Iva Greenwald

Presenilin is required for activity and nuclear access of Notch in Drosophila

Presenilins are membrane proteins with multiple transmembrane domains that are thought to contribute to the development of Alzheimers disease by affecting the processing of β-amyloid precursor protein. Presenilins also facilitate the activity of transmembrane receptors of the LIN-12/Notch family. After ligand-induced processing, the intracellular domain of LIN-12/Notch can enter the nucleus and participate in the transcriptional control of downstream target genes. Here we show that null mutations in the Drosophila Presenilin gene abolish Notch signal transduction and prevent its intracellular domain from entering the nucleus. Furthermore, we provide evidence that presenilin is required for the proteolytic release of the intracellular domain from the membrane following activation of Notch by ligand.

The lin-12 locus specifies cell fates in caenorhabditis elegans

We describe two classes of mutations in the lin-12 locus of the nematode Caenorhabditis elegans. Ten semidominant mutations (lin-12[d]) appear to elevate the level of lin-12 activity. Thirty-two recessive alleles (lin-12[0]), including two amber mutations, appear to eliminate gene activity. The lin-12(d) and lin-12(0) mutations result in reciprocal homeotic transformations in the fates of defined cells in several different tissues. Gene dosage studies suggest that a high level of lin-12 activity specifies one cell fate and a low level specifies an alternative fate. Temperature-shift experiments indicate that lin-12 acts at the time cell fate is determined in wild type. We propose that lin-12 functions as a binary switch to control decisions between alternative cell fates during C. elegans development.

Intrinsic activity of the lin-12 and Notch intracellular domains in vivo

The lin-12 gene of C. elegans and the Notch gene of D. melanogaster encode structurally related transmembrane proteins that mediate intercellular signaling. We show that truncated forms of these proteins consisting of only the intracellular domains cause cell fate transformations associated with constitutive activity in their respective organisms. This activity does not depend on endogenous gene function. Our results indicate that the intracellular domains of Lin-12 and Notch have intrinsic activity and that the principal role of the extracellular domains in the intact proteins is to regulate this activity. Our results also suggest that equivalent truncated forms of lin-12/Notch family members in vertebrates, including known oncogenes, are similarly active.

Making a difference : the role of cell-cell interactions in establishing separate identities for equivalent cells

Iva Greenwald’ and Gerald M. Rublnf *Department of Molecular Biology Princeton University Princeton, New Jersey 08844 tDepartment of Molecular and Cell Biology Howard Hughes Medical Institute and University of California Berkeley, California 94720 Introduction Equivalent cells have the same set of potential fates and must choose one of the available options. In many cases, the choice is influenced by intercellular signaling events. If the signal originates outside of the population of equivalent cells (equivalence group), the signaling process is termed induction. If the signal arises within the equivalence group, the signaling process is termed lateral specification (or lateral inhibition). Cells that do not receive and respond to the signal express a default fate, while cells that receive and respond to the signal express an alternative fate. In this review, we describe in detail one example of each process in Caenorhabditiselegans and Drosophila. In both organisms, powerful methods of genetic analysis and sin- gle cell resolution have facilitated the identification of mol- ecules involved in induction and lateral specification, and are beginning to lead to an understanding of the biochemi- cal circuitry that mediates cellular decisions. Common fea- tures emerge from a comparison of the worm and fly exam- ples that are immediately applicable to higher organisms. Key components of identified signaling systems have proven to be members of highly conserved gene families, implying that similar molecular mechanisms underlie cell- cell interactions that specify cell fate decisions in all animals. Induction Cells of an equivalence group may express different fates in response to a signal emanating from cells that are not part of the equivalence group. We will consider two such inductive events in detail. One example is R7 photorecep- tor induction in Drosophila, when a signal from an R8 pho- toreceptor cell causes one of an apparently equivalent group of five bipotential cells to become an R7 photorecep- tor cell rather than a nonneuronal cone cell. The other example is vulva1 induction in C. elegans, when a signal from the anchor cell (AC) of the gonad causes 3 of 8 equi- potential cells to generate vulva1 cells rather than cells that join the hypodermal syncytium. In both cases, the activation of a Ras protein in the induced cell appears to be a critical event. Ras activation is a consequence of the reception of the inductive signal by a receptor with a protein tyrosine kinase (PTK) domain. R7 Photoreceptor Induction The induction of the R7 photoreceptor fate during Dro- sophila eye development is the simplest possible example of induction, since it involves only two cells. One cell, the R8 photoreceptor cell, sends an inductive signal to an uncommitted cell that has a choice between becoming the R7 photoreceptor cell or a nonneuronal cone cell. This inductive event is the last of a series of sequential induc- tions that occur to specify photoreceptor cell identity. The adult Drosophila eye is made up of an array ap- proximately 800 twenty-ceil units called ommatidia. Each ommatidium contains eight photoreceptor neurons, Rl- R8, as well four lens-secreting cone cells and eight other accessory cells. The cells that make up the eye ini- tially all appear to be members of one equivalence group (Ready et al., 1978; Lawrence and Green, 1979). Devel- oping ommatidia are spaced in this field of cells by a pro- cess involving lateral specification (lateral specification is discussed in detail below). Then the cells that make up each ommatidium are thought to be recruited by a series of local cell-cell interactions, with differentiating cells in- ducing their immediate neighbors to adopt particular fates (for recent reviews see Tomlinson, 1988; Ready, 1989; Rubin, 1991; Banerjee and Zipursky, 1990). The number of these inductive events is not known. It may be that the ultimate fate of a cell in the developing eye is not specified by a single inductive signal, or even combination simultaneous signals. Rather, a series of inductive events may progressively limit the options available to a cell; that is, the equivalence group to which the cell belongs may be sequentially reduced in size and developmental potential. (Superficially similar processes in vertebrate development are discussed by Gurdon, 1992, this issue.) The R7 photoreceptor is the last of the eight photorecep- tors to be recruited to the developing ommatidium (see Figure 1A). The R7 photoreceptor cell fate is induced by a signal from the R8 cell (Figure IA). The generation of this signal requires the function of the boss gene in the R8 cell (Reinke and Zipursky, 1988) and its reception requires thefunctionof thesevenlessgenein theR7cell(Tomlinson and Ready, 1987). In aboss or sevenless mutant the signal is apparently not received by the presumptive R7 cell, and it differentiates as a nonneuronal cone cell. Many cells in the developing eye express the Sevenless protein, a transmembrane PTK receptor (Tomlinson et al., 1987; Banerjee et al., 1987) yet only one cell becomes an R7 cell. This specificity is accomplished by a combination of two mechanisms. First, the apparent ligand for Sev- enless receptor, the product of boss gene (Reinke and Zipursky, 1988) is expressed only on the surface of R8 cell (Kramer et al., 1991). Thus, cells must contact to receive the signal. Second, some cells that both contact R8 and express Sevenless do not show a response to the Sevenless-mediated signal. Presumably, a prior develop- mental choice has removed these cells from the equiva- lence group of cells that can respond to the Sevenless- mediated signal by becoming R7 cells (see below). The cells that are competent to respond activation of the Sevenless receptor have been defined in two ways.

Membrane Topology of the C. elegans SEL-12 Presenilin

Mutant presenilins cause Alzheimers disease. Presenilins have multiple hydrophobic regions that could theoretically span a membrane, and a knowledge of the membrane topology is crucial for deducing the mechanism of presenilin function. By analyzing the activity of beta-galactosidase hybrid proteins expressed in C. elegans, we show that the C. elegans SEL-12 presenilin has eight transmembrane domains and that there is a cleavage site after the sixth transmembrane domain. We examine the presenilin sequence in view of the predicted topology and discuss possible mechanisms for presenilin function.

OrthoList: A Compendium of C. elegans Genes with Human Orthologs

Background C. elegans is an important model for genetic studies relevant to human biology and disease. We sought to assess the orthology between C. elegans and human genes to understand better the relationship between their genomes and to generate a compelling list of candidates to streamline RNAi-based screens in this model. Results We performed a meta-analysis of results from four orthology prediction programs and generated a compendium, “OrthoList”, containing 7,663 C. elegans protein-coding genes. Various assessments indicate that OrthoList has extensive coverage with low false-positive and false-negative rates. Part of this evaluation examined the conservation of components of the receptor tyrosine kinase, Notch, Wnt, TGF-ß and insulin signaling pathways, and led us to update compendia of conserved C. elegans kinases, nuclear hormone receptors, F-box proteins, and transcription factors. Comparison with two published genome-wide RNAi screens indicated that virtually all of the conserved hits would have been obtained had just the OrthoList set (∼38% of the genome) been targeted. We compiled Ortholist by InterPro domains and Gene Ontology annotation, making it easy to identify C. elegans orthologs of human disease genes for potential functional analysis. Conclusions We anticipate that OrthoList will be of considerable utility to C. elegans researchers for streamlining RNAi screens, by focusing on genes with apparent human orthologs, thus reducing screening effort by ∼60%. Moreover, we find that OrthoList provides a useful basis for annotating orthology and reveals more C. elegans orthologs of human genes in various functional groups, such as transcription factors, than previously described.

The lateral signal for LIN-12/Notch in C. elegans vulval development comprises redundant secreted and transmembrane DSL proteins.

The vulval precursor cells (VPCs) are spatially patterned by a LET-23/EGF receptor-mediated inductive signal and a LIN-12/Notch-mediated lateral signal. The lateral signal has eluded identification, so the mechanism by which lateral signaling is activated has not been known. Here, we computationally identify ten genes that encode potential ligands for LIN-12, and show that three of these genes, apx-1, dsl-1, and lag-2, are functionally redundant components of the lateral signal. We also show that transcription of all three genes is initiated or upregulated in VPCs in response to inductive signaling, suggesting that direct transcriptional control of the lateral signal by the inductive signal is part of the mechanism by which these cell signaling events are coordinated. In addition, we show that DSL-1, which lacks a predicted transmembrane domain, is a natural secreted ligand and can substitute for the transmembrane ligand LAG-2 in different functional assays.

Endocytosis-mediated downregulation of LIN-12/Notch upon Ras activation in Caenorhabditis elegans.

The coordination of signals from different pathways is important for cell fate specification during animal development. Here, we define a novel mode of crosstalk between the epidermal growth factor receptor/Ras/mitogen-activated protein kinase cascade and the LIN-12/Notch pathway during Caenorhabditis elegans vulval development. Six vulval precursor cells (VPCs) are initially equivalent but adopt different fates as a result of an inductive signal mediated by the Ras pathway and a lateral signal mediated by the LIN-12/Notch pathway. One consequence of activating Ras is a reduction of LIN-12 protein in P6.p (ref. 2), the VPC believed to be the source of the lateral signal. Here we identify a ‘downregulation targeting signal’ (DTS) in the LIN-12 intracellular domain, which encompasses a di-leucine-containing endocytic sorting motif. The DTS seems to be required for internalization of LIN-12, and on Ras activation it might mediate altered endocytic routing of LIN-12, leading to downregulation. We also show that if LIN-12 is stabilized in P6.p, lateral signalling is compromised, indicating that LIN-12 downregulation is important in the appropriate specification of cell fates in vivo.

LIN-12/notch activation leads to microRNA-mediated down-regulation of vav in C. elegans

Cell-cell interactions and cross-talk between signaling pathways specify Caenorhabditis elegans vulval precursor cells (VPCs) to adopt a spatial pattern: a central “1°” VPC, in which epidermal growth factor receptor (EGFR)–mitogen-activated protein kinase (MAPK) activity is high and LIN-12/Notch activity is low, flanked by two “2°” VPCs, in which LIN-12/Notch activity is high and EGFR-MAPK activity is low. Here, we identify a microRNA gene, mir-61, as a direct transcriptional target of LIN-12 and show that expression of mir-61 promotes the 2° fate. We also identify vav-1, the ortholog of the Vav oncogene, as a target of mir-61, and show that down-regulation of VAV-1 promotes lin-12 activity in specifying the 2° fate. Our results suggest that lin-12, mir-61, and vav-1 form a feedback loop that helps maximize lin-12 activity in the presumptive 2° VPCs.

Structure/function studies of lin-12/Notch proteins.

The lin-12/Notch proteins appear to act as transmembrane receptors for intercellular signals that specify cell fates during animal development. Recent structure/function studies have shown that the lin-12/Notch intracellular domain alone has the intrinsic signal-transducing activity of the intact protein, and that the role of the extracellular domain is to regulate this intrinsic activity. These studies have also suggested that the different lin-12/Notch proteins in a given organism are interchangeable biochemically and have addressed the role of lin-12/Notch genes in development.