1,721,035 research outputs found
Emergence of cardiac function and cell types during early embryogenesis
Get PDFThe heart is the first organ to form and function during embryonic development. Over the past few years, research on cardiac development has focused on uncovering the genetic mechanism controlling cardiac differentiation and clarifying the embryonic origin of the different cardiac lineages. The study of the physiological mechanisms behind cardiac function has proven instrumental for our understanding of heart disease and the interplay between different cardiac cells. However, there is not an extensive body of literature on the role of physiology on early embryonic development, especially on cardiac development. In this thesis, I present my work on the early stages of heart development, focusing on the events occurring from the formation of the cardiac crescent to the linear heart tube stage. This thesis is divided in 3 result chapters. In the first result chapter (chapter 3), I present the methods I developed to perform live imaging of mouse embryos, including calcium imaging, and to extract quantitative information.. On the second result chapter (chapter 4), I described the work I performed investigating the physiological mechanisms of early heart development, their role in cardiac differentiation, and a detailed single-cell RNA sequencing analysis of early heart development. On the last result chapter (chapter 5), I explore preliminary data on a potential contribution of endodermal cells to the formation of the heart. The work presented here has led to the discovery of Spontaneous Asynchronous Calcium Oscillations (SACOs) in the early cardiac mesoderm and a key role for NCX1 in regulating these oscillations and cardiac differentiation. Single-cell RNA sequencing analysis suggests a potential role for Naca and Fbxo32 in regulating early cardiac function. I have also identified potential new markers for both the First and Second Heart Field. Lastly, I found evidence that the endoderm may be contributing to the Second Heart Field, although the function of these cells is still unclear
Going Beyond Counting First Authors in Author Co-citation Analysis
Get PDFThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Investigating the molecular regulation and morphological dynamics of the Visceral Endoderm
Get PDFCell migration is of huge importance in the field of development, where the correct positioning of signaling centers and functional structures is of paramount importance. An example of this occurs within the Visceral Endoderm (VE), an extra-embryonic epithelium that is involved in patterning the embryo. A specialized subset of VE cells known as the anterior visceral endoderm (AVE) is induced at the distal tip of the egg∼cylinder, and is responsible for specifying anterior embryo pattern. AVE cells migrate in a stereotypic manner towards the prospective anterior. Failure of this migration leads to dramatic mis-patterning of the primary body axis and renders the embryo unviable. The main aim of my D.Phil is to investigate cell movements throughout the VE, in order to understand the behaviour of both the AVE and non-AVE cells and to give insight into the global cell dynamics of reorganizing epithelial tissues during development.
After using DIC and fluorescence microscopy to visualize migratory processes I then applied cell-tracking analysis to the resultant data. Confirming and furthering work that quantified cell movement on the anterior face of the embryo, I performed time∼lapse microscopy to visualize the entire surface of the embryo as the AVE migrated, in higher temporal and axial resolution than has been demonstrated to date. Cell∼tracking analysis of this data indicates that these areas exhibit different cell behaviour to that previously quantified for the anterior side of the embryo. Further to this, I have developed a novel method of imaging the distal tip of the egg∼cylinder embryo. Using this technique, I have generated high definition time∼lapse sequences of induced AVE cells prior to migration, allowing previously uncharacterized behavior to be described.
In order to fully understand the cell dynamics of the VE, I took extremely high spatial resolution fluorescence microscopy of the VE from live embryos in culture and manually segmented the tissue in 3D. This allowed the description of regional differences of cell shape within the tissue.
The Wnt/PCP pathway has been implicated in coordinating AVE migration. The second major aim of my project is to investigate how Planar Cell Polarity (PCP) signaling controls the behaviour of cells in the VE. I investigated the PCP core component Vangl2 using mice that contain a dominant negative version of this protein, and determined that there was no overt AVE migration defects in mutant embryos. In order to investigate the role of the Vangl2-binding PCP component Dvl-2, specifically examining whether localized recruitment of Dvl-2 in the VE is obligatory for normal AVE migration, I have generated genetic tools for use in transient transgenics. Further to this, I have demonstrated a role for the Dvl-2-binding protein β- arrestin2 in AVE migration, using mice null for this protein.</p
ETS2 regulation of tissue size in early mouse embryogenesis
Get PDFETS2 is a transcription factor with both oncogenic and tumour suppressive roles, yet
the mechanisms underlying these opposing effects remain unclear. I have analysed an Ets2-
loss-of-function mouse mutant that displays a diminished extra-embryonic ectoderm (ExE) on
one hand and a thickened anterior visceral endoderm (AVE) on the other. The opposing effects
of ETS2 on tissue size, relevant to its tumour suppressive and oncogenic properties, appear to
manifest in this mutant. Here I showed that the ExE defect is due to increased peri-implantation
apoptosis, verified by phenotypic rescue via apoptosis inhibition. I demonstrated that the AVE
transforms from a simple to stratified epithelium due to increased proliferation and
perpendicular cell divisions, reminiscent of neoplastic transformation. Inhibition of MEK,
which acts upstream of ETS2, phenocopied the AVE defect in wild type (WT) embryos. To
identify ETS2 transcriptional targets that cause these AVE defects, I conducted single-cell
RNA-sequencing (scRNA-seq), which showed that all cell types in the Ets2-mutant retain
normal character; and revealed upregulation of several WNT/β-CATENIN pathway agonists
in the mutant AVE. Pharmacological activation of the WNT/β-CATENIN pathway in WT
recapitulated the mutant AVE phenotype, supporting scRNA-seq results. 2C-HR-CRIPSR
mediated misexpression of candidate WNT agonists in the VE of WT embryos was attempted
to verify whether the novel ETS2-WNT axis of AVE tissue size regulation is relevant in vivo.
Overall, I have demonstrated the opposing effects of ETS2 on tissue size in early mouse
embryogenesis: ETS2 protects the peri-implantation ExE from apoptosis, while dampens
WNT/β-CATENIN signalling in the AVE to prevent overproliferation and maintain simple
epithelial integrity.</p
Variations on the Author
Get PDF“Variations on the Author” discusses two of Eduardo Coutinho’s recent films (Um Dia na Vida, from 2010, and Últimas Conversas, posthumously released in 2015) and their contribution to the general question of documentary authorship. The director’s filmography is characterized by a consistent yet self-effacing form of authorial self-inscription: Coutinho often features as an interviewer that rather than express opinions propels discourses; an interviewer that is good at listening. This mode of self-inscription characterizes him as an author who is not expressive but who is nonetheless markedly present on the screen. In Um Dia na Vida, however, Coutinho is completely absent form the image, while Últimas Conversas, on the contrary, includes a confessional prologue that moves the director from the margins to the center of his films. This article examines the ways in which these works stand out in the filmography of a director who offers new insights into the notion of cinematic authorship
Appropriate Similarity Measures for Author Cocitation Analysis
Get PDFWe provide a number of new insights into the methodological discussion about author cocitation analysis. We first argue that the use of the Pearson correlation for measuring the similarity between authors’ cocitation profiles is not very satisfactory. We then discuss what kind of similarity measures may be used as an alternative to the Pearson correlation. We consider three similarity measures in particular. One is the well-known cosine. The other two similarity measures have not been used before in the bibliometric literature. Finally, we show by means of an example that our findings have a high practical relevance.information science;Pearson correlation;cosine;similarity measure;author cocitation analysis
Investigating the role of ASPP2 in post-implantation mouse embryos
Get PDFMy research focuses on the role of ASPP2 (Apoptosis-stimulating of p53 protein 2, encoded by the TP53BP2 gene) during early post-implantation development. ASPP2 has multiple important biological functions, including roles in the control of cell polarity, tight junction formation, cell proliferation, differentiation and apoptosis.
To study the molecular basis of ASPP2 function in post-implantation development, I generated a mouse line in which exon 4 of ASPP2 is flanked by LoxP sites. Using genetic crossings, this allowed me to generate various mouse lines that were used to study the function of ASPP2 in the whole embryo and in specific tissues.
My results indicate that gastrulation is able to proceed in ASPP2-/- embryos – all 3 germ layers appear to form correctly. However, the embryo becomes progressively more disordered as development proceeds, resulting in embryonic lethality by E9.5. Somites are present at E8.5 and E9.5, showing that mesoderm-derived structures can form. The somites, however, are smaller than their wildtype counterparts. Additionally, instead of the normal structure of a single layer of somitic cells surrounding a central cavity, the somites have a disorganised structure with multiple cell layers and often lack a cavity.
Heart formation is also affected in these mutant embryos. By E9.5, there is no heart structure, with the absence of a beating heart. I crossed the ASPP2flox/flox mouse line to a line expressing CRE under the Mesp1 promoter, which controls early cardiac progenitor formation. These mice developed normal hearts at E8.5 and E9.5, implying that the perturbation in cardiac development is a secondary defect, likely caused by disturbances to the surrounding tissues meaning that the cardiac progenitors do not receive the signals they require to continue developing.
Thus, my research has indicated that ASPP2 is essential for post-implantation embryonic development. The mesoderm initially forms correctly but as the embryo continues to develop, the mesoderm-derived somites become disrupted, indicating a role for ASPP2 in regulating the epithelial architecture of the somites. Similarly, as seen in the heart, the disorganisation of structures leads to secondary defects later in development, ultimately resulting in embryonic lethality. These results demonstrate the importance of ASPP2 and shed light on some of the functions that it may play in development
Study of sex determination in mice lacking candidate genes identified by human exome sequencing
Get PDFDisorders of sex development (DSD) comprise a heterogeneous group of conditions in which human sex development is atypical. The research described in this thesis aims at improving our understanding of DSDs, in particular those characterised by abnormal gonad development (gonadal dysgenesis, GD), using the mouse as a model of human sex development. I discuss four genes (TLE3, SEC31A, NCOR2 and ZNRF3) identified by exome sequencing screens of individuals with 46,XY GD and experiments aimed at assessing their candidacy as human testis-determining genes and, in the case of one (ZNRF3), exploring mechanisms that lead to mouse embryonic gonadal sex reversal in their absence.
Sexual development in the mouse starts at around 10 days post coitum (dpc), with the formation of the bipotential gonadal primordium on the surface of the mesonephros. At around 11.5 dpc, the gonad acquires a fate determined by the chromosomal sex of the individual – a process known as gonadal sex determination. In XY embryos, SRY expression from the Y chromosome results, by 14.5 dpc, in Sertoli cell differentiation and testis development. In XX embryos, in the absence of SRY, canonical WNT signals and FOXL2 cause granulosa cell specification and ovary development. Therefore, I examined mouse lines that lack function of the four genes discussed above during the period 11.5 to 14.5 dpc.
Loss of the transcriptional co-repressor TLE3 did not result in any overt gonadal phenotype. By contrast, loss of SEC31A, a component of the coat protein complex II (COPII), led to very early embryonic lethality, and I investigate the molecular and cellular basis of this lethality. NCOR2 encodes a nuclear receptor co-repressor that mediates transcriptional silencing of target genes. Exome sequencing identified sequence variants in unrelated 46,XY and 46,XX individuals with DSD, across 3 continents, making it the strongest of the candidate genes. I describe subtle but clear defects in testis and ovary development in a mouse line lacking NCOR2 function, dependent on the mouse genetic background in which the loss-of-function phenotype was analysed. These data support a role for NCOR2 in human sexual development and DSD, and warrant further investigations.
Finally, I describe experiments aimed at understanding the role of the anti-WNT ubiquitin ligase ZNRF3 in mouse testis determination, a role that was reported during the research period covered by this thesis. I show cell proliferation defects in the Znfr3 null XY gonad and discuss the relevance of these to the sex reversal phenotype. I also describe experiments aimed at being able to detect endogenous ZNRF3 protein, using antibody generation and CRISPR/Cas9-mediated epitope tagging of the endogenous gene. Finally, I describe data from an RNA sequencing (RNAseq) experiment aimed at identifying the transcriptional consequences of Znrf3 ablation at the sex-determining stage of gonadogenesis.
I conclude by discussing the benefits and limitations of using the mouse model for study of primary sex determination in humans, the importance of genetic background and suggest ways in which the success rate of this approach might be improved in future, by incorporating new models
PIERCE1: a determinant of left-right asymmetry in mammals
Get PDFThe study of mice with heart and situs defects can provide useful models for improving our understanding of congenital heart defects in humans. This project characterised the involvement of 1700007K13Rik (PIERCE1) in left-right patterning. A proportion of homozygotes with a mutation in this gene were identified by the International Mouse Phenotyping Consortium as exhibiting situs inversus totalis and heart defects, which are indicative of faulty left-right patterning. We hypothesised that this gene is involved in the establishment of ciliary motility at the embryonic node. Motile cilia are also found in the trachea, and if the tracheal cilia of knock-out mice also have motility defects, C9orf116 (the human orthologue of 1700007K13Rik) may be a new candidate gene for primary ciliary dyskinesia, a heterogeneous recessive condition.
The evaluation of nodal ciliary motility and nodal flow in mice homozygous for a Pierce1-null allele demonstrated that aberrant motility, leading to a lack of effective leftwards nodal flow, underlies the left-right patterning defects in these mice. In addition to this, studies of tracheal ciliary and sperm flagellar motility showed that PIERCE1 is required for correct motility in all classes of motile cilia and flagellar evaluated. Transmission electron microscopy studies point towards a role for PIERCE1 in the assembly or trafficking of dynein arms and their components; however, protein interaction studies have proved inconclusive thus far at placing PIERCE1 within the network of known dynein arm assembly factors.</p
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