Because survivin-null embryos die at an early embryonic stage, the role of survivin in thymocyte development is unknown. We have investigated the role by deleting the survivin gene only in the T lineage and show here that loss of survivin blocks the transition from CD4− CD8− double negative (DN) thymocytes to CD4+ CD8+ double positive cells. Although the pre–T cell receptor signaling pathway is intact in survivin-deficient thymocytes, the cells cannot respond to its signals. In response to proliferative stimuli, cycling survivin-deficient DN cells exhibit cell cycle arrest, a spindle formation defect, and increased cell death. Strikingly, loss of survivin activates the tumor suppressor p53. However, the developmental defects caused by survivin deficiency cannot be rescued by p53 inactivation or introduction of Bcl-2. These lines of evidence indicate that developing thymocytes depend on the cytoprotective function of survivin and that this function is tightly coupled to cell proliferation but independent of p53 and Bcl-2. Thus, survivin plays a critical role in early thymocyte development.

The mitochondrial proapoptotic protein Smac/DIABLO has recently been shown to potentiate apoptosis by counteracting the antiapoptotic function of the inhibitor of apoptosis proteins (IAPs). In response to apoptotic stimuli, Smac is released into the cytosol and promotes caspase activation by binding to IAPs, thereby blocking their function. These observations have suggested that Smac is a new regulator of apoptosis. To better understand the physiological function of Smac in normal cells, we generated Smac-deficient (Smac(-/-)) mice by using homologous recombination in embryonic stem (ES) cells. Smac(-/-) mice were viable, grew, and matured normally and did not show any histological abnormalities. Although the cleavage in vitro of procaspase-3 was inhibited in lysates of Smac(-/-) cells, all types of cultured Smac(-/-) cells tested responded normally to all apoptotic stimuli applied. There were also no detectable differences in Fas-mediated apoptosis in the liver in vivo. Our data strongly suggest the existence of a redundant molecule or molecules capable of compensating for a loss of Smac function.

Functional inactivation of the tumor susceptibility gene tsg101 in NIH 3T3 fibroblasts results in cellular transformation and the ability to form metastatic tumors in nude mice. The N-terminal region of tsg101 protein is structurally similar to the catalytic domain of ubiquitin-conjugating enzymes, suggesting a potential role of tsg101 in ubiquitin-mediated protein degradation. The C-terminal domain of TSG101 can function as a repressor of transcription. To investigate the physiological function of tsg101, we generated a null mutation of the mouse gene by gene targeting. Homozygous tsg101-/- embryos fail to develop past day 6.5 of embryogenesis (E6.5), are reduced in size, and do not form mesoderm. Mutant embryos show a decrease in cellular proliferation in vivo and in vitro but no increase in apoptosis. Although levels of p53 transcripts were not affected in tsg101-/- embryos, p53 protein accumulated dramatically, implying altered posttranscriptional control of p53. In addition, transcription of the p53 effector, cyclin-dependent kinase inhibitor p21(WAF-1/CIP-1), was increased 5- to 10-fold, whereas activation of MDM2 transcription secondary to p53 elevation was not observed. Introduction of a p53 null mutation into tsg101-/- embryos rescued the gastrulation defect and prolonged survival until E8.5. These results demonstrate that tsg101 is essential for the proliferative burst before the onset of gastrulation and establish a functional connection between tsg101 and the p53 pathway in vivo.

Bcl10, a CARD-containing protein identified from the t(1;14)(p22;q32) breakpoint in MALT lymphomas, has been shown to induce apoptosis and activate NF-kappaB in vitro. We show that one-third of bcl10-/- embryos developed exencephaly, leading to embryonic lethality. Surprisingly, bcl10-/- cells retained susceptibility to various apoptotic stimuli in vivo and in vitro. However, surviving bcl10-/- mice were severely immunodeficient and bcl10-/- lymphocytes are defective in antigen receptor or PMA/Ionomycin-induced activation. Early tyrosine phosphorylation, MAPK and AP-1 activation, and Ca2+ signaling were normal in mutant lymphocytes, but antigen receptor-induced NF-kappaB activation was absent. Thus, Bcl10 functions as a positive regulator of lymphocyte proliferation that specifically connects antigen receptor signaling in B and T cells to NF-kappaB activation.

The hexosamine pathway provides UDP‐N‐acetylhexosamine donor substrates used in cytosolic and Golgi‐mediated glycosylation of proteins and for formation of glycosylphosphatidylinositol (GPI) anchors, which tether proteins to the outer plasma membrane. We have recently identified the murine glucosamine‐6‐phosphate (GlcN6P) acetyltransferase, EMeg32, as a developmentally regulated enzyme on the route to UDP‐N‐acetylglucosamine (UDP‐GlcNAc). Here we describe embryos and cells that have the EMeg32 gene inactivated by homologous recombination. Homozygous mutant embryos die at around embryonic day (E) 7.5 with a general proliferative delay of development. In vitro differentiated EMeg32−/− ES cells show reduced proliferation. Mouse embryonic fibroblasts (MEFs) deficient for EMeg32 exhibit defects in proliferation and adhesiveness, which could be complemented by stable re‐expression of EMeg32 or by nutritional restoration of intracellular UDP‐GlcNAc levels. Reduced UDP‐GlcNAc levels predominantly translated into decreased O‐GlcNAc modifications of cytosolic and nuclear proteins. Interestingly, growth‐impaired EMeg32−/− MEFs withstand a number of apoptotic stimuli and express activated PKB/AKT. Thus, EMeg32‐dependent UDP‐GlcNAc levels influence cell cycle progression and susceptibility to apoptotic stimuli.

Mutation of Caspase 9 (Casp9) results in embryonic lethality and defective brain development associated with decreased apoptosis. Casp9 (-/-) embryonic stem cells and embryonic fibroblasts are resistant to several apoptotic stimuli, including UV and gamma irradiation. Casp9(-/-) thymocytes are also resistant to dexamethasone- and gamma irradiation-induced apoptosis, but are surprisingly sensitive to apoptosis induced by UV irradiation or anti-CD95. Resistance to apoptosis is accompanied by retention of the mitochondrial membrane potential in mutant cells. In addition, cytochrome c is translocated to the cytosol of Casp9(-/-) ES cells upon UV stimulation, suggesting that Casp9 acts downstream of cytochrome c. Caspase processing is inhibited in Casp9(-/-) ES cells but not in thymocytes or splenocytes. Comparison of the requirement for Casp9 and Casp3 in different apoptotic settings indicates the existence of at least four different apoptotic pathways in mammalian cells.

Mutations in the SMAD4/DPC4 tumor suppressor gene, a key signal transducer in most TGFbeta-related pathways, are involved in 50% of pancreatic cancers. Homozygous Smad4 mutant mice die before day 7.5 of embryogenesis. Mutant embryos have reduced size, fail to gastrulate or express a mesodermal marker, and show abnormal visceral endoderm development. Growth retardation of the Smad4-deficient embryos results from reduced cell proliferation rather than increased apoptosis. Aggregation of mutant Smad4 ES cells with wild-type tetraploid morulae rescues the gastrulation defect. These results indicate that Smad4 is initially required for the differentiation of the visceral endoderm and that the gastrulation defect in the epiblast is secondary and non-cell autonomous. Rescued embryos show severe anterior truncations, indicating a second important role for Smad4 in anterior patterning during embryogenesis.

Mutations of the tumor suppressor gene BRCA2 are associated with predisposition to breast and other cancers. Homozygous mutant mice in which exons 10 and 11 of the Brca2 gene were deleted by gene targeting (Brca2(10-11)) die before day 9.5 of embryogenesis. Mutant phenotypes range from severely developmentally retarded embryos that do not gastrulate to embryos with reduced size that make mesoderm and survive until 8.5 days of development. Although apoptosis is normal, cellular proliferation is impaired in Brca2(10-11) mutants, both in vivo and in vitro. In addition, the expression of the cyclin-dependent kinase inhibitor p21 is increased. Thus, Brca2(10-11) mutants are similar in phenotype to Brca1(5-6) mutants but less severely affected. Expression of either of these two genes was unaffected in mutant embryos of the other. This study shows that Brca2, like Brca1, is required for cellular proliferation during embryogenesis. The similarity in phenotype between Brca1 and Brca2 mutants suggests that these genes may have cooperative roles or convergent functions during embryogenesis.