Live Imaging during Annelid Sequential Segmentation
In addition to vertebrates, both annelids and most arthropods also display sequential segment formation from a posterior region (similar to the PSM), in this case called the posterior growth zone or the segment addition zone (SAZ). Annelids are extremely variable in their morphology, and segmentation in this phylum has been understudied compared to vertebrates and arthropods (Balavoine 2014; see Chapter 4). Few studies have included long-term live imaging in their analysis. For example, Zattara and colleagues (2016; also see Chapter 10) evaluated cell migration during anterior and posterior regeneration of the adult freshwater annelid Pristina leidyi, based on cell morphology and migratory characteristics using DIC microscopy on unlabeled tissue. Recently, Ozpolat and coworkers (2017) performed time-lapse imaging at single-cell resolution of embryonic and larval stages of the polychaete Platynereis dumerilii, by injecting different constructs in order to label cell components such as nuclei (HistoneH2A-mCherry) and the cell membrane (EGFP-caax) or to visualize cell cycle progression. For the latter they developed a fluorescent cell cycle reporter based on FUCCI, a fluorescent ubiquitination-based cell cycle indicator. Pd-FUCCI was made by two constructs, the HistoneH2A-mCherry that directed the expression of mCherry to all cell nuclei and the mVenus-Cdtl(aal-147), composed by a truncated form of Cdtl (a cell cycle protein) that lacked sequences for DNA binding domains but contained an endogenous ubiquitination site that conferred the necessary instability for the dynamic expression of the reporter, fused to the fluorescent protein mVenus. The cyclic expression was observed during late-G2/ mitosis/Gl phases and allowed the authors to perform germline and mesodermal cell lineage analysis that showed the cellular origin of the early swimming larva mesodermal segments (formed by simultaneous segmentation) as well as the origin of the posterior growth zone, from where the SAZ is formed and sequential segmentation proceeds later during juvenile development (Ozpolat et al. 2017).