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NIH

Each year, millions of individuals receive repetitive head injuries (RHI) from playing contact sports or through military service. RHIs are a type of traumatic brain injury characterized by mild, mostly non-concussive injuries that, in the context of a sport like American football, can occur dozens of times a day for years. In addition to long-term disability and dysfunction, individuals who have a history of RHI are at an increased risk of developing neurodegenerative diseases, likely stemming from a neuroinflammatory environment that results from the constant low-level head trauma. To that end, our preliminary single nucleus RNA sequencing (snRNAseq) studies have demonstrated inflammatory SPP1+/Hif1a+ microglia are observed in cases with exposure to RHI but have not yet developed pathology, suggesting inflammation precedes protein deposition. Interestingly, it has become clear that neuropathologies like Alzheimer’s disease (AD) in the context of RHI are not identical to their non-RHI counter parts. In contrast to the normal confluent appearance of cortical hyperphosphorylated tau (ptau) distribution observed in AD, individuals with AD and an RHI history (AD+RHI) were observed to have a sulcal predominance of ptau. Additionally, preliminary bulk sequencing studies suggest AD+RHI cases have an altered neuroinflammatory environment. These observations raise the question if other “classical AD” features are altered during RHI. The implications for differences are significant, as imaging biomarkers for AD diagnosis rely on understanding the known progression pattern of neuropathology but altered pathology makes it harder to diagnose AD. Also, the altered neuropathologic burden and distribution may skew clinical symptoms, which complicates clinical assessment and results in improper treatment strategies. To better examine RHI-specific changes in AD, we will deeply characterize the neurodegeneration, pathologic protein regional progression, and clinical phenotypes observed in individuals with a history of RHI and a neuropathologic diagnosis of AD. We will use our unique resource—the NIA-funded P30 Boston University AD Research Center brain bank, the largest RHI brain bank in the world, to compare 7 groups: individuals either with Low, Intermediate, or High AD neuropathologic changes with or without exposure to RHI (n = 379), and a no pathology/no RHI control group. Overall, we hypothesize that the exposure to RHI will result in altered AD neuropathology, which will also have direct associations with clinical outcomes. Using digital pathology and artificial intelligence toolsets, we will perform disease progression modeling using a machine learning tool called subtype and stage inference algorithm (SuStaIn), to examine whole-slide digital images taken from 19 brain regions per case that are stained for ptau, Aβ, TDP43, and a-syn. SuStaIn uses spectrums of neuropathology data to predict progression patterns of diseases through the brain and can determine cases that fall outside the normal range identifying subtypes. We will compare progression patterns between AD and AD+RHI cases encompassing early, late, and end stage disease. We will also determine if specific SuStaIn subtypes correlate with distinct clinical symptom presentation. Next, we will perform snRNAseq to compare how cell population might be altered in our 7 groups using tissue taken from the dorsolateral frontal, temporal, and calcarine cortex representing early, late, and end stage pathology. In addition, we will use proximal frozen tissue to perform pTau and Aβ histology to measure pathologic accumulation directly related with the cells assayed by snRNAseq. Finally, we will perform a spatial histologic analysis across AD and AD+RHI cases to investigate how local cell populations are directly related to pathology. We will again utilize tissue from the dorsolateral frontal, temporal, and calcarine cortex to capture the full spectrum of disease related changes and investigate if previously observed RHI-induced neuroinflammatory cell populations are present in brain structures most susceptible to RHI damage. Overall, these findings will be critical in advancing our understanding of how RHI alters neuropathology and shapes clinical presentations. The direct impact of this work will be enhanced criteria and methods to better distinguish AD- and RHI-specific changes.

NIAMS - National Institute of Arthritis and Musculoskeletal and Skin Diseases

Project Summary Cutaneous capillary malformation (CM, a.k.a, Port Wine Birthmark, PWB) is a defect of primitive capillaries in the skin during development. It mainly appears on the face with an estimated prevalence of 0.3-0.9% in newborns and is highly associated with many complicated types of vascular malformations and outgrowth syndromes, such as Sturge-Weber syndrome (SWS), Parkes-Weber syndrome, Klippel-Trenaunay syndrome, arteriovenous malformations (AVM), and CLOVES syndrome. Photobiological-based modalities, such as pulsed dye laser (PDL) or photodynamic therapy (PDT), are the common treatment options for CM; unfortunately, complete removal occurs in less than 10% of patients treated. In addition, about 20% of lesions have no response to laser, e.g., laser-resistant (PDL- or PDT-resistant) CM. The inadequate outcomes are unmet clinical barriers. This study will tackle these challenging clinical barriers by repurposing an FDA-approved anti- diabetic medicine sitagliptin to target laser-resistant CMs. In this proposal, a series of clinically relevant in vitro and in vivo models including CM-derived inducible pluripotent stem cells (iPSCs), their induced endothelial cells (iECs), vascular organoids (VOs), and mouse models with xenograft of CM iECs will be used. We will determine the therapeutic window of sitagliptin on laser-resistant CM iECs in vitro. We will dissect sitagliptin-mediated inhibition of antioxidant pathways as a novel mechanistic action to sensitize CM lesions. We will characterize how sitagliptin-modulated GPX activity, GSH and ROS levels in response to PDT. We will test if sitagliptin improves the outcomes of laser therapy on VOs in vitro and determine if oral administration of sitagliptin at non-toxic doses enhances the efficacy of PDT in xenograft mouse models. This study is highly translational. First, sitagliptin is among the top list of compounds generated through our data-driven drug repurposing pipline using the multiome datasets of CM iPSCs, iECs and patients’ biopsies. Second, sitagliptin is an FDA-approved drug, thus repurposing sitagliptin would be easier, cheaper, and more feasible than other candidate compounds. Third, PK/PD, safety, and real-world data of sitagliptin are available for its’ repurposing. Third, sitagliptin shows efficacy in nanomolar ranges, displaying a wide therapeutic window for pediatric patients. The successful completion of this proof-of-concept study will serve as a milestone and basis for a Go/No-Go decision for the further repositioning sitagliptin for off-label trials in patients.

NIMH - National Institute of Mental Health

Recent advances in human genetics defined hundreds of causal variants for Autism Spectrum Disorder (ASD) and other neurodevelopmental disorders (NDDs). However, substantial effort is now required to identify downstream disease processes and guide development of interventions. One such new NOD gene is ZNF292: mutations were recently associated with ASD and other NDDs in humans, but ZNF292 requirements for brain development and consequences of pathogenic mutation have not been studied in any animal or human cellular model. Here, we propose a comprehensive mechanistic investigation of ZNF292, using both established cuttingedge workflows and innovative new approaches to study the consequences of both deficiency and pathogenic mutation at the molecular, cellular, structural, and functional and behavioral circuit levels. We use two complementary experimental systems, mouse models and human pluripotent stem cell (hPSC)-derived neurons, to define ZNF292 requirements for neurodevelopment. We focus initially on hPSC models carrying six pathogenic ZNF292 variants identified in patients with ASD, intellectual disability, and other NOD clinical phenotypes (e.g., microcephaly, epilepsy), and comparison with hPSC lines with constitutive or inducible ZNF292 deficiency. Our preliminary work has already demonstrated shared disruptions of neurodevelopment, transcriptional regulation, and neuronal network function stemming from either ZNF292 deficiency or pathogenic mutation in these models. We also developed conditional and non-conditional mouse knockout models of ZNF292 deficiency and demonstrated that these exhibit NOD-relevant circuit disruptions and, accordingly, altered behavior. Further, we developed cutting-edge tools for temporally controlled reversal of ZNF292 deficiency in our hPSC models, enabling us to investigate the effects of restoring ZNF292 gene function either during development or in mature neurons. Related landmark experiments profoundly changed our understanding of other NDDs by demonstrating that a substantial proportion of chronic NOD phenotypes were reversible, thus spurring the development of therapeutics based on either restoring gene expression or reversing its chronic consequences for neuronal function. In complementary efforts, we continue to model additional cases in this rapidly expanding patient population, linking NOD clinical phenotypes to shared, reversible endophenotypes related to altered neuronal network function. Based upon our preliminary data, we hypothesize that impaired circuit and neuronal network function stemming from chronic reduction of ZNF292 activity contributes substantially to NDDs in this patient population. These deficits may be tractable for molecular or pharmacological treatment to develop interventions. Therefore, together, the experiments performed here will elucidate requirements for and mechanisms by which ZNF292 normally controls brain development and function, will determine how these are disrupted by pathogenic ZNF292 mutation, and could also chart a course towards ZNF292-targeted therapies.

Dept of the Army -- Materiel Command

Under the authority of 10 U.S.C. 4144, in alignment with Section 223 of the FY 2026 National Defense Authorization Act (NDAA), funding in the FY 2026 defense appropriations, and, Presidential Executive Order 14332, Improving Oversight of Federal Grantmaking, the DoW announces this funding opportunity for the acquisition of equipment/instrumentation at Historically Black Colleges and Universities (HBCUs) and Tribal Colleges and Universities (TCUs).The program is executed under policy and guidance of the Office of the Under Secretary of War for Research and Engineering (OUSW(R&E)) and is administered by ARL, ONR, and AFOSR, hereafter called the Agencies. This Notification of Funding Opportunity (NOFO) aims to (a) enhance research and education programs and capabilities in scientific and engineering disciplines critical to the national security functions of DoW; (b) enhance the capacity of HBCUs and TCUs to participate in DoW research programs and activities; and (c) increase the number of graduates in the fields of science, technology, engineering, and mathematics (STEM). The DoW is announcing this research solicitation based on 10 U.S.C. 4144 and Section 223 of the FY 2026 NDAA (https://www.congress.gov/119/bills/s1071/BILLS-119s1071enr.pdf), which directs for fiscal year 2026 and each fiscal year thereafter, the Secretary of Defense to ensure that funding solicitations through the DoW HBCU/MI Program Element include separate funding solicitations each focused toward: (1) Historically Black Colleges and Universities and Tribal Colleges; and (2) Minority-Serving Institutions that are not HBCUs or TCUs. Also informing this solicitation is Section 262 of the NDAA for FY 2020 (https://www.congress.gov/116/plaws/publ92/PLAW-116publ92.pdf), section 220 of the NDAA for FY 2022 (https://www.congress.gov/117/plaws/publ81/PLAW-117publ81.pdf), and the publication of the National Academies of Sciences, Engineering, and Medicine titled Defense Research Capacity at Historically Black Colleges and Universities and Other Minority Institutions: Transitioning from Good Intentions to Measurable Outcomes dated April 28, 2022 (https://www.nationalacademies.org/publications/26399).The DoW is soliciting applications (also referred to as proposals ) for the acquisition of equipment/instrumentation under the Fiscal Year 2027 Research and Education Program for HBCU/MI. The Research and Education Program is designed to enhance the research capabilities of HBCUs and MIs and to strengthen their STEM education programs. The purpose of the funding under this NOFO is to (1) support the acquisition of equipment/instrumentation to augment existing capabilities or to develop new capabilities in research areas of interest to DoW, and (2) attract students to pursue studies leading to STEM careers. Although funding provided under this NOFO cannot be used for student support, in order to further DoW s objective of attracting students to pursue studies leading to STEM careers, applicants must address the impact of the requested equipment/instrumentation on student participation in research. The proposals must align with the research areas of interest to the DoW (which includes the USW(R&E) and the Agencies) and enhance the education of students in areas of STEM that align with the DoW research interests.

U.S. National Science Foundation

The Research in Disabilities Education (RDE) program seeks to broaden the participation and achievement of people with disabilities in all fields of science, technology, engineering, and mathematics (STEM) education and associated professional careers. The RDE program has been funding this objective since 1994 under the prior name "Program for Persons with Disabilities." Particular emphasis is placed on contributing to the knowledge base by addressing disability related differences in secondary and post-secondary STEM learning and in the educational, social and pre-professional experiences that influence student interest, academic performance, retention in STEM degree programs, STEM degree completion, and career choices. Projects also investigate effective practices for transitioning students with disabilities across critical academic junctures, retaining students in undergraduate and graduate STEM degree programs, and graduating students with STEM associate, baccalaureate and graduate degrees. Research project results inform the delivery of innovative, transformative and successful practices employed by the Alliances for Students with Disabilities in STEM to increase the number of students with disabilities completing associate, undergraduate and graduate degrees in STEM and to increase the number of students with disabilities entering our nation's science and engineering workforce. RDE projects contribute to closing the gaps occurring for people with disabilities in STEM fields by successfully disseminating findings, project evaluation results, and proven good practices and products to the public.Innovation through Institutional Integration (I3) projects enable faculty, administrators and others in institutions to think and act strategically about the creative integration of NSF-funded awards, with particular emphasis on awards managed through programs in the Directorate for Education and Human Resources (EHR), but not limited to those awards. For Fiscal Year 2010 proposals are being solicited in nine EHR programs that advance I3 goals: CREST, GSE, HBCU-UP, ITEST, LSAMP, MSP, Noyce, RDE, and TCUP.

Naval Supply Systems Command

This FOA is intended for proposals related to basic and applied research in the STEM categories, and not related to the development of a specific system or hardware procurement.The proposal submission process has two stages:I. Applicants are encouraged to submit a white paper first to Grants@NPS.edu, ; andII. Applicants must submit a full proposal through Grants.gov (do not submit white papers through Grants.gov, only full proposals)Applicants are encouraged to submit a White Paper in advance of a Full Proposal to minimize the labor and other costs associated with the production of detailed full proposals that have little chance of being selected for funding. Based on an assessment of the white papers, the responsible Research Program Officer will provide informal feedback notification to the prospective awardees to encourage or discourage submission proposals. The Research Program Officer may also, on occasion, provide feedback encouraging re-work to strengthen a proposal. White Papers may be submitted at any time during the open period of this FOA. A separate White Paper is required for each research proposal.

Naval Supply Systems Command

<p>This FOA is intended for proposals related to basic and applied research in the STEM categories, and not related to the development of a specific system or hardware procurement.</p><p><br></p><p>The proposal submission process has two stages:</p><p>I.&nbsp;Applicants&nbsp;<u>are encouraged&nbsp;</u>to submit a white paper first to Grants@NPS.edu, ; and</p><p>II. Applicants must submit a full proposal through Grants.gov (do not submit white papers through Grants.gov, only full proposals)</p><p>Applicants are encouraged to submit a White Paper in advance of a Full Proposal to minimize the labor and other costs associated with the production of detailed full proposals that have little chance of being selected for funding.&nbsp;Based on an assessment of the white papers, the responsible Research Program Officer will provide informal feedback notification to the prospective awardees to encourage or discourage submission proposals.&nbsp;The Research Program Officer may also, on occasion, provide feedback encouraging re-work to strengthen a proposal.&nbsp;</p><p><br></p><p>White Papers may be submitted at any time during the open period of this FOA. A separate White Paper is required for each research proposal.&nbsp;</p>

NIMH - National Institute of Mental Health

PROJECT SUMMARY The brain is an extraordinarily complex organ containing many millions of neurons and non-neuronal cells that are organized into highly specialized yet intricately integrated circuits controlling various activities, such as sensory perception, motor control, and cognitive processes. The brainstem is a critical region responsible for regulating essential bodily functions, like heart rate, blood pressure, and digestion, and thus maintaining body homeostasis. The individual neurons in brainstem can be classified into types based on shared characteristics like gene expression. Binning individual cells into cell types is fundamental for advancing our understanding of complex biological systems, like the brain, from normal tissue function to disease processes. The characterization of cell types enables creation of tools to gain genetic access to groups of cells, it enables dissection of cellular heterogeneity, identify key players in various contexts like disease and aging, and lay the groundwork for targeted interventions and therapies. We recently built a comprehensive, high-resolution atlas of cell types across the entire adult mouse brain and the cell type diversity in brainstem exceeded our expectations. Brainstem is home to a highly heterogeneous group of neurons that does not share a specific gene module, yet these neurons are highly similar to one another. In addition, these cell types intermingle in various regions and their function is strongly determined by their input/output relationship. This suggests that a high-dimensional combinatorial gene expression code is needed to resolve the unique transcriptomic cell types in this region. Our goal is to create a refined atlas of cell types in brainstem using a combination of single cell transcriptomic profiling, spatial transcriptomic profiling, and mapping of projection patterns to transcriptomic cell types in brainstem. In addition, we will computationally align brain stem cell types from mouse, non-human primate, and human to define a cross-species consensus atlas of brain stem. Cell type homologies across species can be established based on conserved marker expression. This enables inference of cellular properties, such as long- range projection targets, that are difficult to measure in humans. The proposed efforts will lead to a significantly improved understanding of brainstem cell types and their function and lay the foundation for a better understanding of disease processes related to that region.

NICHD - Eunice Kennedy Shriver National Institute of Child Health and Human Development

Project Summary The mammalian female reproductive tract (FRT), consisting of the oviducts, uterus, cervix and vagina, is essential for pregnancy establishment and a frequent site of human disease, including infertility and cancer. The embryonic origin of FRT is the Müllerian ducts, a pair of epithelial tubes with a surrounding mesenchyme. Once formed, the Müllerian duct regionally divides into segments along the anterior- posterior axis with each segment developing into distinct structure (oviduct, uterus, cervix and upper vagina). Classical tissue recombination experiments have shown that the Müllerian duct epithelial cell fate determination along the anterior-posterior axis is mediated by signals from the underlying stroma and retinoic acid (RA) signaling has been proposed to be the stromal cue for uterine epithelial cell fate determination. Despite advances in the molecular understanding of MDE specification during FRT development, there remains a striking knowledge gap in our understanding of how adult uterine cell fate is maintained. Recently a group of uterine epithelial cells at the intersection of luminal and glandular epithelium has been proposed to be a uterine stem cell population capable of differentiating into either luminal or glandular cells. However, little is known how these stem/progenitor cells acquire and maintain uterine cell fate and whether stromal signals are required for their specification. Our preliminary studies provide strong genetic data supporting the idea that RA signaling is continually required to maintain uterine epithelial cell fate during adult uterine homeostasis. In addition, our data suggest a model in which an antagonistic relationship between RA and estrogen signaling regulates Müllerian duct epithelial cytodifferentiation. These hypotheses will be tested in two aims. Aim 1 will examine the cell-autonomous function of retinoic acid receptors in FRT development and in adult uterine cell fate determination. Aim 2 will test the hypothesis that an RA-estrogen antagonism drives FRT cell fate determination both in mouse and in human. Successful completion of these studies may provide novel insights into female infertility and uterine cancer as a result of signaling imbalance between RA and estrogen, which could have a long-lasting impact in multiple research fields including development, fertility and cancer research.

NINDS - National Institute of Neurological Disorders and Stroke

Project Summary Neural stem cells (NSCs) in the brain generate newborn neurons throughout life, providing an endogenous stem cell pool that can be harnessed to improve cognitive function during aging and neurodegenerative disease. Thus, understanding how adult neurogenesis is regulated may provide new targets and opportunities for therapeutic upregulation in these conditions. Intermediate filament (IF) proteins such as nestin and glial fibrillary acidic protein (GFAP) have been invaluable as markers for NSCs, increasing our understanding of the different cell states and cell types of the neurogenic niche. The IF protein vimentin is also expressed in NSCs, but due to variability in antibody quality and the lack of reporter mouse lines, very little is known about when and where it is expressed in the neurogenesis cascade. Recently, my lab has demonstrated many unique aspects of vimentin’s function and regulation in adult hippocampal NSCs in vitro. We found that vimentin mRNA is stabilized during quiescence, yet translationally repressed through an RNA-binding protein interaction with vimentin mRNA’s 3’UTR. As qNSCs activate, repression is removed, resulting in a rapid increase in vimentin protein. Additionally, as qNSCs activate, they traffic accumulated proteins that need to be degraded to the centrosome to form an aggresome. Vimentin collapses around the aggresome, forming a vimentin cage, bringing with it interacting proteins such as proteasomes. During cell division, the aggresome, vimentin cage, and associated proteins are asymmetrically segregated into one daughter cell. The daughter which inherits these cargoes has a decreased proliferation rate, whereas the non-inheriting daughter has a normal proliferation rate, resulting in a rejuvenative asymmetry between daughter cells. Vimentin is also required for efficient quiescence exit both in vitro and in vivo, further suggesting that vimentin’s role in NSCs is not only as a potential marker, but also as a key component to intrinsic mechanisms of quiescence exit. However, most of these findings were largely performed in vitro, thus we do not know if this process is conserved in the adult brain, nor how this asymmetric inheritance would affect the outcome of daughter cells in the neurogenic niche itself. To address these open questions, we created a novel transgenic mouse with endogenous vimentin fused to linker-mScarlet. We will characterize vimentin-mScarlet expression at the mRNA and protein level in cells of the hippocampus, and through prospective sorting followed by cell behavior analyses, we will reveal when and where vimentin mRNA and protein are expressed in the hippocampus. Importantly, we will also perform chronic in vivo imaging in cranial windows in these mice to visualize vimentin-mScarlet protein during quiescence exit and its asymmetric segregation during divisions, following the consequence of this inheritance in vivo. These studies will not only provide an important novel tool to the scientific community, but also reveal new knowledge on NSC subpopulation dynamics, answering critical questions about how NSCs rejuvenate their niche.

NIDCR - National Institute of Dental and Craniofacial Research

PROJECT SUMMARY/ABSTRACT Dental enamel is formed by ameloblasts that are derived from dental epithelium. How dental epithelial stem cells (DESCs) commit to ameloblast lineage remains elusive. A potential critical factor that drives the commitment of DESCs to the ameloblast lineage is retinoic acid (RA). The complex system that drives ameloblast lineage commitment and the abundance of molecules that determine the RA signaling system require us to examine the transcriptomics of developing teeth at single-cell and spatial levels, followed by biological validations. Our preliminary data showed the diversifications of DESCs and the continuous differentiation of ameloblasts in a mouse incisor scRNAseq dataset. We sorted the differentiating ameloblasts into novel clusters in both mouse and human incisor scRNAseq datasets and replicated these findings using RNAscope. Our findings also suggest that RA signaling is inhibited in pre-secretory stage ameloblasts but activated when ameloblasts transition into the secretory stage. We identified potential RA response elements (RARE) in genes that are critical for secretory stage enamel formation. Therefore, we hypothesize that the sequential ameloblast differentiation is specified by critical genes through the retinoic acid (RA) signaling. By integrating scRNA-seq data analyses with molecular validations in developing teeth, we can 1) elucidate the differentiation trajectory of ameloblasts and 2) reveal critical roles of RA signaling in ameloblast differentiation. Aim 1. Determine the ameloblast differentiation trajectory. We will integrate the scRNAseq data obtained from 14 studies in different tooth types from mice, rats, and humans. We will conduct cluster analysis within tooth types and species to identify each phase of the continuous differentiation paths predicted by trajectory inference. We will compare the ameloblast differentiation trajectory across tooth types and species, with an emphasis on molecules relevant to RA degradation and signaling activation. To validate the trajectory defined by bioinformatic analyses, we will perform RNAscope HiPlex assay on mouse developing teeth using identified genes. Aim 2. Determine the critical roles of RA signaling at the onset of secretory stage enamel formation. First, we will conduct spatial transcriptomics and consequent bioinformatic analyses to map spatial distributions of molecules involved in RA synthesis, signaling activation, and metabolism, together with potential target genes of the RA signaling, in mouse enamel organ epithelium and adjacent dental mesenchyme. Second, we will perform the CUT&Tag sequencing to identify RAREs in RA signaling targets across the genome in mouse secretory ameloblasts. We will validate these findings by analyzing spatial transcriptomic and existing scRNAseq data. The completion of this project will allow us to identify critical factors in ameloblast differentiation. These insights will shed light on the mechanisms of tooth morphogenesis and congenital tooth disorders. This project will provide essential information to the development of bioengineering strategies for tooth regeneration.

NIDCD - National Institute on Deafness and Other Communication Disorders

Project Summary One of the most common medical conditions in any aging society is presbycusis, or age-related hearing loss. Approximately one third of American adults suffer from this condition typically starting in their middle ages, and about half of adults over 70 years have a substantial hearing impairment. One of the mechanisms of presbycusis happens in the central nervous system and is termed central hearing loss. Older adults with central hearing loss may have normal or near normal audiograms yet have problems carrying on a conversation in acoustically complex environments where multiple sound sources are active at the same time, such as a busy restaurant, a public place, or any situation where background noises are active. The main reason for this difficulty is that affected individuals have trouble perceptually isolating sound sources of interest (e.g., the voice of the speaker they want to listen to) effectively from other sources, presumably because the neural mechanisms that perform this computation are less effective. Our laboratories' recent work suggests that one key age-related subcellular change in the sound localization pathway contributes to this phenomenology: A demyelination of afferent fibers to the sound localization pathway. In Mongolian gerbils, we propose to experimentally isolate this mechanism by re-creating it in young animals – effectively “making young animals old”. Furthermore, we will test an approach using a pharmaceutical agent in combination with sound stimulation to reverse this age-related change – effectively “making old animals young”. Finally, we will investigate the mechanisms driving the age-related demyelination by studying oligodendrocytes, the glia cells that produce myelin, in young and old animals. The expected results from this study will help determine the role of demyelination in central hearing loss, a common medical condition which is still poorly understood. The expected results will also suggest a potential treatment for this condition.

OD - NIH Office of the Director

Metastases cause 90% of cancer deaths, and can occur years to decades after eradication of the primary tumor. This is due to the remarkable ability of cancer stem cells to migrate to different tissues and transition into a dormant state, providing protection from cytotoxic therapies. Both cell intrinsic processes, as well as extrinsic signals from surrounding cells and the extracellular matrix (the niche), instruct stem cell dormancy, but the nature of these signals and the mechanisms that allow a cell to survive in a dormant state are not understood. Years later, dormant metastatic cells can awaken, but the signals regulating their re-activation are also not understood. In addition, in long-lived organisms, populations of tissue stem cells maintain themselves in a quiescent state, yet remain poised to re- activate when needed. Understanding these mechanisms have major implications for studies in both homeostatic and injury-induced tissue regeneration, and how they are dysregulated in disease and ageing. A major limitation in dissecting these processes is the absence of a system that provides the opportunity to experimentally trigger the induction and exit from a dormant state. We have developed a model in which we can precisely control when and where stem cell dormancy occurs. This organism, called Botrylloides diegensis, responds to environmental stress by resorbing its entire body, leaving only a small (3-5 mm2) vascular mat that contains both pluripotent stem cells and a complex cellular and acellular niche that supports them. Dormancy can be induced by multiple environmental triggers, including heat, cold, starvation and hypoxia, and the vascular mat can survive for months in these severe conditions. Upon return to a normal environment, the pluripotent stem cells within the vascular niche are rapidly re-activated and regenerate the entire body within days, in a process called whole body regeneration (WBR). Simply put, we can stick an animal in the refrigerator, and it will go dormant in 72 h. When we move it back to normal conditions, the stem cells are reactivated and WBR will be completed in 7 days. The stem cells can be isolated by FACS, labeled and transplanted, and the vascular mat can be live imaged. In addition, the stem cells can be selectively ablated with no effect on the ability of the niche to support WBR, allowing us to study the stem cells and niche independently. This ORIP R21 will develop a new model organism that allows studies of the interaction between stem cells and their niche at unprecedented temporal and visual resolution. These interactions are critical for understanding homeostatic and injury-induced tissue regeneration, how stem cells respond to different environmental insults, age-dependent changes in stem cell biology, and metastatic cancer recurrence.

NINDS - National Institute of Neurological Disorders and Stroke

ABSTRACT Moyamoya disease (MMD), a progressive vasculopathy leading to narrowing and ultimate occlusion of the intracranial internal carotid arteries, is an important cause of childhood stroke. Our lack of knowledge of the etiopathogenesis of MMD hampers the development of preventive and therapeutic strategies. MMD can be genetically triggered and is highly genetically heterogeneous. The gene most commonly associated with MMD is RNF213, due to a founder variant in Asian individuals that confers a low penetrant risk of disease. We identified de novo RNF213 rare variants in a limited region of the protein that lead to early onset, severe, and progressive MMD in infants and toddlers. However, the function of the RNF213 protein and its role in MMD pathogenesis is poorly understood. Our prior work supports the hypothesis that incomplete differentiation of smooth muscle cells (SMCs) from neural crest progenitor cells (NCPCs) leads to increased migration into the lumen and proliferation that fills the occlusive lesion. We identified that decreased oxidative phosphorylation is a consequence of incomplete differentiation and that treatments to increase mitochondrial respiration can rescue the differentiation defect in vitro and prevent MMD-like lesions in vivo in a mouse model. Our goals in this study are to assess whether a highly penetrant RNF213 pathogenic variant, p.F4120L, conforms to this hypothesis and to identify specific molecular mechanisms linking RNF213 to SMC phenotype. The best evidence for RNF213 function comes from a study using mCherry- tagged RNF213, which showed localization of the protein to intracellular lipid droplets (LDs). RNF213 prevents localization of the lipolysis enzyme ATGL to LDs and thus regulates LD turnover. NCPCs have highly variable numbers of LDs, and increasing LDs by lipid loading affects both cell fate and cellular metabolism. We therefore hypothesize that RNF213 in NCPCs is required to modulate LDs to permit SMC differentiation and that the RNF213 p.F4120L variant increases lipolysis of LDs, preventing complete SMC differentiation and leading to increased proliferation and migration and thus occlusive lesion formation. We will test this hypothesis in two specific aims by using (1) an in vitro system of genetically edited induced pluripotent stem cells differentiated to NCPCs then SMCs and (2) a novel Rnf213F4120L/+ knock-in mouse model. Completion of these aims will yield novel and critical insights into MMD pathogenesis, RNF213 function, and the role of LDs in SMC differentiation. We will generate resources that will be made freely available to the research community with the goal to accelerate discovery and testing of potential therapeutic options to prevent, diagnose, and treat MMD.

NASA Ames Research Center

NASA Ames Research Center (ARC) Robotics Alliance Project (RAP) intends to issue a Cooperative Agreement Notice (CAN) soliciting proposals to design and administer the Robotics Outreach Competition (ROC) Program. This announcement, leading to the award of a Cooperative Agreement, will be issued pursuant to title 14 CFR Part 1260 for educational and nonprofit institutions. The ROC addresses the critical shortage in Science, Technology, Engineering and Mathematics (STEM) fields that the Nation is facing by providing hands-on robotics competition events while working with engineers and talented faculty from universities and high schools in the U.S. It is the strategic intent of this program that students will be inspired and motivated to pursue degrees that meet NASA's robotics competency requirements. A draft CAN was released September 23, 2010 and is available electronically through http://nspires.nasaprs.com . Do not submit a proposal in response to the draft notice. It is issued as an acquisition planning tool and as a means of soliciting industry comments for use in developing the final notice. Interested Parties are encouraged to furnish comments by September 29, 2010. The final CAN is expected to be released on or around September 30, 2010 and will be available electronically through http://nspires.nasaprs.com . Eligible organizations may submit proposals that provide evidence of the capability and proven experience necessary to provide both the technical and administrative framework required to implement a national, high caliber outreach program, including a high-quality national level robotics competition experience that leverages hands-on experiences in a technical environment. Electronically submitted Notices of Intent to propose are requested by October 12, 2010. The proposal due date is anticipated to be October 29, 2010. The electronic submission of each proposal in its entirety is required by the due date for proposal submission. Notwithstanding the posting of this opportunity at FedBizOpps.gov, Grants.gov, or at both sites, NASA reserves the right to determine the appropriate award instrument for each proposal selected pursuant to this announcement. Oral communications are not acceptable in response to this notice.

NHLBI - National Heart Lung and Blood Institute

Grant title: Role of CYB5R3 in erythropoiesis Funding mechanism: NIH/NHLBI-F32 PROJECT SUMMARY/ABSTRACT Cytochrome b5 reductase 3 (CYB5R3) plays a critical role in regulating redox signaling in both physiological and pathological contexts. Key biological substrates of CYB5R3 include hemoproteins such as hemoglobin, myoglobin, and soluble guanylate cyclase (sGC), as well as vitamin E and coenzyme Q. Our recent findings demonstrate that the CYB5R3 T117S variant, which results in approximately 50% loss of reductase activity, serves as a modifier of hydroxyurea-induced fetal hemoglobin (HbF) production—a widely used therapeutic approach for managing anemia in sickle cell disease (SCD). Using CYB5R3 knockout CD34+ hematopoietic stem cells (HSC)s, we found depleted baseline hemoglobin and impaired hydroxyurea- induced HbF production and erythroid maturation. In CYB5R3-deleted human erythroleukemic K562 cells, we similarly found depleted baseline hemoglobin, but in the presence of heme deficiency. The role of CYB5R3 in erythropoiesis remains unexplored despite. Based on our preliminary and published data, we hypothesize that CYB5R3 plays a previously unrecognized role in erythropoiesis by regulating heme biosynthesis. This hypothesis will be tested using two specific aims: Aim 1 will investigate the impact of CYB5R3 depletion on hemoglobin synthesis and erythropoiesis using human K562 cells. Aim 2 will employ a novel mouse model with hematopoietic compartment-specific deletion of CYB5R3 to assess its effects on erythropoiesis in vivo, both under baseline conditions and following hypoxic conditions. Successful completion of this study will provide novel insights into the significance of CYB5R3 in erythropoiesis, with potential applications in drug development, regenerative medicine, and the improved management of genetic anemias. Moreover, this research—plan combined with mentored training under Drs. Straub and Wood, interdisciplinary collaborations, and a structured career development plan within the outstanding training environment at the University of Pittsburgh School of Medicine—will equip the applicant with the expertise necessary to achieve a long-term career in biomedical research.

NIEHS - National Institute of Environmental Health Sciences

Project Summary Humans are continuously exposed to micro- and nanoplastics (MNPs) by accidental ingestion (e.g. cracked plastic utensils) or ingestion of food and water contaminated with MPs. Our recent findings (resulting from a multi-institutional effort) evidence that human tissues and organs including placenta, kidney, and brain in fact accumulate MNPs – the threat of MNPs to human health is a reality. However, the reactivity, bioaccumulation, and toxicity of MNPs after ingestion is largely unknown. In the 2022-2025 funding phase of our R15 AREA award, we recruited and mentored eight undergraduate students, retained one postbaccalaureate student for graduate school, supported two graduate students already enrolled in the Environmental Engineering and Chemistry graduate programs, and obtained a supplement to hire a postdoctoral fellow. Our key findings from the previous funding period indicate that 1) UV aging modifies the capacity of polyethylene (PE) and polyvinyl chloride (PVC) MPs to adsorb cadmium (Cd) and hydrophobic organic compounds; 2) PVC and PE MPs preserved their sorption capacity and decreased the available Cd in solution in an in vitro digestion system; 3) UV aging enhances the release of phthalates from PVC MPs into aqueous media; 4) MNPs attenuated the cytotoxicity and reduced the bioavailability of Cd, lindane, and (1,1-dichloro-2,2-bis(p-chlorophenyl)ethylene (DDE), and 5) the toxicity increased in assays supplied with NPs. In this renewal application, we build on the findings from the previous funding phase to advance the key knowledge barrier of our understanding of the transformation that MNPs undergo in the digestive system. Our overall goal is to recruit and develop strong research skills in undergraduate researchers in an interdisciplinary environmental engineering and toxicology project that studies MNP transformation, bioavailability, and toxicity to encourage them to pursue a graduate degree and career in STEM. We propose to use the rainbow trout gut intestinal cells (RTgutGC) in vitro digestion system as it resembles the human digestion system. We hypothesize that MNPs will continue transforming during the digestion process and they will release plastic additives (e.g., phthalates and flame retardants) from the bulk plastic and contaminants sorbed onto their surface into the GI fluids; additionally, the compounds released into the GI fluids will affect bioavailability and induce toxic effects. To test these hypotheses we propose to: Specific Aim 1: Evaluate the release (leaching capacity) of plastic additives from MNPs into GI fluids; Specific Aim 2: Asses the desorption of chemicals previously sorbed onto MNPs into GI fluids; and Specific Aim 3: Evaluate the bioavailability and toxicity of MNPs leachates and pollutants desorbed from MNPs to RTgutGC following GI processes. Our results will bridge the knowledge gap between chemical transformations of MPs in the GI system and will serve as a platform for integrating undergraduate students into multidisciplinary research and will allow them to acquire multiple skills spanning from environmental chemistry to cell biology and toxicology.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Project Summary/Abstract In vertebrates, self-renewing hematopoietic stem cells (HSCs) are produced from a developmental event called endothelial-to-hematopoietic transition (EHT). EHT consists of a cellular and transcriptional reprogramming that allows hemogenic endothelial cells (HECs) from a subset of embryonic arteries to leave the vessel and become blood stem cells. HSCs have the capacity to replace and restore the complete blood system upon transplant, making HSC transplant the only curative therapy available for blood diseases like leukemia and lymphoma. Given this therapeutic need, great effort has focused on the development of in vitro protocols that attempt to recapitulate the conditions of EHT for clinical expansion or de novo production of stem cells in the dish. To date none efficiently produce long-lived multipotent HSCs, suggesting that one or more developmental signals for this process remain to be defined. Mechanical forces from blood flow are an essential cue for HSC production via EHT, and the zebrafish Danio rerio provides an excellent animal model in which to study this contribution to hematopoiesis due to conserved molecular genetics of EHT in this species and the ability to observe live embryos with active circulation. Flow-driven EHT is mediated in part by the Yes-associated protein (YAP) transcription factor (TF), a transcriptional coregulator that has roles in organ growth, nutrient regulation and cell fate specification. YAP can be directed to the nucleus as a direct result of physical forces acting on the cell, but the molecular mechanisms by which this promotes EHT and HSC production are unclear. In preliminary data generated under K01 support, single-cell transcriptional analysis of wildtype, yap -/- and YAP-overexpressing HECs from zebrafish point to a role for YAP in regulating a battery of self-renewal hematopoietic TFs, cell cycling and metabolic processes. In examining these YAP gain- and loss-of-function (GOF/LOF) transcriptomes, gene module scores suggest an impaired glycolysis-to-oxidative phosphorylation rewiring in HECs. Genes related to lipid metabolism are also dysregulated by YAP perturbation and can be identified in ‘no flow’ datasets from mouse models. This R03 application will investigate the role of force-directed lipid metabolism in developmental EHT using zebrafish as a model. We hypothesize that hemodynamic forces alter lipid usage in HE to drive the metabolically intensive process of EHT. In the first aim, an unbiased approach of mass spectrometry-based lipidomic profiling will be used to quantify the abundance of lipid species in wildtype and YAP gain or loss of function (GOF/LOF) whole-embryo and sorted endothelial cell populations to determine those metabolites that are YAP-regulated (as a proxy for a major cellular transducer of mechanical force). In the second aim a candidate pathway, the secreted sphingosine-1-phosphate lipid mediator, will be studied for its role in EHT by live-imaging, chemical perturbation and state-of-the-art genome editing technologies to create tissue-specific LOF zebrafish lines. Findings from this proposal will uncover force-driven metabolic responses that might enhance production of HSCs via EHT and generate critical preliminary data to support R01 applications.

NIDDK - National Institute of Diabetes and Digestive and Kidney Diseases

Metabolic dysfunction-associated steatotic liver disease (MASLD) is the most common chronic liver disease in children. Progression from fatty liver (FL) to inflammation/steatohepatitis (MASH), significantly increases the risk of cirrhosis, hepatocellular carcinoma and liver failure. Lack of effective treatments for this significant public health burden for children stem from gaps in MASLD pathophysiology specific to children. A unique aspect in children is ongoing liver growth, which is more pronounced prepubertally, and during which hepatocytes depend on nuclear and mitochondrial DNA integrity. We and others have found that hepatic inflammation and fibrosis is more severe in prepubertal children. Similarly, when compared for equal duration of high fat diet, weanling/prepubertal mice develop MASH/fibrosis much more rapidly than post pubertal mice. Increased MASLD severity correlated with greater hepatocyte nuclear and mitochondrial DNA damage, greater cell cycle arrest and restriction of liver regeneration in prepubertal mice and children. This heightened susceptibility to DNA damage in the prepubertal period was due to dysregulation of the Ataxia Telangiectasia Mutated (ATM) pathway, which oversees the cellular DNA damage/repair response. Identification of molecular mechanisms that influence the ATM pathway and DNA damage/repair would therefore be highly significant for pediatric MASLD. We found that inhibition of the ATM kinase significantly worsened MASLD outcomes in prepubertal mice but had little effect in post pubertal mice. As pubertal status profoundly impacted the disease modifying effect of the ATM pathway on MASLD, we hypothesize that pediatric MASLD progression beyond steatosis to MASH and fibrosis during childhood liver growth is linked to Growth Hormone (GH) regulation of hepatic DNA integrity via the ATM pathway. In our preliminary studies, GH protected HuH-7 cells from lipotoxicity, implying liver cell specific effect. This correlated with reduced nuclear and mitochondrial DNA damage and increased ATM gene and protein expression. Conversely, GH receptor antagonism exacerbated lipotoxicity, increased nuclear and mitochondrial DNA damage and reduced ATM expression in HuH-7 cells and prepubertal mice, additionally with rapid progression to MASH and fibrosis in prepubertal, but not in adult mice. Liver tissue of children and prepubertal mice with MASLD showed decreased expression of the liver specific GH targets: STAT5B and IGF1, in MASH vs fatty liver. GH has systemic pleiotropic effects, but we will elucidate its liver specific roles in impacting the ATM pathway for hepatic DNA damage/repair in pediatric MASLD. We will use genetically modified mice, cells, and clinical samples collected during my K08 award to the examine the hepatic GH-Jak/STAT5B axis in Aim 1 and the hepatic GH-STAT5B-IGF1/IGF1R axis in Aim 2. The clinical and research expertise of our investigative team and outstanding institutional environment will ensure completion of this project. We expect to identify downstream targets of GH and ATM in the liver specifically important for pediatric MASLD, which can be further studied in future R01 applications, to bridge critical gaps that currently impact pediatric MASLD.

NHLBI - National Heart Lung and Blood Institute

Project Summary Lung respiratory diseases remain a leading cause of global morbidity and mortality, often linked to impaired alveolar repair and regeneration. Severe forms of interstitial lung diseases (ILDs) and idiopathic pulmonary fibrosis (IPF) emerge when alveolar cells fail to mount effective responses to repeat lung injury, resulting in excessive extracellular matrix (ECM) deposition, fibrotic tissue scarring, and loss of alveolar type 1 (AT1) cells. While AT1 cells are essential for gas exchange, their role in lung remodeling and fibrosis is not well understood. Alveolar type 2 (AT2) cells, the primary alveolar progenitor stem cells, can proliferate and differentiate to replace damaged or dead AT1 cells. In ILD, AT2 cells may become impaired, leading to the abnormal formation of a heterogeneous population of pro-fibrotic KRT8+ alveolar differentiation intermediate (ADI) cells. Our work identified the let-7 microRNA family as a key regulator that restrains excessive ADI cell formation and hinders proper AT1 differentiation. Additionally, we found that impaired let-7 activity in AT2 cells promotes the ectopic expression of pro-fibrotic oncogenes EZH2/BACH1. Our preliminary data also support the novel hypothesis that let-7 plays a pro-fibrotic signaling role in AT1 and AT2 cells through double negative feedback loop via EZH2 and BACH1. Our proposal has three specific aims: (1) to elucidate the role of the let-7/Ezh2/Bach1 axis in maintaining AT1 cell homeostasis during alveolar injury and fibrotic remodeling; (2) to investigate the contribution of the let-7/Ezh2/Bach1 feedback circuit in lung injury and fibrotic remodeling in AT2 and AT1 cells; and (3) to examine the conservation and function of LET-7/EZH2/BACH1 regulatory axis on human AT2-to-AT1 cell differentiation. This project will address a critical knowledge gap in lung repair and regeneration processes and aims to uncover new therapeutic targets for fibrotic lung diseases.

NHLBI - National Heart Lung and Blood Institute

Project Summary The ability to migrate is a hallmark of hematopoietic stem cells (HSCs), which travel to different hematopoietic sites during development and continue to be released from the adult bone marrow throughout life. The migratory property of HSCs has facilitated their collection from blood and their transplantation for curative anticancer therapies. Our recent studies revealed that a large subset of HSCs display macrophage-associated markers, including F4/80 and CD169, on their surface. Remarkably, macrophage marker–presenting HSCs are selectively retained in the bone marrow (BM), whereas HSCs without detectable macrophage marker are exclusively mobilized into the peripheral blood upon forced mobilization. We discovered that HSC use trogocytosis – a rapid and highly effective transfer mechanisms enabling acquisition of membrane-bound proteins from adjacent cells – to license their BM residence and retention. Mobilized HSCs are intravenously injected into recipients previously conditioned with irradiation and/or chemotherapy to reestablish hematopoietic system. Homing of HSCs to the BM niche is the first and critical step that precedes successful transplantation. Preliminary data supporting this application provided evidence that BM-resident macrophages interact with HSCs and guide their homing to the BM niche post-transplantation. These results raise important new questions as to how macrophages interact with HSCs to initiate the transfer, what is transferred, and whether macrophages guide HSC homing via transfer-dependent or -independent mechanisms. In the Specific Aim 1, we propose to define the mechanisms of HSC trogocytosis. Our preliminary results suggest that macrophage markers along with retention machinery, including CXCR4, are transferred from macrophages to HSCs and that HSC trogocytic activity strongly correlates with c-Kit levels on HSCs. We will assess the role of CXCR4 transfer and c-Kit signaling in trogocytosis-mediated transfer between macrophages and HSCs using pharmacologic and genetic means. We will elucidate the molecular underpinnings of a putative stem cell synapse with macrophages. In the Specific Aim 2, we will investigate how macrophages interact with HSCs to regulate their homing and/or engraftment post-transplantation. We will use co-culture system in vitro to manipulate the interactions between macrophages and HSCs and test models to explain their functional consequences with respect to stem cell transplantation. We will further investigate the macrophage heterogeneity and their location and interactions with HSCs during homing and engraftment using single cel technologies and immunofluorescent imaging. These studies will provide foundational insights into the macrophage niches and their contributions to HSC homing, retention, and mobilization, all key to improving stem cell transplant therapy for patients with blood disorders.

NIMH - National Institute of Mental Health

PROJECT SUMMARY/ABSTRACT Schizophrenia (SZ) remains one of the most debilitating psychiatric disorders and mechanistic understanding of the disease is minimal. Glutamatergic neurotransmission may play a role in SZ, but this has been difficult to study directly as the developing brain is inaccessible and complicated psychiatric syndromes cannot be easily replicated in non-human animals. Work using neurons derived from patient stem cells (iNeurons) has demonstrated a way to circumvent these limitations, and data from Dr. Anderson’s lab suggest the variable penetrance SZ in a well-defined neurodevelopmental disorder (22q11.2 deletion syndrome; 22qDS) corresponds to impaired oxidative phosphorylation (OXPHOS) in mitochondria. This proposal addresses the fundamental question of how alterations in mitochondrial function lead to complex psychiatric symptoms of SZ. AIM1 will test the hypothesis that mitochondrial dysfunction in presynaptic compartments results in diminished glutamate release in iNeurons from 22qDS individuals with SZ (22q+SZ) relative to those without SZ [22q(-)SZ]. Optical imaging of glutamate (AIM1.1) and synaptic vesicle cycling (AIM1.2) will be used to assess glutamate release from individual nerve terminals by independent methods under both high and low frequency neuronal stimulation. AIM2 will use computational modeling to access how alterations in presynaptic mitochondrial function contribute to circuit connectivity and entropy (disorder) in (22q+SZ) and [22q(-)SZ] iNeurons. This proposal fits within NIMH’s Strategic Plan to define brain mechanisms underlying behavior and mental illness and is expected to generate insights into mitochondrial influence on presynaptic function and glutamatergic neurotransmission in the emergence of SZ symptoms in 22qDS while also establishing a robust model of human cellular, synaptic, and circuit neuropathology in patient derived cells. Dr. Rossano will receive training in induced pluripotent stem cell models, medium-to-high throughput synaptic imaging in patient-derived cells, computational modeling of circuit connectivity, and integration of mitochondrial biology with synaptic physiology. Drs. Anderson and Goldberg possess complementary expertise and are uniquely suited for this proposal. A thoughtfully selected advisory committee will provide further scientific and career mentorship. Together with the world-class resources available at the Children’s Hospital of Philadelphia and the University of Pennsylvania the proposed scientific and training objectives will allow Dr. Rossano to establish an independent research program focused on synaptic neuroscience and mitochondrial biology as therapeutically targetable mechanisms underlying symptoms in SZ, autism, and additional neurodevelopmental disorders.

NIGMS - National Institute of General Medical Sciences

PROJECT SUMMARY/ABSTRACT All mammalian embryos undergo a highly conserved sequence of developmental events, but different species complete this sequence at significantly different speeds. Astonishingly, embryonic development is completed in 15 days in mice, 56 days in humans, and 116 days in elephants. How species-specific developmental rates are set represents a major unanswered question in developmental biology, for which the answer would yield multiple translational opportunities. Acceleration of developmental rate would allow faster production of mature cell types from human induced pluripotent stem cells for cell-based therapies and disease modeling; whereas its deceleration would provide new avenues to halt tumor growth and slow down the aging process. The overarching goal of our research program is to elucidate the regulatory mechanisms that give rise to species-specific developmental speed and develop strategies for its manipulation in translational settings. We previously established an in vitro system that recapitulates the 2-3 fold difference in developmental speed between mouse and human embryos. This system relies on the differentiation of mouse and human pluripotent stem cells to presomitic mesoderm, a cell type that harbors a molecular oscillator known as the segmentation clock. This clock provides a high-resolution, quantitative proxy for developmental speed. Using this system, we recently identified mitochondrial metabolism as an important regulator of developmental speed, with higher mitochondrial respiration giving rise to accelerated development in mouse cells compared to human cells. This regulation was not mediated by mitochondrial ATP production but rather by flux through the electron transport chain independently of oxidative phosphorylation. Our proposed research program consists of three main research directions that will dissect the role of mitochondrial metabolism in determining species-specific developmental rates. The first direction will identify the molecular mechanisms that underpin the connection between mitochondrial respiration and developmental speed. We will evaluate NAD(H) redox homeostasis and pyrimidine biosynthesis as potential rate-limiting factors working downstream of the electron transport chain. Our second research direction will pinpoint the factors that give rise to differential levels of mitochondrial respiration between species. We hypothesize that a combination of differences in mitochondrial ultrastructure and mitochondrial biogenesis are responsible for increased mitochondrial capacity in mouse cells compared to human cells. Finally, our third research direction will test whether mitochondrial metabolism can efficiently regulate developmental speed in more complex developmental settings comprised of multiple lineages. Using genetic tools to boost mitochondrial respiration, we will track the impact of elevated electron transport chain flux on the developmental progression of spatially coupled axial organoids in vitro and as well as mouse embryos in vivo. Our long-term goal is to leverage the insights gleaned from this project to enhance the utility of human pluripotent stem cells for therapeutic applications by devising strategies for the acceleration of developmental rate.

NHLBI - National Heart Lung and Blood Institute

PROJECT SUMMARY Cardiac development is a complex process that occurs early during embryonic development and requires precise regulation. Early cardiac development, especially of the endocardial and myocardial lineages, is implicated in the development of congenital heart diseases (CHDs). Unfortunately, despite CHDs being the most frequent birth defect affecting approximately one in 100 live births, very few mechanisms are understood about many different forms of CHDs. Proper cardiac development requires precise post-transcriptional regulation, such as alternative splicing, translation initiation, and mRNA modification. These functions are mediated by RNA-binding proteins (RBPs), and unsurprisingly, mutations to many RBPs are implicated in cardiac development and formation of CHDs. One such RBP is DDX3X, which is known from clinical evidence to have implications in CHDs: patients with mutations in DDX3X exhibit DDX3X syndrome, marked by neurodevelopmental disorders and increased risk of CHDs. However, DDX3X has not been well-studied to date in the context of heart development. My proposed research will study the dosage-sensitive effects and mechanisms of DDX3X in the endocardial lineage during cardiac development. In Aim 1 of this proposal, I will determine the effects of reduced DDX3X levels on endocardium formation and function. Using transgenic mice, I will conditionally delete DDX3X in male and female mouse endocardium. Expression level of DDX3X in the endocardium will be quantified using immunofluorescent imaging and flow cytometry. I will then characterize resulting phenotypes by examining phenotypic onset, structural and functional consequences, and cellular and molecular consequences in the heart using a combination of brightfield and immunofluorescent imaging, weight measurements, and echocardiograms. In Aim 2 of this proposal, I will identify the molecular mechanisms of DDX3X in the endocardium. Using immunofluorescent imaging, I will characterize the subcellular localization of DDX3X in the endocardium. I will then identify the direct regulatory network of DDX3X in the endocardial lineage using RNA-seq and Ribo-seq. Targets will be validated against pre-existing eCLIP data as well as in an in vitro system of human pluripotent stem cell-derived endocardial cells. The results of this research will contribute to our understanding about RNA biology, gene dosage, development and disease.