University Of California Santa Barbara

NIH Research Projects · FY 2026 · 2022-06

Project Summary In the first two weeks of development, the human embryo breaks symmetry twice, transforming itself from a uniform ball of cells into a highly-patterned, spatially organized set of tissues. Although decades of elegant genetics and biochemistry in non-human model organisms have uncovered many of the essential signaling proteins and pathways for embryonic patterning at this early stage, a lack of tools for directly manipulating the signaling in time and space, as well as limitations to working with human embryos, have limited our ability to understand how these early patterning events arise in humans. Thus, my laboratory seeks to develop engineering strategies to (1) understand what patterns of signaling activity encode extracellular information, and (2) determine how these patterns are decoded at the tissue level to drive high fidelity collective cell fate decisions in human embryonic stem cells. This proposal brings together many recent advances in stem cell and molecular engineering to decode how spatiotemporal signaling and embryo size instruct tissue fate patterning. We leverage advances in 2D micropatterning, 3D bioprinting, cellular optogenetic control over developmental signaling pathways, and CRISPR-based reporters of cell signaling pathways. Together, these technologies give us unprecedented control over and visualization of microengineered models of human gastrulation, thereby enabling us to investigate the principles of environmental information transmission and potential mechanisms of pregnancy loss and developmental anomalies that arise with a surprisingly high frequency (10-20% by some estimates) in the early human embryo. In addition, our platform does not face the same ethical barriers that have limited human embryo research, allowing us to ascertain how physical and information-bearing parameters of the embryo lead to stereotyped patterning of the germ layers during human gastrulation. In this proposal, we focus on the role of canonical developmental signaling pathways by dissecting the effects of spatiotemporal signaling and variance of the Wnt pathway on germ layer fate positioning in 2D (Aim 1); examining the role of Erk signaling on positioning, dynamics, and coordination of cells during the development of the mesoderm/trophectoderm boundary (Aim 2); and examining the effect of cell number and tissue size in 3D gastrulating models of the human epiblast and amniotic sac (Aim 3).

NIH Research Projects · FY 2026 · 2022-05

Neural circuitry processes information at the cellular level by filtering, amplifying, and integrating electrical signals generated and modulated by synaptic input and voltage-gated mechanisms. This project focuses on uncovering the underpinnings of such processes using electrophysiology, two-photon imaging, and optogenetics. The key aim is to understand the role of neuronal dendrites in processing information during in vivo circuit activity. Dendrites can fire regenerative electrical spikes much like axons, and this potentially provides a critical aspect to information processing at the cellular level. How such an active mechanism is engaged and plays a functional role in a behaving animal remains unclear. By directly recording intracellular electrical activity from fine distal dendrites and their parent somas during sensory processing in vivo, along with two-photon imaging of calcium dynamics at synaptic inputs and dendrites, we seek to understand how active dendritic mechanisms contribute to synaptic integration, and how their modulation affects sensory integration. In this study, we will address the following questions in vivo: 1) What is the relationship between dendritic synaptic inputs and dendritic spiking, 2) how effective are dendritic spikes in triggering axonal action potentials, and 3) what is the functional role of dendritic spikes in sculpting the receptive field properties of neuronal output. Findings from the project will both further our understanding of the fundamental mechanisms of cellular information processing and provide a foothold to decipher how neural circuitry is affected in conditions such as Alzheimer’s Disease and other neurophysiological disorders.

NIH Research Projects · FY 2025 · 2022-03

PROJECT SUMMARY Several neurodegenerative diseases, such as Alzheimer's disease (AD), are characterized by the spread and aggregation of the protein tau. Tau aggregates or neurofibrillary tangles (NFTs) accumulate throughout the brain of patients and lead to dementia. No effective treatments currently exist for tauopathies. Those approaches that have been considered, such as tau antibodies and antisense oligonucleotides, directly target tau. The complexity of tau cell biology, however, makes this approach challenging. A completely novel approach is to take advantage of the tau spreading pathway. The spread of NFTs correlate with disease progression and is a likely mediator for the observed neurotoxicity. Recently, we identified a cellular receptor, LRP1 (Low-density lipoprotein Receptor-related Protein 1), that regulates the tau spread pathway. Knockdown of LRP1 prevents tau spread in human iPS neurons and the mouse brain, suggesting that the tau-LRP1 interaction could be an important entry point for disease intervention. Therefore, the main objective of this project is to identify small-molecule chemical probes that prevent the binding of tau to LRP1, with the hypothesis that these molecules would serve as key starting points for novel therapeutics. In preliminary work, we have identified the primary interaction surface for tau on LRP1 and have developed a TR-FRET high-throughput screening (HTS) assay to identify compounds that can disrupt this interaction. We have optimized the assay to 1536-well format and conducted a pilot screen of 5,000 compounds with excellent performance, Z'~0.7 and a hit rate of ~0.4%. Analysis of the 20 hits from this screen in primary, artifact, and orthogonal assays identified several compounds with modest potency in dose response. To fully develop this work, we propose three aims. In Aim 1, we will use the TR-FRET assay to screen a 420,000 chemical library and, in parallel, conduct an affinity screen of a 4.4-billion- member DNA encoded library leveraging the DELopen platform (WuXi AppTec). In Aim 2, we will narrow our hit selection using orthogonal and novel biochemical profiling assays to determine mechanism of action. Finally, in Aim 3 we will validate hits with advanced biophysical and cell-based assays. We expect our multi-pronged approach will identify multiple chemical series with different mechanisms of action. Subsequent hit expansion efforts will produce chemical probes with properties suitable to test our hypothesis that small molecule LRP1-tau inhibitors can prevent tau uptake and spread. In future studies we intend to develop these probes into drugs that prevent tau spreading in tauopathies such as AD. As the critical path testing funnel is in place, we anticipate we can rapidly obtain and evaluate selective in vitro hits, explore their activity, and ultimately their suitability as starting points for hit-to-lead studies and for future in vivo evaluation in animal models and eventually patients.

NIH Research Projects · FY 2025 · 2021-09

Abstract The mosquito Aedes aegypti spreads diseases such as dengue, Zika, yellow fever, and others that afflict >100 million people each year. These mosquitoes rely on multiple keen senses to locate human hosts for blood meals, and for finding conspecifics for mating. Currently, we have only a rudimentary understanding of the receptors that control these critical behaviors. The goal of the proposed research is to address this gap. The unifying theme of this proposal is to test the idea that opsins and TRP channels are two key classes of signaling proteins that have broad roles in sensation and in controlling behavior in Ae. aegypti. Rhodopsins are the founding G-protein coupled receptors (GPCRs). We recently discovered that opsins are multi-modal sensory receptors, challenging 100 years of dogma that they detect only light. To find humans, female Ae. aegypti integrate information from diverse stimuli, including CO2, visual cues, organic molecules, and convection heat from skin. We discovered another cue. Aim 1 builds on our preliminary data that Ae. aegypti use infrared (IR) radiation as an additional host stimulus. We outline experiments to reveal the roles of opsins and the TRPA1 channel in IR detection. We propose to identify the IR-sensing neurons that express the opsins and TRPA1, and to test a model to explain the role of opsins in IR sensation. To pursue this aim, we devised a highly effective assay for monitoring IR attraction and a new molecular genetic approach to bypass difficulties in combining multiple genetic elements. Aim 2 takes advantage of a mutation that we created in another TRP (TRPV-A), which renders males and females deaf. We will test the roles of hearing and TRPV-A in swarm formation, in mating, and in finding humans. Aim 2 will also build on the observations that male mating requires audition mediated by TRPV-A to devise a strategy to overcome a major impediment limiting the efficacy of the sterile insect technique (SIT). SIT is a promising strategy to suppress Ae. aegypti. It involves inundating a local population with sterile males, which then render females sterile upon mating. An obstacle to using SIT is that wild-type males outcompete sterile males. We propose that manipulation of the activity of the TRPV-A- expressing auditory neurons elevates sterile male mating success, and will thereby increase the efficacy of SIT in suppressing Ae. aegypti. Aim 3 concerns identifying the sensory receptors for repellents. We propose to test the idea that an opsin functions as a highly sensitive receptor for insect repellents. If confirmed by the proposed experiments, this would demonstrate that opsins comprise a new class of olfactory receptor. To accomplish our goals, we have developed an extensive repertoire of state-of-the-art approaches. These include new molecular genetic tools, a suite of behavioral assays, original video tracking software, and in vivo electrophysiological recordings. In summary, this project will reveal the roles of opsins and TRP channels in allowing mosquitoes to sense humans and conspecific mates. The insights gleaned from this work have exciting potential to lead to innovative strategies to control Ae. aegypti and reduce insect-borne disease.

NIH Research Projects · FY 2024 · 2021-09

PROJECT SUMMARY (See instructions): Proteins are the drivers of molecular activity in the cell, but we still lack a comprehensive understanding of the mechanisms by which cells regulate protein abundances. A long-standing question has focused specifically on the mechanisms and degree of post-transcriptional regulation, which also determines the degree to which mRNA levels can be used to predict protein abundances. For example, mRNA levels generally correlate with protein abundance across genes, but that mRNA-protein correlations can vary significantly within genes across conditions, due in part to post-transcriptional regulation, such as regulation of protein synthesis and degradation. Post-transcriptional regulation has been analyzed in bulk samples composed of heterogeneous cell types, but it remains largely unexplored in single cells. To enable systematic analysis of post-transcriptional regulation at single-cell resolution, we need a novel analytic framework which 1) accounts for single-cell measurement error and technical bias, which are convolved with the relevant biological signal for both mRNA and protein abundance 2) leverages probabilistic models which pool information across genes in functional groups or account for the determinants contributing to protein abundance, like ribosomal binding proteins 3) models abundance-dependent missing data in single-cell mRNA and protein data and 4) associates observed post-transcriptions regulation with likely regulatory mechanisms. To achieve these goals, we build upon our long-standing collaboration and propose the following aims: Aim 1. To develop methods for inferring post-transcriptional regulation in single cells. These methods will employ hierarchical models which account for similarities between genes with common regulatory mechanisms. We will explicitly model non-ignorable missing data and account for measurement error in the data. Aim 2. To apply and validate the methodology from Aim 1. We will analyze single-cell mRNA and protein measurements from human immune cells and testis with a focus on identifying and validating functionally related genes regulated by common RNA binding proteins. The research will be integrated into the education programs at the Pl's institutions, with a particular focus on capstone experiences. Reproducible software implementations for new tools will be developed.

NIH Research Projects · FY 2025 · 2021-09

Project Summary Key questions of the 21st century in medicinal and biochemical areas are focused on the intimate operation and functionality of biomolecule at the molecular level. By understanding the physical properties of biomolecules at the nanoscale we can envision how to control them and use them for various applications. The proposal below is aimed to promote these key questions via the use of nanoscale electrochemistry. I am very excited to share with you some of our preliminary results and future vision dealing with both the applied and fundamental sides of Bioelectrochemistry. The first part of the proposal is focused on merges the lab expertise in nanoelectrochemistry and charge transport phenomena and focuses on an innovative method to measure the electronic response of individual enzymes during catalysis. By that, we hope to unravel by electronic means the way that enzymes operate and fluctuate with respect to their catalytic activity at the molecular level. Gained information can guide us towards the control and design of enzymes both from fundamental aspects and industrial applications. The second part of the research program offers a template for a paradigm shift in the way we think about electrochemical biosensors. We propose to construct a chemically amplified electrochemical system that enables the detection and analytical sizing of insulating materials, one at a time. We plan to use this experimental approach for designing a sensor for rapid pathogen detection. The main goal here is to innovate a sensor that can be used in a personalized fashion, without the need of complex device operation. Realization of such sensor can potentially mitigate the number of unnecessary antibiotics given at the point of care.

NIH Research Projects · FY 2024 · 2021-09

Project Summary Epigenetic modifications are essential chemical modifications that play critical roles in gene regulation, development, and diseases. Therefore, understanding how epigenetic changes between species occur and how they affect gene regulation has potential to advance our knowledge of regulatory evolution. However, the details of epigenetic evolution are sparse, and how epigenetic evolution correlates with phenotype evolution is even less understood. The proposed research will address this gap of knowledge by integrating novel data on DNA methylation with primate brain evolution. Studies of human brains have demonstrated that distinctive brain cell types have substantially different DNA methylation and gene expression, and analyses without separating these cell types can yield misleading results. Additionally, comparative studies of primates and other mammals have shown that the anatomical and cellular structure of brain regions evolve at varying rates as a result of differences in neurodevelopmental events linked to overall brain size. DNA methylation is a key molecular mechanism to record and affect development, and shows difference between brain regions. Therefore, the proposed research will test a novel hypothesis that DNA methylation of distinctive cell types in human and non-human primate brains shows variation consistent with brain size evolution. Moreover, validation studies will be performed for specific candidate genes and genomic regions that show DNA methylation and gene expression difference related to brain region differences and species differences. Specifically, evolutionary histories will be constructed for DNA methylation (Specific Aim 1) and gene expression (Specific Aim 2) from two major subclasses of neurons (excitatory and inhibitory neurons) as well as oligodendrocytes (a major non-neuronal cell) of brains from diverse anthropoid primates, including humans, apes, and monkeys. Highly divergent brain regions in terms of function and anatomy (e.g., prefrontal cortex and the pons) will be compared to connect changes at the phenotypic level to molecular changes. Some of these candidate genes will be further investigated in deeper histological resolution (Specific Aim 3). This study will generate novel data to expand our understanding of epigenetic evolution of brains, and to infer functionally important positions of noncoding genomic regions. Furthermore, it will also provide knowledge on how epigenome changes during evolution and how epigenome evolution correlates with phenotype, which is a fundamental yet currently little understood topic.

NIH Research Projects · FY 2025 · 2021-08

DNA double strand break (DSB) repair pathways resolve DNA lesions that arise during cellular metabolism or as the by-product of cell damage. Human DSB repair pathways fall into two distinct categories: end joining (EJ) pathways that rejoin the DSB molecule, and homology directed repair (HDR) pathways that use a template molecule to repair the DSB molecule. The factors that cells use to decide between EJ and HDR repair pathways remain incompletely defined. Many studies have shown that the cell cycle regulates DSB pathway choice, yet cultures arrested at points in the cell cycle that favor HDR still repair the majority of DSBs using EJ. The long-term goal of the research in my lab is to comprehensively define factors that bias DSB repair in sufficient detail that we can predict DSB repair outcomes based on the initial conditions inside a cell. Pursuit of this goal will improve our understanding of DNA repair and related processes, enable new generations of gene editing reagents with greatly increased efficacy, and suggest new strategies to diagnose and treat human DNA repair pathologies, including cancer and aging. Over the next five years, we will develop a holistic model for DSB repair that describes DNA repair events occurring on the DSB and template molecules. Our goals in generating this model are to define the irreversible commitment step between EJ/HDR and to understand if cells sense their capacity to perform HDR before they pass commitment. These are important challenges for the cell, because inappropriate HDR can cause cell death or genomic instability. We hypothesize that cells have the heretofore unmeasured ability to develop DSB repair complexes in parallel, and that parallel maturation of DSB repair complexes plays a role both in the EJ/HDR commitment and as a checkpoint for these repair pathways. Parallel development of EJ and HDR complexes either on the DSB molecule or split between the DSB and template molecule would allow cells to simultaneously develop different types of repair before committing to one or the other. The ability to generate mature repair complexes prior to commitment would make DNA repair substantially less risky. Our practical approach is to develop genomic and proteomic techniques that allow us to measure DSB repair intermediates with unprecedented temporal and spatial resolution. We will use these techniques to define how protein complexes associate with chromatin over time and, crucially, the strandedness of DNA bound to DSB repair proteins. Measuring this latter parameter will allow us to determine when events occur in relation to the EJ/HDR decision and thus understand when and how this decision is made. We also explore mechanisms of communication between multiple DSB repair complexes assembled in parallel onto chromatin. Parallel events are especially informative because they indicate a dynamic system in which cells simultaneously explore multiple DSB repair pathways, thereby preserving choice until repair is nearly complete. For example, events on the template molecule may act as a checkpoint for events on the DSB molecule, or vice versa. This work will enable new tools that leverage our understanding of DSB repair to influence gene editing outcomes and to improve therapeutic workflows. We also anticipate that our work will open new fields of inquiry, for example defining how DSB repair complexes assembled interact with each other and with cell-wide signaling mechanisms.

NIH Research Projects · FY 2025 · 2021-07

This project proposes to investigate neurophysiological circuit mechanisms supporting spatial cognition and episodic memory. The retrosplenial cortex (RSC) is critical in these cognitive processes as RSC dysfunction is associated with spatial disorientation and learning and memory deficits, as well as Alzheimer’s disease pathology. One prominent idea is that RSC facilitates spatial transformations between coordinate systems, wherein egocentric spatial information encoded relative to the animal itself is related to allocentric spatial information encoded relative to the external world. This computation is required for navigation and episodic memory, as both require information experienced via sensory organs to be represented relative to the broader environment. RSC possesses the requisite anatomy and activity patterns to facilitate spatial transformations, but there has been no direct evidence of the computation occurring within the region. This gap at least partly arises from a general lack of knowledge regarding RSC base function; it is unknown if the region is flexibly recruited as a consequence of ongoing behavior, where functionally-defined RSC sub-populations project, or how afferent inputs contribute to known forms of spatial coding within the area. I will learn techniques for high-density extracellular recordings, in vivo neuroimaging, and projection- specific optogenetics to test the role of retrosplenial circuit dynamics in spatial transformations. First, I will provide the first characterization of spatial coding differences and task-based recruitment of distinct RSC sub-regions that have biased projections to egocentric and allocentric spatial processing streams. From these large populations of simultaneously recorded neurons, I will test for intra- and extra-regional internal network states that reflect computation of spatial transformations. Next, I will utilize in vivo imaging of large RSC populations longitudinally to examine if neurons are prewired or learn their spatial receptive fields as a function of task demands. I will utilize projection-specific imaging to test if specific spatial signals are transmitted to specific efferent targets in support of spatial transformations. Finally, I will pair the aforementioned methods for observing activity of large neuronal populations with projection-specific optogenetic circuit manipulations to test the role of different afferent inputs on different forms of RSC spatial coding. By utilizing innovative experimental approaches, these projects will provide important insights regarding the function of RSC in spatial transformations underlying spatial navigation and episodic memory. Results from these studies will establish RSC circuit mechanisms that mediate these cognitive processes in both healthy and pathological states. The scientific expertise and career/laboratory management tools that I will develop during the mentored phase of this award will be vital for my success as I transition into a faculty position and pursue my own independent research program.

NIH Research Projects · FY 2025 · 2021-07

Project Summary/Abstract This application seeks funding for a new Interdisciplinary Predoctoral Training Program in Quantitative Mechanobiology at University of California, Santa Barbara (UCSB). The Program brings biologists, physicists, and engineers together to pursue fundamental understanding of the biophysical principles underlying human health spanning molecular to organismal level processes of mechanosignaling in development, homeostasis and disease. The focus of this initiative is cross-disciplinary training in mechanobiology, i.e., relationships between physical forces and biological structure and function, to produce a new cadre of bioscientists who will develop quantitative solutions to biology’s most challenging problems. If funded, the Training Program in Quantitative Mechanobiology will be UCSB’s first training grant and will serve as a flagship predoctoral training program that nucleates a connected community across the many labs already conducting mechanobiology research at UCSB through peer-to-peer interactions, cross-lab training, seminars, courses, and an annual symposium/retreat. The Program will provide support for Fellows to undertake three research rotations in their first year and opportunities for unique cross-training in mechanobiology. Each year, six Mechanobiology Fellows will be admitted with a steady state Program size of twelve predoctoral training slots. The Program will recruit and support Fellows from six graduate degree programs and augment their PhD programs with training in quantitative bioscience methods, engineering models and devices, and multi-disciplinary cross-training to develop and apply quantitative approaches to problems in mechanobiology. To provide optimal interdisciplinary mentorship and research training experiences, each Fellow will work with their project PI and will also be assigned a Program Mentor with complementary seniority, quantitative, experimental, and analytical approaches. The Program Mentor will also serve on the Fellow’s dissertation committee. This chain of mentorship is supported by formal Mentor trainings and leverages our mix of junior and senior Faculty Mentors to support both our Fellows and our junior Faculty Mentors. Fellows will benefit from formalized interactions with the diverse community of faculty and peers, as well as resources, associated with Program and affiliated graduate programs. Key components of the Program include formalized training in Responsible Conduct of Research, Quantitative Experiments, didactic and hands-on training in methods and analytical techniques in a Mechanobiology Methods course and quarterly “open door” lab sessions in Faculty Mentor labs. The Program will feature monthly seminars with peers, visiting researchers, and Faculty Mentors as well as career development activities, and an annual symposium/retreat uniting the entire UCSB mechanobiology community. Trainees from diverse disciplines, backgrounds, and groups will be prepared to lead rigorous biomedical research programs and promote scientific advances in academia, industry, national labs, and technology and policy careers.

NIH Research Projects · FY 2025 · 2021-04

Psychomotor-stimulant Use Disorder (PUD) is a chronic relapsing disorder, characterized by a high propensity for relapse even during protracted abstinence. In both humans with PUD & animal models, the intensity of cue- elicited drug craving & drug-seeking behavior increases or “incubates” during protracted withdrawal. The neurochemical underpinnings of drug craving & its incubation are not well understood. Drug cue-induced increase in metabolic hyperactivity within the prefrontal cortex (PFC) is correlated with the intensity of drug- craving in humans. Consistent with this, we have reported a link between the magnitude of drug-seeking in a rat model of cocaine-taking & a number of abnormalities in glutamate (GLU) transmission within the ventromedial aspect of the PFC (vmPFC). Notably, incubated cocaine-seeking is associated with a time-dependent increase in the capacity of drug-predictive cues to increase GLU levels, primarily within the prelimbic (PL) subregion. We theorize this cue-elicited rise in GLU might underpin cue-elicited increases in metabolic hyperactivity observed within PFC of PUD patients. Importantly: (1) the increased GLU responsiveness to drug-predictive cues is selective for rats with a cocaine-taking history; (2) the magnitude of the GLU increase predicts the vigor of cocaine-seeking behavior; & (3) neuropharmacological inhibition of GLU transmission within the PL eliminates cocaine-incubated responding. Intriguingly, the incubated cue-responsiveness of vmPFC GLU is inversely related to cue-elicited changes in vmPFC dopamine (DA). This inverse neurochemical relation has led to the over-arching hypothesis to be tested in this proposal: the incubation of cue-elicited drug-seeking behavior results from dysregulated GLU-DA interactions w ithin the PL subregion of the vmPFC . Aim 1 of this proposal employs neuropharmacological approaches to systematically target & dissect the relative contribution of postsynaptic AMPA & NMDA GLU receptor subtypes to the manifestation of incubated cocaine-seeking & examine for the generalization of pharmacological effects to a highly prevalent psychomotor-stimulant, methamphetamine (MA), as well as the non-drug reinforcer, sucrose. It is hypothesized in Aim 1 that the incubation of COC craving is driven by GLU-mediated activation of ionotropic GLU receptors within vmPFC. Aim 2 will employ a combination of in vivo microdialysis & neuropharmacological approaches to examine the role for D1- & D3-type DA receptors & their regulation of GLU, DA & GABA release within the vmPFC in incubated cocaine-, MA- & sucrose-seeking. It is hypothesized in Aim 2 that the incubation of cue- elicited GLU release, cellular hyperactivity & drug-seeking reflect time-dependent anomalies in DA signaling within PL. The proposal presents a series of theoretically innovative experiments designed to address the biobehavioral underpinnings of incubated craving, which will advance our basic understanding of the neurobiology of relapse.

NIH Research Projects · FY 2025 · 2021-04

Project summary Vision plays a key role in our ability to navigate through the environment, from identifying landmarks and obstacles to determining location and heading. While studies of visual cortex have provided an understanding of properties such as orientation selectivity and object recognition, much less is known about how cortical circuitry extracts and processes features from the visual scene to support navigation. In particular, there are two challenges. First, the nature of the visual stimulus is dramatically different in navigation, where the subject's movement through the world creates a complex and dynamic visual input, in contrast to standard synthetic stimuli presented to stationary subjects. Second, the types of visual features and computations that must be performed are different in navigation than in standard detection or discrimination paradigms. Our goal in this proposal is to determine how the brain extracts relevant visual features from the rich, dynamic visual input that typifies active exploration, and investigate how the neural representation of these features can support visual navigation. We will investigate this through three parallel aims, that build up from the representation of the visual scene in V1 during freely moving navigation, to the computation of specific variables needed for navigation. In our first aim, we will measure the visual input in freely moving mice using miniature head-mounted cameras, together with neural activity in V1, to determine how neural dynamics represent the visual scene during natural navigation. In our second aim, we will use large field-of-view two-photon imaging of multiple cortical areas, while mice navigate in a naturalistic open-world virtual reality system, to determine how visual features are represented across visual cortical areas. In our third aim, we will use 2-photon imaging in mice in a rotational arena to determine how visual input is used to dynamically update a key navigational variable: heading direction. Together, this project bridges foundational measurements in freely moving animals with mechanistic circuit investigations, to provide insights into an important aspect of visual system function.

NIH Research Projects · FY 2026 · 2021-01

Project Summary/Abstract: There are currently two research thrusts in the PI’s laboratories: a) the development of bifunctional biaryl-2-ylphosphine ligands to achieve metal-ligand cooperation in gold(I) catalysis and catalysis by other metals, and b) the development of SN2 glycosylation chemistry that is applicable to the construction of a broad range of glycosidic linkages. By employing chiral bifunctional ligands readily prepared from chiral binols, the former thrust has led to unique solutions to challenging asymmetric gold catalysis for the past several years. Considering the potent and versatile catalysis in which gold(I) could render alkyne/allene substrates, our approach has led to expedient and highly enantioselective access to functional structures that are valuable in biomedical research. Additional efforts in this designed ligand-driven research started to bear fruits in asymmetric Pd catalysis. The latter thrust aims to address a long-standing challenge in carbohydrate chemistry, that is, the lack of a general stereoselective/stereospecific method for the construction of every type of glycosylic linkages. Over the past 7 years, the PI’s lab has advanced an SN2 glycosylation strategy termed the directing-group-on-leaving-group (DGLG) strategy. As the term suggests, this strategy engineers the leaving group of a glycosyl donor to strategically accommodate a directing group, which can promote the attack of an alcoholic acceptor at the anomeric center and opposite to the leaving group. The PI has published two implementations of the strategy and demonstrated the feasibility of the design. A variety of challenging 1,2-cis glycosidic linkages can be prepared in excellent yields and with complete inversion of anomeric configurations. However, more challenging glycosidic bonds such as those in β-mannosides, 2-deoxy sugars, and α-sialosides remain to be tackled by newer designs/implementations of the DGLG strategy. The following are the research areas that the PI’s research group would pursue: a) to further advance enantioselective gold catalysis by harnessing asymmetric gold-ligand cooperation; b) to expand the utility of the designed bifunctional biaryl-2-ylphosphines to asymmetric catalysis by other transition metals such as Pd. c) to develop new approaches of DGLG to achieving stereospecific synthesis of challenging glycosides including α-sialosides. d) to develop efficient gold catalysis to access biologically valuable structures whenever possible. This includes expedient access to unnatural amino acids and quaternary amino acids. The application of these amino acids in the development of peptide therapeutics and biomaterials will be subsequently pursued via collaborations. Overall, the mission of the PI’s lab is to employ innovative strategies to address important synthetic needs and facilitate their applications in biomedical research and drug development.

NIH Research Projects · FY 2025 · 2021-01

Abstract Our lab works at the intersection of immunology, stem cell biology and regeneration, and the grants funding this work (GM123267 and GM 123255) which we are requesting to merge in the MIRA program have provided numerous insights into the molecular mechanisms underlying both self/non-self recognition, as well as a genetically determined cell competition event that occurs between mobile germline stem cells for niche occupancy. In addition, we have recently found that these same germline stem cells, which are lineage restricted under normal conditions, are responsible for a regenerative response to injury called Whole Body Regeneration, during which entire bodies, including all cardiovascular, GI, central and peripheral nervous, endocrine and germline tissues are regenerated de novo from isolated vascular fragments, and we propose to extend our research efforts into this robust model system of chordate regeneration. As described in the proposal, in the last 18 months, these studies have led to a number of exciting findings we will follow-up on during the upcoming funding period, including: dissecting the molecular basis for allorecognition specificity and its conservation with vertebrate immunity; a novel mechanism of autocrine stimulation that is required for homing of germline stem cells and likely plays a role in the competitive phenotype; and rescue and lineage tracing assays for whole body regeneration that have revealed that a single germline stem cell can give rise to an entire body- a result which may have major implications for understanding germ cell tumors, and also provides a unique opportunity for rapidly creating genetically modified lines of Botryllus. Our long-term goals are to utilize the unique biological properties of Botryllus to carry out innovative molecular mechanistic studies, and a MIRA award would allow us to redirect our efforts from funding to carrying out more and better innovative research on these biomedically important topics.

NIH Research Projects · FY 2024 · 2020-09

The complex interaction between Alzheimer drivers and aging Project Summary/Abstract (30 lines of text) The greatest risk for Alzheimer’s disease is age. This extremely tight correlation with age has no explanation. However, most of what know about Alzheimer’s comes from early onset cases. We will utilize the 100 cases of early onset AD stored in the Colombian brain bank all with the same PSEN1[E280A] mutation to determine the full range of pathology observed due to a monogenic defect and compare these data to sporadic older onset disease. Some of the Colombian individuals in the bank have had amyloid and tau PET studies. In sporadic disease among the elderly, brain changes related to aging are frequent and in the absence of their clear delineation, treatments targeted solely at dominant genetic forms of the disease may be ineffective. Factors which might distinguish and promote AD in the elderly include inflammation, compromised brain vasculature, excessive microgliosis, cellular aging such as break down of the nuclear membrane and consequently escape of TDP-43 from the nucleus and possible contributions of synuclein. We will explore interactions of these factors with aging through descriptive neuropathology and experimental neuropathology methods. Comparisons will utilize sporadic AD post-mortem from several brain banks. These studies include state of the art single cell RNAseq and advanced assessment of inflammation markers. Cellular models will be explored using human induced pluripotent stem cell-derived neurons that harbor the PSEN1[E280A] mutation and are intended to discover downstream pathways affected by the mutation. Co-cultures with microglia to capture autonomous and non-autonomous effects of the mutation will be determined. To accomplish the aims we have assembled a multi-institutional international team with a long history of collaboration. Dr. Lopera, who first recognized the families and manages the Alzheimer Prevention Trial, heads the team in Colombia, an NIH supported project. To support the neuropathology effort we have enlisted the expertise of Dr. Eric Huang. Kosik has a nearly 30 year collaboration with Lopera, is familiar with the conduct of research in Colombia and recently traveled to visit the Colombian brain bank with Eric Huang. Kosik is closely connected institutionally with UCSF through his role as co-director of the Tau Consortium along with Bruce Miller. Kosik has published with Ellisman and serves on the review board for the National Center for Microscopy and Imaging Research center at UCSD. Thus the project consists of a strong, experienced and highly integrated team capable of conducting a complex project and dealing with any of the obstacles that will inevitably arise.

NIH Research Projects · FY 2025 · 2020-09

ABSTRACT Shape is critical for proper organ function. From genetic model animals, we have learned much about the principles of how morphogens setup body axes, and trigger a cascade of regulatory factors to setup a coordinate system of fate patterning the organism, and endowing each cell with a unique fate. Pioneering work demonstrated that the fate system controls shape. But how these processes control shape remains elusive. Mechanobiology demonstrated that shape emerges from physical interactions between cells. Therefore, morphogenesis is also a problem of physics, leading to the question: How does developmental biology control the physics of morphogenesis? From single cell studies in vitro, we have learned how cells utilize fundamentally dynamic processes that involve their cytoskeleton to generate forces. However, how forces are coordinated across tissues to reliably generate form remains elusive. Progress requires extending molecular analysis to investigation of cellular dynamics at the organ level, to study the interplay of forces and cell behaviors. The polymath D’Arcy Thompson already pointed out that quantitative analysis of morphogenesis promises new biological insights. His pioneering ideas came before the genetic revolution, that provided much of what we know about the molecular picture of morphogenesis. Since then, new tools such as fluorescence live imaging have emerged. In this proposal, we will lay the foundations for quantitative morphogenesis across length scales, connecting molecular level information with the dynamic processes that govern shape. Quantitative analysis of tissue dynamics at the organ scale, combined with emerging tools from physics will lead to the development of new, experimentally testable hypotheses for the dynamic rules of morphogenesis. We develop new quantitative tools to bring this interdisciplinary approach to fruition, and lead the way to the principles of morphogenesis. Multi view light sheet microscopy and emerging super resolution methods pave the way to interrogate the dynamics of molecular processes across entire embryos. Our biophysical image informatics approach extracts quantitative measurements of morphogenetic processes from microscopy data. These quantitative measurements are summarized in the powerful language of tensor morphogens, an emerging concept from physics. These concepts dovetail with theory development, and novel machine learning approaches – aimed to formulate a comprehensive framework that predicts how a genotype determines the outcome of morphogenesis. We begin with basic morphogenetic processes such as axis elongation, or folding. Both are wide spread strategies to shape the body across the animal kingdom. Our approach will open up new paths to a quantitative understanding of the physics of living matter, from the molecular level to the whole embryo.

NIH Research Projects · FY 2026 · 2020-06

PROJECT SUMMARY We propose a Maximizing Access to Research Careers at the University of California Santa Barbara (MARC-UCSB) program that builds on the strengths of our current MARC-UCSB program, and expands the reach and organizational capacity of our institution for increasing retention and matriculation into PhD and MD/PhD programs of all undergraduates (but especially underrepresented [UR] and disadvantaged students) interested in pursuing careers in biomedical research. As the first Association of American Universities (AAU) to reach Hispanic Serving Institution (HSI) status, and one of now six HSI-designated UC campuses, UCSB is among a growing group of research-intensive institutions serving a large number of Hispanic undergraduates and well-placed to incubate and share biomedical training innovations. In the nine years of MARC-UCSB, 25 Scholars have graduated, of which 100% have remained active in STEM and 68% (17) have enrolled in PhD or MD/PhD programs. With this proposal, we aim to incorporate a growing MARC alumni network with new campus training partnerships to expand our impact. We include a series of initial engagement points for MARC-eligible students that stimulate new points of entry into research and scientific community, and integrate into existing infrastructure at UCSB: gateway classroom outreach and early research experience; a mentoring network of peers, graduate students/postdoctoral scholars, and faculty; and an introduction to research course “Practice of Science.” As they build community and increase awareness of various research and career opportunities, students will be prepared to further develop as researchers through the two-year MARC Scholars program and accompanying ecosystem initiatives open to STEM students. Nine diverse motivated students will be selected as MARC Scholars each year. They will acquire extensive research experience at UCSB as well as extramural research sites, grow their communication and leadership skills through well-defined professional activities, serve as mentors for lower division students, and increase awareness of biomedical research careers for themselves and their peers. In addition to the 45 students who will receive MARC scholarships, hundreds more will be impacted by our activities and will be well-prepared to apply for other synergistic research internship programs and eventually to advanced degrees and careers in biomedical research. Underpinning this Scholar retention and success will be a suite of faculty and graduate student/postdoc mentor training initiatives and resources integrated into the expanding training landscape for sustained impact. Our MARC-UCSB program goals are to: a) ensure students are immersed into a cohesive social, academic, and research community that values their contributions from an early stage; b) cultivate students’ confidence and skills to enable them to matriculate into and succeed in top PhD programs (with at least 80% of MARC Scholars matriculating into PhD or MD/PhD programs); and c) increase our organizational capacity for mentoring excellence and an inclusive environment for our entire STEM campus community. To achieve our goals, we will: 1) apply “proposed innovations” to meet our objectives and address challenges in our current program, 2) expand “effective practice elements” that have been core features of our current successes, and 3) leverage “synergistic infrastructure” from institutional elements that contribute to achieving MARC-UCSB objectives.

NIH Research Projects · FY 2024 · 2020-03

PROJECT SUMMARY Targeted internal radionuclide therapy is a highly efficacious form of cancer treatment that employs administered radiopharmaceutical agents to destroy malignant cells. Radiopharmaceutical agents of this type require a biological targeting vector, which selectively recognizes and binds to receptors that are overexpressed on cancer cells, and a bifunctional chelating agent, which stably binds to and attaches the radionuclide to the targeting vector. The radionuclide can be chosen and altered to possess different nuclear decay properties, potentially rendering it valuable for theranostic applications, as long as its coordination chemistry is compatible with the bifunctional chelator. Conventionally, beta particle emitters have been leveraged for therapeutic applications, but recent clinical studies have revealed the efficacious nature of alpha particle emitters for this application. The use of alpha emitters is hindered, however, by a lack of gamma photon or positron emissions that can be leveraged for theranostic applications and by their unconventional coordination chemistries, making it challenging to find a suitable bifunctional chelator. This project will address both of these challenges in alpha particle emitter targeted internal therapy by designing new bifunctional chelators that can be simultaneously used with diagnostic gamma- or positron-emitting radionuclides. In Specific Aim 1, new bifunctional chelators for the established highly promising alpha emitter actinium-225 will be developed. These chelators will be designed so that they can also effectively accommodate the widely available imaging radionuclide indium-111. With these new chelators, theranostic agents based on actinium- 225 can be easily accessed. Specific Aim 2 will focus on the short-lived alpha emitters lead-212 and bismuth- 213. To make theranostic agents from these radionuclides, bifunctional chelators that can be easily functionalized with the versatile positron-imaging radionuclide fluorine-18 will be synthesized. Lastly, in Specific Aim 3, chelators for the unconventional newly arising radionuclides uranium-230, and vanadium-48 will be developed. Uranium-230 is a therapeutic alpha emitter and vanadium-48 is a diagnostic positron emitter. We will explore chelating agents that are mutually compatible for these radiometals to enable their use as a theranostic pair. Through these three aims, this work will increase the diversity of radionuclides with distinct half-lives and decay chains that can be used for this application. Furthermore, it will give rise to diagnostic partners for these therapeutic alpha emitters, which is critically important for predicting patient dosimetry, disease staging, and response. Collectively, the successful execution of this project will give rise to theranostic alpha-emitting therapeutic agents that can cure and diagnose human disease.

NIH Research Projects · FY 2026 · 2018-09

Early detection through screening mammography has decreased death rates from breast cancer. There are approximately 39 million mammogram procedures conducted each year in the US. However, there are still alarmingly high error rates in radiological interpretations, with missed cancer rates ranging from 10-18 percent and false positive rates as high as 67% over a 10-year period. To reduce error rates, digital breast tomosynthesis, a new 3D imaging technology intended to make cancers more visible to the radiologist, is rapidly being introduced throughout clinics in the US. The widespread adoption of new 3D technologies has dramatically increased the data volume that radiologists must scrutinize and has fundamentally altered how they search, relying on vision away from where they are fixating (peripheral vision), eye movements, and image (slice) scrolls to find potential disease. Yet, we are missing a theoretical and empirical understanding of how radiologists might best search through 3D clinical images to maximize target detection while maintaining reasonable reading times. Furthermore, what metrics to use to optimize image processing and acquisition parameters to maximize radiologists’ performance with the new 3D technologies? The last 30 years of the field of medical imaging have been shaped by task-based image quality metrics based on ideal and model observers that mimic human performance. And yet, these often omit human bottlenecks of peripheral visual processing and do not work with 3D search with clinical images or phantoms. In this context, the overall goal of the current proposal is to combine recent Deep Neural Network developments and biologically-plausible models of human-foveated vision to create a model that learns about anatomy and optimally (performance maximizing) programs eye movements and scrolls to find lesions in digital phantoms and clinical images (foveated search DNN, FS-DNN). If successful, the FS-DNN could be used to evaluate in what way a particular radiologist is not adequately examining the 3D clinical, estimate the accuracy costs of the scroll/search inefficiency, and determine how they might improve. The project will implement learning protocols based on FS-DNN/human comparisons and assess their impact on improving diagnostic accuracy. If successful, the FS-DNN could be a new powerful metric of image quality for 3D search that, unlike previous models, can be applied to phantoms and clinical images. A collaboration with the Food and Drug Administration (FDA) aims at integrating the developed FS-DNN model with the FDA Virtual Imaging Clinical Trial for Regulatory Evaluation (VICTRE) pipeline that is made available to academic researchers and industry technology developers. Together, these advances can potentially help reduce radiological errors with digital breast tomosynthesis and also help evaluate and optimize new technologies. Although the proposed work is developed for breast cancer and DBT, the approach, framework and concepts investigated are potentially applicable to other areas of 3D medical images in radiology.

NIH Research Projects · FY 2025 · 2017-09

Summary. Our overarching goal is to render therapeutic drug monitoring as convenient and highly time resolved as the continuous glucose monitor has rendered the monitoring of blood sugar. The realization of this goal would transform many aspects of both biomedical research and clinical practice. It would, for example, enable personalized dosing based on a patient’s accurately determined, rather than poorly predicted, drug metabolism, an outcome of high relevance to the treatment of infectious diseases, which commonly employs drugs of dangerously narrow therapeutic index, and to improving women’s health, as pharmacokinetic sex differences lead to a doubling of adverse pharmacotherapeutic outcomes in females. Ultimately such a technology could enable feedback-controlled drug dosing, which, by responding in real time to metabolic variations, would improve the safety and efficacy of drugs that suffer from dose-limiting toxicity. To achieve our goal, however, requires two significant innovations: (1) a technology able to monitor arbitrary drug molecules in situ in the intestinal fluid (ISF) of the subcutaneous space and (2) vastly improved knowledge regarding how the pharmacokinetics of drugs in the ISF relate to the pharmacokinetics seen in plasma. Under the prior round of grant funding, we achieved the first of these necessary advances. Specifically, we demonstrated that minimally-invasive Electrochemical, Aptamer-Based (EAB) sensors support the seconds-resolved, real-time measurement of drugs in situ in the plasma (venous), cerebrospinal fluid (brain), and ISF (subcutaneous space) of our live rat animal model. Here we propose to tackle the second innovation. That is, using intravenous and subcutaneous EAB sensors we propose to advance understanding of the relationships between plasma and ISF pharmacokinetics across a diverse set of antimicrobial and immunosuppressant drugs for which therapeutic drug monitoring is an important element of the standard of care. We believe the resulting orders of magnitude improvement in measuring these relationships is a critical step towards our long-range goal of rendering high-precision therapeutic drug monitoring convenient and cost effective.

NIH Research Projects · FY 2026 · 2017-06

Abstract Opsins are classical G-protein coupled receptors, which for more than a century have been thought to function exclusively as light detectors. However, using Drosophila, we have uncovered multiple, light-independent roles for opsins including detection of bitter tastants. This discovery raises exciting questions, which we propose to address here. Do opsins only respond to bitter compounds, or do they function more broadly in taste sensation? Of particular interest, do opsins serve as taste receptors in other animals, including the mosquito, Aedes (Ae.) aegypti? This vector spreads viruses that cause dengue, yellow fever, and other diseases that afflict millions of people each year. Both male and female Aedes use taste to identify nectar and avoid toxic chemicals, while females use many senses to identify people for blood meals. However, the receptors required for mosquito taste are largely unknown. Another fascinating question is whether opsins serve as taste receptors in mammals. To address these questions, we propose to employ an extensive suite of approaches including electrophysiology, Ca2+ imaging, behavioral assays, cell biology, an in vitro receptor activation assay, and state-of-the art molecular genetics. We will also bring to bear our expertise in Drosophila taste, opsins, and our recent experience performing molecular genetics and behavior in Ae. aegypti and in the mouse. Aim 1 will define the role of a Drosophila opsin in amino acid taste, which in flies is a poorly understood taste modality. Attraction to amino acids increases if the flies are kept on an amino acid deficient diet. We outline experiments to define the contribution of the opsin to this dynamic change in amino acid appeal. We will also test the proposal that the opsin is activated directly by amino acids. Aim 2 will build on our preliminary data that two Ae. aegypti opsins are expressed in the major taste organ, and are required for sensing a flavonoid. The goals of this aim are to reveal the chemical specificity of these opsins in vitro and in vivo, determine whether they act independently or together, and identify the gustatory receptor neurons in which these opsins function. Aim 3 will characterize the roles of opsins in mammalian taste. This aim is supported by preliminary data showing that multiple mouse opsins are expressed in the mouse tongue, and are activated in vitro by bitter compounds, including a flavonoid for which there is no known receptor. In addition, we present preliminary data that the response to this flavonoid is impaired in the opsin mutant. We propose to test the hypothesis that this opsin functions broadly in sensing flavonoids, identify the taste receptor cells that express this and the other “gustatory opsins,” and perform behavioral tests with mutants to characterize the requirements for each opsin for sensing bitter compounds. The results from this project would establish opsins as a new class of taste receptor conserved from insects to mammals. We propose that defining opsins as the first bitter receptors in mosquitoes offers the potential to harness this information to manipulate mosquito behavior and thereby reduce their populations and thus insect-borne disease.

NIH Research Projects · FY 2026 · 2017-04

PROJECT SUMMARY The bottleneck for tauopathy therapy development is the lack of validated tauopathy models, mouse, cell or in vitro. This is reflected in the current reality that tauopathy-specific fibril structures solved by cryo-EM from post mortem patient brain tissue have never been replicated outside a patient, i.e. not in a mouse, cell or in vitro. While the patient- derived tauopathy fibrils offer critical goal posts, they are not in and of themselves viable therapeutic targets. For example, the development of Positron Emission Tomography (PET) ligands to diagnose and track Alzheimer’s disease (AD) or corticobasal degeneration (CBD) disease progression relies on screening small molecule binding to CBD- or AD-phenotypic fibrils—the very construct that nobody knows how to build yet. There are many more factors to consider for replicating the pathological pathway of tau aggregation, but replicating disease phenotypic tau fibrils is a minimal and necessary requirement, and so far an unattained tool for therapy development. The major knowledge gap that this proposal aims to close is the mechanism and tools to replicate tauopathy specific fibrils in vitro (Aim 1), and the key cellular and molecular factors that initiate misfolding of tau in cell to disease phenotypic shapes and facilitate aggregation (Aim 2). If we can successfully replicate any one tauopathy-phenotypic tau fold, or even a part of a folded tau structure, such as a mini-hairpin fold of CBD or AD with seeding competency, it will have an immediate impact on ongoing therapy developments, such as on the development of tauopathy-specific PET ligands, antibodies and small molecule drugs. This team will employ an innovative set of structural biology tools encompassing pulsed double electron-electron resonance (DEER), TEM and cryo-EM, as well as computational tools to focus on capturing the full folding and aggregation pathway of the tau protein ensemble from its intrinsically disordered to partially folded and fully converged fibril states. This team will concurrently use innovative cell biological tools with a strong premise of the knowledge of a dedicated tau receptor and transporter that can enhance tau seeding by endosomal escape and the knowledge that enhanced hydrophobicity of the local environment of tau is a potent factor to initiate misfolding, aggregation and propagation. While discovering the defining property of a competent seed and achieving shape propagation with seeds developed in this proposal will be a breakthrough, independent of this success, we will have developed experimental and computational tools to evaluate whether seeded shape propagation has occurred, or whether all, part, or none of the shape propagates. To have the tools to evaluate the mechanism of shape propagation will be a game changer. The lack of progress in closing the above-described knowledge gap is not due to a lack of investment by top notch laboratories around the world, but due to shortcomings of existing concepts and tools. Han and Kosik, together with Shea, will rely on the convergence of their respective fields and investing concerted effort using innovative tools to address long-standing questions in tauopathy research.

NIH Research Projects · FY 2026 · 2015-04

SUMMARY The major objectives of the proposed research are to illuminate the cellular and molecular mechanisms that control developmental plasticity and to investigate how post-mitotic differentiated cells in an intact animal can be reprogrammed and remodeled into new cell types in the process of transdifferentiation (Td). The well-described pathway for endoderm development in C. elegans will be applied to molecularly dissecting Td and “transorganogenesis” (conversion of one organ into another). One component in this pathway, the C. elegans ELT-7 GATA transcription factor, is capable of converting differentiated, post-mitotic cells of two organs, the pharynx and uterus, into cells with gene expression patterns and ultrastructural characteristics of normal gut cells. Highly dynamic changes in the transcriptome occur during this remarkable process, and six stages (promiscuity, attenuation, extinction, rebound, persistence, and remodeling) can be resolved during Td based on gene expression and altered cellular and organ morphology. These events define Td-competent (Td+) cells that convert to gut-like cells and Td-resistant (Td-) cells that only transiently permit ELT-7 to activate its target. Among the genes whose expression undergoes upregulation during Td are sets of genes associated with protein turnover, autophagy, and the intracellular pathogen response (IPR), suggesting that these pathways play a role in Td. On the basis of ELT-7-induced developmental arrest, two genetic selections were developed that identify large numbers of mutants defective in Td. With these preliminary findings in hand, we will probe the mechanisms of Td through three Specific Aims. In SA1, we will investigate the dynamics of cellular remodeling, test the hypothesis that turnover processes of protein degradation and autophagy participate in key stages of Td and remodeling, assess the role of the IPR in Td, and investigate the action of cell-cycle exit in the Td process. In SA2, we will evaluate whether differences between single Td+ and Td- cells of varied differentiated cell types can be ascribed to their unique transcriptome dynamics and will test the hypothesis that changes in chromatin architecture of Td- cells, assessed by ATAC-seq, initially and transiently proceed through similar patterns to those of Td+ cells, with elastic reversion to the original state. In SA3, we will characterize mutants from two genetic selections that are defective in ELT-7-mediated developmental arrest and Td and will use them to investigate interdependence of the six stages of Td. We will identify the causal gene(s) underlying large numbers of mutants defective in Td by a high-throughput method based on statistical analysis of variants in pooled genome-wide sequences, including those coding for proteins and non-coding RNAs. These studies may advance our understanding of mechanisms involved in pre-cancerous metaplasias of the digestive tract. They will also provide insights into the mechanisms of organ malformation in birth defects and could lead to methods for reprogramming differentiation for organ generation in regenerative medicine.

NIH Research Projects · FY 2024 · 2015-04

SUMMARY The major objective of the proposed research is to illuminate the molecular basis underlying genetic and epigenetic variation in a major developmental gene regulatory network (GRN). Studies from this and other labs have identified a cascade of “core” zygotically expressed GATA-type transcription factors, and maternal regulatory inputs, that activate the GRN controlling development of the endoderm in C. elegans. The latter include the maternally supplied SKN-1/Nrf2 transcription factor and a triply redundant Wnt, MAPK, and src signaling system that acts through the LIT-1/NLK kinase and the POP-1/Tcf/Lef transcription factor to initiate endoderm development. Removal of any one of these inputs results in an impenetrant phenotype, reflecting a bistable state that shows wide variation between genetically distinct isotypes. Analysis of reciprocal crosses between isotypes with quantitatively different requirements for SKN-1 in this process revealed that endoderm GRN output is also influenced by long-term heritable epigenetic states that differ between natural C. elegans isotypes. This transgenerational epigenetic inheritance (TEI) requires genes involved in piRNA function, the nuclear RNAi pathway, and histone H3K9 methylation. These findings provide a springboard for unveiling the molecular basis for genetic and epigenetic plasticity in the regulation of the endoderm GRN. In Aim 1, we will evaluate hypotheses regarding the mechanisms of action of three genes that differentially alter the requirements for SKN-1 and Wnt signaling. We will assess how expression of the core regulators of endoderm development is influenced by quantitative variation in the requirement for the maternal GRN inputs. We will assess how variation in the requirement for LIT-1 kinase is accommodated in the mechanism that controls asymmetric cell division leading to activation of the endoderm GRN and will test the hypothesis that quantitative variation in the requirement for LIT-1 extends to its global action in many asymmetric cell divisions. In Aim 2, we will develop and implement high-resolution, high-throughput approaches to identifying causal genes underlying variation in the requirement for the major endoderm regulatory inputs. We will test candidate genes for modulation of the SKN-1-dependent activation of the endoderm GRN. In Aim 3, we will analyze the molecular basis for transgenerational inheritance (TEI) of GRN output. We will assess the stages in the endoderm GRN that are modulated by this TEI and test the hypothesis that epigenetic differences in SKN-1 requirement between selected isotypes extends to other regulatory inputs. We will test the hypothesis that TEI results from differences in chromatin states of endoderm genes and that differential expression of non-coding RNAs and endoderm regulatory genes is associated with TEI. Findings from this research will help to illuminate mechanisms of birth defects and can provide a paradigm for understanding relationships between an individual’s genotype and responsiveness to pharmacological agents, of importance to advancing precision medicine. They will also reveal factors that alter the outcome of Wnt signaling, a major regulatory mechanism associated with many cancers.

NIH Research Projects · FY 2025 · 2006-07

Abstract Insects infect hundreds of millions of people annually with deadly viruses and parasites. The goal of the proposed project is to decipher the molecular and cellular mechanisms mediating the insect sense of taste, with the long-term goal of using these insights to develop new insect control strategies. The proposed work will exploit the sophisticated arsenal of tools available for studying the fruit fly, Drosophila melanogaster, to address fundamental questions in the insect taste field. The aims will employ a wide diversity of approaches, including electrophysiology, Ca2+ imaging, behavioral assays, molecular genetics, and cell biology. The project will leverage insights from Drosophila to attack similar problems in the mosquito, Aedes aegypti. The sense of taste is not only essential for mosquito survival, allowing them to discriminate nutritious from dangerous foods, but assists them in making the final decision to bite a human or fly away. Currently, the receptors that function in mosquito taste are largely unknown. In this proposal, we outline experiments focusing on three poorly- understood areas in insect taste: fatty acid taste, amino acid taste, and acid taste. Drosophila find low levels of fatty acids attractive and high levels aversive. The goal of aim 1 is to decipher the receptor and neuronal mechanisms that underlie this discrimination. We will test the model that low fatty acid levels stimulate feeding by activating an amplification cascade initiated by a G-protein coupled receptor and culminating with a TRP channel in cells that stimulate attraction, whereas high levels of fatty acids directly activate a TRP channel in cells that cause repulsion. Fatty acids are potent antifeedants that suppress biting by Aedes. We will test whether the TRP homolog in Aedes aegypti mediates this effect. Aim 2 is to define the receptor and neuronal mechanisms underlying amino acid taste. This is important because flies must consume essential amino acids for growth and egg production. We will test the ideas that: 1) within taste neurons of the fly tongue, a Drosophila TRP senses amino acids in food, and 2) the same TRP detects circulating levels of amino acids in the brain. We will also test the idea that the Aedes TRP homolog is an amino acid sensor. Aim 3 addresses the identity of the proton channel that functions in acid taste. Low levels of some acids are attractive, but high levels are toxic. We will test the idea that a H+ sensor is required in multiple types of taste neurons in the Drosophila proboscis to prevent ingestion of dangerous levels of acids. Acid taste is also important in Aedes to suppress consumption of toxic acids in food, yet carboxylic acids are present in human sweat. We will test whether the Aedes homolog of the Drosophila H+ sensor functions in acid taste. In summary, the proposed research will reveal the receptor mechanisms underlying poorly understood areas in Drosophila taste, and includes the development of a key new molecular genetic tool to facilitate work in Aedes. This project has the long-term potential to lead to the development of innovative strategies to deter biting and reduce devastating mosquito-borne diseases.