Abstract
In this study, we aimed to identify molecular mechanisms involved in the specification of the 4d (mesentoblast) lineage in Platynereis dumerilii. We employ RT-PCR and in situ hybridization against the Platynereis dumerilii twist homolog (Pdu-twist) to reveal mesodermal specification within this lineage. We show that Pdu-twist mRNA is already maternally distributed. After fertilization, ooplasmatic segregation leads to relocation of Pdu-twist transcripts into the somatoblast (2d) lineage and 4d, indicating that the maternal component of Pdu-twist might be an important prerequisite for further mesoderm specification but does not represent a defining characteristic of the mesentoblast. However, after the primordial germ cells have separated from the 4d lineage, zygotic transcription of Pdu-twist is exclusively observed in the myogenic progenitors, suggesting that mesodermal specification occurs after the 4d stage. Previous studies on spiral cleaving embryos revealed a spatio-temporal correlation between the 4d lineage and the activity of an embryonic organizer that is capable to induce the developmental fates of certain micromeres. This has raised the question if specification of the 4d lineage could be connected to the organizer activity. Therefore, we aimed to reveal the existence of such a proposed conserved organizer in Platynereis employing antibody staining against dpERK. In contrast to former observations in other spiralian embryos, activation of MAPK signaling during 2d and 4d formation cannot be detected which questions the existence of a conserved connection between organizer function and specification of the 4d lineage. However, our experiments unveil robust MAPK activation in the prospective nephroblasts as well as in the macromeres and some micromeres at the blastopore in gastrulating embryos. Inhibition of MAPK activation leads to larvae with a shortened body axis, defects in trunk muscle spreading and improper nervous system condensation, indicating a critical function for MAPK signaling for the reorganization of embryonic tissues during the gastrulation process.
Highlights
Development in the marine polychaete annelid Platynereis dumerilii follows a canonical spiral cleavage mode leading to blastomeres with distinct volumes and cytoplasmatic compositions [1,2]
After the first unequal cleavage, the majority of the Pdu-twist mRNA and the clear cytoplasm are distributed into the CDblastomere (Fig. 1D), while the second unequal medial cleavage results in accumulation of the majority of the maternally inherited Pdu-twist transcripts in the largest of the four blastomeres, the Dblastomere (Fig. 1E)
The somatoblast 2d has formed (Fig. 1F) [1,9]. This is the largest of the micromeres in Platynereis and contains the highest amount of clear cytoplasm and Pdu-twist mRNA when compared to the other micromeres (Fig. 1F)
Summary
Development in the marine polychaete annelid Platynereis dumerilii follows a canonical spiral cleavage mode leading to blastomeres with distinct volumes and cytoplasmatic compositions [1,2]. The Dblastomere will give rise to the D-quadrant including the somatoblast (2d micromere) and mesentoblast (4d micromere) that represent the progenitors of most trunk-forming cells in Platynereis. Both cells receive remarkably high amounts of clear cytoplasm which makes them distinguishable from all other, rather yolky blastomeres [1]. Altering the regular cytoplasmatic composition of the early blastomeres or provoking equalized cleavages by experimental manipulation leads to characteristic Janus malformations of the trunk suggesting that the distribution of early morphogenetic determinants is crucial for later trunk and axis formation [5,6,7] These early molecular determinants decisive in the specification of lineage fates are still unknown in Platynereis
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