Modular, cascade-like transcriptional program of regeneration in Stentor

Abstract

The giant ciliate Stentor coeruleus is a classical model system for studying regeneration and morphogenesis at the level of a single cell. The anterior of the cell is marked by an array of cilia, known as the oral apparatus, which can be induced to shed and regenerate in a series of reproducible morphological steps, previously shown to require transcription. If a cell is cut in half, each half will regenerate an intact cell, including a new oral apparatus in the posterior half. We used RNAseq to assay the dynamic changes in Stentor's transcriptome during regeneration, after both oral apparatus shedding and bisection, allowing us to identify distinct temporal waves of gene expression including kinases, RNA binding proteins, centriole biogenesis factors, and orthologs of human ciliopathy genes implicated in Meckel and Joubert syndromes. By comparing transcriptional profiles of different regeneration events in the same species, we were able to identify distinct modules of gene expression corresponding to oral apparatus regeneration, posterior holdfast regeneration, and recovery after wounding. By measuring gene expression in cells in which translation is blocked, we show that the sequential waves of gene expression involve a cascade mechanism in which later waves of expression are triggered by translation products of early-expressed genes. Among the early-expressed genes, we identified an E2F transcription factor and the conserved RNA binding protein Pumilio as potential regulators of regeneration based on the expression pattern of their predicted target genes. RNAi mediated knockdown experiments indicate that Pumilio is required for regenerating oral structures of the correct size. We show that E2F is involved in the completion of regeneration but is dispensable for earlier steps. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.

The nuclear transport factor CSE1 drives macronuclear volume increase and macronuclear node coalescence inStentor coeruleus

“…Next, we looked for potential candidates in the list of top differentially expressed genes during regeneration. 6 Because nuclear transport has been shown to affect overall nuclear size, we focused on genes encoding potential nuclear transport factors. 14,15 Among the differentially expressed genes predicted to encode nuclear transport-related proteins, the gene CSE1 stood out as a candidate because it is highly expressed early in regeneration before the macronuclear shape change cycle takes place ( Figure 2B ).…”

“…Both SteCoe_28374 and SteCoe_28918 are up-regulated during regeneration. 18 In the following experiments we used the Stentor gene SteCoe_28374 to create RNAi constructs because the complete gene was represented in the RNA-seq data (Supplemental Figure S7 and S8B-C).…”

“…1 Previous work from the 1960’s - 1980’s demonstrated that the macronuclear shape change is a highly regulated part of cell division and regeneration 2,3 , but the molecular pathways driving these changes are unknown. With the recent availability of a sequenced Stentor genome, a transcriptome during regeneration, and molecular tools like RNAi 4–6 , it is now possible to investigate the molecular mechanisms that drive macronuclear shape change. We found that the volume of the macronucleus increases during coalescence, suggesting an inflation-based mechanism.…”

Genome-wide analysis of anterior-posterior mRNA regionalization inStentor coeruleusreveals a role for the microtubule cytoskeleton

“…But Stentor was never developed as a molecular model system, and we do not currently know the molecular basis for how it is able to regenerate its structures. In order to exploit the unique opportunities presented by this organism, we sequenced the Stentor genome [9], developed methods for RNAi by feeding [10], and analyzed the transcriptional program of regeneration [11].…”

Hydrodynamic dissection ofStentor coeruleusin a microfluidic cross junction

“…17 This trait may allow even a small fragment of the cell to carry sufficient copies of the genome to enable regeneration. 8,18,19,21,26,27 However, the detailed mechanisms of wound healing and regeneration in Stentor remain incompletely understood. One of the major experimental challenges is the lack of a high-throughput and repeatable method to wound and/or to dissect the cells.…”