Evidence for a cordal, not ganglionic, pattern of cephalopod brain neurogenesis
© Shigeno et al. 2015
Received: 30 January 2015
Accepted: 22 July 2015
Published: 7 September 2015
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.
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.
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.
Molluscs are highly successful animals with a great diversity of body plans. Exceptional even among this group are the cephalopods, with the elaborate sensory systems, flexible learning abilities, and sophisticated motor outputs expected of an accomplished predator (reviewed in [1–3]). The behavioral capacities of cephalopods are reflected in the size and complexity of the cephalopod brain, with its dozens of functionally distinct lobes and associated complex of neural interconnections . How cephalopod brain organization corresponds with patterns of neural tissue organization in other molluscs remains uncertain.
One approach to studying cephalopod brain evolution has been to investigate nautiloid cephalopods, an ancient lineage that includes the only extant non-coleoid cephalopods . Young  described the adult brain of Nautilus as consisting of cerebral, pedal, and visceral cords surrounding the esophagus and suggested a homology between these cords and those of a polyplacophoran nervous system, such as that of a chiton [8, 9]. A problem for this interpretation is that the lobes of the Nautilus brain, unlike those of coleoid brains, show a mixing of neuronal somata and processes that does not follow the typical invertebrate histology of somata situated peripherally around a central core of neuropil. It is possible that the cord-like appearance of the Nautilus brain is a derived feature reflecting the absence of standard ganglionic structuring in the mature nautiloid brain. Bullock , for example, has specifically proposed for the Nautilus brain that “a superficial resemblance to primitive medullary cords” may have been produced during development by a secondary spread of neurons into commissures and the process-rich cores of ganglia.
A second approach to the problem of cephalopod brain origins is to ask how the cephalopod brain develops. Is its central nervous system initially generated as a system of cords or as a spatially restricted set of nodal ganglia? Indeed, Bullock’s criticism of Young’s Nautilus analysis, that a commissural and longitudinal spread of neurons from paired ganglia could readily account for any cordal appearance of the adult cephalopod nervous system , is in essence a speculation about development.
Octopus and squid nervous system development has been studied using reconstructions of histological sections [11–16]. These reports concluded that coleoid brain development is ganglionic (Fig. 1 Right Upper Panel), with neural progenitors arising in ectodermal thickenings and ingressing from the surface ectoderm. These ingressing cells form four pairs of clusters of neuronal cell bodies that initially lack processes. The anterior clusters, bilateral to the mouth, give rise to the cerebral ganglia, which later fuse across the midline. The other three ganglionic pairs become (1) the optic ganglia adjoining the embryonic eye field, (2) the pedal ganglia near the base of the arms, and (3) the palliovisceral or visceral ganglia situated along the collar between the mantle and the arms. In these histological reconstructions, the cerebral, pedal and palliovisceral clusters appear spherical or nodular in shape. Only the prospective optic ganglia display an elongated C-shape reminiscent of a medullary cord. The ganglia expand in size with the accumulation of additional neurons, eventually forming the masses of the adult cephalopod brain (Fig. 1 Right Lower Panel).
Development of the coleoid brain from a system of cords would appear very different (Fig. 1 Middle Upper Panel). The early allocation of neurons would be not to nodular clusters, but to extensive, rope-like territories in the neurectoderm. Some of these neuronal territories would be expected to span the midline early in embryogenesis, rather than populate the midline by ganglionic fusion later in development. The size of the cordal territories would increase over time, but adult brain morphology would arise from global transitions of the embryonic body plan that centralize the peripherally arranged cords, rather than relatively local and more constrained migrations of neuronal somata.
Because traditional silver staining methods for recognizing neurons do not work effectively on early cephalopod embryos, the reconstruction data supporting a ganglionic origin for the coleoid brain were based on material stained with alum-haematoxylin and toluidine blue [11–16]. Consequently, definitive cell-type identification of embryonic neurons was not possible, making the often-repeated conclusion of ganglionic rather than cordal origins of the coleoid brain less secure. In this report, we have revisited the issue of coleoid brain development by studying the expression of molecular markers of neuronal cell types in wholemount preparations of octopus embryos, which permits direct demonstration, rather than section reconstruction, of three-dimensional patterns.
elav (embryonic lethal, abnormal visual system) is a RNA binding protein gene identified in a Drosophila mutant screen as required by neurons from their birth . Molecular analysis has demonstrated that Drosophila ELAV protein is expressed by young neurons, but not neuronal progenitors . Independent studies in mammals identified the genes for the autoimmune antigens HuC and HuD as elav homologs that are also expressed in postmitotic neurons (ELAVL3 and ELAVL4; [19–21]). It is now clear that ELAV exhibits highly conserved neuron-specific expression during development across animals as disparate as flies, worms, and vertebrates [22, 23]. Nomaksteinsky et al.  took specific advantage of the restricted neuronal expression of elav to demonstrate that the nervous system of adult acorn worms is centralized into dorsal and ventral cords.
NEUROD and NEUROGENIN (NEUROG) are members of the atonal basic helix-loop-helix transcription factor family involved in the neuronal differentiation and cell-type specification of the developing vertebrate nervous system . In Xenopus and mouse, Neurogenin directly activates the transcriptional targets NeuroD1 and NeuroD4 to induce regulators of neuronal signaling, differentiation, and migration . Both NEUROD and NEUROG are highly conserved across many metazoan lineages including sponges and sea anemones . In the annelid worm Platynereis dumerilii, for example, Neurogenin has been shown to be expressed principally in the apical-most layer of cells of the prospective ventral nerve cord, while annelid elav is abundant in more basal cells .
We describe here the pattern of neurogenesis in octopus embryos and assess the processes by which the octopus brain is assembled during embryogenesis. Our findings, based on ELAV, NEUROD, and NEUROG gene expression and immunolabeling using the neuronal territory marker Distalless-like (DLL), provide support for the conclusions that (1) the cephalopod brain develops from concentric cords, and (2) a system of nerve cords is a feature of molluscan neuroembryogenesis shared by octopuses, monoplacophorans, polyplacophorans and some early branching gastropod clades, such as those in Patellogastropoda and Vetigastropoda .
Materials and methods
Collection of animals
ELAV gene isolation
An 849bp PCR product, recovered from stage 18 octopus embryo cDNA, was identified as representing an octopus ELAV ortholog based on 71 % nucleotide and 68 % amino acid identity with the ELAV sequence from the annelid Platynereis dumerilii (GenBank EF384209). The PCR employed two rounds of nested primers. Primers were designed with Blockmaker and CODEHOP algorithms [35, 36] using Drosophila, mouse and Amphioxus amino acid sequences as inputs. First-round pairs were the forward primer 5′-GTGAACTACCTGCCCCARAMNATGAC-3′ (VNYLPQTMT) and the reverse primer 5′-GCGGCCTCGTCGTARTKNGYCAT-3′ (MTHYDEAA). Second-round nested primers were 5′-GGGCTACGGCTTCGTNAAYTAYRT-3′ (LGYGFVNY) and 5′-CACGGCGCCGAANGGNCCRAA-3′ (FGPFGAV). Primers were used at a final concentration of 3 μM. The PCR protocol for both rounds featured a 4-min denaturation step at 94 °C and 35 rounds of 35 s at 94 °C, 35 s at 42 °C, and 50 s at 72 °C. The amplified 849 bp band was excised from a 2 % low melt agarose gel and subcloned into the pCRII-TOPO vector (Invitrogen).
Neuronal differentiation gene isolation
A NEUROG cDNA was recovered serendipitously during a quality control screen of 5′ RACE-ready O. bimaculoides cDNA synthesized with the SMART RACE kit (Clontech). An O. bimaculoides NEUROD fragment was amplified with the non-degenerate primers 5′-TTGGTGACAGCAATAGCG-3′ and 5′-GCGGATGATGACAGAATCTC-3′. NEUROG and NEUROD were subcloned into pCRII-TOPO (Invitrogen) and pGEM-T EASY (Promega), respectively.
Phylogenetic analyses of gene families
Phylogenetic analyses of NEUROD, NEUROG, ELAV, and ELAV-like genes were performed to determine the orthology of the genes isolated. Related genes from other species were obtained from GenBank and aligned using MUSCLE . Approximately maximum likelihood phylogenetic trees were generated with FastTree  and illustrated with FigTree (http://tree.bio.ed.ac.uk/software/figtree).
Wholemount in situ hybridization
Embryos inside their egg cases were fixed by immersion in 4 % paraformaldehyde in phosphate-buffered saline (PBS) overnight at 4 °C. After removal of the chorions with fine forceps, the embryos were staged, sorted, and stored overnight at 4 °C in fresh fixative. Older animals (stage 20 embryos and hatchlings) were anesthetized in 3 % ethanol in seawater before fixation. Embryos and hatchlings were dehydrated through a methanol series prior to hybridization. Wholemount in situ hybridization was performed with digoxigenin-labeled riboprobes following previously established methods  with the following modifications: (1) embryos were bleached in 6 % hydrogen peroxide in PBS-1 % Tween (PBST) for one hour following rehydration, and (2) after detergent treatment young embryos were postfixed in 4 % paraformaldehyde-PBS without glutaraldehyde. Labeled RNA probes were detected with antibody-phosphatase conjugates (Roche Molecular Biochemicals, Indianapolis, IN), and enzyme-catalyzed deposition of chloride 5-bromo-4-chloro-3-indolyl phosphate (BCIP) and nitroblue tetrazolium (NBT). Control embryo wholemounts hybridized with ELAV sense probes did not display labeling above background (not illustrated).
Combined immunostaining and gene expression histochemistry
Paraformaldehyde-fixed hatchlings were cryoprotected in 20 % sucrose-PBST, embedded in 10 % gelatin blocks, and cut into 30-micron thick sections on a freezing microtome. Sections were mounted on glass slides and processed for in situ hybridization . After the tetrazolium histochemistry, the sections were rinsed in PBST, blocked in 10 % bovine serum-PBST and incubated for 3-6 hours at room temperature with the mouse monoclonal antibody 6-11B-1, which recognizes acetylated alpha-tubulin (1:5000 dilution in 1 % serum-PBST; Sigma, T6793). Antibody labeling was detected with goat anti-mouse IgG conjugated to Alexa 594 (Sigma, A11005).
Paraformaldehyde-fixed embryos (stage 8) were rinsed in PBST, blocked in 10 % bovine serum-PBST and incubated overnight at 4 °C with a rabbit polyclonal antibody against DISTALLESS (DLL) homeodomain  diluted 1:2000 (kindly provided by Dr. Claudia Farfán) and with the mouse monoclonal antibody for acetylated alpha-tubulin. Secondary antibody labeling for DLL employed a goat anti-rabbit IgG conjugated to Alexa 488 (Sigma, A11008). 40,6-diamidino-2-phenylindole dihydrochloride (DAPI, Sigma, 5 μg ml−1 in PBS) served as a fluorescent nuclear marker to illustrate the overall morphology of the embryo. Embryos were incubated in DAPI solution for 30 min, washed in PBS, and studied with UV stereo epifluorescence microscopy. Immunostained samples were analyzed with a TIRF Live Cell Microscope (Olympus) or Leica TCS SP2 AOBS laser scanning confocal microscope.
Results and discussion
Staging of O. bimaculoides development
The embryos of O. bimaculoides display the direct development mode typical of cephalopods, without the cataclysmic metamorphosis seen in other molluscan larvae [41, 42]. The placement of the early embryo is telolecithal, and the movements of gastrulation are epibolic. We adopted the morphological criteria defined for Octopus vulgaris  and Eledone cirrosa [43, 44] to stage O. bimaculoides development, recording Naef stages I–XX as 1-20 (Fig. 2). The embryo is first visible as an apical disc on top of the large yolk (Fig. 2a). As epiboly proceeds, concentrically placed organ primordia form, with the mantle at the center and the four pairs of arm buds at the periphery (Fig. 2b). In the intermediate region, the primordia of the collar and funnel are visible as distinct folds between the mantle and the arms (Fig. 2b, e, and h). Also visible in the intermediate region are the ectodermal thickenings that give rise to three major sensory organs: the eyes, the olfactory organs, and the statocysts (Fig. 2b and c). The morphogenetic movements that generate the adult octopus anatomy are extensive, but maintain the organ topology found in the organogenetic stage (stage 8) embryo. The mantle at the center of the disc extends upwards and the periphery of the disc contracts, establishing an arm crown around the mouth (Fig. 2d-n) and forming a circumesophageal brain comprising a supraesophageal mass with paired optic lobes (Fig. 2o) and a subesophageal mass with brachial, pedal, and palliovisceral lobes .
ELAV mRNA labels somata but not processes in octopus brain
ELAV gene expression in octopus brain development
The cerebral cord
The cerebral cord is the anlage of the supraesophageal brain, including the learning and memory centers (frontal–vertical lobes) and the higher motor centers (basal lobes). This cord remains anterior throughout development. Previous descriptions suggested that anterior neurons are initially organized as paired spherical cerebral ganglia situated lateral to the foregut [12–14]. ELAV gene expression wholemounts demonstrated instead a cord-like arrangement as early as stage 7, when a band of ELAV-rich cells spans the midline immediately posterior to the mouth (Fig. 5a, b and i). Sites of early neurogenesis were also seen at the lateral limits of the prospective optic lobes, where ELAV gene expression identified a bilateral pair of swoosh-like territories. Between these three concentrations of early neurons, the densities of labeled cells appeared to decline gradually. By stage 10 the cerebral and optic lobe precursor territories extended circumferentially to form a nearly continuous cord of young neurons (Fig. 5d). Between stages 11 and 15, cerebral cord tissue condensed to form a supraesophageal mass flanked by the optic lobes (Fig. 5e–g; see also Summary Fig. 6).
The palliovisceral cord
The palliovisceral cord gives rise to the posterior subesophageal mass, which innervates all dorsal organs, including the mantle, visceral mass, and collar, through the pallial, visceral, and posterior funnel nerves . ELAV gene expression at stages 7 and 8 demonstrated concentrations of prospective palliovisceral neurons beneath the presumptive mucous funnel organ at the posterior midline and in a bilateral pair of multiple cell-clusters just inside the collar folds (Fig. 5a, c, and j). In addition, low-density fields of ELAV-positive cells extended away from these concentrations in a graded fashion. By stage 10, ELAV expression clearly described a U-shaped palliovisceral cord encircling the prospective mantle, which was capped by the C-shaped cerebral cord (Fig. 5d). This neural architecture is strikingly different from the spherical ganglionic forms described for the developing palliovisceral nervous systems of Octopus vulgaris and the oegopsid squid Todarodes pacificus [13, 16], but is in closer accord with the report of an elongated palliovisceral primordium in the pygmy squid Idiosepius paradoxus . At later stages, the palliovisceral cord condenses to form a pair of ovoid masses, which then centralize to produce a single posterior subesophageal lobe (Fig. 5e–h).
The pedal cord
This cord, also sometimes called the brachiopedal cord, forms a large brain center for the arms and anterior part of the funnel. The pedal cord is initially situated close to the arm buds and the prospective axial nerve cord of each arm, as previously reported . By stage 8, ELAV gene expression demonstrated neurons extending from the prospective axial nerve cords to the pedal cord (Fig. 5c). ELAV labeling established that the pedal cord is continuous across the posterior midline, extending under the funnel folds (Fig. 5a, c). At later stages, the ELAV-rich pedal cord centralizes to form the compact structure comprising the anterior and middle subesophageal masses (see Summary Fig. 6).
Markers of neurogenetic territories
Acetylated alpha-tubulin-positive processes are prominent in the palliovisceral territories, in the pedal cord close to the statocysts, and in the optic lobes and retina, but not in the cerebral cord (Fig. 9e–h), raising the possibility of an asynchronous maturation of neuronal cells and processes, reminiscent of that described for neurogenesis in the cuttlefish Sepia officinalis [46, 47]. In the Buresi et al. report , the expression of Sof-elav1 in S. officinalis embryos showed an asynchronous maturation of neurons across different brain centers. The Sof-elav1 expression appeared first in the palliovisceral centers, and only subsequently in the cerebral and optic lobe territories . Our findings on octopus acetylated alpha-tubulin immunoreactivity do indicate asynchrony in neuronal development between the cerebral and pedal cords. However, in contrast to the cuttlefish study, we identified a coincident onset of ELAV expression for the cerebral and palliovisceral cords at stage 7, suggesting that the timing of neurogenesis is different in these species, with postmitotic events in the octopus proceeding rapidly during stage 8.
Our study of neurogenetic gene expression in octopus embryo wholemounts indicates that the anlagen of the octopus brain follow a multi-cordal plan rather than one of multiple spherical ganglia such as those seen in the adult nervous system architecture of gastropod species, such as Aplysia, Lymnaea, and Helix . This finding, together with the cord-like organization postulated recently for Nautilus embryos , strongly suggests that a multiple cord origin for the cephalopod brain is a shared feature of extant cephalopods. Recent molecular phylogenetic studies establish a close relationship between cephalopods and monoplacophorans [29, 49, 50] and ratify fossil evidence that cephalopods evolved from a monoplacophoran-like mollusc [6, 51]. The cord-like organization of the embryonic cephalopod nervous system can therefore be directly compared with the cerebral, palliovisceral (or pleurovisceral) and pedal medullary cords described in embryonic and adult monoplacophoran and polyplacophoran nervous systems [52–59]. One compelling interpretation of these data is that cephalopods, despite the large size and compact organization of their brains, have retained an ancestral molluscan nervous system plan of multiple neural cords [55, 57, 59], and that this plan is particularly conspicuous in the embryo.
Octopus bimaculoides ELAV, NEUROD and NEUROG sequences are deposited as GenBank FJ861207, KR153584 and HM369392.
We employ the terms “cords” and “ganglia” as defined by Richter et al. . Neural structures are cords if they are organized as longitudinally and transversely distributed neuronal somata; ganglia are tightly packed, clearly demarcated clusters of neuronal somata with centrally placed neuropils. The central nervous system of chitons, for example, is considered to be composed of a primitive type of neural cord, lacking true ganglia , as illustrated in Fig. 1 Left Lower Panel.
This research was supported by a Research Abroad Award of the Japanese Society for the Promotion of Science to SS and by funding from the NSF (IOS-1354898) to CWR.
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