University Of California Riverside

NIH Research Projects · FY 2026 · 2020-07

Project Summary/Abstract A central aspect of parasitic nematode success and prevalence is their ability to modify host biology and evade and/or subvert the host’s immune response. In some cases, humans can host thousands of nematode parasites with little to no pathology, yet our understanding of this incredible evasion or suppression of the immune system remains limited. Modulation of host biology and the pathology they cause is largely driven by the release of proteins and small molecules that interact with host cells and tissues. There are hundreds of proteins released in nematode spit during an infection and only a few have been studied in any detail. My lab is focused on understanding host-parasite interactions, with an emphasis on elucidating the molecules that parasites release into the host, characterizing their interaction with host signaling pathways to modulate host biology, and learning from the evolution of the parasite arsenal how to manipulate the immune system. Over the next five years my lab will undertake in-depth studies of specific proteins released by parasitic nematodes. Our specific focus will be to 1) leverage pipelines for identifying novel parasite-derived proteins and small molecules that modulate host biology, 2) determine the effects of the molecules we identify, beginning with members of the fatty acid- and retinol-binding (FAR) protein family, 3) elucidate molecular interactions between parasite molecules and host pathways, and 4) further characterize eicosanoid signaling in Drosophila melanogaster, a genetic model of immunity. A major strategy of my lab’s research is to combine in silico, in vitro, and in vivo experimental approaches with an established infection model that leverages our deep understanding of fruit fly biology and its powerful genetics, to reveal not only the binding targets of parasite proteins and molecules in an active infection, but also to define their effect on infection outcomes. Our overall goal is to understand how nematode parasites modify host biology in order to successfully infect them. This includes parasites’ ability to evade and/or suppress host immunity, which is important to human health in at least two ways. First, nematode infections continue to be a major source of global morbidity and mortality, affecting more than 25% of the world’s population. Increasing drug resistance and recurring infections compound this problem. And second, there is mounting evidence that the immunomodulatory effects of nematode infections can dampen or even eliminate the pathologies that define autoimmune disorders such as Crohn’s disease, inflammatory bowel disease, and Celiac disease. Understanding how nematodes suppress the immune system will lead to new treatment and vaccination strategies against nematode infection, and may reveal new avenues for treating autoimmune disorders. We will employ a powerful model system to probe immune modulation by nematodes to identify specific secreted proteins and small molecules as well as the signaling pathways they target to effectively manipulate host immunity.

NIH Research Projects · FY 2026 · 2020-06

ABSTRACT Alkylating agents are ubiquitously present in the environment, and they can alkylate DNA and RNA directly or after metabolic activation. The focus of the present R35 application is placed on the chemistry and biology of DNA and RNA alkylation, with the overarching goal of understanding the molecular mechanisms through which alkylating agents and other environmental chemicals exert their adverse human health effects. In particular, we will examine the roles of translesion synthesis DNA polymerases in the transcriptional bypass of DNA lesions in cultured human cells, identify DNA damage recognition proteins for alkylated DNA lesions, and investigate their roles in DNA damage response signaling and repair. We will also assess the implications of RNA methylation in the molecular pathology of trinucleotide repeat expansion diseases. The proposed research is a culmination of our established expertise in the areas of DNA damage and repair, proteomics and epitranscriptomics. Completion of the proposed research will lead to an unprecedented level of understanding about the human health consequences of exposure to alkylating agents and other environmental toxicants, and will ultimately result in approaches for the prevention and mitigation of adverse human health consequences arising from environmental exposure.

NIH Research Projects · FY 2026 · 2020-02

Abstract The control of organellar gene expression is critical for the cellular programming of all eukaryotic organisms. While perturbing mitochondrial gene expression leads to human pathologies, including cancer, altering plastid gene expression can kill plants. However, the cell signaling mechanisms that control organellar gene expression remain poorly understood. The long-term goal of the PI’s laboratory is to utilize the light-induced plastid differentiation into photosynthetically active chloroplasts in Arabidopsis as a genetic model to interrogate cell signaling mechanisms for controlling organellar gene expression. The current data support the central hypothesis that the red and far-red photoreceptor phytochrome B promotes the degradation of a small family of phytochrome-interacting basic/helix-loop-helix transcription factors in the nucleus to generate nucleus-to-plastid (anterograde) signals that trigger the assembly and activation of the multisubunit, bacterial-type plastid RNA polymerase for transcribing plastid photosynthesis genes. Here the PI propose to utilize a combination of molecular genetics, biochemistry, and genomics approaches to (1) determine the mechanism initiating anterograde signaling in the nucleus, (2) determine the mechanism of the assembly of the multisubunit plastid RNA polymerase complex, and (3) determine the mechanism activating the plastid RNA polymerase. The proposed research is innovative because it utilizes photoreceptor signaling and chloroplast biogenesis in Arabidopsis as a genetic model to elucidate a previously uncharacterized anterograde signaling pathway. The PI has developed new forward genetic approaches and biochemical assays and identified critical components that define the framework of anterograde signaling. The proposed research is significant, because it is expected to uncover the light signaling mechanism for initiating chloroplast biogenesis - a long-standing gap in our knowledge of plant light signaling and the regulation of photosynthesis. Because the control of transcription in plastids shares intrinsic similarities with that in mitochondria, what we learn in the plastid model is expected to enhance the understanding of the general principles of cell signaling mechanisms in controlling organellar gene expression, including the regulation of mitochondrial gene expression, and therefore, will ultimately contribute to the understanding of the mechanisms underlying the misregulations of mitochondrial gene expression in human diseases.

NIH Research Projects · FY 2026 · 2019-08

ABSTRACT The fate and function of individual cells and tissues is often dictated at the transcriptional level by the activity of cell- and tissue-specific transcription factors, as well as by RNA Binding Proteins (RBPs) that determine aspects of mRNA processing such as alternative splicing, stability, and localization. The physiological importance of RBPs in regulating RNA processing is illustrated by many human diseases, for example Spinal Muscular Atrophy and ALS/FTD, which can result from RBP mutation or dysregulation. A major open question for many of these diseases is: why does disruption of a widely-expressed RBP result in highly cell-specific phenotypes? For example, in SMA, why does mutation of the ubiquitously-expressed SMN1 result specifically in motor neuron degeneration? A second major question is: which target(s) of a given RBP are responsible for a specific phenotype? And a third related question: are the physiologically-relevant targets of a given RBP different in different cell or tissue types? Collectively these open questions illustrate that much remains unresolved about the relationship between RBP regulation and function in specific cells and tissues. Thus, we will ask how broadly-expressed RBPs result in highly specific functional outcomes, focusing on cell- specific regulatory mechanisms of two model RBPs, MEC-8/RBPMS and SMN-1. In addition to well-established roles for RBPs in regulating processes such as RNA stability and alternative splicing, the known universe of RNA regulation modalities continues to expand. Our lab is particularly interested in novel types of alternative splicing, aiming to dissect new and emerging types of alternative splicing at the cell-specific level, focusing on (1) formation of circular RNAs (circRNAs) due to back-splicing as opposed to canonical forward splicing, and (2) alternative splicing of exons that are not multiples of 3 nucleotides, and thus may lead to unusual outcomes including amino acid recoding, alternative C-termini lengths, and regulated RNA degradation. Together these experiments will address a significant gap in our understanding of the regulation and function of RNA processing at the cell- and tissue-specific level.

NIH Research Projects · FY 2025 · 2019-01

PROJECT SUMMARY/ABSTRACT In order to survive, animals develop fear responses to dangerous situations. The neural mechanism of learned fear has great survival value for animals, who must predict danger from seemingly neutral contexts. For adaptive fear, the brain discriminates between different contexts and associates only relevant contexts with aversive events. Dysregulation of this process leads to maladaptive overgeneralized fear in PTSD, which affects 7 percent of the U.S. population. A fundamental gap in understanding the mechanism underlying the specificity and persistence of contextual fear memory limits research on developing effective neuromodulatory strategies to prevent long-lasting maladaptive fear in PTSD. The overall objective in this application is to determine the mechanism by which specific contextual fear memory is encoded and consolidated in a network of the hippocampus, neocortex, and amygdala. The rationale for the proposed studies is to fill a critical void in the understanding of fundamental principles of adaptive fear responses to relevant contexts and to provide new insight into developing strategies to attenuate persistent pathological fear in PTSD. The central hypothesis, based on the applicant’s preliminary data, is that (1) the acquisition and consolidation of contextual fear memory require strengthening of engram cell pathways, which connect populations of memory engram cells in a distributed network, and (2) selective weakening of the engram cell pathways prevents conditioned fear responses to a context that predicts danger. This hypothesis will be tested by pursuing three specific aims: (A) Determine how contextual fear memory is encoded in the hippocampal–amygdala circuit and how it is consolidated for permanent storage in prefrontal neocortical circuits (Aims 1 and 2), and (B) Determine input- output connectivity of memory engram cells for contextual fear memory (Aim 3). The first and second aims will investigate synaptic changes in the context-specific hippocampal–amygdala pathway as well as excitatory connections between prefrontal neocortical engram cells during the acquisition and consolidation of contextual fear memory. To accomplish this, the applicant recently developed a novel approach by combining engram cells labeling, optogenetic, and electrophysiological techniques. Under the third aim, engram cells labeling and trans- synaptic tracing will be used to determine how memory engram cells are connected to neurons conveying contextual and aversive signals and neurons generating defensive behavior. The proposed research is innovative because it will use novel combined approaches to efficiently identify the neural correlates of associative memory encoded in functionally heterogeneous neural circuits, which has been unattainable through conventional approaches. The proposed research is significant because it will elucidate how fear memory for a relevant context is encoded and consolidated in a distributed network of memory engram cells and provide insight into developing a novel approach to attenuate chronic maladaptive fear in PTSD without affecting adaptive emotional memories.

NIH Research Projects · FY 2026 · 2018-09

In higher eukaryotes, mitochondria play multiple roles in bioenergetics, metabolism, and signaling. The mitochondrial DNA (mtDNA) genome is indispensable for mitochondrial function because it encodes protein subunits of the oxidative phosphorylation system and a set of transfer and ribosomal RNAs. mtDNA degradation is an essential mechanism in mitochondrial genomic maintenance and cell signaling. The knowledge regarding the mechanism of mtDNA degradation remains limited, representing a significant knowledge gap. Such knowledge is fundamental to the understanding of mitochondrial genomic maintenance and pathology because mtDNA degradation may contribute to the etiology of mtDNA depletion syndromes and inflammatory and immunological diseases triggered by cytosolic and cell-free mtDNA. The objective of this project is to delineate the chemical and molecular basis of damaged mtDNA degradation by identifying the proteins factors and molecular triggers in mtDNA degradation and characterizing the released mtDNA products. Addressing these critical knowledge barriers will facilitate the PI’s long-term goal of unraveling the basis of mtDNA turnover and its role in mitochondrial pathobiology. The application builds on the PI’s expertise in DNA and protein biochemistry, mechanistic enzymology, and quantitative analysis, and accelerates the progress in an exciting, productive area of research into mitochondrial biology. The proposed research is grounded in progress from the PI’s and other laboratories in the field. Recent research from the PI’s laboratory has provided strong chemical, molecular, and cellular evidence for the involvement of mitochondrial transcription factor A (TFAM) in the degradation of damaged mtDNA containing abasic (AP) sites. AP sites are ubiquitous DNA lesions and central DNA repair intermediates. Enzymology studies from the PI’s laboratory have also provided insights into catalytic and kinetic mechanisms of key proteins in mtDNA maintenance. The proposed research will focus on (i) identifying unknown protein factors in mtDNA degradation using proteomics and siRNA-based approaches, (ii) clarifying the molecular triggers of mtDNA degradation using biochemical and cellular assays, and (iii) characterizing the chemical and molecular properties of fragmented mtDNA using mass spectrometry-based methods. The expected outcome is that the results from this research will provide new insights into the molecular basis of mtDNA degradation and shed light on the characteristics of subsequent mtDNA products. The project is significant because it addresses a critical barrier in the field by providing fundamental knowledge at the molecular level. In addition, the PI’s commitment to enhancing diversity in the biomedical workforce further the significance at a minority- and Hispanic-serving institution. Considering the importance of mtDNA in cell signaling and innate immunity, new insights into mtDNA degradation will not only advance the understanding of mtDNA biology and unveil potential drug targets for manipulating mtDNA for therapeutic purposes, but have broad implications for understanding the pathobiology of inflammatory and immunological diseases.

NIH Research Projects · FY 2026 · 2018-07

Abstract Amyotrophic lateral sclerosis (ALS) is a progressive degenerative disease that affects motor neurons. Mutations in the gene SOD1 (superoxide dismutase 1) and in chromosome 9 seem the most prevalent in those affected by the disease. Despite tremendous efforts aimed at identifying contributing factors for ALS, the mechanisms underlying motor neuron death have not yet been fully elucidated and consequently no effective treatment is currently available for ALS. Several clinical trials have been initiated based on drugs selected from animal studies, however, these ultimately failed. Obviously among the possible reasons for such failures is the lack of a proper drug target responsible for the onset and progression of ALS and associated pharmacological tools. In this regard, numerous recent studies clearly suggest that the EphA4 receptor tyrosine kinase is a potential drug target for ALS and that targeting its ligand–binding domain may provide a possible avenue to novel and effective therapeutics. Based on these premises, we have previously recently obtained the first bona fide EphA4 agonistic agents targeting its ligand binding domain, that are brain penetrant and show protection in cellular and animal models of the disease. Our studies aimed at further optimizing and characterizing this series will provide critical pharmacological tools and mechanistic insights on the role of the EphA4 modulation in the progression of ALS, and the data gathered in this study will be critical in supporting the development of these agents into innovative targeted therapeutics for ALS.

NIH Research Projects · FY 2025 · 2016-08

Mechanistic Insights into Mammalian DNA Methylation ABSTRACT DNA methylation in mammals is a major epigenetic mechanism that is essential for transcriptional silencing of retrotransposons, genomic imprinting, and X-chromosome inactivation. Aberrant DNA methylation leads to genomic and chromosomal instabilities and silencing of tumor suppressor genes, which contribute to the development of cancers and many other human diseases. Mammalian DNA methylation is established and maintained by two groups of DNA methyltransferases (DNMTs): de novo DNMTs (DNMT3A and DNMT3B), which are responsible for establishing the DNA methylation patterns during gametogenesis and early embryogenesis, and maintenance DNMT (DNMT1), which propagates DNA methylation during mitotic division. We have a long-standing interest in mechanistic understanding of mammalian DNA methylation, which has led us to unravel the molecular basis of DNMT1-mediated maintenance DNA methylation and DNMT3A/3B- mediated de novo DNA methylation. The activities of these DNMTs are governed not only by their intrinsic enzymatic specificities, but also by an intricate network of cellular factors, such as histone modifications and chromatin modifiers. However, the mechanism underlying the functional regulation of the DNA methylation machinery remains poorly understood, partly due to the lack of structural and dynamic information on DNMTs under the chromatin environment. Our research program focuses on addressing this important challenge through an approach that integrates structural biology with biochemistry, molecular biology, and cell biology. We have two long-terms goals: to provide a comprehensive understanding of the structure and mechanism of DNA methylation machinery, and to identify the relationship between DNA methylation, gene regulation, and human diseases. Our work in the past has led to structure-function understanding of the protein-protein and protein-DNA interactions underpinning discrete steps of mammalian DNA methylation. We have identified multilayered mechanisms, involving intricate interplay between intramolecular and intermolecular interactions, for the substrate specificity and chromatin association of both maintenance and de novo DNA methylation machinery. Our goal in the next five years is to gain a mechanistic understanding of mammalian DNA methylation at the chromatin level, with emphasis on four new directions: (i) structural understanding of mammalian DNA methylation in the chromatin context, (ii) dynamic characterization of mammalian DNA methylation, (iii) protein engineering targeting specific DNA methylation pathways, and (iv) deciphering the regulatory network of mammalian DNA methylation. Together, these combined studies promise to lead us toward a comprehensive mechanistic understanding of mammalian DNA methylation.

NIH Research Projects · FY 2026 · 2016-03

Port wine stain (PWS) is a congenital and progressive malformations of the dermal capillaries. Pulsed dye laser (POL) irradiation in the visible wavelength range of 585-600 nm remains as the gold standard of treatment. The underlying treatment principle is based on the absorption of POL light by hemoglobin to induce irreversible photothermal coagulation of the vasculature. However, therapeutic efficacy with POLs remains limited due to insufficient penetration of light in skin, and non-specific absorption by the epidermal melanin pigments. Clinically acceptable outcomes are achieved in only about 20% of patients with diminishing returns beyond five treatment sessions. Our long-term objective is the development of a new therapeutic approach based on intravascular administration of optical micro-particles, fabricated from erythrocytes, as targets for pulsed near infrared (NIR) laser treatment at 755 nm. These micro-particles are doped with indocyanine green (ICG), the only FOA-approved NIR chromophore. The underlying premise is based on reduced absorption of light by melanin, strong ICG absorption, and availability of dermatological lasers at 755 nm. A particularly innovative feature of these micro-particles is that their membrane is enriched with cholesterol to prevent the flipping of phosphatidylserine from the inner to the outer leaflet of the membrane, which would otherwise serve as a signal for removal of the particles from the vasculature. We refer to these micro-particles as c⁺- µNETs. By using c⁺-µNETs, we aim to prolong the circulation time of ICG, and increase its availability in the lesion vasculature so that more sites can be treated during a given session, ultimately leading to minimal therapeutic sessions to clear the stain. Another innovative aspect is the use of transgenic mice whose melanin content can be varied in a controllable manner to simulate the epidermal response of PWS with different pigmentations to 755 nm laser irradiation. We will use these mice to determine the threshold values of the laser radiant exposures for epidermal injury and blood vessels photocoagulation in conjunction with c⁺-µNETs. We will also use a rabbit model to characterize the circulation and biodistribution dynamics of c⁺-µNETs, determine the therapeutic window of time when using c⁺-µNETs, and evaluate the vascular response as it relates to laser irradiation parameters and dose of c⁺-µNETs. SA 1: Fabricate and characterize c⁺-µNETs. SA 2: Characterize the circulation and biodistribution dynamics of c⁺-µNETs. SA 3: Evaluate the therapeutic efficacy of c⁺-µNETs in conjunction with pulsed NIR laser irradiation. A key outcome of our proposed studies is that we will know the maximum length of time over which effective blood vessels photocoagulation can be achieved when using c⁺-µNETs, in addition to finding the appropriate radiant exposure levels for vascular photocoagulation in skins with various pigmentations. This knowledge is not currently available, but is essential towards development of safe and effective protocols for laser treatment of PWS patients. Proposed studies are consistent with the scientific themes of NIAMS in developing effective therapies for PWS.

NIH Research Projects · FY 2026 · 2012-04

SUMMARY/ABSTRACT Compromised intestinal barrier function and alterations in intestinal microbes are critical factors contributing to many autoinflammatory diseases such as Inflammatory Bowel Disease (IBD), celiac disease and Type 1 diabetes, and affect ~24 million Americans (www.niehs.nih.gov). Genetic contributions to these diseases include the increased association with loss-of-function single-nucleotide polymorphisms (SNPs) in the protein tyrosine phosphatase non-receptor type 2 (PTPN2) gene. Moreover, PTPN2 was identified as a major influence on microbiome composition across multiple patient cohorts.In mice constitutively lacking Ptpn2, we identified substantial changes in gut microbiota populations highlighted by increased abundance of a novel mouse adherent-invasive E. coli (AIEC). This mouse AIEC was able to colonize mouse intestine, exacerbate colitis onset, and delay recovery from colitis. Moreover, we now report that PTPN2 loss compromises Paneth cells which have critical roles in preserving intestinal mucosal-microbial homeostasis. We also identify that epithelial PTPN2 deletion reduces Paneth cell antimicrobial peptide expression, and increases susceptibility to pathogen infection. Thus, we hypothesize that PTPN2 serves as a “microbial modulator” by regulating innate defense mechanisms of epithelial cells to protect the intestine against bacterial ‘dysbiosis’, including expansion of, and colonization with, the disease-relevant pathobiont, AIEC. The goals of this proposal are to determine how loss of PTPN2 activity disrupts i) Paneth cell antimicrobial properties; and ii) how does PTPN2 regulate other (non-Paneth cell) features of epithelial antimicrobial defense and intracellular bacterial handling. Expected Outcomes & Impact: This proposal will increase our broader understanding of the molecular basis by which host factors preserve the intestinal barrier and microbial homeostasis, and lead to development of new approaches and targets to restore host-microbe relationships in diseases such as IBD.

NIH Research Projects · FY 2026 · 2011-09

Project Summary: Temporal lobe epilepsy (TLE) develops in a third of over 300,000 patients and lead to long term memory and cognitive disorders which impact quality of life. The dentate gyrus, a circuit severely impacted in hippocampal sclerosis in TLE, serves as the first node in the flow of cortical information to the hippocampus. The dentate gyrus is a critical for episodic memory function and is proposed to discriminate highly similar inputs through a process known as pattern separation, the underlying mechanisms of which are not fully understood. The dentate receives highly structured inputs with lateral entorhinal cortical projections with content or object related information targeting the distal dendrites of the granule cells through the lateral perforant path and spatial and contextual information from the medial entorhinal cortex reaching the middle dendrites through the medial perforant path. However, the circuit mechanisms underlying how these inputs streams are processed to discriminate subtle differences in each modality, how they are associated to form episodic memory representations and their contribution to dentate electrical activity patterns associated memory consolidation and recall are not known. We propose that input specific recruitment of inhibitory neurons and their layer specific inhibition of granule cell dendrites is critical for the ability of the dentate to decorrelate distinct input streams. Since dendrite targeting dentate interneuron subtypes undergo extensive structural and functional plasticity in epilepsy, we further propose that seizure induced changes in dendritic inhibition compromise input specific recruitment of inhibition and undermine input output transformations. Combining electro- and optophysiology in ex vivo slices from transgenic mice subject to experimental epilepsy and behavioral and multisite recordings with optogenetic interrogation will allow us to address these questions. Aim 1 will develop a fundamental understanding of input specific and recruitment, and frequency dependent dynamics of dentate interneuron subtypes and how it is altered in epilepsy. Aim 2 will determine the role of somatic and dendritic inhibition in dentate decorrelation of layer specific inputs and their transformations in epilepsy at the circuit level. Finally, Aim 3 will examine determine the role of inhibitory neuron subtypes to behavioral spatial and object discrimination and electrical correlates of dentate memory processing in control and epileptic mice. in line with the proprieties established in the 2021 AES/NINDS Epilepsy Research Benchmarks, these studies will address mechanisms underlying neuropsychiatric comorbidities in epilepsy and is of broader relevance to understanding memory processing and decline in ageing and Alzheimer’s Disease.

NIH Research Projects · FY 2025 · 2010-07

ABSTRACT In this competitive renewal of the Ruth L. Kirschstein NRSA Institutional Research Training Grant Application, we request funds to support the research training in environmental toxicology at the University of California Riverside. The proposed interdisciplinary research training program is built upon the solid foundation of the existing curriculum structure of the environmental toxicology graduate program. The eighteen preceptors are from the Division of Biomedical Sciences as well as the Departments of Chemistry, Biochemistry, Botany and Plant Science, MCSB (Molecular, Cell and Systems Biology), and Environmental Sciences. They have been very actively engaged in training pre- and postdoctoral individuals in environmental toxicology, and their research projects have been well funded. The research programs of these faculty members include genetic toxicology and epigenetics, analytical chemistry and proteomics, developmental toxicology, and endocrine disruption and metabolism. Pre-doctoral trainees will be selected from the participating Chemistry and Environmental Toxicology graduate programs. An Internal Steering Committee is established to select trainees and monitor progress of trainees, and an External Advisory Board is also named to assist the management of the training program. A total of 9 trainees per year (7 pre-doctorates + 2 postdoctorates) are requested. The trainees will be exposed to multi-disciplinary research involving a variety of cutting-edge technologies in nanofabrication, genomics, proteomics, metabolomics, etc. The request for continued funding of this research training grant is justified by the relevance of the proposed research training to environmental health, the excellent training environment, the superb training experience of the preceptors, the strong institutional commitment, the diverse graduate student body at the University, as well as our success in recruiting, appointing and nurturing the success of trainees in the last two funding cycles.

NIH Research Projects · FY 2025 · 2007-08

Project Summary/Abstract. Neural crest cells (NC) are unique to vertebrates, arise early in development, emigrate from the dorsal aspect of the neural tube, and differentiate into a plethora of derivatives throughout the body, contributing neurons and glia of the peripheral nervous system, melanocytes, and craniofacial bone and cartilage amongst other derivatives. Failed NC development is associated with a large number of conditions known as Neurocristopathies, which include common orofacial clefts, aggressive cancers and rare syndromes. The long term goal of this research program is to advance our understanding of the earliest signaling events responsible for the formation of the NC. Currently signaling by WNT, FGF and BMP are amongst a few of key inputs recognized to be critical for the formation of NC, yet our understanding of the molecular mechanisms by which they execute their effects remain limited. To improve our knowledge of this events, it is critical to identify the effectors modulated by these pathways and to characterize their effects. The objectives of this proposal are: 1) to establish and characterize for the first time the role of TGFb /SMAD2-SMAD3 signaling during early facets of NC formation, prior to their allocation into the neural tube, migration or differentiation; and 2) to identify the effectors of the WNT and/or TGFb and characterize their role during early NC formation. Our Hypotheses are that the TGFb /SMAD2-SMAD3 signaling is required to launch the earliest events in NC formation, and that a combination of TGFb and WNT responsive molecules direct the specific acquisition of the NC lineage. To address these hypotheses our proposal is guided by 3 specific aims: 1) to demonstrate the requirement and contribution of the TGFb /SMAD2-SMAD3 signaling during early facets of NC formation, 2) to identify novel candidate effectors of WNT and/or TGFb and characterize their specific contributions to the acquisition of the NC fate, and 3) to assess their function in vivo in a vertebrate embryo. This innovative proposal takes advantage of a robust model of human NC formation based in pluripotent stem cells and classic chick embryology to, for the first time address the possible contribution of TGFb signaling (not BMPs) during this process and pursues an integrative approach addressing the combinatorial signaling of WNT and TGFb. The proposal is significant because the gains in this area will have impact in key aspects of fate acquisition and differentiation applicable to other cell types, and should improve our capacity to manage the many pathologies associated with NC.