IST Austria Thesis

Blood – this is what animals use to heal wounds fast and efficient. Plants do not have blood circulation and their cells cannot move. However, plants have evolved remarkable capacities to regenerate tissues and organs preventing further damage. In my PhD research, I studied the wound healing in the Arabidopsis root. I used a UV laser to ablate single cells in the root tip and observed the consequent wound healing. Interestingly, the inner adjacent cells induced a division plane switch and subsequently adopted the cell type of the killed cell to replace it. We termed this form of wound healing “restorative divisions”. This initial observation triggered the questions of my PhD studies: How and why do cells orient their division planes, how do they feel the wound and why does this happen only in inner adjacent cells. For answering these questions, I used a quite simple experimental setup: 5 day - old seedlings were stained with propidium iodide to visualize cell walls and dead cells; ablation was carried out using a special laser cutter and a confocal microscope. Adaptation of the novel vertical microscope system made it possible to observe wounds in real time. This revealed that restorative divisions occur at increased frequency compared to normal divisions. Additionally, the major plant hormone auxin accumulates in wound adjacent cells and drives the expression of the wound-stress responsive transcription factor ERF115. Using this as a marker gene for wound responses, we found that an important part of wound signalling is the sensing of the collapse of the ablated cell. The collapse causes a radical pressure drop, which results in strong tissue deformations. These deformations manifest in an invasion of the now free spot specifically by the inner adjacent cells within seconds, probably because of higher pressure of the inner tissues. Long-term imaging revealed that those deformed cells continuously expand towards the wound hole and that this is crucial for the restorative division. These wound-expanding cells exhibit an abnormal, biphasic polarity of microtubule arrays before the division. Experiments inhibiting cell expansion suggest that it is the biphasic stretching that induces those MT arrays. Adapting the micromanipulator aspiration system from animal scientists at our institute confirmed the hypothesis that stretching influences microtubule stability. In conclusion, this shows that microtubules react to tissue deformation and this facilitates the observed division plane switch. This puts mechanical cues and tensions at the most prominent position for explaining the growth and wound healing properties of plants. Hence, it shines light onto the importance of understanding mechanical signal transduction

Computing motion in the primate's visual system

Computing motion on the basis of the time-varying image intensity is a difficult problem for both artificial and biological vision systems. We will show how one well-known gradient-based computer algorithm for estimating visual motion can be implemented within the primate's visual system. This relaxation algorithm computes the optical flow field by minimizing a variational functional of a form commonly encountered in early vision, and is performed in two steps. In the first stage, local motion is computed, while in the second stage spatial integration occurs. Neurons in the second stage represent the optical flow field via a population-coding scheme, such that the vector sum of all neurons at each location codes for the direction and magnitude of the velocity at that location. The resulting network maps onto the magnocellular pathway of the primate visual system, in particular onto cells in the primary visual cortex (V1) as well as onto cells in the middle temporal area (MT). Our algorithm mimics a number of psychophysical phenomena and illusions (perception of coherent plaids, motion capture, motion coherence) as well as electrophysiological recordings. Thus, a single unifying principle ‘the final optical flow should be as smooth as possible’ (except at isolated motion discontinuities) explains a large number of phenomena and links single-cell behavior with perception and computational theory

The Monitoring and Assessment Plan (MAP) Greater Everglades Wetlands Module- Landscape Pattern- Ridge, Slough, and Tree Island Mosaics: Year 1 Annual Report

In the current managed Everglades system, the pre-drainage, patterned mosaic of sawgrass ridges, sloughs and tree islands has been substantially altered or reduced largely as a result of human alterations to historic ecological and hydrological processes that sustained landscape patterns. The pre-compartmentalization ridge and slough landscape was a mosaic of sloughs, elongated sawgrass ridges (50-200m wide), and tree islands. The ridges and sloughs and tree islands were elongated in the direction of the water flow, with roughly equal area of ridge and slough. Over the past decades, the ridge-slough topographic relief and spatial patterning have degraded in many areas of the Everglades. Nutrient enriched areas have become dominated by Typha with little topographic relief; areas of reduced flow have lost the elongated ridge-slough topography; and ponded areas with excessively long hydroperiods have experienced a decline in ridge prevalence and shape, and in the number of tree islands (Sklar et al. 2004, Ogden 2005)

echinus, required for interommatidial cell sorting and cell death in the Drosophila pupal retina, encodes a protein with homology to ubiquitin-specific proteases

Background: Programmed cell death is used to remove excess cells between ommatidia in the Drosophila pupal retina. This death is required to establish the crystalline, hexagonal packing of ommatidia that characterizes the adult fly eye. In previously described echinus mutants, interommatidial cell sorting, which precedes cell death, occurred relatively normally. Interommatidial cell death was partially suppressed, resulting in adult eyes that contained excess pigment cells, and in which ommatidia were mildly disordered. These results have suggested that echinus functions in the pupal retina primarily to promote interommatidial cell death. Results: We generated a number of new echinus alleles, some of which are likely null mutants. Analysis of these alleles provides evidence that echinus has roles in cell sorting as well as cell death. echinus encodes a protein with homology to ubiquitin-specific proteases, which cleave ubiquitin-conjugated proteins at the ubiquitin C-terminus. The echinus locus encodes multiple splice forms, including two proteins that lack residues thought to be critical for deubiquitination activity. Surprisingly, ubiquitous expression in the eye of versions of Echinus that lack residues critical for ubiquitin specific protease activity, as well as a version predicted to be functional, rescue the echinus loss-of-function phenotype. Finally, genetic interactions were not detected between echinus loss and gain-of-function and a number of known apoptotic regulators. These include Notch, EGFR, the caspases Dronc, Drice, Dcp-1, Dream, the caspase activators, Rpr, Hid, and Grim, the caspase inhibitor DIAP1, and Lozenge or Klumpfuss. Conclusions: The echinus locus encodes multiple splice forms of a protein with homology to ubiquitin-specific proteases, but protease activity is unlikely to be required for echinus function, at least when echinus is overexpressed. Characterization of likely echinus null alleles and genetic interactions suggests that echinus acts at a novel point(s) to regulate interommatidial cell sorting and/or cell death in the fly eye

Doctor of Philosophy

dissertationToday, we are implanting electrodes into many different parts of the peripheral and central nervous systems for the purpose of restoring function to people with nerve injury or disease. As technology and manufacturing continue to become more advanced, ne

Engineered skeletal muscle from human pluripotent stem cells to model muscle disease and regeneration

Skeletal muscle disease modeling offers the unique opportunity to investigate devastating muscle diseases like Duchenne Muscle Dystrophy in vitro but requires advanced three-dimensional (3D) model systems reflecting the characteristics of human muscle in vivo. The aim of this study was to generate engineered models of skeletal muscle from human pluripotent stem cells (hPSCs) with physiological properties by recapitulating specific stages of muscle development. To allow for robust skeletal muscle tissue engineering first a directed differentiation protocol was established in 2D culture under serum-free conditions. Comparison of hPSC differentiation to embryonic muscle development confirmed significant overlap with characteristic signatures of paraxial mesoderm, dermatomyotome, and somite stage. The protocol robustly directed multiple hPSC lines into skeletal muscle cells in 2D culture as well as in a collagen-1/Matrigel® hydrogel in 3D generating bioengineered skeletal muscle (BSM) organoid. By identifying additional maturation cues (creatine, triiod-L-thyronine) hPSC-derived skeletal myogenic cells embedded into a collagen-1/Matrigel® hydrogel generated engineered skeletal muscle (ESM) with compact muscle syncytia, anisotropically arranged sarcomeres, properly localized dystrophin-associated complex proteins, and contractile function of developing fast muscle. Importantly, Pax7-positive cells were found adjacent to muscle fibers underneath a laminin-positive basal lamina in a satellite-like cell position. Cardiotoxin injury of ESM induced a regenerative response with recovery of tetanic force after complete loss of function. Finally, modelling of Duchenne Muscular Dystrophy (DMD) in ESM demonstrates “proof of concept” for efficacy of CRISPR/Cas9 based exon skipping. Collectively, human BSM and ESM models provide unprecedented opportunities to study muscle development, maturation, and regeneration in vitro and may serve as preclinical test bed for novel therapies of skeletal muscle disease.2021-07-0

Inducible photoreceptor degeneration model in goldfish

2011 Summer.Includes bibliographical references.Photoreceptor degenerative diseases are among the leading causes of vision loss and there is presently no known cure. The future success of biological and prosthetic vision rescue approaches following photoreceptor loss remains questionable, due to the morphological and functional changes occurring in the remaining retinal circuitry. In the current study we sought to establish a chemically-induced photoreceptor degenerative model in goldfish, based on the ability of teleost to regenerate their retina following damage. N-methyl-N-nitrosourea (MNU) was chosen to chemically induce the photoreceptor degeneration, because it has been found to be potent, and selective in mammalian studies. We hypothesized that MNU would induce selective and complete photoreceptor loss in the goldfish retina as well as the consequent morphological changes observed in mammalian retinas. Under anesthesia, fish received a direct, intraocular injection of MNU into the posterior chamber of one eye whereas the contralateral eye served as sham-injected control. The effects of MNU were determined by standard immunohistochemical methods using known, well-established molecular markers of retinal cells. The MNU induced unilateral, selective, and dose-dependent photoreceptor degeneration: up to ~60% of photoreceptors lost the injected eye of the goldfish within 7 days, followed by nearly complete regeneration by ~50 days post-injection. Repeated MNU treatments did not increase the magnitude of degeneration, but delayed the regeneration. Unlike in mammals, MNU did not destroy all of the photoreceptors in fish. The incomplete photoreceptor degeneration together with the quick regeneration may be responsible for preventing the development of chronic morphological and functional consequences. However, the regeneration observed after MNU treatment is promising. Inducing total photoreceptor degeneration in fish retina, possibly by combining MNU with other factors shown to destroy photoreceptors (i.e. strong light) could provide an all-encompassing natural model for studying the potential of stem cell-based vision rescue approaches after photoreceptor loss