Lens complementation system for the genetic analysis of growth, differentiation, and apoptosis in vivo.

Abstract

A genetic approach has been established that combines the advantages of blastocyst complementation with the experimental attributes of the developing lens for the functional analysis of genes governing cellular proliferation, terminal differentiation, and apoptosis. This lens complementation system (LCS) makes use of a mutant mouse strain, aphakia (ak), homozygotes of which fail to develop an ocular lens. We demonstrate that microinjection of wild-type embryonic stem (ES) cells into ak/ak blastocysts produces chimeras with normal ES-cell-derived lenses and that microinjection of Rb-/-ES cells generates an aberrant lens phenotype identical to that obtained through conventional gene targeting methodology. Our determination that a cell autonomous defect underlies the aphakia condition assures that lenses generated through LCS are necessarily ES-cell-derived. LCS provides for the rapid phenotypic analysis of loss-of-function mutations, circumvents the need for germ-line transmission of null alleles, and, most significantly, facilitates the study of essential genes whose inactivation is associated with early lethal phenotypes.The capacity to manipulate the genetic composition of the mouse has allowed for significant advances in the study of developmental and cancer biology. Much of this progress is the result of methods that permit the precise structural alteration of genes through homologous recombination in embryonic stem (ES) cells followed by germ-line transmission of these targeted alleles through chimera formation (1). Successful implementation of gene targeting requires maintenance of ES-cell pluripotency to passage the mutant allele through the germ line. A potential impediment to the analysis of loss-offunction mutations generated through the classical knockout approach is attendant embryonic lethality. This outcome is particularly problematic in cancer-relevant models wherein early lethality contracts the time available for the emergence of additional genetic lesions that cooperate to bring about a fully transformed state. Procedural modifications developed to circumvent some of these obstacles include the generation of chimeras from homozygous null ES cells (2) and the use of a modified Cre-LoxP approach for the production of a celltype-specific nullizygous condition (3). However, the former approach requires a detailed in situ .assessment of whether tissues of the chimera are ES-or host blastocyst-derived, and the latter can be complicated by mosaicism caused by inefficient Cre-mediated deletion (A. Nagy, personal communication).To overcome these problems, Chen et al. (4) developed a method for evaluating gene function in the immune system that makes use of host blastocysts derived from RAG-2-deficient mice, which are incapable of variable-diversity-joining (VDJ) recombination and thus lack mature B and T lymphocytes. Injection of normal or genetically modified ES cells into theseThe publication costs of this article were defrayed in part by page charge payment. This article must therefore be here...

An essential role for FGF receptor signaling in lens development

“…One of these involved the production of chimeric embryos by the aggregation of LacZ marked FGFR1 null ES cells with embryos homozygous for the aphakia mutation. This lens complementation system had previously been shown to result in completely ES cell derived lenses in chimeric mice [141]. In a few cases, chimeric embryos developed morphologically normal FGFR1 null lenses displaying typical expression patterns for α-, β-, and γ-crystallins [142].…”

Cooperation between the Cdk inhibitors p27KIP1 and p57KIP2 in the control of tissue growth and development

“…Formation of this structure involves spatially controlled proliferation and differentiation events that are dependent on the retinoblastoma (Rb) gene product, a critical target of Cdks. Loss of Rb leads to defects in cell cycle arrest and differentiation, as well as increased p53-dependent apoptosis (Morgenbesser et al 1994;Liegeois et al 1996). In other differentiation systems such as skeletal muscle, Rb appears to play a dual role; it acts as a growth suppressor facilitating G 1 arrest and is also required for activating the transcriptional program that brings about differentiation (for review, see Mulligan and Jacks 1998), possibly through physical association with critical transcription factors (Gu et al 1993;Chen et al 1996;Nead et al 1998).…”

Analysis of mouse eye development with chimeras and mosaics

“…There was failure of Rb -/-lens cell differentiation in the chimeras, and ectopic proliferation of retinoblasts, suggesting a failure of Rb -/-cells to respond to differentiation and growth-arrest signals. Liégeois et al (1996) used their 'lens complementation assay' described above to investigate the role of Rb in the lens.…”

“…The Tg(Hbb-b1)83Clo β-globin transgene (incorporated into the genome as approximately a thousand contiguous copies) was used by Liégeois et al (1996) in examining the action of the recessive aphakia (ak) mutation. Homozygous ak/ak mice do not develop lenses and ak is almost certainly an allele of Pitx3 (Semina et al, 2000;Rieger et al, 2001).…”