Freshwater planaria are a very attractive model system for stem cell biology, tissue homeostasis, and regeneration. The genome of the planarian Schmidtea mediterranea has recently been sequenced and is estimated to contain >20,000 protein-encoding genes. However, the characterization of its transcriptome is far from complete. Furthermore, not a single proteome of the entire phylum has been assayed on a genome-wide level. We devised an efficient sequencing strategy that allowed us to de novo assemble a major fraction of the S. mediterranea transcriptome. We then used independent assays and massive shotgun proteomics to validate the authenticity of transcripts. In total, our de novo assembly yielded 18,619 candidate transcripts with a mean length of 1118 nt after filtering. A total of 17,564 candidate transcripts could be mapped to 15,284 distinct loci on the current genome reference sequence. RACE confirmed complete or almost complete 5′ and 3′ ends for 22/24 transcripts. The frequencies of frame shifts, fusion, and fission events in the assembled transcripts were computationally estimated to be 4.2%–13%, 0%–3.7%, and 2.6%, respectively. Our shotgun proteomics produced 16,135 distinct peptides that validated 4200 transcripts (FDR ≤1%). The catalog of transcripts assembled in this study, together with the identified peptides, dramatically expands and refines planarian gene annotation, demonstrated by validation of several previously unknown transcripts with stem cell-dependent expression patterns. In addition, our robust transcriptome characterization pipeline could be applied to other organisms without genome assembly. All of our data, including homology annotation, are freely available at SmedGD, the S. mediterranea genome database.

In recent years, it has become clear that phase separation represents an important class of subcellular compartmentalization. However, relatively little is known about how the formation or disassembly of such compartments is regulated. In zebrafish, the Balbiani body (Bb) and the germ plasm (Gp) are phase-separated structures essential for germ cell specification and home to many germ cell-specific mRNAs and proteins. Throughout development, these structures range from a single large aggregate (Bb), to a dispersed state and back to relatively large assemblies (Gp). Formation of the Bb requires Bucky ball (Buc), a protein with prion-like properties. We found that the multi-tudor domain-containing protein Tdrd6a interacts directly with Buc, affecting its mobility and aggregation properties. Importantly, lack of this regulatory interaction leads to significant defects in germ cell development. Our work presents a new mechanism for how prion-like protein-aggregations can be regulated and highlights the biological relevance of such regulatory events.

Keywords 20Gastrulation, mouse embryo, Eomes, definitive endoderm, mesoderm, lineage 21 specification 22 Summary statement 24 Cells lineages are specified in the mouse embryo already within the primitive streak 25 where Mesp1+ mesoderm and Foxa2+ endoderm are generated in a spatial and 26 temporal sequence from unbiased progenitors. Abstract 29 Anterior mesoderm (AM) and definitive endoderm (DE) progenitors represent the 30 earliest embryonic cell types that are specified during germ layer formation at the 31 primitive streak (PS) of the mouse embryo. Genetic experiments indicate that both 32 lineages segregate from Eomes expressing progenitors in response to different 33 NODAL signaling levels. However, the precise spatiotemporal pattern of the 34 emergence of these cell types and molecular details of lineage segregation remain 35 unexplored. We combined genetic fate labeling and imaging approaches with scRNA-36 seq to follow the transcriptional identities and define lineage trajectories of Eomes 37 dependent cell types. All cells moving through the PS during the first day of 38 gastrulation express Eomes. AM and DE specification occurs before cells leave the 39 PS from discrete progenitor populations that are generated in distinct spatiotemporal 40 patterns. Importantly, we don't find evidence for the existence of progenitors that co-41 express markers of both cell lineages suggesting an immediate and complete 42 separation of AM and DE lineages.43 44 48 embryo under the influence of elevated levels of the instructive signals of 49 TGFß/NODAL, WNT and FGF. These signals induce an epithelial-to-mesenchymal 50 transition (EMT) of epiblast cells at the primitive streak (PS) leading to their 51 delamination and the formation of the mesoderm and DE cell layer. The nascent 52 mesoderm layer rapidly extends towards the anterior embryonic pole by cell migration 53 between the epiblast and the visceral endoderm (VE) (reviewed by (Arnold and 54 Robertson 2009; Rivera-Pérez et al. 2003)). DE progenitors migrate from the epiblast 55 together with mesoderm cells, before they eventually egress into the VE layer to 56 constitute the DE (reviewed by (Rivera-Pérez and Hadjantonakis 2014; Viotti, Foley, 57 et al. 2014)). 58 Current concepts suggest that different cell fates are specified according to the time 59 and position of cell ingression through the PS reflecting different instructive signaling 60 environments (Rivera-Pérez and Hadjantonakis 2014). However, the precise 61 morphogenetic mechanisms guiding the emergence of various cell types along the 62 PS still remain uncertain. This is at least in parts due to the lack of detailed knowledge 63 about the precise timing and location of individual cells becoming lineage specified, 64 and the challenge to exactly determine the signaling pathway activities during fate 65 commitment. 66 Clonal cell labeling and transplantation experiments have proposed the gross patterns 67 and dynamics of cell specification during gastrulation, which have been represented 68 in fate maps...