75 research outputs found

    Quantifying the Frequency and Orientation of Mitoses in Embryonic Epithelia

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    The miraculous birth of a new life starts by the formation of an embryo. The process by which an embryo is formed, embryogenesis, has been studied and shown to consist of three types of processes: mitosis, cell differentiation and morphogenetic movements. Scientists and medical doctors are still at a loss to explain the fundamental forces driving embryo development and the causes of birth defects remain largely unknown. Recent efforts by the Embryo Biomechanics Lab at the University of Waterloo have shown a relationship between morphogenetic movements that occur during embryo formation and the frequency and orientation of mitosis. To further study this relationship a means of automatically identifying the frequency and orientation of mitosis on time-lapse images of embryo epithelia is needed. Past efforts at identifying mitosis have been limited to the study of cell cultures and stained tissue segments. Two methods for identifying mitosis in contiguous sheets of cells are developed. The first method is based on local motion analysis and the second method is based on intensity analysis. These algorithms were tested on images of early and late stage embryos of the axolotl (Ambystoma mexicanum), a type of amphibian. The performance of the algorithms were measured using the F-Measure. The F-Measure determines the performance of the algorithm as the true mitosis detection rate penalized by the false mitosis detection rate. The motion based algorithm had performance rates of 68.2% on an early stage image set and 66.7% on a late stage image set, whereas the intensity based algorithm had a performance rates of 73.9% on early stage image set and 90.0% on late stage image set. The mitosis orientation errors for the motion based algorithm were 27.3 degrees average error with a standard deviation (std.) of 19.8 degrees for early stage set and 34.8 degrees average error with a std. of 23.5 degrees for the late stage set. For the intensity based algorithm the orientation errors were 39.8 degrees average with std. of 28.9 degrees for the early stage image set and 15.7 degrees average with std. of 18.9 degrees for the late stage image set. The intensity based algorithm had the best performance of the two algorithms presented, and the intensity based algorithm performs best on high-magnification images. Its performance is limited by mitoses in adjacent cells and by the presence of natural cell pigment variations. The algorithms presented here offer a powerful new set of tools for evaluating the role of mitoses in embryo morphogenesis

    Rapid mechanosensitive migration and dispersal of newly divided mesenchymal cells aid their recruitment into dermal condensates

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    Embryonic mesenchymal cells are dispersed within an extracellular matrix but can coalesce to form condensates with key developmental roles. Cells within condensates undergo fate and morphological changes and induce cell fate changes in nearby epithelia to produce structures including hair follicles, feathers, or intestinal villi. Here, by imaging mouse and chicken embryonic skin, we find that mesenchymal cells undergo much of their dispersal in early interphase, in a stereotyped process of displacement driven by 3 hours of rapid and persistent migration followed by a long period of low motility. The cell division plane and the elevated migration speed and persistence of newly born mesenchymal cells are mechanosensitive, aligning with tissue tension, and are reliant on active WNT secretion. This behaviour disperses mesenchymal cells and allows daughters of recent divisions to travel long distances to enter dermal condensates, demonstrating an unanticipated effect of cell cycle subphase on core mesenchymal behaviour

    Junctional complexes and cell-cell signalling in zebrafish morphogenesis

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    Intercellular junctions are composed of tight, gap and adherens junctions and have been shown to play many roles in embryonic morphogenesis. I have been studying the role that intercellular junctions play in zebrafish development. First I have studies the organisation of apical neuroepithelial junctions in the developing brain. By quantifying size and segmental patterning of the junctional arrangement in the hindbrain and elsewhere I have described a level of organisation that is characteristic of compartment boundaries. I describe a distinct pattern of apical junctions in both boundary and non-boundary regions. This pattern appears to be in part regulated by the Notch signalling pathway since apical junctions distribution is different in hdac1-/- zebrafish, which have deficient Notch-Delta signalling. Second I have established that boundary cells prevent the exchange of small molecular weight, gap junction permeable dyes between adjacent CNS compartments, suggesting that the compartments are separate developmental units. Third I have studies the role of gap junction mediated intercellular communication in the propagation of calcium waves and in the coordination of cell divisions in the zebrafish blastocyst. Calcium activity is cyclical, with cells producing a greater number of calcium transients and intercellular waves during cytokinesis than during interphase. In control conditions distinct waves of cell divisions spread across the embryo in an animal to vegetal progression. I show that both the calcium activity and the cell division waves require gap junction communication because pharmacological blockade of coupling reduces the frequency of calcium activity and disrupts cell division waves


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    Oriented cell divisions contribute to tissue morphogenesis and homeostasis. Planar divisions occurring with the spindle within the epithelial plane enlarge sheets and tubules, while asymmetric cell divisions with the spindle aligned to the apico-basal polarity axis sustain differentiation programs. Several pathways have been involved in establishing correct spindle orientation, both in cultured cells and in vivo. Most of these pathways impinge on the evolutionarily conserved G\u3b1i/LGN/NuMA complexes that orient the spindle by generating pulling forces on astral microtubules (MTs), via direct interaction of NuMA with the MT-motors Dynein/Dynactin. My PhD projects focused on the molecular mechanisms underlying the spindle orientation function of Afadin, and on the relevance of NuMA phosphorylation by Aurora-A for spindle orientation. During planar cell divisions, G\u3b1i/LGN/NuMA assemblies are restricted to the lateral cortex, for molecular reasons that are still unclear. Studies conducted during this thesis indicate that LGN interacts directly with the junctional and F-actin binding protein Afadin, and define the TPR domain of LGN (hereon LGNTPR) and a C-terminal peptide of Afadin (AfadinPEPT) as the minimal interacting regions retaining micromolar binding affinity. The crystal structure of the LGNTPR-AfadinPEPT fusion protein shows that the AfadinPEPT threads along the LGNTPR superhelix with opposite chain directionality, similarly to what observed for LGN in complex with other ligands, including NuMA. Consistently, we provided evidence that Afadin competes with NuMA for binding to LGN. Afadin knock-down in HeLa cells leads to reduced LGN cortical levels, and unexpectedly also to complete loss of cortical NuMA and Dynein/Dynactin, and hence spindle misorientation. Importantly, we discovered that Afadin interacts concomitantly with F-actin and LGN in vitro. Furthermore, we showed that loss of Afadin impairs correct cystogenesis of Caco-2 cells, suggesting that it plays essential functions in epithelial planar cell divisions. Altogether our data suggest a model whereby in metaphase Afadin mediates cortical recruitment of Dynein/Dynactin, by targeting LGN at the lateral cortex via direct and concomitant interaction with LGN and with cortical F-actin. Later, LGN engages with NuMA and Dynein/Dynactin to exert pulling forces on the mitotic spindle. Thus, Afadin represents the first described mechanical anchor between the acto-myosin cell cortex and the Dynein/Dynactin MT-motors. Besides being spatially regulated, the cortical recruitment of G\u3b1i/LGN/NuMA is timely controlled by mitotic kinases coordinating spindle orientation with mitotic progression. It was reported that the activity of the mitotic kinase Aurora-A is required for correct spindle orientation in human cells in culture, and that NuMA is among its phosphorylation targets. However, whether NuMA is phosphorylated directly by Aurora-A and how molecularly its kinase activity affects spindle orientation was still unknown when we started our studies. Analyses in HeLa and RPE-1 cells revealed that, in metaphase, depletion or inhibition of Aurora-A leads to aberrant accumulation of NuMA at the spindle poles and loss from the cortex, despite LGN localizes normally at the cortex. FRAP experiments revealed that Aurora-A governs the dynamic exchange between the cytoplasmic and the spindle pole-localized pools of NuMA. Our experiments in vitro and in cells showed that Aurora-A phosphorylates directly three serine residues on the C-terminus of NuMA, and mutation of Ser1969 into alanine recapitulates the aberrant polar accumulation of NuMA and the spindle orientation defects observed upon Aurora-A inhibition. Thus we concluded that phosphorylation on Ser1969 of NuMA by Aurora-A controls NuMA distribution between the spindle poles and the overlying cortex, and allows proper spindle orientation. Intriguingly, Ser1969 lies within a previously characterized microtubule (MT)-binding domain. However, in vitro co-sedimentation and bundling assays revealed that the binding affinity of NuMA for MTs is unaltered by Aurora-A-mediated phosphorylation, suggesting that unphosphorylated NuMA accumulates at spindle poles via a receptor other than MTs. Most interestingly, with our experiments we also identified a new MT-binding domain of NuMA positioned downstream of the LGN binding motif. This result is consistent with our finding that NuMA can simultaneously interact with LGN and MTs. Based on these data, we propose that in metaphase the MT-binding activity of NuMA may contribute to anchor astral MT +TIPs at cortical sites together with LGN

    Cytokinesis in the mouse preimplantation embryo : mechanism and consequence of failure

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    Essentiel au maintien d‚Äôun organisme sain, la division cellulaire est un processus biologique compos√©e de deux phases : la mitose et la cytokin√®se. Au cours de la mitose, un fuseau mitotique bipolaire est assembl√© et les chromosomes s‚Äôalignent au niveau de la plaque m√©taphasique par l‚Äôattachement des kin√©tochores aux microtubules du fuseau. Une fois les chromosomes align√©s, les chromatides soeurs sont s√©par√©es par les microtubules pendant l'anaphase et sont s√©gr√©gu√©es entre les cellules filles. La cytokin√®se est initi√©e peu apr√®s le d√©but de l'anaphase, marquant ainsi la fin de la division cellulaire en s√©parant le cytoplasme en deux nouvelles cellules filles. Une ex√©cution pr√©cise de la mitose et de la cytokin√®se est essentielle pour le maintien de l'int√©grit√© du g√©nome. L'√©chec de l'un de ces processus affecte la fid√©lit√© g√©n√©tique. Les erreurs de s√©gr√©gation des chromosomes durant la mitose peuvent entra√ģner un gain ou une perte de chromosomes entiers, appel√© aneuplo√Įdie. Tandis que l'√©chec de la cytokin√®se conduit √† la formation d'une cellule binucl√©√©e avec un g√©nome enti√®rement dupliqu√©, appel√© t√©traplo√Įdie. Dans les cellules somatiques, la t√©traplo√Įdie peut conduire √† l'arr√™t du cycle cellulaire, √† la mort cellulaire, ou provoquer une instabilit√© chromosomique (CIN), favorisant ainsi la prolif√©ration de cellules avec un potentiel tumorig√®ne. Par cons√©quent, il est essentiel de bien comprendre la r√©gulation et les causes potentielles de l‚Äô√©chec de la cytokin√®se en particulier dans le contexte des syst√®mes multicellulaires comme l‚Äôembryon. En effet, dans ces syst√®mes, la r√©duction progressives de la taille des cellules co√Įncident avec les principaux √©v√®nements du d√©veloppement. De plus, la binucl√©ation est fr√©quemment observ√©e dans les cliniques de fertilit√© chez les embryons humains. Cependant, l‚Äôimpact de la binucl√©ation sur les divisions pr√©implantatoires demeure inexpliqu√© √† ce jour. Afin de d√©terminer les cons√©quences de la t√©traplo√Įdie, nous avons utilis√© l'embryon de souris pour mod√®le et r√©alis√© des exp√©riences d'immunofluorescence √† haute r√©solution et une imagerie sur cellules vivantes. Nous avons d√©couvert que la t√©traplo√Įdie chez les embryons de souris provoque une CIN et l'aneuplo√Įdie par un m√©canisme diff√©rent de celui des cellules somatiques. Dans les cellules somatiques, la formation des fuseaux multipolaires caus√©e par des centrosomes surnum√©raires est le principal m√©canisme conduisant √† la t√©traplo√Įdie et ainsi, √† une CIN. En revanche, chez les embryons de souris, qui ne poss√®dent pas de centrosomes, la t√©traplo√Įdie ne conduit pas √† la formation des fuseaux multipolaires. Les embryons t√©traplo√Įdes de souris d√©veloppent une CIN en raison d‚Äôune r√©duction du renouvellement des microtubules et d‚Äôune alt√©ration de l‚Äôactivit√© de correction d‚Äôerreurs dans l‚Äôattachement des kin√©tochores aux microtubules. Ainsi, une mauvaise correction de l‚Äôattachement des kin√©tochores aux microtubules entra√ģne des niveaux √©lev√©s d'erreurs de s√©gr√©gation chromosomique. Dans le cadre d'une √©tude de suivi, nous avons ensuite utilis√© des diff√©rentes exp√©riences d'imageries sur des cellules vivantes et d'immunofluorescences. Celles-ci furent coupl√©es √† des micromanipulations de la taille des cellules, des techniques modifiant l'adh√©sion cellulaire et des approches de knock-down des prot√©ines pour √©tudier les m√©canismes de r√©gulation de la cytokin√®se. Les exp√©riences d'imageries sur cellules vivantes et les micromanipulations du volume cytoplasmique ont d√©montr√© que la taille des cellules d√©termine la vitesse de constriction de l'anneau contractile, c'est-√†-dire que la vitesse de constriction devient progressivement plus lente √† mesure que la taille des cellules diminue. Cependant, ce ph√©nom√®ne n'a lieu que lorsque les embryons atteignent le stade de 16 cellules ce qui sugg√®re qu'une limite sup√©rieure de vitesse de constriction peut exister pour restreindre l‚Äôaugmentation de cette vitesse quand les cellules sont trop grandes. La taille des cellules √©tant un d√©terminant de la progression de la cytokin√®se, nos exp√©riences de knock-down des prot√©ines ont, de plus, d√©montr√© que la formation de la polarit√© cellulaire a un impact n√©gatif sur l'assemblage et la constriction de l'anneau contractile dans les cellules externes au stade de morula. Plus pr√©cis√©ment, nous avons constat√© que la polarit√© limite le recrutement des composants de la cytokin√®se sp√©cifiquement d'un c√īt√© de l'anneau contractile, provoquant ainsi un d√©s√©quilibre de l‚Äôingression du sillon de clivage et r√©duisant la vitesse de constriction dans les cellules externes. Nous sp√©culons que la polarit√© cellulaire agit comme un obstacle √† la progression de la cytokin√®se, rendant ainsi les cellules externes plus sensibles √† un √©chec de la cytokin√®se. Ces √©tudes ont d√©montr√© un nouveau m√©canisme par lequel la t√©traplo√Įdie conduit √† l‚Äôinstabilit√© chromosomique et √† l‚Äôaneuplo√Įdie chez les embryons. Ainsi un d√©faut de la dynamique de correction de l‚Äôattachement des kin√©tochores aux microtubules entra√ģne une mauvaise s√©gr√©gation des chromosomes ind√©pendamment √† la formation des fuseaux multipolaires. Ce travail a mis en √©vidence un r√īle inhibiteur de la polarit√© apicale inattendu sur la machinerie cytokin√©tique. Cette inhibition pourrait fournir une explication m√©canistique de l‚Äôincidence √©lev√©e de la binucl√©ation dans le trophectoderme. Dans l'ensemble, ces r√©sultats contribuent √† notre compr√©hension du contr√īle spatio-temporel de la cytokin√®se au cours du d√©veloppement embryonnaire et fournissent de nouvelles informations m√©canistiques sur les origines et les cons√©quences biologiques de la t√©traplo√Įdie chez les embryons pr√©implantatoires. Les r√©sultats pr√©sent√©s dans cette th√®se ont des implications cliniques importantes, puisqu‚Äôils fournissent des preuves d√©finitives que la t√©traplo√Įdie g√©n√©r√©e par un √©chec de la cytokin√®se est d√©l√©t√®re pour le d√©veloppement embryonnaire. Ces travaux mettent ainsi en lumi√®re que la binucl√©ation est un crit√®re de s√©lection embryonnaire important √† consid√©rer lors des traitements de fertilit√©.Cell division is comprised of mitosis and cytokinesis and is an essential biological process for the maintenance of healthy organisms. During mitosis, a bipolar spindle is assembled, and the chromosomes are aligned at the metaphase plate via the attachment of kinetochores to spindle microtubules. Once chromosome alignment is achieved, the sister chromatids are pulled apart by the microtubules during anaphase and segregated into the nascent daughter cells. Cytokinesis is initiated after anaphase onset and marks the completion of cell division by partitioning the cytoplasm of the dividing cell into two new daughter cells. Successful and timely completion of both mitosis and cytokinesis is key for the maintenance of genome integrity, and failure in either one of these processes affects genetic fidelity. Whereas chromosome segregation errors in mitosis can lead to whole chromosome gains or losses, termed aneuploidy, cytokinesis failure leads to the formation of a binucleated cell with an entirely duplicated genome, termed tetraploidy. In somatic cells, tetraploidy can either lead to cell cycle arrest and death or cause chromosomal instability (CIN), thereby promoting the proliferation of cells with high tumorigenic potential. Therefore, understanding cytokinesis regulation and the potential causes of cytokinesis failure is key, especially in the context of multicellular embryonic systems, wherein progressive cell size reductions coincide with developmental transitions. Moreover, binucleation is frequently observed in human embryos in fertility clinics, and whether binucleation impacts early divisions remains elusive. To elucidate the consequences of tetraploidy, we used the mouse embryo as a model and employed high-resolution immunofluorescence and live-cell imaging experiments. We found that tetraploidy in mouse embryos causes CIN and aneuploidy by a mechanism distinct from that of somatic cells. Whereas in somatic cells multipolar spindle formation caused by supernumerary centrosomes is the major mechanism by which tetraploidy leads to CIN, in mouse embryos - which are acentriolar ‚Äď tetraploidy does not lead to multipolar spindle formation. Instead, mouse tetraploid embryos develop CIN due to reduced microtubule turnover and impaired error correction activity, which prevents the timely resolution of kinetochore-microtubule mis-attachments, thereby leading to high levels of chromosome segregation errors. As a follow-up study, we next employed live imaging and immunofluorescence experiments, coupled with micromanipulations of cell size, cell adhesion and protein knockdown approaches to investigate the regulatory mechanisms of cytokinesis. Live imaging experiments and micromanipulations of cytoplasmic volume demonstrated that cell size determines the speed of contractile ring constriction i.e., constriction speed becomes progressively slower as the cells decrease in size. However, this phenomenon takes place only when embryos reach the 16-cell stage, suggesting that an upper limit of constriction speed may exist to restrict the scalability of ring constriction to cell size. In addition to cell size being a powerful determinant of cytokinesis progression, our loss-of-function experiments revealed that the emergence of cell polarity negatively impacts contractile ring assembly and constriction in outer cells at the morula stage. More specifically, we found that polarity limits the recruitment of cytokinesis components specifically to one side of the contractile ring, thereby causing unbalanced furrow ingression and reducing constriction speed in outer cells. We speculate that cell polarity may act as an obstacle for cytokinesis progression and render outer cells to be more susceptible to cytokinesis failure. These studies have revealed a novel mechanism by which tetraploidy leads to chromosomal instability and aneuploidy in embryos, wherein defective kinetochore-microtubule dynamics cause chromosome mis-segregation in a manner independent of multipolar spindle formation. In addition, this work unravelled an unexpected inhibitory role of apical polarity on the cytokinetic machinery that might provide a mechanistic explanation for the high incidences of binucleation in the outer layer of blastocysts. Altogether, these findings contribute to our understanding of the spatiotemporal control of cytokinesis during embryonic development and provide new mechanistic insights into the origins and biological consequences of tetraploidy in preimplantation embryos. The results presented in this thesis have substantial clinical implications, as they provide definitive evidence that tetraploidy generated by cytokinesis failure is deleterious to embryonic development, therefore underlining binucleation as an important embryo selection criterion to be considered during fertility treatments

    Doctor of Philosophy

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    dissertationMechanosensory hair cell damage leads to permanent hearing loss. The zebrafish mechanosensory organs (neuromasts) are an excellent model for the study of hair cell regeneration. Neuromasts have a central group of sensory hair cells surrounded by support cells and the outermost mantle cells. While adult mammalian hair cells cannot regenerate, neuromast hair cells have the ability to do so via support cell proliferation. In this study, we used proliferation assays and lineage analyses that demonstrated the existence of proliferative compartments in the posterior lateral line neuromasts where support cells either self-renew or differentiate. These spatially restricted lineage decisions within the progenitor pool resemble the spatial heterogeneities within the intestinal crypt or the hair follicle niche that guide stem cell transit amplification or differentiation. In addition, we identified the mantle cells as a quiescent support cell pool that do not proliferate in response to selective hair cell ablation, but that re-enter the cell cycle when hair cell and support cells numbers are drastically reduced. By combining our lineage analysis with gene expression analyses, genetic manipulations, and the use of chemical inhibitors, we were able to dissect the roles of Notch and Wnt signaling during homeostasis and regeneration. We demonstrate that Notch signaling restricts hair cell differentiation and maintains the spatial pattern of support cell proliferation through Wnt signaling inhibition. iv Thus, Notch-Wnt signaling interactions are required to maintain pools of amplifying support cells in the poles and maintain tissue homeostasis by balancing self-renewal and differentiation

    Design and implementation of transgenic tools to visualise cell cycle progression in mammalian development

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    Cell cycle progression is the series of steps a cell has to take in order to duplicate its DNA and produce two daughter cells. Correct spatial and temporal coordination of the cell cycle is key for the normal development of any organ or tissue and is stringently controlled during embryogenesis and homeostasis. Misregulation of cell cycle progression is causal in many developmental disorders and diseases such as microcephaly and cancer. Fucci (Fluorescent Ubiquitination based Cell Cycle Indicator) is a system that allows for the visualisation of cell cycle progression by the use of two differently coloured fluorescent probes whose abundance is regulated reciprocally during the cell cycle. The probes contain the E3 ligase recognition domains of Cdt1 and Geminin fused to the fluorophores mCherry (red fluorescence) and mVenus (yellow fluorescence) respectively. Cells are therefore labelled red during G1, yellow in the G1/S transition and green during late S/G2 and M phases of the cell cycle. In order to study development and tissue homoeostasis a Fucci expressing mouse line was developed however this has several key limitations: First, the two Fucci probes are expressed from separate loci complicating mouse colony maintenance. Second, the constructs were not inducible, making it impossible to follow cell cycle progression in specific cell lineages and third the mice were generated by random transgenesis which is prone to silencing and can exhibit variation in expression between different tissues. Here I have characterised an improved version of the original Fucci system known as Fucci2a designed by Dr Richard Mort (University of Edinburgh) to overcome these limitations. The Fucci2a genetic construct contains both Fucci probes fused with the Thosea asigna virus self-cleaving peptide sequence T2A. This allows expression of both probes as a single bicistronic mRNA with subsequent cleavage by ribosomal ‚Äėskipping‚Äô during translation to yield separate proteins. A Fucci2a mouse (R26Fucc2aR) was generated by homologous recombination into the ROSA26 locus using the strong, ubiquitous CAG promoter to drive expression and incorporating a floxed-Neo stop cassette. This allows tissue specific activation by Cre recombinase when combined with a second Cre expressing mouse line. Building on the bicistronic Fucci2a technology I have gone on to develop and characterise four new tricistronic reporter constructs which allow for the dual visualisation of cell cycle progression with apoptosis, cytokinesis and ciliogenesis. In each case an additional fluorescent probe was added to the original Fucci2a construct separated by the self-cleaving peptide P2A and the construct characterised in 3T3 stable cell lines. The combination of a dual cilia and cell cycle reporter construct proved fruitful and I have gone on to investigate the relationship between cell cycle progression and ciliogenesis in 3T3 cells and have generated and characterised the R26Arl13b-Fucci2aR mouse line. I have also illustrated the utility of the R26Fucci2aR mouse for generating quantitative data in development research in two development situations; melanocyte development and lung branching morphogenesis. Melanocytes are specialised melanin producing cells responsible for the pigmentation of the hair, skin and eyes. Their precursors, melanoblasts, are derived from the neural crest where they migrate and proliferate before becoming localised to hair follicles and their study provides a good model for understanding the development of other neural crest derived lineages such as the peripheral nervous system. Using time-lapse imaging of ex vivo skin cultures in which melanoblasts are labelled with the Fucci probes I have characterised melanoblast migration and proliferation. In addition, I have shown that Kit signalling, which is necessary for melanoblast migration and survival, controls melanoblast proliferation in a density dependent manner and that melanoblast migration is more persistent in S/G2/M phases of the cell cycle. Lung branching morphogenesis requires constant proliferation at the apical tip of a growing epithelial branch. Loss of epithelial symmetry through an unidentified mechanism (requiring BMP, FgF10, Shh and Wnt signalling) within a branch is required to initiate branching either latterly from the side of a elongating branch by domain branching or by bifurcation of the tip. In the final section of this thesis I performed a comparative analysis of the behaviour of the developing lung epithelium using proliferative status (Fucci2a expression) to categorise each cell. Using a combination of live imaging and immunohistochemistry I have identified a transition zone 100-150őľm from the tip of the branching lung epithelium where epithelial cells become stationary and drop out of the cell cycle corresponding with the onset of proximal bronchial progenitor marker Sox2. A comparative gene expression analysis of the proliferating and non-proliferating regions using Fucci2a to distinguish them has eluded to several interesting genes which could influence branching morphogenesis during lung development

    Funkńćn√≠ role SOX2 v neurosenzorick√©m v√Ĺvoji vnitŇôn√≠ho ucha

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    Neurony a senzorick√© buŇąky, kter√© jsou hlavn√≠mi funkńćn√≠mi buŇąkami vnitŇôn√≠ho ucha, vznikaj√≠ ze spoleńćn√© embryon√°ln√≠ epiteli√°ln√≠ neurosenzorick√© dom√©ny. K pochopen√≠ patofyziologie ztr√°ty sluchu je kl√≠ńćov√© identifikovat geny, kter√© se pod√≠l√≠ na specifikaci a diferenciaci senzorick√Ĺch bunńõk a neuronŇĮ ze spoleńćn√©ho prekurzoru. Nńõkter√© z tńõchto faktorŇĮ jsou nezbytn√© nejen pro vnitŇôn√≠ ucho, ale tak√© pro v√Ĺvoj dalŇ°√≠ch smyslŇĮ, jako jsou zrakov√Ĺ a ńćichov√Ĺ syst√©m. C√≠lem t√©to pr√°ce bylo popsat dŇĮleŇĺitost jednoho z tńõchto faktorŇĮ, transkripńćn√≠ho faktoru SOX2, ve v√Ĺvoji vnitŇôn√≠ho ucha za pouŇĺit√≠ myŇ°√≠ho modelu s rŇĮzn√Ĺmi podm√≠nńõn√Ĺmi delecemi genu Sox2. Gen Sox2 byl deletov√°n pomoc√≠ rekombinańćn√≠ho syst√©mu cre-loxP. V myŇ°√≠ linii Isl1-cre, Sox2 CKO vznikalo pouze mal√© mnoŇĺstv√≠ vl√°skov√Ĺch bunńõk v nńõkter√Ĺch org√°nech vnitŇôn√≠ho ucha (utrikulus, sakulus a b√°ze kochley), zat√≠mco ve zb√Ĺvaj√≠c√≠ch org√°nech se vl√°skov√© buŇąky nediferencovaly vŇĮbec (kristy a apex kochley). ńĆasnńõ se diferencuj√≠c√≠ neurony vestibul√°rn√≠ho ganglia a neurony inervuj√≠c√≠ b√°zi kochley kr√°tce po vzniku apopticky zanikly v dŇĮsledku chybńõj√≠c√≠ch neurotrofick√Ĺch faktorŇĮ produkovan√Ĺch senzorick√Ĺmi buŇąkami. Naopak pozdnńõ vznikaj√≠c√≠ neurony v apexu kochley se u tohoto mutanta vŇĮbec netvoŇôily. Delece Sox2 u Foxg1-cre, Sox2 CKO zpŇĮsobila vznik velmi redukovan√©ho...The main functional cells of the inner ear are neurons and sensory cells that are formed from a common embryonic epithelial neurosensory domain. Discovering genes important for specification and differentiation of sensory cells and neurons in the inner ear is a crucial basis for understanding the pathophysiology of hearing loss. Some of these factors are necessary not only for the inner ear but also for the development of other neurosensory systems such as the visual and olfactory system. The aim of this work was to reveal functions of transcription factor SOX2 in inner ear development by using mouse models with different conditional deletions of Sox2 gene. Sox2 gene was deleted by cre-loxP recombination. In Isl1-cre, Sox2 CKO mutant, reduced number of hair cells differentiated only in some inner ear organs (utricle, saccule and cochlear base) and not in others (cristae and cochlear apex). Early forming inner ear neurons in the vestibular ganglion and neurons innervating the cochlear base developed in these mutants but died by apoptosis due to the lack of neurotrophic support from sensory cells. Late forming neurons in the cochlear apex never formed. In Foxg1-cre, Sox2 CKO mutant, only rudimental ear with no sensory cells was formed. The initial formation of vestibular ganglion with peripheral and...Katedra genetiky a mikrobiologieDepartment of Genetics and MicrobiologyFaculty of SciencePŇô√≠rodovńõdeck√° fakult
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