Reconstruction of the Global Polarity of an Early Spider Embryo by Single-Cell and Single-Nucleus Transcriptome Analysis

Abstract

Patterning along an axis of polarity is a fundamental step in the development of a multicellular animal embryo. In the cellular field of an early spider embryo, Hedgehog signaling operates to specify a “fuzzy” French-flag-like pattern along the primary axis, which is related to the future anterior–posterior (A–P) axis. However, details regarding the generation and development of a diversity of cell states based on the embryo polarity are not known. To address this issue, we applied single-cell RNA sequencing to the early spider embryo consisting of approximately 2,000 cells. Our results confirmed that this technique successfully detected 3 cell populations corresponding to the germ layers and some transient cell states. We showed that the data from dissociated cells had sufficient information for reconstruction of a correct global A–P polarity of the presumptive ectoderm, without clear segregation of specific cell states. This outcome is explained by the varied but differentially overlapping expression of Hedgehog-signal target genes and newly identified marker genes. We also showed that the data resources generated by the transcriptome analysis are applicable to a genome-wide search for genes whose expression is spatially regulated, based on the detection of pattern similarity. Furthermore, we performed single-nucleus RNA sequencing, which was more powerful in detecting emerging cell states. The single-cell and single-nucleus transcriptome techniques will help investigate the pattern-forming processes in the spider model system in an unbiased, comprehensive manner. We provided web-based resources of these transcriptome datasets for future studies of pattern formation and cell differentiation.

Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

“…In a recent separate study, we performed single-nucleus and single-cell RNA-seq analysis of late stage 5 embryos in P. tepidariorum [ 61 ]. In this study, clearly isolated endodermal cell clusters, which were marked by a known early endoderm marker 012_A08 [ 56 ], were identified (Additional file 3 : Fig.…”

Lineage-specific, fast-evolving GATA-like gene regulates zygotic gene activation to promote endoderm specification and pattern formation in the Theridiidae spider

“…In a recent separate study, we performed single-nucleus and single-cell RNA-seq analysis of late stage 5 embryos in P. tepidariorum [60]. In this study, clearly isolated endodermal cell clusters, which were marked by a known early endoderm marker 012_A08 [56], were identified (Additional file3: Fig.…”

“…S9). Strong fuchi expression was detected in these clusters, with weaker levels of fuchi expression being additionally detected in mesodermal (marked by Pt-twi )[58] and dorsal germ-disc (marked by Pt-GATA1 )[60] cell populations. At the same time, the endoderm-specific expression of Pt-GATA4 and Pt-GATA5 was confirmed.…”

“…fuchi homologs may aid in identifying the existence of an evolutionarily conserved abembryonic cell type in other spider species. Complicatedly, the P. tepidariorum genome has a paralog of fuchi that was presumably generated by This study has produced genome-wide datasets related to molecular and genetic regulation of early embryonic development in P. tepidariorum, which can be integrated with single-nucleus and single-cell RNA-seq datasets established in another study [61]. These data resources, as well as the research strategies presented, will facilitate our efforts to dissect the early developmental processes at molecular resolution and establish an independent paradigm of genetic programs for early arthropod development.…”

Virtual spherical-shaped multicellular platform for simulating the morphogenetic processes of spider-like body axis formation

“…Such a regulatory connection between the emerging rough positional information and cell mechanical parameters should be incorporated to improve our vertex model. Quantitative data on cell position, cell behavior, and gene expression can now be obtained from spider embryos using live imaging, multicolor fluorescent in situ hybridization, and single-cell/nuclear transcriptomes (Hemmi et al, 2018;Akiyama-Oda et al, 2022). Analyses of such quantitative data may help us understand the mechanical regulations and genetic networks underlying the patternforming processes in the spider embryo.…”