Cell Death and the p53 Enigma During Mammalian Embryonic Development.

Twelve forms of programmed cell death (PCD) have been described in mammalian cells, but which of them occurs during embryonic development and the role played by the p53 transcription factor and tumor suppressor remains enigmatic. Although p53 is not required for mouse embryonic development, some studies conclude that PCD in pluripotent embryonic stem cells from mice (mESCs) or humans (hESCs) is p53-dependent whereas others conclude that it is not. Given the importance of pluripotent stem cells as models of embryonic development and their applications in regenerative medicine, resolving this enigma is essential. This review reconciles contradictory results based on the facts that p53 cannot induce lethality in mice until gastrulation and that experimental conditions could account for differences in results with ESCs. Consequently, activation of the G2-checkpoint in mouse ESCs is p53-independent and generally, if not always, results in noncanonical apoptosis. Once initiated, PCD occurs at equivalent rates and to equivalent extents regardless of the presence or absence of p53. However, depending on experimental conditions, p53 can accelerate initiation of PCD in ESCs and late-stage blastocysts. In contrast, DNA damage following differentiation of ESCs in vitro or formation of embryonic fibroblasts in vivo induces p53-dependent cell cycle arrest and senescence.

Cell cycle arrest and apoptosis are not dependent on p53 prior to p53-dependent embryonic stem cell differentiation.

Previous efforts to determine whether or not the transcription factor and tumor suppressor protein p53 is required for DNA damage-induced apoptosis in pluripotent embryonic stem cells (ESCs) produced contradictory conclusions. To resolve this issue, p53+/+ and p53-/- ESCs derived by two different methods were used to quantify time-dependent changes in nuclear DNA content; annexin-V binding; cell permeabilization; and protein expression, modification, and localization. The results revealed that doxorubicin (Adriamycin [ADR]) concentrations 10 to 40 times less than commonly used in previous studies induced the DNA damage-dependent G2-checkpoint and completed apoptosis within the same time frame, regardless of the presence or absence of p53, p21, and PUMA. Increased ADR concentrations delayed initiation of apoptosis in p53-/- ESCs, but the rates of apoptosis remained equivalent. Similar results were obtained by inducing apoptosis with either staurosporine inhibition of kinase activities or WX8 disruption of lysosome homeostasis. Differentiation of ESCs by LIF deprivation revealed p53-dependent formation of haploid cells, increased genomic stability, and suppression of the G2-checkpoint. Minimal induction of DNA damage now resulted in p53-facilitated apoptosis, but regulation of pluripotent gene expression remained p53-independent. Primary embryonic fibroblasts underwent p53-dependent total cell cycle arrest (a prelude to cell senescence), and p53-independent apoptosis occurred in the presence of 10-fold higher levels of ADR, consistent with previous studies. Taken together, these results reveal that the multiple roles of p53 in cell cycle regulation and apoptosis are first acquired during pluripotent stem cell differentiation.

And-1 coordinates with Claspin for efficient Chk1 activation in response to replication stress.

The replisome is important for DNA replication checkpoint activation, but how specific components of the replisome coordinate with ATR to activate Chk1 in human cells remains largely unknown. Here, we demonstrate that And-1, a replisome component, acts together with ATR to activate Chk1. And-1 is phosphorylated at T826 by ATR following replication stress, and this phosphorylation is required for And-1 to accumulate at the damage sites, where And-1 promotes the interaction between Claspin and Chk1, thereby stimulating efficient Chk1 activation by ATR. Significantly, And-1 binds directly to ssDNA and facilitates the association of Claspin with ssDNA. Furthermore, And-1 associates with replication forks and is required for the recovery of stalled forks. These studies establish a novel ATR-And-1 axis as an important regulator for efficient Chk1 activation and reveal a novel mechanism of how the replisome regulates the replication checkpoint and genomic stability.

TEAD4 establishes the energy homeostasis essential for blastocoel formation.

It has been suggested that during mouse preimplantation development, the zygotically expressed transcription factor TEAD4 is essential for specification of the trophectoderm lineage required for producing a blastocyst. Here we show that blastocysts can form without TEAD4 but that TEAD4 is required to prevent oxidative stress when blastocoel formation is accompanied by increased oxidative phosphorylation that leads to the production of reactive oxygen species (ROS). Both two-cell and eight-cell Tead4(-/-) embryos developed into blastocysts when cultured under conditions that alleviate oxidative stress, and Tead4(-/-) blastocysts that formed under these conditions expressed trophectoderm-associated genes. Therefore, TEAD4 is not required for specification of the trophectoderm lineage. Once the trophectoderm was specified, Tead4 was not essential for either proliferation or differentiation of trophoblast cells in culture. However, ablation of Tead4 in trophoblast cells resulted in reduced mitochondrial membrane potential. Moreover, Tead4 suppressed ROS in embryos and embryonic fibroblasts. Finally, ectopically expressed TEAD4 protein could localize to the mitochondria as well as to the nucleus, a property not shared by other members of the TEAD family. These results reveal that TEAD4 plays a crucial role in maintaining energy homeostasis during preimplantation development.

Geminin is Essential to Prevent DNA Re-Replication-Dependent Apoptosis in Pluripotent Cells, but not in Differentiated Cells.

Geminin is a dual-function protein unique to multicellular animals with roles in modulating gene expression and preventing DNA re-replication. Here, we show that geminin is essential at the beginning of mammalian development to prevent DNA re-replication in pluripotent cells, exemplified by embryonic stem cells, as they undergo self-renewal and differentiation. Embryonic stem cells, embryonic fibroblasts, and immortalized fibroblasts were characterized before and after geminin was depleted either by gene ablation or siRNA. Depletion of geminin under conditions that promote either self-renewal or differentiation rapidly induced DNA re-replication, followed by DNA damage, then a DNA damage response, and finally apoptosis. Once differentiation had occurred, geminin was no longer essential for viability, although it continued to contribute to preventing DNA re-replication induced DNA damage. No relationship was detected between expression of geminin and genes associated with either pluripotency or differentiation. Thus, the primary role of geminin at the beginning of mammalian development is to prevent DNA re-replication-dependent apoptosis, a role previously believed essential only in cancer cells. These results suggest that regulation of gene expression by geminin occurs only after pluripotent cells differentiate into cells in which geminin is not essential for viability.

The dual roles of geminin during trophoblast proliferation and differentiation.

Geminin is a protein involved in both DNA replication and cell fate acquisition. Although it is essential for mammalian preimplantation development, its role remains unclear. In one study, ablation of the geminin gene (Gmnn) in mouse preimplantation embryos resulted in apoptosis, suggesting that geminin prevents DNA re-replication, whereas in another study it resulted in differentiation of blastomeres into trophoblast giant cells (TGCs), suggesting that geminin regulates trophoblast specification and differentiation. Other studies concluded that trophoblast differentiation into TGCs is regulated by fibroblast growth factor-4 (FGF4), and that geminin is required to maintain endocycles. Here we show that ablation of Gmnn in trophoblast stem cells (TSCs) proliferating in the presence of FGF4 closely mimics the events triggered by FGF4 deprivation: arrest of cell proliferation, formation of giant cells, excessive DNA replication in the absence of DNA damage and apoptosis, and changes in gene expression that include loss of Chk1 with up-regulation of p57 and p21. Moreover, FGF4 deprivation of TSCs reduces geminin to a basal level that is required for maintaining endocycles in TGCs. Thus, geminin acts both like a component of the FGF4 signal transduction pathway that governs trophoblast proliferation and differentiation, and geminin is required to maintain endocycles.

Selective Termination of Autophagy-Dependent Cancers.

The goal of cancer research is to identify characteristics of cancer cells that allow them to be selectively eliminated without harming the host. One such characteristic is autophagy dependence. Cancer cells survive, proliferate, and metastasize under conditions where normal cells do not. Thus, the requirement in cancer cells for more energy and macromolecular biosynthesis can evolve into a dependence on autophagy for recycling cellular components. Recent studies have revealed that autophagy, as well as different forms of cellular trafficking, is regulated by five phosphoinositides associated with eukaryotic cellular membranes and that the enzymes that synthesize them are prime targets for cancer therapy. For example, PIKFYVE inhibitors rapidly disrupt lysosome homeostasis and suppress proliferation in all cells. However, these inhibitors selectively terminate PIKFYVE-dependent cancer cells and cancer stem cells with not having adverse effect on normal cells. Here, we describe the biochemical distinctions between PIKFYVE-sensitive and -insensitive cells, categorize PIKFYVE inhibitors into four groups that differ in chemical structure, target specificity and efficacy on cancer cells and normal cells, identify the mechanisms by which they selectively terminate autophagy-dependent cancer cells, note their paradoxical effects in cancer immunotherapy, and describe their therapeutic applications against cancers.

PIKFYVE inhibitors trigger interleukin-24-dependent cell death of autophagy-dependent melanoma.

Inhibitors specifically targeting the 1-phosphatidylinositol 3-phosphate 5-kinase (PIKFYVE) disrupt lysosome homeostasis, thereby selectively terminating autophagy-dependent human cancer cells in vivo as well as in vitro without harming the viability of nonmalignant cells. To elucidate the mechanism by which PIKFYVE inhibition induces cell death, autophagy-dependent melanoma cells were compared with normal foreskin fibroblasts. RNA sequence profiling suggested that PIKFYVE inhibitors upregulated an endoplasmic reticulum (ER) stress response involving interleukin-24 (IL24; also known as MDA7) selectively in melanoma cells. Subsequent biochemical and genetic analyses confirmed these results and extended them to tumor xenografts in which tumor formation and expansion were inhibited. IL24 expression was upregulated by the DDIT3/CHOP/CEBPz transcription factor, a component of the PERK-dependent ER-stress response. Ectopic expression of IL24-induced cell death in melanoma cells, but not in foreskin fibroblasts, whereas ablation of the IL24 gene in melanoma cells prevented death. IL24 upregulation was triggered specifically by PIKFYVE inhibition. Thus, unlike thapsigargin and tunicamycin, which induce ER-stress indiscriminately, PIKFYVE inhibitors selectively terminated PIKFYVE-sensitive melanoma by inducing IL24-dependent ER-stress. Moreover, induction of cell death by a PIKFYVE inhibitor together with ectopic expression of IL24 protein was cumulative, thereby confirming the therapeutic potential of PIKFYVE inhibitors in the treatment of melanoma.

The involvement of acidic nucleoplasmic DNA-binding protein (And-1) in the regulation of prereplicative complex (pre-RC) assembly in human cells.

DNA replication in all eukaryotes starts with the process of loading the replicative helicase MCM2-7 onto chromatin during late mitosis of the cell cycle. MCM2-7 is a key component of the prereplicative complex (pre-RC), which is loaded onto chromatin by the concerted action of origin recognition complex, Cdc6, and Cdt1. Here, we demonstrate that And-1 is assembled onto chromatin in late mitosis and early G(1) phase before the assembly of pre-RC in human cells. And-1 forms complexes with MCM2-7 to facilitate the assembly of MCM2-7 onto chromatin at replication origins in late mitosis and G(1) phase. We also present data to show that depletion of And-1 significantly reduces the interaction between Cdt1 and MCM7 in G(1) phase cells. Thus, human And-1 facilitates loading of the MCM2-7 helicase onto chromatin during the assembly of pre-RC.

Regulating the licensing of DNA replication origins in metazoa.

Eukaryotic DNA replication is a highly conserved process; the proteins and sequence of events that replicate animal genomes are remarkably similar to those that replicate yeast genomes. Moreover, the assembly of prereplication complexes at DNA replication origins ('DNA licensing') is regulated in all eukaryotes so that no origin fires more than once in a single cell cycle. And yet there are significant differences between species both in the selection of replication origins and in the way in which these origins are licensed to operate. Moreover, these differences impart advantages to multicellular animals and plants that facilitate their development, such as better control over endoreduplication, flexibility in origin selection, and discrimination between quiescent and proliferative states.

High-throughput screening for genes that prevent excess DNA replication in human cells and for molecules that inhibit them.

High-throughput screening (HTS) provides a rapid and comprehensive approach to identifying compounds that target specific biological processes as well as genes that are essential to those processes. Here we describe a HTS assay for small molecules that induce either DNA re-replication or endoreduplication (i.e. excess DNA replication) selectively in cells derived from human cancers. Such molecules will be useful not only to investigate cell division and differentiation, but they may provide a novel approach to cancer chemotherapy. Since induction of DNA re-replication results in apoptosis, compounds that selectively induce DNA re-replication in cancer cells without doing so in normal cells could kill cancers in vivo without preventing normal cell proliferation. Furthermore, the same HTS assay can be adapted to screen siRNA molecules to identify genes whose products restrict genome duplication to once per cell division. Some of these genes might regulate the formation of terminally differentiated polyploid cells during normal human development, whereas others will prevent DNA re-replication during each cell division. Based on previous studies, we anticipate that one or more of the latter genes will prove to be essential for proliferation of cancer cells but not for normal cells, since many cancer cells are deficient in mechanisms that maintain genome stability.

PIP5K1C phosphoinositide kinase deficiency distinguishes PIKFYVE-dependent cancer cells from non-malignant cells.

Although PIKFYVE phosphoinositide kinase inhibitors can selectively eliminate PIKFYVE-dependent human cancer cells in vitro and in vivo, the basis for this selectivity has remained elusive. Here we show that the sensitivity of cells to the PIKFYVE inhibitor WX8 is not linked to PIKFYVE expression, macroautophagic/autophagic flux, the BRAFV600E mutation, or ambiguous inhibitor specificity. PIKFYVE dependence results from a deficiency in the PIP5K1C phosphoinositide kinase, an enzyme required for conversion of phosphatidylinositol-4-phosphate (PtdIns4P) into phosphatidylinositol-4,5-bisphosphate (PtdIns[4,5]P2/PIP2), a phosphoinositide associated with lysosome homeostasis, endosome trafficking, and autophagy. PtdIns(4,5)P2 is produced via two independent pathways. One requires PIP5K1C; the other requires PIKFYVE and PIP4K2C to convert PtdIns3P into PtdIns(4,5)P2. In PIKFYVE-dependent cells, low concentrations of WX8 specifically inhibit PIKFYVE in situ, thereby increasing the level of its substrate PtdIns3P while suppressing PtdIns(4,5)P2 synthesis and inhibiting lysosome function and cell proliferation. At higher concentrations, WX8 inhibits both PIKFYVE and PIP4K2C in situ, which amplifies these effects to further disrupt autophagy and induce cell death. WX8 did not alter PtdIns4P levels. Consequently, inhibition of PIP5K1C in WX8-resistant cells transformed them into sensitive cells, and overexpression of PIP5K1C in WX8-sensitive cells increased their resistance to WX8. This discovery suggests that PIKFYVE-dependent cancers could be identified clinically by low levels of PIP5K1C and treated with PIKFYVE inhibitors.Abbreviations: DMSO: dimethylsulfoxide; ELISA: enzyme-linked immunosorbent assay; LC3-I: microtubule associated protein light chain 3-I; LC3-II: microtubule associated protein light chain 3-II; MS: mass spectrometry; PtdIns: phosphatidylinositol; PtdIns3P: PtdIns-3-phosphate; PtdIns4P: PtdIns-4-phosphate; PtdIns5P: PtdIns-5-phosphate; PtdIns(3,5)P2: PtdIns-3,5-bisphosphate; PtdIns(4,5)P2/PIP2: PtdIns-4,5-bisphosphate; PtdIns(3,4,5)P3/PIP3: PtdIns-3,4,5-trisphosphate; PIKFYVE: phosphoinositide kinase, FYVE-type zinc finger containing; PIK3C3: phosphatidylinositol 3-kinase catalytic subunit type 3; PI4KA: phosphatidylinositol 4-kinase alpha; PI4KB: phosphatidylinositol 4-kinase beta; PI4K2A: phosphatidylinositol 4-kinase type 2 alpha; PI4K2B: phosphatidylinositol 4-kinase type 2 beta; PIP4K2A: phosphatidylinositol-5-phosphate 4-kinase type 2 alpha; PIP4K2B: phosphatidylinositol-5-phosphate 4-kinase type 2 beta; PIP4K2C: phosphatidylinositol-5-phosphate 4-kinase type 2 gamma; PIP5K1A: phosphatidylinositol-4-phosphate 5-kinase type 1 alpha; PIP5K1B: phosphatidylinositol-4-phosphate 5-kinase type 1 beta; PIP5K1C: phosphatidylinositol-4-phosphate 5-kinase type 1 gamma; WX8: 1H-indole-3-carbaldehyde (4-anilino-6-[4-morpholinyl]-1,3,5-triazin-2-yl)hydrazone.

Assembly of the human origin recognition complex occurs through independent nuclear localization of its components.

Initiation of eukaryotic genome duplication begins when a six-subunit origin recognition complex (ORC) binds to DNA. However, the mechanism by which this occurs in vivo and the roles played by individual subunits appear to differ significantly among organisms. Previous studies identified a soluble human ORC(2-5) complex in the nucleus, an ORC(1-5) complex bound to chromatin, and an Orc6 protein that binds weakly, if at all, to other ORC subunits. Here we show that stable ORC(1-6) complexes also can be purified from human cell extracts and that Orc6 and Orc1 each contain a single nuclear localization signal that is essential for nuclear localization but not for ORC assembly. The Orc6 nuclear localization signal, which is essential for Orc6 function, is facilitated by phosphorylation at its cyclin-dependent kinase consensus site and by association with Kpna6/1, nuclear transport proteins that did not co-purify with other ORC subunits. These and other results support a model in which Orc6, Orc1, and ORC(2-5) are transported independently to the nucleus where they can either assemble into ORC(1-6) or function individually.

Selective elimination of pluripotent stem cells by PIKfyve specific inhibitors.

Inhibition of PIKfyve phosphoinositide kinase selectively kills autophagy-dependent cancer cells by disrupting lysosome homeostasis. Here, we show that PIKfyve inhibitors can also selectively eliminate pluripotent embryonal carcinoma cells (ECCs), embryonic stem cells, and induced pluripotent stem cells under conditions where differentiated cells remain viable. PIKfyve inhibitors prevented lysosome fission, induced autophagosome accumulation, and reduced cell proliferation in both pluripotent and differentiated cells, but they induced death only in pluripotent cells. The ability of PIKfyve inhibitors to distinguish between pluripotent and differentiated cells was confirmed with xenografts derived from ECCs. Pretreatment of ECCs with the PIKfyve specific inhibitor WX8 suppressed their ability to form teratocarcinomas in mice, and intraperitoneal injections of WX8 into mice harboring teratocarcinoma xenografts selectively eliminated pluripotent cells. Differentiated cells continued to proliferate, but at a reduced rate. These results provide a proof of principle that PIKfyve specific inhibitors can selectively eliminate pluripotent stem cells in vivo as well as in vitro.

PIKfyve-specific inhibitors restrict replication of multiple coronaviruses in vitro but not in a murine model of COVID-19.

The ongoing COVID-19 pandemic has claimed more than 6 million lives and continues to test the world economy and healthcare systems. To combat this pandemic, the biological research community has shifted efforts to the development of medical countermeasures, including vaccines and therapeutics. However, to date, the only small molecules approved for the treatment of COVID-19 in the United States are the nucleoside analogue Remdesivir and the protease inhibitor Paxlovid, though multiple compounds have received Emergency Use Authorization and many more are currently being tested in human efficacy trials. One such compound, Apilimod, is being considered as a COVID-19 therapeutic in a Phase II efficacy trial. However, at the time of writing, there are no published efficacy data in human trials or animal COVID-19 models. Here we show that, while Apilimod and other PIKfyve inhibitors have potent antiviral activity in various cell lines against multiple human coronaviruses, these compounds worsen disease in a COVID-19 murine model when given prophylactically or therapeutically.

Spotlight on geminin.

In the previous issue of Breast Cancer Research, Gardner and co-workers describe a novel interaction between Geminin, a protein that prevents reinitiation of DNA replication, and Topoisomerase IIα (TopoIIα), an enzyme essential for removing catenated intertwines between sister chromatids. Geminin facilitates the action of TopoIIα, thereby promoting termination of DNA replication at the same time it inhibits initiation. In this manner, Geminin ensures that cells duplicate their genome once, but only once, each time they divide. Remarkably, either depletion of Geminin or over-expression of Geminin inhibits the action of TopoIIα, thereby making Geminin an excellent target for cancer chemotherapy.

Contributions of the two accessory subunits, RNASEH2B and RNASEH2C, to the activity and properties of the human RNase H2 complex.

Eukaryotic RNase H2 is a heterotrimeric enzyme. Here, we show that the biochemical composition and stoichiometry of the human RNase H2 complex is consistent with the properties previously deduced from genetic studies. The catalytic subunit of eukaryotic RNase H2, RNASEH2A, is well conserved and similar to the monomeric prokaryotic RNase HII. In contrast, the RNASEH2B and RNASEH2C subunits from human and Saccharomyces cerevisiae share very little homology, although they both form soluble B/C complexes that may serve as a nucleation site for the addition of RNASEH2A to form an active RNase H2, or for interactions with other proteins to support different functions. The RNASEH2B subunit has a PIP-box and confers PCNA binding to human RNase H2. Unlike Escherichia coli RNase HII, eukaryotic RNase H2 acts processively and hydrolyzes a variety of RNA/DNA hybrids with similar efficiencies, suggesting multiple cellular substrates. Moreover, of five analyzed mutations in human RNASEH2B and RNASEH2C linked to Aicardi-Goutières Syndrome (AGS), only one, R69W in the RNASEH2C protein, exhibits a significant reduction in specific activity, revealing a role for the C subunit in enzymatic activity. Near-normal activity of four AGS-related mutant enzymes was unexpected in light of their predicted impairment causing the AGS phenotype.

Developmental Acquisition of p53 Functions.

Remarkably, the p53 transcription factor, referred to as "the guardian of the genome", is not essential for mammalian development. Moreover, efforts to identify p53-dependent developmental events have produced contradictory conclusions. Given the importance of pluripotent stem cells as models of mammalian development, and their applications in regenerative medicine and disease, resolving these conflicts is essential. Here we attempt to reconcile disparate data into justifiable conclusions predicated on reports that p53-dependent transcription is first detected in late mouse blastocysts, that p53 activity first becomes potentially lethal during gastrulation, and that apoptosis does not depend on p53. Furthermore, p53 does not regulate expression of genes required for pluripotency in embryonic stem cells (ESCs); it contributes to ESC genomic stability and differentiation. Depending on conditions, p53 accelerates initiation of apoptosis in ESCs in response to DNA damage, but cell cycle arrest as well as the rate and extent of apoptosis in ESCs are p53-independent. In embryonic fibroblasts, p53 induces cell cycle arrest to allow repair of DNA damage, and cell senescence to prevent proliferation of cells with extensive damage.

Efficacy of a small molecule inhibitor of the transcriptional cofactor PC4 in prevention and treatment of non-small cell lung cancer.

The human positive coactivator 4 (PC4) was originally identified as a multi-functional cofactor capable of mediating transcription activation by diverse gene- and tissue-specific activators. Recent studies suggest that PC4 might also function as a novel cancer biomarker and therapeutic target for different types of cancers. siRNA knockdown studies indicated that down-regulation of PC4 expression could inhibit tumorigeneicity of A549 non-small cell lung cancer tumor model in nude mice. Here we show that AG-1031, a small molecule identified by high throughput screening, can inhibit the double-stranded DNA binding activity of PC4, more effectively than its single-stranded DNA binding activity. AG-1031 also specifically inhibited PC4-dependent transcriptional activation in vitro using purified transcription factors. AG-1031 inhibited proliferation of several cultured cell lines derived from non-small cell lung cancers (NSCLC) and growth of tumors that formed from A549 cell xenografts in immuno-compromised mice. Moreover, pre-injection of AG-1031 in these mice not only reduced tumor size, but also prevented tumor formation in 20% of the animals. AG-1031 treated A549 cells and tumors from AG-1031 treated animals showed a significant decrease in the levels of both PC4 and VEGFC, a key mediator of angiogenesis in cancer. On the other hand, all tested mice remained constant weight during animal trials. These results demonstrated that AG-1031 could be a potential therapy for PC4-positive NSCLC.

The multifunctional RNA-binding protein La is required for mouse development and for the establishment of embryonic stem cells.

The La protein is a target of autoantibodies in patients suffering from Sjögren's syndrome, systemic lupus erythematosus, and neonatal lupus. Ubiquitous in eukaryotes, La functions as a RNA-binding protein that promotes the maturation of tRNA precursors and other nascent transcripts synthesized by RNA polymerase III as well as other noncoding RNAs. La also associates with a class of mRNAs that encode ribosome subunits and precursors to snoRNAs involved in ribosome biogenesis. Thus, it was surprising that La is dispensable in the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe, the organisms from which it has been characterized most extensively. To determine whether La is essential in mammals and if so, at which developmental stage it is required, mice were created with a disrupted La gene, and the offspring from La+/-intercrosses were analyzed. La-/- offspring were detected at the expected frequency among blastocysts prior to implantation, whereas no nullizygotes were detected after implantation, indicating that La is required early in development. Blastocysts derived from La+/- intercrosses yielded 38 La+/+ and La+/- embryonic stem (ES) cell lines but no La-/- ES cell lines, suggesting that La contributes a critical function toward the establishment or survival of ES cells. Consistent with this, La-/- blastocyst outgrowths revealed loss of the inner cell mass (ICM). The results indicate that in contrast to the situation in yeasts, La is essential in mammals and is one of a limited number of genes required as early as the development of the ICM.