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Embryonic Structures Commons

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Enhanced Cas12a Editing In Mammalian Cells And Zebrafish, Pengpeng Liu, Kevin Luk, Masahiro Shin, Feston Idrizi, Samantha F. Kwok, Benjamin P. Roscoe, Esther Mintzer, Sneha Suresh, Kyle Morrison, Josias B. Frazao, Mehmet Fatih Bolukbasi, Karthikeyan Ponnienselvan, Jeremy Luban, Lihua Julie Zhu, Nathan D. Lawson, Scot A. Wolfe 2019 University of Massachusetts Medical School

Enhanced Cas12a Editing In Mammalian Cells And Zebrafish, Pengpeng Liu, Kevin Luk, Masahiro Shin, Feston Idrizi, Samantha F. Kwok, Benjamin P. Roscoe, Esther Mintzer, Sneha Suresh, Kyle Morrison, Josias B. Frazao, Mehmet Fatih Bolukbasi, Karthikeyan Ponnienselvan, Jeremy Luban, Lihua Julie Zhu, Nathan D. Lawson, Scot A. Wolfe

Open Access Articles

Type V CRISPR-Cas12a systems provide an alternate nuclease platform to Cas9, with potential advantages for specific genome editing applications. Here we describe improvements to the Cas12a system that facilitate efficient targeted mutagenesis in mammalian cells and zebrafish embryos. We show that engineered variants of Cas12a with two different nuclear localization sequences (NLS) on the C terminus provide increased editing efficiency in mammalian cells. Additionally, we find that pre-crRNAs comprising a full-length direct repeat (full-DR-crRNA) sequence with specific stem-loop G-C base substitutions exhibit increased editing efficiencies compared with the standard mature crRNA framework. Finally, we demonstrate in zebrafish embryos that the ...


Defects In Fetal Mouth Movement And Pharyngeal Patterning Underlie Cleft Palate Caused By Retinoid Deficiency., Regina Friedl 2019 University of Louisville

Defects In Fetal Mouth Movement And Pharyngeal Patterning Underlie Cleft Palate Caused By Retinoid Deficiency., Regina Friedl

Electronic Theses and Dissertations

Cleft palate is a common birth defect. Etiologic mechanisms of palate cleft include defects in palate morphogenesis, mandibular growth, or spontaneous fetal mouth movement. Cleft palate linked to deficient fetal mouth movement has been demonstrated directly only in a single experimental model of loss of neurotransmission. Here, using retinoid deficient mouse embryos, we demonstrate directly for the first time that deficient fetal mouth movement and cleft palate occurs as a result of mis-patterned development of pharyngeal peripheral nerves and cartilages. Retinoid deficient embryos were generated by inactivation of retinol dehydrogenase 10 (Rdh10), which is critical for production of Retinoic Acid ...


Ouabain Enhances Cell-Cell Adhesion Mediated By Beta1 Subunits Of The Na(+),K(+)-Atpase In Cho Fibroblasts, Claudia Andrea Vilchis-Nestor, Maria Luisa Roldan, Angelina Leonardi, Juan G. Navea, Teresita Padilla-Benavides, Liora Shoshani 2019 University of Massachusetts Medical School

Ouabain Enhances Cell-Cell Adhesion Mediated By Beta1 Subunits Of The Na(+),K(+)-Atpase In Cho Fibroblasts, Claudia Andrea Vilchis-Nestor, Maria Luisa Roldan, Angelina Leonardi, Juan G. Navea, Teresita Padilla-Benavides, Liora Shoshani

Open Access Articles

Adhesion is a crucial characteristic of epithelial cells to form barriers to pathogens and toxic substances from the environment. Epithelial cells attach to each other using intercellular junctions on the lateral membrane, including tight and adherent junctions, as well as the Na(+),K(+)-ATPase. Our group has shown that non-adherent chinese hamster ovary (CHO) cells transfected with the canine beta1 subunit become adhesive, and those homotypic interactions amongst beta1 subunits of the Na(+),K(+)-ATPase occur between neighboring epithelial cells. Ouabain, a cardiotonic steroid, binds to the alpha subunit of the Na(+),K(+)-ATPase, inhibits the pump activity and induces ...


Single-Cell Rna-Sequencing-Based Crispri Screening Resolves Molecular Drivers Of Early Human Endoderm Development, Ryan M. Genga, Eric M. Kernfeld, Krishna M. Parsi, Teagan J. Parsons, Michael J. Ziller, Rene Maehr 2019 University of Massachusetts Medical School

Single-Cell Rna-Sequencing-Based Crispri Screening Resolves Molecular Drivers Of Early Human Endoderm Development, Ryan M. Genga, Eric M. Kernfeld, Krishna M. Parsi, Teagan J. Parsons, Michael J. Ziller, Rene Maehr

Open Access Articles

Studies in vertebrates have outlined conserved molecular control of definitive endoderm (END) development. However, recent work also shows that key molecular aspects of human END regulation differ even from rodents. Differentiation of human embryonic stem cells (ESCs) to END offers a tractable system to study the molecular basis of normal and defective human-specific END development. Here, we interrogated dynamics in chromatin accessibility during differentiation of ESCs to END, predicting DNA-binding proteins that may drive this cell fate transition. We then combined single-cell RNA-seq with parallel CRISPR perturbations to comprehensively define the loss-of-function phenotype of those factors in END development. Following ...


Arterial Distribution Of The Human Aorta: An Examination Of The Evolutionary, Developmental, And Physiological Bases Of Asymmetry., Brandon Oddo, Cooker Storm 2019 Pepperdine University

Arterial Distribution Of The Human Aorta: An Examination Of The Evolutionary, Developmental, And Physiological Bases Of Asymmetry., Brandon Oddo, Cooker Storm

Seaver College Research And Scholarly Achievement Symposium

The study of anatomy contends that “form follows function”; a disciplinary theme purporting that anatomical structures (i.e., cells, tissues, and organs) have a shape that serves its proper function. With this in mind, it is unclear why human arterial distribution off the aortic arch is asymmetrical, while the corresponding venous anatomy is symmetrical. We investigated the evolutionary, developmental, and physiological bases for the asymmetry of aortic arch branches in humans. First, we investigated the cardiovascular anatomy of ancestral species to determine if, and at what level, anatomical divergence (from aortic symmetry to asymmetry) occurs. Second, we examined the formation ...


Entosis Controls A Developmental Cell Clearance In C. Elegans, Yongchan Lee, Jens C. Hamann, Mark Pellegrino, Joanne Durgan, Marie-Charlotte Domart, Lucy M. Collinson, Cole M. Haynes, Oliver Florey, Michael Overholtzer 2019 Sloan Kettering Institute for Cancer Research

Entosis Controls A Developmental Cell Clearance In C. Elegans, Yongchan Lee, Jens C. Hamann, Mark Pellegrino, Joanne Durgan, Marie-Charlotte Domart, Lucy M. Collinson, Cole M. Haynes, Oliver Florey, Michael Overholtzer

Open Access Articles

Metazoan cell death mechanisms are diverse and include numerous non-apoptotic programs. One program called entosis involves the invasion of live cells into their neighbors and is known to occur in cancers. Here, we identify a developmental function for entosis: to clear the male-specific linker cell in C. elegans. The linker cell leads migration to shape the gonad and is removed to facilitate fusion of the gonad to the cloaca. We find that the linker cell is cleared in a manner involving cell-cell adhesions and cell-autonomous control of uptake through linker cell actin. Linker cell entosis generates a lobe structure that ...


Complement Targets Newborn Retinal Ganglion Cells For Phagocytic Elimination By Microglia, Sarah R. Anderson, Jianmin Zhang, Michael R. Steele, Cesar O. Romero, Amanda G. Kautzman, Dorothy P. Schafer, Monica L. Vetter 2019 University of Utah

Complement Targets Newborn Retinal Ganglion Cells For Phagocytic Elimination By Microglia, Sarah R. Anderson, Jianmin Zhang, Michael R. Steele, Cesar O. Romero, Amanda G. Kautzman, Dorothy P. Schafer, Monica L. Vetter

Neurobiology Publications and Presentations

Microglia play important roles in shaping the developing CNS, and at early stages they have been proposed to regulate progenitor proliferation, differentiation, and neuronal survival. However, these studies reveal contradictory outcomes, highlighting the complexity of these cell-cell interactions. Here, we investigate microglia function during embryonic mouse retina development, where only microglia, progenitors, and neurons are present. In both sexes, we determine that microglia primarily interact with retinal neurons and find that depletion of microglia via conditional KO of the Csf1 receptor results in increased density of retinal ganglion cells (RGCs). Pharmacological inhibition of microglia also results in an increase in ...


Receptor Interacting Protein Kinase 3 (Rip3) Regulates Ipscs Generation Through Modulating Cell Cycle Progression Genes, Ahmad Al-Moujahed, Bo Tian, Nikolaos E. Efstathiou, Eleni K. Konstantinou, Mien Hoang, Haijiang Lin, Joan W. Miller, Demetrios G. Vavvas 2019 Harvard Medical School

Receptor Interacting Protein Kinase 3 (Rip3) Regulates Ipscs Generation Through Modulating Cell Cycle Progression Genes, Ahmad Al-Moujahed, Bo Tian, Nikolaos E. Efstathiou, Eleni K. Konstantinou, Mien Hoang, Haijiang Lin, Joan W. Miller, Demetrios G. Vavvas

Open Access Articles

The molecular mechanisms involved in induced pluripotent stem cells (iPSCs) generation are poorly understood. The cell death machinery of apoptosis-inducing caspases have been shown to facilitate the process of iPSCs reprogramming. However, the effect of other cell death processes, such as programmed necrosis (necroptosis), on iPSCs induction has not been studied. In this study, we investigated the role of receptor-interacting protein kinase 3 (RIP3), an essential regulator of necroptosis, in reprogramming mouse embryonic fibroblast cells (MEFs) into iPSCs. RIP3 was found to be upregulated in iPSCs compared to MEFs. Deletion of RIP3 dramatically suppressed the reprogramming of iPSCs (~82%). RNA-seq ...


Two Contrasting Classes Of Nucleolus-Associated Domains In Mouse Fibroblast Heterochromatin, Anastassiia Vertii, Jianhong Ou, Jun Yu, Aimin Yan, Hervé Pagès, Haibo Liu, Lihua Julie Zhu, Paul D. Kaufman 2018 University of Massachusetts Medical School

Two Contrasting Classes Of Nucleolus-Associated Domains In Mouse Fibroblast Heterochromatin, Anastassiia Vertii, Jianhong Ou, Jun Yu, Aimin Yan, Hervé Pagès, Haibo Liu, Lihua Julie Zhu, Paul D. Kaufman

University of Massachusetts Medical School Faculty Publications

In interphase eukaryotic cells, almost all heterochromatin is located adjacent to the nucleolus or to the nuclear lamina, thus defining Nucleolus Associated Domains (NADs) and Lamina Associated Domains (LADs), respectively. Here, we determined the first genome-scale map of murine NADs in mouse embryonic fibroblasts (MEFs) via deep sequencing of chromatin associated with purified nucleoli. We developed a Bioconductor package called NADfinder and demonstrated that it identifies NADs more accurately than other peak-calling tools, due to its critical feature of chromosome-level local baseline correction. We detected two distinct classes of NADs. Type I NADs associate frequently with both the nucleolar periphery ...


Dynamic Placement Of The Linker Histone H1 Associated With Nucleosome Arrangement And Gene Transcription In Early Drosophila Embryonic Development, Jian Hu, Liang Gu, Youqiong Ye, Meizhu Zheng, Zhu Xu, Jing Lin, Yanhua Du, Mengxue Tian, Lifang Luo, Beibei Wang, Xiaobai Zhang, Zhiping Weng, Cizhong Jiang 2018 Tongji University

Dynamic Placement Of The Linker Histone H1 Associated With Nucleosome Arrangement And Gene Transcription In Early Drosophila Embryonic Development, Jian Hu, Liang Gu, Youqiong Ye, Meizhu Zheng, Zhu Xu, Jing Lin, Yanhua Du, Mengxue Tian, Lifang Luo, Beibei Wang, Xiaobai Zhang, Zhiping Weng, Cizhong Jiang

Open Access Articles

The linker histone H1 is critical to maintenance of higher-order chromatin structures and to gene expression regulation. However, H1 dynamics and its functions in embryonic development remain unresolved. Here, we profiled gene expression, nucleosome positions, and H1 locations in early Drosophila embryos. The results show that H1 binding is positively correlated with the stability of beads-on-a-string nucleosome organization likely through stabilizing nucleosome positioning and maintaining nucleosome spacing. Strikingly, nucleosomes with H1 placement deviating to the left or the right relative to the dyad shift to the left or the right, respectively, during early Drosophila embryonic development. H1 occupancy on genic ...


Identifying The 5’End Of The Camta1 Genes In Zebrafish, Morgan Fronk 2018 Brigham Young University

Identifying The 5’End Of The Camta1 Genes In Zebrafish, Morgan Fronk

Undergraduate Honors Theses

Congenital heart diseases (CHDs) are a significant cause of infant death and are frequently caused by mutations in transcription factors. Camta1 (calmodulin binding transcription activator 1) is a transcription factor that has been proposed as a modulator in embryonic heart development and a possible cause of CHDs. The only other known member of its family in vertebrates is involved in activating a hypertrophy gene program in adult heart failure. Unlike camta2, camta1 is expressed in the embryonic heart during heart looping. However, few studies have been done on camta1. In zebrafish, there are two camta1 ohnologs (homologs created through a ...


Small Rnas Gained During Epididymal Transit Of Sperm Are Essential For Embryonic Development In Mice, Colin C. Conine, Fengyun Sun, Lina Song, Jaime A. Rivera-Perez, Oliver J. Rando 2018 University of Massachusetts Medical School

Small Rnas Gained During Epididymal Transit Of Sperm Are Essential For Embryonic Development In Mice, Colin C. Conine, Fengyun Sun, Lina Song, Jaime A. Rivera-Perez, Oliver J. Rando

University of Massachusetts Medical School Faculty Publications

The small RNA payload of mammalian sperm undergoes dramatic remodeling during development, as several waves of microRNAs and tRNA fragments are shipped to sperm during post-testicular maturation in the epididymis. Here, we take advantage of this developmental process to probe the function of the sperm RNA payload in preimplantation development. We generated zygotes via intracytoplasmic sperm injection (ICSI) using sperm obtained from the proximal (caput) vs. distal (cauda) epididymis, then characterized development of the resulting embryos. Embryos generated using caput sperm significantly overexpress multiple regulatory factors throughout preimplantation development, and subsequently implant inefficiently and fail soon after implantation. Remarkably, microinjection ...


Profiling Of Pluripotency Factors In Individual Stem Cells And Early Embryos, Sarah J. Hainer, Ana Boskovic, Oliver J. Rando, Thomas G. Fazzio 2018 University of Massachusetts Medical School

Profiling Of Pluripotency Factors In Individual Stem Cells And Early Embryos, Sarah J. Hainer, Ana Boskovic, Oliver J. Rando, Thomas G. Fazzio

University of Massachusetts Medical School Faculty Publications

Major cell fate decisions are governed by sequence-specific transcription factors (TFs) that act in small cell populations within developing embryos. To understand how TFs regulate cell fate it is important to identify their genomic binding sites in these populations. However, current methods cannot profile TFs genome-wide at or near the single cell level. Here we adapt the CUT&RUN method to profile chromatin proteins in low cell numbers, mapping TF-DNA interactions in single cells and individual pre-implantation embryos for the first time. Using this method, we demonstrate that the pluripotency TF NANOG is significantly more dependent on the SWI/SNF ...


C. Elegans Multidrug Resistance Protein 5 (Mrp-5) Transports Vitamin B12 From The Intestine To The Gonad To Support Embryonic Development, Huimin Na, Olga Ponomarova, Gabrielle E. Giese, Albertha J. M. Walhout 2018 University of Massachusetts Medical School

C. Elegans Multidrug Resistance Protein 5 (Mrp-5) Transports Vitamin B12 From The Intestine To The Gonad To Support Embryonic Development, Huimin Na, Olga Ponomarova, Gabrielle E. Giese, Albertha J. M. Walhout

University of Massachusetts Medical School Faculty Publications

Vitamin B12 functions as a cofactor for methionine synthase to produce the anabolic methyl donor S-adenosylmethionine (SAM) and for methylmalonyl-CoA mutase to catabolize the short chain fatty acid propionate. In the nematode Caenorhabditis elegans, maternally supplied vitamin B12 is required for the development of her offspring. However, the mechanism for exporting vitamin B12 from the mother to her offspring is not yet known. Here, we use RNAi of more than 200 transporters with a vitamin B12-sensor transgene to identify the ABC transporter MRP-5 as a candidate vitamin B12 exporter. We show that injection of vitamin B12 into the gonad of ...


Small Rnas Are Trafficked From The Epididymis To Developing Mammalian Sperm, Upasna Sharma, Fengyun Sun, Brian Reichholf, Veronika Herzog, Stefan Ameres, Oliver J. Rando 2017 University of Massachusetts Medical School

Small Rnas Are Trafficked From The Epididymis To Developing Mammalian Sperm, Upasna Sharma, Fengyun Sun, Brian Reichholf, Veronika Herzog, Stefan Ameres, Oliver J. Rando

University of Massachusetts Medical School Faculty Publications

RNAs present in mature mammalian sperm are delivered to the zygote at fertilization, where they have the potential to affect early development. The biogenesis of the small RNA payload of mature sperm is therefore of great interest, as it may be a target of signaling pathways linking paternal conditions to offspring phenotype. Recent studies have suggested the surprising hypothesis that the small RNA payload carried by mature sperm may include RNAs that were not synthesized during testicular spermatogenesis, but that are instead delivered to sperm during the process of post-testicular maturation in the epididymis. To further test this hypothesis, we ...


Embryonic Transcription Factor Expression In Mice Predicts Medial Amygdala Neuronal Identity And Sex-Specific Responses To Innate Behavioral Cues., Julieta E Lischinsky, Katie Sokolowski, Li Peijun, Shigeyuki Esumi, Yasmin Kamal, Meredith Goodrich, Livio Oboti, Timothy R Hammond, Meera Krishnamoorthy, Daniel Feldman, Molly Huntsman, Judy Liu, Joshua G Corbin 2017 George Washington University

Embryonic Transcription Factor Expression In Mice Predicts Medial Amygdala Neuronal Identity And Sex-Specific Responses To Innate Behavioral Cues., Julieta E Lischinsky, Katie Sokolowski, Li Peijun, Shigeyuki Esumi, Yasmin Kamal, Meredith Goodrich, Livio Oboti, Timothy R Hammond, Meera Krishnamoorthy, Daniel Feldman, Molly Huntsman, Judy Liu, Joshua G Corbin

Pediatrics Faculty Publications

The medial subnucleus of the amygdala (MeA) plays a central role in processing sensory cues required for innate behaviors. However, whether there is a link between developmental programs and the emergence of inborn behaviors remains unknown. Our previous studies revealed that the telencephalic preoptic area (POA) embryonic niche is a novel source of MeA destined progenitors. Here, we show that the POA is comprised of distinct progenitor pools complementarily marked by the transcription factors Dbx1 and Foxp2. As determined by molecular and electrophysiological criteria this embryonic parcellation predicts postnatal MeA inhibitory neuronal subtype identity. We further find that Dbx1-derived and ...


Mechanism For Generation Of Left Isomerism In Ccdc40 Mutant Embryos, Kelsey Sugrue, Irene E. Zohn 2017 George Washington University

Mechanism For Generation Of Left Isomerism In Ccdc40 Mutant Embryos, Kelsey Sugrue, Irene E. Zohn

Pediatrics Faculty Publications

Leftward fluid flow in the mouse node is generated by cilia and is critical for initiating asymmetry of the left-right axis. Coiled-coil domain containing-40 (Ccdc40) plays an evolutionarily conserved role in the assembly of motile cilia and establishment of the left-right axis. Approximately one-third of Ccdc40lnks mutant embryos display situs defects and here we investigate the underlying mechanism. Ccdc40lnks mutants show delayed induction of markers of the left-lateral plate mesoderm (L-LPM) including Lefty1, Lefty2 and Nodal. Consistent with defective cilia motility compromising fluid flow across the node, initiation of asymmetric perinodal Cerberus like-2 (Cerl2) expression is delayed and ...


Dreadlocks (Dock) Is Necessary To Regulate Growth Of The Germline Ring Canals In The Developing Drosophila Melanogaster Egg Chamber, Olivia Crowe 2017 Butler University

Dreadlocks (Dock) Is Necessary To Regulate Growth Of The Germline Ring Canals In The Developing Drosophila Melanogaster Egg Chamber, Olivia Crowe

Undergraduate Honors Thesis Collection

Infertility is a prevalent issue in the United States, impacting 1.5 million women (1). A possible cause of infertility is defects in gametogenesis, or the formation of sperm and egg. Therefore, understanding the basic mechanisms that promote normal gamete formation could impact our understanding of infertility. The Drosophila melanogaster egg develops from an organ-like structure called an egg chamber. The egg chamber is composed of a central cluster of 16 germ cells that are connected to one another by intercellular bridges, called ring canals. These ring canals are composed of filamentous actin and allow the transfer of materials from ...


A Low-Cost Time-Lapse Imaging System Developed For Documentation Of Human Pre-Implantation Embryogenesis In Clinical Ivf Settings, Christine E. Hennigan Miss 2017 University of Colorado Boulder

A Low-Cost Time-Lapse Imaging System Developed For Documentation Of Human Pre-Implantation Embryogenesis In Clinical Ivf Settings, Christine E. Hennigan Miss

Undergraduate Honors Theses

Infertility is a disease of the reproductive system that affects many women of various ages, races, and socioeconomic factors across the world (CDC, WHO, 2017). The expensive nature of treatments for infertility, such as in vitro fertilization (IVF), has been combated with a simplified culture system (SCS) developed in the Van Blerkom lab (Van Blerkom et al., 2013). This recent advancement in clinical IVF procedures has prompted an effort to develop additional, low-cost technology able to help assess pre-implantation embryogenesis development and competence. This study examines the development and application of a low-cost, simplified, time-lapse imaging system that has shown ...


The Arp2/3 Complex Is Essential At Multiple Stages Of Neural Development., Fu-Sheng Chou, Pei-Shan Wang 2016 Children's Mercy Hospital

The Arp2/3 Complex Is Essential At Multiple Stages Of Neural Development., Fu-Sheng Chou, Pei-Shan Wang

Manuscripts, Articles, Book Chapters and Other Papers

During development of the nervous system, radial glial cells perform self-renewing asymmetric divisions and give rise to intermediate progenitor cells (IPC) and neurons. The neuronally committed IPC subsequently undergo multiple rounds of transient amplification and migrate outwards to form cortical layers as they continue to differentiate into mature neurons. Maturing neurons extend protrusions on their cell surface to form neurites, a process called neuritogenesis. Neurite formation results in the establishment of dendrites and axons for synapse formation, which is essential for sensory and motor functions and even higher-level functioning including memory formation and cognitive function, as well as shaping of ...


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