Neuronal segmentation in cephalopod arms.

The prehensile arms of the cephalopod are among these animals most remarkable features, but the neural circuitry governing arm and sucker movements remains largely unknown. We studied the neuronal organization of the adult axial nerve cord (ANC) of Octopus bimaculoides with molecular and cellular methods. The ANCs, which lie in the center of every arm, are the largest neuronal structures in the octopus, containing four times as many neurons as found in the central brain. In transverse cross section, the cell body layer (CBL) of the ANC wraps around its neuropil (NP) with little apparent segregation of sensory and motor neurons or nerve exits. Strikingly, when studied in longitudinal sections, the ANC is segmented. ANC neuronal cell bodies form columns separated by septa, with 15 segments overlying each pair of suckers. The segments underlie a modular organization to the ANC neuropil: neuronal cell bodies within each segment send the bulk of their processes directly into the adjoining neuropil, with some reaching the contralateral side. In addition, some nerve processes branch upon entering the NP, forming short-range projections to neighboring segments and mid-range projections to the ANC segments of adjoining suckers. The septa between the segments are employed as ANC nerve exits and as channels for ANC vasculature. Cellular analysis establishes that adjoining septa issue nerves with distinct fiber trajectories, which across two segments (or three septa) fully innervate the arm musculature. Sucker nerves also use the septa, setting up a nerve fiber "suckerotopy" in the sucker-side of the ANC. Comparative anatomy suggests a strong link between segmentation and flexible sucker-laden arms. In the squid Doryteuthis pealeii, the arms and the sucker-rich club of the tentacles have segments, but the sucker-poor stalk of the tentacles does not. The neural modules described here provide a new template for understanding the motor control of octopus soft tissues. In addition, this finding represents the first demonstration of nervous system segmentation in a mollusc.

Signaling Ligand Heterogeneities in the Peduncle Complex of the Cephalopod Mollusc Octopus bimaculoides.

INTRODUCTION: The octopus peduncle complex is an agglomeration of neural structures with remarkably diverse functional roles. The complex rests on the optic tract, between the optic lobe and the central brain, and comprises the peduncle lobe proper, the olfactory lobe, and the optic gland. The peduncle lobe regulates visuomotor behaviors, the optic glands control sexual maturation and maternal death, and the olfactory lobe is thought to receive input from the olfactory organ. Recent transcriptomic and metabolomic studies have identified candidate peptide and steroid ligands in the Octopus bimaculoides optic gland. METHODS: With gene expression for these ligands and their biosynthetic enzymes, we show that optic gland neurochemistry extends beyond the traditional optic gland secretory tissue and into lobular territories. RESULTS: A key finding is that the classically defined olfactory lobe is itself a heterogeneous territory and includes steroidogenic territories that overlap with cells expressing molluscan neuropeptides and the synthetic enzyme dopamine beta-hydroxylase. CONCLUSION: Our study reveals the neurochemical landscape of the octopus peduncle complex, highlighting the unexpected overlap between traditionally defined regions.

Toward an Understanding of Octopus Arm Motor Control.

Octopuses have the extraordinary ability to control eight prehensile arms with hundreds of suckers. With these highly flexible limbs, they engage in a wide variety of tasks, including hunting, grooming, and exploring their environment. The neural circuitry generating these movements engages every division of the octopus nervous system, from the nerve cords of the arms to the supraesophegeal brain. In this review, the current knowledge on the neural control of octopus arm movements is discussed, highlighting open questions and areas for further study.

Steroid hormones of the octopus self-destruct system.

Among all invertebrates, soft-bodied cephalopods have the largest central nervous systems and the greatest brain-to-body mass ratios, yet unlike other big-brained animals, cephalopods are unusually short lived.1-5 Primates and corvids survive for many decades, but shallow-water octopuses, such as the California two-spot octopus (Octopus bimaculoides), typically live for only 1 year.6,7 Lifespan and reproduction are controlled by the principal neuroendocrine center of the octopus: the optic glands, which are functional analogs to the vertebrate pituitary gland.8-10 After mating, females steadfastly brood their eggs, begin fasting, and undergo rapid physiological decline, featuring repeated self-injury and leading to death.11 Removal of the optic glands completely reverses this life history trajectory,10 but the signaling factors underlying this major life transition are unknown. Here, we characterize the major secretions and steroidogenic pathways of the female optic gland using mass spectrometry techniques. We find that at least three pathways are mobilized to increase synthesis of select sterol hormones after reproduction. One pathway generates pregnane steroids, known in other animals to support reproduction.12-16 Two other pathways produce 7-dehydrocholesterol and bile acid intermediates, neither of which were previously known to be involved in semelparity. Our results provide insight into invertebrate cholesterol pathways and confirm a remarkable unity of steroid hormone biology in life history processes across Bilateria.

Genome and transcriptome mechanisms driving cephalopod evolution.

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 emergence of the brain non-CpG methylation system in vertebrates.

Mammalian brains feature exceptionally high levels of non-CpG DNA methylation alongside the canonical form of CpG methylation. Non-CpG methylation plays a critical regulatory role in cognitive function, which is mediated by the binding of MeCP2, the transcriptional regulator that when mutated causes Rett syndrome. However, it is unclear whether the non-CpG neural methylation system is restricted to mammalian species with complex cognitive abilities or has deeper evolutionary origins. To test this, we investigated brain DNA methylation across 12 distantly related animal lineages, revealing that non-CpG methylation is restricted to vertebrates. We discovered that in vertebrates, non-CpG methylation is enriched within a highly conserved set of developmental genes transcriptionally repressed in adult brains, indicating that it demarcates a deeply conserved regulatory program. We also found that the writer of non-CpG methylation, DNMT3A, and the reader, MeCP2, originated at the onset of vertebrates as a result of the ancestral vertebrate whole-genome duplication. Together, we demonstrate how this novel layer of epigenetic information assembled at the root of vertebrates and gained new regulatory roles independent of the ancestral form of the canonical CpG methylation. This suggests that the emergence of non-CpG methylation may have fostered the evolution of sophisticated cognitive abilities found in the vertebrate lineage.

Evolution of the Chordate Telencephalon.

The dramatic evolutionary expansion of the neocortex, together with a proliferation of specialized cortical areas, is believed to underlie the emergence of human cognitive abilities. In a broader phylogenetic context, however, neocortex evolution in mammals, including humans, is remarkably conservative, characterized largely by size variations on a shared six-layered neuronal architecture. By contrast, the telencephalon in non-mammalian vertebrates, including reptiles, amphibians, bony and cartilaginous fishes, and cyclostomes, features a great variety of very different tissue structures. Our understanding of the evolutionary relationships of these telencephalic structures, especially those of basally branching vertebrates and invertebrate chordates, remains fragmentary and is impeded by conceptual obstacles. To make sense of highly divergent anatomies requires a hierarchical view of biological organization, one that permits the recognition of homologies at multiple levels beyond neuroanatomical structure. Here we review the origin and diversification of the telencephalon with a focus on key evolutionary innovations shaping the neocortex at multiple levels of organization.

Homology, neocortex, and the evolution of developmental mechanisms.

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.

Multiple optic gland signaling pathways implicated in octopus maternal behaviors and death.

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 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 upregulation and downregulation 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.

Molecular anatomy of the alligator dorsal telencephalon.

The evolutionary relationships of the mammalian neocortex and avian dorsal telencephalon (DT) nuclei have been debated for more than a century. Despite their central importance to this debate, nonavian reptiles remain underexplored with modern molecular techniques. Reptile studies harbor great potential for understanding the changes in DT organization that occurred in the early evolution of amniotes. They may also help clarify the specializations in the avian DT, which comprises a massive, cell-dense dorsal ventricular ridge (DVR) and a nuclear dorsal-most structure, the Wulst. Crocodilians are phylogenetically and anatomically attractive for DT comparative studies: they are the closest living relatives of birds and have a strikingly bird-like DVR, but they also possess a highly differentiated reptile cerebral cortex. We studied the DT of the American alligator, Alligator mississippiensis, at late embryonic stages with a panel of molecular marker genes. Gene expression and cytoarchitectonic analyses identified clear homologs of all major avian DVR subdivisions including a mesopallium, an extensive nidopallium with primary sensory input territories, and an arcopallium. The alligator medial cortex is divided into three components that resemble the mammalian dentate gyrus, CA fields, and subiculum in gene expression and topography. The alligator dorsal cortex contains putative homologs of neocortical input, output, and intratelencephalic projection neurons and, most notably, these are organized into sublayers similar to mammalian neocortical layers. Our findings on the molecular anatomy of the crocodilian DT are summarized in an atlas of the alligator telencephalon.

Neocortical Association Cell Types in the Forebrain of Birds and Alligators.

The avian dorsal telencephalon has two vast territories, the nidopallium and the mesopallium, both of which have been shown to contribute substantially to higher cognitive functions. From their connections, these territories have been proposed as equivalent to mammalian neocortical layers 2 and 3, various neocortical association areas, or the amygdala, but whether these are analogies or homologies by descent is unknown. We investigated the molecular profiles of the mesopallium and the nidopallium with RNA-seq. Gene expression experiments established that the mesopallium, but not the nidopallium, shares a transcription factor network with the intratelencephalic class of neocortical neurons, which are found in neocortical layers 2, 3, 5, and 6. Experiments in alligators demonstrated that these neurons are also abundant in the crocodilian cortex and form a large mesopallium-like structure in the dorsal ventricular ridge. Together with previous work, these molecular findings indicate a homology by descent for neuronal cell types of the avian dorsal telencephalon with the major excitatory cell types of mammalian neocortical circuits: the layer 4 input neurons, the deep layer output neurons, and the multi-layer intratelencephalic association neurons. These data raise the interesting possibility that avian and primate lineages evolved higher cognitive abilities independently through parallel expansions of homologous cell populations.

Cadherin genes and evolutionary novelties in the octopus.

All animals with large brains must have molecular mechanisms to regulate neuronal process outgrowth and prevent neurite self-entanglement. In vertebrates, two major gene families implicated in these mechanisms are the clustered protocadherins and the atypical cadherins. However, the molecular mechanisms utilized in complex invertebrate brains, such as those of the cephalopods, remain largely unknown. Recently, we identified protocadherins and atypical cadherins in the octopus. The octopus protocadherin expansion shares features with the mammalian clustered protocadherins, including enrichment in neural tissues, clustered head-to-tail orientations in the genome, and a large first exon encoding all cadherin domains. Other octopus cadherins, including a newly-identified cadherin with 77 extracellular cadherin domains, are elevated in the suckers, a striking cephalopod novelty. Future study of these octopus genes may yield insights into the general functions of protocadherins in neural wiring and cadherin-related proteins in complex morphogenesis.

Evidence for a cordal, not ganglionic, pattern of cephalopod brain neurogenesis.

INTRODUCTION: From the large-brained cephalopods to the acephalic bivalves, molluscs show a vast range of nervous system centralization patterns. Despite this diversity, molluscan nervous systems, broadly considered, are organized either as medullary cords, as seen in chitons, or as ganglia, which are typical of gastropods and bivalves. The cephalopod brain is exceptional not just in terms of its size; its relationship to a molluscan cordal or ganglionic plan has not been resolved from the study of its compacted adult structure. One approach to clarifying this puzzle is to investigate the patterns of early cephalopod brain neurogenesis, where molecular markers for cephalopod neural development may be informative. RESULTS: We report here on early brain pattern formation in the California two-spot octopus, Octopus bimaculoides. Employing gene expression analysis with the pan-bilaterian neuronal marker ELAV and the atonal-related neuronal differentiation genes NEUROGENIN and NEUROD, as well as immunostaining using a Distalless-like homeoprotein antibody, we found that the octopus central brain forms from concentric cords rather than bilaterally distributed pairs of ganglia. CONCLUSION: We conclude that the cephalopod brain, despite its great size and elaborate specializations, retains in its development the hypothesized ancestral molluscan nervous system plan of medullary cords, as described for chitons and other aculiferan molluscs.

The octopus genome and the evolution of cephalopod neural and morphological novelties.

Coleoid cephalopods (octopus, squid and cuttlefish) are active, resourceful predators with a rich behavioural repertoire. They have the largest nervous systems among the invertebrates and present other striking morphological innovations including camera-like eyes, prehensile arms, a highly derived early embryogenesis and a remarkably sophisticated adaptive colouration system. To investigate the molecular bases of cephalopod brain and body innovations, we sequenced the genome and multiple transcriptomes of the California two-spot octopus, Octopus bimaculoides. We found no evidence for hypothesized whole-genome duplications in the octopus lineage. The core developmental and neuronal gene repertoire of the octopus is broadly similar to that found across invertebrate bilaterians, except for massive expansions in two gene families previously thought to be uniquely enlarged in vertebrates: the protocadherins, which regulate neuronal development, and the C2H2 superfamily of zinc-finger transcription factors. Extensive messenger RNA editing generates transcript and protein diversity in genes involved in neural excitability, as previously described, as well as in genes participating in a broad range of other cellular functions. We identified hundreds of cephalopod-specific genes, many of which showed elevated expression levels in such specialized structures as the skin, the suckers and the nervous system. Finally, we found evidence for large-scale genomic rearrangements that are closely associated with transposable element expansions. Our analysis suggests that substantial expansion of a handful of gene families, along with extensive remodelling of genome linkage and repetitive content, played a critical role in the evolution of cephalopod morphological innovations, including their large and complex nervous systems.

Levels of homology and the problem of neocortex.

The neocortex is found only in mammals, and the fossil record is silent on how this soft tissue evolved. Understanding neocortex evolution thus devolves to a search for candidate homologous neocortex traits in the extant nonmammalian amniotes. The difficulty is that homology is based on similarity, and the six-layered neocortex structure could hardly be more dissimilar in appearance from the nuclear organization that is so conspicuous in the dorsal telencephalon of birds and other reptiles. Recent molecular data have, however, provided new support for one prominent hypothesis, based on neuronal circuits, that proposes the principal neocortical input and output cell types are a conserved feature of amniote dorsal telencephalon. Many puzzles remain, the greatest being understanding the selective pressures and molecular mechanisms that underlie such tremendous morphological variation in telencephalon structure.

The gyri of the octopus vertical lobe have distinct neurochemical identities.

The cephalopod vertical lobe is the largest learning and memory structure known in invertebrate nervous systems. It is part of the visual learning circuit of the central brain, which also includes the superior frontal and subvertical lobes. Despite the well-established functional importance of this system, little is known about neuropil organization of these structures and there is to date no evidence that the five longitudinal gyri of the vertical lobe, perhaps the most distinctive morphological feature of the octopus brain, differ in their connections or molecular identities. We studied the histochemical organization of these structures in hatchling and adult Octopus bimaculoides brains with immunostaining for serotonin, octopus gonadotropin-releasing hormone (oGNRH), and octopressin-neurophysin (OP-NP). Our major finding is that the five lobules forming the vertical lobe gyri have distinct neurochemical signatures. This is most prominent in the hatchling brain, where the median and mediolateral lobules are enriched in OP-NP fibers, the lateral lobule is marked by oGNRH innervation, and serotonin immunostaining heavily labels the median and lateral lobules. A major source of input to the vertical lobe is the superior frontal lobe, which is dominated by a neuropil of interweaving fiber bundles. We have found that this neuropil also has an intrinsic neurochemical organization: it is partitioned into territories alternately enriched or impoverished in oGNRH-containing fascicles. Our findings establish that the constituent lobes of the octopus superior frontal-vertical system have an intricate internal anatomy, one likely to reflect the presence of functional subsystems within cephalopod learning circuitry.

NSF workshop report: discovering general principles of nervous system organization by comparing brain maps across species.

Efforts to understand nervous system structure and function have received new impetus from the federal Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative. Comparative analyses can contribute to this effort by leading to the discovery of general principles of neural circuit design, information processing, and gene-structure-function relationships that are not apparent from studies on single species. We here propose to extend the comparative approach to nervous system 'maps' comprising molecular, anatomical, and physiological data. This research will identify which neural features are likely to generalize across species, and which are unlikely to be broadly conserved. It will also suggest causal relationships between genes, development, adult anatomy, physiology, and, ultimately, behavior. These causal hypotheses can then be tested experimentally. Finally, insights from comparative research can inspire and guide technological development. To promote this research agenda, we recommend that teams of investigators coalesce around specific research questions and select a set of 'reference species' to anchor their comparative analyses. These reference species should be chosen not just for practical advantages, but also with regard for their phylogenetic position, behavioral repertoire, well-annotated genome, or other strategic reasons. We envision that the nervous systems of these reference species will be mapped in more detail than those of other species. The collected data may range from the molecular to the behavioral, depending on the research question. To integrate across levels of analysis and across species, standards for data collection, annotation, archiving, and distribution must be developed and respected. To that end, it will help to form networks or consortia of researchers and centers for science, technology, and education that focus on organized data collection, distribution, and training. These activities could be supported, at least in part, through existing mechanisms at NSF, NIH, and other agencies. It will also be important to develop new integrated software and database systems for cross-species data analyses. Multidisciplinary efforts to develop such analytical tools should be supported financially. Finally, training opportunities should be created to stimulate multidisciplinary, integrative research into brain structure, function, and evolution.

NSF workshop report: discovering general principles of nervous system organization by comparing brain maps across species.

Efforts to understand nervous system structure and function have received new impetus from the federal Brain Research through Advancing Innovative Neurotechnologies (BRAIN) Initiative. Comparative analyses can contribute to this effort by leading to the discovery of general principles of neural circuit design, information processing, and gene-structure-function relationships that are not apparent from studies on single species. We here propose to extend the comparative approach to nervous system 'maps' comprising molecular, anatomical, and physiological data. This research will identify which neural features are likely to generalize across species, and which are unlikely to be broadly conserved. It will also suggest causal relationships between genes, development, adult anatomy, physiology, and, ultimately, behavior. These causal hypotheses can then be tested experimentally. Finally, insights from comparative research can inspire and guide technological development. To promote this research agenda, we recommend that teams of investigators coalesce around specific research questions and select a set of 'reference species' to anchor their comparative analyses. These reference species should be chosen not just for practical advantages, but also with regard for their phylogenetic position, behavioral repertoire, well-annotated genome, or other strategic reasons. We envision that the nervous systems of these reference species will be mapped in more detail than those of other species. The collected data may range from the molecular to the behavioral, depending on the research question. To integrate across levels of analysis and across species, standards for data collection, annotation, archiving, and distribution must be developed and respected. To that end, it will help to form networks or consortia of researchers and centers for science, technology, and education that focus on organized data collection, distribution, and training. These activities could be supported, at least in part, through existing mechanisms at NSF, NIH, and other agencies. It will also be important to develop new integrated software and database systems for cross-species data analyses. Multidisciplinary efforts to develop such analytical tools should be supported financially. Finally, training opportunities should be created to stimulate multidisciplinary, integrative research into brain structure, function, and evolution.