Facilitadores Genéticos/genética , Regulação da Expressão Gênica no Desenvolvimento/genética , Regiões Promotoras Genéticas/genética , Drosophila/genética , Diferenciação Celular/genéticaRESUMO
The basic helix-loop-helix transcription factor twist plays a key role during mesoderm development in Bilateria. In this study, we identified a twist ortholog in the polychaete annelid Platynereis dumerilii and analyze its expression during larval development, postlarval growth up to the adult stage, and caudal regeneration after amputation of posterior segments. At late larval stages, Pdu-twist is expressed in the mesodermal anlagen and in developing muscles. During adulthood and caudal regeneration, Pdu-twist is expressed in the posterior growth zone, in mesodermal cells within the newly forming segments and budding parapodia. Our results indicate that Pdu-twist is involved in mesoderm formation during larval development, posterior growth, and caudal regeneration.
Assuntos
Poliquetos/embriologia , Poliquetos/metabolismo , Proteína 1 Relacionada a Twist/genética , Proteína 1 Relacionada a Twist/metabolismo , Sequência de Aminoácidos , Animais , Clonagem Molecular , Embrião não Mamífero/metabolismo , Regulação da Expressão Gênica no Desenvolvimento , Mesoderma/metabolismo , Dados de Sequência Molecular , Filogenia , Proteína 1 Relacionada a Twist/químicaRESUMO
HLH54F, the Drosophila ortholog of the vertebrate basic helix-loop-helix domain-encoding genes capsulin and musculin, is expressed in the founder cells and developing muscle fibers of the longitudinal midgut muscles. These cells descend from the posterior-most portion of the mesoderm, termed the caudal visceral mesoderm (CVM), and migrate onto the trunk visceral mesoderm prior to undergoing myoblast fusion and muscle fiber formation. We show that HLH54F expression in the CVM is regulated by a combination of terminal patterning genes and snail. We generated HLH54F mutations and show that this gene is crucial for the specification, migration and survival of the CVM cells and the longitudinal midgut muscle founders. HLH54F mutant embryos, larvae, and adults lack all longitudinal midgut muscles, which causes defects in gut morphology and integrity. The function of HLH54F as a direct activator of gene expression is exemplified by our analysis of a CVM-specific enhancer from the Dorsocross locus, which requires combined inputs from HLH54F and Biniou in a feed-forward fashion. We conclude that HLH54F is the earliest specific regulator of CVM development and that it plays a pivotal role in all major aspects of development and differentiation of this largely twist-independent population of mesodermal cells.
Assuntos
Fatores de Transcrição Hélice-Alça-Hélice Básicos/metabolismo , Movimento Celular , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/embriologia , Drosophila melanogaster/metabolismo , Mesoderma/metabolismo , Músculo Esquelético/embriologia , Músculo Esquelético/metabolismo , Sequência de Aminoácidos , Animais , Fatores de Transcrição Hélice-Alça-Hélice Básicos/química , Fatores de Transcrição Hélice-Alça-Hélice Básicos/genética , Padronização Corporal , Diferenciação Celular , Proteínas de Drosophila/química , Proteínas de Drosophila/genética , Drosophila melanogaster/citologia , Drosophila melanogaster/genética , Mesoderma/citologia , Dados de Sequência Molecular , Músculo Esquelético/citologia , Filogenia , Alinhamento de SequênciaRESUMO
Genesis of syncytial muscles is typically considered as a paradigm for an irreversible developmental process. Notably, transdifferentiation of syncytial muscles is naturally occurring during Drosophila development. The ventral longitudinal heart-associated musculature (VLM) arises by a unique mechanism that revokes differentiation states of so-called alary muscles and comprises at least two distinct steps: syncytial muscle cell fragmentation into single myoblasts and successive reprogramming into founder cells that orchestrate de novo fiber formation of the VLM lineage. Here, we provide evidence that the mesodermal master regulator twist plays a key role during this reprogramming process. Acting downstream of Drosophila Tbx1 (Org-1), Twist is regulating the activity of the Hippo pathway effector Yorkie and is required for the initiation of syncytial muscle dedifferentiation and fragmentation. Subsequently, fibroblast growth factor receptor (FGFR)-Ras-mitogen-activated protein kinase (MAPK) signaling in resulting mononucleated myoblasts maintains Twist expression, thereby stabilizing nuclear Yorkie activity and inducing their lineage switch into founder cells of the VLM.
Assuntos
Reprogramação Celular/fisiologia , Proteínas de Drosophila/metabolismo , Coração/embriologia , Miocárdio/citologia , Proteína 1 Relacionada a Twist/metabolismo , Proteínas de Sinalização YAP/metabolismo , Animais , Linhagem da Célula/fisiologia , Transdiferenciação Celular/fisiologia , Drosophila melanogasterRESUMO
Drosophila melanogaster is a powerful, long-standing model for metazoan development and gene regulation. We profiled chromatin accessibility in almost 1 million and gene expression in half a million nuclei from overlapping windows spanning the entirety of embryogenesis. Leveraging developmental asynchronicity within embryo collections, we applied deep neural networks to infer the age of each nucleus, resulting in continuous, multimodal views of molecular and cellular transitions in absolute time. We identify cell lineages; infer their developmental relationships; and link dynamic changes in enhancer usage, transcription factor (TF) expression, and the accessibility of TFs' cognate motifs. With these data, the dynamics of enhancer usage and gene expression can be explored within and across lineages at the scale of minutes, including for precise transitions like zygotic genome activation.
Assuntos
Proteínas de Drosophila , Drosophila melanogaster , Desenvolvimento Embrionário , Regulação da Expressão Gênica no Desenvolvimento , Animais , Linhagem da Célula/genética , Proteínas de Drosophila/genética , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/embriologia , Drosophila melanogaster/genética , Desenvolvimento Embrionário/genética , Elementos Facilitadores Genéticos , Redes Neurais de Computação , Análise de Célula ÚnicaRESUMO
Early lineage-specific master regulators are essential for the specification of cell types. However, once cells are committed to a specific fate, it is critical to restrict the activity of such factors to enable differentiation. To date, it remains unclear how these factors are silenced. Using the Drosophila mesoderm as a model and a comparative genomic approach, we identify the Hox transcription factor Ultrabithorax (Ubx) to be critical for the repression of the master regulator Twist. Mesoderm-specific Ubx loss-of-function experiments using CRISPR-Cas9 and overexpression studies demonstrate that Ubx majorly impacts twist transcription. A mechanistic analysis reveals that Ubx requires the NK-homeodomain protein Tinman to bind to the twist promoter. Furthermore, we find these factor interactions to be critical for silencing by recruiting the Polycomb DNA binding protein Pleiohomeotic. Altogether, our data reveal that Ubx is a critical player in mediating the silencing of Twist, which is crucial for coordinated muscle differentiation.
Assuntos
Proteínas de Drosophila/metabolismo , Drosophila melanogaster/metabolismo , Proteínas de Homeodomínio/metabolismo , Mesoderma/metabolismo , Desenvolvimento Muscular , Fatores de Transcrição/metabolismo , Proteína 1 Relacionada a Twist/metabolismo , Animais , Proteínas de Drosophila/genética , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Regulação da Expressão Gênica no Desenvolvimento , Proteínas de Homeodomínio/genética , Proteínas do Grupo Polycomb/metabolismo , Regiões Promotoras Genéticas , Fatores de Transcrição/genética , Proteína 1 Relacionada a Twist/genéticaRESUMO
Lineage reprogramming has received increased research attention since it was demonstrated that lineage-restricted transcription factors can be used in vitro for direct reprogramming. Recently, we reported that the ventral longitudinal musculature of the adult Drosophila heart arises in vivo by direct lineage reprogramming from larval alary muscles, a process that starts with the dedifferentiation and fragmentation of syncytial muscle cells into mononucleate myoblasts and depends on Org-1 (Drosophila Tbx1). Here, we shed light on the events occurring downstream of Org-1 in this first step of transdifferentiation and show that alary muscle lineage-specific activation of Yorkie plays a key role in initiating the dedifferentiation and fragmentation of these muscles. An additional necessary input comes from active dJNK signaling, which contributes to the activation of Yorkie and furthermore activates dJun. The synergistic activities of the Yorkie/Scalloped and dJun/dFos transcriptional activators subsequently initiate alary muscle fragmentation as well as up-regulation of Myc and piwi, both crucial for lineage reprogramming.
Assuntos
Linhagem da Célula , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/citologia , Drosophila melanogaster/metabolismo , MAP Quinase Quinase 4/metabolismo , Músculos/citologia , Mioblastos/citologia , Proteínas Nucleares/metabolismo , Proteínas com Domínio T/metabolismo , Transativadores/metabolismo , Animais , Músculos/metabolismo , Mioblastos/metabolismo , Proteínas de Sinalização YAPRESUMO
Although muscle development has been widely studied in Drosophila melanogaster there are still many gaps in our knowledge, and it is not known to which extent this knowledge can be transferred to other insects. To help in closing these gaps we participated in a large-scale RNAi screen that used the red flour beetle, Tribolium castaneum, as a screening platform. The effects of systemic RNAi were screened upon double-stranded RNA injections into appropriate muscle-EGFP tester strains. Injections into pupae were followed by the analysis of the late embryonic/early larval muscle patterns, and injections into larvae by the analysis of the adult thoracic muscle patterns. Herein we describe the results of the first-pass screens with pupal and larval injections, which covered â¼8,500 and â¼5,000 genes, respectively, of a total of â¼16,500 genes of the Tribolium genome. Apart from many genes known from Drosophila as regulators of muscle development, a collection of genes previously unconnected to muscle development yielded phenotypes in larval body wall and leg muscles as well as in indirect flight muscles. We then present the main candidates from the pupal injection screen that remained after being processed through a series of verification and selection steps. Further, we discuss why distinct though overlapping sets of genes are revealed by the Drosophila and Tribolium screening approaches.
Assuntos
Genes de Insetos , Desenvolvimento Muscular/genética , Tribolium/genética , Animais , Clonagem Molecular , Genoma de Inseto , Interferência de RNA , Tribolium/crescimento & desenvolvimentoRESUMO
Only few examples of transdifferentiation, which denotes the conversion of one differentiated cell type to another, are known to occur during normal development, and more often, it is associated with regeneration processes. With respect to muscles, dedifferentiation/redifferentiation processes have been documented during post-traumatic muscle regeneration in blastema of newts as well as during myocardial regeneration. As shown herein, the ventral longitudinal muscles of the adult Drosophila heart arise from specific larval alary muscles in a process that represents the first known example of syncytial muscle transdifferentiation via dedifferentiation into mononucleate myoblasts during normal development. We demonstrate that this unique process depends on the reinitiation of a transcriptional program previously employed for embryonic alary muscle development, in which the factors Org-1 (Drosophila Tbx1) and Tailup (Drosophila Islet1) are key components. During metamorphosis, the action of these factors is combined with cell-autonomous inputs from the ecdysone steroid and the Hox gene Ultrabithorax, which provide temporal and spatial specificity to the transdifferentiation events. Following muscle dedifferentiation, inductive cues, particularly from the remodeling heart tube, are required for the redifferentiation of myoblasts into ventral longitudinal muscles. Our results provide new insights into mechanisms of lineage commitment and cell-fate plasticity during development.
Assuntos
Transdiferenciação Celular , Proteínas de Drosophila/genética , Drosophila melanogaster/fisiologia , Regulação da Expressão Gênica no Desenvolvimento , Metamorfose Biológica , Proteínas com Domínio T/genética , Fatores de Transcrição/genética , Animais , Padronização Corporal , Proteínas de Drosophila/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/crescimento & desenvolvimento , Coração/crescimento & desenvolvimento , Larva/genética , Larva/crescimento & desenvolvimento , Larva/fisiologia , Desenvolvimento Muscular , Pupa/genética , Pupa/crescimento & desenvolvimento , Pupa/fisiologia , Proteínas com Domínio T/metabolismo , Fatores de Transcrição/metabolismoRESUMO
A 200- to 1000-fold higher affinity for sialyltransferase is shown by compounds 1 and 2 relative to the natural substrate. These inhibitors, which are derived from the transition state of SN 1-type sialyltransfer, contain a flat ring that is attached through a carbon atom with a phosphonate and a cytidine monophosphate group.
RESUMO
In this study, we aimed to identify molecular mechanisms involved in the specification of the 4d (mesentoblast) lineage in Platynereis dumerilii. We employ RT-PCR and in situ hybridization against the Platynereis dumerilii twist homolog (Pdu-twist) to reveal mesodermal specification within this lineage. We show that Pdu-twist mRNA is already maternally distributed. After fertilization, ooplasmatic segregation leads to relocation of Pdu-twist transcripts into the somatoblast (2d) lineage and 4d, indicating that the maternal component of Pdu-twist might be an important prerequisite for further mesoderm specification but does not represent a defining characteristic of the mesentoblast. However, after the primordial germ cells have separated from the 4d lineage, zygotic transcription of Pdu-twist is exclusively observed in the myogenic progenitors, suggesting that mesodermal specification occurs after the 4d stage. Previous studies on spiral cleaving embryos revealed a spatio-temporal correlation between the 4d lineage and the activity of an embryonic organizer that is capable to induce the developmental fates of certain micromeres. This has raised the question if specification of the 4d lineage could be connected to the organizer activity. Therefore, we aimed to reveal the existence of such a proposed conserved organizer in Platynereis employing antibody staining against dpERK. In contrast to former observations in other spiralian embryos, activation of MAPK signaling during 2d and 4d formation cannot be detected which questions the existence of a conserved connection between organizer function and specification of the 4d lineage. However, our experiments unveil robust MAPK activation in the prospective nephroblasts as well as in the macromeres and some micromeres at the blastopore in gastrulating embryos. Inhibition of MAPK activation leads to larvae with a shortened body axis, defects in trunk muscle spreading and improper nervous system condensation, indicating a critical function for MAPK signaling for the reorganization of embryonic tissues during the gastrulation process.