Speed vs. Completeness: A Comparative Study of Solitary and Colonial Tunicate Embryogenesis

Chiara Anselmi ^1* Katherine J Ishizuka ² Karla J Palmeri ² Paolo Burighel ¹ Ayelet Voskoboynik ^2,3 Kohji Hotta ⁴ Lucia Manni ¹

• ¹ Department of Biology, School of Sciences, University of Padua, Padua, Veneto, Italy
• ² Department of Biology, Stanford University, Palo Alto, United States
• ³ Institute for Stem Cell Biology and Regenerative Medicine, School of Medicine, Stanford University, Stanford, California, United States
• ⁴ Department of Biosciences and Informatics, Keio University, Minato, Japan

Solitary ascidians, such as Ciona robusta, have been used for over a century as a model system for embryological studies. These species are oviparous, producing many relatively small and transparent eggs, which are released and fertilized outside the parent body. Embryos develop rapidly in a stereotyped way and reach the larva stage in less than one day (at 20oC). The larvae then settle and metamorphose into sessile juveniles in about two days. On the other hand, colonial ascidians are ovoviviparous, with heavily yolked eggs developing inside the parent body. In the colonial Botryllus schlosseri, embryos are connected to the parental body via a cup-like placenta and develop into larvae in a week (at 20oC). The larvae, which possess both typical larval organs and prospective juvenile organs, are released into the seawater where they settle very rapidly, even after only fifteen minutes of free swimming. Then, they metamorphose into juvenile oozooids. The ability to study embryo development in colonial ascidians within the parent body is limited. To counter this, we developed a method for in vitro culturing B. schlosseri embryos outside the parental body and combined it with time-lapse microscopy and confocal microscopy to describe the embryonic developmental stages. Moreover, we used histology based on serial sections for a deep analysis of late development, when embryo opaqueness made other techniques unhelpful. We recognized 19 stages of development, from the fertilized egg to the swimming larva, and described the stage of organ appearance and differentiation. When we compared the timeline of embryo development in B. schlosseri vs that of C. robusta, we found heterochrony in development, regarding both the timing of some organ appearance and their growth rate. We hypothesize that this difference in maturation timing between solitary and colonial ascidians is the expression of a shift in the regulation of key developmental pathways that contributed to ascidian diversification. This heterochronic evolution likely facilitated a significant (approximately four-fold) shortening in the time of metamorphosis in B. schlosseri by allowing embryos to remain in a safe ovoviviparous environment five times longer than those in C. robusta before hatching.