Visual perception of photographs of rotated 3D objects in goldfish (Carassius auratus)

© The Author(s), 2022. This article is distributed under the terms of the Creative Commons Attribution License. The definitive version was published in Wegman, J. J., Morrison, E., Wilcox, K. T., & DeLong, C. M. Visual perception of photographs of rotated 3D objects in goldfish (Carassius auratus). Animals, 12(14), (2022): 1797, https://doi.org/10.3390/ani12141797.This study examined goldfishes’ ability to recognize photographs of rotated 3D objects. Six goldfish were presented with color photographs of a plastic model turtle and frog at 0° in a two-alternative forced-choice task. Fish were tested with stimuli at 0°, 90°, 180°, and 270° rotated in the picture plane and two depth planes. All six fish performed significantly above chance at all orientations in the three rotation planes tested. There was no significant difference in performance as a function of aspect angle, which supported viewpoint independence. However, fish were significantly faster at 180° than at +/−90°, so there is also evidence for viewpoint-dependent representations. These fish subjects performed worse overall in the current study with 2D color photographs (M = 88.0%) than they did in our previous study with 3D versions of the same turtle and frog stimuli (M = 92.6%), although they performed significantly better than goldfish in our two past studies presented with black and white 2D stimuli (M = 67.6% and 69.0%). The fish may have relied on color as a salient cue. This study was a first attempt at examining picture-object recognition in fish. More work is needed to determine the conditions under which fish succeed at object constancy tasks, as well as whether they are capable of perceiving photographs as representations of real-world objectsThis work was supported with a RIT College of Liberal Arts Faculty Development Grant to CMD and the RIT Paul A. and Francena L. Miller Research Fellowship awarded to CMD from the Rochester Institute of Technology

Machine learning-based automated segmentation with a feedback loop for 3D synchrotron micro-CT

Die Entwicklung von Synchrotronlichtquellen der dritten Generation hat die Grundlage für die Untersuchung der 3D-Struktur opaker Proben mit einer Auflösung im Mikrometerbereich und höher geschaffen. Dies führte zur Entwicklung der Röntgen-Synchrotron-Mikro-Computertomographie, welche die Schaffung von Bildgebungseinrichtungen zur Untersuchung von Proben verschiedenster Art förderte, z.B. von Modellorganismen, um die Physiologie komplexer lebender Systeme besser zu verstehen. Die Entwicklung moderner Steuerungssysteme und Robotik ermöglichte die vollständige Automatisierung der Röntgenbildgebungsexperimente und die Kalibrierung der Parameter des Versuchsaufbaus während des Betriebs. Die Weiterentwicklung der digitalen Detektorsysteme führte zu Verbesserungen der Auflösung, des Dynamikbereichs, der Empfindlichkeit und anderer wesentlicher Eigenschaften. Diese Verbesserungen führten zu einer beträchtlichen Steigerung des Durchsatzes des Bildgebungsprozesses, aber auf der anderen Seite begannen die Experimente eine wesentlich größere Datenmenge von bis zu Dutzenden von Terabyte zu generieren, welche anschließend manuell verarbeitet wurden. Somit ebneten diese technischen Fortschritte den Weg für die Durchführung effizienterer Hochdurchsatzexperimente zur Untersuchung einer großen Anzahl von Proben, welche Datensätze von besserer Qualität produzierten. In der wissenschaftlichen Gemeinschaft besteht daher ein hoher Bedarf an einem effizienten, automatisierten Workflow für die Röntgendatenanalyse, welcher eine solche Datenlast bewältigen und wertvolle Erkenntnisse für die Fachexperten liefern kann. Die bestehenden Lösungen für einen solchen Workflow sind nicht direkt auf Hochdurchsatzexperimente anwendbar, da sie für Ad-hoc-Szenarien im Bereich der medizinischen Bildgebung entwickelt wurden. Daher sind sie nicht für Hochdurchsatzdatenströme optimiert und auch nicht in der Lage, die hierarchische Beschaffenheit von Proben zu nutzen. Die wichtigsten Beiträge der vorliegenden Arbeit sind ein neuer automatisierter Analyse-Workflow, der für die effiziente Verarbeitung heterogener Röntgendatensätze hierarchischer Natur geeignet ist. Der entwickelte Workflow basiert auf verbesserten Methoden zur Datenvorverarbeitung, Registrierung, Lokalisierung und Segmentierung. Jede Phase eines Arbeitsablaufs, die eine Trainingsphase beinhaltet, kann automatisch feinabgestimmt werden, um die besten Hyperparameter für den spezifischen Datensatz zu finden. Für die Analyse von Faserstrukturen in Proben wurde eine neue, hochgradig parallelisierbare 3D-Orientierungsanalysemethode entwickelt, die auf einem neuartigen Konzept der emittierenden Strahlen basiert und eine präzisere morphologische Analyse ermöglicht. Alle entwickelten Methoden wurden gründlich an synthetischen Datensätzen validiert, um ihre Anwendbarkeit unter verschiedenen Abbildungsbedingungen quantitativ zu bewerten. Es wurde gezeigt, dass der Workflow in der Lage ist, eine Reihe von Datensätzen ähnlicher Art zu verarbeiten. Darüber hinaus werden die effizienten CPU/GPU-Implementierungen des entwickelten Workflows und der Methoden vorgestellt und der Gemeinschaft als Module für die Sprache Python zur Verfügung gestellt. Der entwickelte automatisierte Analyse-Workflow wurde erfolgreich für Mikro-CT-Datensätze angewandt, die in Hochdurchsatzröntgenexperimenten im Bereich der Entwicklungsbiologie und Materialwissenschaft gewonnen wurden. Insbesondere wurde dieser Arbeitsablauf für die Analyse der Medaka-Fisch-Datensätze angewandt, was eine automatisierte Segmentierung und anschließende morphologische Analyse von Gehirn, Leber, Kopfnephronen und Herz ermöglichte. Darüber hinaus wurde die entwickelte Methode der 3D-Orientierungsanalyse bei der morphologischen Analyse von Polymergerüst-Datensätzen eingesetzt, um einen Herstellungsprozess in Richtung wünschenswerter Eigenschaften zu lenken

Modulation of the Wnt pathway at single-cell level uncovers diverging functional domains in the ciliary marginal zone of medaka

The continuous life-long growth of the fish retina is fuelled by neural stem cells located within the ciliary marginal zone (CMZ). These stem cells are characterised by their multipotency and ability to self-renew. Through asymmetric divisions, the neural stem cells give rise to progenitor cells with restricted proliferation potential that ultimately commit to terminally differentiated neurons of the mature retina. In this process, pharmacological manipulation suggests that Wnt signalling acts on both cell proliferation and differentiation, but a refined analysis is missing. Therefore, I present here a detailed analysis of Wnt signalling localisation and function within different cell types and their lineages in the post-embryonic CMZ of medaka. In this thesis, I show that Wnt ligands are expressed by cells of the retinal pigment epithelium, which is located directly adjacent to the CMZ. Wnt/β-catenin signalling activity is restricted to stem cells, whereas β-catenin independent Wnt/LRP6 signalling extends to dividing progenitor cells. To address the role of Wnt signalling in proliferation and differentiation of specific stem and progenitor cells, I created transgenic lines that allow inducible clonal labelling combined with upregulation of Wnt signalling in individual cells. My lineage tracing experiments suggest that Wnt upregulation has diverging effects on stem and progenitor cells. First, stem cells lose stemness characteristics presumably through induction of apoptosis or symmetric division. Second, progenitor cells reacquire the capacity to self-renew, but their pre-existing fate restrictions are irreversible. Finally, committed progenitors shift their fate and/or change their division mode and proliferation characteristics upon Wnt upregulation. Taken together, my results indicate that Wnt signalling functionally divides the CMZ into stem cells, non-committed and committed progenitors, which has far reaching implications for Wnt functions in other stem cell niches

Removing striping artifacts in light-sheet fluorescence microscopy: a review

In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented along the light sheet propagation direction. Several methods have been developed to address this issue, ranging from fully optical solutions to entirely digital post-processing approaches. In this work, we present them, outlining their advantages, performance and limitations

Mechanisms of growth control in the postembryonic medaka retina

Postembryonic stem cell niches are present throughout the vertebrate clade to facilitate development, homeostasis, regeneration and growth. While teleosts and amphibians display sustained stem cell activity in most organs after embryogenesis, higher vertebrates retain stem cell activity only in specific tissues. Despite these differences, similar challenges are imposed on all vertebrate organisms: new tissue has to be generated to expand or replace existent one while simultaneously ensuring integrity and functionality of the organ. Tight control of stem and progenitor cell proliferation is necessary to avoid aberrant growth such as in cancer. In the retina of the teleost medaka (Oryzias latipes), retinal stem (RSC) and progenitor (RPC) cells are located in the ciliary marginal zone (CMZ) and mediate postembryonic growth and neurogenesis. Since function and shape of the eye are intimately linked, the activity of RSCs and RPCs is tightly coordinated to establish proper cell type composition and number. In this thesis I addressed intrinsic and extrinsic regulation mechanisms of the RSC niche. I hypothesised that retinal growth underlies intrinsically active growth factor signaling, and that immune cells safeguard the RSC niche in homeostasis and injury. To analyse intrinsic regulation of RSC proliferation, I assessed the function of insulin-like growth factor (Igf) signaling in the CMZ using gain- and loss- of-function approaches. I found that Igf1 receptor over-activation increased cell cycle speed, RPC number and consequently retinal size, while simultaneously preserving the stereotypical retinal architecture. Strikingly, RSCs were not susceptible to mitogenic stimuli, indicating that RPC amplification is the determinant of retinal size and composition. To understand the extrinsic regulation of the RSC niche, I examined the interplay of immune cells and RSCs. I found that Ccl25b-positive RSCs are phagocytosed by Ccr9a-positive immune cells located in the CMZ. Ccl25b mutation abrogates reactivation of immune cells upon RSC injury, implicating Ccl25b–Ccr9a signaling in the immune–stem cell interaction during homeostatic surveillance and injury response. In summary, my results propose that accurate postembryonic growth and tissue integrity depend on both cell intrinsic and extrinsic mechanisms of growth control in the RSC niche of medaka

The Teleost Retina as a Model for Developmental and Regeneration Biology

Retinal development in teleosts can broadly be divided into three epochs. The first is the specification of cellular domains in the larval forebrain that give rise to the retinal primordia and undergo early morphogenetic movements. The second is the neurogenic events within the retina proper—proliferation, cell fate determination, and pattern formation—that establish neuronal identities and form retinal laminae and cellular mosaics. The third, which is unique to teleosts and occurs in the functioning eye, is stretching of the retina and persistent neurogenesis that allows the growth of the retina to keep pace with the growth of the eye and other tissues. The first two events are rapid, complete by about 3 days postfertilization in the zebrafish embryo. The third is life-long and accounts for the bulk of retinal growth and the vast majority of adult retinal neurons. In addition, but clearly related to the retina's developmental history, lesions that kill retinal neurons elicit robust neuronal regeneration that originates from cells intrinsic to the retina. This paper reviews recent studies of retinal development in teleosts, focusing on those that shed light on the genetic and molecular regulation of retinal specification and morphogenesis in the embryo, retinal neurogenesis in larvae and adults, and injury-induced neuronal regeneration.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/63365/1/zeb.2004.1.257.pd

Removing striping artifacts in light-sheet fluorescence microscopy: a review

In recent years, light-sheet fluorescence microscopy (LSFM) has found a broad application for imaging of diverse biological samples, ranging from sub-cellular structures to whole animals, both in-vivo and ex-vivo, owing to its many advantages relative to point-scanning methods. By providing the selective illumination of sample single planes, LSFM achieves an intrinsic optical sectioning and direct 2D image acquisition, with low out-of-focus fluorescence background, sample photo-damage and photo-bleaching. On the other hand, such an illumination scheme is prone to light absorption or scattering effects, which lead to uneven illumination and striping artifacts in the images, oriented along the light sheet propagation direction. Several methods have been developed to address this issue, ranging from fully optical solutions to entirely digital post-processing approaches. In this work, we present them, outlining their advantages, performance and limitations

Characterisation and computational modelling of retinal stem cells in medaka (Oryzias latipes)

The central functional unit of the vertebrate eye is the retina, composed of neural retina (NR), retinal pigmented epithelium (RPE), and non-visual retina (NVR). In amphibians and fish, the retina grows throughout life via different pools of stem cells (SCs). In this work, I combined experimental and computational approaches to elucidate SC dynamics in the three retinal tissues of the teleost fish medaka (Oryzias latipes). I developed a cell centred agent based model to recapitulate post-embryonic growth of the NR and RPE. By accounting for 3D tissue geometry and continuous growth, the model reconciled conflicting hypotheses, demonstrating that competition between SCs is not mutually exclusive with lifelong coexistence of multiple SC lineages. To understand how NR and RPE regulate their proliferative output to coordinate growth rates, I developed quantitative methods to compare experiment and simulation. I tested the experimental data against simulations implementing two modes of feedback between cell proliferation and organ growth. Thus, I identified that the NR acts upstream to set the growth pace by sending an inductive growth signal, while the RPE responds downstream to this signal. Leveraging the model, I showed that NR SCs compete for niche space, but tissue geometry biases cells at certain positions to win this competition. Further, NR SCs modulate division axes and proliferation rate to change organ shape and retinal topology. Motivated by model predictions, I experimentally characterised the large SC population of the RPE, which consisted of both cycling and non-cycling quiescent cells. Putative sister cells exhibited similar temporal dynamics in local clusters, indicating that quiescence was the major mechanism for regulating proliferative output in the RPE. Finally, I experimentally showed that the NVR grows post-embryonically from a primordium, and shared all known markers for NR SCs in the same spatial distribution. Unlike NR and RPE, the NVR lacked a dedicated niche, instead proliferative cells were distributed throughout the tissue. Lineage tracing revealed a continuous relationship between RPE, NVR, and NR. Thus, the SCs of NR and RPE, and all cells of the NVR displayed plastic multipotency capable of generating all retinal tissues. By taking advantage of the positive feedback loop between experiment and simulation, this work shines a new light into a fundamental problem – growth coordination of different SC populations in a complex vertebrate organ

Role of sedlin, a TRAPP complex subunit, in membrane trafficking and in the pathogenesis of Spondyleopyphiseal Dysplasia Tarda

Genetic defects occurring in the sedlin gene, a conserved component of TRAPP complex, cause Spondyloepiphyseal Dysplasia Tarda (SEDT), a rare progressive condition characterised by impaired chondrogenesis resulting in short stature, flattening of the vertebrae, and premature osteoarthritis. The role of sedlin in the pathogenesis of SEDT disease so far is still unknown. Prompted by the consideration that sedlin is ubiquitously expressed but that sedlin mutations cause cartilaginous-restricted dysfunctions, I hypothesized that sedlin might exert a role in membrane trafficking generally but in particular in the transport of chondrocyte- specific cargoes, such as type II procollagen (PCII). This hypothesis was reinforced by the fact that mutations in PCII give rise to autosomal dominant forms of spondyloepiphiseal dysplasia. I tested this hypothesis by analyzing the involvement of sedlin in the transport of different classes of secretory cargoes and found that sedlin is selectively required for PCII to exit the ER, while it is not essential for ER exit of small soluble and membrane-associated cargoes. I have also identified the molecular mechanism underlying this role of sedlin in its ability to bind the GTPase Sarl and to control the membrane-cytosol cycle of Sarl itself and of the COPIl coat complex at the level of the ER exit sites. Sedlin depletion and/or mutation in SEDT patients slows down the Sar1 cycle and prolongs the membrane association of Sar1-GTP at the ER exit sites, thus inducing constriction and premature fission of nascent carriers which fail to incorporate the large PC protofibrils but are still competent for smaller secretory cargoes. All together these findings provide new insights not only into understanding the role of sedlin but also shed new light on the molecular mechanisms underlying the onset of the SEDT disease

Technical implementations of light sheet microscopy

Fluorescence-based microscopy is among the most successful methods in biological studies. It played a critical role in the visualization of subcellular structures and in the analysis of complex cellular processes, and it is nowadays commonly employed in genetic and drug screenings. Among the fluorescence-based microscopy techniques, light sheet fluorescence microscopy (LSFM) has shown a quite interesting set of benefits. The technique combines the speed of epi-fluorescence acquisition with the optical sectioning capability typical of confocal microscopes. Its unique configuration allows the excitation of only a thin plane of the sample, thus fast, high resolution imaging deep inside tissues is nowadays achievable. The low peak intensity with which the sample is illuminated diminishes phototoxic effects and decreases photobleaching of fluorophores, ensuring data collection for days with minimal adverse consequences on the sample. It is no surprise that LSFM applications have raised in just few years and the technique has been applied to study a wide variety of samples, from whole organism, to tissues, to cell clusters, and single cells. As a consequence, in recent years numerous set-ups have been developed, each one optimized for the type of sample in use and the requirements of the question at hand. Hereby, we aim to review the most advanced LSFM implementations to assist new LSFM users in the choice of the LSFM set-up that suits their needs best. We also focus on new commercial microscopes and do-it-yourself strategies; likewise we review recent designs that allow a swift integration of LSFM on existing microscopes