Understanding vertebrate embryonic development under conditions present in outer space

The ability of humans to survive, thrive, and sustain life in outer space requires that human embryos develop normally under conditions such as microgravity. We are using the zebrafish model system due to the fact that they share similarities with humans during development. The larval zebrafish will be used to assess embryonic and neural development under simulated microgravity conditions, with specific focus on neural development which has not been well studied. Before performing these experiments, it is critical that histological procedures, including cryosectioning and immunohistochemistry, are up and running in our lab for stages of early zebrafish development. This includes immunohistochemistry for the neuronal marker HuC/D (commonly used to label neurons in zebrafish), and RNA-binding proteins Rbfox1l and Rbfox2 (label neuronal populations). Cryosectioning of 7 day post-fertilization (dpf) larval zebrafish was performed, followed by immunohistochemistry for HuC/D in conjunction with Rbfox labeling, and samples will be imaged on a compound fluorescent microscope to determine whether the protocol is working. Our work aims to better understand vertebrate embryonic development under conditions present in outer space

Understanding vertebrate embryonic development under conditions present in outer space

It is currently unknown whether humans can survive and thrive in outer space, which includes human embryonic development. In this study we investigate vertebrate embryonic development under conditions present in outer space including microgravity and an altered day-and-night cycle using zebrafish and chicken model systems. Zebrafish and chicken model systems are widely used in developmental biology research given their similarity as vertebrates to humans. We aim to analyze the development of brain, muscle and other tissues under conditions of microgravity in both zebrafish and chicken embryos. Additionally, we will analyze zebrafish development under an altered day-and-night cycle (16 sunrises and 16 sunsets per day), and determine whether adult zebrafish can survive and reproduce under these conditions. Zebrafish and chicken embryos will be placed onto a clinostat, which is a device used to simulate a microgravity environment. Embryos will be harvested between 2 and 14 days of incubation on the clinostat, and markers of cell proliferation, death, and differentiation will be analyzed on tissue sections of the brain, skeletal muscle, and other tissues. We expect that our results may allow us to better understand embryonic development under conditions present in outer space, which may shed light on this process in humans

Role of RNA-binding proteins Rbfox1l and Rbfox2 in neuronal development and behavior in zebrafish

Rbfox proteins are RNA-binding proteins that play a significant role in the alternative splicing of neuronal transcripts in the central nervous system (CNS). Rbfox proteins are required for proper brain development and function. In humans, RBFOX1 has been implicated in a variety of neurological disorders, including autism, anxiety, epilepsy, and schizophrenia. Rbfox2 is involved in cerebellar development in mammals. The zebrafish is used as a model system for studies in neurobiology given their neuroanatomical conservation with mammals, and remarkable capability to regenerate parts of their CNS. Rbfox1l (Rbfox1-like) and Rbfox2 have been identified in neurons of the adult zebrafish brain. Rbfox1l was found in a restricted population of dorsal telencephalic neurons, and Rbfox2 was found broadly throughout the brain. Both genes have been found in Purkinje cells of the cerebellum. Utilizing antibody staining on zebrafish brain tissue sections, we will analyze expression of Rbfox1l and Rbfox2 at larval stages and stages leading up to adulthood. Furthermore, we will use rbfox1l and rbfox2 mutant zebrafish (in collaboration with Ohio State University) to better understand the role of rbfox1l in behavior and determine whether rbfox2 is necessary for the regeneration of the cerebellum. Understanding the role of the Rbfox proteins in neural development, regeneration, and behavior may lead to a substantial advancement in the research field and health care

Role of RNA-binding proteins Rbfox1l and Rbfox2 in neuronal development and behavior in zebrafish

Rbfox proteins are RNA-binding proteins that play a significant role in the alternative splicing of neuronal transcripts in the central nervous system (CNS). Rbfox proteins are required for proper brain development and function. In humans, RBFOX1 has been implicated in a variety of neurological disorders, including autism, anxiety, epilepsy, and schizophrenia. Rbfox2 is involved in cerebellar development in mammals. The zebrafish is used as a model system for studies in neurobiology given their neuroanatomical conservation with mammals, and remarkable capability to regenerate parts of their CNS. Rbfox1l (Rbfox1-like) and Rbfox2 have been identified in neurons of the adult zebrafish brain. Rbfox1l was found in a restricted population of dorsal telencephalic neurons, and Rbfox2 was found broadly throughout the brain. Both genes have been found in Purkinje cells of the cerebellum. Utilizing antibody staining on zebrafish brain tissue sections, we will analyze expression of Rbfox1l and Rbfox2 at larval stages and stages leading up to adulthood. Furthermore, we will use rbfox1l and rbfox2 mutant zebrafish (in collaboration with Ohio State University) to better understand the role of rbfox1l in behavior and determine whether rbfox2 is necessary for the regeneration of the cerebellum. Understanding the role of the Rbfox proteins in neural development, regeneration, and behavior may lead to a substantial advancement in the research field and health care

Next-generation sequencing reveals deep intronic cryptic ABCC8 and HADH splicing founder mutations causing hyperinsulinism by pseudoexon activation

Copyright © 2013 The American Society of Human Genetics. Published by Elsevier Inc.Next-generation sequencing (NGS) enables analysis of the human genome on a scale previously unachievable by Sanger sequencing. Exome sequencing of the coding regions and conserved splice sites has been very successful in the identification of disease-causing mutations, and targeting of these regions has extended clinical diagnostic testing from analysis of fewer than ten genes per phenotype to more than 100. Noncoding mutations have been less extensively studied despite evidence from mRNA analysis for the existence of deep intronic mutations in >20 genes. We investigated individuals with hyperinsulinaemic hypoglycaemia and biochemical or genetic evidence to suggest noncoding mutations by using NGS to analyze the entire genomic regions of ABCC8 (117 kb) and HADH (94 kb) from overlapping ~10 kb PCR amplicons. Two deep intronic mutations, c.1333-1013A>G in ABCC8 and c.636+471G>T HADH, were identified. Both are predicted to create a cryptic splice donor site and an out-of-frame pseudoexon. Sequence analysis of mRNA from affected individuals' fibroblasts or lymphoblastoid cells confirmed mutant transcripts with pseudoexon inclusion and premature termination codons. Testing of additional individuals showed that these are founder mutations in the Irish and Turkish populations, accounting for 14% of focal hyperinsulinism cases and 32% of subjects with HADH mutations in our cohort. The identification of deep intronic mutations has previously focused on the detection of aberrant mRNA transcripts in a subset of disorders for which RNA is readily obtained from the target tissue or ectopically expressed at sufficient levels. Our approach of using NGS to analyze the entire genomic DNA sequence is applicable to any disease

One Step Nucleic Acid Amplification (OSNA) - a new method for lymph node staging in colorectal carcinomas

<p>Abstract</p> <p>Background</p> <p>Accurate histopathological evaluation of resected lymph nodes (LN) is essential for the reliable staging of colorectal carcinomas (CRC). With conventional sectioning and staining techniques usually only parts of the LN are examined which might lead to incorrect tumor staging. A molecular method called OSNA (One Step Nucleic Acid Amplification) may be suitable to determine the metastatic status of the complete LN and therefore improve staging.</p> <p>Methods</p> <p>OSNA is based on a short homogenisation step and subsequent automated amplification of cytokeratin 19 (CK19) mRNA directly from the sample lysate, with result available in 30-40 minutes. In this study 184 frozen LN from 184 patients with CRC were investigated by both OSNA and histology (Haematoxylin & Eosin staining and CK19 immunohistochemistry), with half of the LN used for each method. Samples with discordant results were further analysed by RT-PCR for CK19 and carcinoembryonic antigen (CEA).</p> <p>Results</p> <p>The concordance rate between histology and OSNA was 95.7%. Three LN were histology+/OSNA- and 5 LN histology-/OSNA+. RT-PCR supported the OSNA result in 3 discordant cases, suggesting that metastases were exclusively located in either the tissue analysed by OSNA or the tissue used for histology. If these samples were excluded the concordance was 97.2%, the sensitivity 94.9%, and the specificity 97.9%. Three patients (3%) staged as UICC I or II by routine histopathology were upstaged as LN positive by OSNA. One of these patients developed distant metastases (DMS) during follow up.</p> <p>Conclusion</p> <p>OSNA is a new and reliable method for molecular staging of lymphatic metastases in CRC and enables the examination of whole LN. It can be applied as a rapid diagnostic tool to estimate tumour involvement in LN during the staging of CRC.</p

Understanding the role of fezf2 in adult neural stem cell maintenance and fate: A study in zebrafish

Adult neurogenesis, or the birth of new neurons in the mature brain, is a process that occurs continuously and robustly from neural stem cells located in two discreet regions of the adult mammalian brain, the subventricular zone (SVZ) of the lateral ventricles, and the subgranular zone (SGZ) of the dentate gyrus region of the hippocampus. Though some progress has been made in understanding the factors which regulate the maintenance and fate of adult neural stem cells, our understanding remains limited. Fezf2, a conserved forebrain-specific transcription factor, is expressed during development in both zebrafish and mouse in regions where neural progenitor cells are present, and previous studies point to a role for Fezf2 in embryonic neurogenesis in both systems. Here, we show that fezf2 is expressed in the adult zebrafish forebrain, most notably in radial glial-like cells of the telencephalic ventricular zone, which label with markers of neural stem cells and proliferation. Further analysis using a fezf2-GFP transgenic zebrafish line indicates that these fezf2-expressing cells have neural stem cell-like properties, as they are able to self-renew and can likely give rise to glutamatergic neurons in the adult zebrafish telencephalon. Analysis of too few (Fezf2) homozygous mutant zebrafish crossed to our fezf2-GFP reporter line reveals a previously unreported adult telencephalic phenotype, including an increase in proliferation of fezf2-GFP+ cells, and an increase in adult neurogenesis. No major differences are observed in the mutant telencephalon at early larval stage, suggesting that the phenotype occurs largely at the late-larval to adult stage. Moreover, transplantation of fezf2-GFP+ mutant cells into wiltype hosts reveals a cell-autonomous role for fezf2 in maintaining the non-proliferative state of telencephalic radial glial cells. Taken together, our findings suggest a role for fezf2 in regulating the proliferation and differentiation of neural progenitor cells in the adult vertebrate telencephalon

Understanding the role of fezf2 in adult neural stem cell maintenance and fate: A study in zebrafish

Adult neurogenesis, or the birth of new neurons in the mature brain, is a process that occurs continuously and robustly from neural stem cells located in two discreet regions of the adult mammalian brain, the subventricular zone (SVZ) of the lateral ventricles, and the subgranular zone (SGZ) of the dentate gyrus region of the hippocampus. Though some progress has been made in understanding the factors which regulate the maintenance and fate of adult neural stem cells, our understanding remains limited. Fezf2, a conserved forebrain-specific transcription factor, is expressed during development in both zebrafish and mouse in regions where neural progenitor cells are present, and previous studies point to a role for Fezf2 in embryonic neurogenesis in both systems. Here, we show that fezf2 is expressed in the adult zebrafish forebrain, most notably in radial glial-like cells of the telencephalic ventricular zone, which label with markers of neural stem cells and proliferation. Further analysis using a fezf2-GFP transgenic zebrafish line indicates that these fezf2-expressing cells have neural stem cell-like properties, as they are able to self-renew and can likely give rise to glutamatergic neurons in the adult zebrafish telencephalon. Analysis of too few (Fezf2) homozygous mutant zebrafish crossed to our fezf2-GFP reporter line reveals a previously unreported adult telencephalic phenotype, including an increase in proliferation of fezf2-GFP+ cells, and an increase in adult neurogenesis. No major differences are observed in the mutant telencephalon at early larval stage, suggesting that the phenotype occurs largely at the late-larval to adult stage. Moreover, transplantation of fezf2-GFP+ mutant cells into wiltype hosts reveals a cell-autonomous role for fezf2 in maintaining the non-proliferative state of telencephalic radial glial cells. Taken together, our findings suggest a role for fezf2 in regulating the proliferation and differentiation of neural progenitor cells in the adult vertebrate telencephalon

Understanding vertebrate embryonic development under conditions present in outer space

It is currently unknown whether humans can survive and thrive in outer space, which includes human embryonic development. In this study we investigate vertebrate embryonic development under conditions present in outer space including microgravity and an altered day-and-night cycle using zebrafish and chicken model systems. Zebrafish and chicken model systems are widely used in developmental biology research given their similarity as vertebrates to humans. We aim to analyze the development of brain, muscle and other tissues under conditions of microgravity in both zebrafish and chicken embryos. Additionally, we will analyze zebrafish development under an altered day-and-night cycle (16 sunrises and 16 sunsets per day), and determine whether adult zebrafish can survive and reproduce under these conditions. Zebrafish and chicken embryos will be placed onto a clinostat, which is a device used to simulate a microgravity environment. Embryos will be harvested between 2 and 14 days of incubation on the clinostat, and markers of cell proliferation, death, and differentiation will be analyzed on tissue sections of the brain, skeletal muscle, and other tissues. We expect that our results may allow us to better understand embryonic development under conditions present in outer space, which may shed light on this process in humans