Dynamic expression of the basic helix-loop-helix transcription factor neuroD in the rod and cone photoreceptor lineages in the retina of the embryonic and larval zebrafish

NeuroD is a basic helix-loop-helix (bHLH) transcription factor critical for determining neuronal cell fate and regulating withdrawal from the cell cycle. We showed previously that, in goldfish, neuroD is expressed in the rod photoreceptor lineage, and we inferred that neuroD is also expressed in a subset of amacrine cells and nascent cone photoreceptors. Here we extended that study by examining the temporal and spatial expression pattern of neuroD in the embryonic and larval zebrafish and by identifying the cell types that express this gene. NeuroD expression in the developing zebrafish retina is dynamic, spanning early retinogenesis and the maturation of cone photoreceptors. In early retinogenesis neuroD expression expands from a small patch in the ventronasal retina, through the remaining retinal neuroepithelium. As retinogenesis progresses, neuroD expression becomes restricted to amacrine cells, immature cones, and cells of rod and cone lineages. This expression achieves an adult pattern by 96 hours postfertilization (hpf), whereupon the temporal pattern of neuroD expression in central retina is spatially recapitulated at the germinative margin. The cellular pattern of expression suggests that neuroD regulates aspects of rod and cone genesis, but through separate cellular lineages. Furthermore, neuroD is coexpressed with the cone-rod-homeobox transcription factor (Crx) in putative cone progenitors and nascent cone photoreceptors, suggesting that, in the zebrafish retina, as in other vertebrate retinas, similar genetic cascades regulate photoreceptor genesis and maturation. J. Comp. Neurol. 501:1–12, 2007. © 2007 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/55874/1/21150_ftp.pd

Expression and Function of the Basic Helix-Loop-Helix Transcription Factor NeuroD in the Teleost Retina.

NeuroD is a basic helix-loop-helix (bHLH) transcription factor critical for regulating withdrawal from the cell cycle and determining neuronal cell fate. In adult teleosts, neuroD is expressed in proliferating cells of rod and cone photoreceptor lineages and in differentiating cone photoreceptors, suggesting its potential role in photoreceptor genesis. The zebrafish has become a powerful tool for reverse genetic approaches to determine gene function. In the first part of my dissertation, I showed that, in the developing zebrafish retina, neuroD is expressed in proliferating cells of rod and cone photoreceptor lineages and transiently in nascent cone photoreceptors. In the second part of my dissertation, I used complementary gain and loss-of-function approaches to test the hypothesis that NeuroD promotes cell cycle withdrawal among rod and cone progenitor cells and promotes photoreceptor genesis. I created a line of zebrafish transgenic for Hsp70/4:nrd-EGFP, which allowed for temporal control of NeuroD induction following heat shock. I examined proliferation using a panel of cell cycle markers and investigated potential mechanisms utilizing probes for cell cycle regulatory proteins. Compared to controls, transgenic retinas show a significant decrease in the number of mitotically-active cells, indicating that NeuroD promotes cell cycle withdrawal. Induction of NeuroD results in the upregulation of p27 and p57, and downregulation of Cyclin B1, Cyclin D1, and Cyclin E2. Using a panel of cell type-specific antibodies, I showed that NeuroD promotes photoreceptor genesis and inhibits gliogenesis. I used morpholino oligonucleotides to block the translation of NeuroD in cells of the rod and cone photoreceptor lineages. In the absence of NeuroD, although cell type-specific markers show that retinal development is otherwise normal, cells of the rod and cone photoreceptor lineages continue to proliferate and fail to exit the cell cycle. In addition, the absence of NeuroD results in the upregulation of Cyclin D1. Taken together, the cellular pattern of neuroD expression and results from gain and loss-of-function experiments reveal that NeuroD promotes cell cycle withdrawal among photoreceptor progenitors. NeuroD may also function in early aspects of cone photoreceptor maturation. These experiments are the first in vivo demonstration of cell cycle regulation by NeuroD in the retina.Ph.D.NeuroscienceUniversity of Michigan, Horace H. Rackham School of Graduate Studieshttp://deepblue.lib.umich.edu/bitstream/2027.42/57632/2/ochocins_1.pd

Modulation of cell proliferation in the embryonic retina of zebrafish ( Danio rerio )

We describe light-microscopically the development of the embryonic zebrafish eye with particular attention to cell number, cell proliferation, and cell death. The period from 16 to 36 hr post fertilization (hpf) comprises two phases; during the first (16–27 hpf) the optic vesicle becomes the eye cup, and during the second (27–36 hpf) the eye cup begins to differentiate into the neural retina and pigmented epithelium. All cells in the eye primordium are proliferative prior to 28 hpf, and the length of the cell cycle has been estimated to be 10 hr at 24–28 hpf (Nawrocki, 1985 ). Our cell counts are consistent with that estimate at that age, but not at earlier ages. A 10-hr cell cycle predicts that the cell number should increase by 7% per hr, but during 16–24 hpf the cell number increased by only 1.5% per hr. Despite the low rate of increase, all cells labeled with bromo-deoxyuridine, so all were proliferative. We considered three possible explanations for the nearly-constant cell number in the first phase: proliferation balanced by cell emigration from the eye, proliferation balanced by cell death, and low proliferation caused by a transient prolongation of the cell cycle. We excluded the first two, and found direct support for the third. Previous examinations of the cell cycle length in vertebrate central nervous system have concluded that it increases monotonically, in contrast to the modulation that we have shown. Modulation of the cell cycle length is well-known in flies, but it is generally effected by a prolonged arrest at one phase, in contrast to the general deceleration that we have shown. © 2000 Wiley-Liss, Inc.Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/35168/1/1063_ftp.pd

Using the Tg(nrd:egfp)/albino Zebrafish Line to Characterize In Vivo Expression of neurod

In this study, we used a newly-created transgenic zebrafish, Tg(nrd:egfp)/albino, to further characterize the expression of neurod in the developing and adult retina and to determine neurod expression during adult photoreceptor regeneration. We also provide observations regarding the expression of neurod in a variety of other tissues. In this line, EGFP is found in cells of the developing and adult retina, pineal gland, cerebellum, olfactory bulbs, midbrain, hindbrain, neural tube, lateral line, inner ear, pancreas, gut, and fin. Using immunohistochemistry and in situ hybridization, we compare the expression of the nrd:egfp transgene to that of endogenous neurod and to known retinal cell types. Consistent with previous data based on in situ hybridizations, we show that during retinal development, the nrd:egfp transgene is not expressed in proliferating retinal neuroepithelium, and is expressed in a subset of retinal neurons. In contrast to previous studies, nrd:egfp is gradually re-expressed in all rod photoreceptors. During photoreceptor regeneration in adult zebrafish, in situ hybridization reveals that neurod is not expressed in Müller glial-derived neuronal progenitors, but is expressed in photoreceptor progenitors as they migrate to the outer nuclear layer and differentiate into new rod photoreceptors. During photoreceptor regeneration, expression of the nrd:egfp matches that of neurod. We conclude that Tg(nrd:egfp)/albino is a good representation of endogenous neurod expression, is a useful tool to visualize neurod expression in a variety of tissues and will aid investigating the fundamental processes that govern photoreceptor regeneration in adults

Resource: A multi‐species multi‐timepoint transcriptome database and webpage for the pineal gland and retina

The website and database https://snengs.nichd.nih.gov provides RNA sequencing data from multi-species analysis of the pineal glands from zebrafish (Danio rerio), chicken (White Leghorn), rat (Rattus novegicus), mouse (Mus musculus), rhesus macaque (Macaca mulatta), and human (Homo sapiens); in most cases, retinal data are also included along with results of the analysis of a mixture of RNA from tissues. Studies cover day and night conditions; in addition, a time series over multiple hours, a developmental time series and pharmacological experiments on rats are included. The data have been uniformly re-processed using the latest methods and assemblies to allow for comparisons between experiments and to reduce processing differences. The website presents search functionality, graphical representations, Excel tables, and track hubs of all data for detailed visualization in the UCSC Genome Browser. As more data are collected from investigators and improved genomes become available in the future, the website will be updated. This database is in the public domain and elements can be reproduced by citing the URL and this report. This effort makes the results of 21st century transcriptome profiling widely available in a user-friendly format that is expected to broadly influence pineal research.Fil: Chang, Eric. National Instituto of Child Health & Human Development; Estados UnidosFil: Fu, Cong. National Instituto of Child Health & Human Development; Estados UnidosFil: Coon, Steven L.. National Instituto of Child Health & Human Development; Estados UnidosFil: Alon, Shahar. No especifíca;Fil: Bozinoski, Marjan. No especifíca;Fil: Breymaier, Matthew. National Instituto of Child Health & Human Development; Estados UnidosFil: Bustos, Diego Martin. Consejo Nacional de Investigaciones Científicas y Técnicas. Centro Científico Tecnológico Conicet - Mendoza. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos. Universidad Nacional de Cuyo. Facultad de Ciencias Médicas. Instituto de Histología y Embriología de Mendoza Dr. Mario H. Burgos; ArgentinaFil: Clokie, Samuel J.. National Instituto of Child Health & Human Development; Estados UnidosFil: Gothilf, Yoav. No especifíca;Fil: Esnault, Caroline. National Instituto of Child Health & Human Development; Estados UnidosFil: Iuvone, P. Michael. Emory University School of Medicine; Estados UnidosFil: Mason, Christopher E.. No especifíca;Fil: Ochocinska, Margaret J.. National Instituto of Child Health & Human Development; Estados UnidosFil: Tovin, Adi. No especifíca;Fil: Wang, Charles. Loma Linda University; Estados UnidosFil: Xu, Pinxian. No especifíca;Fil: Zhu, Jinhang. No especifíca;Fil: Dale, Ryan. National Instituto of Child Health & Human Development; Estados UnidosFil: Klein, David C.. National Instituto of Child Health & Human Development; Estados Unido

NIH Workshop 2018: Towards Minimally Invasive or Noninvasive Approaches to Assess Tissue Oxygenation Pre- and Post-transfusion

Because blood transfusion is one of the most common therapeutic interventions in hospitalized patients, much recent research has focused on improving the storage quality in vitro of donor red blood cells (RBCs) that are then used for transfusion. However, there is a significant need for enhancing our understanding of the efficacy of the transfused RBCs in vivo. To this end, the NIH sponsored a one-and-a-half-day workshop that brought together experts in multiple disciplines relevant to tissue oxygenation (eg, transfusion medicine, critical care medicine, cardiology, neurology, neonatology and pediatrics, bioengineering, biochemistry, and imaging). These individuals presented their latest findings, discussed key challenges, and aimed to identify opportunities for facilitating development of new technologies and/or biomarker panels to assess tissue oxygenation in a minimally-invasive to non-invasive fashion, before and after RBC transfusion

NIH Workshop 2018: Towards Minimally-invasive or Non-invasive Approaches to Assess Tissue Oxygenation Pre- and Post-Transfusion

Because blood transfusion is one of the most common therapeutic interventions in hospitalized patients, much recent research has focused on improving the storage quality in vitro of donor red blood cells (RBCs) that are then used for transfusion. However, there is a significant need for enhancing our understanding of the efficacy of the transfused RBCs in vivo. To this end, the NIH sponsored a one-and-a-half-day workshop that brought together experts in multiple disciplines relevant to tissue oxygenation (e.g., transfusion medicine, critical care medicine, cardiology, neurology, neonatology and pediatrics, bioengineering, biochemistry, and imaging). These individuals presented their latest findings, discussed key challenges, and aimed to construct recommendations for facilitating development of new technologies and/or biomarker panels to assess tissue oxygenation in a minimally-invasive to non-invasive fashion, before and after RBC transfusion. The workshop was structured into four sessions: (1) Global Perspective; (2) Organ Systems; (3) Neonatology; and (4) Emerging Technologies. The first day provided an overview of current approaches in the clinical setting, both from a global perspective, including the use of metabolomics for studying RBCs and tissue perfusion, and from a more focused perspective, including tissue oxygenation assessments in neonates and in specific adult organ systems. The second day focused on emerging technologies, which could be applied pre- and post-RBC transfusion, to assess tissue oxygenation in minimally-invasive or non-invasive ways. Each day concluded with an open-microphone discussion among the speakers and workshop participants. The workshop presentations and ensuing interdisciplinary discussions highlighted the potential of technologies to combine global “omics” signatures with additional measures (e.g., thenar eminence measurements or various imaging methods) to predict which patients could potentially benefit from a RBC transfusion and whether the ensuing RBC transfusion was effective. The discussions highlighted the need for collaborations across the various disciplines represented at the meeting to leverage existing technologies and to develop novel approaches for assessing RBC transfusion efficacy in various clinical settings. Although the Workshop took place in April, 2018, the concepts described and the ensuing discussions were, perhaps, even more relevant in April, 2020, at the time of writing this manuscript, during the explosive growth of the COVID-19 pandemic in the United States. Thus, issues relating to maintaining and improving tissue oxygenation and perfusion are especially pertinent because of the extensive pulmonary damage resulting from SARS-CoV-2 infection [1], compromises in perfusion caused by thrombotic-embolic phenomena [2], and damage to circulating RBCs, potentially compromising their oxygen-carrying capacity [3]. The severe end organ effects of SARS-CoV-2 infection mandate even more urgency for improving our understanding of tissue perfusion and oxygenation, improve methods for measuring and monitoring them, and develop novel ways of enhancing them

Current challenges and future directions for engineering extracellular vesicles for heart, lung, blood and sleep diseases.

Extracellular vesicles (EVs) carry diverse bioactive components including nucleic acids, proteins, lipids and metabolites that play versatile roles in intercellular and interorgan communication. The capability to modulate their stability, tissue-specific targeting and cargo render EVs as promising nanotherapeutics for treating heart, lung, blood and sleep (HLBS) diseases. However, current limitations in large-scale manufacturing of therapeutic-grade EVs, and knowledge gaps in EV biogenesis and heterogeneity pose significant challenges in their clinical application as diagnostics or therapeutics for HLBS diseases. To address these challenges, a strategic workshop with multidisciplinary experts in EV biology and U.S. Food and Drug Administration (USFDA) officials was convened by the National Heart, Lung and Blood Institute. The presentations and discussions were focused on summarizing the current state of science and technology for engineering therapeutic EVs for HLBS diseases, identifying critical knowledge gaps and regulatory challenges and suggesting potential solutions to promulgate translation of therapeutic EVs to the clinic. Benchmarks to meet the critical quality attributes set by the USFDA for other cell-based therapeutics were discussed. Development of novel strategies and approaches for scaling-up EV production and the quality control/quality analysis (QC/QA) of EV-based therapeutics were recognized as the necessary milestones for future investigations.Funding information: National Heart, Lung, and Blood Institute, Grant/Award Numbers: HL 122596, HL124021, HL124074, HL128297, HL141080, HL155346-01, R35HL150807, R56HL141206 Prithu Sundd was supported by NIH-NHLBI R01 grants (HL128297 and HL141080) and 18TPA34170588 from American Heart Association. Stephen Y. Chan was supported by NIH grants R01 HL124021 and HL 122596 as well as AHA grant 18EIA33900027. SuamyaDaswas supported by NIH grants R35HL150807, UH3 TR002878 andAHASFRN35120123. ZhenjiaWangwas supported by NIH grant (R01EB027078). Pilar Martín was supported by MCIN-ISCIII-Fondo de Investigación Sanitaria grant PI22/01759. KennethW.Witwer was supported in part by NIH grants R01AI144997, R01DA047807, R33MH118164 andUH3CA241694. Tianji Chen was supported by AHA Career Development Award 18CDA34110301, Gilead Sciences Research Scholars Program in PAH, NIH-NHLBI grant R56HL141206 and Chicago Biomedical ConsortiumCatalyst Award. EduardoMarbán was supported byNIH R01 HL124074 and HL155346-01.S

Derivation of Human Differential Photoreceptor-like Cells from the Iris by Defined Combinations of CRX, RX and NEUROD

Examples of direct differentiation by defined transcription factors have been provided for beta-cells, cardiomyocytes and neurons. In the human visual system, there are four kinds of photoreceptors in the retina. Neural retina and iris-pigmented epithelium (IPE) share a common developmental origin, leading us to test whether human iris cells could differentiate to retinal neurons. We here define the transcription factor combinations that can determine human photoreceptor cell fate. Expression of rhodopsin, blue opsin and green/red opsin in induced photoreceptor cells were dependent on combinations of transcription factors: A combination of CRX and NEUROD induced rhodopsin and blue opsin, but did not induce green opsin; a combination of CRX and RX induced blue opsin and green/red opsin, but did not induce rhodopsin. Phototransduction-related genes as well as opsin genes were up-regulated in those cells. Functional analysis; i.e. patch clamp recordings, clearly revealed that generated photoreceptor cells, induced by CRX, RX and NEUROD, responded to light. The response was an inward current instead of the typical outward current. These data suggest that photosensitive photoreceptor cells can be generated by combinations of transcription factors. The combination of CRX and RX generate immature photoreceptors: and additional NEUROD promotes maturation. These findings contribute substantially to a major advance toward eventual cell-based therapy for retinal degenerative diseases