Cell cycle dynamics during diapause entry and exit in an annual killifish revealed by FUCCI technology
Authors: Dolfi Luca , Ripa Roberto , Antebi Adam , Valenzano Dario Riccardo , Cellerino Alessandro 
Published: January 2019

Background

Annual killifishes are adapted to surviving and reproducing over alternating dry and wet seasons. During the dry season, all adults die and desiccation-resistant embryos remain encased in dry mud for months or years in a state of quiescence, delaying hatching until their habitats are flooded again. Embryonic development of annual killifishes deviates from canonical teleost development. Epiblast cells disperse during epiboly, and a “dispersed phase” precedes gastrulation. In addition, annual fish have the ability to enter diapause and block embryonic development at the dispersed phase (diapause I), mid-somitogenesis (diapause II) and the final phase of development (diapause III).

Developmental transitions associated with diapause entry and exit can be linked with cell cycle events. Here we set to image this transitions in living embryos.

Results

To visibly explore cell cycle dynamics during killifish development in depth, we created a stable transgenic line in Nothobranchius furzeri that expresses two fluorescent reporters, one for the G1 phase and one for the S/G2 phases of the cell cycle, respectively (fluorescent ubiquitination based cell cycle indicator, FUCCI). Using this tool, we observed that, during epiboly, epiblast cells progressively become quiescent and exit the cell cycle. All embryos transit through a phase where dispersed cells migrate, without showing any mitotic activity, possibly blocked in the M phase (diapause I).

Thereafter, exit from diapause I is synchronous and cells enter directly into the S phase without transiting through G1. The developmental trajectories of embryos entering diapause and of those that continue to develop are different. In particular, embryos entering diapause have reduced growth along the medio-lateral axis. Finally, exit from diapause II is synchronous for all cells and is characterized by a burst of mitotic activity and growth along the medio-lateral axis such that, by the end of this phase, the morphology of the embryos is identical to that of direct-developing embryos.

Conclusions

Our study reveals surprising levels of coordination of cellular dynamics during diapause and provides a reference framework for further developmental analyses of this remarkable developmental quiescent state.

List of Abbreviations

In this paper, we will refer to several developmental stages or morphological structures using abbreviations. To make the reading easier, we resume here a list of all the abbreviations, to which the reader can refer at any time.

WS

Wourms Stage. Developmental stage referring to the embryonic description made by Wourms for the killifish species Austrofundulus limneus.

YSL

Yolk syncytial layer. A layer of cells that form a syncytium and that are in direct contact with the yolk. This is the most internal layer, through this layer nutrients from the yolk can be delivered to the upper layers.

EL

Epiblast layer: A layer of cells composed by blastomeres that divides actively during development and will take part in the generation of the several embryonic and fish major structures like head tail trunk and organs.

EVL

Enveloping layer. A thin layer of cells that envelopes all the embryo. It is the most external layer. The cells belonging to this layer are big with big nuclei that do not divide.

DI

Diapause I. A dormancy stage peculiar of annual killifish species that occurs after the completion of epiboly, during the dispersed phase.

DII

Diapause 2. The second and most important dormancy stage of annual killifish species. Fish can stop in DII only entering a different developmental trajectory after the reaggregation phase. The final developmental block occurs at the mid somitogenesis stage.

DC

Diapause Committed embryo. An embryo that undertook the Diapause II trajectory of development and that will stop for sure in Diapause II during the somitogenesis stage.

DD

Direct Developing embryo. An embryo that is following the not diapause II developmental trajectory. These embryos grow more in lateral size during somitogenesis and never stop their development in this phase.