Min Han - US grants

The long term goal of this research is to understand the mechanism of cell communication in controlling developmental pattern formation. Vulval development in the nematode Caenorhabditis elegans is a simple example of inductive pattern formation: three of six equipotent precursor cells are induced to generate vulval cells by a signal from the gonad. A number of genes including the let-60 ras proto-oncogene play key roles in a genetic pathway that specifies the vulval cell fates in response to the inductive signal. The proposed research uses both molecular and classical genetics to dissect the vulval signal transduction pathway. In addition to our continued characterization of the let-60 ras gene, we will identify and characterize genes acting downstream of ras including its effector. The research plan consists of three major parts. (1) Analysis of let-60 ras. The expression pattern of let-60 ras gene during development will be examined by whole mount staining of let-60 ras and its fusion gene products. The relationship between let-60 ras structure and function will be studied by analyzing mutations that alter its specific activities or interactions with other factors. (2) Analysis of the lin-45 gene which likely acts downstream of let-60 ras in the signal transduction pathway. The function and structure of the lin-45 gene will be determined by analysis of mutants and molecular cloning. The site of action of lin-45 during vulval development will be examined by mosaic analysis. The expression pattern of lin-45 and the relationship between its structure and function will also be studied. (3) Identification and characterization of new genes acting downstream of let-60 ras in the signal transduction pathway. Novel genetic screens will be carried out to isolate mutations that suppress the loss-of-function or gain-of-function, mutant phenotype caused by let-60 ras mutations. Genetic and molecular analysis of genes defined by these suppressors will lead to determination of their structures and functions. By using vulval development as a model system and the power of C. elegans genetics, the in vivo function of ras oncogene product and its interacting factors in signal transduction can be elucidated. Study of the molecular basis of cell interaction in specifying developmental pattern is thus closely related to the study of the of cancer. The analysis of suppressors of activated let-60 ras might also allow us to discover new ways to suppress ras-mediated cancer.

DESCRIPTION (provided by applicant): Developmental decisions are controlled by the combined action of multiple regulatory pathways. One critical aspect of regulation is the controlled timing of the onset of specific developmental programs that render cells competent for responding to other signals. Our goal is to understand the genetic and biochemical mechanisms by which several genes regulate developmental timing and to understand the link between the regulatory pathway and specific cellular events such as cell divisions and differentiations. Research under this grant has used C. elegans vulval induction as a model system to investigate the functions and interactions of multiple pathways, including the Ras-MAP kinase pathway. Recently, by searching for genes acting downstream of Ras signaling, we have identified four genes that are involved in regulating the timing of vulval cell divisions. Among them, LIN-66 represses the expression of a key timing regulator LIN-28, and AIN-1 binds to ALG-1, Dicer and microRNA of the microRNA induced complexes (miRICS) and targets ALG-1 to specific cytoplasmic foci. A transcription factor LIN-31 that acts downstream of Ras-MAPkinase is also involved in timing regulation. We will carry out a series molecular and genetic experiments to understand how LIN-66 regulates lin-28 expression and what biochemical properties AIN-1 provides to miRISCs. Through identifying the miRNAs and their targets associated with AIN-1, we will determine if AIN-1 is associated with only a subset of miRNAs for their functions, as well as learning about these specific functions carried by these targets. We also plan to study the functions of AIN-2, which has structural and functional similarities to AIN-1, as well as two other genes. Finally, we will search for targets of LIN-31 to learn about what factors execute the regulatory role of LIN-31 in timing regulatory and in mediated Ras signaling. microRNA functions and Ras signaling are conserved between worms and human, and are involved in many human developmental processes and diseases, such as cancers. Studies in C. elegans have made a huge impact on the research in related fields. The proposed research intends to provide new insights into cellular processes involved in these newly identified genes.

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. The subproject and investigator (PI) may have received primary funding from another NIH source, and thus could be represented in other CRISP entries. The institution listed is for the Center, which is not necessarily the institution for the investigator. PURPOSE: Dynamic contrast breast MRI requires both high temporal and spatial resolution. To increase temporal resolution in bilateral breast imaging which needs to cover two large volumes, we combine rapid 3D spiral imaging and temporal sensitivity-encoding (TSENSE) acceleration. METHODS: A dual-band spectral-spatial pulse was used to excite both breasts for providing robust fat suppression and an independent spatial and spectral profile for each breast volume. The "stack-of-spirals" imaging trajectory was used for a rapid k-space acquisition. We accelerated an imaging speed by applying TSENSE in the slab direction. All even kz-planes are acquired before all odd kz-planes for time frame. The odd and even frames together can be used to estimate coil sensitivity functions. Then sensitivity-encoding (SENSE) can be used to fill in missing planes in both the odd and even sets, to accelerate the frame rate by a factor of two.

ACT2; AT744.1; Act-2; Biochemical; Biogenesis; CCL4; CCL4 gene; CRISP; Classification; Complex; Computer Retrieval of Information on Scientific Projects Database; Funding; Gene Expression; Grant; Immune Precipitation; Immunoprecipitation; Institution; Investigators; LAG1; MIP-1-beta; MIP1B; Mass Spectrum; Mass Spectrum Analysis; Messenger RNA; Micro RNA; MicroRNAs; Microarray Analysis; Microarray-Based Analysis; NIH; National Institutes of Health; National Institutes of Health (U.S.); Numbers; Origin of Life; Photometry/Spectrum Analysis, Mass; Property; Property, LOINC Axis 2; Protein Family; Proteins; RNA, Messenger; Research; Research Personnel; Research Resources; Researchers; Resources; SCYA4; Source; Spectrometry, Mass; Spectroscopy, Mass; Spectrum Analyses, Mass; Spectrum Analysis, Mass; Systematics; United States National Institutes of Health; gene product; insight; mRNA; miRNA; microarray technology

This subproject is one of many research subprojects utilizing the resources provided by a Center grant funded by NIH/NCRR. Primary support for the subproject and the subproject's principal investigator may have been provided by other sources, including other NIH sources. The Total Cost listed for the subproject likely represents the estimated amount of Center infrastructure utilized by the subproject, not direct funding provided by the NCRR grant to the subproject or subproject staff. Partial saturation of an outer slab can reduce inflow enhancement and pulsatile ghost artifacts by preparing flowing spins to a steady state before entering the imaging slab, while dephasing their signal [1]. However, the partial saturation requires an additional RF pulse and spoilers, increasing TR. Here, we present a short RF pulse that simultaneously excites the imaging slab while partially saturating the outer slab. To read about other projects ongoing at the Lucas Center, please visit http://rsl.stanford.edu/ (Lucas Annual Report and ISMRM 2011 Abstracts)

DESCRIPTION (provided by applicant): MicroRNAs (miRNAs) commonly repress gene expression by binding to the 3' untranslated region of target mRNAs, and thousands of mRNAs have been predicted to be targets of hundreds of miRNAs in animals. Despite intense studies in the past decade, the majority of regulatory miRNA::mRNA interactions for a broad spectrum of physiological functions remain to be elucidated. In C. elegans, genetic analyses have only linked a small percentage of miRNAs or miRNA families to prominent roles in development. The difficulty encountered in functional studies of miRNAs may be attributed in part to that multiple miRNAs from different families may collaborate to regulate a set of target genes for specific physiological functions. We may also hypothesize that a large number of miRNAs may function in cellular processes involved in animals' responses to environmental changes such as nutrient availability, stress conditions and pathogen infection. Our goal is to gain a global view of dynamic miRNA::target interactions in different tissues and under different physiological conditions to obtain insights regarding miRNA-mediated functions in stress and infection responses. We propose to combine novel systematic approaches with individual gene- based analysis to tackle the problem, using the nematode C. elegans as a model system. We will first evaluate physiological functions of miRNAs by disrupting activities of all miRNAs in selected tissues and examine the developmental and non- developmental consequences, including responses to stresses and infections. We will then identify and analyze miRNA::target interactions in selected tissues during development by applying AIN-2 immunoprecipitation, high-throughput RNA analysis, and computational methods. This approach will further be used to identify dynamic miRNA::target interactions during animals' responses to food deprivation, pathogen infection and other stresses. Finally, we will carry out experiments to verify miRNA::target interactions for selected pairs and analyze their functions in stress and pathogen responses. The results of this study should provide important insights regarding important functions of miRNA regulations and mechanisms of animals' defense against environmental stresses and infections.

Project Summary How animals regulate growth, development and behaviors in response to changes in nutrient availability and metabolic status is a fundamental biological problem that is closely related to human diseases and health. While major advances have been made over the last decade in understanding TOR, insulin receptor and other signaling systems that sense the levels of sugar and amino acids to control various cellular and physiological functions, the study of the mechanisms by which cells and animals respond to changes in nucleotide levels to regulate development and other physiological functions is lacking, even though nucleotide levels have been shown to impact several cellular processes including cancer progression and certain human disorders. The goal of this proposal is to discover and analyze the mechanism of such a nucleotide-responsive regulatory system. By using genes involved in nucleotide metabolism in both sthe worm and its food source E. coli, we have established a unique model system where we observe a profound impact of uridine and thymidine (referred to as U/T) on germline development. Results from our preliminary study, including a suppressor screen, have indicated two key factors in the U/T sensing system leading to the hypothesis that an unknown system involving a ribonuclease acts upstream of a Notch signaling pathway to regulate germline proliferation and pyrimidine metabolic pathways. We will test this hypothesis by studies under three specific aims: (1) determine how Notch receptor (glp-1) expression is regulated by U/T levels, including the identification of the U/T-responsive element and potential upstream regulator; (2) determine if a novel ribonuclease (scdd-1), identified from our genetic screen, mediates the impact of U/T level on glp-1 expression; (3) investigate the potential regulation of pyrimidine metabolic pathways by U/T levels scdd-1; and (4) investigate the mechanism of scdd-1 function. By connecting nucleotide availability to developmental programs and metabolism, the results from these studies will likely present an important conceptual advance in the fields of nucleotide metabolism, development and nutritional science. Such findings may also provoke others to evaluate the impact of nucleotide quality in food or nutritional supplements on human health, especially for those with genetic risks for certain metabolic diseases.

Project Summary Due to their essential roles in fundamental biological processes (e.g., energy production and membrane formation), the availability of fatty acids (FAs) profoundly impacts the initiation and progression of various cellular and developmental events in animals. In particular, fat or FA levels have long been proposed to promote reproductive development, as well as neuronal and muscle functions for foraging ability under fasting conditions. Extensive studies have also revealed a cause/effect relationship between abnormal FA metabolism and pathologic conditions, including age-related neurological and muscular diseases. However, mechanisms underlying the impact of FA levels on specific physiological functions, especially functions regulated by FA-sensing mechanisms in specific tissues, have been underexplored. Our recent study found that an acyl-coA synthetase and protein myristoylation act as a FA sensor in the germline to regulate the onset of oogenesis by modulating the sex-determination. Based on this finding, we propose to elucidate the mechanisms by which FA availability regulates muscle maintenance and provide insights into the pathogenesis of FA metabolism-related degenerative diseases. We have obtained extensive preliminary data for a hypothesis where myristoylation deficiency of two ARF GTPases, and other proteins in muscle, mediate the impact of FA deficiency on sarcomere integrity by inducing ER stress and unfolded protein responses (UPR) that are known to be involved in the genesis of major diseases. We proposed three specific research aims to further investigate this hypothesis and the underlying mechanism. In Aim 1, we will use both molecular and genetic approaches to determine the role of myristoylation in muscle to maintain sarcomere integrity. Myristoylation level and subcellular localization of specific regulatory factors will be examined for roles in mediating the effect of FA level change on muscle functions. In Aim 2, we will analyze the role of ER stress and UPR in mediating the impact of myristoylation deficiency on muscle maintenance. We will first characterize ER stress and changes in the 3 UPR pathways in responding to FA and myristoylation deficiency. We will then test if experimentally inducing ER stress causes muscle defects similar to that from myristoylation deficiency, and whether repressing ER stress can rescue the muscle functions under myristoylation deficiency. For Aim 3, we will turn our attention to understanding how myristoylation and ER stress impact muscle integrity. We will use two systematic approaches, one expression analysis-initiated and one based on a suppressor screen, to search for factors that act downstream of or in parallel to UPRER in FA/myristoylation deficiency-induced muscle defects. We have already started analyzing one promising candidate, UNC-97/PINCH, that appears to play a significant role in the process. The proposed research will make significant advances in our understanding of the impact of FA metabolism on muscle functions.

Project Summary/Abstract Despite its long history, the nutrient biology field is still facing a plethora of outstanding questions closely related to human health, two of which will be explored in this proposal. First, the nutrient value of many specific molecules in our diet, or the beneficial impact of many specific bacterial metabolites on human physiology (as predicted by the symbiotic relationship between commensal microbes and host animals), remain unclear. Second, whereas great advances have been made in the last decades on understanding signaling systems that sense the levels of glucose, amino acids and other well-studied nutrients to regulate various physiological events, the mechanisms that respond to the level of many other specific nutrients, including certain fatty acids, nucleotide variants and micronutrients, are largely unexplored. About 6 years ago, our lab boldly moved the major research direction to study problems related to nutrient functions and sensing, focusing mainly on under-explored fatty acid variants, nucleotides and bacterial metabolites, using the nematode C. elegans as the primary model with additional analysis in mice and mammalian cells. In one aspect, we developed innovative assays to identify the unknown beneficial impact of bacterial metabolites, including the siderophore enterobactin and bacterial cell wall components peptidoglycan (PG), on animal development and behaviors. Under this MIRA grant, we will carry out a thorough investigation of the newly discovered beneficial roles of PG fragments that have mainly been the subject of immune defense studies in the past. By analyzing the structure of potent PG fragments, their impacts on various aspects of animal physiology, and the interacting host factors, we aim to uncover the mechanism of this fascinating new role of bacterial PG. In the other aspect, our effort in recent years has uncovered four novel regulatory systems that sense the deprivation of specific fatty acid and nucleotide variants to regulate developmental and behavioral events to protect animals? reproductive fitness. In particular, our study under the existing GM R01 grant uncovered an intestine-initiated pathway that regulates germ cell proliferation and metabolism in response to pyrimidine deficiency. Under this MIRA grant, we will address critical mechanistic questions surrounding the roles of an obscure endonuclease that increases its expression in response to nucleotide imbalance and that acts in the intestine to regulate metabolic and developmental events. Past research in the C. elegans field has indicated that this organism is best used to make novel discoveries that present important conceptual advances in biology. With promising preliminary data, the projects described in this MIRA application have great potential to make paradigm-shifting discoveries that would impact our understanding of the nutritional value of microbiota-produced molecules and the diversity of nutrient-sensing mechanisms.