California Institute Of Technology

NIH Research Projects · FY 2025 · 2021-07

Proposal Summary Neural crest cells are an important stem-like cell population characterized by their multipotency and migratory ability. Originating within the forming central nervous system, neural crest cells undergo a spatiotemporally regulated epithelial-to-mesenchymal transition (EMT) to leave the neural tube and become migratory. They then migrate extensively throughout the developing embryo, giving rise to a wide range of derivatives as diverse as elements of the craniofacial skeleton and peripheral nervous system. In the post- migratory phase, neural crest cells condense into different structures, a process that involves loss of migratory characteristics, perhaps reflecting the reverse of the EMT process. While neural crest EMT has been studied extensively, the mechanisms underlying the condensation of neural crest cells to form final derivatives is far less well characterized. To address this knowledge gap, we propose to identify transcriptional changes that occur during gangliogenesis with the goal of identifying those mediating alterations in intercellular adhesion required for neural crest condensation into peripheral ganglia. Our hypothesis is that the gene regulatory mechanisms that play a role during peripheral ganglion formation may reflect a reversal of the EMT process. The goal is to uncover the molecular mechanisms that drive condensation of neural crest cells into ganglia. These may in turn lead to clues regarding the underlying cause of certain types of neurocristopathies like familial dysautonomia and neural crest-derived cancers like neuroblastoma and pheochromocytoma. Aim 1: RNA-sequencing of pure populations of post-migratory cranial neural crest cells: RNA-sequencing of isolated condensing cranial neural crest cells will allow us to identify novel transcription factors and adhesion molecules that may drive neural crest condensation into cranial ganglia. Aim 2: Functional analysis of genes selectively upregulated upon condensation to form ganglia: Identified upregulated genes in condensing cranial neural crest cells will be validated by in situ hybridization and Hybridization Chain Reaction. We will then perform systematic loss-of-function and ectopic expression experiments on selected genes to examine their role in regulating condensation into and differentiation of peripheral ganglia. Aim 3: Characterization of cis-regulatory elements modulating gene expression during ganglion condensation: To identify putative enhancers driving gene expression during cranial neural crest condensation, we will perform ATAC-sequencing to identify conserved noncoding regions in the genome that are accessible to transcription factors during cranial neural crest condensation.

NIH Research Projects · FY 2025 · 2021-06

PROJECT SUMMARY The progression of T cell precursors from multipotency to commitment occurs after multiple cell cycles in the thymus. These early, pre-commitment cell cycles are important for expansion of the precursors to generate enough pro-T cells to survive later selection events. However, the early stages are poorly understood, and in particular it has not been clear what controls the precise trajectory of the cells' differentiation nor the timing or irreversibility of commitment when it occurs. Genomic regulatory elements that are active after commitment tend to be characterized by motifs for basic helix-loop-helix (bHLH) E proteins, E2A or HEB, whereas those that are active before commitment have other signatures, suggesting that there is a major increase in E protein activity across this transition. In fact, the expression of E proteins is almost equally high before commitment, but our recent data show that they are occupied in substantially different roles before commitment, in complexes with other heterodimerization partners. This proposal is based on recent evidence that the alternative complex, containing Lmo2 and Lyl1 dimerized with E2A or HEB, may actually be a major controller of the commitment transition in early T cells. Our recent evidence shows that expression of Lmo2 and Lyl1 is sufficient to make committed pro-T cells reverse their differentiation in terms of gene expression. Not only does the Lmo2/Lyl1/E2A complex bind to different genomic sites than E protein dimers, but also the addition of Lmo2 and Lyl1 to committed pro-T cells is sufficient to remove E proteins from sites that they occupy after commitment and shift them to sites that are normally active only before commitment. The implication is that the kinetics of downregulation of Lmo2 and Lyl1 in normal T-cell differentiation could be vital for determining the timing of commitment and of the maturation of the pro-T cells. Lmo2 and Lyl1 have been considered as T- lineage proto-oncogenes, but our evidence suggests a potent role in normal development. This proposal is to determine the mechanism of how this works and to test its significance for the actual developmental dynamics of normal early T cells. Our preliminary work identifies the signature target genes affected by Lmo2+Lyl1 and their overlap with genes expressed in normal pro-T cells. We now propose to define: (1) the distinct molecular mechanisms that control different subsets of these signature genes, based on genome-wide mapping of the chromatin state changes caused by Lmo2+Lyl1/E protein complex binding as compared to pure E protein dimer binding; (2) whether endogenous Lmo2/Lyl1/E protein complexes indeed control differentiation kinetics and commitment of normal pro-T cells, based on acute CRISPR and monitoring in vitro and in vivo; and (3) the gene regulatory network architecture, involving factors regulated by Lmo2+Lyl1, through which Lmo2+Lyl1 exert their surprisingly broad impacts on T cell development. The results should determine whether and how a biochemical mechanism of transcription factor heterodimerization partner switching may explain a central unsolved problem in the dynamics of T cell development.

NIH Research Projects · FY 2025 · 2021-06

Project Summary Developing sustainable approaches to the synthesis of molecular therapeutics will be important for the continued evolution and success of medicinal and pharmaceutical chemistries. A major component of drug synthesis involves transition metal catalyzed C–X (X = C, N, O, etc.) bond formation reactions. While precious metals such as Pd are used for these reactions, first row transition metals are becoming more widely adopted, as they are abundant and open new mechanistic pathways involving one- and multi-electron transfer reactivity, which can potentially work in concert with ligand noninnocence and multireference electronic structure to form transformative structure/function relationships. The merger of thermal catalysis with photochemistry also provides new mechanistic possibilities for cross-couplings that harness the energy of light to drive bond-formation reactions that would not occur in ground states. However, the nature of inorganic intermediates and the important ultrafast transition metal excited state relaxation processes in ground and excited state cross-coupling reactions are not well understood. This proposal therefore applies physical inorganic approaches to develop a fundamental knowledge base of the geometric and electronic structures of the critical inorganic species formed in Cu- and Ni- catalyzed cross-coupling reactions, as well as the time and energy evolution of photoinduced electronic states involved in excited state catalysis. This knowledge base will ultimately guide the development of a molecular engineering approach to ligand development and catalyst discovery. We will bring new spectroscopic methods to the field, including variable temperature variable field magnetic circular dichroism (VTVH MCD) and X-ray absorption and emission spectroscopies, which will be critical to quantitatively define transition metal electronic structure, including multireference character. Ultrafast optical and X-ray spectroscopic approaches will also be used to define the key photonic energy distribution pathways that define photocatalyst efficiency and further guide ligand perturbations to control the excited state potential energy surfaces (PESs) of photocatalysts. Spectral features of isolable species will be used to experimentally calibrate computational methods to define the critical frontier molecular orbitals and bonding interactions that activate metal centers for reactivity, especially those that are fleeting but critical to catalysis. Electronic structure calculations will also allow for the translation of our understanding of resting states, intermediates, and excited states to reaction coordinates in catalysis and the PESs governing relaxation pathways. In concert with collaborative methodological studies, the proposed research will help inform chemists how to leverage the ground and excited state electronic structures of first-row transition metal complexes and thus guide academic and industry research toward sustainable approaches for bond constructions in drug synthesis.

NIH Research Projects · FY 2026 · 2021-05

The proposed research aims to obtain scientific knowledge on the visuomotor transformations in posterior parietal cortex (PPC) and motor cortex (MC) by characterizing the functional similarities and differences between both regions in tetraplegic participants enrolled in a clinical trial designed to advance the development of neural prosthetics. In experiments from the last grant period, we demonstrated that the functional characteristics of these brain regions remain intact over time, even following spinal cord injury and prolonged use of brain-machine interfaces (BMIs). We identified overlapping neural populations in both areas that encode motor variables for effectors throughout the body using mixed selectivity, a coding scheme that combines multiple variables within the same neurons. Despite these similarities, we found that the encoding properties of MC and PPC exhibit essential differences. While MC preferentially encodes the contralateral hand, PPC shows similar encoding strength for all effectors across the body. Furthermore, PPC is actively involved in planning and executing motor actions, whereas MC shows mainly activation during the execution of movements. PPC also shows a compositional code for observed and felt somatosensory stimuli. This renewal proposal seeks to broaden our understanding of the neural encoding mechanisms within PPC and MC by exploring how context affects the previously unexamined sensorimotor processes of navigation, drawing, and simultaneous, coordinated movements . In Aim 1, we will study the neural representations of allocentric and egocentric cognitive maps during two virtual reality navigation tasks performed under BMI control. We hypothesize that PPC encodes both allocentric and egocentric cognitive maps, while MC only encodes egocentric motor execution during these navigational tasks. Aim 2 will examine whether the brain uses compositional encoding of visuomotor variables for drawing. Our hypothesis is that PPC uses compositional encoding, which involves the combination of basic movements to draw strokes to create more complex character representations during planning and execution of multi-stroke character drawing. On the other hand, MC represents motor variables related only to the stroke drawing movement. Finally, in Aim 3, we will explore the representation of hand-eye coordination. Our hypothesis is that during simultaneous movements, PPC encodes information about both effectors, while MC encodes motor information about the preferred effector. This proposal emphasizes the potential to harness mixed selectivity and compositional coding strategies to enhance neuroprosthetic interfaces for complex and multi-functional motor applications.

NIH Research Projects · FY 2025 · 2021-04

Project Summary Many women struggle with infertility with only around 30% of pregnancies progressing to live birth and the remainder failing through spontaneous abortions, the majority of which are associated with aneuploidy. Here we use a mouse model for mosaic aneuploidy to study the effects of chromosome mosaicism on development of the conceptus through the pre-, peri-and early post-implantation stages. We have previously shown that aneuploidy results in two different responses in different adjacent tissues of the pre-implantation embryo: cell cycle delay in the extra-embryonic trophectoderm that will establish the placenta and apoptosis in the inner cell mass that will establish the foetus. We will now determine the fate of the majority of aneuploid cells that persist into implantation stages and how, in many cases, these can be eliminated without compromising implantation morphogenesis and the associated transition in the state of pluripotency. We will determine the extent to which the embryo can compensate for lost aneuploid cells to ensure development and determine the mechanisms employed by different post-implantation tissues to cope with aneuploidy and protect the pluripotent lineage that generates all germ layers and germline. As studies in diverse organisms indicate that global gene expression and translation levels correlate with the degree of aneuploidy, we will determine whether aneuploidy in the embryo results in proteomic imbalance leading to a common set of proteotoxic stress responses that induce autophagy. We will elucidate the role of autophagy in the elimination of aneuploid cells from the embryo and determine the roles of p53 and mTOR in this process. Our study will shed light onto competition between aneuploid and diploid cells in development and will uncover new pathways that regulate embryo growth and plasticity. It will inform IVF strategies in the clinic by building a working knowledge of circumstances in which human embryos diagnosed as mosaic should or should not be discarded. It will enable a more accurate assessment of the developmental potential of mosaic aneuploid embryos and permit the development of better methods to assess the probability of successful pregnancy.

NIH Research Projects · FY 2026 · 2021-03

PROJECT SUMMARY The centriole is a conserved organelle of metazoans that is found at the core of microtubule organizing centers, centrosomes, and at the base of cilia and flagellae. Centriole dysfunction leads to a wide range of diseases including the developmental defects of ciliopathies; defective brain development in microcephaly; and in cancer where supernumerary or defective centrosomes are associated with poor prognosis. Centriole duplication is promoted by Plk4, which phosphorylates cartwheel proteins to mediate their assembly. We and others have found that induction of Plk4-mediated centriole amplification results in hyperplasia of several tissues and increases the susceptibility to tumorigenesis in the mouse in the absence of the p53 tumor suppressor. Both the loss and acquisition of extra centrosomes normally block cell proliferation. However, the pathway that responds to loss of centrosomes differs from several pathways that respond to supernumerary centrosomes. Therefore, to identify proteins that signal or respond to the presence of extra centrosomes, we carried out a genome-wide screen for genes that when deleted or knocked- down permit the proliferation of cells that have elevated levels of Plk4. This has identified new pathways whereby cells respond to supernumerary centrosomes: a previously unknown involvement of specific Rac-mediated signaling that regulates centriole duplication; proteins that regulate the elongation, disengagement and separation of centrioles; and negative regulators of ciliogenesis. Here we follow three approaches to understand how centriole number is regulated in proliferating cells. First, we will determine how the Arh15gap GTPase Activating Protein acts upon the Pak1/2 protein kinases to regulate Plk4 levels or activity and upon Arp2/3 to regulate spindle associated actin to permit an increase in centriole- and cell-cycle arrest. Second, we examine how known components of the centriole affect the execution of the centriole and cell cycles at unexpected points. We will determine how the Usp33 deubiquitinase regulates the centriole capping protein complex to protect the nascent procentriole and how proteins required in the centriole cycle for both centriole disengagement and centrosome separation contribute to the block to cell proliferation in the presence of supernumerary centrosomes. Finally, we will explore how negative regulators of ciliogenesis block primary cilium formation in the presence of extra centrioles in such a way as to arrest cell proliferation. We anticipate that this research will advance our understanding of the multiple ways in which cells respond to supernumerary centrosomes. It will identify pathways that can be targeted for restoring centriole numbers or cell cycle regulation in tumor cells or for targeting such cells for apoptosis. This will find translational application in developing multiple drug strategies for cancer treatment.

NIH Research Projects · FY 2024 · 2020-09

PROJECT SUMMARY Lysosome-Related Organelles (LROs) contain both lysosomal proteins and cell-type specific proteins in an acidic lumen. They are enlarged in Chediak-Higashi Syndrome (CHS) patients resulting from either excessive fusion or inhibition of their fission. The mutated gene in CHS encodes the lysosomal trafficking regulator (LYST) protein, whose function is poorly understood. Defects in microtubule behavior and centrosome behavior are seen at the immunological synapse of CHS patients but whether microtubule nucleation is affected directly in CHS cells is controversial. To determine LYST's function in LROs and clarify its requirements at microtubules, we will use a Drosophila model in which mutants of the LYST counterpart, encoded by the mauve (mv) gene, show enlarged LROs (yolk granules) and microtubule defects in mitosis and in maintaining nuclei at the correct position in the embryo. Mauve co-immunoprecipitates from Drosophila embryos with factors involved in maturation of endosomes; a factor enabling dissociation of the SNARE complex from mature vesicles; Dynein/Dynactin, which have roles in vesicle trafficking and at microtubules; and several centrosome-associated molecules. Thus, this stage of Drosophila development is highly amenable to study the role of LYST/Mauve in the biogenesis of LROs and at microtubules and centrosomes. To establish the role of the Mauve/LYST complex in regulating LRO size and trafficking, we will follow yolk granule biogenesis in wild-type and mv-mutant females; determine the effects of constitutively active and dominant-negative forms of the enodcytotic regulators Rab5, Rab7 and NSF1. To discover the role of Mauve/LYST complex in regulating microtubule dynamics, we will determine microtubule defects in mv- derived embryos and establish the genetic interactions between mv and genes for microtubule associated proteins with which it associates and physical interactions between these gene products. By determining how Mauve directs the centrosomal association of Minispindles protein; how together with Rab5 and Dynein, it promotes accumulation of microtubule associated proteins at the centrosome; and how Mauve's partner proteins participate in recruitment of microtubule organizing molecules at centrosomes we will uncover how vesicle trafficking associated proteins can participate in promoting centrosomal maturation. We anticipate that this will define the dual role of Mauve/LYST in regulating vesicle fission/fusion and in the trafficking of proteins important for microtubule nucleation and centrosome maturation. We anticipate our findings will translate to human cells where they will have potential to unlock doors for the development of therapeutic agents to treat the immunological defects of CHS patients.

NIH Research Projects · FY 2024 · 2020-09

Most human pregnancies fail around the time of embryo implantation. Yet, the developmental mechanisms of this stage and how they go awry remain a mystery, because the implanted embryo is inaccessible to analysis within the body of the mother. Uncovering these mechanisms is of critical importance to overcome existing barriers to fertility and proper development. We have successfully generated systems that enable development of natural mouse and human embryos from pre- to post-implantation stages in vitro, and built stem cell-derived synthetic mouse embryos that can mimic some aspects of early post-implantation development. But approaches to study development continuously through the implantation stage and beyond gastrulation are lacking. We now propose to create a maternal-like environment that permits the long-term survival of both natural and synthetic mouse embryos. Our first challenge will be to engineer synthetic pre-implantation blastocysts with an expanded ability to generate the full range of correctly functioning extra-embryonic tissues. This breakthrough is expected to enable their implantation and development in utero, and may eventually transform approaches for engineering genetically modified mice. We will use these new tools to determine the precise cellular and molecular mechanisms that allow synthetic blastocysts to interact with the uterus in foster mothers. Our second challenge will be to generate artificial substrates, comprising hydrogels and proteins of the decidual extra-cellular matrix, to facilitate implantation events. In parallel, we will engineer synthetic placental-like structures for natural and synthetic embryo development using organoids derived from trophoblast and endometrial tissue. These systems would allow investigations and tracking of how insults to pre- and peri-implantation development, such as the exposure to pathogens, toxins, or teratogens affect subsequent development and life. Our third challenge will be to utilize these systems to discover the molecular events that accompany implantation. We will take advantage of our in vitro placental systems to investigate the chemical and physical signalling events that are key for development and determine how improved extra-embryonic contributions affect embryonic development until neurulation. These innovations will allow us to finally decipher a stage of development that is currently out of reach and of which our knowledge is greatly lacking. This will bring insight into a time of development when most pregnancies fail and thereby lead to advances in assisted reproductive technology; it will offer new screening routes for drug testing and environmental safety; and it will advance our knowledge of the use of stem cells in organogenesis and regenerative medicine.

NIH Research Projects · FY 2024 · 2020-09

SUMMARY Cellular differentiation involves tightly coupled changes in gene expression, chromatin state, and sub-nuclear arrangements of chromosomes. Understanding and controlling differentiation requires understanding how each of these processes occurs dynamically within the same cell and how they influence one another. Existing techniques can provide genome scale analysis of interactions or spatial organization of a few chromosomal positions. However, we have lacked a generalizable framework for simultaneous reconstruction of the overall dynamics of the nucleus across all three levels. Recent work from our labs has opened up the possibility of achieving such coupled analysis. Our track first, identify later approach allows many DNA to be simultaneously tracked in living cells. RNA and DNA seqFISH allows a large number of transcripts and DNA loci to be imaged in single fixed cells and MEMOIR allows lineage information to be recovered from endpoint measurements. In this project, we propose to combine live imaging, multiplexed RNA, DNA, and immunofluorescence measurements, and MEMOIR lineage tracking to capture whole- genome dynamics of chromosomal loci and chromatin states. Using mouse embryonic stem cells (mESCs) as a model system, we will study the transition from the pluripotent state to an earlier 2- cell (2C) like state which shows drastic chromosome re-arrangement and changes in nascent gene expression patterns. In addition, we will study the chromosomal dynamics of X-inactivation based on the initial observations that sister X chromosomes are in contact with each other during early phases of the inactivation process. Both of these biological questions require tracking chromosomal dynamics and chromatin state simultaneously in single cells. The “Track First and ID later” approach allows a large number of loci to be tracked in living cells. The combined MEMOIR approach with multiplex immunofluorescence allows us to infer the kinetics of chromatin states transitions. Bringing these tools to study X inactivation and 2C state transition will demonstrate the capability of this approach for addressing a broad range of cell fate decision questions. We will also develop analysis and visualization tools to integrate genomics (SPRITE) and imaging data. The technology developed in this project can be readily implemented in human cell lines and adopted by other labs in the 4DN consortium.

NIH Research Projects · FY 2024 · 2020-09

Autism spectrum disorder (ASD) is caused by both environmental and genetic factors, with the genetic contribution estimated at 60-80%. Dozens of genes that increase risk for ASD have been identified, most based on de novo mutations, but these mutations are predicted to account for only 15-20% of ASD cases. Thus, the majority of the genetic contribution to ASD is predicted to result from common and rare inherited variation, but few such genes have been identified. Recently, using whole genome sequencing, we reported genome wide evidence for >60 ASD risk genes, 26 of them novel for ASD, with signals derived from inherited and de novo protein truncating or missense mutations. The functions of most of these genes are unknown, so a crucial and necessary next step is to explore their impact on neurodevelopment and neuronal function using a model organism. The current pace of translating genetic risk factors into phenotypes, mechanisms and therapies is limited in part by inefficiencies with in vivo mammalian model systems, which makes them impractical for creating and behaviorally testing large numbers of mutant lines. Here, we leverage the zebrafish, which occupies a unique niche as a vertebrate model with features amenable to both in vivo screening and mechanistic understanding, including ex utero development, transparency, small size, rapid development, a conserved yet relatively simple vertebrate brain, behaviors relevant to ASD, and cost-effectiveness relative to mammalian models. While the zebrafish cannot recapitulate ASD and has limitations for modeling a human disorder, an emerging literature supports the notion that it is a useful model to study the functions of genes that contribute to ASD risk. Rather than assess ASD-risk genes one at a time, we will accelerate progress towards mechanistic understanding via high-throughput assays and analyses. In Specific Aim 1 we will generate null mutations in the zebrafish orthologs of 24 high confidence, novel, genome-wide significant ASD risk genes, and systematically test each mutant for neurodevelopmental, behavioral, neuronal network, and transcriptomic phenotypes. In Specific Aim 2, we will use transcriptomic analyses, at the whole brain and single cell levels, to integrate ASD risk genes into functional networks, and test for convergence across genes and species, including ASD post mortem brain. We will also test for functional associations among behavioral phenotypes that are often co-morbid in ASD, such as disrupted sleep and social behavioral deficits. In Specific Aim 3 we will perform mechanistic studies to understand how mutation of specific ASD-risk genes leads to phenotypes. This project will efficiently and cost-effectively create and characterize vertebrate animal models for a large number of novel ASD risk genes. These animal models will be a valuable resource for the community, particularly for large-scale in vivo drug screens to identify new therapies for ASD.

NIH Research Projects · FY 2024 · 2020-09

ABSTRACT (30 Lines) The BRAIN initiative (RFA-EB-19-002) has called for the development of entirely new or next-generation noninvasive human brain imaging tools and methods that will lead to transformative advances in our understanding of the human brain. Functional MRI (fMRI) at ultrahigh fields has made tremendous improvements in spatiotemporal resolution, allowing brain function to be studied on the level of cortical layers and columns. However, fMRI is generally considered to have a low sensitivity and strong tissue background for detection of function. Positron emission tomography provides powerful metabolic imaging through radioactive tracers but suffers low spatial resolution, as is diffuse optical tomography despite its advantages in speed, cost, and portability. Ultrasound-only imaging cannot image adult human brains because the ultrasonic waves are attenuated and aberrated twice by the skull due to the round-trip propagation. To address these issues, we propose to develop 3D photoacoustic computed tomography (PACT) for fast and ultrafast large-scale neural activity imaging in human brains. PACT is especially well suited for detecting hemodynamic changes related to neural activities. It offers comparable spatial resolution but can be made much faster than fMRI. It is directly sensitive to both oxy- and deoxy-hemoglobin linearly with a low tissue background. Other potential benefits of PACT over fMRI include open imaging platforms, minimal site requirements, quiet and bedside operation, magnet-free environment, and low system maintenance. In the last two decades, we have developed photoacoustic technology at multiple spatial scales ranging from microscopic (subcellular and cellular) to macroscopic (whole rodent, whole human breast, ex vivo adult human skull, and preliminary single-channel 2D and 64-channel 3D in vivo adult human brain) imaging. We have revealed hemodynamic response in the rodent brain to whisker or electrical stimulation and mapped the resting- state functional connectivity of the rat brain in the deep thalamic region. We have also developed sophisticated numerical methods for simulating photoacoustic wave propagation in heterogeneous media and developed frameworks for image reconstruction in acoustically heterogeneous media. Further, we have successfully demonstrated ex vivo PACT through adult human skulls and acquired preliminary images of human heads in vivo. We propose to translate these advances in PACT to human brain imaging through two specific aims: Aim 1: Develop massively parallel high-speed 3D PACT for in vivo fast and ultrafast functional human brain imaging. Aim 2: Validate functional PACT in adult humans in vivo by comparing with ultrahigh-field 7 T fMRI.

NIH Research Projects · FY 2024 · 2020-09

Project Summary Gene regulation is a highly complex process that involves the recruitment of numerous regulatory factors as well as dynamic 3-dimensional spatial rearrangements of DNA, RNA, and protein molecules that are important for quantitatively controlling the rate of gene regulation. Yet, how these various components interact simultaneously and what their quantitative contributions are to gene regulation remains unresolved largely because of the lack of methods that can integrate combinatorial molecular binding, spatial information, and quantitative measurements of various aspects of gene regulation within the same individual cell. Here we will develop highly innovative methods that will allow us to generate dynamic molecular movies that monitor the movement of DNA, RNA. and protein molecules at high resolution in a manner that provides information about the spatial arrangement of molecules along with simultaneous information about transcription rates and mRNA splicing rates within single cells. To achieve this, we will develop pioneering new genomic methods for measuring the spatial interactions of RNA, DNA, and protein with single cell capabilities and build novel quantitative and computational modeling approaches to generate high resolution temporal “movies” from snapshots derived from tens of thousands of synchronized single cells. We will use these approaches to quantitatively understand the dynamic assembly of RNA-protein complexes, localization to DNA, and structural dynamics of genomic DNA and how these integrated components impact gene regulation across time. Specifically, we will dissect the dynamics of three RNA-mediated processes that link dynamic 3D nuclear structure and gene regulation in unique regulatory paradigms in biology and disease: (i) chromosome-wide transcriptional silencing, (ii) kinetic coupling of mRNA transcription and splicing, and (iii) RNA-induced aggregation and cellular toxicity in neurodegenerative disorders. Together, the results of this proposal will generate highly innovative new approaches for quantitatively measuring molecular and spatial dynamics of various regulators and their role in gene regulation.

NIH Research Projects · FY 2024 · 2020-08

SUMMARY Collective cell migration is essential to the progression of normal embryonic development and organogenesis, and is a tightly-regulated process that can involve the interplay between two or more signaling pathways to drive forward movement of cell cohorts. Additionally, patterning an organ often requires selective apoptosis and compensatory proliferation of cells. Errors in collective migration and cell death programs can have serious consequences, including complete developmental arrest, abnormal organ function, and tumorigenesis. In this proposed research plan, we will use the Drosophila embryonic caudal visceral mesoderm (CVM), a small population of muscle precursor cells that undergo highly stereotyped directional movement, as a model for collective cell migration and survival. As the longest migration of embryogenesis, CVM cells must receive input via signaling cues from other cells in order to navigate the changing environment of the developing embryo. We have previously determined an important role for FGF signaling as both chemotropic and survival cue, and that FGF receptor is specifically expressed in a subset of migratory cells. However, loss of FGF signaling does not completely ablate collective migration, suggesting the existence of additional, as-of-yet uncharacterized cues. The objective of this study is to gain a comprehensive understanding of the spatiotemporally-regulated cues that guide directional movement of the CVM, and subsequent survival or apoptosis of distinct subsets of cells. Our central hypothesis is that FGF signaling cooperates with additional signaling cues in order to drive forward movement and cell survival, and involves defining specialized subsets of cells within each CVM cohort to promote spatial organization driving forward movement. To test this hypothesis, we will pursue the following specific aims: (AIM 1) Investigate roles for spatially-localized genes within the migrating CVM collective in promoting cell migration; (AIM 2) Investigate mechanism of CVM attraction to PGCs; and (AIM 3) Investigate the relationship between BMP and FGF signaling in regulating CVM cell migration and survival. To accomplish these aims, we will employ an innovative combination of established genetics and immunostaining techniques with elegant optogenetics and in vivo live imaging approaches to manipulate and visualize migratory cells, as well as quantify spatiotemporal activation of the cell death program. We believe this study is significant because it would not only demonstrate a mechanism for signaling cross-talk in an emerging yet poorly-characterized cell migration system, but considering the large number of functions and diseases attributed to signaling pathways such as BMP and FGF, elucidating the interaction between multiple pathways in the context of the genetically-tractable and conserved Drosophila model system has the potential to identify more specific therapeutic targets. Therefore, this study will be impactful by contributing to a more comprehensive understanding of collective cell migration, the mechanisms underlying organogenesis, as well as the cell migration and survival programs implicated in normal development and cancer.

NIH Research Projects · FY 2026 · 2020-08

How do individual cells self-organize into an epithelialized structure during early mammalian development, while simultaneously establishing distinct lineage identities? At the 8-cell stage, blastomeres undergo compaction and establish apical-basal polarity. During the transition to the 16-cell stage, polarized and apolar cells sort into distinct positions, forming the outer trophectoderm (TE) and inner cell mass (ICM). These processes rely on coordinated cell adhesion, cytoskeletal forces, and transcriptional programs activated during zygotic genome activation (ZGA). Despite extensive knowledge of mechanical signaling from other developmental contexts, how these cues interface with transcriptional programs during the earliest morphogenetic event of the mammalian embryo, compaction, remains incompletely understood. Our central hypothesis is that α-catenin functions as a mechanical gatekeeper during compaction, translating junctional tension into actomyosin polarization, while ZGA-activated transcription factors, such as Tfap2c and Tead4, coordinate with these mechanical cues to drive epithelial structure and lineage specification. Together, these systems couple mechanical and transcriptional signals to orchestrate the emergence of structure and cell fate. Aim 1 will define how junctional tension regulates actomyosin polarization during compaction. Using α-catenin conformational biosensors and mechanosensory- deficient mutants, we will test whether α-catenin senses mechanical forces at adherens junctions to modulate RhoA activity and apical contractility, revealing how cytoskeletal asymmetry and epithelial structure are established in response to force. Aim 2 will determine how α-catenin–mediated mechanosensation integrates spatial position with lineage identity. As blastomeres sort into inner and outer compartments, fate is directed by apical polarity, mechanical asymmetries, and Hippo signaling. This aim will assess how α-catenin conformation influences YAP localization and TE vs. ICM fate, using a combination of live imaging, FRET-based reporters, and computational modeling to infer and manipulate force landscapes. Aim 3 will determine how ZGA-induced transcription factors Tfap2c and Tead4 regulate compaction and apical domain formation. These factors are required and, with active RhoA, sufficient for precocious compaction and polarization. We will dissect their downstream effectors using gain- and loss-of-function approaches, and screen ~60 RNA-seq–identified targets for roles in cytoskeletal remodeling, adhesion, and polarity. Together, these studies will establish how gene expression and mechanical forces integrate to initiate epithelial morphogenesis and lineage segregation. By combining high-resolution imaging, biosensors, and targeted genetic perturbations, this work aims to clarify the earliest physical and transcriptional mechanisms underlying tissue organization in the early mammalian embryo. This research has broad relevance for developmental biology, cell biology, reproductive medicine, and stem cell engineering. Defects in compaction and polarization contribute to implantation failure and infertility; mechanistic insights may inform assisted reproductive technologies and improve stem cell–derived embryo models.

NIH Research Projects · FY 2026 · 2020-04

Project Summary: Accurate Molecular Decision Making during Protein Biogenesis Accurate protein biogenesis is a pre-requisite for the generation and maintenance of a functional proteome and, by extension, for human health. Our long-term goal is to understand the molecular mechanisms by which diverse protein biogenesis pathways in the cell accurately select nascent protein substrates and ensure their correct folding, localization, maturation, and quality control. Three major components define our research program in the next grant cycle. Firstly, we will define how nascent proteins acquire the correct set of chemical modifications during translation, which play essential roles in the maturation, localization, and stability of the proteome. These studies will include N-terminal methionine excision by methionine aminopeptidases, protein acetylation by multiple classes of N-acetyltransferases, and protein acylation by N-terminal myristoyltransferases. In addition to establishing the cotranslational mechanisms of these essential enzymes for the first time, these enzymes will provide a case study for how diverse protein biogenesis machineries gain timely and selective access to the nascent proteome in the crowded environment of the ribosome exit site. Secondly, we will decipher how cotranslational chaperones guide nascent proteins through the most productive biogenesis pathways. We will focus on two goals: (i) Test the role of the nascent polypeptide-associated complex (NAC) as a master regulator of diverse cotranslational protein biogenesis machineries; and (ii) Decipher the mechanism by which the ribosome- associated complex (RAC) together with Hsp70 facilitate cotranslational protein folding. These studies will establish a new model to understand the spatiotemporal coordination of diverse protein biogenesis pathways at the ribosome and probe the mechanisms by which the Hsp70 machinery cooperates with the translating ribosome to reshape the folding trajectory of nascent proteins. Thirdly, we will probe the mechanism of mitochondrial biogenesis and protein quality control. We will use selective ribosome profiling to uncover and understand cotranslational protein targeting to mitochondria. We will decipher the molecular mechanism of the recently discovered AAA+ chaperone Skd3, which displays multiple distinct chaperone activities that may be particularly suited to its role as the only general chaperone in the mitochondrial intermembrane space (IMS). These studies will address long-standing questions about protein targeting to mitochondria and protein folding in the mitochondrial IMS and provide insights into the molecular principles governing these processes. The proposed research will not only generate high-resolution understandings of many protein biogenesis pathways, but also establish valuable conceptual frameworks to understand how nascent proteins are accurately selected into their appropriate biogenesis pathways in the crowded cytosolic environment.

NIH Research Projects · FY 2025 · 2019-09

SUMMARY Model organisms are essential experimental systems for investigating and defining protein and genetic networks, discovering new gene functions, and uncovering the functional consequences of human genome variation. The Alliance of Genome Resources (aka, the Alliance) is a consortium of seven model organism databases (MODs; Drosophila, C. elegans, budding yeast, zebrafish, laboratory mouse, laboratory rat, Xenopus) and the Gene Ontology Consortium (GOC) with a shared mission of facilitating use of biological insights from model organisms to understand the genetic and genomic basis of human health and disease. The Alliance seeks to serve a diverse community of biomedical researchers including basic scientists, clinicians, and data scientists. The Alliance is organized as two interdependent units: Alliance Central and Alliance Knowledge Centers. Alliance Central serves as a software platform developed using modular infrastructure and common data models to and for the coordination of data harmonization and data modeling activities across the Knowledge Centers. Alliance Knowledge Centers including MODs and the GOC are responsible for expert curation and for submission of annotations to Alliance Central using community standards for knowledge representation. Alliance Central represents a next generation extensible software platform for knowledgebases capable of adapting to the rapidly changing data landscape and conforming to modern standards for data management. Alliance Central provides the biomedical research community with unprecedented support for comparative genomics via unified user interfaces and APIs for common data types and promotes sustainability and operational efficiencies of core biodata resources. This U24 application describes the plans for the enhancement and management of Alliance Central building on the significant accomplishments of the Alliance consortium since it was launched in 2016. The focus for Specific Aim 1 will be on expanding the Alliance Central infrastructure for ingesting, storing, and accessing the harmonizing biological annotations from contributing Knowledge Centers. We will continue software development practices that reflect our long-standing commitment to data management practices that align with FAIR (Findable, Accessible, Interoperable, Reusable) principles. In Specific Aim 2 we describe our plans to continue development of a state of the art literature curation system that can be adapted for use by a wide range of biomedical model organism databases. The deliverables for this aim will include the incorporation of machine learning, natural language processing, and artificial intelligence designed to enhance scalability and efficiency of expert curation. In Specific Aim 3 we describe our plans for implementation and/or adoption of user interfaces that advance the mission of the Alliance to facilitate comparative genomics to gain insights into the function of the human genome. Finally, in Specific Aim 4, we describe the management and organization of the Alliance as well as extensive activities for user support and community engagement.

NIH Research Projects · FY 2026 · 2019-07

Project Summary T cells are generated in the thymus from gestation through much of the lifespan in mice and humans, and in some ways the products of fetal and postnatal T cell development are highly similar. However, there are several distinct waves of precursors that enter the T-cell program from mid-gestation to puberty, and successive waves make T cells with distinctive, specific differences in their developmental kinetics and qualitative lineage outputs. First-wave fetal thymocytes, for example, generate unique innate lymphoid cell subsets (ILCs) as well as T cells, give rise to unique gd T cell subsets, limit their diversity of T-cell receptor (TCR) gene rearrangements, and differentiate quickly, reaching early maturation milestones in a fraction of the time of postnatal thymocytes. In all these ways they differ from postnatal thymocytes. Importantly, these differences in T-cell development are not only observed in vivo, where the thymic structure itself is undergoing changes, but also, when they differentiate in a common microenvironment, as distinct waves of precursors maintain most of their unique developmental features in vitro in cocultures with Notch ligand-presenting stromal cells (e.g. OP9-DLL1). Thus, fetal and adult precursors are intrinsically programmed to differentiate with strikingly different kinetics and some differences in pathway, despite many similar outputs. Our goal is to determine the regulatory mechanisms that endow the cells with these different versions of the T-cell program. This proposal is for a renewal of a systems biology grant that focused on identifying T-cell developmental speed controllers. Our goal now is to determine how these and other mechanisms may be responsible for ontogenic control of T cell program differences. It is based on new results from our recent single-cell transcriptome analyses, ATAC-seq analyses, in vitro differentiation studies, and novel views of the cells’ regulatory states using specialized transgenic reporter mouse strains. We focus on three proposed mechanisms to explain the difference between first-wave fetal and postnatal precursors: the potential role of genome-wide chromatin states and accessibility; the role of transcription factor Meis1; and the potential modulation of differentiation by a select group of other regulatory factors. We will exploit novel knock-in fluorescent reporter mouse strains to identify previously unidentified developmental branchpoints unique to the first-wave fetal T-cell precursors. In addition, we will use lineage tracing to track whether the cells that give rise to the most fetal-unique descendants also give rise to conventional postnatal-type T cells or whether the fetal thymus is populated by a mosaic of precursors with different lineage biases. Our aims will be: (1) To determine the chromatin states across the genome that distinguish fetal and postnatal T cell precursors (2) To define the impact of Meis1 and select additional trans-acting factors on fetal T cell development (3) To determine whether common or distinct lineage precursors give rise to conventional vs. fetal-restricted cell fates, and to determine the developmental branchpoints when lineage-biasing mechanisms are deployed.

NIH Research Projects · FY 2025 · 2019-07

This proposal will extend a program for Predoctoral Training in Quantitative Neurosciences at Caltech. The field of neuroscience is currently experiencing explosive growth, driven by a plethora of new technologies for observing and manipulating the brain. To apply these methods fruitfully and interpret the resulting flood of data, modern neuroscientists need a sophistication in mathematical and computational approaches that traditional neuroscience training programs cannot provide. At the same time we are seeing a convergence of understanding at different levels of brain organization, ranging from the molecules that specify neural connections to the dynamic function of large circuits in the human brain. Thus, there is an urgent need to train young neuroscientists with an integrated perspective that spans many levels – from molecules to behavior to computation – which have traditionally been taught in separate programs. Caltech is in an ideal position to answer these demands. It is an institution almost entirely dedicated to scientific research, routinely ranked among the top few universities in the world. Caltech is well known for its rigorous training in physics, mathematics, and engineering, but it also has a distinguished history in biological research, and in the neurosciences in particular. The small size supports a climate in which scientific collaboration across disciplines is effortless. In 2019, we created an exciting environment for training in quantitative neuroscience. The training program is focused on PhD students in years 1 and 2. The strategy begins with selective recruiting of candidates from a broad range of undergraduate majors, ranging from biochemistry to psychology to computer science. First-year students get introduced to faculty research during three rotations of 3 months each, after which they choose a primary mentor to supervise their PhD research. Trainees also complete a rigorous 2-year course curriculum that ensures core competency in the following six areas: (i) molecular and cellular neuroscience; (ii) systems and circuit neuroscience; (iii) human neuroscience and brain disorders; (iv) tools and technology for neuroscience; (v) applied mathematics and statistics; (vi) scientific programming and data analysis. Trainees get writing experience through a mentored process of fellowship applications. They also give numerous oral presentations in forums on and off campus. After a candidacy examination at the end of year 2, trainees focus on PhD research, with the aim of authoring one or more major publications, and graduating by year 6 or earlier. Each trainee’s trajectory through the program is accompanied by individualized advising, adjusting coursework to ensure broad competency while also challenging the student in a domain of special expertise. In summary, the proposed program in quantitative neurosciences responds in a timely manner to an urgent demand for a new type of graduate training in this rapidly evolving discipline.

NIH Research Projects · FY 2026 · 2019-05

The neural crest is a uniquely vertebrate cell type that is thought to have played an important role in vertebrate evolution by forming peripheral ganglia and jaws, thus facilitating predation and expansion of the brain. We recently identified a “cranial-specific” neural crest transcriptional subcircuit in jawed vertebrates (gnathostomes) that imbues this neural crest population with the unique ability, absent from trunk neural crest, to form craniofacial cartilage that is critical for jaw formation. Here, we propose to examine whether homologous genes are expressed in the cranial premigratory neural crest of lamprey, a jawless basal vertebrate (agnathan). Our preliminary results suggest that many of these genes are “missing” from the lamprey's premigratory cranial neural crest, thus challenging the hypothesis that invention of neural crest in vertebrates gave rise to a “New Head”. Accordingly, we hypothesize that there was progressive expansion of neural crest derivatives during the course of vertebrate evolution. This likely occurred by addition of new enhancer elements into the premigratory neural crest that conferred novel developmental potential onto this cell population. As case in point, lamprey lack a vagal neural crest that in gnathostomes forms the enteric nervous system. To test this hypothesis, we will perform lineage analysis, analyze transcription factors and regulatory regions of selected genes across agnathan and gnathostomes. We will identify putative enhancers, dissect their regulatory inputs, and test the ability of these regions to drive reporter expression in lamprey as well as cross-species, in the zebrafish. The results promise to elucidate how new cell types arose during vertebrate evolution under the umbrella of the neural crest.

NIH Research Projects · FY 2024 · 2018-08

ABSTRACT (30 Lines Max) Objective: Develop two innovative high-speed 3D breast photoacoustic computed tomography (PACT) systems to diagnose benign, atypical, and malignant lesions — leading to a more streamlined and accurate workup that reduces unnecessary follow-up imaging and benign breast biopsies. Clinical significance: Abnormal findings detected by screening mammography lead to workups including additional imaging, usually in the form of extra mammogram views, tomosynthesis, and/or ultrasound, and possibility of breast biopsy. Biopsies have considerable side effects such as pain and bleeding, and are unnecessary in the majority (60 – 70%) of cases because of the high false-positive rate of mammography. The side effects, costs, and delays of the workups cause considerable stress to patients. PACT combines the functional optical contrast of diffuse optical tomography and the high spatial resolution of ultrasonography without speckle artifacts. The attenuation coefficient of near-IR light in breast tissue is only twice that of mammographic x-ray—enabling adequate optical penetration, but light has far higher soft-tissue contrast than x-ray. With rich functional contrast at high spatial and temporal resolutions and without using ionizing radiation or exogenous contrast agents, 3D breast PACT has the potential to reduce unnecessary benign breast biopsies by serving as a diagnostic tool adjunct to mammography. Possible long-term breast imaging applications include not only diagnosis, but also screening, assessment of response to pre-operative systemic therapy, and definitive surgical planning. Challenges: Numerous innovations are required to develop a PACT imaging system effective for clinical breast imaging. Previous PACT breast imagers possess significant limitations, including suboptimal light delivery, limited detection view, non-isotropic spatial resolution, and long scanning times. There remains an imperative need for more advanced PACT breast imaging technologies. Solutions: Encouraged by the preliminary deep and clear in vivo breast images acquired by our newly developed PACT system, we propose further innovations for 3D functional breast imaging to overcome the above-mentioned limitations. Aim 1: To develop a single-breath-hold 3D breast imaging system with nearly isotropic 3D resolutions and dual-wavelength contrasts (Model I). Aim 2: To develop a snapshot 3D breast PACT system (Model II) using the concept of acoustic ergodicity. It can reach the ultimate imaging speed (single laser shot), desirable for 3D high-resolution functional and dynamic imaging without motion artifacts. Both systems can perform elastography. Contrasts to be imaged include vascularity, concentration and oxygen saturation of hemoglobin, elasticity, and tumor volume and shape. Aim 3: To test PACT for the diagnosis of benign, atypical, and malignant breast lesions by comparing with the gold standard of tissue pathology.

NIH Research Projects · FY 2025 · 2018-07

Project Summary/Abstract Mitochondria are best known as the “powerhouses” of the cell, due to their predominant role in generating cellular energy through the tricarboxylic acid cycle, fatty acid metabolism, and oxidative phosphorylation (OXPHOS). Beyond energy generation, these essential organelles also play central roles in apoptosis, calcium handing, innate immunity, cell signaling, and iron-sulfur cluster assembly. Human health therefore depends critically on mitochondrial function. The research program proposed here seeks to understand three homeostatic mechanisms that regulate mitochondrial health. The first mechanism is mitochondrial fusion and fission dynamics. Mitochondria are dynamic organelles whose physiology is regulated by the balance between the opposing processes of membrane fusion and fission. Second, the quality of the mitochondrial population is maintained by selective degradation of excessive or defective mitochondria through mitophagy, the autophagic pathway that shuttles mitochondria to the lysosome for destruction. Finally, mitochondrial quantity and function are regulated by biogenesis programs that control expression of mitochondrial genes, ensuring that appropriate levels of mitochondria are maintained in response to a specific cellular state. This research project targets key gaps in knowledge in each of these fundamental homeostatic mechanisms. For mitochondrial fusion and fission, we are using mouse genetics and cell biology to understand how mitochondrial dynamics regulates mitochondrial function. The least understood of the core mitochondrial dynamics genes is Fis1. We have found that Fis1 plays essential functions in neurons, astrocytes, and oligodendrocytes in the central nervous system. We are pinpointing the cellular functions of Fis1 and determining which function is responsible for the in vivo phenotypes. Our preliminary data suggest that Fis1 serves as a link between mitochondria and the actin cytoskeleton. Moreover, we are determining the elusive mechanism through which the balance between fusion and fission is regulated. There is evidence that each fusion event is coupled to a future fission event, and we will determine the molecular mechanism of this coupling. To study mitochondrial degradation, we are performing whole- genome CRISPR interference screens and have discovered the integrated stress response as a key regulator of mitophagy. We will elucidate the molecular mechanism through which this cell stress pathway tunes the level of mitophagy. Finally, we are using CRISPR interference screens to identify how mitochondrial density and function are regulated. These studies have identified two chromatin remodeling complexes as critical for mitochondrial function. By determining how these chromatin remodeling complexes regulate mitochondrial biogenesis through control of gene expression, we will gain insight into how cells dynamically adjust mitochondrial density to fit cellular demands. All together, these studies will provide a comprehensive understanding of how mitochondrial health in cells is maintained.

NIH Research Projects · FY 2026 · 2018-02

Both neural crest progenitors and ectodermal placode cells arise from the neural plate border (NPB). While the neural crest gives rise to the craniofacial skeleton, the placodes form the lens, ear and olfactory system; both contribute to cranial sensory ganglia. However, rather than being fixed to a neural crest or placodal fate, our results show that cells in the neural plate border appear to coexpress transcription factors characteristic of multiple lineages, ranging from neural crest to neural to placodal. Moreover, we find there is a stem cell niche within the dorsal neural tube that expresses pluripotency factors including Sall4, Nanog, Oct4, Klf4, and cMyc as confirmed in our single cell RNA-seq data. Thus, the question of what maintains stem cells with the potential to form neural crest, placode and neural tube derivatives at the neural plate border remains open. Our preliminary data suggest that Sall4 may form a feed-back loop with other multipotency genes, including Pou5f3/Oct4 and Sall1. Moreover, overexpression of Sall4 prevents neural crest specification and upregulates Oct4. To test the hypothesis that these pluripotency factors maintain multipotency of the neural plate border and that their downregulation is necessary for neural crest and/or placode specification, we will explore the effects of their gain and loss of function, identify enhancers that mediate their expression and examine dynamic changes in their gene expression during neural plate border maturation. To this end, the following aims will be performed. Aim 1: Effects of ectopic maintenance or loss of pluripotency factors on neural crest and placode development. We will test the role of pluripotency factors Sall4, Oct4, Nanog and Klf4 in vivo in neural plate border development using gain and loss of function approaches coupled with single cell RNA-seq. In particular, we will test the hypothesis that these pluripotency factors maintain the multipotency of the neural plate border and their downregulation is necessary for completion of neural crest specification. We will also examine other transcription factors to test their role in driving lineage specification at the neural plate border. Aim 2: Identification and cis-regulatory analysis of putative enhancers mediating expression of pluripotency factors and neural plate border transcriptional regulators. Detailed cis-regulatory analysis allows identification of active enhancers and their direct inputs, both positive and negative, thus informing upon gene regulatory network connections. We propose to use single cell ATAC-seq to identify putative regulatory elements active at the forming neural plate border, particularly those mediating expression of pluripotency factors and transcriptional regulators. Putative enhancers will be tested for inputs and their ability to drive expression. Aim 3: Dynamic analysis of enhancer-mediated expression during maturation of the neural plate border. By coupling live imaging with enhancer driven reporter expression, we propose to test dynamic changes in gene expression mediated by pluripotency genes and transcription factors. We will initially focus on enhancers that mediate expression of Pax7, Sox2 and Six1, characteristic of neural crest, neural and placodes, respectively.

NIH Research Projects · FY 2025 · 2017-05

PROJECT SUMMARY Pseudomonas aeruginosa is an opportunistic pathogen found in acute infections (burns, wounds, ventilator associated pneumonia, eye infections) and chronic infections of the foot (diabetic ulcers) and lung (cystic fibrosis). This bacterium commonly survives in these contexts as a biofilm, the formation and high-level antibiotic tolerance of which interferes with effective patient treatment. A defining aspect of P. aeruginosa is its ability to make pyocyanin, a colorful redox-active pigment that contributes to biofilm development and its fitness in the context of infection. Pyocyanin has been detected at appreciable concentrations in skin wounds and in cystic fibrosis sputum, and pyocyanin has been shown to be a virulence factor in animal infection models. Pyocyanin exerts a range of effects over the cells that produce it, ranging from toxic in the presence of oxygen to beneficial in its absence; under oxygen-limited conditions, pyocyanin serves as an electron acceptor that promotes redox-balancing and long-term survival. These toxic and beneficial roles are important at different times in biofilm development, with early pyocyanin -promoted lysis generating extracellular DNA (eDNA), a key component of the biofilm matrix together with exopolysaccharides. Recently, we determined that eDNA underpins pyocyanin’s ability to promote extracellular electron transfer (EET) within biofilms, facilitating metabolic activity in the oxygen-limited interior. We found that eDNA enables both the retention of pyocyanin and charge transfer to pyocyanin. Critical to making these discoveries was our development of new bioelectrochemical technologies and approaches and the application of advanced spectroscopic techniques to directly probe EET in biofilms. We now seek to extend our interdisciplinary work to gain a mechanistic understanding of how biofilm EET efficiency is tuned by the composition of the matrix and the consequences this may have for antibiotic tolerance. Does the ratio of certain exopolysaccharides (Pel, Psl) to eDNA modulate pyocyanin diffusivity in the matrix, controlling EET efficiency? Does EET efficiency correlate with the rate of redox balancing in the biofilm interior? How do pyocyanin-mediated cellular effects, including EET, contribute to antibiotic tolerance in biofilms, and do these mechanisms differ according to the amount of oxygen in the microenvironment? Does the relative sensitivity of diverse pyocyanin-producing P. aeruginosa isolates to antibiotics correlate with their matrix composition and EET efficiency? Aim1 will explore how the matrix composition, particularly the ratio of Pel and Psl exopolysaccharides to eDNA, determines EET efficiency. Aim 2 will test the hypothesis that pyocyanin is a versatile intrinsic tolerance factor, where PYO-EET helps P. aeruginosa biofilms tolerate mechanistically distinct and clinically important antibiotic classes via bioenergetic effects and/or by inducing defense mechanisms; we predict the dominant mechanism by which PYO impacts tolerance will differ as a function of oxygen concentration. Attainment of these objectives will lay the foundation of basic knowledge necessary to design better strategies to control P. aeruginosa biofilms.

NIH Research Projects · FY 2025 · 2016-08

Regulation of gene expression along the dorsal-ventral (DV) axis of Drosophila embryos serves as a paradigm of developmental patterning. Comparative studies of cis-regulatory elements that support expression along the DV axis from many research groups have made it clear that combinatorial input into enhancers by multiple transcription factors drives distinct spatial-outputs of gene expression. A pivotal regulator of this patterning process is the maternally-provided transcription factor Dorsal (Dl), homolog of NFKB. Dl functions as a morphogen to activate target gene expression in a concentration-dependent manner along the DV axis, contributing to the initiation of zygotic gene expression at the maternal-to-zygotic transition (MZT). Using live imaging, we quantified the Dl gradient in embryos and found, surprisingly, that levels change not only in space but also build in time. Our focus during the previous funding period was to study the impact of these Dl dynamics on target gene expression using quantitative approaches involving analysis of live imaging or fixed embryo time-series data to provide insight. In the current proposal, we follow three new and exciting directions, which relate to the timing of cell actions in early embryos and arose as a result of the previous work. Project 1 involves studying how broadly-expressed activators and repressors cooperate to control the onset of zygotic gene expression during the MZT. We hypothesize that broadly-expressed repressors are equally important to pioneer activators in the control of chromatin accessibility and thereby also regulate initiation of zygotic gene expression. Project 2 focuses on dissecting the function of short-transcripts for long genes that are expressed specifically in the early syncytial embryo. We hypothesize that these short transcripts act to regulate timing of cell signaling pathway activation by functioning as dominant-negative variants of signaling molecules. Project 3 focuses on identifying the mechanism by which FGF signaling regulates adherens junctions (AJs) and their interaction with the actin cytoskeleton to contribute to the first epithelial-to-mesenchymal transition (EMT) in embryos; in particular, to understand how a degron associated with one FGF ligand, Pyramus, limits signaling time. The overarching goal of the proposed research program is to understand how the timing of these cell activities - patterning, signaling, and movement - are controlled in developing Drosophila embryos and to provide general insights applicable to higher animals. While many studies have focused on spatial outputs of gene expression, less is known about the temporal dynamics of patterning. Drosophila embryos are a tractable system to study MZT as it occurs in 3-4 hours, in contrast to taking days in preimplantation mammalian embryos. The Drosophila embryo is also amenable to live in vivo imaging and tracking analyses making it well-suited to the study of nascent transcription and cell morphology. Lastly, our proposed studies will provide general insight into early embryo development of higher animals as many regulatory mechanisms are likely conserved.

NIH Research Projects · FY 2024 · 2016-08

Project Summery Equipment supplement – same as the parent grant