Reprogramming of cells during embryonic transfating: overcoming a reprogramming block

Regulative development, a remarkable ability demonstrated by many animal embryos, allows for the replacement of missing cells or parts. However, the molecular mechanisms underlying this ability remain largely unexplored. This study investigates the phenomenon of “transfating” in sea urchin embryos, where the removal of micromeres (skeletogenic cell progenitors) at the 16-cell stage triggers a sequential switch in cell fates. In the absence of micromeres, other mesoderm cells are initially absent due to their dependence on micromere signaling. While most mesoderm cells eventually reappear through transfating, pigment cells do not. Using single-cell RNA sequencing (scRNA-seq) tracked over time, we reveal the reprogramming sequence of these replacements. Cells transition from an early endoderm specification state through endomesoderm and mesoderm stages, eventually differentiating into distinct skeletogenic and blastocoelar cell types, but not pigment cells. Methods scRNA-seq and Computational Analysis: The 10x Genomics v3.1 gene expression kit was used for each time point, with library quality verified using the Agilent 2100 Bioanalyzer. Libraries were pooled and sequenced on a NovaSeq6000 S-Prime 50bp PE Full flow cell, achieving over 50,000 reads per cell. Sequenced libraries were demultiplexed using 10x Genomics Cell Ranger 7.1.0, and the resulting RNA count matrices were processed in R using Seurat v4.3. After merging Seurat objects for uniform quality control and normalization, SCTransform was applied to normalize and remove technical variation. The final dataset included 18,688 cells with 4,000 variable features from micromereless embryos over seven time points. Data Visualization and Clustering: UMAP (Uniform Manifold Approximation and Projection) was used to visualize the multi-dimensional scRNA-seq data in two dimensions. Graph-based Louvain Clustering identified 43 clusters, annotated using sea urchin GRN genes and in situ hybridization patterns. Developmental Trajectories: Developmental trajectories were inferred using Waddington-OT, with cell division rates estimated based on lineage knowledge. Triangle plots illustrated lineage trajectories, showing probabilities of cells reaching specific gene expression states. They can be found in the zipped directories Our findings indicate that the rescue of pigment cells is contingent on the timing of signaling: pigment cells return if Delta is expressed before Nodal. This study demonstrates that transfating operates through a series of gene regulatory state transitions and highlights that reprogramming fails when endogenous negative signals precede positive signals in the reprogramming sequence.

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