Conserved Genes in Highly Regenerative Metazoans Are Associated with Planarian Regeneration

Abstract

Metazoan species depict a wide spectrum of regeneration ability which calls into question the evolutionary origins of the underlying processes. Since species with high regeneration ability are widely distributed throughout metazoans, there is a possibility that the metazoan ancestor had an underlying common molecular mechanism. Early metazoans like sponges possess high regenerative ability, but, due to the large differences they have with Cnidaria and Bilateria regarding symmetry and neuronal systems, it can be inferred that this regenerative ability is different. We hypothesized that the last common ancestor of Cnidaria and Bilateria possessed remarkable regenerative ability which was lost during evolution. We separated Cnidaria and Bilateria into three classes possessing whole-body regenerating, high regenerative ability, and low regenerative ability. Using a multiway BLAST and gene phylogeny approach, we identified genes conserved in whole-body regenerating species and lost in low regenerative ability species and labeled them Cnidaria and Bilaterian regeneration genes. Through transcription factor analysis, we identified that Cnidaria and Bilaterian regeneration genes were associated with an overabundance of homeodomain regulatory elements. RNA interference of Cnidaria and Bilaterian regeneration genes resulted in loss of regeneration phenotype for HRJDa, HRJDb, DUF21, DISP3, and ARMR genes. We observed that DUF21 knockdown was highly lethal in the early stages of regeneration indicating a potential role in wound response. Also, HRJDa, HRJDb, DISP3, and ARMR knockdown showed loss of regeneration phenotype after second amputation. The results strongly correlate with their respective RNA-seq profiles. We propose that Cnidaria and Bilaterian regeneration genes play a major role in regeneration across highly regenerative Cnidaria and Bilateria.

Keywords: comparative genomics; planarian; regeneration.

Fig. 1.

Reclassification of regenerative species. a) Early metazoans, Cnidaria, and Bilateria were classified according to the presence of symmetry and neuronal systems. b) Cnidarians and Bilaterians were classified into three groups: WBR, HRA, and LRA. Multiway mutual BLAST hits across HRA species were contrasted with LRA species to identify genes conserved across HRA species and lost in evolution across LRA species. Silhouettes were obtained from PhyloPic (http://phylopic.org/).

Fig. 2.

Characterization of CBR genes. a) Expression profile of all CBR genes. Expression values used are FPKM values. SMEDWI1, SMEDWI2, and SMEDWI3 were included as contrast. b) TF domain enrichment across different upstream sequence sizes. TFBSs were identified in upstream sequences, and the corresponding transcription factor domains were tallied and plotted on the x axis. Heatmap was generated using Morpheus (https://software.broadinstitute.org/morpheus/).

Fig. 3.

RNAi mortality and phenotype in unamputated individuals. a) Percentage mortality in unamputated planaria treated with dsRNA (N = 10). The y axis denotes percent mortality. b) HRJDa dsRNA treatment after 10, 12, and 14 d post last feeding showing loss of head phenotype.

Fig. 4.

RNAi mortality and phenotype in amputated individuals. a) and b) Percentage mortality observed after first (N = 20 each for head, trunk, and tail) and second amputation (N = surviving fragments post first amputation), respectively. The y axis denotes the percent mortality. Error bars generated using standard deviation of multiple experiments. c) and d) Mortality phenotype observed in regenerating heads and tails, respectively.