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Cellular, Cytoskeletal, and Biophysical Mechanisms of Spiral Cleavage during Platynereis dumerilii EmbryogenesisHsieh, Yu-Wen 20 November 2020 (has links)
Embryogenesis is one of the most delicate biological processes which requires precise control in various levels, including molecular distribution and gene expression, cellular orientation and specification, and tissue dynamics giving rise to proper morphology. The diverse animal morphology can be resulted from the difference during early embryonic cleavages. Spiral cleavage is a conserved embryonic patterning strategy used in the majority of the animal clade Spiralia. The specific cell positioning during cell division and quadrant-based clonal domain formation make the embryos with the blastomeres orientated in a spiral manner when viewing from the animal pole. Although spiral cleavage is conserved in many phyla, the detailed cellular, molecular and biophysical mechanisms for this left-right symmetry breaking event remain unclear.
Here I studied the early development of the prototypic annelid spiral-cleaver Platynereis dumerilii, which performs two unequal embryonic cleavages followed by the first dextral spiral cleavages, and compared the mechanisms to other spiralians or to other cleavage types. First, I described the morphology of each cell cycle from the zygote until 64-cell stage by imaging the fluorescently labeled fixed embryos. Second, with mRNA injection, whole-embryo live-imaging with Selective Plane Illumination Microscopy (SPIM), and in silico cell tracking, I monitored these cleavages in 4-D, constructed the early cell lineages, and revealed the subtle asynchrony of the four quartets. Third, together with the spindle inclination angle measurement, I discovered the leading role of the D macromere during P. dumerilii spiral cleavage. I also confirmed that the dextral micromere orientation is neither affected by the eggshell nor the presence of all the neighbor macromeres, suggesting that this cellular property may be achieved by cell autonomous molecular mechanisms.
In order to quantify the candidate cytoskeletal dynamics during spiral cleavage, I optimized the construction of the injected mRNAs and the injection protocol to achieve the highest translational level of the fluorescent protein within a given developmental time. Beside mRNA injection, I also established a protein expression and injection protocol for P. dumerilii protein injection in order to visualize the target gene as early as possible. Both techniques didn’t dramatically influence embryogenesis and allow for quantification of the protein dynamics. With these strategies, I discovered and measured the chiral counter rotational flow of cortical actomyosin in each spiral cleavage and revealed that it’s present in the first two spiral cleavages, especially of the macromeres. The biophysical force generated by actomyosin contributes in the cell deformation and spindle inclination, resulting in proper dextral micromere positioning, during the first spiral cleavage, confirmed by the chemical treatment to the P. dumerilii embryos. The asymmetric actomyosin distribution, nuclei migration, and the change of the cell axes during cytokinesis in the macromeres also suggests that the macromeres may play critical roles to lead spiral cleavage.
This work is built on the knowledge of the spiral cleavage machinery and has extended it in multiple dimensions. The detailed phase-by-phase description of each cleavage increases the information of P. dumerilii embryogenesis. The established labeling and imaging techniques in this thesis are the important basis for investigation and comparisons of different spiralian development in the future. More broadly, the discovery of actomyosin dynamics shows conservation to the left-right symmetry breaking events of the animals which does not belong to Spiralia. These together bring insights to a global evolutionary speculation: a conserved mechanical force generation pathway, tuned by the upstream molecular signals, may be the key of the miscellaneous cleavage types, resulting in the astonishing variety of embryo patterning.
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The embryonic development of Elminius modestus Darwin, 1854Ponomarenko, Ekaterina 04 August 2014 (has links)
Die vorliegende Arbeit behandelt die Embryonalentwicklung des Rankenfußkrebses Elminius modestus (Thecostraca: Cirripedia). Der Entwicklungsprozess wurde mithilfe unterschiedlicher Methoden wie 4D Mikroskopie, in vivo Einzelzellmarkierungen, Fluoreszenzhistochemie und konfokaler Laserscanningmikroskopie in Verbindung mit 3D Rekonstruktionen untersucht. Die Furchung von E. modestus ist total, inequal in Bezug auf die Dotterzelle und asynchron mit einem anterior-posterioren Gradienten. Der gesamte Prozess folgt einem strengen Teilungsmuster mit nur sehr geringer Variabilität. Eine davon stellt das Auftreten spiegelbildlicher Embryonen ab dem 4-Zell. Die Keimblattdifferenzierung wurde vor allem mittels in vivo Zellmarkierungen untersucht. Die Trennung der endodermalen und endomesodemalen Keimblätter erfolgt nach der vierten Furchungsteilung, die Trennung des Ectomesoderm nach der sechsten Teilung. Die Urkeimzellen sind aller Wahrscheinlichkeit nach ein Produkt der siebten Furchungsteilung der Dotterzellen (3Da und 3Dp). Im Zuge der Untersuchung konnte die Zelllinie jedes Keimblattes rekonstruiert werden, die Zellschicksale der Abkömmlinge der Quadranten wurde bis zum 16-Zell Stadium beschrieben. Das Ectoderm entspringt allen vier Quadranten, ebenso das Ectomesoderm (die letzten identifizierten Mesectoblasten sind 3A, 3B, 3C, 1drp und 1dlp). Endoderm und Endomesoderm entwickeln sich aus einzelnen Vorläuferzellen im 16-Zell Stadium (2D bzw. 2d). Das Auftreten nur eines einzelnen Endoblasten stellt eine mögliche Apomorphie aller Ecdysozoa dar. Das Vorhandensein eines einzelnen Mesendoblasten wird als mögliches Merkmal des Grundmusters aller Protostomia in Betracht gezogen. / The present work is devoted to the embryonic development of the thoracican barnacle Elminius modestus (Thecostraca: Cirripedia). The developmental process was investigated by means of different techniques like 4D microscopy, in vivo labelling, fluorescent histochemistry, and confocal laser scanning microscopy combined with 3D reconstructions. The cleavage of E. modestus is total, unequal with regards to the yolky cell, and asynchronous with an anterior-posterior gradient. The entire process appears to follow a strict pattern of divisions with very little variability, one of which includes the occurrence of mirror image embryos from the 4-cell stage on. The germ layer differentiation was mainly studied by means of in vivo labelling. The segregation of the endodermal and the endomesodemal germ layers are shown to happen after the fourth division, whereas the ectomesoderm segregates after the sixth division. The primordial germ cells are suggested to be a product of the seventh cleavage division of the yolky cells (3Da and 3Dp). During the research the cell lineage of each germ layer was established, the fates of the quadrant descendants are described up to the 16-cell stage. The ectoderm originates from four quadrants, as does the ectomesoderm (the last identified mesectoblasts are 3A, 3B, 3C, 1drp, and 1dlp). The endoderm and the endomesoderm develop from single precursors at the 16-cell stage (2D and 2d, respectively). The presence of only a single endoblastic cell, might represent an apomorphy for the entire group of Ecdysozoa. A singular mesendoblast is suggested to be a possible feature in the developmental ground pattern of all Protostomia.
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