Functions of the growth arrest specific 1 gene in the development of the mouse embryo

The growth arrest specific 1 (gas1) gene is highly expressed in quiescent m ammalian cells (Schneider et al., 1988, Cell 54, 787-793). Overexpression o f gas1 in normal and some cancer cell lines could inhibit G(0)/G(1) transit ion. Presently, we have examined the functions of this gene in the developi ng mouse embryo. The spatial-temporal expression patterns for gas1 were est ablished in 8.5- to 14.5-day-old embryos by immunohistochemical staining an d in situ hybridization. Gas1 was found heterogeneously expressed in most o rgan systems including the brain, heart, kidney, limb, lung, and gonad. The antiproliferative effects of gas1 on 10.5 and 12.5 day limb cells were inv estigated by flow cytometry. In 10.5 day limbs cells, gas1 overexpression c ould not prevent G(0)/G(1) progression. It was determined that gas1 could o nly induce growth arrest if p53 was also coexpressed. In contrast, gas1 ove rexpression alone was able to induce growth arrest in 12.5 day limb cells. We also examined the cell cycle profile of gas1-expressing and nonexpressin g cells by immunochemistry and flow cytometry. For 10.5 day Gas1-expressing heart and limb cells, we did not find these cells preferentially distribut ed at G(0)/G(1), as compared with Bas1-negative cells. However, in the 12.5 day heart and limb, we did find significantly more Gas1-expressing cells d istributed at G(0)/G(1) phase than Gas1-negative cells. These results impli ed that Gas1 alone, during the early stages of development, could not inhib it cell growth. This inhibition was only established when the embryo grew o lder. We have overexpressed gas1 in subconfluent embryonic limb cells to de termine the ability of gas1 to cross-talk with various response elements of important transduction pathways. Specifically, we have examined the intera ction of gas1 with Ap-l, NF kappaB, and c-myc responsive elements tagged wi th a SEAP reporter. In 10.5 day limb cells, gas1 overexpression had little effect on Ap-l, NF kappaB, and c-myc activities. In contrast, gas1 overexpr ession in 12.5 day limb cells enhanced AP-1 response while it inhibited NF kappaB and c-myc activities. These responses were directly associated with the ability of gas1 to induce growth arrest in embryonic limb cells. In the 12.5 day hindlimb, gas1 was found strongly expressed in the interdigital t issues. We overexpressed gas1 in these tissues and discovered that it promo ted interdigital cell death. Our in situ hybridization studies of limb sect ions and micromass cultures revealed that, during the early stages of chond rogenesis, only cells surrounding the chondrogenic condensations expressed gas1. The gene was only expressed by chondrocytes after the cartilage start ed to differentiate. To understand the function of gas1 in chondrogenesis, we overexpressed the gene in limb micromass cultures. It was found that cel ls overexpressing gas1/GFP could not participate in cartilage formation, un like cells that lust express the GFP reporter. We speculated that the reaso n gas1 was expressed outside the chondrogenic nodules was to restrict cells from being recruited into the nodules and thereby defining the boundary be tween chondrogenic and nonchondrogenic forming regions. (C) 2001 Academic P ress.