Joel H. Rothman

Baculovirus p35 prevents developmentally programmed cell death and rescues a ced-9 mutant in the nematode Caenorhabditis elegans

Programmed cell death, or apoptosis, occurs throughout the course of normal development in most animals and can also be elicited by a number of stimuli such as growth factor deprivation and viral infection. Certain morphological and biochemical characteristics of programmed cell death are similar among different tissues and species. During development of the nematode Caenorhabditis elegans, a single genetic pathway promotes the death of selected cells in a lineally fixed pattern. This pathway appears to be conserved among animal species. The baculovirus p35‐encoding gene (p35) is an inhibitor of virus‐induced apoptosis in insect cells. Here we demonstrate that expression of p35 in C. elegans prevents death of cells normally programmed to die. This suppression of developmentally programmed cell death results in appearance of extra surviving cells. Expression of p35 can rescue the embryonic lethality of a mutation in ced‐9, an endogenous gene homologous to the mammalian apoptotic suppressor bcl‐2, whose absence leads to ectopic cell deaths. These results support the hypothesis that viral infection can activate the same cell death pathway as is used during normal development and suggest that baculovirus p35 may act downstream or independently of ced‐9 in this pathway.

Restriction of Mesendoderm to a Single Blastomere by the Combined Action of SKN-1 and a GSK-3β Homolog Is Mediated by MED-1 and -2 in C. elegans

The endoderm and much of the mesoderm arise from the EMS cell in the four-cell C. elegans embryo. We report that the MED-1 and -2 GATA factors specify the entire fate of EMS, which otherwise produces two C-like mesectodermal progenitors. The meds are direct targets of the maternal SKN-1 transcription factor; however, their forced expression can direct SKN-1-independent reprogramming of non-EMS cells into mesendodermal progenitors. We find that SGG-1/GSK-3beta kinase acts both as a Wnt-dependent activator of endoderm in EMS and an apparently Wnt-independent repressor of the meds in the C lineage, indicating a dual role for this kinase in mesendoderm development. Our results suggest that a broad tissue territory, mesendoderm, in vertebrates has been confined to a single cell in nematodes through a common gene regulatory network.

The TBP-like Factor CeTLF Is Required to Activate RNA Polymerase II Transcription during C. elegans Embryogenesis

Metazoans possess two TATA-binding protein homologs, the general transcription factor TBP and a related factor called TLF. Four models have been proposed for the role of TLF in RNA polymerase II (Pol II) transcription: (1) TLF and TBP function redundantly, (2) TLF antagonizes TBP, (3) TLF is a tissue-specific TBP, or (4) TLF and TBP have distinct activities. Here we report that CeTLF is required to express a subset of Pol II genes and associates with at least one of these genes in vivo. CeTLF is also necessary to establish bulk transcription during early embryogenesis. Since CeTLF and CeTBP are expressed at comparable levels in the same cells, these findings suggest CeTLF performs a unique function in activating Pol II transcription distinct from that of CeTBP.

dad-1, an endogenous programmed cell death suppressor in Caenorhabditis elegans and vertebrates

Programmed cell death (apoptosis) is a normally occurring process used to eliminate unnecessary or potentially harmful cells in multicellular organisms. Recent studies demonstrate that the molecular control of this process is conserved phylogenetically in animals. The dad‐1 gene, which encodes a novel 113 amino acid protein, was originally identified in a mutant hamster cell line (tsBN7) that undergoes apoptosis at restrictive temperature. We have identified a dad‐1 homologue in Caenorhabditis elegans (Ce‐dad‐1) whose predicted product is > 60% identical to vertebrate DAD‐1. A search of the sequence databases indicated that DAD‐1‐like proteins are also expressed in two plant species. Expression of either human dad‐1 or Ce‐dad‐1 under control of a C.elegans heat‐shock‐inducible promoter resulted in a reduction in the number of programmed cell death corpses visible in C.elegans embryos. Extra surviving cells were present in these animals, indicating that both the human and C.elegans dad‐1 genes can suppress developmentally programmed cell death. Ce‐dad‐1 was found to rescue mutant tsBN7 hamster cells from apoptotic death as efficiently as the vertebrate genes. These results suggest that dad‐1, which is necessary for cell survival in a mammalian cell line, is sufficient to suppress some programmed cell death in C.elegans.

A POP-1 repressor complex restricts inappropriate cell type-specific gene transcription during Caenorhabditis elegans embryogenesis

In Caenorhabditis elegans, histone acetyltransferase CBP‐1 counteracts the repressive activity of the histone deacetylase HDA‐1 to allow endoderm differentiation, which is specified by the E cell. In the sister MS cell, the endoderm fate is prevented by the action of an HMG box‐containing protein, POP‐1, through an unknown mechanism. In this study, we show that CBP‐1, HDA‐1 and POP‐1 converge on end‐1, an initial endoderm‐determining gene. In the E lineage, an essential function of CBP‐1 appears to be the activation of end‐1 transcription. We further identify a molecular mechanism for the endoderm‐suppressive effect of POP‐1 in the MS lineage by demonstrating that POP‐1 functions as a transcriptional repressor that inhibits inappropriate end‐1 transcription. We provide evidence that POP‐1 represses transcription via the recruitment of HDA‐1 and UNC‐37, the C.elegans homolog of the co‐repressor Groucho. These findings demonstrate the importance of the interplay between acetyltransferases and deacetylases in the regulation of a critical cell fate‐determining gene during development. Furthermore, they identify a strategy by which concerted actions of histone deacetylases and other co‐repressors ensure maximal repression of inappropriate cell type‐specific gene transcription.

Suppression of CED-3-independent apoptosis by mitochondrial βNAC in Caenorhabditis elegans

To ensure cell survival, it is essential that the ubiquitous pro-apoptotic machinery is kept quiescent. As death is irreversible, cells must continually integrate developmental information with regulatory inputs to control the switch between repressing and activating apoptosis. Inappropriate activation or suppression of apoptosis can lead to degenerative pathologies or tumorigenesis, respectively. Here we report that Caenorhabditis elegans inhibitor of cell death-1 (ICD-1) is necessary and sufficient to prevent apoptosis. Loss of ICD-1 leads to inappropriate apoptosis in developing and differentiated cells in various tissues. Although this apoptosis requires CED-4, it occurs independently of CED-3—the caspase essential for developmental apoptosis—showing that these core pro-apoptotic proteins have separable roles. Overexpressing ICD-1 inhibits the apoptosis of cells that are normally programmed to die. ICD-1 is the β-subunit of the nascent polypeptide-associated complex (βNAC) and contains a putative caspase-cleavage site and caspase recruitment domain. It localizes primarily to mitochondria, underscoring the role of mitochondria in coordinating apoptosis. Human βNAC is a caspase substrate that is rapidly eliminated in dying cells, suggesting that ICD-1 apoptosis-suppressing activity may be inactivated by caspases.

Regulation of developmental rate and germ cell proliferation in Caenorhabditis elegans by the p53 gene network

Caenorhabditis elegans CEP-1 activates germline apoptosis in response to genotoxic stress, similar to its mammalian counterpart, tumor suppressor p53. In mammals, there are three p53 family members (p53, p63, and p73) that activate and repress many distinct and overlapping sets of genes, revealing a complex transcriptional regulatory network. Because CEP-1 is the sole p53 family member in C. elegans, analysis of this network is greatly simplified in this organism. We found that CEP-1 functions during normal development in the absence of stress to repress many (331) genes and activate only a few (28) genes. In response to genotoxic stress, 1394 genes are activated and 942 are repressed, many of which contain p53-binding sites. Comparison of the CEP-1 transcriptional network with transcriptional targets of the human p53 family reveals considerable overlap between CEP-1-regulated genes and homologues regulated by human p63 and p53, suggesting a composite p53/p63 action for CEP-1. We found that phg-1, the C. elegans Gas1 (growth arrest-specific 1) homologue, is activated by CEP-1 and is a negative regulator of cell proliferation in the germline in response to genotoxic stress. Further, we find that CEP-1 and PHG-1 mediate the decreased developmental rate and embryonic viability of mutations in the clk-2/TEL2 gene, which regulates lifespan and checkpoint responses.

Caenorhabditis elegans as a model for stem cell biology

We review the application of Caenorhabditis elegans as a model system to understand key aspects of stem cell biology. The only bona fide stem cells in C. elegans are those of the germline, which serves as a valuable paradigm for understanding how stem‐cell niches influence maintenance and differentiation of stem cells and how somatic differentiation is repressed during germline development. Somatic cells that share stem cell–like characteristics also provide insights into principles in stem‐cell biology. The epidermal seam cell lineages lend clues to conserved mechanisms of self‐renewal and expansion divisions. Principles of developmental plasticity and reprogramming relevant to stem‐cell biology arise from studies of natural transdifferentiation and from analysis of early embryonic progenitors, which undergo a dramatic transition from a pluripotent, reprogrammable condition to a state of committed differentiation. The relevance of these developmental processes to our understanding of stem‐cell biology in other organisms is discussed. Developmental Dynamics 239:1539–1554, 2010.

Essential embryonic roles of the CKI-1 cyclin-dependent kinase inhibitor in cell-cycle exit and morphogenesis in C. elegans

Following a phase of rapid proliferation, cells in developing embryos must decide when to cease division and then whether to survive and differentiate or instead undergo programmed death. In screens for genes that regulate embryonic patterning of the endoderm in Caenorhabditis elegans, we identified overlapping chromosomal deletions that define a gene required for these decisions. These deletions result in embryonic hyperplasia in multiple somatic tissues, excessive numbers of cell corpses, and profound defects in morphogenesis and differentiation. However, cell-cycle arrest of the germline is unaffected. Cell lineage analysis of these mutants revealed that cells that normally stop dividing earlier than their close relatives instead undergo an extra round of division. These deletions define a genomic region that includes cki-1 and cki-2, adjacent genes encoding members of the Cip/Kip family of cyclin-dependent kinase inhibitors. cki-1 alone can rescue the cell proliferation, programmed cell death, and differentiation and morphogenesis defects observed in these mutants. In contrast, cki-2 is not capable of significantly rescuing these phenotypes. RNA interference of cki-1 leads to embryonic lethality with phenotypes similar to, or more severe than, the deletion mutants. cki-1 and -2 gene reporters show distinct expression patterns; while both are expressed at around the time that embryonic cells exit the cell cycle, cki-2 also shows marked expression starting early in embryogenesis, when rapid cell division occurs. Our findings demonstrate that cki-1 activity plays an essential role in embryonic cell cycle arrest, differentiation and morphogenesis, and suggest that it may be required to suppress programmed cell death or engulfment of cell corpses.

The maternal-to-zygotic transition in embryonic patterning of Caenorhabditis elegans.

Maternal factors laid down in the oocyte regulate blastomere identities in the early Caenorhabditis elegans embryo by activating zygotic patterning genes and restricting their expression to the appropriate lineages. A number of early-acting zygotic genes that specify various cell fates have been identified recently and their temporal and spatial regulation by maternal factors has begun to be elucidated.