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Cell behaviors influencing the mode of cell division of retinal progenitors: a role for the Zebrafish Anillin protein

Paolini, Alessio

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Abstract

Understanding molecular and cellular events influencing the neural progenitor mode of cell division and fates of daughter cells is central to Developmental Biology. Members of the cytokinetic machinery have received increasing attention as possible molecular mediators of this event: their inheritance or retention appears to influence proliferation versus differentiation of daughter cells in cancer and stem cells. I therefore took under investigation the F‐actin binding protein Anillin, shown to be essential for cytokinesis progression and completion in invertebrate model systems. I found Anillin downregulation to be mediated by the bHLH transcription factor Atoh7, which is essential for the generation of Retinal Ganglion Cells (RGCs). I therefore first investigated the expression and in vivo dynamics of Anillin in the zebrafish neural retina. This analysis revealed that Anillin expression correlates with proliferative states of retinal progenitor and, concomitantly, Anillin knock down promoted RGCs and early born retinal cell type generation at the expense of the late born ones, such as bipolar and muller glia cells. These effects on cell fate were due to change in mode of cell division promoted by Anillin knock‐down Anillin morpholino injected clones tend to undergo symmetric neurogenic division, generating two Atoh7 positive cells or two RGCs, while the control clones tend to undergo asymmetric division. These results reveal a role for Anillin in neurogenesis by affecting cell division outcomes. I then asked if anillin knockdown effect on cell division outcome correlates with putative roles in cytokinesis. By developing a zebrafish transgenic line recapitulating Anillin‐GFP expression in the in vivo developing embryo, I could analyze Anillin distribution at cellular level. Anillin protein highlights the progression of the cytokinetic furrow, as well as midbody formation and postmitotic inheritance in dividing retinal progenitor cells. Research in invertebrates and vertebrates suggests a colocalization of Anillin and its main interactor, F­‐actin, during cytokinesis progression. Accordingly, Anillin and F‐actin co‐localized at the cleavage furrow and midbody of cycling retinal progenitors. Midbody and F‐actin spot remnant are both inherited by the daughter cell that differentiates as RGC, suggesting for the first time a correlation between apical F‐actin/midbody distribution and fate. I further aimed at elucidating the effect of Anillin knockdown on F‐actin dynamics during cytokinesis. Results show that symmetric outcome of cell division produced by Anillin knock down correlate with symmetric apical F‐actin inheritance at the daughter cell’s apical domain. These results indicate that Anillin links cytokinesis progression with apical F­‐actin distribution between the daughter cells. In addition to slowing down cytokinesis progression, Anillin appears to influence the basal to apical interkinetic nuclear migration of progenitor cells, which I found slowed down in Anillin morpholino­‐injected embryos, along with an increase in the progenitors cell cycle length. This results in an longer permanence of the nuclei close to the basal side of the neuroepithelium namely, far away from proliferative signal. Likewise, longer permanence of nuclei in this basal side of the neuroepithelium have been associated with symmetric neurogenic divisions of retinal progenitors. Controversial studies showed how correlation between mode and orientation of cell division in vertebrates is not clear. In the present study I found that increase in symmetric fate outcome of daughter cells (in term of Atoh7 expression) correlates with a shift in the orientation of cell division from radial to circumferential. The biological significance of this switch remains to be investigated. These functional studies combined with in vivo imaging provide evidences indicating that Anillin affects neurogenesis by maintaining the progenitor pool and influencing the mode of cell division. By directly assessing correlations between F­‐actin, midbody inheritance, orientation of cell division and daughter cell fate, our study also provides insight into the mechanism whereby this might operate in vivo.

Item Type: Dissertation
Supervisor: Wittbrodt, Prof. Dr. Joachim
Date of thesis defense: 11 July 2014
Date Deposited: 18 Dec 2014 06:51
Date: 2014
Faculties / Institutes: The Faculty of Bio Sciences > Dean's Office of the Faculty of Bio Sciences
Subjects: 500 Natural sciences and mathematics
570 Life sciences
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