Neural Development of Cephalopods

Why Cephalopods?

Cephalopods are excellent models for studying neural development in higher vertebrates, especially humans, because they display relatively high complexity of the nervous system among invertebrates (Budelmann 1995). They also have enlarged axons, which makes it easier to observe and study.

Questions to ask:

1.  What are the stages of neural development?

2. How does this development occur?

3. How can this information be applied to higher vertebrates and humans?

Picture 1 (Left). Sepioteuthis lessoniana

Picture 2. Loligo edulis (Right)

Two of the species of squids used in the 2001 Shigeno studies of embryonic neural development.

The Shigeno lab has extensively studied the neural development during embryogenesis of cephalopods, particularly in sepiolids, which can be broken down into three basic phases (Shigeno 2001):

(1) Accumulation of the ganglia: After the differentiation of the eyes, neuroblasts separate from the ectoderm, migrate, and accumulate, forming four pairs of cell clusters without yet any defined axons or dendrites.

(2) Differentiation of the lobes: These four clusters of ganglia then undergo differentiation into three different masses (subesophageal, periesophageal, and supraesophageal) which will further differentiate into the cerebral, visceral, pedal, and optic lobes. The neuropils, dense networks of axons, dendrites, and glial branches, first form in the ganglia of the subesophageal and periesophageal masses, then in the basal lobe system, then the interior frontal lobes and superior frontal and vertical lobe systems.

Figure 1. A diagram of the ganglia cell clusters in different stages of development.

(3) Growth of the neuropils: In this phase, the lobes start to form the shapes of the developed brain and become more distinct. There is growth of the neuropils, especially in the vertical lobe.

A proposed explanation of the difference in the timing of the formation of the neuropils for different areas of the brain is due to function (Shigeno 2001). For example, in the study conducted by the Shigeno lab, the subesophageal mass grew faster than any other parts of the brain. In its early postembryonic stages, the sepiolid depends on the lower motor centers of the subesophageal area for swimming, while any other motor activity or learning processes are developed later on (Segowa et. al 1988). Therefore, the functional necessities of movement and respiration is developed before learning and memory is.

Figure 3. Diagrams of the S. officinalis embryos through organogenesis (Baratte 2009)

However, one of the weaknesses with the initial Shigeno studies was that the process of how the neuroblasts enter and accumulate in the ganglionic accumulation phase could not be determined precisely, because it was difficult to cut the serial sections of the embryos that had large yolk masses. Also, how the ganglia form connections precisely with each other could not be determined through the studies either. These observations were mostly taken from histological observations with scanning electron microscopes, so the Shigeno studies can answer the questions “What happens?” and “In what order does development occur” but not necessarily answer all the questions of “How do these processes occur?”

Figure 2. Tubulin immunostaining throughout stages of development in Baratte's study. (Baratte 2009)

The Baratte lab took another approach in 2009 and studied the these processes by using in toto immunostainings to mark against alpha tubulin antibodies and tyrosine hydroxylase, enzymes known to catalyze differentiation, to study different stages of neural development in Sepia officinalis embryos. Embryogenesis in this species is about 30 days long (Lemaire 1970). First, after fertilization, a discoidal cleavage occurred at the animal pole of the egg. Then, the potential mantle starts to delineate as an invagination occurs centrally and initiates the shell sac. It is not until the mantle region is established that the embryonic ganglia converge and differentiate into various brain lobes. Two main factors are responsible for starting the mantle network, the stellate ganglia, which attracts the nervous processes including sensory and motor, and the bilateral symmetry line, which repulses and attracts growing axons. This was the first study to show that although first thought to differentiate after lobes form, the neural ganglia actually differentiate before being fused into prospective lobes in the brain (Baratte 2009).

What research has been done on what’s responsible for path of development?

Unfortunately, there is still lots of research left to be done on the specific functional roles of certain neurotransmitters and genes that are responsible for the neural development pathway.

Wolleson has conducted studies with the expression of two neurotransmitters, serotonin (5-HT-ir) and FMRFamide in the CNS during embryogenesis (Wolleson 2010). However, even with these studies, it is not perfectly clear of the specific function that these play in the neural development process.  It is hypothesized that because these neurotransmitters are involved in cognitive processes in the adult brain, such as learning, sleep modulation, sensory processing, circadian rhythms, etc. that these do not directly play a role in the development of the CNS but rather in the post embryonic development processes. Further research is continuing in this area.

Pax 6 is an identified gene known to be expressed at early stages of the embryo and is thought to contribute to the development of cerebral and optic ganglia but functionally not known in too much detail until further research is done (Genoscope 2011) .

Why is this significant?

The importance of research into the neural development of cephalopods involves the ability to study various degenerative diseases and neuromuscular disorders on a molecular level, and to identify the differences and similarities during evolution in development of neural structures. Expression of several families of neurotransmitters and neuronal genes are conserved across various species of cephalopods as well as seen in higher vertebrates (Farfan 2009).


Baratte, S, and L Bonnaud. “Evidence of Early Nervous Differentiation and Early Catecholaminergic Sensory System During Sepia Officinalis Embryogenesis.” The Journal of Comparative Neurology, 517.4 (2009): 539-549.

Budelmann, B.U. “The cephalopod nervous system: What evolution has made of the molluscandesign”. In: O. Breidbach and W. Kutsch (eds.). The nervous system of invertebrates: an evolutionaryand comparative approach. Birkhäuser, Basel. (1995): 115–138.

“ESTs Library of Sepia Officinalis Embryos.” Site Du Genoscope. Genoscope. Web. 02 May 2011. <>.

Farfán, Claudia, Shuichi Shigeno, Marie-Therese Nödl, and H Gert de Couet. “Developmental Expression of Apterous/Lhx2/9 in the Sepiolid Squid Euprymna Scolopes Supports an Ancestral Role in Neural Development.”Evol Dev, 11.4: 354.

Lemaire J. 1970. Table de developpement embryonnaire de Sepia officinalis. L. (Mollusque Ce´ phalopode). Bull Soc Zool 95:773–782.

Segawa S, Yang WT, Marthy HJ, Hanlon RT. 1988. Illustrated embryonicstages of the Eastern Atlantic squid Loligo forbesi. Veliger 30:230–243.

Shigeno, S, K Tsuchiya, and S Segawa. “Embryonic and Paralarval Development of the Central Nervous System of the Loliginid Squid Sepioteuthis Lessoniana.”J Comp Neurol, 437.4 (2001): 449.

Shigeno, Shuichi, Takenori Sasaki, Takeya Moritaki, Takashi Kasugai, Michael Vecchione, and Kiyokazu Agata. “Evolution of the Cephalopod Head Complex by Assembly of Multiple Molluscan Body Parts: Evidence from Nautilus Embryonic Development.” J Morphol, 269.1 (2008): 1.


Wollesen, Tim, Bernard M Degnan, and Andreas Wanninger. “Expression of Serotonin (5-HT) During CNS Development of the Cephalopod Mollusk, Idiosepius Notoides.” Cell Tissue Res, 342.2 (2010): 161.

Wollesen, T, SF Cummins, BM Degnan, and A Wanninger. “FMRFamide Gene and Peptide Expression During Central Nervous System Development of the Cephalopod Mollusk, Idiosepius Notoides.” Evolution & Development, 12.2 (2010): 113-130.

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