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Open Access Publications from the University of California

Scholarly Works (19 results)

  • Thesis
  • Peer Reviewed
UC San Diego Electronic Theses and Dissertations (2017)

Every cell in the body arises from a single fertilized cell that, through many cell divisions and transitions from one type to another, travels a landscape of possible fate decisions during development. Though adult cell types have been well cataloged molecularly and functionally, the states a cell can pass through and the genes that regulate these choices remain unclear. Recent advances in quantitative cellular measurements for monitoring global gene regulation simultaneously in hundreds to thousands of single cells from an experiment is allowing for the discovery of cell types and cell states as well as the trajectories linking them. This dissertation is comprised of investigation of, and applies towards, the developmental states and genetic regulation of mouse mesoderm.

The goal of the first study was to catalog cell types and states during mouse mesodermal developmental by gene expression at the single cell level (Chapter 2). We determined developmental trajectories for six mesodermal cell types, providing putative novel cellular intermediates with previously undocumented gene expression profiles. The goal of the second study was to functionally investigate a cellular intermediate identified in the first study that had not been previously described.

These studies describe new findings concerning the cellular and molecular hierarchy of mesodermal development that will enable further study of genetic regulation of developing cell types. Mapping developmental landscapes may promote the discovery of genes and pathways that govern cell fate decisions and transitions essential for advancements in understanding the etiology of disease and the development of medical therapies.

    Cover page: Defining Mesodermal Cell Types, Branching States, and Origins by Single Cell Genomics
    • Multimedia
    • Peer Reviewed
    UC San Diego Electronic Theses and Dissertations (2024)

    Advancements in cardiac regenerative therapies require understanding the early developmental decisions involved in cardiomyocyte (CM) specification and differentiation. While traditional CM differentiation approaches employ monolayer/2D protocols, they are unable to capture the complexity observed in vivo. This limitation has prompted the development of organoid/3D protocols, which is capable of recapitulating functional, structural, and metabolic features, as well as CM subtypes seen in vivo. Here, we aimed to characterize and genomically interrogate cardiac developmental models to elucidate and validate the mechanisms governing early CM and ventricular CM (vCM) development in order to direct key cardiac cell sources for cardiac therapies. Firstly, drawing upon scientific literature, we illustrated how developing cellularly complex cardiac models with the proper stimuli can be used for heart modeling and disease treatment. Secondly, we integrated multi-modal single-cell genomics studies with functional genetic screening to systematically discover and validate the coordinated gene regulatory networks and transcriptional regulators controlling the development of distinct human CM populations. Lastly, we extended these multi-modal genomic techniques to unravel the mechanisms underlying the specification and differentiation of vCMs for cell therapy-based heart failure treatment. Overall, these studies significantly enhance our understanding of early cardiac development, driving advancements in the field and paving the way for therapeutic intervention.

    • 8 supplemental ZIPs
    • Article
    • Peer Reviewed
    UC San Diego Previously Published Works (2021)
    Organ maturation entails the reshaping of simple tissues into more complex structures critical for function. In a recent issue of Nature, Priya et al. show how tension heterogeneity between developing cardiomyocytes can coordinate the cell behaviors that remodel the architecture of the cardiac chamber wall.
      Cover page: Cardiac Morphogenesis: Crowding and Tension Resolved through Social Distancing
      • Article
      • Peer Reviewed
      UC Irvine Previously Published Works (2014)
      Understanding the regenerative capacity of small vertebrate models has provided new insights into the plasticity of injured myocardium. Here, we demonstrate the application of flexible microelectrode arrays (MEAs) in elucidating electrophysiological phenotypes of zebrafish and neonatal mouse models of heart regeneration. The 4-electrode MEA membranes were designed to detect electrical signals in the aquatic environment. They were micro-fabricated to adhere to the non-planar body surface of zebrafish and neonatal mice. The acquired signals were processed to display an electrocardiogram (ECG) with high signal-to-noise-ratios, and were validated via the use of conventional micro-needle electrodes. The 4-channel MEA provided signal stability and spatial resolution, revealing the site-specific electrical injury currents such as ST-depression in response to ventricular cryo-injury. Thus, our polymer-based and wearable MEA membranes provided electrophysiological insights into long-term conduction phenotypes for small vertebral models of heart injury and regeneration with a translational implication for monitoring cardiac patients. © 2014 the Partner Organisations.
        Cover page: Wearable multi-channel microelectrode membranes for elucidating electrophysiological phenotypes of injured myocardium
        • Article
        • Peer Reviewed
        UC San Diego Previously Published Works (2015)
        Angiogenesis relies on specialized endothelial tip cells to extend toward guidance cues in order to direct growing blood vessels. Although many of the signaling pathways that control this directional endothelial sprouting are well known, the specific cellular mechanisms that mediate this process remain to be fully elucidated. Here, we show that Polo-like kinase 2 (PLK2) regulates Rap1 activity to guide endothelial tip cell lamellipodia formation and subsequent angiogenic sprouting. Using a combination of high-resolution in vivo imaging of zebrafish vascular development and a human umbilical vein endothelial cell (HUVEC) in vitro cell culture system, we observed that loss of PLK2 function resulted in a reduction in endothelial cell sprouting and migration, whereas overexpression of PLK2 promoted angiogenesis. Furthermore, we discovered that PLK2 may control angiogenic sprouting by binding to PDZ-GEF to regulate RAP1 activity during endothelial cell lamellipodia formation and extracellular matrix attachment. Consistent with these findings, constitutively active RAP1 could rescue the endothelial cell sprouting defects observed in zebrafish and HUVEC PLK2 knockdowns. Overall, these findings reveal a conserved PLK2-RAP1 pathway that is crucial to regulate endothelial tip cell behavior in order to ensure proper vascular development and patterning in vertebrates.
          Cover page: Polo-like kinase 2 regulates angiogenic sprouting and blood vessel development.
          • Article
          • Peer Reviewed
          UC San Francisco Previously Published Works (2022)

          Motivation

          Unsupervised clustering of single-cell transcriptomics is a powerful method for identifying cell populations. Static visualization techniques for single-cell clustering only display results for a single resolution parameter. Analysts will often evaluate more than one resolution parameter but then only report one.

          Results

          We developed Cell Layers, an interactive Sankey tool for the quantitative investigation of gene expression, co-expression, biological processes and cluster integrity across clustering resolutions. Cell Layers enhances the interpretability of single-cell clustering by linking molecular data and cluster evaluation metrics, providing novel insight into cell populations.

          Availability and implementation

          https://github.com/apblair/CellLayers.
            Cover page: Cell Layers: uncovering clustering structure in unsupervised single-cell transcriptomic analysis
            • Article
            • Peer Reviewed
            UC Irvine Previously Published Works (2013)
            Peristaltic contraction of the embryonic heart tube produces time- and spatial-varying wall shear stress (WSS) and pressure gradients (∇P) across the atrioventricular (AV) canal. Zebrafish (Danio rerio) are a genetically tractable system to investigate cardiac morphogenesis. The use of Tg(fli1a:EGFP) (y1) transgenic embryos allowed for delineation and two-dimensional reconstruction of the endocardium. This time-varying wall motion was then prescribed in a two-dimensional moving domain computational fluid dynamics (CFD) model, providing new insights into spatial and temporal variations in WSS and ∇P during cardiac development. The CFD simulations were validated with particle image velocimetry (PIV) across the atrioventricular (AV) canal, revealing an increase in both velocities and heart rates, but a decrease in the duration of atrial systole from early to later stages. At 20-30 hours post fertilization (hpf), simulation results revealed bidirectional WSS across the AV canal in the heart tube in response to peristaltic motion of the wall. At 40-50 hpf, the tube structure undergoes cardiac looping, accompanied by a nearly 3-fold increase in WSS magnitude. At 110-120 hpf, distinct AV valve, atrium, ventricle, and bulbus arteriosus form, accompanied by incremental increases in both WSS magnitude and ∇P, but a decrease in bi-directional flow. Laminar flow develops across the AV canal at 20-30 hpf, and persists at 110-120 hpf. Reynolds numbers at the AV canal increase from 0.07±0.03 at 20-30 hpf to 0.23±0.07 at 110-120 hpf (p< 0.05, n=6), whereas Womersley numbers remain relatively unchanged from 0.11 to 0.13. Our moving domain simulations highlights hemodynamic changes in relation to cardiac morphogenesis; thereby, providing a 2-D quantitative approach to complement imaging analysis.
              Cover page: Moving Domain Computational Fluid Dynamics to Interface with an Embryonic Model of Cardiac Morphogenesis
              • Article
              • Peer Reviewed
              UC San Diego Previously Published Works (2019)
              Pacemaker cardiomyocytes that create the sinoatrial node are essential for the initiation and maintenance of proper heart rhythm. However, illuminating developmental cues that direct their differentiation has remained particularly challenging due to the unclear cellular origins of these specialized cardiomyocytes. By discovering the origins of pacemaker cardiomyocytes, we reveal an evolutionarily conserved Wnt signaling mechanism that coordinates gene regulatory changes directing mesoderm cell fate decisions, which lead to the differentiation of pacemaker cardiomyocytes. We show that in zebrafish, pacemaker cardiomyocytes derive from a subset of Nkx2.5+ mesoderm that responds to canonical Wnt5b signaling to initiate the cardiac pacemaker program, including activation of pacemaker cell differentiation transcription factors Isl1 and Tbx18 and silencing of Nkx2.5. Moreover, applying these developmental findings to human pluripotent stem cells (hPSCs) notably results in the creation of hPSC-pacemaker cardiomyocytes, which successfully pace three-dimensional bioprinted hPSC-cardiomyocytes, thus providing potential strategies for biological cardiac pacemaker therapy.
                Cover page: Canonical Wnt5b Signaling Directs Outlying Nkx2.5+ Mesoderm into Pacemaker Cardiomyocytes
                • Article
                • Peer Reviewed
                UC San Francisco Previously Published Works (2008)
                Vertebrate hearts depend on highly specialized cardiomyocytes that form the cardiac conduction system (CCS) to coordinate chamber contraction and drive blood efficiently and unidirectionally throughout the organism. Defects in this specialized wiring system can lead to syncope and sudden cardiac death. Thus, a greater understanding of cardiac conduction development may help to prevent these devastating clinical outcomes. Utilizing a cardiac-specific fluorescent calcium indicator zebrafish transgenic line, Tg(cmlc2:gCaMP)(s878), that allows for in vivo optical mapping analysis in intact animals, we identified and analyzed four distinct stages of cardiac conduction development that correspond to cellular and anatomical changes of the developing heart. Additionally, we observed that epigenetic factors, such as hemodynamic flow and contraction, regulate the fast conduction network of this specialized electrical system. To identify novel regulators of the CCS, we designed and performed a new, physiology-based, forward genetic screen and identified for the first time, to our knowledge, 17 conduction-specific mutations. Positional cloning of hobgoblin(s634) revealed that tcf2, a homeobox transcription factor gene involved in mature onset diabetes of the young and familial glomerulocystic kidney disease, also regulates conduction between the atrium and the ventricle. The combination of the Tg(cmlc2:gCaMP)(s878) line/in vivo optical mapping technique and characterization of cardiac conduction mutants provides a novel multidisciplinary approach to further understand the molecular determinants of the vertebrate CCS.
                  Cover page: Genetic and Physiologic Dissection of the Vertebrate Cardiac Conduction System
                  • Article
                  • Peer Reviewed
                  UCLA Previously Published Works (2016)
                  Hemodynamic shear forces are intimately linked with cardiac development, during which trabeculae form a network of branching outgrowths from the myocardium. Mutations that alter Notch signaling also result in trabeculation defects. Here, we assessed whether shear stress modulates trabeculation to influence contractile function. Specifically, we acquired 4D (3D + time) images with light sheets by selective plane illumination microscopy (SPIM) for rapid scanning and deep axial penetration during zebrafish morphogenesis. Reduction of blood viscosity via gata1a morpholino oligonucleotides (MO) reduced shear stress, resulting in downregulation of Notch signaling and attenuation of trabeculation. Arrest of cardiomyocyte contraction either by troponin T type 2a (tnnt2a) MO or in weak atriumm58 (wea) mutants resulted in reduced shear stress and downregulation of Notch signaling and trabeculation. Integrating 4D SPIM imaging with synchronization algorithm demonstrated that coinjection of neuregulin1 mRNA with gata1 MO rescued trabeculation to restore contractile function in association with upregulation of Notch-related genes. Crossbreeding of Tg(flk:mCherry) fish, which allows visualization of the vascular system with the Tg(tp1:gfp) Notch reporter line, revealed that shear stress-mediated Notch activation localizes to the endocardium. Deleting endocardium via the clochesk4 mutants downregulated Notch signaling, resulting in nontrabeculated ventricle. Subjecting endothelial cells to pulsatile flow in the presence of the ADAM10 inhibitor corroborated shear stress-activated Notch signaling to modulate trabeculation.
                    Creative Commons 'BY' version 4.0 license
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