Ann Rougvie - US grants

The proper form of a multicellular organism requires precise control of terminal differentiation events. This research will address how a single terminal differentiation event is executed during the development of a simple model organism, the nematode C. elegans. The analysis will focus on the terminal differentiation of lateral hypodermal "seam" cells, which terminally differentiate during the final (4th) molt. Several distinct cellular processes are coordinately controlled during this terminal differentiation event, including cell cycle, cell fusion, and the stage-specific regulation of cuticle gene expression. A gene called lin-29 has been identified as a pivotal component in programming the execution of seam cell terminal differentiation. Loss of lin-29 function causes hypodermal cells of the adult stage to reiterate indefinitely the larval program of cell divisions instead of exiting the cell cycle and differentiating. lin-29 encodes a putative transcription factor of the (Cys)2-(His)2 class, and, thus likely programs these events by regulating the transcription of other genes. The major objective of this work is to identify genes controlled by the lin-29 protein. Three approaches will be employed to identify lin-29-regulated genes. ?1! Interactions of lin-29 protein with predicted target genes: The stage-specific expression of the cuticle collagen genes, col-17 and col-19, will be analyzed. col-17 expression is transcriptionally repressed during the larval/adult switch, whereas col-19 expression is activated. The role of lin-29 in the alternative repression and activation of these genes will be tested. ?2! Differential hybridization screens to identify genes whose expression is influenced by the presence or absence of lin-29 protein: Genes isolated in this fashion will be examined for direct regulation by lin-29, and those directly regulated will be characterized further. ?3! Development of a protein-DNA UV crosslinking method to isolate DNA sequences bound by a specific protein in vivo: In the future, this method will be used to identify genes bound by lin-29 protein in vivo, and should also be applicable to the isolation of genes controlled by other C. elegans gene-regulatory proteins. Genes involved in cell cycle exit and cell fusion, as well as additional stage-specifically expressed cuticle genes, should be identified by these methods. Analysis of the types of genes regulated by lin-29 in vivo will lead to an understanding of how a complex terminal differentiation program is orchestrated at a specific time during development.

The aim of this work is to understand the timing mechanisms that govern specific cell fate decisions during development. Genetic and molecular methods will be used to dissect the mechanism that restricts the terminal differentiation of lateral hypodermal "seam" cells to the final molt in nematode C. elegans. At that time, the seam cells exit the proliferative larval seam cell program and execute the adult terminal differentiation program, a process called the larval-to-adult switch (L/A switch). The transcription factor LIN-29 is the most direct known regulator of the L/A switch, and its activity is timed by the heterochronic genes lin-4, lin-14, and lin- 28. The genes lin-4, line-14, lin-28 and lin-29 are not the complete set of regulators that time seam cell terminal differentiation. Genetic screens will be used to identify additional genes that time the L/A switch. These screens have already identified lin-42 as an additional important temporal regulator. Genetic and molecular analysis of lin-42, and other newly identified heterochronic genes, will be performed to determine their roles in the timing pathway. When worms reinitiate development following the developmentally-arrested "dauer larva" stage the suspends the continuous timing program and the L/A switch must be rescheduled in the post-dauer worm. Genetic screens will be performed to test if there are genes that time lin-29 activity specifically during post-dauer development. The molecular mechanism by which the upstream genes control the timing of lin-29 activity will be determined. Sequences critical for lin-29 regulation will be defined by transformation rescue and the patterns of accumulation of lin-29 gene products will be determined. Lin-29 activity is also required for proper vulva formation during the L4 molt. Whether this requirement reflects a role for lin-29 in vulva/somatic gonad cells and/or in the surrounding hypodermis will be tested by mosaic analysis. Loss of lin-29 function causes cells of the adult stage to indefinitely reiterate the larval program of cell divisions instead of exciting the cell cycle and differentiating. Thus, lin-29 can be thought of in general terms as an anti-oncogene: a gene whose function is required for cell cycle exit. Temporal control of lin-29 thus provides a model for how cells within an organism are instructed to cease dividing at a specific time in development and differentiate. Knowledge about this control should aid in our understanding of the problems that occur when growth controls go awry, such as the inappropriate resumption of cell divisions that occurs in some cancers.

DESCRIPTION (provided by applicant): The aim of this work is to understand the timing mechanisms that govern specific cell fate decisions during metazoan development. The heterochronic genes of the nematode C. elegans are global temporal regulators that control the sequence and timing of diverse events during post embryonic development This research will investigate how one of these events, the terminal differentiation of lateral hypodermal "seam" cells, becomes temporally restricted to the final molt in wild-type animals. Mutations in the heterochronic genes cause seam cell terminal differentiation to occur earlier or later than normal. Nearly a dozen heterochronic genes have been identified and they can be roughly divided into two classes based upon their known or proposed times of action. The early acting genes specify developmental progression through the first three larval stages (L1-L3) and then the late acting genes take over and direct the remainder of development (late L3-Adult). This research will investigate the molecular roles of four heterochronic genes, the early acting genes, lin-42 and lin-58 and the late acting genes, lin-57 and lin-29. The experiments described in this proposal will seek to understand the functions of these genes in the context of the other genes that comprise the heterochronic gene pathway. Genetic and molecular epistasis experiments will be performed and the expression patterns of these genes will be determined. lin-42 encodes a protein with similarity to Drosophila Period, a protein involved in a second type of biological timing mechanism, control of circadian rhythms. Proposed experiments will investigate the functional relevance of this sequence homology and the yeast two-hybrid system will be employed to discover UN-42-interacting proteins. lin-58 will be cloned, and the information gleaned from its identity will be used to design molecular tests of its function. Because only a single, hypomorphic allele of lin-57 exists, genetic screens will be performed to identify lin-57(null) mutations. These alleles will be characterized and the hypothesis that lin.57 is temporally controlled by the 21 nt let-7 regulatory RNA will be tested. lin-29 plays a key role as the most downstream regulator of seam cell terminal differentiation. The molecular mechanism that times lin-29 activity will be determined and sequences critical for lin-29 regulation will be defined. Finally, genetic screens will be employed to search for addition heterochronic genes. Loss of lin-29 function causes cells of the adult stage to indefinitely reiterate the larval program of cell divisions rather than exiting the cell cycle and differentiating. Thus, lin-29 can be thought of in general terms as an anti-oncogene: a gene whose function is required for cell cycle exit Temporal control of lin-29 provides a model for how cells within an organism are instructed to cease dividing at a specific time in development and differentiate. This knowledge should aid in our understanding of the problems that occur when growth controls go awry, such as the inappropriate resumption of cell divisions that occurs in some cancers.

Nutritional control of developmental programs

The long term goal of the proposed research is to elucidate the mechanism(s) by which nutritional cues are linked to the initiation of developmental programs in animals. This problem is fundamental to animal development, but has received relatively little attention at the molecular level. The simplicity of C. elegans and its ease of growth and manipulation makes it an ideal system for these studies. In particular, food availability during postembryonic development is simply controlled, and the worm's transparency allows developmental responses to food to be easily monitored. The proposal focuses on dissecting two genetic pathways: 1) a daf-18-mediated signaling pathway that actively controls the initiation of postembryonic development in response to food, and 2) the heterochronic gene pathway that times certain postembryonic events and is influenced by environmental conditions, including food availability. The presence of food initiates and coordinates timing of postembryonic developmental programs in diverse tissues of the worm. If nematodes are hatched in the absence of a food source, they arrest development. Work in the Rougvie laboratory indicates that DAF-18, the worm homolog of the tumor suppressor PTEN, is required for arrest of postembryonic developmental programs under starvation conditions. Components of the daf-18-mediated nutritional response pathway will be defined by testing candidate genes for involvement and by conducting genetic screens to identify novel components. Nutritional inputs also impinge upon the heterochronic gene pathway, but the mechanism by which these external signals influence timing genes is unknown. Temporal progression of postembryonic cell fates is triggered by the accumulation of the lin-4 microRNA which subsequently acts to down-regulate target genes. In the absence of food, lin-4 expression is not activated and development does not progress. In addition, another heterochronic gene, lin-42, is extremely sensitive to environmental conditions during development; its mutant phenotype is suppressed by adverse conditions. Genes will be identified that link environmental cues to the heterochronic gene pathway via lin-4 or lin-42. Broader impacts: This project will contribute to the training of a postdoctoral fellow, graduate and undergraduate students. Emphasis will be placed on developing testable hypotheses and critical interpretation of data. Past undergraduates in the laboratory have co-authored papers, and it is anticipated that undergraduate trainees supported with award will make similar contributions while conducting their research projects.

DESCRIPTION (provided by applicant): Control of developmental time is of fundamental importance to all multicellular organisms and is achieved with astonishing precision. The aim of this work is to understand the timing mechanisms that govern specific cell fate decisions during metazoan development, using the nematode C. elegans as a model organism. The heterochronic genes of C. elegans are global temporal regulators that control the sequence and timing of diverse events during postembryonic development. Mutations in these genes alter the relative timing of developmental programs, causing certain events to occur too early or too late. This research will primarily investigate the molecular roles of three key temporal regulatory genes: lin-58, lin-42, and hbl-1. Emphasis will be directed at understanding how these genes receive temporal information from genes acting upstream in the hierarchy and in turn relay that information to correctly time expression of downstream targets, lin-58 mutations define temporal regulatory role for the mir-48 microRNA, which acts early in development, while lin-42 and hbl-1 are distinct in that their analysis reveals both early and late roles suggesting they have a more global timing function. Molecular mechanism(s) of action of these genes will be determined, including analysis of conserved homology domains, and they will be positioned with respect to others in the pathway through genetic analysis. A key goal is to identify molecules that interact directly with the products of these genes and to determine their function. A variety of molecular and biochemical techniques will be employed to achieve this aim. Genetic screens will identify additional temporal regulatory genes. Relevance to human health: Understanding the mechanisms by which these genes act provides a model for how cells within an organism are instructed to cease dividing at a specific time in development and differentiate. Knowledge about this control should aid in the understanding of the problems that occur when growth controls go awry, such as the inappropriate resumption of cell divisions that occurs in some cancers.

The Caenorhabditis Genetics Center (CGC) is the sole comprehensive repository and distribution center for the nematode Caenorhabditis elegans, a premier model organism for biomedical research studies. The overall objective of this animal resource is to promote research on C. elegans by acquiring, maintaining, and distributing genetically characterized nematode stocks. Researchers throughout the world use genetic stocks obtained from the CGC in diverse basic and applied research endeavors, as well as for hand-on teaching of experimental science. Studies using this premier model organism have led to fundamental insights into basic biological mechanisms, including the genetic basis of programmed cell death, the discovery of microRNAs, and the mechanism of RNA interference in animals. The nematode has also proved important for understanding mechanisms of cancer progression and other diseases including Alzheimer's and Parkinson's, as well as for revealing basic mechanisms underlying human development. In addition, C. elegans serves as a key model for illuminating our understanding of parasitic nematodes with relevance to human and livestock health. As the only general stock center for C. elegans, the CGC is an extremely important international research resource, supporting research in these diverse areas and educational endeavors. The CGC provides more than 30,000 strains are distributed per year to thousands of laboratories; with a collection of over 19,000 unique strains, still expanding in proportion to the growth of the field, the CGC not only facilitates research, but also ensures that valuable strains are preserved. The CGC distributes strains upon request through an on-line ordering system. A scheme of user fees helps to defray costs and support CGC activities. The CGC also includes a research component aimed at enhancing the CGC collection. Our close monitoring of user needs, ties with the C. elegans community, and focus on genetic tools has given us a unique perspective in devising a research component. Two aims will be pursued, one focused on expanding the genetic tool-kit by generating intrachromosomal inversions for use as crossover suppressors. The other aim is to complete the collection of null mutations in microRNA genes.

Project Summary/Abstract?Applied Research Component The goal of the Applied Research Component of the Caenorhabditis Genetics Center (CGC) is to enhance the CGC strain collection. The CGC aims to curate strains of importance to the biomedical research field, including null mutations in all protein coding genes as well as ncRNAs, certain transgenic strains, and genetic tools. The C. elegans molecular genetic tool-kit has a deep foundation built upon identified mutations in many thousands of genes, genome-wide RNAi libraries, easy and efficient methods of transgenesis, well developed and rapidly evolving methods for genome modification using Crispr-Cas9 techniques, mechanisms for tissue-specific gene activation and protein destruction, and others. The applied research component will use Crispr-Cas9 genome modification methods to expand the strain collection. In Aim 1, we propose to improve the C. elegans genetic balancer collection. Genetic balancers are chromosomal rearrangements that act as crossover suppressors. They are essential useful tools for working efficiently with lethal or sterile mutations, allowing them to be maintained easily as heterozygotes. They also facilitate genetic crosses of mutants with subtle phenotypes. A particularly useful class of crossover suppressors encompasses intrachromosomal inversions that are also marked so as to allow different progeny classes to be readily identified. Recently developed Crispr-Cas9 strategies allow creation of targeted chromosomal rearrangements allowing us to make designer inversions. We will build a set of inversion-based crossover suppressors that fully covers the C. elegans genome, marked with red or green-fluorescent reporters. In Aim 2, to support the goal of curating a null mutation in every gene, the CGC will make a collection of mutations in miRNA genes. microRNAs (miRNAs) are small regulatory RNAs, first discovered through basic research in C. elegans, that have profoundly changed our understanding of eukaryotic gene regulation in processes including development, metabolism, stem cell maintenance, and cancer; with demonstrated roles in human development and disease, they are being developed as diagnostic as well as therapeutic tools. To facilitate community research on the functions of this important gene class, we will make deletion alleles of this remaining set of miRNAs, again using established Crispr-Cas9 methods. All strains generated in this work will be curated by the CGC and immediately available through the CGC website.

Project Summary/Abstract The objective of this project is to extend the C. elegans genetic toolkit available to researchers by providing publicly available gene knockout (KO) mutations in genes of high value to those interested in human biology and disease. C. elegans is a proven model for discovery of gene function and for embedding genes into functional pathways, many of which were discovered in this transparent animal and are conserved in humans. Loss-of-function mutations are the gold-standard for genetic analysis and allow inferences of gene function and interaction. A relatively recent trend is that large-scale screens (e.g., RNA-seq, proteomics, natural variation studies) generate long candidate gene lists for researchers to sort through and functionally validate, requiring loss of function mutants in many genes to be examined. Having a community-driven resource generate KOs in such genes provides reproducibility and an economy of scale that benefits all. We have developed an efficient and high throughput CRISPR-Cas9 based gene knockout (KO) project to provide a set of precisely edited gene knockout strains to the communities of C. elegans researchers and human geneticists. We seek to continue the production phase of this project, thereby transformatively increasing the efficiency and impact of C. elegans molecular genetics. We employ a highly coordinated three-site production strategy to generate KOs. Each gene edit is carefully confirmed, and validated strains are grossly phenotyped and deposited into the Caenorhabditis Genetics Center (CGC) strain collection along with detailed strain information for distribution to the community. Strains are advertised through both the CGC and WormBase websites. We have generated over 1,000 KOs to date; these KOs have supported a variety of research projects ? spanning from mechanistic studies of human variants to metabolomics ? funded by at least 14 NIH Institutes and Centers, demonstrating the high impact of our resource. We propose to continue to use our established pipelines to target an additional 2500 C. elegans orthologs of human genes. We will prioritize known or suspected human disease genes, druggable gene classes, as well as understudied genes that are conserved to humans but that have no actionable information. This is a multi-PI project which includes the lead-PIs of the CGC and of WormBase and a subcontractor who maintains multiple community-centered databases, including a CRISPR guide RNA selection site.