Modular, Cascade-like Transcriptional Program of Regeneration inStentor

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 Stentors 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. This work allows us to classify regeneration genes into groups based on their potential role for regeneration in distinct cell regeneration paradigms, and provides new insight into how a single cell can coordinate complex morphogenetic pathways to regenerate missing structures.

Regeneration in Stentor coeruleus

“…as a cascade, such that a small number of early genes must be translated in order to trigger transcription of the later genes (Sood et al, 2021).…”

“…In Stentor, the cortical cilia generate a coherent flow over the whole body (Wan et al, 2020) which I hypothesize, based on the role of flow in PCP, might serve as a signal to help align the cortical rows during recovery after minceration or other disarrangements. It is interesting to note that Stentor regeneration is accompanied by expression of genes whose products are known to be involved in coupling ciliary orientation to planar cell polarity proteins in animals (Sood et al, 2021). Likewise, the Stentor cortex contains contractile fibers built of centrin-like EF hand proteins, and I hypothesize that mechanical tension generated by these fibers might play a role in transmitting long range spatial information to help enforce a common polarity among cortical units.…”

Proteomic Dissection of a Giant Cell

“…However, within the cell body samples, this protein was only detected in the anterior half and not the posterior half, consistent with it being associated with an anterior structure, possibly the MB itself. The possibility that SteCoe_22878 is a bona fide MB protein is further supported by the fact that this gene is upregulated during MB regeneration in sucrose-shocked cells (Sood 2021). In the soluble fraction of isolated MB (Table S3), one of the most highly enriched proteins (SteCoe_13619) is one of the GAS2-related proteins also detected in the MB specific protein list.…”

“…It is harder to reliably calculate an enrichment value for less abundant proteins, hence we applied a set of multiple criteria to add additional proteins to our MB protein list. As detailed in methods, we applied a joint criterion in which we required proteins to be enriched in the MB sample relative to intact cells, depleted in cell bodies from which the MB was removed, and encoded by genes that were upregulated during MB regeneration based on our previous RNAseq results (Sood 2021). This additional set contained 123 proteins.…”

“…As a final test of selectivity in the anterior vs posterior half-cell experiment , we compared differential gene expression in the anterior and posterior halves of bisected Stentor cells (Sood 2021) to our differential protein abundance data. When cells are bisected, posterior half-cells regenerate new anterior structures such as the MB, while anterior half cells regenerate new posterior structures such as the holdfast.…”