No abstract

No abstract

Fibroblast cell lines were established from mouse embryos homozygous for a targeted disruption of the lgflr gene, encoding the type 1 receptor for insulin-like growth factor I (IGF-I) and from their wild-type littermates. The cells from the wild-type embryos (W cells) grow in serum-free medium supplemented with platelet-derived growth factor, epidermal growth factor, and IGF-I, whereas the cells from Igflr(-/-) embryos (R-cells) do not, although they grow at a reduced rate in 10% fetal calf serum. The simian virus 40 (SV40) large T antigen, expressed from a transfected plasmid, can trnsform W cells, which form foci in monolayer cultures and colonies in soft agar (anchorage-independent growth). In contrast, the SV40 large tumor antigen, although normally expressed from the transfected template, is unable to transform R-cells, which remain contact-inhibited and fail to grow in soft agar. The transformed phenotype is restored if the R-cells carrying the SV40 large tumor antigen are stably transfected with a plasmid expressing the human IGF-I receptor. These results demonstrate that signaling via the IGF-I receptor is an indispensable component ofthe SV40 transformation pathway. This conclusion is further supported from the results of antisense RNA experiments with tumor cell lines showing that interference with the function of the IGF-I receptor has a profound effect on anchorage-independent growth, even under conditions that only modestly affect growth in monolayers.Interaction of the insulin-like growth factor type I (IGF-I) receptor (IGF-IR) with its ligands (IGF-I, IGF-II, and insulin at supraphysiological concentrations) plays a pivotal role in embryonal development (1) and in the proliferation of several types of cells in culture (2-5). The simian virus 40 (SV40) large tumor antigen (TAg) is one of the most effective transforming agents of mouse cells in culture (6, 7), lowering the growth factor requirements and inducing the ability to grow in soft agar (6,(8)(9)(10)(11) (1), using DNA prepared from their tails. Wild-type and homozygous Igflr(-/-) mutant littermates were used to establish primary cultures of embryonic fibroblasts as described (14). Briefly, the embryos were minced, and after treatment with trypsin for 15 min, the cells of the resulting suspension were plated onto 100-mm culture dishes and cultured in Dulbecco's modified Eagle's medium (DMEM; GIBCO/BRL)/10% fetal bovine serum. The cultures were maintained at subconfluent levels by treating with trypsin every 3 days and reseeding at a density of 1.5 x 103 cells per cm2, following the same protocol used to generate 3T3 cell lines (15). Primary cultures underwent crisis after 2-4 weeks in culture. R-cultures entered crisis later than the wild-type cells due to the relatively slow doubling rate.T98G cells (16) are a human glioblastoma cell line that produces large amounts of IGF-I. Two other cell lines were generated from T98G cells, expressing, respectively, a sense and an antisense RNA to the human IGF-IR RNA. Preparation of the express...

The expansion of CAG triplet repeats in the translated region of the human HD gene, encoding a protein (huntingtin) of unknown function, is a dominant mutation leading to manifestation of Huntington's disease. Targeted disruption of the homologous mouse gene (Hdh), to examine the normal role of huntingtin, shows that this protein is functionally indispensable, since nullizygous embryos become developmentally retarded and disorganized, and die between days 8.5 and 10.5 of gestation. Based on the observation that the level of the regionalized apoptotic cell death in the embryonic ectoderm, a layer expressing the Hdh gene, is much higher than normal in the null mutants, we propose that huntingtin is involved in processes counterbalancing the operation of an apoptotic pathway.

The organization of neurons into columns is a prominent feature of central nervous system structure and function. In many regions of the central nervous system the grouping of neurons into columns links cell-body position to axonal trajectory, thus contributing to the establishment of topographic neural maps. This link is prominent in the developing spinal cord, where columnar sets of motor neurons innervate distinct targets in the periphery. We show here that sequential phases of Hox-c protein expression and activity control the columnar differentiation of spinal motor neurons. Hox expression in neural progenitors is established by graded fibroblast growth factor signalling and translated into a distinct motor neuron Hox pattern. Motor neuron columnar fate then emerges through cell autonomous repressor and activator functions of Hox proteins. Hox proteins also direct the expression of genes that establish motor topographic projections, thus implicating Hox proteins as critical determinants of spinal motor neuron identity and organization.