A high-resolution, single-molecule study directly assesses the prevalence and dynamics of DNA looping in gene regulation in live E. coli cells.
DNA looping mediated by transcription factors plays critical roles in prokaryotic gene regulation. The “genetic switch” of bacteriophage λ determines whether a prophage stays incorporated in the E. coli chromosome or enters the lytic cycle of phage propagation and cell lysis. Past studies have shown that long-range DNA interactions between the operator sequences OR and OL (separated by 2.3 kb), mediated by the λ repressor CI (accession number P03034), play key roles in regulating the λ switch. In vitro, it was demonstrated that DNA segments harboring the operator sequences formed loops in the presence of CI, but CI-mediated DNA looping has not been directly visualized in vivo, hindering a deep understanding of the corresponding dynamics in realistic cellular environments. We report a high-resolution, single-molecule imaging method to probe CI-mediated DNA looping in live E. coli cells. We labeled two DNA loci with differently colored fluorescent fusion proteins and tracked their separations in real time with ∼40 nm accuracy, enabling the first direct analysis of transcription-factor-mediated DNA looping in live cells. Combining looping measurements with measurements of CI expression levels in different operator mutants, we show quantitatively that DNA looping activates transcription and enhances repression. Further, we estimated the upper bound of the rate of conformational change from the unlooped to the looped state, and discuss how chromosome compaction may impact looping kinetics. Our results provide insights into transcription-factor-mediated DNA looping in a variety of operator and CI mutant backgrounds in vivo, and our methodology can be applied to a broad range of questions regarding chromosome conformations in prokaryotes and higher organisms.
One mechanism cells use to regulate gene expression is DNA looping, whereby two distant DNA sites are brought together by regulatory proteins. The looping then either enhances interactions between other regulatory proteins bound at the separate sites or brings those regulatory proteins close to RNA polymerase at the promoter. Recent work in bacteriophage λ has suggested that DNA looping mediated by a transcription factor called λ repressor CI plays a critical role in regulating the expression of λ genes and consequently in determining the fate of the host E. coli bacterial cells. CI-mediated DNA looping has been directly demonstrated in vitro, but it has only been indirectly inferred in vivo. For the current study we developed a method to visualize CI-mediated DNA looping in individual live E. coli cells. We labeled two DNA sites—one each side of the proposed loop—with differently colored fluorescent fusion proteins, allowing us to measure their separation with an accuracy of a few tens of nanometers. Using this method, we directly analyzed CI-mediated DNA looping, providing insight into how transcription factor-mediated DNA looping influences gene regulation in live E. coli cells. Our methodology can be applied to a broad range of questions regarding chromosome conformation in prokaryotes and higher organisms.
How does basic cell biology contribute to biomedicine? A new series of Features in JCB provides a cross section of compelling examples of how basic cell biology findings can lead to therapeutics. These articles highlight the fruitful, essential, and increasingly prominent bridge that exists between cell biology and the clinic.
In yeast and humans, interaction of a nuclear pore protein with promoters alters chromatin structure and allows RNA polymerase II to bind, poising them for faster reactivation for several generations.
The interaction of nuclear pore proteins (Nups) with active genes can promote their transcription. In yeast, some inducible genes interact with the nuclear pore complex both when active and for several generations after being repressed, a phenomenon called epigenetic transcriptional memory. This interaction promotes future reactivation and requires Nup100, a homologue of human Nup98. A similar phenomenon occurs in human cells; for at least four generations after treatment with interferon gamma (IFN-γ), many IFN-γ-inducible genes are induced more rapidly and more strongly than in cells that have not previously been exposed to IFN-γ. In both yeast and human cells, the recently expressed promoters of genes with memory exhibit persistent dimethylation of histone H3 lysine 4 (H3K4me2) and physically interact with Nups and a poised form of RNA polymerase II. However, in human cells, unlike yeast, these interactions occur in the nucleoplasm. In human cells transiently depleted of Nup98 or yeast cells lacking Nup100, transcriptional memory is lost; RNA polymerase II does not remain associated with promoters, H3K4me2 is lost, and the rate of transcriptional reactivation is reduced. These results suggest that Nup100/Nup98 binding to recently expressed promoters plays a conserved role in promoting epigenetic transcriptional memory.
Cells respond to changes in nutrients or signaling molecules by altering the expression of genes. The rate at which genes are turned on is not uniform; some genes are induced rapidly and others are induced slowly. In brewer's yeast, previous experience can enhance the rate at which genes are turned on again, a phenomenon called “transcriptional memory.” After repression, such genes physically interact with the nuclear pore complex, leading to altered chromatin structure and binding of a poised RNA polymerase II. Human genes that are induced by interferon gamma show a similar behavior. In both cases, the phenomenon persists through several cell divisions, suggesting that it is epigenetically inherited. Here, we find that yeast and human cells utilize a similar molecular mechanism to prime genes for reactivation. In both species, the nuclear pore protein Nup100/Nup98 binds to the promoters of genes that exhibit transcriptional memory. This leads to an altered chromatin state in the promoter and binding of RNA polymerase II, poising genes for future expression. We conclude that both unicellular and multicellular organisms use nuclear pore proteins in a novel way to alter transcription based on previous experiences.
One of the major forces driving the birth of the field of cell biology was the application of electron microscopy to cells. Today, virtual nanoscopy has brought electron microscopy and the cell biology community to a new frontier in biological imaging and cell biological inquiry. The Journal of Cell Biology is pleased to announce that the JCB DataViewer is “going big” to host electron microscopy data at a whole new scale.
The nucleus is unique amongst cellular organelles in that it contains a myriad of discrete suborganelles. These nuclear bodies are morphologically and molecularly distinct entities, and they host specific nuclear processes. Although the mode of biogenesis appears to differ widely between individual nuclear bodies, several common design principles are emerging, particularly, the ability of nuclear bodies to form de novo, a role of RNA as a structural element and self-organization as a mode of formation. The controlled biogenesis of nuclear bodies is essential for faithful maintenance of nuclear architecture during the cell cycle and is an important part of cellular responses to intra- and extracellular events.
The different nuclear bodies in the cell nucleus have distinct components and are sites for different processes. But they share the ability to self-organize, form de novo in certain conditions, and use RNA as a structural element.
The kinesin motor protein KIF4 performs essential functions in mitosis. Like other mitotic kinesins, loss of KIF4 causes spindle defects, aneuploidy, genomic instability and ultimately tumor formation. However, KIF4 is unique among molecular motors in that it resides in the cell nucleus throughout interphase, suggesting a non-mitotic function as well. Here we identify a novel cellular function for a molecular motor protein by demonstrating that KIF4 acts as a modulator of large-scale chromatin architecture during interphase. KIF4 binds globally to chromatin and its absence leads to chromatin decondensation and loss of heterochromatin domains. KIF4-dependent chromatin decondensation has functional consequences by causing replication defects and global mis-regulation of gene expression programs. KIF4 exerts its function in chromatin architecture via regulation of ADP-ribosylation of core and linker histones and by physical interaction and recruitment of chromatin assembly proteins during S-phase. These observations document a novel function for a molecular motor protein in establishment and maintenance of higher order chromatin structure.
There is a troubling trend in scientific publishing for manuscripts to undergo multiple, often lengthy, rounds of review, resulting in significant delays to publication. JCB is announcing new procedures to streamline its editorial process and eliminate unnecessary delays.
There is no doubt that genomes are organized nonrandomly in the nucleus of higher eukaryotes. But what is the functional relevance of this nonrandomness? In this Essay, we explore the biological meaning of spatial gene positioning by examining the functional link between the activity of a gene and its radial position in the nucleus.
DNA repair and maintenance of genome stability are crucial to cellular and organismal function, and defects in these processes have been implicated in cancer and ageing. Detailed molecular, biochemical and genetic analyses have outlined the molecular framework involved in cellular DNA-repair pathways, but recent cell-biological approaches have revealed important roles for the spatial and temporal organization of the DNA-repair machinery during the recognition of DNA lesions and the assembly of repair complexes. It has also become clear that local higher-order chromatin structure, chromatin dynamics and non-random global genome organization are key factors in genome maintenance. These cell-biological features of DNA repair illustrate an emerging role for nuclear architecture in multiple aspects of genome maintenance.
Correct segmentation is critical to many applications within automated microscopy image analysis. Despite the availability of advanced segmentation algorithms, variations in cell morphology, sample preparation, and acquisition settings often lead to segmentation errors. This manuscript introduces a ranked-retrieval approach using logistic regression to automate selection of accurately segmented nuclei from a set of candidate segmentations. The methodology is validated on an application of spatial gene repositioning in breast cancer cell nuclei. Gene repositioning is analyzed in patient tissue sections by labeling sequences with fluorescence in situ hybridization (FISH), followed by measurement of the relative position of each gene from the nuclear center to the nuclear periphery. This technique requires hundreds of well-segmented nuclei per sample to achieve statistical significance. Although the tissue samples in this study contain a surplus of available nuclei, automatic identification of the well-segmented subset remains a challenging task.
Logistic regression was applied to features extracted from candidate segmented nuclei, including nuclear shape, texture, context, and gene copy number, in order to rank objects according to the likelihood of being an accurately segmented nucleus. The method was demonstrated on a tissue microarray dataset of 43 breast cancer patients, comprising approximately 40,000 imaged nuclei in which the HES5 and FRA2 genes were labeled with FISH probes. Three trained reviewers independently classified nuclei into three classes of segmentation accuracy. In man vs. machine studies, the automated method outperformed the inter-observer agreement between reviewers, as measured by area under the receiver operating characteristic (ROC) curve. Robustness of gene position measurements to boundary inaccuracies was demonstrated by comparing 1086 manually and automatically segmented nuclei. Pearson correlation coefficients between the gene position measurements were above 0.9 (p < 0.05). A preliminary experiment was conducted to validate the ranked retrieval in a test to detect cancer. Independent manual measurement of gene positions agreed with automatic results in 21 out of 26 statistical comparisons against a pooled normal (benign) gene position distribution.
Accurate segmentation is necessary to automate quantitative image analysis for applications such as gene repositioning. However, due to heterogeneity within images and across different applications, no segmentation algorithm provides a satisfactory solution. Automated assessment of segmentations by ranked retrieval is capable of reducing or even eliminating the need to select segmented objects by hand and represents a significant improvement over binary classification. The method can be extended to other high-throughput applications requiring accurate detection of cells or nuclei across a range of biomedical applications.
Genomes are organized into complex higher-order structures by folding of the DNA into chromatin fibers, chromosome domains, and ultimately chromosomes. The higher-order organization of genomes is functionally important for gene regulation and control of gene expression programs. Defects in how chromatin is globally organized are relevant for physiological and pathological processes. Mutations and transcriptional misregulation of several global genome organizers are linked to human diseases and global alterations in chromatin structure are emerging as key players in maintenance of genome stability, aging, and the formation of cancer translocations.
Defects in proteins that control chromatin organization generate architectural changes that increase sensitivity to DNA damage, leading to cancer and various developmental disorders.
The temporal order of replication of mammalian chromosomes appears to be linked to their functional organization, but the process that establishes and modifies this order during cell differentiation remains largely unknown. Here, we studied how the replication of the Igh locus initiates, progresses, and terminates in bone marrow pro-B cells undergoing B cell commitment. We show that many aspects of DNA replication can be quantitatively explained by a mechanism involving the stochastic firing of origins (across the S phase and the Igh locus) and extensive variations in their firing rate (along the locus). The firing rate of origins shows a high degree of coordination across Igh domains that span tens to hundreds of kilobases, a phenomenon not observed in simple eukaryotes. Differences in domain sizes and firing rates determine the temporal order of replication. During B cell commitment, the expression of the B-cell-specific factor Pax5 sharply alters the temporal order of replication by modifying the rate of origin firing within various Igh domains (particularly those containing Pax5 binding sites). We propose that, within the Igh CH-3′RR domain, Pax5 is responsible for both establishing and maintaining high rates of origin firing, mostly by controlling events downstream of the assembly of pre-replication complexes.
Each time a mammalian cell duplicates its genome in preparation for cell division it activates thousands of so called “DNA origins of replication.” The timely and complete duplication of the genome depends on careful orchestration of origin activation, which is modified when cells differentiate to perform a specific function. We currently lack a universally accepted model of origin regulation that can explain the replication dynamics in complex eukaryotes. Here, we studied the mouse immunoglobulin heavy-chain locus, one of the antibody-encoding portions of the genome, where origins change activity when antibody-producing B cells differentiate in the bone marrow. We show that multiple aspects of DNA replication initiation, progression, and termination can be explained mathematically by the interplay between randomly firing origins and two independent variables: the speed of progression of replication forks and the firing rate of origins along the locus. The rate of origin firing varies extensively along the locus during B cell differentiation and, thus, is a dominant factor in establishing the temporal order of replication. A differentiation factor called Pax5 can alter the temporal order of replication by modifying the rate of origin firing across various parts of the locus.
Activated RAS promotes dimerization of members of the RAF kinase family1-3. ATP-competitive RAF inhibitors activate ERK signaling4-7 by transactivating RAF dimers4. In melanomas with mutant BRAF(V600E), levels of RAS activation are low and these drugs bind to BRAF(V600E) monomers and inhibit their activity. This tumor-specific inhibition of ERK signaling results in a broad therapeutic index and RAF inhibitors have remarkable clinical activity in patients with melanomas that harbor mutant BRAF(V600E)8. However, resistance invariably develops. Here, we identify a novel resistance mechanism. We find that a subset of cells resistant to vemurafenib (PLX4032, RG7204) express a 61kd variant form of BRAF(V600E) that lacks exons 4-8, a region that encompasses the RAS-binding domain. p61BRAF(V600E) exhibits enhanced dimerization in cells with low levels of RAS activation, as compared to full length BRAF(V600E). In cells in which p61BRAF(V600E) is expressed endogenously or ectopically, ERK signaling is resistant to the RAF inhibitor. Moreover, a mutation that abolishes the dimerization of p61BRAF(V600E) restores its sensitivity to vemurafenib. Finally, we identified BRAF(V600E) splicing variants lacking the RAS-binding domain in the tumors of six of 19 patients with acquired resistance to vemurafenib. These data support the model that inhibition of ERK signaling by RAF inhibitors is dependent on levels of RAS-GTP too low to support RAF dimerization and identify a novel mechanism of acquired resistance in patients: expression of splicing isoforms of BRAF(V600E) that dimerize in a RAS-independent manner.
The lamins are the major architectural proteins of the animal cell nucleus. Lamins line the inside of the nuclear membrane, where they provide a platform for the binding of proteins and chromatin and confer mechanical stability. They have been implicated in a wide range of nuclear functions, including higher-order genome organization, chromatin regulation, transcription, DNA replication and DNA repair. The lamins are members of the intermediate filament (IF) family of proteins, which constitute a major component of the cytoskeleton. Lamins are the only nuclear IFs and are the ancestral founders of the IF protein superfamily. Lamins polymerize into fibers forming a complex protein meshwork in vivo and, like all IF proteins, have a tripartite structure with two globular head and tail domains flanking a central α-helical rod domain, which supports the formation of higher-order polymers. Mutations in lamins cause a large number of diverse human diseases, collectively known as the laminopathies, underscoring their functional importance.
Mutations in the A-type lamins A and C, two major components of the nuclear lamina, cause a large group of phenotypically diverse diseases collectively referred to as laminopathies. These conditions often involve defects in chromatin organization. However, it is unclear whether A-type lamins interact with chromatin in vivo and whether aberrant chromatin–lamin interactions contribute to disease. Here, we have used an unbiased approach to comparatively map genome-wide interactions of gene promoters with lamin A and progerin, the mutated lamin A isoform responsible for the premature aging disorder Hutchinson–Gilford progeria syndrome (HGPS) in mouse cardiac myoytes and embryonic fibroblasts. We find that lamin A-associated genes are predominantly transcriptionally silent and that loss of lamin association leads to the relocation of peripherally localized genes, but not necessarily to their activation. We demonstrate that progerin induces global changes in chromatin organization by enhancing interactions with a specific subset of genes in addition to the identified lamin A-associated genes. These observations demonstrate disease-related changes in higher order genome organization in HGPS and provide novel insights into the role of lamin–chromatin interactions in chromatin organization.
Electronic supplementary material
The online version of this article (doi:10.1007/s00412-012-0376-7) contains supplementary material, which is available to authorized users.
Continued examination of substituted 6-arylquinazolin-4-amines as Clk4 inhibitors resulted in selective inhibitors of Clk1, Clk4, Dyrk1A and Dyrk1B. Several of the most potent inhibitors were validated as being highly selective within a comprehensive kinome scan.
Clk1; Clk2; Clk3; Clk4; Dyrk1A; Dyrk1B; Pre-mRNA splicing; kinase inhibition; quinazoline
NUP1, the first example of a nuclear lamin analog in nonmetazoans, performs roles similar to those of lamins in maintaining the structure and organization of the nucleus in Trypanosoma brucei.
A unifying feature of eukaryotic nuclear organization is genome segregation into transcriptionally active euchromatin and transcriptionally repressed heterochromatin. In metazoa, lamin proteins preserve nuclear integrity and higher order heterochromatin organization at the nuclear periphery, but no non-metazoan lamin orthologues have been identified, despite the likely presence of nucleoskeletal elements in many lineages. This suggests a metazoan-specific origin for lamins, and therefore that distinct protein elements must compose the nucleoskeleton in other lineages. The trypanosomatids are highly divergent organisms and possess well-documented but remarkably distinct mechanisms for control of gene expression, including polycistronic transcription and trans-splicing. NUP-1 is a large protein localizing to the nuclear periphery of Trypanosoma brucei and a candidate nucleoskeletal component. We sought to determine if NUP-1 mediates heterochromatin organization and gene regulation at the nuclear periphery by examining the influence of NUP-1 knockdown on morphology, chromatin positioning, and transcription. We demonstrate that NUP-1 is essential and part of a stable network at the inner face of the trypanosome nuclear envelope, since knockdown cells have abnormally shaped nuclei with compromised structural integrity. NUP-1 knockdown also disrupts organization of nuclear pore complexes and chromosomes. Most significantly, we find that NUP-1 is required to maintain the silenced state of developmentally regulated genes at the nuclear periphery; NUP-1 knockdown results in highly specific mis-regulation of telomere-proximal silenced variant surface glycoprotein (VSG) expression sites and procyclin loci, indicating a disruption to normal chromatin organization essential to life-cycle progression. Further, NUP-1 depletion leads to increased VSG switching and therefore appears to have a role in control of antigenic variation. Thus, analogous to vertebrate lamins, NUP-1 is a major component of the nucleoskeleton with key roles in organization of the nuclear periphery, heterochromatin, and epigenetic control of developmentally regulated loci.
Eukaryotes—fungi, plants, animals, and many unicellular organisms—are defined by the presence of a cell nucleus that contains the chromosomes and is enveloped by a lipid membrane lined on the inner face with a protein network called the lamina. Among other functions, the lamina serves as an anchorage site for the ends of chromosomes. In multicellular animals (metazoa), the lamina comprises a few related proteins called lamins, which are very important for many functions related to the nucleus; abnormal lamins result in multiple nuclear defects and diseases, including inappropriate gene expression and premature aging. Until now, however, lamins had been found only in metazoa; no protein of equivalent function had been identified in plants, fungi, or unicellular organisms. Here, we describe a protein from African trypanosomes—the single-cell parasites that cause sleeping sickness—that fulfils many lamin-like roles, including maintaining nuclear structure and organizing the chromosomes of this organism. We show that this protein, which we call NUP-1 for nuclear periphery protein-1, is vital for the antigenic variation mechanisms that allow the parasite to escape the host immune response. We propose that NUP-1 is a lamin analogue that performs similar functions in trypanosomes to those of authentic lamins in metazoa. These findings, we believe, have important implications for understanding the evolution of the nucleus.
A combination of cis-regulatory elements can impose the formation of an early replicating domain in a naturally late replicating region and might constitute the basic unit of early replicating domains.
The nuclear genomes of vertebrates show a highly organized program of DNA replication where GC-rich isochores are replicated early in S-phase, while AT-rich isochores are late replicating. GC-rich regions are gene dense and are enriched for active transcription, suggesting a connection between gene regulation and replication timing. Insulator elements can organize independent domains of gene transcription and are suitable candidates for being key regulators of replication timing. We have tested the impact of inserting a strong replication origin flanked by the β-globin HS4 insulator on the replication timing of naturally late replicating regions in two different avian cell types, DT40 (lymphoid) and 6C2 (erythroid). We find that the HS4 insulator has the capacity to impose a shift to earlier replication. This shift requires the presence of HS4 on both sides of the replication origin and results in an advance of replication timing of the target locus from the second half of S-phase to the first half when a transcribed gene is positioned nearby. Moreover, we find that the USF transcription factor binding site is the key cis-element inside the HS4 insulator that controls replication timing. Taken together, our data identify a combination of cis-elements that might constitute the basic unit of multi-replicon megabase-sized early domains of DNA replication.
All eukaryotic organisms must duplicate their genome precisely once before cell division. This occurs according to an established temporal program during S-phase (when DNA synthesis takes place) of the cell cycle. In vertebrates, this program is regulated at the level of large chromosomal domains ranging from 200 kb to 2 Mb, but the molecular mechanisms that control the temporal firing order of animal replication origins are not clearly understood. Using the genetically tractable chicken DT40 cell system, we identified a minimal combination of cis-regulatory DNA elements that is able to shift the timing of a naturally “mid-late” replicated region to “mid-early.” This critical group of elements is composed of one strong replication origin flanked by binding sequences for the upstream stimulatory factor (USF) protein. The additional presence of a strongly transcribed gene shifted the region towards an even earlier replication time, suggesting cooperation between cis-elements when establishing temporal programs of replication. We speculate that USF binding sequences cooperate with sites of replication initiation and transcribed genes to promote the establishment of early replicating domains along vertebrate genomes.
New technologies and approaches in cell biology research necessitate new venues for information sharing and publication. JCB continues its support of innovation in publishing with the launch of Tools, a new article type for the description of methods and high-throughput datasets, and of a new interface for the JCB DataViewer for hosting high-content screening datasets in their entirety.
Cancer stem cells (CSCs) have been implicated in the maintenance and progression of several types of cancer. The origin and cellular properties of human CSCs are poorly characterized. Here we show that CSC-like cells can be generated in vitro by oncogenic reprogramming of human somatic cells during neoplastic transformation. We find that in vitro transformation confers stem cell properties to primary differentiated fibroblasts, including the ability to self-renew and to differentiate along multiple lineages. Tumours induced by transformed fibroblasts are hierarchically-organized and the cells which act as CSCs to initiate and maintain tumour growth are marked by the stage-specific embryonic antigen SSEA-1. Heterogeneous lineages of cancer cells in the bulk of the tumour arise through differentiation of SSEA-1+ fibroblasts and differentiation is associated with loss of tumorigenic potential. These findings establish an experimental system to characterize cellular and molecular properties of human CSCs and demonstrate that somatic cells have the potential to de-differentiate and acquire properties of CSCs.
Protein Quality Control (PQC) pathways are essential to maintain the equilibrium between protein folding and the clearance of misfolded proteins. In order to discover novel human PQC factors, we developed a high-content, high-throughput cell-based assay to assess PQC activity. The assay is based on a fluorescently tagged, temperature sensitive PQC substrate and measures its degradation relative to a temperature insensitive internal control. In a targeted screen of 1591 siRNA genes involved in the Ubiquitin-Proteasome System (UPS) we identified 25 of the 33 genes encoding for 26S proteasome subunits and discovered several novel PQC factors. An unbiased genome-wide siRNA screen revealed the protein translation machinery, and in particular the EIF3 translation initiation complex, as a novel key modulator of misfolded protein stability. These results represent a comprehensive unbiased survey of human PQC components and establish an experimental tool for the discovery of genes that are required for the degradation of misfolded proteins under conditions of proteotoxic stress.
Alternative splicing plays critical roles in differentiation, development and disease and is a major source for protein diversity in higher eukaryotes. Traditionally, analysis of alternative splicing regulation has focused on RNA sequence elements and their associated factors, but recent provocative studies point to a key function of chromatin structure and histone modifications in alternative splicing regulation. These insights suggest that epigenetic regulation not only determines what parts of the genome are expressed, but also how they are spliced.
Defects in induction signaling and response underlie the nucleocytoplasmic incompatibility between two evolutionarily distant frog species, while specific treatments partially restore this response in explants and whole embryos.
Incompatibilities between the nucleus and the cytoplasm of sufficiently distant species result in developmental arrest of hybrid and nucleocytoplasmic hybrid (cybrid) embryos. Several hypotheses have been proposed to explain their lethality, including problems in embryonic genome activation (EGA) and/or nucleo-mitochondrial interactions. However, conclusive identification of the causes underlying developmental defects of cybrid embryos is still lacking. We show here that while over 80% of both Xenopus laevis and Xenopus (Silurana) tropicalis same-species androgenetic haploids develop to the swimming tadpole stage, the androgenetic cybrids formed by the combination of X. laevis egg cytoplasm and X. tropicalis sperm nucleus invariably fail to gastrulate properly and never reach the swimming tadpole stage. In spite of this arrest, these cybrids show quantitatively normal EGA and energy levels at the stage where their initial gastrulation defects are manifested. The nucleocytoplasmic incompatibility between these two species instead results from a combination of factors, including a reduced emission of induction signal from the vegetal half, a decreased sensitivity of animal cells to induction signals, and differences in a key embryonic protein (Xbra) concentration between the two species, together leading to inefficient induction and defective convergence-extension during gastrulation. Indeed, increased exposure to induction signals and/or Xbra signalling partially rescues the induction response in animal explants and whole cybrid embryos. Altogether, our study demonstrates that the egg cytoplasm of one species may not support the development promoted by the nucleus of another species, even if this nucleus does not interfere with the cytoplasmic/maternal functions of the egg, while the egg cytoplasm is also capable of activating the genome of that nucleus. Instead, our results provide evidence that inefficient signalling and differences in the concentrations of key proteins between species lead to developmental defects in cybrids. Finally, they show that the incompatibilities of cybrids can be corrected by appropriate treatments.
When two species evolve separately for several million years, their respective genomes accumulate many small changes that together are responsible for the differences in their characters. Some of these affect the way eggs are prepared inside the germline, and/or how embryos develop, such that the egg cytoplasm of a given species can only support development promoted by its own genome or nucleus. Thus, developmental incompatibility arises between the cytoplasm and the nucleus of distant species during evolution and we don't know its mechanism. We have studied this phenomenon in an advantageous system using two evolutionarily distant frog species (Xenopus laevis and Xenopus tropicalis). We found that hybrid frog embryos with X. laevis cytoplasm and X. tropicalis nuclei are always defective in an important process that is necessary to generate morphogenetic cell movements during development. Through a series of experiments in which we dissect out and/or recombine parts of such hybrid embryos and observe their behaviour in culture, we show that this phenomenon occurs because of malfunctions in the signalling cascade that is responsible for generating these cell movements. Thus, we postulate that inefficient molecular signalling contributes to the death of such hybrids.
The next revolution in microscopy is upon us: it is high-throughput imaging (HTI). In HTI large numbers of images from many samples are acquired and analyzed. This has become possible due to the confluence of dramatic progress in microscope engineering enabling efficient image collection and the availability of high computing power for data analysis. As recently exemplified by Neumann, Ellenberg and colleagues, combining HTI with genome-wide RNA interference-based gene-knockdown technology offers a powerful approach for unbiased discovery of cellular mechanisms.
A-type lamins are a major component of the nuclear lamina. Mutations in the LMNA gene, which encodes the A-type lamins A and C, cause a set of phenotypically diverse diseases collectively called laminopathies. While adult LMNA null mice show various symptoms typically associated with laminopathies, the effect of loss of lamin A/C on early post-natal development is poorly understood. Here we developed a novel LMNA null mouse (LMNAGT−/−) based on genetrap technology and analyzed its early post-natal development. We detect LMNA transcripts in heart, the outflow tract, dorsal aorta, liver and somites during early embryonic development. Loss of A-type lamins results in severe growth retardation and developmental defects of the heart, including impaired myocyte hypertrophy, skeletal muscle hypotrophy, decreased amounts of subcutaneous adipose tissue and impaired ex vivo adipogenic differentiation. These defects cause death at 2 to 3 weeks post partum associated with muscle weakness and metabolic complications, but without the occurrence of dilated cardiomyopathy or an obvious progeroid phenotype. Our results indicate that defective early post-natal development critically contributes to the disease phenotypes in adult laminopathies.
laminopathies; lamin A; LMNA; knock-out mouse; cardiac hypertrophy; muscular dystrophy; differentiation