MicroRNAs are deeply linked to the emergence of the complex octopus brain

Abstract

Soft-bodied cephalopods such as octopuses are exceptionally intelligent invertebrates with a highly complex nervous system that evolved independently from vertebrates. Because of elevated RNA editing in their nervous tissues, we hypothesized that RNA regulation may play a major role in the cognitive success of this group. We thus profiled messenger RNAs and small RNAs in three cephalopod species including 18 tissues of the Octopus vulgaris. We show that the major RNA innovation of soft-bodied cephalopods is an expansion of the microRNA (miRNA) gene repertoire. These evolutionarily novel miRNAs were primarily expressed in adult neuronal tissues and during the development and had conserved and thus likely functional target sites. The only comparable miRNA expansions happened, notably, in vertebrates. Thus, we propose that miRNAs are intimately linked to the evolution of complex animal brains.

Fig. 1.. RNA profiling of the common octopus O. vulgaris.

(A) Schematic representation of tissues sampled in the study. Neuronal and non-neuronal tissues are colored in blue and yellow, respectively. Inset (B): Brain and surrounding structures. (C) Main sequencing methods and computational analyses used in this study.

Fig. 2.. Expansion of the miRNA repertoire in cephalopods.

(A) Phylogeny of several animal groups with the branch lengths between nodes, or from a node to an extant species, reflecting the gains of miRNA families minus the losses (Materials and Methods). Vertical lines at the end of the branches indicate the shared complement of the indicated taxon as deposited in MirGeneDB (21); the other branches lead to single species (sponge: A. queenslandica; sea anemone: N. vectensis; flatworm: S. mediterranea; annelid: C. teleta; oyster: C. gigas; limpet: L. gigantea). (B) Number of miRNA families (excluding species-specific novel families) versus median 3′UTR length in selected animals. For instance, “Human” represents the number of miRNA families annotated in genus Homo. Median lengths of 3′UTRs were computed from genome annotations (Materials and Methods).

Fig. 3.. Novel, conserved octopus miRNAs are specifically expressed in neuronal tissues.

A simplified phylogenetic tree showing the number of miRNAs that evolved from the time bilaterians split from cnidarians to the last common ancestor of the two considered Octopus species. Color code as in Fig. 2. For each miRNA (columns), its expression distribution across tissues (rows) in both neural and non-neural tissues and the corresponding Z scores were computed. Columns within each bin were hierarchically clustered on the basis of the Z scores (extended version: fig. S3A). PSG, posterior salivary gland.

Fig. 4.. Novel, conserved octopus miRNAs are highly expressed during development.

Proportions of miRNA transcriptomes dedicated to miRNAs of different phylogenetic nodes of origin. Samples were obtained by developmental stage of O. vulgaris (). These samples cover the organogenic stages of O. vulgaris development (stage XI to stage XVIII) when most of the embryonic growth occurs, as well as the whole body and brain of 1-day-old paralarvae when the growth of the larval brain commences. “L + P” refers to the collective miRNAs that evolved in lophotrochzoans and platytrochozoans (see Fig. 2).

Fig. 5.. Target sites of novel miRNAs are conserved and coexpressed with the respective miRNA.

(A) Definition of MREs (or “8-mer”) and their evolutionary conservation. The 8-mer conservation rate is defined as the percentage of occurrences in 3′UTRs, where a particular 8-mer (red) is matched by exactly the same 8-mer at the same position in the aligned orthologous 3′UTR. (B) Shown here, for novel octopus miRNAs (conserved between O. vulgaris and O. bimaculoides), is the MRE conservation rate in units of a standard Z score. Coexpression is defined as an mRNA with an MRE and the respective miRNA codetected in at least one tissue at 10 and 100 counts per million, respectively (Materials and Methods). Coexpressed miRNA-MRE pairs are statistically more highly (P < 0.001) conserved than non-coexpressed pairs or control 8-mers which were not related to any MRE in the octopus. (C) As expected, MRE conservation rates are higher for evolutionarily older miRNA families. In (B) and (C), statistical significances: Mann-Whitney U test with Bonferroni correction for multiple hypothesis testing (n.s., P > 0.05 and ***P < 0.001).