Transgenic mammals in the biology of development

Cells and chromosomes of the embryo can be labeled by integrating foreign DNA into its genome. Genes coding for some cytotoxic products can selectively destroy embryonic rudiments in which these genes are expressed. Insertional mutations arise in transgenic animals and this facilitates identification and cloning of the genes that affect the development. Embryos carrying mutations in certain genes were produced using transformed stem cells. Some kinds of mutations can be corrected by introducing the cloned genes into mutant oocytes. Use of transgenic animals substantially increases the properties of methods employed in developmental genetics and experimental embryology of mammals.     

Cancer initiation and progression: involvement of stem cells and the microenvironment

The molecular events that lead to the cancer-initiating cell involve critical mutations in genes regulating normal cell growth and differentiation. Cancer stem cells, or cancer initiating cells have been described in the context of acute myeloid leukemia, breast, brain, bone, lung, melanoma and prostate. These cells have been shown to be critical in tumor development and should harbor the mutations needed to initiate a tumor. The origin of the cancer stem cells is not clear. They may be derived from stem cell pools, progenitor cells or differentiated cells that undergo trans-differentiation processes. It has been suggested that cell fusion and/or horizontal gene transfer events, which may occur in tissue repair processes, also might play an important role in tumor initiation and progression. Fusion between somatic cells that have undergone a set of specific mutations and normal stem cells might explain the extensive chromosomal derangements seen in early tumors. Centrosome deregulation can be an integrating factor in many of the mechanisms involved in tumor development. The regulation of the balance between cell renewal and cell death is critical in cancer. Increased knowledge of developmental aspects in relation to self-renewal and differentiation, both under normal and deregulated conditions, will probably shed more light on the mechanisms that lead to tumor initiation and progression.     

Generation of multipurpose alleles for the functional analysis of the mouse genome.

A novel generation of retroviral gene-trap vectors has been developed with the ability to induce conditional mutations in most genes expressed in mouse embryonic stem (ES) cells. The vectors rely on directional site-specific recombination systems, which can repair and re-induce the gene-trap mutations when activated in succession. After the gene-trap insertions are passaged into mice, this system enables the induction of temporally and spatially restricted mutations in somatic cells. In addition to their conditional features, the vectors create multipurpose alleles amenable to a wide range of post-insertional modifications. These vectors have been used to assemble the largest library of ES cell lines with conditional mutations in single genes, presently totalling 1724 unique genes. Due to their efficiency, the vectors are part of the core technologies to be used by EUCOMM for establishing a complete collection of conditional null mutations in mice.     

Insertional versus targeted mutagenesis in mice.

Recent innovations in mutagenesis techniques for mice have the potential to revolutionize the molecular genetic analysis of mouse development. Insertional mutagenesis by the introduction of exogenous DNA into the mouse germline hs permitted the molecular cloning and analysis of several novel genes important for early embryonic development. Targeted mutagenesis by homologous recombination in embryonic stem cells permits, in theory, the production of mutations in any cloned gene. The complementary information being obtained from these two mutagenesis procedures is shedding new light on the genes important for early mouse development, and the roles these genes play in that process.     

DNA-mediated genetic transformation of mouse embryos and bone marrow--a review

In recent years, new gene transfer systems have been developed which allow molecularly cloned genetic material to be introduced into whole organisms. These systems include the microinjection of DNA into mammalian embryos, transfection of DNA into mouse bone marrow cells, and the infection of early embryos with retroviruses. Exogenous DNA appears to integrate randomly into the host genome. The production of transgenic mice by injection of DNA into mouse embryos has rapidly gained importance as an experimental tool for the study of gene regulation during development. Through this technique, recombinant molecules of any type can be introduced into one-celled embryos, and thus can be used to study development from its earliest stages. DNA sequences have been shown to integrate and transmit through the germ line to subsequent generations as mendelian traits. Transgenic mice carrying various gene constructs have been successfully exploited for the elucidation of factors which determine tissue specificity of gene expression as well as the level of gene control. Phenotypic changes related to expression of foreign genes have also been observed. This experimental approach thus promises to rapidly solve many of the heretofore most challenging problems in developmental genetics. Insertion of foreign genes has also made possible the creation of insertional mutants which manifest themselves most frequently as recessives. Such mutations can be readily studied at the molecular level by using the transferred material as a probe for recovery of the affected host sequence from genomic libraries. Many of these same problems have been addressed by introducing retroviral DNA into mouse embryos. Here, the sequences used for transfer have been limited to retroviral genes, but nonetheless these experiments have been profitably exploited for studies both of gene regulation and mutagenesis. Gene transfer systems are being developed allowing the experimenter to transfer DNA into bone marrow cells of mice, after which the recipient cells can be reintroduced into lethally irradiated histocompatible animals. This system has the advantage that selection can be applied during the gene transfer process such that the expression of the foreign material is assured. In addition, these experiments have created a model system for production of animals carrying a subpopulation of cells which is highly resistant to a toxic agent. This system has the potential for therapeutic application to man.     

Gene targeting and gene trap screens using embryonic stem cells: new approaches to mammalian development.

Mouse embryonic stem cell lines offer an attractive route for introducing rare genetic alternations into the gene pool since the cells can be pre-screened in culture and the mutations then transmitted into the germline through chimera production. Two applications of this technique that seem ideally suited for a genetic analysis of development are enhancer and gene trap screens for loci expressed during gastrulation and production of targeted mutations using homologous recombination. These approaches should greatly increase the number of mouse developmental mutants available and help to elucidate the genetic hierarchy controlling embryogenesis.     

New Components of Drosophila Leg Development Identified through Genome Wide Association Studies

The adult Drosophila melanogaster body develops from imaginal discs, groups of cells set-aside during embryogenesis and expanded in number during larval stages. Specification and development of Drosophila imaginal discs have been studied for many years as models of morphogenesis. These studies are often based on mutations with large developmental effects, mutations that are often lethal in embryos when homozygous. Such forward genetic screens can be limited by factors such as early lethality and genetic redundancy. To identify additional genes and genetic pathways involved in leg imaginal disc development, we employed a Genome Wide Association Study utilizing the natural genetic variation in leg proportionality found in the Drosophila Genetic Reference Panel fly lines. In addition to identifying genes already known to be involved in leg development, we identified several genes involved in pathways that had not previously been linked with leg development. Several of the genes appear to be involved in signaling activities, while others have no known roles at this time. Many of these uncharacterized genes are conserved in mammals, so we can now begin to place these genes into developmental contexts. Interestingly, we identified five genes which, when their function is reduced by RNAi, cause an antenna-to-leg transformation. Our results demonstrate the utility of this approach, integrating the tools of quantitative and molecular genetics to study developmental processes, and provide new insights into the pathways and networks involved in Drosophila leg development.     

Epigenetic stability of embryonic stem cells and developmental potential

Recent studies highlight the tremendous potential of human embryonic stem (ES) cells and their derivatives as therapeutic tools for degenerative diseases. However, derivation and culture of ES cells can induce epigenetic alterations, which can have long lasting effects on gene expression and phenotype. Research on human and mouse stem cells indicates that developmental, cancer-related genes, and genes regulated by genomic imprinting are particularly susceptible to changes in DNA methylation. Together with the occurrence of genetic alterations, epigenetic instability needs to be monitored when considering human stem cells for therapeutic and technological purposes. Here, we discuss the maintenance of epigenetic information in cultured stem cells and embryos and how this influences their developmental potential.     

Enhanced gene trapping in mouse embryonic stem cells

Gene trapping is used to introduce insertional mutations into genes of mouse embryonic stem cells (ESCs). It is performed with gene trap vectors that simultaneously mutate and report the expression of the endogenous gene at the site of insertion and provide a DNA tag for rapid identification of the disrupted gene. Gene traps have been employed worldwide to assemble libraries of mouse ESC lines harboring mutations in single genes, which can be used to make mutant mice. However, most of the employed gene trap vectors require gene expression for reporting a gene trap event and therefore genes that are poorly expressed may be under-represented in the existing libraries. To address this problem, we have developed a novel class of gene trap vectors that can induce gene expression at insertion sites, thereby bypassing the problem of intrinsic poor expression. We show here that the insertion of the osteopontin enhancer into several conventional gene trap vectors significantly increases the gene trapping efficiency in high-throughput screens and facilitates the recovery of poorly expressed genes.     

Proto-oncogenes in mammalian development.

The phenotypic analysis of mice carrying germline mutations in protooncogenes is beginning to provide convincing genetic evidence for the important role that these genes play in mammalian development and differentiation. Two approaches are being taken to elucidate the biological function of proto-oncogenes in vivo. The first involves the molecular analysis of existing mouse developmental mutants, while the second approach involves the generation of specific germline mutations by gene targeting using homologous recombination in embryonic stem cells. Several key points have already emerged from these genetic approaches. First, many proto-oncogenes are important to more than one cell lineage and function both during embryogenesis and in the adult. Second, the patterns of expression of these genes provide only a guide to their biological function. Third, mutant phenotypes are generally less severe than would be expected from their expression patterns, suggesting that there may be functional overlap between two or more members of a gene family.     

Neutrality, compensation, and negative selection during evolution of B-cell development transcriptomes

B cells develop in the mammalian bone marrow through a sequence of precursor stages, which can be ordered by the recombination status of their immunoglobulin loci. This developmental pathway is functionally similar between mice and man. However, whether this similarity is based on usage of the same genes is unknown. We show that large-scale gene expression patterns differ substantially between human and mouse B-cell development. Among 644 genes which were differentially expressed in 4 early stages of human B-cell development, only 48, 86, and 75 genes could be identified, which are upregulated in both human and mouse pre-BI, large pre-BII, and small pre-BII cells, respectively. A comparison of mouse B- and T-cell development reveals that gene expression patterns of early murine B- and T-cell precursors are most similar, whereas in more differentiated precursors, human and mouse B cells have a more similar gene expression profile. We conclude that large-scale differences in gene expression patterns between human and mouse B-cell precursors may stem from either selective neutrality or compensatory evolution, whereas the few similarities may stem from negative selection. Gene expression patterns are shaped by ontogenic relationships in early and by functional specialization in later stages of development.     

Cancer stem cells: lessons from leukaemia

Increasing evidence suggests that leukaemias are sustained by leukaemic stem cells. Leukaemia can indeed be viewed as aberrant haematopoietic processes initiated by rare leukaemic stem cells that have maintained or re-acquired the capacity for indefinite proliferation through accumulated mutations and/or epigenetic changes. Yet, despite their critical importance, much remains to be learned about the developmental origin of leukaemic stem cells and the molecular pathways underlying the transformation of normal cells into leukaemic stem cells. This report will review our current knowledge on leukaemic stem cells development and finally demonstrate how these discoveries provide a paradigm for identification of cancer stem cells from solid tumours.     

多能干细胞体外分化为类神经管模型的研究进展

近年来多能干细胞(PSCs)的体外培养与分化技术发展迅速,并广泛应用于再生医学和发育生物学等领域。PSCs能够在体外神经诱导的条件下分化为类神经管模型,这为探索体内早期神经发育与中枢神经系统发育疾病的形成机制提供了全新的实验平台。本文总结了近年来应用小鼠和人PSCs建立体外类神经管模型的研究进展,其中体外模型主要包括在不同培养体系下诱导获得的二维(2D)与三维(3D)类神经管模型,并针对早期类神经管模型在神经系统发育性疾病机制研究中的前景和挑战作进一步探讨,同时为疾病预防和治疗提供新的思路。     

Genetic mosaic techniques for studying Drosophila development

Genetic screens for recessive mutations continue to provide the basis for much of the modern work on Drosophila developmental genetics. However, many of the mutations isolated in these screens cause embryonic or early larval lethality. Studying the effects of such mutations on later developmental events is still possible, however, using genetic mosaic techniques, which limit losses or gains of genetic function to specific tissues and cells, and to selected stages of development. A variety of genetic mosaic techniques have been developed, and these have led to key insights into developmental processes in the fly. Variations on these techniques can also be used to screen for novel genes that are involved in non-embryonic patterning and growth.     

From mouse to man: generating megabase chromosome rearrangements

Experimental approaches for deciphering the function of human genes rely heavily on our ability to generate mutations in model organisms such as the mouse. However, because recessive mutations are masked by the wild-type allele in the diploid context, conventional mutagenesis and screening is often laborious and costly. Chromosome engineering combines the power of gene targeting in embryonic stem (ES) cells with Cre--loxP technology to create mice that are functionally haploid in discrete portions of the genome. Chromosome deletions, duplications and inversions can be tagged with visible markers, facilitating strain maintenance. These approaches allow for more refined mutagenesis screens that will greatly accelerate functional mouse genomics and generate mammalian models for developmental processes and cancer.     

Embryonic stem cell differentiation models: cardiogenesis, myogenesis, neurogenesis, epithelial and vascular smooth muscle cell differentiation in vitro

Embryonic stem cells, totipotent cells of the early mouse embryo, were established as permanent cell lines of undifferentiated cells. ES cells provide an important cellular system in developmental biology for the manipulation of preselected genes in mice by using the gene targeting technology. Embryonic stem cells, when cultivated as embryo-like aggregates, so-called ‘embryoid bodies’, are able to differentiate in vitro into derivatives of all three primary germ layers, the endoderm, ectoderm and mesoderm. We established differentiation protocols for the in vitro development of undifferentiated embryonic stem cells into differentiated cardiomyocytes, skeletal muscle, neuronal, epithelial and vascular smooth muscle cells. During differentiation, tissue-specific genes, proteins, ion channels, receptors and action potentials were expressed in a developmentally controlled pattern. This pattern closely recapitulates the developmental pattern during embryogenesis in the living organism. In vitro, the controlled developmental pattern was found to be influenced by differentiation and growth factor molecules or by xenobiotics. Furthermore, the differentiation system has been used for genetic analyses by ‘gain of function’ and ‘loss of function’ approaches in vitro. This revised version was published online in July 2006 with corrections to the Cover Date.     

The identification of new genes: Gene trapping in transgenic mice

Ongoing efforts to clone, sequence and map genes in the mouse have far exceeded our ability to define their functional role. The generation of mutations is an important first step towards understanding the function of genes in normal mouse development and physiology. Gene trapping in embryonic stem cells provides an efficient method to identify, clone and mutate genes at random, permitting the functional analysis of new genes in mice.