Highlights d Large mitotic centrosome identification (246.44 ± 11.93 mm 2 ) in the zebrafish embryo d Decreases in cell size scales closely with mitotic centrosome size d Zebrafish mitotic centrosomes within a spindle are asymmetric in size d PLK1 and PLK4 activity is required for asymmetric mitotic centrosome positioning

Summary During the earliest division stages, zebrafish embryos have large cells that divide rapidly and synchronously to create a cellular layer on top of the yolk. Here, we describe a protocol for monitoring spindle dynamics during these early embryonic divisions. We outline techniques for injecting zebrafish embryos with small-molecule inhibitors toward polo-like kinases, preparing and mounting embryos for three-dimensional imaging using confocal microscopy. These techniques are used to understand how the early zebrafish embryo’s centrosome constructs the mitotic spindle. For complete details on the use and execution of this protocol, please refer to Rathbun et al. (2020) .

Factors that regulate mitotic spindle positioning have been elucidated in vitro, however it remains unclear how a spindle is placed within the confines of extremely large cells. Our studies identified a uniquely large centrosome structure in the early zebrafish embryo (246.44±11.93μm 2 mitotic centrosome in a 126.86±0.35μm diameter cell), whereas C. elegans centrosomes are notably smaller (6.75±0.28μm 2 mitotic centrosome in a 55.83±1.04μm diameter cell). During early embryonic cell divisions, cell size changes rapidly in C. elegans and zebrafish embryos. Notably, mitotic centrosome area scales closely with changing cell size compared to changes in spindle length for both organisms. One interesting difference between the two is that mitotic centrosomes are asymmetric in size across embryonic zebrafish spindles, with the larger mitotic centrosome being 2.14±0.13-fold larger in size than the smaller. The largest mitotic centrosome is placed towards the embryo center in a Polo-Like Kinase (PLK) 1 and PLK4 dependent manner 87.14±4.16% of the time. We propose a model in which uniquely large centrosomes direct spindle placement within the disproportionately large zebrafish embryo cells to orchestrate cell divisions during early embryogenesis.

In hermaphrodites, the allocation of resources to each sex function can influence fitness through mating success. A prediction that arises from sex allocation theory is that in wind-pollinated plants, male fitness should increase linearly with investment of resources into male function but there have been few empirical tests of this prediction. In a field experiment, we experimentally manipulated allocation to male function in Ambrosia artemisiifolia (common ragweed) and measured mating success in contrasting phenotypes using genetic markers. We investigated the effects of morphological traits and flowering phenology on male siring success, and on the diversity of mates. Our results provide evidence for a linear relation between allocation to male function, mating, and fitness. We find earlier onset of male flowering time increases reproductive success, whereas later flowering increases the probability of mating with diverse individuals. Our study is among the first empirical tests of the prediction of linear male fitness returns in wind-pollinated plants and emphasizes the importance of a large investment into male function by wind-pollinated plants and mating consequences of temporal variation in sex allocation.

An essential process for cilia formation during epithelialization is the movement of the centrosome to dock with the cell′s nascent apical membrane. Our study examined centrosome positioning during the development of Danio rerio′s left-right organizer (Kupffer′s Vesicle, KV). We found that when KV mesenchymal-like cells transition into epithelial cells that are organizing into a rosette-like structure, KV cells move their centrosomes from random intracellular positions to the forming apical membrane in a Rab11 and Rab35 dependent manner. During this process, centrosomes construct cilia intracellularly that associated with Myo-Va while the centrosomes repositioned towards the rosette center. Once the centrosomes with associated cilia reach the rosette center, the intracellular cilia recruit Arl13b until they extend into the forming lumen. This process begins when the lumen reaches an area of approximately 300 μm2. Using optogenetic and depletion strategies, we identified that the small GTPases, Rab11 and Rab35, regulate not only cilia formation, but lumenogenesis, whereas Rab8 was primarily involved in regulating cilia length. These studies substantiate both conserved and unique roles for Rab11, Rab35, and Rab8 function in cilia formation during lumenogenesis.