IU Indianapolis ScholarWorks :: Browsing by Subject "Embryology"

Browsing by Subject "Embryology"

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Analysis of integration sites of transgenic sheep generated by lentiviral vectors using next-generation sequencing technology
(2014-07-31) Chen, Yu-Hsiang; Malkova, Anna; Cornetta, Kenneth; Randall, Stephen Karl, 1953-; Atkinson, Simon
The development of new methods to carry out gene transfer has many benefits to several fields, such as gene therapy, agriculture and animal health. The newly established lentiviral vector systems further increase the efficiency of gene transfer dramatically. Some studies have shown that lentiviral vector systems enhance efficiency over 10-fold higher than traditional pronuclear injection. However, the timing for lentiviral vector integration to occur remains unclear. Integrating in different stages of embryogenesis might lead to different integration patterns between tissues. Moreover, in our previous study we found that the vector copy number in transgenic sheep varied, some having one or more copies per cells while other animals having less than one copy per cell suggesting mosaicism. Here I hypothesized that injection of a lentiviral vector into a single cell embryo can lead to integration very early in embryogenesis but can also occur after several cell divisions. In this study, we focus on investigating integration sites in tissues developing from different germ layers as well as extraembryonic tissues to determine when integration occurs. In addition, we are also interested in insertional mutagenesis caused by viral sequence integration in or near gene regions. We utilize linear amplification-mediated polymerase chain reaction (LAM-PCR) and next- generation sequencing (NGS) technology to determine possible integration sites. In this study, we found the evidence based on a series of experiments to support my hypothesis, suggesting that integration event also happens after several cell divisions. For insertional mutagenesis analysis, the closest genes can be found according to integration sites, but they are likely too far away from the integration sites to be influenced. A well-annotated sheep genome database is needed for insertional mutagenesis analysis.
CFP1 governs uterine epigenetic landscapes to intervene in progesterone responses for uterine physiology and suppression of endometriosis
(Springer Nature, 2023-06-03) Yang, Seung Chel; Park, Mira; Hong, Kwon-Ho; La, Hyeonwoo; Park, Chanhyeok; Wang, Peike; Li, Gaizhen; Chen, Qionghua; Choi, Youngsok; DeMayo, Francesco J.; Lydon, John P.; Skalnik, David G.; Lim, Hyunjung J.; Hong, Seok-Ho; Park, So Hee; Kim, Yeon Sun; Kim, Hye-Ryun; Song, Haengseok; Biology, School of Science
Progesterone (P4) is required for the preparation of the endometrium for a successful pregnancy. P4 resistance is a leading cause of the pathogenesis of endometrial disorders like endometriosis, often leading to infertility; however, the underlying epigenetic cause remains unclear. Here we demonstrate that CFP1, a regulator of H3K4me3, is required for maintaining epigenetic landscapes of P4-progesterone receptor (PGR) signaling networks in the mouse uterus. Cfp1f/f;Pgr-Cre (Cfp1d/d) mice showed impaired P4 responses, leading to complete failure of embryo implantation. mRNA and chromatin immunoprecipitation sequencing analyses showed that CFP1 regulates uterine mRNA profiles not only in H3K4me3-dependent but also in H3K4me3-independent manners. CFP1 directly regulates important P4 response genes, including Gata2, Sox17, and Ihh, which activate smoothened signaling pathway in the uterus. In a mouse model of endometriosis, Cfp1d/d ectopic lesions showed P4 resistance, which was rescued by a smoothened agonist. In human endometriosis, CFP1 was significantly downregulated, and expression levels between CFP1 and these P4 targets are positively related regardless of PGR levels. In brief, our study provides that CFP1 intervenes in the P4-epigenome-transcriptome networks for uterine receptivity for embryo implantation and the pathogenesis of endometriosis.
CHAPTER 12 - Non-neoplastic diseases of the testis
(Elsevier, 2020-06-22) Nistal, Manuel; Paniagua, Ricardo; Bostwick, David G.; Cheng, Liang; Pathology and Laboratory Medicine, School of Medicine
Embryology in medical education: a mixed methods study and phenomenology of faculty and first year medical students
(2016-06) Cassidy, Keely Marie; O'Loughlin, Valerie Dean; Brokaw, James; Flinders, David; Husmann, Polly; Kearns, Katherine
The anatomical sciences are experiencing a notable decrease in the time and resources devoted to embryology in North American medical education. With more changes assured, it is necessary to investigate the current trends in curriculum, pedagogy, and related experiences of embryology teachers and learners. To address these concerns, the researcher developed two online mixed methods surveys: one for current anatomy and embryology faculty and another for first year medical students. The faculty survey was followed by interviews with volunteers from that cohort. The researcher used a grounded theory methodology to analyze the qualitative components of the surveys, and descriptive statistics to analyze the quantitative components of the surveys. Both the faculty and student surveys illuminated the vast differences between the explicit, implicit, and null curricular components found in the numerous medical education programs represented. A combined grounded theory methodology and phenomenological approach was used to analyze the interviews with faculty. This generated a lived experience narrative of the phenomenon of teaching embryological content to medical students in the modern world, which led to a better understanding of the needs and challenges that face this subject matter and those who teach it. In this fluid era of medical education reform and integration, the perceptions and experiences of anatomy and embryology faculty and first year medical students are invaluable to assessing the curriculum and pedagogy of this foundational anatomical science and formulating evidence-based recommendations for the future.
Integrated Case-Based Learning Session for Breast and Upper Limb Anatomy
(Association of American Medical Colleges, 2024-05-10) Merritt, Emily; McNulty, Margaret A.; Byram, Jessica N.; Anatomy, Cell Biology and Physiology, School of Medicine
Introduction: Medical students are frequently introduced to medical school curricula through anatomy coursework, which often includes histology and embryology content. As medical education has increasingly emphasized integration of content areas, use of activities such as case-based learning (CBL) sessions has grown. Little published work has demonstrated the effectiveness of CBL sessions in integrating anatomy, embryology, and histology on first-year medical students' ability to improve content mastery and adapt their study techniques. Methods: We developed a CBL session that included anatomy, embryology, and histology content covering the upper extremity and breast pathology that was taught to incoming first-year medical students (N = 51) during a prematriculation program in the summers of 2022 and 2023. The session involved completion of an individual pre- and postsession quiz; group completion of clinical cases involving image interpretation, matching exercises, and construction of diagrams, flowcharts, or tables; and a postsession survey with Likert-style and free-response questions about preparation and session effectiveness. Results: Postsession quiz scores significantly improved (p < .001). On the postsession survey (response rate: 59%), students commented that they enjoyed the real-life application and integration of the cases and that the sessions improved their understanding of the connections between content areas. Other comments demonstrated that students were evaluating and adapting their study approach in preparation for the sessions, often using techniques introduced and practiced in the sessions. Discussion: CBL sessions can provide opportunities to incoming first-year medical students to practice, adapt, and evaluate study techniques while delivering integrated content.
Molecular Basis and Modification of a Neural Crest Deficit in a Down Syndrome Mouse Model
(2013-07-12) Deitz, Samantha L.; Roper, Randall J.; Chang, Hua-Chen; Chernoff, Ellen; Atkinson, Simon
Down syndrome (DS) is the result of trisomy of human chromosome 21 (Hsa 21) and occurs in approximately 1/700 live births. Mouse models of DS have been crucial in understanding the gene-phenotype relationships that underlie many DS anomalies. The Ts65Dn mouse model, trisomic for half of the Hsa 21 orthologs replicates many DS phenotypes including craniofacial alterations such as a small, dysmorphic mandible, midface, and maxilla. Other mouse models, such as the Ts1Rhr which contains a triplication of 33 Hsa 21 orthologs, have been used to better understand the genes responsible for craniofacial alterations. Our laboratory has demonstrated that the postnatal mandibular phenotype found in Ts65Dn mice can be traced back to an original neural crest cell (NCc) deficit in the developing first pharyngeal arch (PA1) at embryonic day 9.5 (E9.5). Furthermore, evidence suggested that both a proliferation deficit in the PA1 and a migration deficit in the NCC from the neural tube (NT) could be the mechanism behind this deficit. However, the molecular mechanisms behind these deficits remain to be elucidated. Due to the involvement of the Hsa 21 genes DYRK1A and RCAN1 in regulation of signaling pathways including NFATc (NFAT2), a transcription factor known to influence cellular proliferation and, later, bone development, we hypothesized that dysregulation of these genes could underlie the cellular deficit in the PA1. Furthermore, we hypothesized that targeting Dyrk1a by decreasing activity or available protein could ameliorate the established deficits. Through the use of RNA isolation techniques and cell culture systems of cell from the PA1 and NT of E9.5 Ts65Dn, Ts1Rhr, and control embryos, we established that trisomic genes Dyrk1a and Rcan1 ara dysregulated in both structures and that these two genes may interact. Furthermore, we established that a proliferation deficit in the Ts65Dn PA1 and a migration deficit in the Ts65Dn PA1 and NT exists at E9.5 and can be rescued to euploid levels in vitro with the addition of the Dyrk1a inhibitor, EGCG, a green tea polyphenol. We also confirmed that harmine, a more highly studied and specific Dyrk1a inhibitor, is capable of similar effects on proliferation of PA1 cell from E9.5 Ts65Dn embryos. Furthermore, when Ts65Dn pregnant mothers were treated with EGCG in vivo, the cellular deficit found in the developing E9.5 embryonic PA1 was rescued to near euploid volume and NCC number. Treatment with EGCG did not adversely impact litter size or embryonic development. Interestingly, euploid embryonic volume increased with EGCG treatment. Expression analysis of the E9.5 PA1 of EGCG treated Ts65Dn and control embryos revealed dysregulation of several genes involved in craniofacial and developmental pathways including Dyrk1a, Rcan1, Ets2 and members of the sonic hedgehog pathways. Our novel results provide a foundation for better understanding the molecular mechanisms of craniofacial development and may provide evidence-based therapeutic options to improve the quality of life for individuals with DS.
Rescuing a broken heart: A tale of two Models of Neural Crest deficiency and its impact on In Utero Heart function and Embryonic Survival via the Beta-Adrenergic pathway
(2011-06-14) Olaopa, Michael A.; Conway, Simon J.; Firulli, Anthony B.; White, Kenneth E.; Rhodes, Simon J.
Congenital heart defects occur in approximately one percent of births every year, which makes it the most frequently occurring congenital defect in patients. The aim of this project was to use two mutant neural crest (NC) mouse models to study the mechanisms underlying congenital heart failure in utero. The first mouse model was a Pax3 systemic knockout, which was lethal by mouse gestational day 14, and had appreciably reduced numbers of migratory NC cells. The second mouse model was a Wnt1Cre-mediated NC genetic cell ablation model, which was surprisingly viable and survived to birth, despite an apparent lack of migratory NC cells. The resultant data indicated that both mouse models had similar heart structural defects including persistent truncus arteriosus, which was due to fewer or no migratory cardiac NC cells. However, in utero heart function was appreciably perturbed in Pax3 mutants when compared to that of the ablated mutant model. The loss of embryonic cardiac function in Pax3 mutants was directly attributed to a substantial decrease in the activity of the beta-adrenergic pathway. This was due to a lack of proper specification of trunk NC cells, leading to diminished levels of circulating catecholamine levels in the embryo. To definitively confirm this conclusion, poor cardiac function was successfully restored by pharmacological stimulation of the beta-adrenergic pathway via administration of isoproterenol and forskolin to pregnant dams, which led to embryonic survival of Pax3 mutants to birth. By comparison of these two mutant mouse models, perturbation in the beta-adrenergic pathway was identified as the underlying mechanism responsible for in utero heart failure and lethality in Pax3 mutant embryos. The results of this study are expected to be significant in developing future therapeutic targets for congenital heart failure in prenatal and newborn patients.
Single cell RNA analysis of the left-right organizer transcriptome reveals potential novel heterotaxy genes
(Springer Nature, 2023-07-01) Bellchambers, Helen M.; Phatak, Amruta R.; Nenni, Mardi J.; Padua, Maria B.; Gao, Hongyu; Liu, Yunlong; Ware, Stephanie M.; Pediatrics, School of Medicine
The establishment of left-right patterning in mice occurs at a transient structure called the embryonic node or left-right organizer (LRO). Previous analysis of the LRO has proven challenging due to the small cell number and transient nature of this structure. Here, we seek to overcome these difficulties to define the transcriptome of the LRO. Specifically, we used single cell RNA sequencing of 0-1 somite embryos to identify LRO enriched genes which were compared to bulk RNA sequencing of LRO cells isolated by fluorescent activated cell sorting. Gene ontology analysis indicated an enrichment of genes associated with cilia and laterality terms. Furthermore, comparison to previously identified LRO genes identified 127 novel LRO genes, including Ttll3, Syne1 and Sparcl1, for which the expression patterns were validated using whole mount in situ hybridization. This list of novel LRO genes will be a useful resource for further studies on LRO morphogenesis, the establishment of laterality and the genetic causes of heterotaxy.
Transgenic use of SMAD7 to suppress TGFß signaling during mouse development
(2010-10-21) Tang, Sunyong; Conway, Simon J.; Harrington, Maureen A.; Skalnik, David Gordon; Rhodes, Simon J.
Neural crest cells (NCC) are a multipotent population of cells that form at the dorsal region of neural tube, migrate and contribute to a vast array of embryonic structures, including the majority of the head, the septum of the cardiac outflow tract (OFT), smooth muscle subpopulations, sympathetic nervous system and many other organs. Anomalous NCC morphogenesis is responsible for a wide variety of congenital defects. Importantly, several individual members of the TGFβ superfamily have been shown to play essential roles in various aspects of normal NCC development. However, it remains unclear what role Smad7, a negative regulator of TGFβ superfamily signaling, plays during development and moreover what the spatiotemporal effects are of combined suppression of TGFβ superfamily signaling during NCC formation and colonization of the developing embryo. Using a cre/loxP three-component triple transgenic system, expression of Smad7 was induced via doxycycline in the majority of pre- and post-migratory NCC lineages (via Wnt1-Cre mice). Further, expression of Smad7 was induced via doxycycline in a subset of post-migratory NCC lineages (via Periostin-Cre mice, after the NCC had reached their target organs and undergone differentiation). Induction of Smad7 within NCC significantly suppressed TGFβ superfamily signaling, as revealed via diminished phosphorylation levels of both Smad1/5/8 and Smad2/3 in vivo. This resulted in subsequent loss of NCC-derived craniofacial, pharyngeal and cardiac OFT cushion tissues. ROSA26r NCC lineage mapping demonstrated that cardiac NCC emigration and initial migration were unaffected, but subsequent colonization of the OFT was significantly reduced. At the cellular level, increased cell death was observed, but cell proliferation and NCC-derived smooth muscle differentiation were unaltered. Molecular analysis demonstrated that Smad7 induction resulted in selective increased phospho-p38 levels, which in turn resulted in the observed initiation of apoptosis in trigenic mutant embryos. Taken together, these data demonstrate that tightly regulated TGFβ superfamily signaling is essential for normal craniofacial and cardiac NCC colonization and cell survival in vivo.

Browsing by Subject "Embryology"

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