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Gas2 Is a Multifunctional Gene Involved in the Regulation of Apoptosis and Chondrogenesis in the Developing Mouse Limb

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Gas2 Is a Multifunctional Gene Involved in the Regulation of Apoptosis and Chondrogenesis in the Developing Mouse Limb Developmental Biology 207, 14–25 (1999) Article ID dbio.1998.9086, available online at http://www.idealibrary.com on View metadata, citation and similar papers at core.ac.uk brought to you by CORE provided by Elsevier - Publisher Connector gas2 Is a Multifunctional Gene Involved in the Regulation of Apoptosis and Chondrogenesis in the Developing Mouse Limb K. K. H. Lee,*,1,2 M. K. Tang,* D. T. W. Yew,* P. H. Chow,* S. P. Yee,† C. Schneider,‡,§ and C. Brancolini‡,§,1 *Department of Anatomy, Faculty of Medicine, Chinese University of Hong Kong, Shatin, Hong Kong, People’s Republic of China; †Cancer Research Laboratories, London Regional Cancer Centre, 790 Commissioners Road East, London, Ontario, Canada; ‡Laboratorio Nazionale Consorzio Interuniversitario Biotecnologie Area, Science Park Padriciano 99, Trieste, Italy; and §Dipartimento Scienze e Tecnologie Biomediche Facolta’ di Medicina, University of Udine, p. Kolbe 4, Udine, Italy The growth-arrest-specific 2 (gas2) gene was initially identified on account of its high level of expression in murine fibroblasts under growth arrest conditions, followed by downregulation upon reentry into the cell cycle (Schneider et al., Cell 54, 787–793, 1988). In this study, the expression patterns of the gas2 gene and the Gas2 peptide were established in the developing limbs of 11.5- to 14.5-day mouse embryos. It was found that gas2 was expressed in the interdigital tissues, the chondrogenic regions, and the myogenic regions. Low-density limb culture and Brdu incorporation assays revealed that gas2 might play an important role in regulating chondrocyte proliferation and differentiation. Moreover, it might play a similar role during limb myogenesis. In addition to chondrogenesis and myogeneis, gas2 is involved in the execution of the apoptotic program in hindlimb interdigital tissues—by acting as a death substrate for caspase enzymes. TUNEL analysis demonstrated that the interdigital tissues underwent apoptosis between 13.5 and 15.5 days. Exactly at these time points, the C-terminal domain of the Gas2 peptide was cleaved as revealed by Western blot analysis. Moreover, pro-caspase-3 (an enzyme that can process Gas2) was cleaved into its active form in the interdigital tissues. The addition of zVAD-fmk, a caspase enzyme inhibitor, to 12.5-day-old hindlimbs maintained in organ culture revealed that the treatment inhibited interdigital cell death. This inhibition correlated with the absence of the Gas2 peptide and pro-caspase-3 cleavage. The data suggest that Gas2 might be involved in the execution of the apoptotic process. © 1999 Academic Press Key Words: apoptosis; chondrogenesis; growth-arrest-specific-2 gene; mouse limb. INTRODUCTION carboxyl-terminal domain by caspase enzymes. These aspartic-specific cysteine protease are the principal effec- gas2 is a member of a family of six genes that are highly tors of the apoptotic program, by cleaving specific cellular expressed in 3T3 fibroblasts during growth arrest (Schneider targets or death substrates (reviewed by Martin and Green, et al., 1988; Brancolini et al., 1992). The products of these 1995; Jacobson et al., 1997). Removal of the carboxyl- genes regulate both negatively and positively cell prolifera- terminal domain of Gas2 dramatically reorganizes the mi- tion and apoptosis (Del Sal et al., 1992; Fabbretti et al., crofilament system and as a result unveils a potent change 1995; Goruppi et al., 1997). The product of gas2 is a in the shape of the affected cells. Therefore, the processing component of the microfilament system and is now be- of Gas2 during apoptosis by caspase enzymes could play an lieved to be a cell death substrate (Brancolini et al., 1992, important role in mediating the complex morphological 1995). During apoptosis, the Gas2 peptide is processed at its changes that characterize cell death. Furthermore, Gas2 is a promising death substrate since its expression is also 1 Both authors contributed equally to this work. coupled to increased susceptibility to apoptosis (Brancolini 2 To whom correspondence should be addressed. Fax: (852) 2603 et al., 1997a). The relationships between microfilament 5031. E-mail: [email protected]. organization and Gas2 have also been demonstrated during 0012-1606/99 $30.00 Copyright © 1999 by Academic Press 14 All rights of reproduction in any form reserved. gas2 in Limb Development 15 G0 3 G1 transition of the cell cycle. Gas2 is rapidly MATERIALS AND METHODS phosphorylated after treatment of growth-arrested cells with various growth factors and the hyperphosphorylated Embryos form of Gas2 is relocalized to the membrane ruffles formed Day-10.5 to Day-14.5 embryos were obtained from pregnant ICR at the edges of the cells (Brancolini and Schneider, 1994). In mice (Chinese University of Hong Kong). The presence of a vaginal vitro studies implicated another potential role for Gas2, in plug was designated as embryonic day 0.5. The mice were killed by the regulation of keratinocytes (Manzow et al., 1996) and cervical dislocation and the embryos were isolated from the de- muscle (Cowled et al., 1994) differentiation. cidua in prewarmed Dulbecco’s phosphate-buffered saline contain- Although a great deal is now known of the physiological ing 0.4% bovine serum albumin (PB1; Sigma, St. Louis, MO). functions of gas2 in vitro, little remains known of its Whole embryos and hindlimbs isolated from embryos were either functional role in vivo. Therefore, we have examined the fixed in 4% cold paraformaldehyde and then used for in situ hybridization studies or directly used for culture studies. expression pattern of gas2 in the mouse embryonic limb during development. The vertebrate limb has traditionally been a favorite model for studying apoptosis (Lee et al., Cell Culture 1993; Zou and Niswander, 1996; Ganan et al., 1996), The distal regions of 12.5-day forelimbs were harvested from chondrogenesis (Fell, 1935; Zimmermann et al., 1982; embryos in PB1 medium. After rinsing the limb fragments with Zanetti and Solursh, 1984; Lee et al., 1994; Goff and Tabin, PBS, they were dissociated by incubation with 0.5% trypsin and 1997), myogenesis (Lee and Sze, 1993; Bladt et al., 1995; 0.25% pancreatin in MEM–Hepes medium (Sigma) containing Webb et al., 1997), tendon development (Patel et al., 1996), 2.2% sodium bicarbonate, for 30 min at 4°C then for 10 min at and tissue patterning (reviewed in Cohn and Tickle, 1996). 37°C followed by trituration with a Pasteur pipet to mechanically disrupt the tissues. The enzymatic reaction was inhibited by the The amenability of the limb to experimental manipulation addition of 10% fetal bovine serum (FBS; Gibco BRL) and all and the vast accumulation of information currently avail- undissociated clumps of cells were removed by filtration through a able regarding the cellular and molecular basis of its devel- nylon filter (21-mm pore size) to produce a single-cell suspension. opment have also made the limb an ideal model for explor- The dissociated cells were pelleted by centrifugation at 250g for 3 ing the developmental function of newly identified genes. min and then resuspended in Dulbecco’s modified essential me- In this study, we have focused mainly on two aspects of dium (DMEM, Sigma) containing 10% FBS. Cell concentration was limb development, interdigital cell death and chondrogen- established with an improved Neubauer hemocytometer and vi- ability was assessed with Trypan blue. The cells were plated out at esis, because gas2 has been implicated in apoptosis, cell 5 5 3 10 cells/ml onto 0.1% gelatin-coated coverslips housed inside growth arrest, and differentiation in vitro. The spatial and four-well tissue culture plates. The cultures were maintained at temporal patterns of programmed cell death in the inter- 37°C and 5% CO2 for 1–7 days. Brdu was added to some of the digital regions of 12.5- to 14.5-day-old mouse autopodia are cultures, 6 h before harvest, so that the extent of cell proliferation now well established (Lee et al., 1994). Bmp-2, -4, and -7 are in these cultures could be established (Brdu cell proliferation kit, now believed to be the initiation signals for the induction of Amersham Life Sciences). Fluorescently-labeled peanut agglutinin interdigital cell death (Zou and Niswander, 1996; Ganan et (TRITC-PNA, Sigma) was used to confirm the presence of chon- drogenic cells in the cultures. PNA is the earliest known marker for al., 1996; Macias et al., 1997). However, no genes have yet chondrogenic cells (Stringa and Tuan, 1996). Briefly, the cultures been identified to be directly involved in the execution of were fixed for1hincold 70% ethanol and then stained with 1 the apoptotic program in the limb. gas2 is a good candidate mg/ml of TRITC-PNA for 30 min at room temperature. The for this role by acting as a death substrate that could be specimens were washed several times in PBS and viewed under a activated by caspase processing (Brancolini et al., 1995; confocal microscope. Porter et al., 1997). The development of cartilage in the limb involves a series of Organ Culture precisely timed acts that include cell condensation, cell move- ment, changes in cell shape, and programmed cell death (Fell, Intact 12.5-day hindlimbs were isolated and placed on a piece of sterile Millipore filter (0.22 mm, Millipore, Bedford, MA) mounted 1935; Ede and Wilby, 1981; Zimmermann et al., 1982; on a non-toxic stainless mesh inside the central well of an organ Solursh, 1989a; Lewinson and Silbemann, 1992). These mor- culture dish (Falcon, Lincon Park, NJ). BGJb was introduced into phogenetic events are accompanied by alteration in the ar- the central well while sterile distal water was placed in the outer rangement of the cell’s cytoskeleton. It has been revealed that well. Some of the limbs were cultured in the presence of 100 mM limb mesenchyme cultured in the presence of cytochalasin D, Z-Val-Ala-Asp-fluoromethylketone (zVAD-fmk, Calbiochem) caspase a drug that disrupts the organization of actin fibers, causes inhibitor.
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