Clifton W. Ragsdale

How genomic changes translate into organismal novelties is often confounded by the multi-layered nature of genome architecture and the long evolutionary timescales over which molecular changes can accumulate. Coleoid cephalopods (squid, cuttlefish, and octopus) provide a unique system to study these processes due to a large-scale chromosomal rearrangement in the coleoid ancestor that gave rise to highly modified karyotypes, followed by lineage-specific repeat expansions, fusions, and translocations. How these events have shaped gene regulatory patterns underlying the evolution of coleoid innovations, including their large and elaborately structured nervous systems, novel organs, and complex behaviours, remains poorly understood. To address this, we integrate Micro-C, RNA-seq, and ATAC-seq across multiple coleoid species, developmental stages, and tissues. We find that while chromatin domains are broadly conserved, hundreds of chromatin loops are species- and context-specific, with distinct regulation signatures and dynamic expression profiles. CRISPR–Cas9 knockout of a putative regulatory sequence within a conserved region demonstrates the role of loops in neural development and the prevalence of long-range interactions that extend beyond chromatin domains. We propose that differential evolutionary constraints across the coleoid 3D genome allow macroevolutionary processes to shape genome topology in distinct ways, facilitating the emergence of novel regulatory entanglements and ultimately contributing to the evolution and maintenance of complex traits in the coleoids.

Octopuses have an incredibly rich behavioral repertoire, exhibiting complex motor acts that require the coordination of eight highly flexible arms, each with hundreds of suckers. These movements are controlled by an axial nerve cord (ANC), equivalent to the spinal cord, situated in the center of the arm musculature. The ANC has a cell body layer which forms a U-shape around its neuropil and is capped aborally, or opposite the sucker, by the cerebrobrachial tract (CBT), a massive fiber bundle known to interconnect the arms and the brain. In vertebrate spinal cords, in addition to the major fiber tracts that interconnect the brain and spinal cord, there are spinospinal connectives that coordinate complex motor behaviors across the appendages. Here, we asked with tract-tracing and immunohistochemistry, whether an octopus arm’s ANC might also have intrinsic longitudinal connections for coordinated arm and sucker movements. We found that the ANC neuropil is enriched in longitudinal fibers. These fibers form distinct tracts, two within the oral (sucker-side) neuropil and two in the aboral (brachial-side) neuropil. In addition, CBT itself demonstrates four major subtracts, and DiI labeling and dextran tracing suggests that (1) the CBT also carries arm-intrinsic longitudinal connections and (2) the CBT and the neuropil tracts can be subcategorized into those that primarily connect with the sucker and those that serve the arm musculature. We also examined the organization of fiber-tracts in the ANC of the arms and tentacles of two species of squid, establishing that an aboral, extra-neuropil tract is a shared feature across all cephalopod species studied. In addition, the squids also had an oral longitudinal tract, though its positioning and size varied with species and appendage. In sum, these findings describe the neural substrate for coordinating motor behaviors across the length of a cephalopod appendage.

Prehensile arms are among the most remarkable features of the octopus, but little is known about the neural circuitry controlling arm movements. Here, we report on the cellular and molecular organization of the arm nervous system, focusing on its massive axial nerve cords (ANCs). We found that the ANC is segmented. In transverse cross sections, the ANC cell body layer wraps around the neuropil with no apparent segregation of sensory and motor neurons. In longitudinal sections, however, ANC neurons form segments, setting up a modular organization to the adjoining neuropil. ANC nerves exit in the septa between segments, and for each sucker, form a spatial topographic map (“suckerotopy”). A strong link between ANC segmentation and flexible sucker-laden arms is confirmed by comparative study of squid arms and tentacles. These ANC modules offer a template for modeling the motor control of soft tissues and provide a compelling example of nervous system segmentation in molluscs.

Cephalopods are known for their large nervous systems, complex behaviors and morphological innovations. To investigate the genomic underpinnings of these features, we assembled the chromosomes of the Boston market squid, Doryteuthis (Loligo) pealeii, and the California two-spot octopus, Octopus bimaculoides, and compared them with those of the Hawaiian bobtail squid, Euprymna scolopes. The genomes of the soft-bodied (coleoid) cephalopods are highly rearranged relative to other extant molluscs, indicating an intense, early burst of genome restructuring. The coleoid genomes feature multi-megabase, tandem arrays of genes associated with brain development and cephalopod-specific innovations. We find that a known coleoid hallmark, extensive A-to-I mRNA editing, displays two fundamentally distinct patterns: one exclusive to the nervous system and concentrated in genic sequences, the other widespread and directed toward repetitive elements. We conclude that coleoid novelty is mediated in part by substantial genome reorganization, gene family expansion, and tissue-dependent mRNA editing.

The six-layered neocortex of the mammalian pallium has no clear homolog in birds or non-avian reptiles. Recent research indicates that although these extant amniotes possess a variety of divergent and nonhomologous pallial structures, they share a conserved set of neuronal cell types and circuitries. These findings suggest a principle of brain evolution: that natural selection preferentially preserves the integrity of information-processing pathways, whereas other levels of biological organization, such as the three-dimensional architectures of neuronal assemblies, are less constrained. We review the similarities of pallial neuronal cell types in amniotes, delineate candidate gene regulatory networks for their cellular identities, and propose a model of developmental evolution for the divergence of amniote pallial structures.

0. Summary statement: Octopus optic glands employ a multiplex progression of signaling molecules to regulate maternal behaviors. 1. Post-reproductive life in the female octopus is characterized by an extreme pattern of maternal care: the mother cares for her clutch of eggs without feeding until her death. These maternal behaviors are completely eradicated if the optic glands, the octopus analog of the vertebrate pituitary gland, are removed from brooding females. Despite the optic gland’s importance in regulating maternal behavior, the molecular features underlying optic gland function are unknown. Here, we identify major signaling systems of the Octopus bimaculoides optic gland. Through behavioral analyses and transcriptome sequencing, we report that the optic gland undergoes remarkable molecular changes that coincide with transitions between behavioral stages. These include the dramatic up- and down-regulation of catecholamine, steroid, insulin, and feeding peptide pathways. Transcriptome analyses in other tissues demonstrate that these molecular changes are not generalized markers of aging and senescence, but instead, specific features of the optic glands. Our results provide strong evidence for the functional conservation of signaling molecules across evolutionarily distant species. For example, elevated levels of insulin growth factor binding proteins are associated with cachexia-like tissue wasting in flies, humans, and, reported here, octopuses. Our study expands the classic optic gland-pituitary gland analogy and indicates that, rather than a single “self-destruct” hormone, the maternal optic glands employ multiple pathways as systemic hormonal signals of behavioral control.

Post-reproductive life in the female octopus is characterized by an extreme pattern of maternal care: the mother cares for her clutch of eggs without feeding until her death. These maternal behaviors are eradicated if the optic glands, the octopus analog of the vertebrate pituitary gland, are removed from brooding females. Despite the optic gland9s importance in regulating maternal behavior, the molecular features underlying optic gland function are unknown. Here, we identify major signaling systems of the Octopus bimaculoides optic gland. Through behavioral analyses and transcriptome sequencing, we report that the optic gland undergoes remarkable molecular changes that coincide with transitions between behavioral stages. These include the dramatic up- and down-regulation of catecholamine, steroid, insulin, and feeding peptide pathways. Transcriptome analyses in other tissues demonstrate that these molecular changes are not generalized markers of senescence, but instead, specific features of the optic glands. Our study expands the classic optic gland-pituitary gland analogy and more specifically, it indicates that, rather than a single “self-destruct” hormone, the maternal optic glands employ multiple pathways as systemic hormonal signals of behavioral regulation.