ABSTRACT
SLC30A9 (ZnT9) is a mitochondria-resident zinc transporter. Mutations in SLC30A9 have been reported in human patients with a novel cerebro-renal syndrome. Here, we show that ZnT9 is an evolutionarily highly conserved protein, with many regions extremely preserved among evolutionarily distant organisms. In Drosophila melanogaster (the fly), ZnT9 (ZnT49B) knockdown results in acutely impaired movement and drastic mitochondrial deformation. Severe Drosophila ZnT9 (dZnT9) reduction and ZnT9-null mutant flies are pupal lethal. The phenotype of dZnT9 knockdown can be partially rescued by mouse ZnT9 expression or zinc chelator TPEN, indicating the defect of dZnT9 loss is indeed a result of zinc dyshomeostasis. Interestingly, in the mouse, germline loss of Znt9 produces even more extreme phenotypes: the mutant embryos exhibit midgestational lethality with severe development abnormalities. Targeted mutagenesis of Znt9 in the mouse brain leads to serious dwarfism and physical incapacitation, followed by death shortly. Strikingly, the GH/IGF-1 signals are almost non-existent in these tissue-specific knockout mice, consistent with the medical finding in some human patients with severe mitochondrial deficiecny. ZnT9 mutations cause mitochondrial zinc dyshomeostasis, and we demonstrate mechanistically that mitochondrial zinc elevation quickly and potently inhibits the activities of respiration complexes. These results reveal the critical role of ZnT9 and mitochondrial zinc homeostasis in mammalian development. Based on our functional analyses, we finally discussed the possible nature of the so far identified human SLC30A9 mutations.
Subject(s)
Cation Transport Proteins , Embryonic Development , Mitochondria , Zinc , Animals , Cation Transport Proteins/metabolism , Cation Transport Proteins/genetics , Humans , Zinc/metabolism , Mice , Mitochondria/metabolism , Embryonic Development/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Evolution, Molecular , Mice, Knockout , Amino Acid Sequence , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Transcription Factors , Cell Cycle ProteinsABSTRACT
Tumor suppressor genes play critical roles in normal tissue homeostasis, and their dysregulation underlies human diseases including cancer. Besides human genetics, model organisms such as Drosophila have been instrumental in discovering tumor suppressor pathways that were subsequently shown to be highly relevant in human cancer. Here we show that hyperplastic disc (Hyd), one of the first tumor suppressors isolated genetically in Drosophila and encoding an E3 ubiquitin ligase with hitherto unknown substrates, and Lines (Lin), best known for its role in embryonic segmentation, define an obligatory tumor suppressor protein complex (Hyd-Lin) that targets the zinc finger-containing oncoprotein Bowl for ubiquitin-mediated degradation, with Lin functioning as a substrate adaptor to recruit Bowl to Hyd for ubiquitination. Interestingly, the activity of the Hyd-Lin complex is directly inhibited by a micropeptide encoded by another zinc finger gene, drumstick (drm), which functions as a pseudosubstrate by displacing Bowl from the Hyd-Lin complex, thus stabilizing Bowl. We further identify the epigenetic regulator Polycomb repressive complex1 (PRC1) as a critical upstream regulator of the Hyd-Lin-Bowl pathway by directly repressing the transcription of the micropeptide drm Consistent with these molecular studies, we show that genetic inactivation of Hyd, Lin, or PRC1 resulted in Bowl-dependent hyperplastic tissue overgrowth in vivo. We also provide evidence that the mammalian homologs of Hyd (UBR5, known to be recurrently dysregulated in various human cancers), Lin (LINS1), and Bowl (OSR1/2) constitute an analogous protein degradation pathway in human cells, and that OSR2 promotes prostate cancer tumorigenesis. Altogether, these findings define a previously unrecognized tumor suppressor pathway that links epigenetic program to regulated protein degradation in tissue growth control and tumorigenesis.
Subject(s)
Carcinogenesis , Drosophila Proteins , Proteolysis , Ubiquitin-Protein Ligases , Animals , Ubiquitin-Protein Ligases/metabolism , Ubiquitin-Protein Ligases/genetics , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Carcinogenesis/genetics , Humans , Tumor Suppressor Proteins/metabolism , Tumor Suppressor Proteins/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/embryology , Genes, Tumor Suppressor , Ubiquitination , Polycomb-Group Proteins/metabolism , Polycomb-Group Proteins/genetics , Polycomb Repressive Complex 1/metabolism , Polycomb Repressive Complex 1/geneticsABSTRACT
In organisms with complex life cycles, life stages that are most susceptible to environmental stress may determine species persistence in the face of climate change. Early embryos of Drosophila melanogaster are particularly sensitive to acute heat stress, yet tropical embryos have higher heat tolerance than temperate embryos, suggesting adaptive variation in embryonic heat tolerance. We compared transcriptomic responses to heat stress among tropical and temperate embryos to elucidate the gene regulatory basis of divergence in embryonic heat tolerance. The transcriptomes of tropical and temperate embryos differed in both constitutive and heat-stress-induced responses of the expression of relatively few genes, including genes involved in oxidative stress. Most of the transcriptomic response to heat stress was shared among all embryos. Embryos shifted the expression of thousands of genes, including increases in the expression of heat shock genes, suggesting robust zygotic gene activation and demonstrating that, contrary to previous reports, early embryos are not transcriptionally silent. The involvement of oxidative stress genes corroborates recent reports on the critical role of redox homeostasis in coordinating developmental transitions. By characterizing adaptive variation in the transcriptomic basis of embryonic heat tolerance, this study is a novel contribution to the literature on developmental physiology and developmental genetics.
Subject(s)
Drosophila melanogaster , Embryo, Nonmammalian , Oxidative Stress , Thermotolerance , Animals , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/physiology , Embryo, Nonmammalian/metabolism , Transcriptome , Heat-Shock Response , Gene Expression Regulation, DevelopmentalABSTRACT
Transcription in developing metazoans is inherently stochastic, involving transient and dynamic interactions among transcriptional machinery. A fundamental challenge with traditional techniques, including fixed-tissue protein and RNA staining, is the lack of temporal resolution. Quantifying kinetic changes in transcription can elucidate underlying mechanisms of interaction among regulatory modules. In this protocol, we describe the successful implementation of a combination of MS2/MCP and PP7/PCP systems in living Drosophila embryos to further our understanding of transcriptional dynamics during development. Our technique can be extended to visualize transcriptional activities of multiple genes or alleles simultaneously, characterize allele-specific expression of a target gene, and quantitatively analyze RNA polymerase II activity in a single-cell resolution.
Subject(s)
Embryonic Development , Gene Expression Regulation, Developmental , Animals , Embryonic Development/genetics , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Embryo, Nonmammalian/metabolism , Drosophila/embryology , Drosophila/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Transcription, Genetic , Drosophila Proteins/genetics , Drosophila Proteins/metabolismABSTRACT
Microfluidic devices support developmental and mechanobiology studies by enabling the precise control of electrical, chemical, and mechanical stimuli at the microscale. Here, we describe the fabrication of customizable microfluidic devices and demonstrate their efficacy in applying mechanical loads to micro-organs and whole organisms, such as Drosophila embryos. The fabrication technique consists in the use of xurography to define channels and chambers using thin layers of thermoplastics and glass. The superposition of layers followed by thermal lamination produces robust and reproducible devices that are easily adapted for a variety of experiments. The integration of deformable layers and glass in these devices facilitates the imaging of cellular and molecular dynamics in biological specimens under mechanical loads. The method is highly adaptable for studies in mechanobiology.
Subject(s)
Embryo, Nonmammalian , Lab-On-A-Chip Devices , Animals , Drosophila/embryology , Biomechanical Phenomena , Stress, Mechanical , Drosophila melanogaster/embryology , Microfluidic Analytical Techniques/instrumentation , Microfluidic Analytical Techniques/methods , Equipment DesignABSTRACT
Transport of macromolecules through the nuclear envelope (NE) is mediated by nuclear pore complexes (NPCs) consisting of nucleoporins (Nups). Elys/Mel-28 is the Nup that binds and connects the decondensing chromatin with the reassembled NPCs at the end of mitosis. Whether Elys links chromatin with the NE during interphase is unknown. Here, using DamID-seq, we identified Elys binding sites in Drosophila late embryos and divided them into those associated with nucleoplasmic or with NPC-linked Elys. These Elys binding sites are located within active or inactive chromatin, respectively. Strikingly, Elys knockdown in S2 cells results in peripheral chromatin displacement from the NE, in decondensation of NE-attached chromatin, and in derepression of genes within. It also leads to slightly more compact active chromatin regions. Our findings indicate that NPC-linked Elys, together with the nuclear lamina, anchors peripheral chromatin to the NE, whereas nucleoplasmic Elys decompacts active chromatin.
Subject(s)
Chromatin , Drosophila Proteins , Interphase , Nuclear Pore Complex Proteins , Nuclear Pore , Animals , Binding Sites , Cell Nucleus/metabolism , Chromatin/metabolism , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Nuclear Pore/metabolism , Nuclear Pore Complex Proteins/metabolism , Nuclear Pore Complex Proteins/geneticsABSTRACT
Embryonic germ cells develop rapidly to establish the foundation for future developmental trajectories, and in this process, they make critical lineage choices including the configuration of their unique identity and a decision on sex. Here, we use single-cell genomics patterns for the entire embryonic germline in Drosophila melanogaster along with the somatic gonadal precursors after embryonic gonad coalescence to investigate molecular mechanisms involved in the setting up and regulation of the germline program. Profiling of the early germline chromatin landscape revealed sex- and stage-specific features. In the male germline immediately after zygotic activation, the chromatin structure underwent a brief remodeling phase during which nucleosome density was lower and deconcentrated from promoter regions. These findings echoed enrichment analysis results of our genomics data in which top candidates were factors with the ability to mediate large-scale chromatin reorganization. Together, they point to the importance of chromatin regulation in the early germline and raise the possibility of a conserved epigenetic reprogramming-like process required for proper initiation of germline development.
Subject(s)
Chromatin Assembly and Disassembly , Chromatin , Drosophila melanogaster , Embryonic Development , Animals , Male , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Chromatin/metabolism , Chromatin/genetics , Chromatin Assembly and Disassembly/genetics , Embryonic Development/genetics , Gene Expression Regulation, Developmental/genetics , Embryonic Germ Cells/metabolism , Embryonic Germ Cells/cytology , Germ Cells/metabolism , Epigenesis, Genetic , Female , Nucleosomes/metabolism , Nucleosomes/genetics , Single-Cell Analysis/methodsABSTRACT
Transcription initiates at the core promoter, which contains distinct core promoter elements. Here, we highlight the complexity of transcriptional regulation by outlining the effect of core promoter-dependent regulation on embryonic development and the proper function of an organism. We demonstrate in vivo the importance of the downstream core promoter element (DPE) in complex heart formation in Drosophila. Pioneering a novel approach using both CRISPR and nascent transcriptomics, we show the effects of mutating a single core promoter element within the natural context. Specifically, we targeted the downstream core promoter element (DPE) of the endogenous tin gene, encoding the Tinman transcription factor, a homologue of human NKX2-5 associated with congenital heart diseases. The 7â bp substitution mutation results in massive perturbation of the Tinman regulatory network that orchestrates dorsal musculature, which is manifested as physiological and anatomical changes in the cardiac system, impaired specific activity features, and significantly compromised viability of adult flies. Thus, a single motif can have a critical impact on embryogenesis and, in the case of DPE, functional heart formation.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Gene Expression Regulation, Developmental , Heart , Promoter Regions, Genetic , Transcription Factors , Animals , Promoter Regions, Genetic/genetics , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Heart/embryology , Transcription Factors/metabolism , Transcription Factors/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Homeobox Protein Nkx-2.5/genetics , Homeobox Protein Nkx-2.5/metabolism , Mutation/genetics , Embryonic Development/genetics , Humans , Transcription, Genetic , Repressor Proteins , Trans-ActivatorsABSTRACT
As development varies greatly across the tree of life, it may seem difficult to suggest a model that proposes a single mechanism for understanding collective cell behaviors and the coordination of tissue formation. Here we propose a mechanism called differentiation waves, which unify many disparate results involving developmental systems from across the tree of life. We demonstrate how a relatively simple model of differentiation proceeds not from function-related molecular mechanisms, but from so-called differentiation waves. A phenotypic model of differentiation waves is introduced, and its relation to molecular mechanisms is proposed. These waves contribute to a differentiation tree, which is an alternate way of viewing cell lineage and local action of the molecular factors. We construct a model of differentiation wave-related molecular mechanisms (genome, epigenome, and proteome) based on bioinformatic data from the nematode Caenorhabditis elegans. To validate this approach across different modes of development, we evaluate protein expression across different types of development by comparing Caenorhabditis elegans with several model organisms: fruit flies (Drosophila melanogaster), yeast (Saccharomyces cerevisiae), and mouse (Mus musculus). Inspired by gene regulatory networks, two Models of Interactive Contributions (fully-connected MICs and ordered MICs) are used to suggest potential genomic contributions to differentiation wave-related proteins. This, in turn, provides a framework for understanding differentiation and development.
Subject(s)
Caenorhabditis elegans , Cell Differentiation , Drosophila melanogaster , Embryonic Development , Animals , Cell Differentiation/genetics , Caenorhabditis elegans/genetics , Caenorhabditis elegans/embryology , Embryonic Development/genetics , Embryonic Development/physiology , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Mice , Gene Regulatory Networks , Saccharomyces cerevisiae/genetics , Models, Biological , Gene Expression Regulation, Developmental , Computational Biology/methodsABSTRACT
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.
Subject(s)
Adherens Junctions , Drosophila Proteins , Embryonic Development , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Adherens Junctions/metabolism , Microtubules/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/metabolism , Embryo, Nonmammalian/cytology , alpha Catenin/metabolism , Actomyosin/metabolism , Morphogenesis , Drosophila/embryology , Cell Shape , Intracellular Signaling Peptides and ProteinsABSTRACT
The transition from oocyte to embryo requires translation of maternally provided transcripts that in Drosophila is activated by Pan Gu kinase to release a rapid succession of 13 mitotic cycles. Mitotic entry is promoted by several protein kinases that include Greatwall/Mastl, whose Endosulfine substrates antagonize Protein Phosphatase 2A (PP2A), facilitating mitotic Cyclin-dependent kinase 1/Cyclin B kinase activity. Here we show that hyperactive greatwallScant can not only be suppressed by mutants in its Endos substrate but also by mutants in Pan Gu kinase subunits. Conversely, mutants in me31B or trailer hitch, which encode a complex that represses hundreds of maternal mRNAs, enhance greatwallScant . Me31B and Trailer Hitch proteins, known substrates of Pan Gu kinase, copurify with Endos. This echoes findings that budding yeast Dhh1, orthologue of Me31B, associates with Igo1/2, orthologues of Endos and substrates of the Rim15, orthologue of Greatwall. endos-derived mutant embryos show reduced Me31B and elevated transcripts for the mitotic activators Cyclin B, Polo and Twine/Cdc25. Together, our findings demonstrate a previously unappreciated conservation of the Greatwall-Endosulfine pathway in regulating translational repressors and its interactions with the Pan Gu kinase pathway to regulate translation and/or stability of maternal mRNAs upon egg activation.
Subject(s)
Drosophila Proteins , Gene Expression Regulation, Developmental , Oocytes , Protein Phosphatase 2 , Animals , Female , DEAD-box RNA Helicases , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Embryo, Nonmammalian/metabolism , Mutation , Oocytes/metabolism , Oocytes/cytology , Protein Biosynthesis , Protein Phosphatase 2/metabolism , Protein Phosphatase 2/genetics , Protein Serine-Threonine Kinases/metabolism , Protein Serine-Threonine Kinases/genetics , RNA Stability , RNA, Messenger, Stored/metabolism , RNA, Messenger, Stored/geneticsABSTRACT
Constitutive heterochromatin is essential for transcriptional silencing and genome integrity. The establishment of constitutive heterochromatin in early embryos and its role in early fruitfly development are unknown. Lysine 9 trimethylation of histone H3 (H3K9me3) and recruitment of its epigenetic reader, heterochromatin protein 1a (HP1a), are hallmarks of constitutive heterochromatin. Here, we show that H3K9me3 is transmitted from the maternal germline to the next generation. Maternally inherited H3K9me3, and the histone methyltransferases (HMT) depositing it, are required for the organization of constitutive heterochromatin: early embryos lacking H3K9 methylation display de-condensation of pericentromeric regions, centromere-centromere de-clustering, mitotic defects, and nuclear shape irregularities, resulting in embryo lethality. Unexpectedly, quantitative CUT&Tag and 4D microscopy measurements of HP1a coupled with biophysical modeling revealed that H3K9me2/3 is largely dispensable for HP1a recruitment. Instead, the main function of H3K9me2/3 at this developmental stage is to drive HP1a clustering and subsequent heterochromatin compaction. Our results show that HP1a binding to constitutive heterochromatin in the absence of H3K9me2/3 is not sufficient to promote proper embryo development and heterochromatin formation. The loss of H3K9 HMTs and H3K9 methylation alters genome organization and hinders embryonic development.
Subject(s)
Chromosomal Proteins, Non-Histone , Heterochromatin , Histones , Animals , Histones/metabolism , Histones/genetics , Heterochromatin/metabolism , Heterochromatin/genetics , Methylation , Chromosomal Proteins, Non-Histone/metabolism , Chromosomal Proteins, Non-Histone/genetics , Chromobox Protein Homolog 5 , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Embryo, Nonmammalian/metabolism , Genome, Insect , Embryonic Development/genetics , Histone-Lysine N-Methyltransferase/metabolism , Histone-Lysine N-Methyltransferase/geneticsABSTRACT
Twisted gastrulation (TWSG1) is an evolutionarily conserved secreted glycoprotein which controls signaling by Bone Morphogenetic Proteins (BMPs). TWSG1 binds BMPs and their antagonist Chordin to control BMP signaling during embryonic development, kidney regeneration and cancer. We report crystal structures of TWSG1 alone and in complex with a BMP ligand, Growth Differentiation Factor 5. TWSG1 is composed of two distinct, disulfide-rich domains. The TWSG1 N-terminal domain occupies the BMP type 1 receptor binding site on BMPs, whereas the C-terminal domain binds to a Chordin family member. We show that TWSG1 inhibits BMP function in cellular signaling assays and mouse colon organoids. This inhibitory function is abolished in a TWSG1 mutant that cannot bind BMPs. The same mutation in the Drosophila TWSG1 ortholog Tsg fails to mediate BMP gradient formation required for dorsal-ventral axis patterning of the early embryo. Our studies reveal the evolutionarily conserved mechanism of BMP signaling inhibition by TWSG1.
Subject(s)
Bone Morphogenetic Proteins , Signal Transduction , Animals , Bone Morphogenetic Proteins/metabolism , Bone Morphogenetic Proteins/genetics , Mice , Humans , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Drosophila Proteins/chemistry , Glycoproteins/metabolism , Glycoproteins/genetics , Intercellular Signaling Peptides and Proteins/metabolism , Intercellular Signaling Peptides and Proteins/genetics , Binding Sites , Protein Domains , Protein Binding , Organoids/metabolism , Organoids/embryology , HEK293 Cells , Gastrulation/genetics , Mutation , Crystallography, X-Ray , Drosophila melanogaster/embryology , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , ProteinsABSTRACT
The MS2-PP7 two-color live-imaging system provides insights into the spatiotemporal dynamics of nascent transcripts at tagged loci. Here, we present a protocol to quantitatively measure the rate of RNA polymerase II elongation for each actively transcribing nucleus in living Drosophila embryos. The elongation rate is calculated by measuring the effective distance and the time elapsed between MS2 and PP7 trajectories. We describe steps for preparing embryo samples, performing live imaging, and measuring the elongation rate. For complete details on the use and execution of this protocol, please refer to Keller et al.1.
Subject(s)
Embryo, Nonmammalian , RNA Polymerase II , Animals , RNA Polymerase II/metabolism , RNA Polymerase II/genetics , Embryo, Nonmammalian/metabolism , Drosophila/embryology , Drosophila/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Drosophila melanogaster/metabolism , Drosophila Proteins/metabolism , Drosophila Proteins/geneticsABSTRACT
The conserved bazooka (baz/par3) gene acts as a key regulator of asymmetrical cell divisions across the animal kingdom. Associated Par3/Baz-Par6-aPKC protein complexes are also well known for their role in the establishment of apical/basal cell polarity in epithelial cells. Here we define a novel, positive function of Baz/Par3 in the Notch pathway. Using Drosophila wing and eye development, we demonstrate that Baz is required for Notch signaling activity and optimal transcriptional activation of Notch target genes. Baz appears to act independently of aPKC in these contexts, as knockdown of aPKC does not cause Notch loss-of-function phenotypes. Using transgenic Notch constructs, our data positions Baz activity downstream of activating Notch cleavage steps and upstream of Su(H)/CSL transcription factor complex activity on Notch target genes. We demonstrate a biochemical interaction between NICD and Baz, suggesting that Baz is required for NICD activity before NICD binds to Su(H). Taken together, our data define a novel role of the polarity protein Baz/Par3, as a positive and direct regulator of Notch signaling through its interaction with NICD.
Subject(s)
Drosophila Proteins , Receptors, Notch , Signal Transduction , Wings, Animal , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Receptors, Notch/metabolism , Wings, Animal/metabolism , Wings, Animal/embryology , Wings, Animal/growth & development , Gene Expression Regulation, Developmental , Protein Binding , Drosophila melanogaster/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/genetics , Eye/embryology , Eye/metabolism , Eye/growth & development , Drosophila/metabolism , Drosophila/embryology , Cell Polarity , Intracellular Signaling Peptides and ProteinsABSTRACT
Movement is a key feature of animal systems, yet its embryonic origins are not fully understood. Here, we investigate the genetic basis underlying the embryonic onset of movement in Drosophila focusing on the role played by small non-coding RNAs (microRNAs, miRNAs). To this end, we first develop a quantitative behavioural pipeline capable of tracking embryonic movement in large populations of fly embryos, and using this system, discover that the Drosophila miRNA miR-2b-1 plays a role in the emergence of movement. Through the combination of spectral analysis of embryonic motor patterns, cell sorting and RNA in situs, genetic reconstitution tests, and neural optical imaging we define that miR-2b-1 influences the emergence of embryonic movement by exerting actions in the developing nervous system. Furthermore, through the combination of bioinformatics coupled to genetic manipulation of miRNA expression and phenocopy tests we identify a previously uncharacterised (but evolutionarily conserved) chloride channel encoding gene - which we term Movement Modulator (Motor) - as a genetic target that mechanistically links miR-2b-1 to the onset of movement. Cell-specific genetic reconstitution of miR-2b-1 expression in a null miRNA mutant background, followed by behavioural assays and target gene analyses, suggest that miR-2b-1 affects the emergence of movement through effects in sensory elements of the embryonic circuitry, rather than in the motor domain. Our work thus reports the first miRNA system capable of regulating embryonic movement, suggesting that other miRNAs are likely to play a role in this key developmental process in Drosophila as well as in other species.
Subject(s)
MicroRNAs , Animals , MicroRNAs/metabolism , MicroRNAs/genetics , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Gene Expression Regulation, Developmental , Movement , Embryo, Nonmammalian/metabolism , Drosophila/genetics , Drosophila/embryology , Drosophila Proteins/genetics , Drosophila Proteins/metabolismABSTRACT
Visual circuit development is characterized by subdivision of neuropils into layers that house distinct sets of synaptic connections. We find that, in the Drosophila medulla, this layered organization depends on the axon guidance regulator Plexin A. In Plexin A null mutants, synaptic layers of the medulla neuropil and arborizations of individual neurons are wider and less distinct than in controls. Analysis of semaphorin function indicates that Semaphorin 1a, acting in a subset of medulla neurons, is the primary partner for Plexin A in medulla lamination. Removal of the cytoplasmic domain of endogenous Plexin A has little effect on the formation of medulla layers; however, both null and cytoplasmic domain deletion mutations of Plexin A result in an altered overall shape of the medulla neuropil. These data suggest that Plexin A acts as a receptor to mediate morphogenesis of the medulla neuropil, and as a ligand for Semaphorin 1a to subdivide it into layers. Its two independent functions illustrate how a few guidance molecules can organize complex brain structures by each playing multiple roles.
Subject(s)
Drosophila Proteins , Morphogenesis , Nerve Tissue Proteins , Neuropil , Optic Lobe, Nonmammalian , Receptors, Cell Surface , Semaphorins , Animals , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Semaphorins/metabolism , Semaphorins/genetics , Nerve Tissue Proteins/metabolism , Nerve Tissue Proteins/genetics , Morphogenesis/genetics , Neuropil/metabolism , Optic Lobe, Nonmammalian/metabolism , Optic Lobe, Nonmammalian/embryology , Receptors, Cell Surface/metabolism , Receptors, Cell Surface/genetics , Drosophila melanogaster/metabolism , Drosophila melanogaster/genetics , Drosophila melanogaster/embryology , Neurons/metabolism , Drosophila/metabolism , Drosophila/embryology , Mutation/geneticsABSTRACT
Although the effects of genetic and environmental perturbations on multicellular organisms are rarely restricted to single phenotypic layers, our current understanding of how developmental programs react to these challenges remains limited. Here, we have examined the phenotypic consequences of disturbing the bicoid regulatory network in early Drosophila embryos. We generated flies with two extra copies of bicoid, which causes a posterior shift of the network's regulatory outputs and a decrease in fitness. We subjected these flies to EMS mutagenesis, followed by experimental evolution. After only 8-15 generations, experimental populations have normalized patterns of gene expression and increased survival. Using a phenomics approach, we find that populations were normalized through rapid increases in embryo size driven by maternal changes in metabolism and ovariole development. We extend our results to additional populations of flies, demonstrating predictability. Together, our results necessitate a broader view of regulatory network evolution at the systems level.
Subject(s)
Drosophila Proteins , Drosophila melanogaster , Gene Dosage , Gene Expression Regulation, Developmental , Gene Regulatory Networks , Animals , Drosophila Proteins/genetics , Drosophila Proteins/metabolism , Female , Drosophila melanogaster/genetics , Drosophila melanogaster/growth & development , Drosophila melanogaster/embryology , Homeodomain Proteins/genetics , Homeodomain Proteins/metabolism , Phenotype , Male , Embryo, Nonmammalian/metabolism , Drosophila/genetics , Drosophila/embryology , Drosophila/metabolism , Mutagenesis , Trans-ActivatorsABSTRACT
The planar orientation of cell division (OCD) is important for epithelial morphogenesis and homeostasis. Here, we ask how mechanics and antero-posterior (AP) patterning combine to influence the first divisions after gastrulation in the Drosophila embryonic epithelium. We analyse hundreds of cell divisions and show that stress anisotropy, notably from compressive forces, can reorient division directly in metaphase. Stress anisotropy influences the OCD by imposing metaphase cell elongation, despite mitotic rounding, and overrides interphase cell elongation. In strongly elongated cells, the mitotic spindle adapts its length to, and hence its orientation is constrained by, the cell long axis. Alongside mechanical cues, we find a tissue-wide bias of the mitotic spindle orientation towards AP-patterned planar polarised Myosin-II. This spindle bias is lost in an AP-patterning mutant. Thus, a patterning-induced mitotic spindle orientation bias overrides mechanical cues in mildly elongated cells, whereas in strongly elongated cells the spindle is constrained close to the high stress axis.
Subject(s)
Cell Division , Cell Polarity , Drosophila melanogaster , Epithelial Cells , Metaphase , Spindle Apparatus , Stress, Mechanical , Animals , Metaphase/physiology , Epithelial Cells/cytology , Epithelial Cells/metabolism , Spindle Apparatus/metabolism , Drosophila melanogaster/embryology , Drosophila melanogaster/cytology , Cell Polarity/physiology , Body Patterning , Myosin Type II/metabolism , Embryo, Nonmammalian/cytology , Drosophila Proteins/metabolism , Drosophila Proteins/genetics , Gastrulation/physiologyABSTRACT
The precise assembly of tissues and organs relies on spatiotemporal regulation of gene expression to coordinate the collective behavior of cells. In Drosophila embryos, the midgut musculature is formed through collective migration of caudal visceral mesoderm (CVM) cells, but how gene expression changes as cells migrate is not well understood. Here, we have focused on ten genes expressed in the CVM and the cis-regulatory sequences controlling their expression. Although some genes are continuously expressed, others are expressed only early or late during migration. Late expression relates to cell cycle progression, as driving string/Cdc25 causes earlier division of CVM cells and accelerates the transition to late gene expression. In particular, we found that the cell cycle effector transcription factor E2F1 is a required input for the late gene CG5080. Furthermore, whereas late genes are broadly expressed in all CVM cells, early gene transcripts are polarized to the anterior or posterior ends of the migrating collective. We show this polarization requires transcription factors Snail, Zfh1 and Dorsocross. Collectively, these results identify two sequential gene expression programs bridged by cell division that support long-distance directional migration of CVM cells.